>1.NF00132420 PMID:11856887 TI - Expression pattern and further characterization of human MAGED2 and identification of rodent orthologues. AB - In a search for genes involved in X-linked mental retardation we have analyzed the expression pattern and genomic structure of human MAGED2. This gene is a member of a new defined MAGE-D cluster in Xp11.2, a hot spot for X-linked mental retardation. Rat and mouse orthologues have been isolated. In contrast to the genes of the MAGE-A, MAGE- B and MAGE-C clusters, MAGED2 is expressed ubiquitously. High expression was detected in specific brain regions and in the interstitium of testes. Five SNPs in the coding region of human MAGED2 were characterized and their allele frequencies determined in a German and Turkish population. >2.NF00118002 PMID:12077706 TI - Mutations in the cone photoreceptor G protein alpha-subunit gene GNAT2 in patients with achromatopsia. AB - Achromatopsia is an autosomal recessively inherited visual disorder that is present from birth and that features the absence of color discrimination. We here report the identification of five independent families with achromatopsia that segregate protein-truncation mutations in the GNAT2 gene, located on chromosome 1p13. GNAT2 encodes the cone photoreceptor-specific alpha-subunit of transducin, a G-protein of the phototransduction cascade, which couples to the visual pigment(s). Our results demonstrate that GNAT2 is the third gene implicated in achromatopsia. >3.NF00118005 PMID:12149458 TI - Chimeric human CstF-77/Drosophila Suppressor of forked proteins rescue suppressor of forked mutant lethality and mRNA 3' end processing in Drosophila. AB - The Suppressor of forked [Su(f)] protein is the Drosophila homologue of CstF-77, a subunit of human cleavage stimulation factor (CstF) that is required for the first step of the mRNA 3' end processing reaction in vitro. We have addressed directly the role of su(f) in the mRNA 3' end processing reaction in vivo. We show that su(f) is required for the cleavage of pre-mRNA during mRNA 3' end formation. Analysis of the functional complementation between Su(f) and CstF-77 shows that most of the Drosophila protein (85%) can be exchanged for the human protein to produce chimeric CstF-77/Su(f) proteins that rescue lethality and cleavage defect during mRNA 3' end formation in su(f) mutants. Interestingly, we show that a domain in human CstF-77 is limiting for the rescue and that this domain is not able to reproduce protein interactions with the CstF subunits of Drosophila. We also show that chimeric CstF-77/Su(f) proteins that rescue lethality of su(f) mutants cannot restore utilization of a regulated poly(A) site in Drosophila. Taken together, these results demonstrate that CstF-77 and Su(f) have the same function in mRNA 3' end formation in vivo, but that these two proteins are not interchangeable for regulation of poly(A) site utilization. >4.NF00106180 PMID:12196012 TI - Identification of neurite outgrowth promoting sites on the laminin alpha 3 chain G domain. AB - Laminins are expressed in specific tissues and are involved in various biological activities including promoting cell adhesion, growth, migration, neurite outgrowth, and differentiation. The laminin alpha3 chain is mainly located in the skin and is also expressed in the floor plate of the developing neural tube. Previously, we showed that the human laminin alpha3 chain LG4 module binds to syndecan-2/4, a membrane-associated proteoglycan, and promotes human fibroblast adhesion. Here, we have evaluated the neurite outgrowth activity of the laminin alpha3 chain LG4 and LG5 modules. Three overlapping recombinant proteins, which contained LG4 and/or LG5 modules of the human laminin alpha3 chain, were prepared using a mammalian cell expression system. Two proteins, rec-alpha3LG4-5 and rec-alpha3LG4, promoted cell attachment and neurite outgrowth of rat pheochromocytoma PC12 cells, but rec-alpha3LG5 was inactive. Twenty-two peptides covering the entire LG4 module were synthesized and tested for cell attachment and neurite outgrowth activity to identify active sites of the LG4 module. A3G75 (KNSFMALYLSKG, alpha3 chain 1411-1422) and A3G83 (GNSTISIRAPVY, alpha3 chain 1476-1487) promoted PC12 cell attachment and neurite outgrowth. Additionally, A3G75 and A3G83 inhibited PC12 cell attachment to rec-alpha3LG4. These results suggest that the A3G75 and A3G83 sites are important for PC12 cell attachment and neurite outgrowth in the laminin alpha3 chain LG4 module. We also conjugated the A3G75 and A3G83 peptides on chitosan membranes to test their potential as bio-materials. These peptide-conjugated chitosan membranes were more active for neurite outgrowth than the peptide-coated plates. These results suggest that the A3G75- and A3G83-conjugated chitosan membranes are applicable as bio-medical materials for neural tissue repair and engineering. >5.NF00133153 PMID:12067979 TI - Aberrant methylation of the CDH13 (H-cadherin) promoter region in colorectal cancers and adenomas. AB - Expression of the cadherin family member CDH13 (H-cadherin) is reduced in several human tumors, and it has been hypothesized that this gene functions as a tumor suppressor gene. Previously, we reported that the 5' region of CDH13 is frequently methylated in breast and lung cancers. Here we confirmed the promoter activity of 5' region of CDH13 by luciferase assay and examined its aberrant methylation in colorectal cancers, cell lines, and adenomas. Methylation status was investigated by methylation-specific PCR (MSP) and by bisulfite DNA sequencing of cloned DNA of PCR amplicons. In cell lines, we examined the correlation between methylation status and mRNA expression by reverse transcription-PCR. Aberrant methylation of CDH13 was present in 7 of 13 (54%) cell lines, and expression was absent in 6 of 13 (46%) cell lines. CDH13 expression was present in six cell lines that showed only the unmethylated form by MSP and in one cell line that showed both the methylated and unmethylated forms. Treatment with 5-aza-2'-deoxycytidine restored CDH13 expression in methylated cell lines. In surgically resected samples, 17 of 35 (49%) cases of primary colorectal cancer, 2 of 33 (6%) cases of corresponding nonmalignant colorectal mucosa, and 8 of 19 (42%) adenomas were methylated. Sequence data after bisulfite treatment indicated that primary cancers and two cell lines with loss of expression were highly methylated compared with nonmalignant colorectal epithelial cells, especially at the attachment sites of primers for MSP, although there was heterogeneity in methylation status. Our results suggest that CDH13 expression is frequently silenced by aberrant methylation in colorectal cancers and adenomas and that methylation of CDH13 commences at an early stage of multistep colorectal tumorigenesis. >6.NF00106425 PMID:11836625 TI - RFX-B, a MHC class II transcription factor, suppressed in human colorectal adenocarcinomas. AB - Regulatory factor X (RFX) is an essential MHC class II transcription factor and contains three distinct subunits of which RFX-B is one. Aberrant expression of MHC class II genes is associated with autoimmunity, tumour growth and failure to mount an immune response. RFX-B protein expression in human colorectal adenocarcinomas and in normal adjacent tissue was analysed in this study. Western blot analysis showed a suppression of nuclear RFX-B protein in the tumour tissue. Immunohistochemistry revealed that the RFX-B protein levels in macrophages were generally lower in colorectal cancerous tissue compared to adjacent non-cancerous tissue and that focally and not frequently tumour and normal epithelial cells were stained weakly for RFX-B. As the expression of MHC class II correlates with the intensity of the immune response system these results may support the idea that cancer is associated with immunodeficiency and that low levels of RFX-B in interstitial macrophages could partly explain this thesis. >7.NF00123516 PMID:12037021 TI - Mutation analysis of the PIG-A gene in Korean patients with paroxysmal nocturnal haemoglobinuria. AB - AIM: Paroxysmal nocturnal haemoglobinuria (PNH) is caused by deficient biosynthesis of the glycosylphosphatidylinositol (GPI) anchor in haemopoietic stem cells. Mutation of the phosphatidylinositol glycan class A (PIG-A) gene, an X linked gene that participates in the first step of GPI anchor biosynthesis, is responsible for PNH. The characteristics of somatic mutation of the PIG-A gene in Korean patients with PNH were studied. METHODS: Twenty four patients with PNH were selected. Ham tests and sucrose haemolysis tests were carried out on all patients. The expression of CD59 in erythrocytes and granulocytes was investigated in 14 and five patients, respectively, to confirm the diagnosis. Dideoxy fingerprinting (ddF) was used to screen mutations, and direct sequencing of DNA was performed to characterise the mutations. RESULTS: Gene mutation was detected in 12 of the 24 patients. The other 12 patients were negative in ddF screening. Ten new mutations and two known mutations were detected. The mutations consisted of five deletions, six substitutions, and one insertion. These mutations resulted in six premature terminations, three abnormal splicings, one missense mutation in exon 2, and two nonsense mutations. Two patients with venous thrombosis showed mutations in exon 3 only. Substitution mutations were seen in six patients and frameshift mutations in the other six. CONCLUSIONS: There were 10 new mutations among the 12 mutations in the Korean patients with PNH and the characteristics of the mutations varied, with no significant hot spots in sites or types. >8.NF00013762 PMID:12223399 TI - COP9 signalosome subunits 4 and 5 regulate multiple pleiotropic pathways in Drosophila melanogaster. AB - The COP9 signalosome (CSN) is an essential eight-subunit repressor of light-regulated development in ARABIDOPSIS: This complex has also been identified in animals, though its developmental role remains obscure. CSN subunits have been implicated in various cellular processes, suggesting a possible role for the CSN as an integrator of multiple signaling pathways. In order to elucidate the function of the CSN in animals, a Drosophila model system has previously been established. Gel-filtration analysis with antibodies against CSN subunits 4, 5 and 7 revealed that these proteins act as a complex in Drosophila that is similar in size to the plant and mammalian complexes. Null mutations in either one of two subunits, CSN4 or CSN5, are larval lethal. Successful embryogenesis appears to be a consequence of maternal contribution of the complex. Biochemical analysis indicates that the different subunits are found in both CSN-dependent and CSN-independent forms, and that these forms are differentially affected by the mutations. Phenotypic characterization of these two mutants indicates that they show both shared and unique phenotypes, which suggest specific roles for each subunit. Both mutants have defective oocyte and embryo patterning, and defects in response to DNA damage, while csn5 mutants develop melanotic tumors and csn4 mutants have phenotypes reminiscent of defects in ecdysone signaling. >9.NF00124085 PMID:11926999 TI - Identification of a domain conferring nucleotide binding to the N-acetyl-d-glucosamine 2-epimerase (Renin binding protein). AB - Renin binding protein (RnBP), a cellular renin inhibitor, has been identified as the enzyme N-acetyl-D-glucosamine (GlcNAc) 2-epimerase. Our recent studies demonstrated that rat GlcNAc 2-epimerase has a ten-times higher affinity for ATP, dATP, and ddATP than the human enzyme [Takahashi, S. et al. (2001) J. Biochem. 130, 815-821]. To identify the domain conferring nucleotide binding to GlcNAc 2-epimerase, we constructed a series of chimeric enzymes successively replacing the three domains of the human enzyme (N-terminal, middle, and C-terminal domains) with the corresponding domains of the rat enzyme. Chimeras were expressed in Escherichia coli JM109 cells under the control of the Taq promoter. The purified chimeric enzymes had GlcNAc 2-epimerase activity and inhibited renin activity in a dose-dependent manner. The recombinant human and rat enzymes required catalytic amounts of ATP with apparent K(m) values of 73 and 5.5 microM, respectively. Chimeric enzymes of HHR, RHH, and RHR (H, human type domain; R, rat type domain) had nearly the same nucleotide specificity as the human GlcNAc 2-epimerase. On the other hand, HRR, HRH, and RRH chimeras had the same nucleotide specificity as the rat enzyme. These results indicate that the middle domain of the GlcNAc 2-epimerase molecule participates in the specificity for and binding of nucleotides, and that nucleotides are essential to form the catalytic domain of the enzyme. >10.NF00132428 PMID:12409310 TI - alpha 1-Adrenergic receptor subtypes differentially control the cell cycle of transfected CHO cells through a cAMP-dependent mechanism involving p27Kip1. AB - Three distinct subtypes of alpha(1)-adrenergic receptors (alpha(1)A-, alpha(1)B-, and alpha(1)D-AR) play a prominent role in cell growth. However, little is known about subtype-specific effects on cell proliferation. The activation of alpha(1)A- or alpha(1)B-AR inhibits serum-promoted cell proliferation, whereas alpha(1)D-AR activation does not show such an inhibitory effect. Notably, cell-cycle progression was blocked at G(1)/S transition after activation of alpha(1)A/alpha(1)B-AR but not of alpha(1)D-AR. In agreement with the differential cell proliferation effect, cAMP production was increased after activation of alpha(1)A/alpha(1)B-AR but not alpha(1)D-AR, whereas all alpha(1)-AR subtypes are associated with inositol 1,4,5-trisphosphate production and mitogen-activated protein kinase activation in a similar fashion. Furthermore, the serum-induced reduction in the levels of the cyclin-dependent kinase inhibitor, p27(Kip1), was blocked after activation of alpha(1)A/alpha(1)B-AR but not alpha(1)D-AR. These results show that alpha(1)-AR subtypes differentially activate the cAMP/p27(Kip1) pathway and thereby have differential inhibitory effects on cell proliferation. Subtype-dependent effects should be taken into consideration when assessing the physiological response of native cells where alpha(1)-AR subtypes are generally co-expressed. >11.NF00088410 PMID:12060656 TI - Distinct sets of adjacent heterogeneous nuclear ribonucleoprotein (hnRNP) A1/A2 binding sites control 5' splice site selection in the hnRNP A1 mRNA precursor. AB - In the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 pre-mRNA, different regions in the introns flanking alternative exon 7B have been implicated in the production of the A1 and A1B mRNA splice isoforms. Among these, the CE1a and CE4 elements, located downstream of common exon 7 and alternative exon 7B, respectively, are bound by hnRNP A1 to promote skipping of exon 7B in vivo and distal 5' splice site selection in vitro. Here, we report that CE1a is flanked by an additional high affinity A1 binding site (CE1d). In a manner similar to CE1a, CE1d affects 5' splice site selection in vitro. Consistent with a role for hnRNP A1 in the activity of CE1d, a mutation that abrogates A1 binding abolishes distal 5' splice site activation. Moreover, the ability of CE1d to stimulate distal 5' splice site usage is lost in an HeLa extract depleted of hnRNP A/B proteins, and the addition of recombinant A1 restores the activity of CE1d. Notably, distal 5' splice site selection mediated by A1 binding sites is not compromised in an extract prepared from mouse cells that are severely deficient in hnRNP A1 proteins. In this case, we show that hnRNP A2 compensates for the A1 deficiency. Further studies with the CE4 element reveal that it also consists of two distinct portions (CE4m and CE4p), each one capable of promoting distal 5' splice site use in an hnRNP A1-dependent manner. The presence of multiple A1/A2 binding sites downstream of common exon 7 and alternative exon 7B probably plays an important role in maximizing the activity of hnRNP A1/A2 proteins. >12.NF00106186 PMID:11884632 TI - Human Rad54B is a double-stranded DNA-dependent ATPase and has biochemical properties different from its structural homolog in yeast, Tid1/Rdh54. AB - The RAD52 epistasis group genes are involved in homologous recombination, and they are conserved from yeast to humans. We have cloned a novel human gene, RAD54B, which is homologous to yeast and human RAD54. Human Rad54B (hRad54B) shares high homology with human Rad54 (hRad54) in the central region containing the helicase motifs characteristic of the SNF2/SWI2 family of proteins, but the N-terminal domain is less conserved. In yeast, another RAD54 homolog, TID1/RDH54, plays a role in recombination. Tid1/Rdh54 interacts with yeast Rad51 and a meiosis-specific Rad51 homolog, Dmc1. The N-terminal domain of hRad54B shares homology with that of Tid1/Rdh54, suggesting that Rad54B may be the human counterpart of Tid1/Rdh54. We purified the hRad54 and hRad54B proteins from baculovirus-infected insect cells and examined their biochemical properties. hRad54B, like hRad54, is a DNA-binding protein and hydrolyzes ATP in the presence of double-stranded DNA, though its rate of ATP hydrolysis is lower than that of hRad54. Human Rad51 interacts with hRad54 and enhances its ATPase activity. In contrast, neither human Rad51 nor Dmc1 directly interacts with hRad54B. Although hRad54B is the putative counterpart of Tid1/Rdh54, our findings suggest that hRad54B behaves differently from Tid1/Rdh54. >13.NF00113477 PMID:12044248 TI - Significance of the parkin gene and protein in understanding Parkinson's disease. AB - Mutations in the parkin gene cause autosomal recessive inherited juvenile parkinsonism (ARJP) and account for the majority of cases of inherited Parkinson's disease (PD) of young onset (<45 years of age). Patients with parkin mutations commonly have atypical clinical features such as dystonia at onset, hyper-reflexia, diurnal fluctuations, and sleep benefit; however, parkin mutation patients with both typical PD symptoms and older age of onset have been identified. Parkin is a ubiquitin protein ligase (E3), a component in the pathway that attaches ubiquitin to specific proteins, designating them for degradation by the proteasome. Several substrates for parkin have been identified (CDCrel-1, o-glycosylated alpha-synuclein, parkin associated endothelin-like cell receptor, and synphilin). The role of these substrates in the pathogenesis of ARJP is under active study. Most patients with parkin mutations lack Lewy bodies, suggesting that functional parkin is involved in the formation of these highly ubiquitinated inclusions. Furthermore, the recognition that parkin mutations can lead to a disorder clinically similar to sporadic PD, but presumably lacking Lewy bodies, calls into question the necessity of Lewy bodies for the diagnosis of PD and nigral cell death. Studies of parkin are increasing the focus on the role of the ubiquitin-proteasome system in the pathogenesis of both familial and sporadic PD. >14.NF00101975 PMID:12027902 TI - Interaction between p21-activated protein kinase and Rac during differentiation of HL-60 human promyelocytic leukemia cell induced by all-trans-retinoic acid. AB - Undifferentiated human promyelocytic leukemia HL-60 cells show little or no superoxide production, but generate a very low O(2)(-) concentration upon incubation with all-trans-retinoic acid (ATRA). Its production reaches a maximum within 20 h, and thereafter is maintained at an almost constant level. The differentiated cells show phorbol 12-myristate 13-acetate (PMA)-stimulated NADPH oxidase activity consistent with the amount of gp91phox (phagocytic oxidase) expressed in the plasma membrane. Three isoforms of p21-activated serine/threonine kinases, PAK68, PAK65 and PAK62, were found in both cytosolic and membrane fractions, and their contents were significantly increased during induced differentiation. The amount of Rac identified in the two fractions was also markedly enhanced by ATRA- induced differentiation. In contrast, neither PAK nor Rac was seen in the plasma membrane of undifferentiated HL-60 or human neutrophil, but they were abundant in the cytoplasmic fraction. Binding of Rac with PAK isoforms was shown in the membrane upon induced differentiation of HL-60 cells. Direct binding of purified Rac1 to PAK68 was quantified using a fluorescent analog of GTP (methylanthraniloyl guanosine-5'-[beta,gamma-imido]triphosphate) bound to Rac as a reporter group. Rac1 bound to PAK68 with a 1 : 1 stoichiometry and with a K(d) value of 6.7 nm. >15.NF01014359 PMID:12244172 TI - Evidence for involvement of a hydrophobic patch in framework region 1 of human V4-34-encoded Igs in recognition of the red blood cell I antigen. AB - The monoclonal IgM cold agglutinins that bind to the I/i carbohydrate Ags on the surface of RBCs all have Ig H chains encoded by the V4-34 gene segment. This mandatory use indicates that distinctive amino acid sequences may be involved in recognition. Critical amino acids exist in framework region 1 (FR1) of V4-34-encoded Ig, and these generate a specific Id determinant which apparently lies close to the I binding site. However, I binding by Id-expressing Ig can be modulated by sequences in complementarity-determining region (CDR)(H)3. Examination of the crystal structure of an anti-I cold agglutinin has revealed a hydrophobic patch in FR1 involving residue W7 on beta-strand A and the AVY motif (residues 23-25) on beta-strand B. In this study we used mutagenesis to show that each of the strand components of the hydrophobic patch is required for binding the I carbohydrate Ag. In addition, the crystal structure reveals that amino acids in the carboxyl-terminal region of CDR(H)3 form a surface region adjacent to the hydrophobic patch. We propose that the I carbohydrate Ag interacts simultaneously with the entire hydrophobic patch in FR1 and with the outside surface of CDR(H)3. This interaction could leave most of the conventional binding site available for binding other Ags. >16.NF00089060 PMID:11743730 TI - Solution structures of the YAP65 WW domain and the variant L30 K in complex with the peptides GTPPPPYTVG, N-(n-octyl)-GPPPY and PLPPY and the application of peptide libraries reveal a minimal binding epitope. AB - The single mutation L30 K in the Hu-Yap65 WW domain increased the stability of the complex with the peptide GTPPPPYTVG (K(d)=40(+/-5) microM). Here we report the refined solution structure of this complex by NMR spectroscopy and further derived structure-activity relationships by using ligand peptide libraries with truncated sequences and a substitution analysis that yielded acetyl-PPPPY as the smallest high-affinity binding peptide (K(d)=60 microM). The structures of two new complexes with weaker binding ligands chosen based on these results (N-(n-octyl)-GPPPYNH(2) and Ac-PLPPY) comprising the wild-type WW domain of Hu-Yap65 were determined. Comparison of the structures of the three complexes were useful for identifying the molecular basis of high-affinity: hydrophobic and specific interactions between the side-chains of Y28 and W39 and P5' and P4', respectively, and hydrogen bonds between T37 (donnor) and P5' (acceptor) and between W39 (donnor) and T2' (acceptor) stabilize the complex.The structure of the complex L30 K Hu-Yap65 WW domain/GTPPPPYTVG is compared to the published crystal structure of the dystrophin WW domain bound to a segment of the beta-dystroglycan protein and to the solution structure of the first Nedd4 WW domain and its prolin-rich ligand, suggesting that WW sequences bind proline-rich peptides in an evolutionary conserved fashion. The position equivalent to T22 in the Hu-Yap65 WW domain sequence is seen as responsible for differentiation in the binding mode among the WW domains of group I. >17.NF00114287 PMID:12606567 TI - Attenuation of cell adhesion in lymphocytes is regulated by CYTIP, a protein which mediates signal complex sequestration. AB - An important theme in molecular cell biology is the regulation of protein recruitment to the plasma membrane. Fundamental biological processes such as proliferation, differentiation or leukocyte functions are initiated and controlled through the reversible binding of signaling proteins to phosphorylated membrane components. This is mediated by specialized interaction modules, such as SH2 and PH domains. Cytohesin-1 is an intracellular guanine nucleotide exchange factor, which regulates leukocyte adhesion. The activity of cytohesin-1 is controlled by phospho inositide-dependent membrane recruitment. An interacting protein was identified, the expression of which is upregulated by cytokines in hematopoietic cells. This molecule, CYTIP, is also recruited to the cell cortex by integrin signaling via its PDZ domain. However, stimulation of Jurkat cells with phorbol ester results in re-localization of CYTIP to the cytoplasm, and membrane detachment of cytohesin-1 strictly requires co-expression of CYTIP. Consequently, stimulated adhesion of Jurkat cells to intracellular adhesion molecule-1 is repressed by CYTIP. These findings outline a novel mechanism of signal chain abrogation through sequestration of a limiting component by specific protein-protein interactions. >18.NF00102867 PMID:12198491 TI - Lon protease preferentially degrades oxidized mitochondrial aconitase by an ATP-stimulated mechanism. AB - Mitochondrial aconitase is sensitive to oxidative inactivation and can aggregate and accumulate in many age-related disorders. Here we report that Lon protease, an ATP-stimulated mitochondrial matrix protein, selectively recognizes and degrades the oxidized, hydrophobic form of aconitase after mild oxidative modification, but that severe oxidation results in aconitase aggregation, which makes it a poor substrate for Lon. Similarly, a morpholino oligodeoxynucleotide directed against the lon gene markedly decreases the amount of Lon protein, Lon activity and aconitase degradation in WI-38 VA-13 human lung fibroblasts and causes accumulation of oxidatively modified aconitase. The ATP-stimulated Lon protease may be an essential defence against the stress of life in an oxygen environment. By recognizing minor oxidative changes to protein structure and rapidly degrading the mildly modified protein, Lon protease may prevent extensive oxidation, aggregation and accumulation of aconitase, which could otherwise compromise mitochondrial function and cellular viability. Aconitase is probably only one of many mitochondrial matrix proteins that are preferentially degraded by Lon protease after oxidative modification. >19.NF00078531 PMID:11745043 TI - Role of multidrug resistance 3 deficiency in pediatric and adult liver disease: one gene for three diseases. AB - Class III multidrug resistance P-glycoproteins, mdr2 in mice and MDR3 in humans, are canalicular phospholipid translocators involved in biliary phospholipid (phosphatidylcholine) excretion. The role of an MDR3 gene defect in liver disease was initially suspected in a subtype of progressive familial intrahepatic cholestasis called PFIC3. Several MDR3 mutations have been identified in children with PFIC3 and are associated with a low level of phospholipids in bile, leading to a high biliary cholesterol saturation index. Mutations leading to a truncated protein are associated with an absence of canalicular MDR3 protein. The phenotypic spectrum of PFIC3 ranges from neonatal cholestasis to cirrhosis in young adults. There is now strong evidence that in addition to PFIC3, an MDR3 defect can be involved in intrahepatic cholestasis of pregnancy and in cholesterol gallstone disease. Therefore, at least three human liver diseases are due to a single gene deficiency. Patients with PFIC3 due to MDR3 deficiency may benefit from ursodeoxycholic acid therapy and could be good candidates for cell therapy in the future. >20.NF00089225 PMID:11558822 TI - Primary congenital glaucoma: three case reports on novel mutations and combinations of mutations in the GLC3A (CYP1B1) gene. AB - PURPOSE: To describe three patients with congenital glaucoma homozygous and compound heterozygous for different mutations and benign sequence variants in the cytochrome P 450 1B1 (CYP1B1) gene. METHODS: All patients were examined by slit-lamp biomicroscopy, gonioscopy, measurement of the cornea and optic disc, ultrasound biometry, and automated static threshold perimetry when possible. Direct sequence analysis was performed on DNA extracted from peripheral blood from the patients and their parents. RESULTS: For patient 1, a newborn boy with buphthalmos and an opaque cornea, a novel homozygous C/T transition in codon 355 (CGA>TGA) led to a predicted nonsense codon Arg355X truncating the protein by 188 amino acids. For patient 2, a 24-year-old man, a compound heterozygous mutation 1410-1422del/1546-1555dup was found. For patient 3, a 34-year-old man, two novel heterozygous missense mutations resulting in an Ala443Gly and a Glu229Lys amino acid exchange and five benign sequence variants were found. CONCLUSION: Our results confirm the crucial role of CYP1B1 mutations for congenital glaucoma. >21.NF00982620 PMID:12019563 TI - Photoreceptor renewal: a role for peripherin/rds. AB - Visual transduction begins with the detection of light within the photoreceptor cell layer of the retina. Within this layer, specialized cells, termed rods and cones, contain the proteins responsible for light capture and its transduction to nerve impulses. The phototransductive proteins reside within an outer segment region that is connected to an inner segment by a thin stalk rich in cytoskeletal elements. A unique property of the outer segments is the presence of an elaborate intracellular membrane system that holds the phototransduction proteins and provides the requisite lipid environment. The maintenance of normal physiological function requires that these postmitotic cells retain the unique structure of the outer segment regions--stacks of membrane saccules in the case of rods and a continuous infolding of membrane in the case of cones. Both photoreceptor rod and cone cells achieve this through a series of coordinated steps. As new membranous material is synthesized, transported, and incorporated into newly forming outer segment membranes, a compensatory shedding of older membranous material occurs, thereby maintaining the segment at a constant length. These processes are collectively referred to as ROS (rod outer segment) or COS (cone outer segment) renewal. We review the cellular and molecular events responsible for these renewal processes and present the recent but compelling evidence, drawn from molecular genetic, biochemical, and biophysical approaches, pointing to an essential role for a unique tetraspanning membrane protein, called peripherin/rds, in the processes of disk morphogenesis. >22.NF00095628 PMID:12060708 TI - A role for ASIC3 in the modulation of high-intensity pain stimuli. AB - Acid-sensing ion channel 3 (ASIC3), a proton-gated ion channel of the degenerins/epithelial sodium channel (DEG/ENaC) receptor family is expressed predominantly in sensory neurons including nociceptive neurons responding to protons. To study the role of ASIC3 in pain signaling, we generated ASIC3 knockout mice. Mutant animals were healthy and responded normally to most sensory stimuli. However, in behavioral assays for pain responses, ASIC3 null mutant mice displayed a reduced latency to the onset of pain responses, or more pain-related behaviors, when stimuli of moderate to high intensity were used. This unexpected effect seemed independent of the modality of the stimulus and was observed in the acetic acid-induced writhing test (0.6 vs. 0.1-0.5%), in the hot-plate test (52.5 and 55 vs. 50 degrees C), and in tests for mechanically induced pain (tail-pinch vs. von Frey filaments). We postulate that ASIC3 is involved in modulating moderate- to high-intensity pain sensation. >23.NF00866395 PMID:11986327 TI - Cloning and characterization of human Siglec-11. A recently evolved signaling that can interact with SHP-1 and SHP-2 and is expressed by tissue macrophages, including brain microglia. AB - Siglecs are sialic acid-recognizing animal lectins of the immunoglobulin superfamily. We have cloned and characterized a novel human molecule, Siglec-11, that belongs to the subgroup of CD33/Siglec-3-related Siglecs. As with others in this subgroup, the cytosolic domain of Siglec-11 is phosphorylated at tyrosine residue(s) upon pervanadate treatment of cells and then recruits the protein-tyrosine phosphatases SHP-1 and SHP-2. However, Siglec-11 has several novel features relative to the other CD33/Siglec-3-related Siglecs. First, it binds specifically to alpha2-8-linked sialic acids. Second, unlike other CD33/Siglec-3-related Siglecs, Siglec-11 was not found on peripheral blood leukocytes. Instead, we observed its expression on macrophages in various tissues, such as liver Kupffer cells. Third, it was also expressed on brain microglia, thus becoming the second Siglec to be found in the nervous system. Fourth, whereas the Siglec-11 gene is on human chromosome 19, it lies outside the previously described CD33/Siglec-3-related Siglec cluster on this chromosome. Fifth, analyses of genome data bases indicate that Siglec-11 has no mouse ortholog and that it is likely to be the last canonical human Siglec to be reported. Finally, although Siglec-11 shows marked sequence similarity to human Siglec-10 in its extracellular domain, the cytosolic tail appears only distantly related. Analysis of genomic regions surrounding the Siglec-11 gene suggests that it is actually a chimeric molecule that arose from relatively recent gene duplication and recombination events, involving the extracellular domain of a closely related ancestral Siglec gene (which subsequently became a pseudogene) and a transmembrane and cytosolic tail derived from another ancestral Siglec. >24.NF00866396 PMID:11546873 TI - Role of the nonsense-mediated decay factor hUpf3 in the splicing-dependent exon-exon junction complex. AB - Nonsense-mediated messenger RNA (mRNA) decay, or NMD, is a critical process of selective degradation of mRNAs that contain premature stop codons. NMD depends on both pre-mRNA splicing and translation, and it requires recognition of the position of stop codons relative to exon-exon junctions. A key factor in NMD is hUpf3, a mostly nuclear protein that shuttles between the nucleus and cytoplasm and interacts specifically with spliced mRNAs. We found that hUpf3 interacts with Y14, a component of post-splicing mRNA-protein (mRNP) complexes, and that hUpf3 is enriched in Y14-containing mRNP complexes. The mRNA export factors Aly/REF and TAP are also associated with nuclear hUpf3, indicating that hUpf3 is in mRNP complexes that are poised for nuclear export. Like Y14 and Aly/REF, hUpf3 binds to spliced mRNAs specifically ( approximately 20 nucleotides) upstream of exon-exon junctions. The splicing-dependent binding of hUpf3 to mRNAs before export, as part of the complex that assembles near exon-exon junctions, allows it to serve as a link between splicing and NMD in the cytoplasm. >25.NF00779079 PMID:11597141 TI - Cloning and chromosomal localization of a gene encoding a novel serine/threonine kinase belonging to the subfamily of testis-specific kinases. AB - Using reverse transcription-polymerase chain reaction (RT-PCR) with degenerate oligonucleotides corresponding to two highly conserved motifs within the protein kinase family of catalytic domains, we isolated a PCR fragment encoding a novel member of the testis-specific serine/threonine kinases (STK) from mouse male mixed germ cell mRNA. This PCR fragment recognized a 1020-bp transcript in male germ cells by northern blot analysis and was used to clone a full-length cDNA from a mouse mixed germ cell cDNA library. This cDNA has an open reading frame of 804 bases encoding a protein of 268 amino acids. This novel gene is almost identical to Stk22c, encoding a recently described testis-specific protein kinase, except for base-pair deletions that result in a shift in the coding region and an alteration of 22 amino acids (residues 109-131). Due to its homology with Stk22c, we have called this protein kinase gene Stk22d. Northern blot analysis revealed that this protein kinase is developmentally expressed in testicular germ cells and is not present in brain, ovary, kidney, liver, or early embryonic cells. We then cloned the human homologue of this protein kinase gene (STK22C) and found it to be expressed exclusively in the testis. Fluorescence in situ hybridization with both the human and mouse cDNA clones revealed syntenic localization on chromosomes 1p34-p35 and 4E1, respectively. >26.NF00142150 PMID:12037680 TI - CK2-dependent phosphorylation of the E2 ubiquitin conjugating enzyme UBC3B induces its interaction with beta-TrCP and enhances beta-catenin degradation. AB - Protein kinase CK2 is a ubiquitous and pleiotropic Ser/Thr protein kinase involved in cell growth and transformation. Here we report the identification by yeast interaction trap of a CK2 interacting protein, UBC3B, which is highly homologous to the E2 ubiquitin conjugating enzyme UBC3/CDC34. UBC3B complements the yeast cdc34-2 cell cycle arrest mutant in S. cerevisiae and transfers ubiquitin to a target substrate in vitro. UBC3B is specifically phosphorylated by CK2 in vitro and in vivo. We mapped by deletions and site directed mutagenesis the phosphorylation site to a serine residue within the C-terminal domain in position 233 of UBC3B and in the corresponding serine residue of UBC3. Following CK2-dependent phosphorylation both UBC3B and UBC3 bind to the F-box protein beta-TrCP, the substrate recognition subunit of an SCF (Skp1, Cul1, F-box) ubiquitin ligase. Furthermore, we observed that co-transfection of CK2alpha' together with UBC3B, but not with UBC3DeltaC, enhances the degradation of beta-catenin. Taken together these data suggest that CK2-dependent phosphorylation of UBC3 and UBC3B functions by regulating beta-TrCP substrate recognition. >27.NF00126031 PMID:12239140 TI - Transgenic overexpression of human IL-17E results in eosinophilia, B-lymphocyte hyperplasia, and altered antibody production. AB - We have identified and cloned a novel human cytokine with homology to cytokines of the interleukin-17 (IL-17) family, which we have termed human IL-17E (hIL-17E). With the identification of several IL-17 family members, it is critical to understand the in vivo function of these molecules. We have generated transgenic mice overexpressing hIL-17E using an apolipoprotein E (ApoE) hepatic promoter. These mice displayed changes in the peripheral blood, particularly, a 3-fold increase in total leukocytes consisting of increases in eosinophils, lymphocytes, and neutrophils. Splenomegaly and lymphoadenopathy were predominant and included marked eosinophil infiltrates and lymphoid hyperplasia. CCR3(+) eosinophils increased in the blood and lymph nodes of the transgenic mice by 50- and 300-fold, respectively. Eosinophils also increased 8- to 18-fold in the bone marrow and spleen, respectively. In the bone marrow, most of the eosinophils had an immature appearance. CD19(+) B cells increased 2- to 5-fold in the peripheral blood, 2-fold in the spleen, and 10-fold in the lymph nodes of transgenic mice, whereas CD4(+) T lymphocytes increased 2-fold in both blood and spleen. High serum levels of the cytokines IL-2, IL-4, IL-5, granulocyte colony-stimulating factor, eotaxin, and interferon gamma were observed. Consistent with B-lymphocyte increases, serum immunoglobulin (Ig) M, IgG, and IgE were significantly elevated. Antigenic challenge of the transgenic mice with keyhole limpet hemocyanin (KLH) resulted in a decrease in anti-KLH IgG accompanied by increases of anti-KLH IgA and IgE. In situ hybridization of transgenic tissues revealed that IL-17Rh1 (IL-17BR/Evi27), a receptor that binds IL-17E, is up-regulated. Taken together, these data indicate that IL-17E regulates hematopoietic and immune functions, stimulating the development of eosinophils and B lymphocytes. The fact that hIL-17E overexpression results in high levels of circulating eosinophils, IL-4, IL-5, eotaxin, and IgE suggests that IL-17E may be a proinflammatory cytokine favoring Th2-type immune responses. >28.NF00108133 PMID:12547166 TI - Altered splicing pattern of TACC1 mRNA in gastric cancer. AB - Transforming acidic coiled-coil (TACC) proteins are centrosome and microtubule-associated proteins that are essential for mitotic spindle function. We identified TACC1 as an immunogenic protein and a potential tumor antigen by applying serological identification of antigens by recombinant expression cloning (SEREX) technique to screen a gastric cancer cDNA library. The 5'RLM-RACE and reverse transcriptase polymerase chain reaction analyses revealed at least six different transcript variants of TACC1 with variable transcription start sites and alternative exon usage (designated TACC1-A-TACC1-F). All transcripts differ in their 5' ends but share an identical 3' region encoding coiled-coil domain. Four transcripts were universally expressed in all normal tissues analyzed but TACC1-D and TACC1-F showed a restricted expression pattern. TACC1-F, a transcript representing the SEREX-identified cDNA clone, was predominantly expressed in brain and gastric tumors to a similar level. TACC1-D was only weakly detectable in kidney and colon but not in other normal tissues, while a relatively strong expression was observed in 50% of gastric cancer tissue samples analyzed. These transcript variants are generated possibly as a result of alterations in efficiency and pattern of alternative splicing; these isoforms may represent genetic markers, for example TACC1-D for gastric cancer. We also propose that inappropriate expression of the isoforms in gastric cancer cells might result in dysfunction of TACC1 thus contributing to the genetic instability. >29.NF00131704 PMID:12058028 TI - The stress-activated protein kinases p38 alpha and JNK1 stabilize p21(Cip1) by phosphorylation. AB - Stress signals activate the SAPK/JNK and p38 MAPK classes of protein kinases, which mediate cellular responses, including steps in apoptosis and the maturation of some cell types. We now show that stress signals initiated by transforming growth factor-beta 1 (TGF-beta 1) induce G(1) arrest through protein stabilization of the CDK inhibitor p21(Cip1). TGF-beta 1 was previously shown to increase p21 protein levels, which in turn mediated G(1) arrest through inactivation of the CDK2-cyclin E complex in HD3 cells (Yan, Z., Kim, G.-Y., Deng, X., and Friedman, E. (2002) J. Biol. Chem. 277, 9870-9879). We now demonstrate that the increase in p21 abundance is caused by a post-transcriptional, SMAD-independent mechanism. TGF-beta1 activated p38 alpha and JNK1, which initiated the phosphorylation of p21. TGF-beta1 treatment increased the half-life of p21 by 3-4-fold. The increase in p21 stability was detected following activation of p38 alpha and JNK1, and treatment of cells with the p38 inhibitor SB203580 prevented this increase in p21 stability. p38 alpha and JNK1 phosphorylated p21 in vivo, and both p38 alpha and JNK1 phosphorylated p21 at Ser(130) in vitro. Peptide mapping demonstrated that both TGF-beta 1 and p38 alpha induced phosphorylation of p21 at Ser(130) in vivo, and mutation of Ser(130) to alanine rendered p21 less stable than wild-type p21. TGF-beta 1 increased the stability of wild-type p21, but not the p21-S130A mutant. These findings demonstrate that SAPKs can mediate cell cycle arrest through post-translational modification of p21. >30.NF00142154 PMID:12493765 TI - Transcriptional repressor germ cell-less (GCL) and barrier to autointegration factor (BAF) compete for binding to emerin in vitro. AB - Emerin belongs to the "LEM domain" family of nuclear proteins, which contain a characteristic approximately 40-residue LEM motif. The LEM domain mediates direct binding to barrier to autointegration factor (BAF), a conserved 10-kDa chromatin protein essential for embryogenesis in Caenorhabditis elegans. In mammalian cells, BAF recruits emerin to chromatin during nuclear assembly. BAF also mediates chromatin decondensation during nuclear assembly. The LEM domain and central region of emerin are essential for binding to BAF and lamin A, respectively. However, two other conserved regions of emerin lacked ascribed functions, suggesting that emerin could have additional partners. We discovered that these "unascribed" domains of emerin mediate direct binding to a transcriptional repressor, germ cell-less (GCL). GCL co-immunoprecipitates with emerin from HeLa cells. We determined the binding affinities of emerin for GCL, BAF, and lamin A and analyzed their oligomeric interactions. We showed that emerin forms stable complexes with either lamin A plus GCL or lamin A plus BAF. Importantly, BAF competed with GCL for binding to emerin in vitro, predicting that emerin can form at least two distinct types of complexes in vivo. Loss of emerin causes Emery-Dreifuss muscular dystrophy, a tissue-specific inherited disease that affects skeletal muscles, major tendons, and the cardiac conduction system. Although GCL alone cannot explain the disease mechanism, our results strongly support gene expression models for Emery-Dreifuss muscular dystrophy by showing that emerin binds directly to a transcriptional repressor, GCL, and by suggesting that emerin-repressor complexes might be regulated by BAF. Biochemical roles for emerin in gene expression are discussed. >31.NF00104652 PMID:11896212 TI - Infantile dilated X-linked cardiomyopathy, G4.5 mutations, altered lipids, and ultrastructural malformations of mitochondria in heart, liver, and skeletal muscle. AB - Mutations in the Xq28 gene G4.5 lead to dilated cardiomyopathy (DCM). Differential splicing of G4.5 results in a family of proteins called "tafazzins" with homology to acyltransferases. These enzymes assemble fatty acids into membrane lipids. We sequenced G4.5 in two kindreds with X-linked DCM and in two unrelated men, one with idiopathic DCM and the other with DCM of arrhythmogenic right ventricular dysplasia. We examined the ultrastructure of heart, liver, and muscle biopsy specimens in these three DCM types; we used gas chromatography to compare fatty acid composition in heart, liver, and muscle autopsy specimens of two patients of kindred 1 with that of controls. In X-linked DCM, G4.5 had a stop codon (E188X), a nonsense mutation, in kindred 1 and an amino acid substitution (G240R), a missense mutation, in kindred 2. In the two men with isolated DCM, G4.5 was not mutated. Ultrastructural mitochondrial malformations were present in the biopsy tissues of the patients with DCM. Cardiac biopsy specimens of both kindreds with X-linked DCM exhibited greatly enlarged mitochondria with large bundles of stacked, compacted, disarrayed cristae that differed from those of the two types of isolated DCM. Autopsy tissue of patients with X-linked DCM had decreased unsaturated and increased saturated fatty acid concentrations. Seven of 13 published G4.5 missense mutations, including the one presented here, occur in acyltransferase motifs. Impaired acyltransferase function could result in increased fatty acid saturation that would decrease membrane fluidity. Mitochondrial membrane proliferation may be an attempt to compensate for impaired function of acyltransferase. Cardiac ultrastructure separates X-linked DCM with G4.5 mutations from the two types of isolated DCM without G4.5 mutations. Electron microscopy of promptly fixed myocardial biopsy specimens has a role in defining the differential diagnosis of DCM. Mutational analysis of the G4.5 gene also serves this purpose. >32.NF00107244 PMID:12054652 TI - Phospholipase C-beta 2 interacts with mitogen-activated protein kinase kinase 3. AB - Phospholipase C (PLC)-beta enzymes (isoenzymes beta 1-beta 4) are activated by G protein subunits, leading to the generation of intracellular messengers which mobilize calcium and activate protein kinase C. It has recently been recognized that these enzymes interact with and are regulated by proteins other than G proteins. Using the yeast two-hybrid technique to screen a leukocyte library we identified mitogen-activated protein kinase kinase 3 (MKK3) as a partner of PLC-beta 2. The interaction was confirmed by co-immunoprecipitation assays which indicated that MKK3 interacts with PLC-beta 2, but not with other PLC-betas. PLC-beta 2 interacted weakly with MKK6, which is related to MKK3, but not with the other MKK3 tested. The region of PLC-beta 2 involved in the interaction with MKK3 was mapped to the C-terminus of PLC-beta 2. p38MAPK also co-immunoprecipitated with PLC-beta 2. The data suggest that PLC-beta 2 serves an unappreciated role assembling components of the p38MAPK signaling module. >33.NF00108136 PMID:12606047 TI - Myeloperoxidase/nitrite-mediated lipid peroxidation of low-density lipoprotein as modulated by flavonoids. AB - In the presence of a H(2)O(2)-generating system, myeloperoxidase (MPO) caused conjugated diene formation in low-density lipoprotein (LDL), indicating lipid peroxidation which was dependent on nitrite but not on chloride. The oxidation of LDL was inhibited by micromolar concentrations of flavonoids such as (-)-epicatechin, quercetin, rutin, taxifolin and luteolin, presumably via scavenging of the MPO-derived NO(2) radical. The flavonoids served as substrates of MPO leading to products with distinct absorbance spectra. The MPO-catalyzed oxidation of flavonoids was accelerated in the presence of nitrite. >34.NF00098301 PMID:11925441 TI - Determinants of the substrate specificity of multidrug resistance protein 1: role of amino acid residues with hydrogen bonding potential in predicted transmembrane helix 17. AB - Human multidrug resistance protein 1 (MRP1) confers resistance to many natural product chemotherapeutic agents and actively transports structurally diverse organic anion conjugates. We previously demonstrated that two hydrogen-bonding amino acid residues in the predicted transmembrane 17 (TM17) of MRP1, Thr(1242) and Trp(1246), were important for drug resistance and 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) transport. To determine whether other residues with hydrogen bonding potential within TM17 influence substrate specificity, we replaced Ser(1233), Ser(1235), Ser(1237), Gln(1239), Thr(1241), and Asn(1245) with Ala and Tyr(1236) and Tyr(1243) with Phe. Mutations S1233A, S1235A, S1237A, and Q1239A had no effect on any substrate tested. In contrast, mutations Y1236F and T1241A decreased resistance to vincristine but not to VP-16, doxorubicin, and epirubicin. Mutation Y1243F reduced resistance to all drugs tested by 2-3-fold. Replacement of Asn(1245) with Ala also decreased resistance to VP-16, doxorubicin, and epirubicin but increased resistance to vincristine. This mutation also decreased E(2)17betaG transport approximately 5-fold. Only mutation Y1243F altered the ability of MRP1 to transport both leukotriene 4 and E(2)17betaG. Together with our previous results, these findings suggest that residues with side chain hydrogen bonding potential, clustered in the cytoplasmic half of TM17, participate in the formation of a substrate binding site. >35.NF00122637 PMID:12393902 TI - MdmX is a RING finger ubiquitin ligase capable of synergistically enhancing Mdm2 ubiquitination. AB - It has been well documented that Mdm2 and its homologue MdmX not only are critical negative regulators of the tumor suppressor p53 but that both Mdm2 and MdmX interact to affect the function of the other. The mechanisms through which these effects are manifested, however, remain unclear. Although Mdm2 has been established as a RING finger ubiquitin ligase, MdmX has not been shown to possess this activity despite the extensive sequence homology between their respective RING finger domains. Here we demonstrate that MdmX acts as a ubiquitin ligase in vitro, being capable of autoubiquitination, as well as mediating the ubiquitination of p53. The addition of Mdm2 to in vitro ubiquitination assays containing MdmX results in a synergistic increase of ubiquitin conjugation. Analysis of the resulting ubiquitin conjugates reveals that this observed synergy reflects an increase in Mdm2 ubiquitination. This study also suggests that ubiquitination of Mdm2 and MdmX may not serve as a signal for degradation, as we show that each are capable of synthesizing non-lysine 48 polyubiquitin chains and, in fact, utilize multiple lysine linkages. Taken together, these findings suggest a more active role for MdmX in the Mdm2-MdmX-p53 regulatory network than has been proposed previously. >36.NF00097330 PMID:12006491 TI - Human SIR2 deacetylates p53 and antagonizes PML/p53-induced cellular senescence. AB - The yeast Sir2 protein mediates chromatin silencing through an intrinsic NAD-dependent histone deacetylase activity. Sir2 is a conserved protein and was recently shown to regulate lifespan extension both in budding yeast and worms. Here, we show that SIRT1, the human Sir2 homolog, is recruited to the promyelocytic leukemia protein (PML) nuclear bodies of mammalian cells upon overexpression of either PML or oncogenic Ras (Ha-rasV12). SIRT1 binds and deacetylates p53, a component of PML nuclear bodies, and it can repress p53-mediated transactivation. Moreover, we show that SIRT1 and p53 co-localize in nuclear bodies upon PML upregulation. When overexpressed in primary mouse embryo fibroblasts (MEFs), SIRT1 antagonizes PML-induced acetylation of p53 and rescues PML-mediated premature cellular senescence. Taken together, our data establish the SIRT1 deacetylase as a novel negative regulator of p53 function capable of modulating cellular senescence. >37.NF00116239 PMID:11856758 TI - Role of integrin-linked kinase in leukocyte recruitment. AB - Chemokines modulate leukocyte integrin avidity to coordinate adhesion and subsequent transendothelial migration, although the sequential signaling pathways involved remain poorly characterized. Here we show that integrin-linked kinase (ILK), a 59-kDa serine-threonine protein kinase that interacts principally with beta(1) integrins, is highly expressed in human mononuclear cells and is activated by exposure of leukocytes to the chemokine monocyte chemoattractant protein-1. Biochemical inhibitor studies show that chemokine-triggered activation of ILK is downstream of phosphoinositide 3-kinase. In functional assays under physiologically relevant flow conditions, overexpression of wild-type ILK in human monocytic cells diminishes beta(1) integrin/vascular cell adhesion molecule-1-dependent firm adhesion to human endothelial cells. These data implicate ILK in the dynamic signaling events involved in the regulation of leukocyte integrin avidity for endothelial substrates. >38.NF00133088 PMID:11809755 TI - Insulin-degrading enzyme rapidly removes the beta-amyloid precursor protein intracellular domain (AICD). AB - The intramembranous gamma-secretase cleavage of the beta-amyloid precursor protein (APP) is dependent on biologically active presenilins (PS). Notch also undergoes a similar PS-dependent gamma-secretase-like cleavage, resulting in the liberation of the Notch intracellular domain (NICD), which is critically required for developmental signal transduction. gamma-Secretase processing of APP results in the production of a similar fragment called AICD (APP intracellular domain), which may function in nuclear signaling as well. AICD, like NICD, is rapidly removed. By using a battery of protease inhibitors we demonstrate that AICD, in contrast to NICD, is degraded by a cytoplasmic metalloprotease. In vitro degradation of AICD can be reconstituted with cytoplasmic fractions obtained from neuronal and non-neuronal cells. Taking into account the inhibition profile and the cytoplasmic localization, we identified three candidate enzymes (neurolysin, thimet oligopeptidase, and insulin-degrading enzyme (IDE), also known as insulysin), which all are involved in the degradation of bioactive peptides in the brain. When insulin, a well characterized substrate of IDE, was added to the in vitro degradation assay, removal of AICD was efficiently blocked. Moreover, overexpression of IDE resulted in enhanced degradation of AICD, whereas overexpression of the inactive IDE E111Q mutant did not affect AICD degradation. Finally, immunodepletion of IDE significantly reduced the AICD degrading activity. Therefore our data demonstrate that IDE, which is one of the proteases implicated in the removal of extracellular Abeta, also removes the cytoplasmic product of gamma-secretase cleaved APP. >39.NF00122477 PMID:12508640 TI - [Mutation analysis of KLF6 gene in human nasopharyngeal carcinomas] AB - BACKGROUND & OBJECTIVE: Nasopharyngeal carcinoma (NPC) is one of the most common cancers in South China and Southeast Asia. The etiological factor is believed to be the interaction between genetic susceptibility, EBV infection and environmental factors, involved in the multi-step process of carcinogenesis and development of NPC. However, the molecular pathology of NPC is unclear yet. Kruppel-like factor 6(KLF6) is a ubiquitously expressed nuclear transcription factor, which is deleted and/or mutated with high frequency in a subset of prostate cancer. This study was designed to investigate KLF6 mutation in NPC tumors and NPC cell lines. METHODS: Genomic DNAs from 19 NPC tumor biospies and 3 NPC cell lines were used in mutation detection of KLF6 coding region and splice sites by PCR-sequencing. 100 chromosomes from 50 random healthy individuals were used as control. RESULTS: In 3 of 19 NPC tissues, 3 different mutations (Glu75Val, Ser136Arg, Arg243 Lys) in KLF6 gene were found by PCR-sequencing. None of the 3 mutations were detected in the 50 random healthy individuals. CONCLUSION: KLF6 gene may be involved in carcinogenesis of sporadic NPC. >40.NF00085672 PMID:11700562 TI - Identification of the cellular receptor for anthrax toxin. AB - The tripartite toxin secreted by Bacillus anthracis, the causative agent of anthrax, helps the bacterium evade the immune system and can kill the host during a systemic infection. Two components of the toxin enzymatically modify substrates within the cytosol of mammalian cells: oedema factor (OF) is an adenylate cyclase that impairs host defences through a variety of mechanisms including inhibiting phagocytosis; lethal factor (LF) is a zinc-dependent protease that cleaves mitogen-activated protein kinase kinase and causes lysis of macrophages. Protective antigen (PA), the third component, binds to a cellular receptor and mediates delivery of the enzymatic components to the cytosol. Here we describe the cloning of the human PA receptor using a genetic complementation approach. The receptor, termed ATR (anthrax toxin receptor), is a type I membrane protein with an extracellular von Willebrand factor A domain that binds directly to PA. In addition, a soluble version of this domain can protect cells from the action of the toxin. >41.NF00098308 PMID:11470784 TI - An isoform-specific inhibitory domain regulates the LHX3 LIM homeodomain factor holoprotein and the production of a functional alternate translation form. AB - The LHX3 LIM homeodomain transcription factor is required for pituitary development and motor neuron specification. The Lhx3 gene encodes two isoforms, LHX3a and LHX3b, that differ in their amino-terminal sequences. Humans and mice with defective Lhx3 genes are deficient in gonadotrope, lactotrope, somatotrope, and thyrotrope pituitary cells. We show that, whereas Lhx3b is highly expressed in these Lhx3-dependent cell types, high levels of Lhx3a expression are restricted to alpha glycoprotein subunit-expressing thyrotropes and gonadotropes. Cross-species comparison reveals the LHX3b-specific domain is more conserved than the LHX3a-specific domain. We demonstrate that the LHX3b-specific domain is a transferable inhibitor that reduces gene activation and DNA binding by homeodomain proteins. In addition, we identify a novel LHX3 protein (M2-LHX3) and determine that this molecule is generated by an internal translation initiation codon. The LHX3a- and LHX3b-specific coding sequences regulate differential usage of this internal start codon. Further, we identify the major activation domain of LHX3 in the carboxyl terminus of the molecule. M2-LHX3 is active because it retains this domain and binds DNA better than LHX3a or LHX3b. Other LIM homeodomain genes, including Lhx4, generate similar truncated proteins. These studies describe how transcriptional regulatory genes can generate multiple functional proteins. >42.NF00094668 PMID:11701952 TI - Comparative FISH mapping of Gab1 and Gab2 genes in human, mouse and rat. AB - Gab1 and Gab2 are members of the Gab family which act as adapters for transmitting various signals in response to stimuli through cytokine and growth factor receptors, and T- and B-cell antigen receptors. We determined chromosome locations of the two genes in human, mouse and rat by fluorescence in situ hybridization. The Gab1 gene was localized to chromosome 4q31.1 in human, 8C3 in mouse and 19q11.1--> q11.2 in rat, and the Gab2 gene was located on chromosome 11q13.4-->q13.5 in human, 7E2 in mouse and 1q33.2-->q33.3 in rat. All human, mouse and rat Gab1 and Gab2 genes were localized to chromosome regions where conserved homology has been identified among the three species. >43.NF00926992 PMID:11799131 TI - Molecular mechanics of cardiac titin's PEVK and N2B spring elements. AB - Titin is a giant elastic protein that is responsible for the majority of passive force generated by the myocardium. Titin's force is derived from its extensible I-band region, which, in the cardiac isoform, comprises three main extensible elements: tandem Ig segments, the PEVK domain, and the N2B unique sequence (N2B-Us). Using atomic force microscopy, we characterized the single molecule force-extension curves of the PEVK and N2B-Us spring elements, which together are responsible for physiological levels of passive force in moderately to highly stretched myocardium. Stretch-release force-extension curves of both the PEVK domain and N2B-Us displayed little hysteresis: the stretch and release data nearly overlapped. The force-extension curves closely followed worm-like chain behavior. Histograms of persistence length (measure of chain bending rigidity) indicated that the single molecule persistence lengths are approximately 1.4 and approximately 0.65 nm for the PEVK domain and N2B-Us, respectively. Using these mechanical characteristics and those determined earlier for the tandem Ig segment (assuming folded Ig domains), we modeled the cardiac titin extensible region in the sarcomere and calculated the extension of the various spring elements and the forces generated by titin, both as a function of sarcomere length. In the physiological sarcomere length range, predicted values and those obtained experimentally were indistinguishable. >44.NF00866488 PMID:12359327 TI - Characterisation and expression analysis of the WDR9 gene, located in the Down critical region-2 of the human chromosome 21. AB - We report the isolation and characterisation of the gene WDR9 (WD Repeat 9), located in the Down Syndrome critical region-2 (DCR-2) from the human chromosome 21. This gene spans 125 kb of genomic sequence and is organised in 41 exons and 40 introns. The WDR9 cDNA has a size of 13 kb and encodes for a putative protein of 2269 amino acids with a potential location in the nucleus. Expression analysis in different human adult tissues and in cultured cell lines indicates that the gene has several tissue-specific transcripts. The more significant protein signatures in the WDR9 protein sequence are for WD repeats, bromodomain, beta-ketoacyl synthases, and ribonucleoprotein (RNP). The WDR9 protein has a high similarity with the Mus musculus neuronal differentiation protein (NDRP) and a region of similarity with the region of the Yotiao protein that has been proposed to bind the NR1 subunit of the NMDA receptor. The presence of protein-protein interaction domains as such the WD repeats, and the similarity of the WDR9 protein to regulatory proteins suggest a potential involvement in some of the clinical features associated to the DCR-2. >45.NF00103850 PMID:12029094 TI - Metallochaperone Atox1 transfers copper to the NH2-terminal domain of the Wilson's disease protein and regulates its catalytic activity. AB - Copper is essential for the growth and development of mammalian cells. The key role in the intracellular distribution of copper belongs to the recently discovered family of metallochaperones and to copper-transporting P-type ATPases. The mutations in the ATPase ATP7B, the Wilson's disease protein (WNDP), lead to intracellular accumulation of copper and severe hepatic and neurological abnormalities. Several of these mutations were shown to disrupt the protein-protein interactions between WNDP and the metallochaperone Atox1, suggesting that these interactions are important for normal copper homeostasis. To understand the functional consequences of the Atox1-WNDP interaction at the molecular level, we produced recombinant Atox1 and characterized its effects on WNDP. We demonstrate that Atox1 transfers copper to the purified amino-terminal domain of WNDP (N-WNDP) in a dose-dependent and saturable manner. A maximum of six copper atoms can be transferred to N-WNDP by the chaperone. Furthermore, the incubation of copper Atox1 with the full-length WNDP leads to the stimulation of the WNDP catalytic activity, providing strong evidence for the direct effect of Atox1 on the function of this transporter. Our data also suggest that Atox1 can regulate the copper occupancy of WNDP. The incubation with apo-Atox1 results in the removal of copper from the metalated N-WNDP and apparent down-regulation of WNDP activity. Interestingly, at least one copper atom remains tightly bound to N-WNDP even in the presence of excess apo-Atox1. We suggest that this incomplete reversibility reflects the functional non-equivalency of the metal-binding sites in WNDP and speculate about the intracellular consequences of the reversible Atox1-mediated copper transfer. >46.NF00116407 PMID:11816009 TI - Development of a rapid and sensitive high-performance liquid chromatographic method to determine CYP2D6 phenotype in human liver microsomes. AB - Dextromethorphan is a probe substrate to determine CYP2D6 phenotype. The conversion of dextromethorphan to dextrorphan by CYP2D6 accounts for approximately 60% of total metabolism. Most analytical methods utilize complicated labor- and time-intensive sample processing methods with several liquid-liquid extraction (LLE) steps. Our goal was to develop a non-LLE based rapid and sensitive HPLC method, to measure dextromethorphan metabolism in human liver microsomes. A solid-phase filtration based reverse-phase HPLC method with fluorescence detection was developed and validated. Human liver (n = 6) microsomal incubations were carried out with dextromethorphan, under optimum conditions. The analytes were separated by one-step centrifugal filtration with Nanosep separation units. The filtrate was injected ( 50 microL) into a Waters Alliance 2690 HPLC system. Metabolic incubations were also conducted to determine levels using LLE for comparisons. The Nanosep separation step reduced the extraction time from 3h to 40 min. The limit of quantitation was 23.8 nM (9.7 ng/mL), recovery was approximately 98%, the mean precision values were <10% RSD for the controls (80, 320 and 640 nM) and mean percentage error was <5%. Michaelis-Menten parameters were determined to distinguish CYP2D6 phenotypes. A rapid and sensitive HPLC method is reported, which may be suitable for automation and allows phenotyping of human liver microsomes. >47.NF00125317 PMID:12359228 TI - Identification of a novel human nicotinamide mononucleotide adenylyltransferase. AB - The enzyme nicotinamide mononucleotide adenylyltransferase is an ubiquitous enzyme catalyzing an essential step in NAD (NADP) biosynthetic pathway. In human cells, the nuclear enzyme, which we will now call NMNAT-1, has been the only known enzyme of this type for over 10 years. Here we describe the cloning and expression of a human cDNA encoding a novel 34.4kDa protein, that shares significant homology with the 31.9kDa NMNAT-1. We propose to call this enzyme NMNAT-2. Purified recombinant NMNAT-2 is endowed with NMN and nicotinic acid mononucleotide adenylyltransferase activities, but differs from NMNAT-1 with regard to chromosomal and cellular localization, tissue-specificity of expression, and molecular properties, supporting the idea that the two enzymes might play distinct physiological roles in NAD homeostasis. >48.NF00131475 PMID:12584323 TI - Furin processing and proteolytic activation of Semliki Forest virus. AB - The alphavirus Semliki Forest virus (SFV) infects cells via a low-pH-dependent membrane fusion reaction mediated by the E1 envelope protein. Fusion is regulated by the interaction of E1 with the receptor-binding protein E2. E2 is synthesized as a precursor termed "p62," which forms a stable heterodimer with E1 and is processed late in the secretory pathway by a cellular furin-like protease. Once processing to E2 occurs, the E1/E2 heterodimer is destabilized so that it is more readily dissociated by exposure to low pH, allowing fusion and infection. We have used FD11 cells, a furin-deficient CHO cell line, to characterize the processing of p62 and its role in the control of virus fusion and infection. p62 was not cleaved in FD11 cells and cleavage was restored in FD11 cell transfectants expressing human furin. Studies of unprocessed virus produced in FD11 cells (wt/p62) demonstrated that the p62 protein was efficiently cleaved by purified furin in vitro, without requiring prior exposure to low pH. wt/p62 virus particles were also processed during their endocytic uptake in furin-containing cells, resulting in more efficient virus infection. wt/p62 virus was compared with mutant L, in which p62 cleavage was blocked by mutation of the furin-recognition motif. wt/p62 and mutant L had similar fusion properties, requiring a much lower pH than control virus to trigger fusion and fusogenic E1 conformational changes. However, the in vivo infectivity of mutant L was more strongly inhibited than that of wt/p62, due to additional effects of the mutation on virus-cell binding. >49.NF00105475 PMID:12359356 TI - Microsomal epoxide hydrolase and glutathione S-transferase polymorphisms in relation to laryngeal carcinoma risk. AB - Two polymorphic sites of the microsomal epoxide hydrolase gene (EPHX1, 113Tyr-->113His, 139His-->139Arg) and four glutathione S-transferase genes (GSTM1, GSTM3, GSTP1, GSTT1) were genotyped in a group of patients with larynx cancer (N=204) and in a group of healthy controls (N=203), all Spanish caucasians. After adjusting for gender, age, and tobacco smoking, none of the polymorphisms alone were found to be associated with larynx cancer risk. The analysis of EPHX1/GST combinations, however, showed a significant over-representation of patients with a combination of 113Tyr/113Tyr EPHX1 and 105Ile/105Ile GSTP1 (adjusted odds ratio (OR): 1.95; 95% confidence interval (CI): 1.02-3.78). The calculation of the predicted epoxide hydrolase (EH) activity also showed an increased risk for the individuals with both predicted high activity EH and 105Ile/105Ile GSTP1 (OR: 2.90; 95% CI: 1.10-7.67). These results on larynx cancer tend to confirm a former study on lung cancer (Cancer Lett. 173 (2001) 155) suggesting the existence of an interaction between variants of EH and GSTpi, both enzymes being involved in the metabolism of aromatic hydrocarbons, that may increase susceptibility to tobacco-related cancers. >50.NF00106528 PMID:11778160 TI - A 117-kb microdeletion removing HOXD9-HOXD13 and EVX2 causes synpolydactyly. AB - Studies in mouse and chick have shown that the 5' HoxD genes play major roles in the development of the limbs and genitalia. In humans, mutations in HOXD13 cause the dominantly inherited limb malformation synpolydactyly (SPD). Haploinsufficiency for the 5' HOXD genes has recently been proposed to underlie the monodactyly and penoscrotal hypoplasia in two children with chromosomal deletions encompassing the entire HOXD cluster. Similar deletions, however, have previously been associated with split-hand/foot malformation (SHFM), including monodactyly. Here we report a father and daughter with SPD who carry a 117-kb microdeletion at the 5' end of the HOXD cluster. By sequencing directly across the deletion breakpoint, we show that this microdeletion removes only HOXD9-HOXD13 and EVX2. We also report a girl with bilateral split foot and a chromosomal deletion that includes the entire HOXD cluster and extends approximately 5 Mb centromeric to it. Our findings indicate that haploinsufficiency for the 5' HOXD genes causes not SHFM but SPD and point to the presence of a novel locus for SHFM in the interval between EVX2 and D2S294. They also suggest that there is a regulatory region, upstream of the HOXD cluster, that is responsible for activating the cluster as a whole. >51.NF00131477 PMID:12050117 TI - Mastermind mediates chromatin-specific transcription and turnover of the Notch enhancer complex. AB - Signaling through the Notch pathway activates the proteolytic release of the Notch intracellular domain (ICD), a dedicated transcriptional coactivator of CSL enhancer-binding proteins. Here we show that chromatin-dependent transactivation by the recombinant Notch ICD-CBF1 enhancer complex in vitro requires an additional coactivator, Mastermind (MAM). MAM provides two activation domains necessary for Notch signaling in mammalian cells and in Xenopus embryos. We show that the central MAM activation domain (TAD1) recruits CBP/p300 to promote nucleosome acetylation at Notch enhancers and activate transcription in vitro. We also find that MAM expression induces phosphorylation and relocalization of endogenous CBP/p300 proteins to nuclear foci in vivo. Moreover, we show that coexpression with MAM and CBF1 strongly enhances phosphorylation and proteolytic turnover of the Notch ICD in vivo. Enhanced phosphorylation of the ICD and p300 requires a glutamine-rich region of MAM (TAD2) that is essential for Notch transcription in vivo. Thus MAM may function as a timer to couple transcription activation with disassembly of the Notch enhancer complex on chromatin. >52.NF00111959 PMID:12209014 TI - Dual localization of human DNA topoisomerase IIIalpha to mitochondria and nucleus. AB - The human TOP3alpha gene encoding DNA topoisomerase IIIalpha (hTop3alpha) has two potential start codons for the synthesis of proteins 1,001 and 976 aa residues in length. The sequence of the N-terminal region of the 1,001-residue form resembles signal peptide sequences for mitochondrial import, and fluorescence microscopy shows that the addition of as few as the first 34 aa of the 1,001-residue form of hTop3alpha to a green fluorescent protein can direct the chimeric protein to mitochondria. Biochemical analyses of subcellular fractions of HeLa cells further demonstrate that a distinctive fraction of hTop3alpha is present inside mitochondria, as evidenced by its resistance to proteinase K. This fraction constitutes several percent of the enzyme in the nuclear fraction, suggesting that the distribution of the mitochondrial and nuclear forms of hTop3alpha is roughly in proportion to the DNA contents of these cellular compartments. The presence of a type IA DNA topoisomerase in the mitochondria of other eukaryotes is supported by an examination of the amino acid sequences of mouse and Drosophila DNA topoisomerase IIIalpha and Schizosaccharomyces pombe DNA topoisomerase III. Given the presence of at least one type IA DNA topoisomerase in all forms of life examined to date, the finding of a type IA enzyme in mitochondria further supports the notion of a key role of such enzymes in DNA transactions. >53.NF00095807 PMID:12140183 TI - Spectrin-like repeats from dystrophin and alpha-actinin-2 are not functionally interchangeable. AB - Mutations in the dystrophin gene result in Duchenne muscular dystrophy (DMD). Dystrophin is a multidomain protein that functions to stabilize the sarcolemmal membrane during muscle contraction. The central rod domain has been proposed to act as a shock absorber, as a force transducer or as a spacer separating important N- and C-terminal domains that interact with actin and the dystrophin-glycoprotein complex (DGC). Structure/function studies demonstrated that deletion of large portions of the rod domain can result in the production of smaller, yet highly functional, dystrophin proteins. In a dramatic example, a 'micro-dystrophin' transgene containing only four dystrophin spectrin-like repeats resulted in complete correction of most of the symptoms associated with dystrophy in the mdx mouse model for DMD. Dystrophin shares considerable homology with the multidomain, actin-crosslinking protein alpha-actinin. To explore the hypothesis that the dystrophin rod domain acts as a spacer region, a chimeric micro-dystrophin transgene containing the four-repeat rod domain of alpha-actinin-2 was expressed in mdx mice. This chimeric transgene was incapable of correcting the morphological pathology of the mdx mouse, but still functioned to assemble the DGC at the membrane and provided some protection from contraction-induced injury. These data demonstrated that different spectrin-like repeats are not equivalent, and reinforced the suggestion that the dystrophin rod domain is not merely a spacer but likely contributes an important mechanical role to overall dystrophin function. >54.NF00087464 PMID:12062429 TI - The dystrophin gene is alternatively spliced throughout its coding sequence. AB - We have analysed splicing patterns in the human dystrophin gene region encoding the rod and cysteine-rich domains in normal skeletal muscle, brain and heart tissues. Sixteen novel alternative transcripts were identified, the majority of them being present in all three tissues. Tissue-specific variants were also identified, suggesting a functional role of transcriptional diversity. Transcript analysis in dystrophinopathic autoptic and bioptic specimens revealed that pre-mRNAs secondary structure formation and relative strength of exon/exon association play little or no role in directing alternative splicing events. This analysis also showed that independent deletion events leading to the loss of the same exons may be associated with transcriptional variability. >55.NF00095564 PMID:12065429 TI - Drf1, a novel regulatory subunit for human Cdc7 kinase. AB - Studies in model organisms have contributed to elucidate multiple levels at which regulation of eukaryotic DNA replication occurs. Cdc7, an evolutionarily conserved serine-threonine kinase, plays a pivotal role in linking cell cycle regulation to genome duplication, being essential for the firing of DNA replication origins. Binding of the cell cycle-regulated subunit Dbf4 to Cdc7 is necessary for in vitro kinase activity. This binding is also thought to be the key regulatory event that controls Cdc7 activity in cells. Here, we describe a novel human protein, Drf1, related to both human and yeast Dbf4. Drf1 is a nuclear cell cycle-regulated protein, it binds to Cdc7 and activates the kinase. Therefore, human Cdc7, like cyclin-dependent kinases, can be activated by alternative regulatory subunits. Since the Drf1 gene is either absent or not yet identified in the genome of model organisms such as yeast and Drosophila, these findings introduce a new level of complexity in the regulation of DNA replication of the human genome. >56.NF01154885 PMID:12075506 TI - Mandibuloacral dysplasia is caused by a mutation in LMNA-encoding lamin A/C. AB - Mandibuloacral dysplasia (MAD) is a rare autosomal recessive disorder, characterized by postnatal growth retardation, craniofacial anomalies, skeletal malformations, and mottled cutaneous pigmentation. The LMNA gene encoding two nuclear envelope proteins (lamins A and C [lamin A/C]) maps to chromosome 1q21 and has been associated with five distinct pathologies, including Dunnigan-type familial partial lipodystrophy, a condition that is characterized by subcutaneous fat loss and is invariably associated with insulin resistance and diabetes. Since patients with MAD frequently have partial lipodystrophy and insulin resistance, we hypothesized that the disease may be caused by mutations in the LMNA gene. We analyzed five consanguineous Italian families and demonstrated linkage of MAD to chromosome 1q21, by use of homozygosity mapping. We then sequenced the LMNA gene and identified a homozygous missense mutation (R527H) that was shared by all affected patients. Patient skin fibroblasts showed nuclei that presented abnormal lamin A/C distribution and a dysmorphic envelope, thus demonstrating the pathogenic effect of the R527H LMNA mutation. >57.NF00866570 PMID:11938494 TI - The TRIM37 gene encodes a peroxisomal RING-B-box-coiled-coil protein: classification of mulibrey nanism as a new peroxisomal disorder. AB - Mulibrey nanism is a rare growth disorder of prenatal onset caused by mutations in the TRIM37 gene, which encodes a RING-B-box-coiled-coil protein. The pathogenetic mechanisms of mulibrey nanism are unknown. We have used transiently transfected cells and antibodies raised against the predicted TRIM37 protein to characterize the TRIM37 gene product and to determine its intracellular localization. We show that the human TRIM37 cDNA encodes a peroxisomal protein with an apparent molecular weight of 130 kD. Peroxisomal localization is compromised in mutant protein representing the major Finnish TRIM37 mutation but is retained in the protein representing the minor Finnish mutation. Colocalization of endogenous TRIM37 with peroxisomal markers was observed by double immunofluorescence staining in HepG2 and human intestinal smooth muscle cell lines. In human tissue sections, TRIM37 shows a granular cytoplasmic pattern. Endogenous TRIM37 is not imported into peroxisomes in peroxin 1 (PEX1(-/-)) and peroxin 5 (PEX5(-/-)) mutant fibroblasts but is imported normally in peroxin 7 (PEX7(-/-)) deficient fibroblasts, giving further evidence for a peroxisomal localization of TRIM37. Fibroblasts derived from patients with mulibrey nanism lack C-terminal TRIM37 immunoreactivity but stain normally for both peroxisomal matrix and membrane markers, suggesting apparently normal peroxisome biogenesis in patient fibroblasts. Taken together, this molecular evidence unequivocally indicates that TRIM37 is located in the peroxisomes, and Mulibrey nanism thus can be classified as a new peroxisomal disorder. >58.NF01119975 PMID:12210501 TI - Blocking the translation elongation factor-1 delta with its antisense mRNA results in a significant reversal of its oncogenic potential. AB - In spite of the strong evidence for the carcinogenic activity of cadmium and its related compounds, the underlying molecular mechanisms that lead to malignant transformation in cells exposed to cadmium remain unknown. Recently, Joseph et al. [J. Biol. Chem. 227:6131-6136, 2002] have identified, cloned, and characterized the mouse Translation Elongation Factor-1 delta sub-unit (TEF-1 delta, GenBank Accession Number AF304351) as a novel cadmium-responsive proto-oncogene. Presently, additional studies regarding the oncogenic potential of TEF-1 delta have been carried out. Transfection of NIH3T3 cells with the pcDNA3.1 expression vector containing the TEF-1 delta cDNA in the sense (5'-->3') orientation resulted in overexpression of the encoded 31 kDa protein. Transfection-mediated overexpression of TEF-1 delta protein resulted in transformation of the cells as evidenced from the appearance of transformed foci. Cotransfection of the cells with a mixture of plasmid DNA consisting of TEF-1 delta cDNA in the sense (5'-->3') and in the antisense (3'-->5') orientation resulted in significant inhibition of translation of the TEF-1 delta protein. Antisense TEF-1 delta mRNA-mediated inhibition of translation of TEF-1 delta protein, furthermore, resulted in inhibition of TEF-1 delta-mediated transformation of NIH3T3 cells as evidenced from the decrease in the number of transformed foci. These results further confirm that overexpression of TEF-1 delta is oncogenic and the antisense TEF-1 delta mRNA expression reverses its oncogenic potential. >59.NF01235968 PMID:11894099 TI - An amino-acid taste receptor. AB - The sense of taste provides animals with valuable information about the nature and quality of food. Mammals can recognize and respond to a diverse repertoire of chemical entities, including sugars, salts, acids and a wide range of toxic substances. Several amino acids taste sweet or delicious (umami) to humans, and are attractive to rodents and other animals. This is noteworthy because L-amino acids function as the building blocks of proteins, as biosynthetic precursors of many biologically relevant small molecules, and as metabolic fuel. Thus, having a taste pathway dedicated to their detection probably had significant evolutionary implications. Here we identify and characterize a mammalian amino-acid taste receptor. This receptor, T1R1+3, is a heteromer of the taste-specific T1R1 and T1R3 G-protein-coupled receptors. We demonstrate that T1R1 and T1R3 combine to function as a broadly tuned L-amino-acid sensor responding to most of the 20 standard amino acids, but not to their D-enantiomers or other compounds. We also show that sequence differences in T1R receptors within and between species (human and mouse) can significantly influence the selectivity and specificity of taste responses. >60.NF00866578 PMID:12081504 TI - Activation mechanism of CDK2: role of cyclin binding versus phosphorylation. AB - Activation of the cyclin-dependent kinases is a two-step process involving cyclin binding followed by phosphorylation at a conserved threonine residue within the kinase activation loop. In this study, we describe the separate roles of cyclin A binding versus phosphorylation in the overall activation mechanism of CDK2. Interaction of CDK2 with cyclin A results in a partially active complex that is moderately defective in the binding of the protein substrate, but not ATP, and severely defective in both phosphoryl group transfer and turnover. Alternatively, phosphorylation of the CDK2 monomer also results in a partially activated species, but one that is severely (> or = 480-fold) defective in substrate binding exclusively. Catalytic turnover in the phosphorylated CDK2 monomer is largely unimpaired (approximately 8-fold lower). Our data support a model for the activation of CDK2 in vivo, in which interaction of unphosphorylated CDK2 with cyclin A serves to configure the active site for ground-state binding of both ATP and the protein substrate, and further aligns ATP in the transition state for phosphoryl transfer. Optimizing the alignment of protein substrates in the phosphoryl transfer reaction is the principal role of phosphorylation at Thr(160). >61.NF00131800 PMID:11840488 TI - Association analysis of polymorphisms at the interleukin-1 locus in essential hypertension. AB - Infection with microorganisms such as Helicobacter pylori and Chlamydia pneumoniae has been associated with coronary heart disease (CAD) and hypertension (HT). Infection increases the release of pro-inflammatory cytokines, thus facilitating interactions that lead to vascular damage and other effects. We hypothesized that genetically determined differences in activity or responsiveness of cytokine(s) might contribute to HT. The interleukin-1 gene (IL1) cluster on chromosome 2q14 contains three related genes (IL1A, IL1B, and IL1RN) located within a 430-kb region. These encode IL-1alpha and IL-1beta, as well as their endogenous receptor antagonist, IL-1ra. The IL1RN gene has a penta-allelic 86-bp tandem repeat in intron 2. Allele IL1RN* 2 is associated with a wide range of chronic inflammatory and autoimmune conditions, and its combination with the -31T variant of an IL1B C(-31)T polymorphism constitutes a pro-inflammatory haplotype that leads to vigorous IL-1beta production. We therefore tested each of these polymorphisms for association with HT. Subjects were white Anglo-Celtic residents of Sydney, Australia. Frequencies of IL1B C(-31)T genotypes CC, CT, and TT were 0.50, 0.40, and 0.10 in normotensive (NT) and 0.46, 0.46, and 0.08 in HT, respectively (chi(2) = 1.2, P = 0.55). T allele frequency in NT (0.30) was similar to that in HT (0.31). For the IL1RN variant, frequencies of alleles IL1RN* 1 and * 2 and combined minor alleles * 3, * 4, and * 5 were 0.61, 0.36, and 0.03 in NT and 0.54, 0.36, and 0.10 in HT, respectively (chi(2) = 11, P = 0.004). In conclusion, no association of the IL1B C(- 31)T with HT was found, whereas combined frequency of the minor alleles of the IL1RN polymorphism was increased in the HT cohort studied. >62.NF00989995 PMID:12084581 TI - Cloning and identification of a new member of water channel (AQP10) as an aquaglyceroporin. AB - Recently, a new member of aquaporins was reported as AQP10 [Biochem. Biophys. Res. Commun. 287 (2001) 814], which is incompletely spliced to lose the sixth transmembrane domain and has poor water and no glycerol/urea permeabilities. Independently, we identified a similar clone in human. Our AQP10 consists of 301 amino acids with a highly conserved sixth transmembrane domain. AQP10 has higher identity with aquaglyceroporins (50% with AQP9, 48% with AQP3, 42% with AQP7) and lower identity with other aquaporins (32% with AQP1 and AQP8). AQP10 is expressed only in the small intestine with (approximately 2 kb). RNase protection assay revealed the absence of the unspliced form, supporting the authenticity of our clone. When expressed in Xenopus oocytes, AQP10 stimulated osmotic water permeability sixfold in a mercury-sensitive manner. Glycerol and urea uptakes were also stimulated, while adenine uptake was not. The genome structure of AQP10 is similar to those of other aquaglyceroporins (AQP3, AQP7, AQP9) with six exons. We conclude that AQP10 represents a new member of aquaglyceroporins functionally as well as structurally. >63.NF00124514 PMID:12461076 TI - A signal peptide derived from hsp60 binds HLA-E and interferes with CD94/NKG2A recognition. AB - Human histocompatibility leukocyte antigen (HLA)-E is a nonclassical major histocompatibility complex (MHC) class I molecule which presents a restricted set of nonameric peptides, derived mainly from the signal sequence of other MHC class I molecules. It interacts with CD94/NKG2 receptors expressed on the surface of natural killer (NK) cells and T cell subsets. Here we demonstrate that HLA-E also presents a peptide derived from the leader sequence of human heat shock protein 60 (hsp60). This peptide gains access to HLA-E intracellularly, resulting in up-regulated HLA-E/hsp60 signal peptide cell-surface levels on stressed cells. Notably, HLA-E molecules in complex with the hsp60 signal peptide are no longer recognized by CD94/NKG2A inhibitory receptors. Thus, during cellular stress an increased proportion of HLA-E molecules may bind the nonprotective hsp60 signal peptide, leading to a reduced capacity to inhibit a major NK cell population. Such stress induced peptide interference would gradually uncouple CD94/NKG2A inhibitory recognition and provide a mechanism for NK cells to detect stressed cells in a peptide-dependent manner. >64.NF00116173 PMID:11741979 TI - Interaction of the C-terminal domain of p43 and the alpha subunit of ATP synthase. Its functional implication in endothelial cell proliferation. AB - Human p43 is associated with macromolecular tRNA synthase complex and known as a precursor of endothelial monocyte-activating polypeptide II (EMAP II). Interestingly, p43 is also secreted to induce proinflammatory genes. Although p43 itself seems to be a cytokine working at physiological conditions, most of the functional studies have been obtained with its C-terminal equivalent, EMAP II. To gain an insight into the working mechanism of p43/EMAP II, we used EMAP II and searched for an interacting cell surface molecule. The level of EMAP II-binding molecule(s) was significantly increased in serum-starved tumor cells. Thus, the EMAP II-binding molecule was isolated from the membrane of the serum-starved CEM cell. The isolated protein was determined to be the alpha subunit of ATP synthase. The interaction of EMAP II and alpha-ATP synthase was confirmed by enzyme-linked immunosorbent assay and in vitro pull down assays and blocked with the antibodies raised against EMAP II and alpha-ATP synthase. The binding of EMAP II to the surface of serum-starved cells was inhibited in the presence of soluble alpha-ATP synthase. EMAP II inhibited the growth of endothelial cells, and this effect was relieved by soluble alpha-ATP synthase. Anti-alpha-ATP synthase antibody also showed an inhibitory effect on the proliferation of endothelial cells mimicking the activity of EMAP II. These results suggest the potential interaction of p43/EMAP II with alpha-ATP synthase and its role in the proliferation of endothelial cells. >65.NF00131727 PMID:11823532 TI - Biological activities of ecalectin: a novel eosinophil-activating factor. AB - Ecalectin, produced by Ag-stimulated T lymphocytes, is a potent eosinophil-specific chemoattractant in vitro as well as in vivo and thus is implicated in allergic responses. Ecalectin differs structurally from other known eosinophil chemoattractants (ECAs); ecalectin belongs to the galectin family defined by their affinity for beta-galactosides and by their conserved carbohydrate recognition domains. These characteristic features suggest that ecalectin has unique activities associated with allergic inflammation besides ECA activity. Conversely, ecalectin may mediate ECA activity by binding to a receptor of a known ECA via affinity for the beta-galactosides present on this receptor. In this study, we have tested whether ecalectin mediates ECA activity by binding to a receptor of a known ECA, and we have assessed its effects on eosinophils. Ecalectin did not mediate ECA activity by binding to the IL-5R or to CCR3. Also, the ECA activity of ecalectin was mainly chemokinetic. In addition, ecalectin induced concentration-dependent eosinophil aggregation, a marker for eosinophil activation. Ecalectin induced concentration-dependent superoxide production from eosinophils but did not induce degranulation; usually these two events are coupled in eosinophil activation. Moreover, ecalectin directly prolonged eosinophil survival in vitro and did not trigger eosinophils to secrete cytokines that prolong eosinophil survival. These results demonstrate that ecalectin has several unique effects on eosinophils. Therefore, we conclude that ecalectin is a novel eosinophil-activating factor. Presumably, these effects allow ecalectin to play a distinctive role in allergic inflammation. >66.NF00105565 PMID:11956185 TI - Transcriptional control of the human thromboxane synthase gene in vivo and in vitro. AB - Thromboxane A(2), a potent mediator of vasoconstriction and platelet aggregation, is synthesized from prostaglandin H(2) by thromboxane synthase (TXAS). We report here on promoter analyses of human TXAS using in vitro transcription and in vivo transfection methods. The 39-bp core promoter, containing both TATA and initiator elements, accurately initiates transcription in an orientation-dependent manner in a cell-free transcription system. Mutation of either TATA or initiator abolished transcriptional activity, but the upstream sequence had no effect on TXAS promoter activities in vitro, suggesting that the core promoter is sufficient for transcriptional activity from a naked DNA template. In contrast, mutation of both elements drastically decreased the promoter activity in vivo. Furthermore, TXAS proximal promoter containing the nucleotides -90 to -56 relative to the transcriptional start site was necessary for promoter transactivation in vivo. Transcriptional activities from 5'-deletion mutants indicated that the effects of the nucleotides -90/-56 were more pronounced in stably transfected cells (a 150-fold difference) than in the transiently transfected cells (an 8-fold difference), reflecting the effects of TXAS transcriptional activation from replicating and nonreplicating DNA templates. Partial micrococcal nuclease digestion indicated that the sequence -90/-56, where the NF-E2 site is located, is associated with alterations of nucleosomal structure at the regions of promoter and reporter gene but not the region further downstream. Mutagenesis and forced expression studies demonstrated a critical role of p45 NF-E2 in controlling TXAS expression under native chromatin conditions. Band shifting and chromatin immunoprecipitation assays indicated that p45 NF-E2 was bound to the TXAS promoter in vitro and in vivo. Indirect end labeling and ligation-mediated PCR analyses further demonstrated that the occupation of TXAS promoter NF-E2 site was associated with disruption of nucleosomal structure. Collectively, these results indicate that binding of NF-E2 is critical both for alteration of the nucleosomal structure and for activation of the TXAS promoter, whereas the TATA and initiator elements are essential for transcription. >67.NF00123385 PMID:11895916 TI - High prevalence of decreased expression of KAI1 metastasis suppressor in human oral carcinogenesis. AB - PURPOSE: KAI1 was originally identified in prostate cancer as a metastasis suppressor gene. Recent studies have shown a frequent down-regulation of KAI1 expression in many tumor types, whereas mutation or hypermethylation of the gene is infrequent. The aim of the present study was to examine whether loss of KAI1 expression that might be caused by genetic or epigenetic alterations could contribute to oral carcinogenesis. EXPERIMENTAL DESIGN : We analyzed mutational and methylation status of the KAI1 gene and both the mRNA and protein level in a series of oral tumors [28 precancerous lesions, 101 primary oral squamous cell carcinomas (OSCCs), and 30 metastatic OSCCs] and OSCC-derived cell lines. We also examined p53 protein expression, which has been reported to be a candidate activator for the KAI1 gene. RESULTS: With the exception of three microsatellite instabilities in the KAI1 gene, we found no mutations in the coding sequence of the KAI1 gene, no loss of heterozygosity, and no hypermethylation of the KAI1 promoter region in all samples investigated. By immunohistochemistry, however, high frequencies of KAI1 down-regulation were evident not only in the metastatic OSCCs [29 of 30 (97%)] but also in the primary OSCCs [83 of 101 (82%)] and in the precancerous lesions [13 of 28 (46%)]. There was a significant relationship between down-regulation of KAI1 protein expression and primary tumors associated with lymph node metastases (P = 0.0115), whereas there was no statistical correlation between p53 status and KAI1 expression. Taken together, reverse transcription-PCR data were consistent with the protein expression status in 16 patients from whom mRNA was available. CONCLUSIONS: Our data suggest that whereas loss of KAI1 protein expression is associated with primary tumors with lymph node metastases, the down-regulation of KAI1 is an early event in the progression of human oral cancer. The down-regulation of KAI1 is not associated with either mutation, allelic loss, methylation of the promoter, or p53 regulation. >68.NF01031557 PMID:12649177 TI - A novel gene, MSI2, encoding a putative RNA-binding protein is recurrently rearranged at disease progression of chronic myeloid leukemia and forms a fusion gene with HOXA9 as a result of the cryptic t(7;17)(p15;q23). AB - The pathogenetic role of the P210 BCR/ABL1 fusion gene in the chronic phase of chronic myeloid leukemia (CML) has been well established.In contrast, the genetic mechanisms underlying the disease progression into the accelerated phase (AP) and the final blast crisis (BC) remain poorly understood. We have previously identified (A. Barbouti et al., Genes Chromosomes Cancer, 35: 127-137, 2002) two cryptic balanced translocations, t(7;17)(p15;q23) and t(7;17)(q32-34;q23), in CML AP/BC using multicolor fluorescence in situ hybridization. In this study, we show that a novel gene in 17q23, Musashi-2 (MSI2), encoding a putative RNA-binding protein, is rearranged in both cases and that a MSI2/HOXA9 fusion gene is formed in the case with the 7p15 breakpoint. The identified in-frame MSI2/HOXA9 fusion transcript retains both of the RNA recognition motif domains of MSI2, which is fused to the homeobox domain of HOXA9, and is likely to play an important role in the disease progression of CML. >69.NF00106457 PMID:11992112 TI - ROCK and Dia have opposing effects on adherens junctions downstream of Rho. AB - Adherens junctions (AJs) are crucial for maintaining the integrity of epithelial tissues and are often disrupted during tumour progression. Rho family proteins have been shown to regulate adherens junctions. We find that activation of the effector kinase ROCK and acto-myosin contraction disrupts AJs downstream of Rho. In contrast, signalling through the Rho effector Dia1 is required to ensure a dynamically stable interface between cells and the maintenance of adherens junction complexes. The ability of Dia1 to regulate the actin network is crucial for the localization of adherens junction components to the cell periphery. >70.NF00130676 PMID:12372421 TI - The human perforin gene is a direct target of STAT4 activated by IL-12 in NK cells. AB - IL-12 activates STAT4 by inducing tyrosine phosphorylation, homo-dimerization, and nuclear translocation in NK cells and thereby stimulates proliferation and activation of these cells. The pore-forming protein perforin is a key effector protein for NK cell- and cytotoxic T lymphocyte-mediated cytolysis. Here we demonstrate that IL-12 induces the expression of the perforin gene in human NK cell line, NKL. Electrophoretic mobility shift assays using a probe containing two putative STAT-binding sequences located at -1085 and -1059 in the human perforin gene showed that STAT4 or STAT5 activated by IL-12 or IL-2, respectively, in NKL cells binds this region. Further analyses using various probes with or without mutated STAT-binding sequences showed that, although either of the two tandem STAT-binding sequences binds STAT4 weakly, the presence of both is required for significant binding of activated STAT4 and for formation of the STAT4-DNA-binding complex with lower electrophoretic mobility. Furthermore, mutation of either of the tandem STAT-binding sequences abolished the IL-12-induced activation of the perforin gene promoter in reporter gene assays. These results indicate that the IL-12-induced expression of the perforin gene in NK cells is directly regulated by STAT4, which binds, most likely as a homo-tetramer, to the tandem STAT-binding sequences in the perforin gene promoter. >71.NF00094921 PMID:11868823 TI - Human bronchial epithelium expresses interleukin-9 receptors and releases neutrophil chemotactic factor. AB - Growing evidence obtained from human genomic analysis and antigen-challenged transgenic mice suggests that interleukin-9 (IL-9) is a candidate factor in immunoglobulin E (IgE) production and thus is thought to be associated with bronchial inflammation and bronchial hyperresponsiveness (BHR). To evaluate the expression of the IL-9 receptor and its effect on the IL-9 human bronchial cell line BEAS-2B cells, reverse transcriptase-polymerase chain reaction (RT-PCR), immunohistochemical investigation, and chemotaxis assay were performed. The components of the IL-9 receptor, consisting of IL-9 receptor alpha (CD129) and IL-2 receptory ((1)132), were expressed on BEAS-2B cells as determined by RT-PCR and flow cytometry. BEAS-2B cells exposed to IL-9 released neutrophil chemotactic activity (NCA) in a time- and dose-dependent manner, and the presence of granulocyte colony-stimulating factor (G-CSF) was also detected. This factor is primarily involved in NCA for the measurement of cytokines and in the inhibition assay of neutrophil chemotaxis. These findings suggest that bronchial epithelial cells may express IL-9 receptors, and that IL-9 may induce airway inflammation through the release of G-CSF from bronchial epithelial cells. >72.NF01196118 PMID:11938437 TI - Merosin-deficient congenital muscular dystrophy, autosomal recessive (MDC1A, MIM#156225, LAMA2 gene coding for alpha2 chain of laminin). AB - Congenital muscular dystrophies (CMDs) are a highly heterogeneous group of neuromuscular disorders. A subgroup displays a specific deficiency in a protein of the extracellular matrix, the alpha2 chain of laminin-2 (merosin). A number of mutations in the gene encoding this protein have been identified in patients who present with a severe phenotype and white matter changes. >73.NF00089173 PMID:11840499 TI - Human metabotropic glutamate receptor 2 gene (GRM2): chromosomal sublocalization (3p21.1-p21.2) and genomic organization. AB - Imbalances in glutamatergic function have been implicated in the pathogenesis of neuropsychiatric disorders. Consequently, glutamate receptors genes are promising candidates in search of susceptibility genes for these disorders. In the present study, we report the chromosomal sublocalization and genomic organization of the human metabotropic glutamate receptor 2 gene (GRM2). Using monochromosomal hybrid cell lines of NIGMS Mapping Panel 2 (Coriell Cell Repository), the GRM2 gene was localized to human chromosome 3, confirming previously reported localization. In addition, using the radiation hybrid panel RH3 (Research Genetics), we sublocalized the GRM2 gene to chromosomal region 3p21.1-p21.2. The genomic organization of the GRM2 gene was established using a premade library of adaptor-ligated, human-specific genomic DNA fragments. The gene consists of 5 exons, with sizes ranging from 74 to 1,076 bp. >74.NF00088609 PMID:11818523 TI - Expression and modulation of Rho kinase in human pregnant myometrium. AB - There is little information outlining the role of Rho kinase, RhoA, and calcium sensitization in regulation of human uterine contractility during pregnancy. The aims of this study were to investigate the expression of RhoA, and the Rho kinases ROCK I and ROCK II in human pregnant myometrium, to evaluate the effects of Rho kinase inhibition on pregnant human myometrial contractility in vitro, and to compare these effects with those of the calcium channel blocker nifedipine. RT-PCR using primers for RhoA, ROCK I and ROCK II was performed on mRNA isolated from human pregnant myometrium. Isometric recording was performed in isolated myometrial strips obtained at Caesarean section. The effects of the Rho kinase inhibitor Y-27632 (1 nmol/l to 10 mmol/l), and nifedipine (1 nmol/l to 10 mmol/l), on oxytocin (0.5 nmol/l) induced contractions were measured and compared. Expression of RhoA, ROCK I and ROCK II mRNA was identified in human pregnant myometrium (n = 3). Y-27632 exerted a potent relaxant effect on myometrial contractility with a pD(2) value (+/- SEM) of 7.63 +/- 0.38 (n = 6). The maximum net relaxant effect (+/- SEM) was 72.3 +/- 6.1% (n = 6). Corresponding values for nifedipine were 7.24 +/- 0.48 (n = 6; P = 0.469) and 93.40 +/- 3.1% (n = 6; P = 0.028). Rho A/Rho kinase-mediated calcium sensitization may play role in the physiology of human parturition, and pharmacological inhibition of this pathway may therefore provide a novel approach to tocolysis for pre-term labour. >75.NF00085856 PMID:11932251 TI - Ran-binding protein 3 links Crm1 to the Ran guanine nucleotide exchange factor. AB - Ran-binding protein 3 (RanBP3) is an approximately 55-kDa protein that functions as a cofactor for Crm1-mediated nuclear export. RanBP3 stimulates export by enhancing the affinity of Crm1 for Ran.GTP and cargo. However, important additional functions for this cofactor may exist. We now report that RanBP3 associates with the Ran-specific guanine nucleotide exchange factor, regulator of chromosome condensation 1 (RCC1). This interaction was stimulated by the addition of Ran; moreover, Ran.GDP, Ran.GTP, and Ran without nucleotide could all stimulate complex formation between RanBP3 and RCC1 even though binding of Ran.GDP to RanBP3 alone was undetectable. RanBP3 could also promote binding of Crm1 to RCC1 in the presence of Ran. Binding of RanBP3 to RCC1 increased the catalytic activity of RCC1 toward Ran, and importantly, the ability of RanBP3 to stimulate RCC1 was not affected by the presence of Crm1. These data indicate that RanBP3 acts as a scaffold protein to promote the efficient assembly of export complexes. By tethering Crm1 to catalytically enhanced RCC1, RanBP3 may lower the entropic barrier for the loading of Ran.GTP onto Crm1. We propose that this provides an additional mechanism by which RanBP3 facilitates export. >76.NF00076948 PMID:11859273 TI - mRNA expression of EGF receptor ligands in atrophic gastritis before and after Helicobacter pylori eradication. AB - BACKGROUND: Epidermal growth factor (EGF) receptor ligands (EGFRL) including transforming growth factor alpha (TGF-alpha), amphiregulin, and heparin binding-EGF (HB-EGF) are involved in gastric mucosal repair in chronic gastritis. Their mRNA expression has been shown to be upregulated after Helicobacter pylori (H.p.)-eradication but little is known about this gene expression in atrophic gastritis. The purpose of our study was to investigate EGFRL mRNA expression in gastric mucosa of patients with atrophic gastritis before and after H.p.-eradication. MATERIAL/METHODS: Antral mucosal biopsies were obtained during endoscopy in 10 H.p. positive patients with atrophic gastritis and in 10 H.p. negative controls with intact mucosa. Total RNA of antral biopsies was extracted and RT-PCR was performed, the PCR-products being measured densitometrically. Values were compared with mRNA expressions in H.p. negative antral mucosa (n=10). RESULTS: Gastric biopsies revealed mRNA expression for TGF-alpha, amphiregulin and HB-EGF, both in H.p. positive atrophic antritis and in H.p. negative healthy mucosa. The mRNA expression of TGF-alpha in atrophic gastritis was significantly upregulated after H.p.-eradication, whereas that of amphiregulin did not change after this eradication. Expression of HB-EGF mRNA was higher in H.p.-infection than after H.p.-eradication or in H.p. negative healthy subjects. CONCLUSIONS: H.p. positive atrophic gastritis is associated with differential mRNA expression of EGF receptor ligands. H.p.-eradication in this entity leads to unequal changes of these growth factor expressions compared to chronic active gastritis without atrophy. >77.NF00100002 PMID:12122017 TI - Head involution defective (Hid)-triggered apoptosis requires caspase-8 but not FADD (Fas-associated death domain) and is regulated by Erk in mammalian cells. AB - The molecular machinery of apoptosis is evolutionarily conserved with some exceptions. One such example is the Drosophila proapoptotic gene Head involution defective (Hid), whose mammalian homologue is not known. Hid is apoptotic to mammalian cells, and we have examined the mechanism by which Hid induces death. We demonstrate for the first time a role for the extracellular signal-related kinase-1/2 (Erk-1/2) in the regulation of Hid function in mammalian cells. Bcl-2 and an inhibitor of caspase-9 blocked apoptosis, indicative of a role for the mitochondrion in this pathway, and we provide evidence for a role for caspase-8 in Hid-induced apoptosis. Thus, apoptosis was blocked by an inhibitor of caspase-8, deletion of caspase-8 rendered cells resistant to Hid-induced apoptosis, and Hid associated with caspase-8 in cell lysates. The Fas-associated death domain (FADD) was dispensable for the apoptotic function of Hid, indicating that Hid does not require extracellular death receptor signaling for the activation of caspase-8. In activated T cells, the cytokine interleukin-2 blocked caspase-8 processing and apoptosis, suggesting that survival cues from trophic factors may target a Hid-like intermediate present in mammalian cells. Thus, this study shows that Hid engages with conserved components of cellular death machinery and suggests that apoptotic paradigms characterized by FADD-independent activation of caspase-8 may involve a Hid-like molecule in mammalian cells. >78.NF00866584 PMID:11992252 TI - Mutations in COL6A3 cause severe and mild phenotypes of Ullrich congenital muscular dystrophy. AB - Ullrich congenital muscular dystrophy (UCMD) is an autosomal recessive disorder characterized by generalized muscular weakness, contractures of multiple joints, and distal hyperextensibility. Homozygous and compound heterozygous mutations of COL6A2 on chromosome 21q22 have recently been shown to cause UCMD. We performed a genomewide screening with microsatellite markers in a consanguineous family with three sibs affected with UCMD. Linkage of the disease to chromosome 2q37 was found in this family and in two others. We analyzed COL6A3, which encodes the alpha3 chain of collagen VI, and identified one homozygous mutation per family. In family I, the three sibs carried an A-->G transition in the splice-donor site of intron 29 (6930+5A-->G), leading to the skipping of exon 29, a partial reduction of collagen VI in muscle biopsy, and an intermediate phenotype. In family II, the patient had an unusual mild phenotype, despite a nonsense mutation, R465X, in exon 5. Analysis of the patient's COL6A3 transcripts showed the presence of various mRNA species-one of which lacked several exons, including the exon containing the nonsense mutation. The deleted splice variant encodes collagen molecules that have a shorter N-terminal domain but that may assemble with other chains and retain a functional role. This could explain the mild phenotype of the patient who was still ambulant at age 18 years and who showed an unusual combination of hyperlaxity and finger contractures. In family III, the patient had a nonsense mutation, R2342X, causing absence of collagen VI in muscle and fibroblasts, and a severe phenotype, as has been described in patients with UCMD. Mutations in COL6A3 are described in UCMD for the first time and illustrate the wide spectrum of phenotypes which can be caused by collagen VI deficiency. >79.NF00135134 PMID:12080057 TI - p52 Mediates XPB function within the transcription/repair factor TFIIH. AB - To further our understanding of the transcription/DNA repair factor TFIIH, we investigated the role of its p52 subunit in TFIIH function. Using a completely reconstituted in vitro transcription or nucleotide excision repair (NER) system, we show that deletion of the C-terminal region of p52 results in a dramatic reduction of TFIIH NER and transcription activities. This mutation prevents promoter opening and has no effect on the other enzymatic activities of TFIIH. Moreover, we demonstrate that intact p52 is needed to anchor the XPB helicase within TFIIH, providing an explanation for the transcription and NER defects observed with the mutant p52. We show that these two subunits physically interact and map domains involved in the interface. Taken together, our results show that the p52/Tfb2 subunit of TFIIH regulates the function of XPB through pair-wise interactions as described previously for p44 and XPD. >80.NF00141372 PMID:12397057 TI - Identification of a specific molecular repressor of the peroxisome proliferator-activated receptor gamma Coactivator-1 alpha (PGC-1alpha). AB - The nuclear co-activator PGC-1alpha is a pivotal regulator of numerous pathways controlling both metabolism and overall energy homeostasis. Inappropriate increases in PGC-1alpha activity have been linked to a number of pathological conditions including heart failure and diabetes mellitus. Previous studies (Puigserver, P., Adelmant, G., Wu, Z., Fan, M., Xu, J., O'Malley, B., and Spiegelman, B. M. (1999) Science 286, 1368-1371) have demonstrated an inhibitory domain within PGC-1alpha that limits transcriptional activity. Using this inhibitory domain in a yeast two-hybrid screen, we demonstrate that PGC-1alpha directly associates with the orphan nuclear receptor estrogen-related receptor-alpha (ERR-alpha). The binding of ERR-alpha to PGC-1alpha requires the C-terminal AF2 domain of ERR-alpha. PGC-1alpha and ERR-alpha have a similar pattern of expression in human tissues, with both being present predominantly in organs with high metabolic needs such as skeletal muscle and kidney. Similarly, we show that in mice physiological stimuli such as fasting coordinately induces PGC-1alpha and ERR-alpha transcription. We also demonstrate that under normal conditions PGC-1alpha is located within discrete nuclear speckles, whereas the expression of ERR-alpha results in PGC-1alpha redistributing uniformly throughout the nucleoplasm. Finally, we show that the expression of ERR-alpha can dramatically and specifically repress PGC-1alpha transcriptional activity. These results suggest a novel mechanism of transcriptional control wherein ERR-alpha can function as a specific molecular repressor of PGC-1alpha activity. In addition, our results suggest that other co-activators might also have specific repressors, thereby identifying another layer of combinatorial complexity in transcriptional regulation. >81.NF00133354 PMID:12409289 TI - Alternative core promoters regulate tissue-specific transcription from the autoimmune diabetes-related ICA1 (ICA69) gene locus. AB - Islet cell autoantigen 69-kDa (ICA69), protein product of the human ICA1 gene, is one target of the immune processes defining the pathogenesis of Type 1 diabetes. We have characterized the genomic structure and functional promoters within the 5'-regulatory region of ICA1. 5'-RNA ligase-mediated rapid amplification of cDNA ends evaluation of ICA1 transcripts expressed in human islets, testis, heart, and cultured neuroblastoma cells reveals that three 5'-untranslated region exons are variably expressed from the ICA1 gene in a tissue-specific manner. Surrounding the transcription initiation sites are motifs characteristic of non-TATA, non-CAAT, GC-rich promoters, including consensus Sp1/GC boxes, an initiator element, cAMP-responsive element-binding protein (CREB) sites, and clusters of other putative transcription factor sites within a genomic CpG island. Luciferase reporter constructs demonstrate that the first two ICA1 exon promoters reciprocally stimulate luciferase expression within islet- (RIN 1046-38 cells) and brain-derived (NMB7) cells in culture; the exon A promoter exhibits greater activity in islet cells, whereas the exon B promoter more efficiently activates transcription in neuronal cells. Mutation of a CREB site within the ICA1 exon B promoter significantly enhances transcriptional activity in both cell lines. Our basic understanding of expression from the functional core promoter elements of ICA1 is an important advance that will not only add to our knowledge of the ICA69 autoantigen but will also facilitate a rational approach to discover the function of ICA69 and to identify relevant ICA1 promoter polymorphisms and their potential associations with disease. >82.NF00124284 PMID:12031497 TI - Human uroplakin lb gene structure and promoter analysis. AB - The uroplakin Ib (UPIb) gene is predominantly expressed in urothelium and is overexpressed in 50% of transitional cell carcinoma (TCC). Molecular cloning of the genomic 5' region and comparison to a chromosome 3q genomic contig determined that the gene spans 31 kb and has eight exons including a noncoding exon 1. Multiple transcription start sites were identified in exon 1 by 5'RACE and ribonuclease protection assay (RPA). In vitro reporter gene analysis was performed with 2.3 kb of genomic DNA sequence flanking the 5' end of UPIb. A 235-bp 5' fragment that included UPIb exon 1 generated strong transcriptional activity in normal and malignant human urothelial cell lines. Established malignant cell lines had greater transcriptional activity from the UPIb promoter than normal human cells. The identification of a functional human UPIb gene promoter may find application in targeting gene therapy strategies for bladder cancer. >83.NF00141618 PMID:12015329 TI - An essential role for DNA methyltransferase DNMT3B in cancer cell survival. AB - Abnormal methylation and associated silencing of tumor suppressor genes is a common feature of many types of cancers. The observation of persistent methylation in human cancer cells lacking the maintenance methyltransferase DNMT1 suggests the involvement of other DNA methyltransferases in gene silencing in cancer. To test this hypothesis, we have evaluated methylation and gene expression in cancer cells specifically depleted of DNMT3A or DNMT3B, de novo methyltransferases that are expressed in adult tissues. Here we have shown that depletion of DNMT3B, but not DNMT3A, induced apoptosis of human cancer cells but not normal cells. DNMT3B depletion reactivated methylation-silenced gene expression but did not induce global or juxtacentromeric satellite demethylation as did specific depletion of DNMT1. Furthermore, the effect of DNMT3B depletion was rescued by exogenous expression of either of the splice variants DNMT3B2 or DNMT3B3 but not DNMT1. These results indicate that DNMT3B has significant site selectivity that is distinct from DNMT1, regulates aberrant gene silencing, and is essential for cancer cell survival. >84.NF00113836 PMID:12359251 TI - Palmitoylation-defective asialoglycoprotein receptors are normal in their cellular distribution and ability to bind ligand, but are defective in ligand uptake and degradation. AB - The hepatic asialoglycoprotein receptor (ASGP-R) is an endocytic recycling receptor that mediates the endocytosis of desialylated glycoproteins. The human ASGP-R is composed of two homologous subunits, H1 and H2, and the cytoplasmic Cys residues in both subunits are palmitoylated. To study the effects of palmitoylation on ASGP-R activity and function, we generated four types of stably transfected cell lines in SK-Hep-1 hepatoma cells, expressing wild-type, or partially or completely palmitoylation-defective ASGP-Rs containing Cys-to-Ser mutations in either one or both subunits. Scatchard analysis showed that all four stable cell lines expressed a similar number of binding sites for asialo-orosomucoid, with comparable dissociation constants of approximately 1-3nM. Immunofluorescence confocal microscopy indicated a normal distribution of the palmitoylation-defective H1 and H2 subunits compared to the wild-type. However, cell lines expressing palmitoylation-defective ASGP-Rs had markedly reduced rates of ligand uptake and degradation compared to cells expressing wild-type ASGP-Rs. We conclude that failure to palmitoylate Cys residues in either or both subunits of human ASGP-Rs results in very inefficient uptake and degradation of ligands. >85.NF00103794 PMID:12200141 TI - RNA polymerase II large subunit is cleaved by caspases during DNA damage-induced apoptosis. AB - UV radiation induces DNA lesions that are repaired by the nucleotide excision repair (NER) pathway. Cells that are NER deficient such as those derived from xeroderma pigmentosum (XP) patients are susceptible to apoptosis after 10J/m(2) UV radiation, a dose largely survivable by repair proficient cells. Herein, we report that RNA polymerase II large subunit (RNAP II-LS) undergoes caspase-mediated cleavage, yielding a 140kDa C-terminal fragment in XP lymphoblasts but not NER proficient lymphoblasts after 10J/m(2) UV irradiation. Cleavage could also be induced by cisplatin or oxaliplatin, but not transplatin, an isomer of cisplatin that does not induce DNA adducts. The cleavage of RNAP II-LS was blocked by a panel of caspase inhibitors but not by proteasomal inhibitors or inhibitors of other proteases. In vitro cleavage with caspase 8 yielded the same 140kDa RNAP II-LS fragment observed in vivo. Using site-directed mutagenesis, the RNAP II-LS cleavage site was localized to an LETD sequence ending at residue 1339, which is near its C-terminal domain. >86.NF00098331 PMID:11750878 TI - Alternate signalling pathways from the interleukin-2 receptor. AB - Interleukin-2 (IL-2) plays a major role in the proliferation of cell populations during an immune reaction. The beta(c) and gamma(c) subunits of the IL-2 receptor (IL-2R) are sufficient and necessary for signal transduction. Despite lacking known catalytic domains, receptor engagement leads to the activation of a diverse array protein tyrosine kinases (PTKs). In resting or anergised T cells, Jak3 is not activated. Signals arising from the PROX domain of the gamma(c) subunit activate p56(lck) (lck) leading to the induction of anti-apoptotic mechanisms. When Jak3 is activated, in primed T cells, other PTKs predominantly mediate the induction of anti-apoptotic mechanisms and drive cellular proliferation. This review intends to suggest a role for these differences within the context of the immune system. >87.NF00087720 PMID:12082086 TI - cGMP-dependent protein kinase I beta physically and functionally interacts with the transcriptional regulator TFII-I. AB - Transcriptional regulation of the fos promoter by nitric oxide and cGMP can occur by nuclear translocation of cGMP-dependent protein kinase I (G-kinase I) (Gudi, T., Lohmann, S. M., and Pilz, R. B. (1997) Mol. Cell. Biol. 17, 5244-5254). To identify nuclear targets of G-kinase I, we performed a yeast two-hybrid screen with G-kinase I beta as bait. We found that G-kinase I beta interacted specifically with TFII-I, an unusual transcriptional regulator that associates with multiple proteins to modulate both basal and signal-induced transcription. By using purified recombinant proteins, the interaction was mapped to the N-terminal 93 amino acids of G-kinase I beta and one of six 95-amino acid repeats found in TFII-I. In baby hamster kidney cells, cGMP analogs enhanced co-immunoprecipitation of G-kinase I beta and TFII-I by inducing co-localization of both proteins in the nucleus, but in other cell types containing cytoplasmic TFII-I the G-kinase-TFII-I interaction was largely cGMP-independent. G-kinase phosphorylated TFII-I in vitro and in vivo on Ser(371) and Ser(743) outside of the interaction domain. G-kinase strongly enhanced TFII-I transactivation of a serum-response element-containing promoter in COS7 cells, and this effect was lost when Ser(371) and Ser(743) of TFII-I were mutated. TFII-I by itself had little effect on a full-length fos promoter in baby hamster kidney cells, but it synergistically enhanced transcriptional activation by G-kinase I beta. Binding of G-kinase to TFII-I may position the kinase to phosphorylate and regulate TFII-I and/or factors that interact with TFII-I at the serum-response element. >88.NF00142189 PMID:11788600 TI - Consequences of mevalonate depletion. Differential transcriptional, translational, and post-translational up-regulation of Ras, Rap1a, RhoA, AND RhoB. AB - Ras-related proteins are small GTPases that are post-translationally modified with mevalonate-derived isoprenoids. Although the effects of inhibition of isoprenylation on protein function have been examined, the consequences of depletion of isoprenoid pools on regulation of expression of isoprenylated proteins have yet to be investigated. In these studies we have shown that depletion of mevalonate results in increased total levels of Ras, Rap1a, RhoA, and RhoB in K562 cells. Cycloheximide and [(35)S]methionine pulse/pulse-chase experiments reveal that mevalonate depletion increases the de novo synthesis of Ras and RhoA and decreases the degradation of existing Ras and RhoA protein. Pretreatment with actinomycin D completely prevents the induced up-regulation of RhoB and only partially prevents the up-regulation of Ras, Rap1a, and RhoA. Although depletion of mevalonate does not alter steady state levels of Ras mRNA, there is an increase in RhoB mRNA. Our results are the first to demonstrate that mevalonate depletion induces up-regulation of Ras and Ras-related proteins by discrete mechanisms that include modulation of transcriptional, translational, and post-translational processes. >89.NF00132388 PMID:12077440 TI - Crystallization and preliminary X-ray crystallographic studies on recombinant human carnitine acetyltransferase. AB - In this paper, the purification, crystallization and preliminary X-ray crystallographic studies of human carnitine acetyltransferase are reported. Recombinant human carnitine acetyltransferase crystals were grown by the hanging-drop vapor-diffusion method and belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 137.65, b = 84.76, c = 57.65 A and one molecule per asymmetric unit. The intensity data were collected from a cryocooled crystal to 1.6 A resolution using a conventional X-ray source. >90.NF00121778 PMID:12082095 TI - Interaction of p58(PITSLRE), a G2/M-specific protein kinase, with cyclin D3. AB - The p58(PITSLRE) is a p34(cdc2)-related protein kinase that plays an important role in normal cell cycle progression. Elevated expression of p58(PITSLRE) in eukaryotic cells prevents them from undergoing normal cytokinesis and appears to delay them in late telophase. To investigate the molecular mechanism of p58(PITSLRE) action, we used the yeast two-hybrid system, screened a human fetal liver cDNA library, and identified cyclin D3 as an interacting partner of p58(PITSLRE). In vitro binding assay, in vivo coimmunoprecipitation, and immunofluorescence cell staining further confirmed the association of p58(PITSLRE) with cyclin D3. This binding was observed only in the G(2)/M phase but not in the G(1)/S phase of the cell cycle; meanwhile, no interaction between p110(PITSLRE) and cyclin D3 was observed in all the cell cycle. The overexpression of cyclin D3 in 7721 cells leads to an exclusively accumulation of p58(PITSLRE) in the nuclear region, affecting its cellular distribution. Histone H1 kinase activity of p58(PITSLRE) was greatly enhanced upon interaction with cyclin D3. Furthermore, kinase activity of p58(PITSLRE) was found to increase greatly in the presence of cyclin D3 using a specific substrate, beta-1,4-galactosyltransferase 1. These data provide a new clue to our understanding of the cellular function of p58(PITSLRE) and cyclin D3. >91.NF00076952 PMID:12391192 TI - Regulatory effect of IFN-kappa, a novel type I IFN, on cytokine production by cells of the innate immune system. AB - IFN-kappa is a recently identified type I IFN that exhibits both structural and functional homology with the other type I IFN subclasses. In this study, we have investigated the effect of IFN-kappa on cells of the innate immune system by comparing cytokine release following treatment of human cells with either IFN-kappa or two recombinant IFN subtypes, IFN-beta and IFN-alpha2a. Although IFN-alpha2a failed to stimulate monocyte cytokine secretion, IFN-kappa, like IFN-beta, induced the release of several cytokines from both monocytes and dendritic cells, without the requirement of a costimulatory signal. IFN-kappa was particularly effective in inhibiting inducible IL-12 release from monocytes. Unlike IFN-beta, IFN-kappa did not induce release of IFN-gamma by PBL. Expression of the IFN-kappa mRNA was observed in resting dendritic cells and monocytes, and it was up-regulated by IFN-gamma stimulation in monocytes, while IFN-beta mRNA was minimally detectable under the same conditions. Monocyte and dendritic cell expression of IFN-kappa was also confirmed in vivo in chronic lesions of psoriasis vulgaris and atopic dermatitis. Finally, biosensor-based binding kinetic analysis revealed that IFN-kappa, like IFN-beta, binds strongly to heparin (K(d): 2.1 nM), suggesting that the cytokine can be retained close to the local site of production. The pattern of cytokines induced by IFN-kappa in monocytes, coupled with the unique induction of IFN-kappa mRNA by IFN-gamma, indicates a potential role for IFN-kappa in the regulation of immune cell functions. >92.NF00078494 PMID:11967569 TI - Structural insights into the pro-apoptotic function of mitochondrial serine protease HtrA2/Omi. AB - HtrA2/Omi, a mitochondrial serine protease in mammals, is important in programmed cell death. However, the underlining mechanism of HtrA2/Omi-mediated apoptosis remains unclear. Analogous to the bacterial homolog HtrA (DegP), the mature HtrA2 protein contains a central serine protease domain and a C-terminal PDZ domain. The 2.0 A crystal structure of HtrA2/Omi reveals the formation of a pyramid-shaped homotrimer mediated exclusively by the serine protease domains. The peptide-binding pocket of the PDZ domain is buried in the intimate interface between the PDZ and the protease domains. Mutational analysis reveals that the monomeric HtrA2/Omi mutants are unable to induce cell death and are deficient in protease activity. The PDZ domain modulates HtrA2/Omi-mediated cell death activity by regulating its serine protease activity. These structural and biochemical observations provide an important framework for deciphering the mechanisms of HtrA2/Omi-mediated apoptosis. >93.NF00100014 PMID:12386154 TI - Absence of calsequestrin 2 causes severe forms of catecholaminergic polymorphic ventricular tachycardia. AB - Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare arrhythmogenic disorder characterized by syncopal events and sudden cardiac death at a young age during physical stress or emotion, in the absence of structural heart disease. We report the first nonsense mutations in the cardiac calsequestrin gene, CASQ2, in three CPVT families. The three mutations, a nonsense R33X, a splicing 532+1 G>A, and a 1-bp deletion, 62delA, are thought to induce premature stop codons. Two patients who experienced syncopes before the age of 7 years were homozygous carriers, suggesting a complete absence of calsequestrin 2. One patient was heterozygous for the stop codon and experienced syncopes from the age of 11 years. Despite the different mutations, there is little phenotypic variation of CPVT for the CASQ2 mutations. Of the 16 heterozygous carriers of these various mutations, 14 were devoid of clinical symptoms or ECG anomalies, whereas 2 of them had ventricular arrhythmias at ECG on exercise tests. In line with this, the diagnosis of the probands was difficult because of the absence of a positive family history. In conclusion, these additional three CASQ2 CPVT families suggest that CASQ2 mutations are more common than previously thought and produce a severe form of CPVT. The full text of this article is available at http://www.circresaha.org. >94.NF00080301 PMID:12483523 TI - Modulation of cystatin C expression impairs the invasive and tumorigenic potential of human glioblastoma cells. AB - Increases in the abundance of cathepsin B transcript and protein with increased tumor grade and changes in subcellular localization and activity of this enzyme. We observed progressive reductions in levels of the protease inhibitor cystatin C, an inhibitor of cathepsin B with corresponding increases in the malignancy of glioma cell lines, implying an inverse correlation between cystatin C and tumor grade. To investigate the role of cystatin C in the invasion of brain tumor cells, we stably transfected SNB19 glioblastoma cells with either a 0.4-kb cDNA construct of human cystatin C in the sense orientation or an empty vector. Clones expressing sense-cystatin C cDNA had higher cystatin C mRNA and protein levels than did control cells. Sense-transfected cells were also markedly less invasive than control cells in a Matrigel invasion assay and in a coculture assay of SNB19 spheroids and fetal rat brain aggregates. Finally, the sense-transfected cells did not form tumors in nude mice upon intracerebral injection. These results strongly implicate cystatin C in the invasiveness of human glioblastoma cells and suggest that sense transcripts of cystatin C may prove useful in cancer therapy. >95.NF00090104 PMID:12138086 TI - Epidermal growth factor and transforming growth factor alpha mimic the effects of insulin in human fat cells and augment downstream signaling in insulin resistance. AB - The ability of the growth factors epidermal growth factor (EGF), transforming growth factor alpha, and platelet-derived growth factor to exert insulin-like effects on glucose transport and lipolysis were examined in human and rat fat cells. No effects were found in rat fat cells, whereas EGF (EC(50) for glucose transport approximately 0.02 nm) and transforming growth factor alpha (EC(50) approximately 0.2 nm), but not platelet-derived growth factor, mimicked the effects of insulin (EC(50) approximately 0.2 nm) on both pathways. EGF receptors, but not EGF, were abundantly expressed in human fat cells as well as in human skeletal muscle. EGF increased the tyrosine phosphorylation of several proteins (the EGF receptor, insulin receptor substrate (IRS)-1, IRS-2, and Grb2-associated binder 1), whereas Shc and Gab2 were only weakly and inconsistently phosphorylated. p85, the regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase), was also found to associate with all of these docking molecules, showing that EGF activated PI 3-kinase pools that were additional to those of insulin. EGF and/or insulin increased protein kinase B/Akt serine phosphorylation to a similar extent, whereas mitogen-activated protein kinase phosphorylation was more pronounced for EGF than for insulin. The impaired insulin-stimulated downstream signaling, measured as protein kinase B/Akt serine phosphorylation, in insulin-resistant cells (Type 2 diabetes) was improved by the addition of EGF. Thus, EGF receptors, but not EGF, are abundantly expressed in human fat cells and skeletal muscle. EGF mimics the effects of insulin on both the metabolic and mitogenic pathways but utilize in part different signaling pathways. Both insulin and EGF increase the tyrosine phosphorylation and activation of IRS-1 and IRS-2, whereas EGF is also capable of activating additional PI 3-kinase pools and, thus, can augment the downstream signaling of insulin in insulin-resistant states like Type 2 diabetes. >96.NF00136114 PMID:12445830 TI - Identification and recombinant production of human laminin alpha4 subunit splice variants. AB - Laminins, the major basement membrane glycoproteins, are composed of three subunits. We identified a splice variant of the human laminin alpha4 subunit transcript containing 21 extra nucleotides. A heptapeptide sequence, MDCPTIS, was inserted close to the two cysteine residues possibly involved in the intersubunit disulfide bonds. Both the authentic alpha4 subunit (alpha4A) and the variant with the heptapeptide insertion (alpha4B) were readily secreted as laminin-8 trimers (alpha4Abeta1gamma1 or alpha4Bbeta1gamma1) upon cotransfection with expression vectors for the beta1 and gamma1 subunits. The purified recombinant laminin-8 containing the alpha4B subunit was more potent in promoting cell spreading than that containing alpha4A, raising the possibility that the alternative splicing of the alpha4 subunit transcript regulates the cell-adhesive activity of laminin-8. Since both alpha4A and alpha4B transcripts were detected by RT-PCR in several human cell lines, these two isoforms of laminin-8 with differing cell-adhesive activities are present in the basement membranes of human tissues. >97.NF00125261 PMID:12008951 TI - Differential effects of fibroblast growth factors on expression of genes of the plasminogen activator and insulin-like growth factor systems by human breast fibroblasts. AB - In breast stroma urokinase plasminogen activator (uPA) is predominantly expressed by fibroblasts located in the near vicinity of tumor cells, and fibroblast-derived insulin-like growth factor-1 (IGF-1) may be involved in inhibiting the expression of uPA in these fibroblasts. To investigate a possible role for fibroblast growth factors (FGFs), we evaluated the expression of components of the PA system and the IGF system in normal and tumor-tissue-derived human breast fibroblasts exposed to various FGFs in vitro. mRNA analysis revealed that FGF-1, FGF-2 and FGF-4 induced the mRNA expression levels of uPA, tPA, uPAR, PAI-1 and PAI-2, and reduced those of IGF-1, IGF-1R, IGF-2R and IGFBP-4, without significantly affecting the levels of IGFBP-3, IGFBP-5 and IGFBP-6 mRNA. Concerning the expression of IGF-2 mRNA, the effects mediated by FGF-1, FGF-2 and FGF-4 were divergent. In general, the effects elicited by FGF-1 on the various mRNA levels studied were rapid and short-term. Those mediated by FGF-2 overall lagged behind but were longer-lasting. For FGF-4 an in between pattern was observed. Blocking transcription and translation demonstrated that a) both the FGF-1 and FGF-2 induced effects were the result of altered gene transcription or mRNA stability, b) the short-term effects mediated by FGF-1 and FGF-2 required de novo protein synthesis, and c) the long-term effects elicited by FGF-2 did not depend on de novo protein synthesis during the first 24 h, but were triggered by proteins produced or made available thereafter. The data presented propose that of the FGFs studied (FGF-1, -2, -4, -5, and -7), FGF-2 is the most attractive target for therapeutical strategies aimed at diminishing the contribution of stromal fibroblasts in the PA-directed breast tumor proteolysis. >98.NF00080307 PMID:12183469 TI - Two highly related p66 proteins comprise a new family of potent transcriptional repressors interacting with MBD2 and MBD3. AB - Methyl-CpG-binding domain proteins (MBD) mediate functional responses of methylated DNA. MBD2 and MBD3 are components of the MeCP1 protein complex, which contains the Mi-2/NuRD complex and includes 66- and 68-kDa polypeptides. Here we identified two highly related 66-kDa proteins in a yeast two-hybrid screen with MBD2b. Based on the high degree of sequence conservation to the previously identified Xenopus p66 subunit of the Mi-2/NuRD complex, we termed these proteins hp66alpha and hp66beta. hp66alpha is the human orthologue of Xenopus p66, whereas hp66beta, previously identified as a component of the human MeCP1 complex, is a second member of a p66 gene family. Coprecipitation of hp66alpha and MBD2 demonstrates their in vivo association. Furthermore, confocal microscopy shows a nuclear colocalization of hp66alpha with hp66beta and MBD2 in a speckled pattern. hp66alpha is a potent transcriptional repressor reducing gene activity about 100-fold and is ubiquitously coexpressed with hp66beta in cell lines and in fetal and adult tissues. We demonstrate direct binding of both p66 family members to MBD2 as well as MBD3. Interestingly, hp66alpha, which binds with a higher affinity than hp66beta, interacts via two interaction domains in contrast to a single interaction domain present in hp66beta. These results demonstrate that two highly related mammalian p66 proteins display overlapping functions and are involved in methylation dependent transcriptional repression. >99.NF00131661 PMID:11713982 TI - Regulation of neuronal cholecystokinin gene transcription. AB - Cholecystokinin (CCK) is a neuroendocrine peptide expressed in I-cells of the small intestine and in central and peripheral neurons. Whereas intestinal CCK is involved in the release of pancreatic enzymes and the contraction of the gallbladder, cerebral CCK is implicated in a variety of functions, such as feeding behaviour, anxiety and memory. The expression of CCK is developmentally regulated. Brain CCK mRNA levels are low before birth, but increase markedly shortly after birth and reach adult like patterns of expression three weeks after birth during the final maturation of the central nervous system. In the adult, several substances induce neuronal CCK mRNA expression via activation of transcription factors binding to regulatory elements in the CCK promoter. Recent studies have examined the signaling pathways, transcription factors and regulatory elements involved in cAMP, fibroblast growth factor-2, and calcium-induced CCK gene transcription in neuronal cells. The review describes the signaling pathways and transcription factors involved in neuronal CCK gene transcription. >100.NF00107282 PMID:10559186 TI - The gamma subunit modulates Na(+) and K(+) affinity of the renal Na,K-ATPase. AB - The Na(+),K(+)-ATPase catalyzes the active transport of ions. It has two necessary subunits, alpha and beta, but in kidney it is also associated with a 7.4-kDa protein, the gamma subunit. Stable transfection was used to determine the effect of gamma on Na, K-ATPase properties. When isolated from either kidney or transfected cells, alphabetagamma had lower affinities for both Na(+) and K(+) than alphabeta. A post-translational modification of gamma selectively eliminated the effect on Na(+) affinity, suggesting three configurations (alphabeta, alphabetagamma, and alphabetagamma*) conferring different stable properties to Na, K-ATPase. In the nephron, segment-specific differences in Na(+) affinity have been reported that cannot be explained by the known alpha and beta subunit isoforms of Na,K-ATPase. Immunofluorescence was used to detect gamma in rat renal cortex. Cortical ascending limb and some cortical collecting tubules lacked gamma, correlating with higher Na(+) affinities in those segments reported in the literature. Selective expression in different segments of the nephron is consistent with a modulatory role for the gamma subunit in renal physiology. >101.NF00103962 PMID:12351790 TI - Role of Hec1 in spindle checkpoint signaling and kinetochore recruitment of Mad1/Mad2. AB - The spindle checkpoint delays sister chromatid separation until all chromosomes have undergone bipolar spindle attachment. Checkpoint failure may result in chromosome mis-segregation and may contribute to tumorigenesis. We showed that the human protein Hec1 was required for the recruitment of Mps1 kinase and Mad1/Mad2 complexes to kinetochores. Depletion of Hec1 impaired chromosome congression and caused persistent activation of the spindle checkpoint, indicating that high steady-state levels of Mad1/Mad2 complexes at kinetochores were not essential for checkpoint signaling. Simultaneous depletion of Hec1 and Mad2 caused catastrophic mitotic exit, making Hec1 an attractive target for the selective elimination of spindle checkpoint-deficient cells. >102.NF00131908 PMID:11590544 TI - Hermansky-Pudlak syndrome type 3 in Ashkenazi Jews and other non-Puerto Rican patients with hypopigmentation and platelet storage-pool deficiency. AB - Hermansky-Pudlak syndrome (HPS), consisting of oculocutaneous albinism and a bleeding diathesis due to the absence of platelet dense granules, displays extensive locus heterogeneity. HPS1 mutations cause HPS-1 disease, and ADTB3A mutations cause HPS-2 disease, which is known to involve abnormal intracellular vesicle formation. A third HPS-causing gene, HPS3, was recently identified on the basis of homozygosity mapping of a genetic isolate of HPS in central Puerto Rico. We now describe the clinical and molecular characteristics of eight patients with HPS-3 who are of non-Puerto Rican heritage. Five are Ashkenazi Jews; three of these are homozygous for a 1303+1G-->A splice-site mutation that causes skipping of exon 5, deleting an RsaI restriction site and decreasing the amounts of mRNA found on northern blotting. The other two are heterozygous for the 1303+1G-->A mutation and for either an 1831+2T-->G or a 2621-2A-->G splicing mutation. Of 235 anonymous Ashkenazi Jewish DNA samples, one was heterozygous for the 1303+1G-->A mutation. One seven-year-old boy of German/Swiss extraction was compound heterozygous for a 2729+1G-->C mutation, causing skipping of exon 14, and resulting in a C1329T missense (R396W), with decreased mRNA production. A 15-year-old Irish/English boy was heterozygous for an 89-bp insertion between exons 16 and 17 resulting from abnormal splicing; his fibroblast HPS3 mRNA is normal in amount but is increased in size. A 12-year-old girl of Puerto Rican and Italian background has the 3,904-bp founder deletion from central Puerto Rico on one allele. All eight patients have mild symptoms of HPS; two Jewish patients had received the diagnosis of ocular, rather than oculocutaneous, albinism. These findings expand the molecular diagnosis of HPS, provide a screening method for a mutation common among Jews, and suggest that other patients with mild hypopigmentation and decreased vision should be examined for HPS. >103.NF00116518 PMID:12401728 TI - Association of IL4R haplotypes with type 1 diabetes. AB - We have investigated, in 282 multiplex Caucasian families (the Human Biological Data Interchange Repository), the association of type 1 diabetes with polymorphisms in the IL4R gene. IL4R encodes a subunit of the interleukin-4 receptor, a molecule critical to T-helper cell development. By genotyping eight different IL4R single-nucleotide polymorphisms (SNPs) and identifying haplotypes (complex alleles) in the multiplex type 1 diabetic families who were stratified for HLA genotype, we have observed significant evidence of linkage and association of the IL4R gene to type 1 diabetes. In particular, we have identified a specific haplotype that appears to be protective and observed that this protective effect is strongest among individuals not carrying the HLA DR3/DR4 genotype (which confers the strongest genetic risk for type 1 diabetes). These findings suggest an important role for the IL4R gene in immune-related disease susceptibility and illustrate the value of using multi-SNP haplotype information in association studies. >104.NF00140656 PMID:12161434 TI - Cytoplasmic serine hydroxymethyltransferase mediates competition between folate-dependent deoxyribonucleotide and S-adenosylmethionine biosyntheses. AB - Folate-dependent one-carbon metabolism is required for the synthesis of purines and thymidylate and for the remethylation of homocysteine to methionine. Methionine is subsequently adenylated to S-adenosylmethionine (SAM), a cofactor that methylates DNA, RNA, proteins, and many metabolites. Previous experimental and theoretical modeling studies have indicated that folate cofactors are limiting for cytoplasmic folate-dependent reactions and that the synthesis of DNA precursors competes with SAM synthesis. Each of these studies concluded that SAM synthesis has a higher metabolic priority than dTMP synthesis. The influence of cytoplasmic serine hydroxymethyltransferase (cSHMT) on this competition was examined in MCF-7 cells. Increases in cSHMT expression inhibit SAM concentrations by two proposed mechanisms: (1) cSHMT-catalyzed serine synthesis competes with the enzyme methylenetetrahydrofolate reductase for methylenetetrahydrofolate in a glycine-dependent manner, and (2) cSHMT, a high affinity 5-methyltetrahydrofolate-binding protein, sequesters this cofactor and inhibits methionine synthesis in a glycine-independent manner. Stable isotope tracer studies indicate that cSHMT plays an important role in mediating the flux of one-carbon units between dTMP and SAM syntheses. We conclude that cSHMT has three important functions in the cytoplasm: (1) it preferentially supplies one-carbon units for thymidylate biosynthesis, (2) it depletes methylenetetrahydrofolate pools for SAM synthesis by synthesizing serine, and (3) it sequesters 5-methyltetrahydrofolate and inhibits SAM synthesis. These results indicate that cSHMT is a metabolic switch that, when activated, gives dTMP synthesis higher metabolic priority than SAM synthesis. >105.NF00943007 PMID:12297480 TI - Expression of trophinin in the cycling endometrium and its association with infertility. AB - OBJECTIVE: To observe the expression of trophinin in the cycling endometrium and investigate its relationship with infertility. METHODS: Trophinin expression in the endometrium was observed in 39 normal cycling women during different menstrual phases and 24 women with infertility during mid-luteal phase by immunohistochemical technique. RESULTS: Trophinin expression was detected in the luteal-phase endometrium of both normal and infertile women, which peaked in the mid-luteal phase. In comparison with normal women, infertile women with endometriosis or unexplained infertility had significantly weakened trophinin expression in the endometrium in the mid-luteal (P<0.001). CONCLUSIONS: Trophinin may play an important role in the process of implantation, and abnormal endometrial trophinin expression might be one of the major causes of infertility. >106.NF00120976 PMID:12242338 TI - External control of Her2 expression and cancer cell growth by targeting a Ras-linked coactivator. AB - Overproduction of the Her2 oncoprotein has been found in approximately 30% of breast tumors, and patients who have Her2 excesses typically have more aggressive disease. Here we show that the expression of the Her2 gene can be decreased by inhibiting the interaction of the two cancer-linked proteins, DRIP130/CRSP130/Sur-2 (a Ras-linked subunit of human mediator complexes) and ESX (an epithelial-restricted transcription factor). Disruption of the interaction by a short cell-permeable peptide reduced the expression of the Her2 gene and specifically impaired the growth and viability of Her2-overexpressing breast cancer cells. The association of ESX with DRIP130 is mediated by a small hydrophobic face of an 8-aa helix in ESX, suggesting a therapeutic approach to incapacitating the Her2 gene by small organic molecules. >107.NF00132559 PMID:11815617 TI - Two proteins essential for apolipoprotein B mRNA editing are expressed from a single gene through alternative splicing. AB - Apolipoprotein B (apoB) mRNA editing involves site-specific deamination of cytidine to form uridine, resulting in the production of an in-frame stop codon. Protein translated from edited mRNA is associated with a reduced risk of atherosclerosis, and hence the protein factors that regulate hepatic apoB mRNA editing are of interest. A human protein essential for apoB mRNA editing and an eight-amino acid-longer variant of no known function have been recently cloned. We report that both proteins, henceforth referred to as ACF64 and ACF65, supported APOBEC-1 (the catalytic subunit of the editosome) equivalently in editing of apoB mRNA. They are encoded by a single 82-kb gene on chromosome 10. The transcripts are encoded by 15 exons that are expressed from a tissue-specific promoter minimally contained within the -0.33-kb DNA sequence. ACF64 and ACF65 mRNAs are expressed in both liver and intestinal cells in an approximate 1:4 ratio. Exon 11 is alternatively spliced to include or exclude 24 nucleotides of exon 12, thereby encoding ACF65 and ACF64, respectively. Recognition motifs for the serine/arginine-rich (SR) proteins SC35, SRp40, SRp55, and SF2/ASF involved in alternative RNA splicing were predicted in exon 12. Overexpression of these SR proteins in liver cells demonstrated that alternative splicing of a minigene-derived transcript to express ACF65 was enhanced 6-fold by SRp40. The data account for the expression of two editing factors and provide a possible explanation for their different levels of expression. >108.NF00015676 PMID:11847339 TI - Rap1 GTPase regulation of adherens junction positioning and cell adhesion. AB - Cell-cell junctions are distributed evenly around the lateral circumference of cells within an epithelium. We find that the even distribution of adherens junctions is an active process that requires the small guanosine triphosphatase Rap1. Cells mutant for Rap1 condensed their adherens junctions to one side of the cell. This disrupted normal epithelial cell behavior, and mutant cell clones dispersed into the surrounding wild-type tissue. Rap1 is enriched at adherens junctions, particularly between newly divided sister cells where it may reseal the adherens junction ring. The regulation of adherens junction positioning could play a role in cell mobility and cell division. >109.NF00104938 PMID:12023287 TI - Molecular basis of the globoside-deficient P(k) blood group phenotype. Identification of four inactivating mutations in the UDP-N-acetylgalactosamine: globotriaosylceramide 3-beta-N-acetylgalactosaminyltransferase gene. AB - The biochemistry and molecular genetics underlying the related carbohydrate blood group antigens P, P(k), and LKE in the GLOB collection and P1 in the P blood group system are complex and not fully understood. Individuals with the rare but clinically important erythrocyte phenotypes P(1)(k) and P(2)(k) lack the capability to synthesize P antigen identified as globoside, the cellular receptor for Parvo-B19 virus and some P-fimbriated Escherichia coli. As in the ABO system, naturally occurring antibodies, anti-P of the IgM and IgG class with hemolytic and cytotoxic capacity, are formed. To define the molecular basis of the P(k) phenotype we analyzed the full coding region of a candidate gene reported in 1998 as a member of the 3-beta-galactosyltransferase family but later shown to possess UDP-N-acetylgalactosamine:globotriaosylceramide 3-beta-N-acetylgalactosaminyltransferase or globoside synthase activity. Homozygosity for different nonsense mutations (C(202) --> T and 538insA) resulting in premature stop codons was found in blood samples from two individuals of the P(2)(k) phenotype. Two individuals with P(1)(k) and P(2)(k) phenotypes were homozygous for missense mutations causing amino acid substitutions (E266A or G271R) in a highly conserved region of the enzymatically active carboxyl-terminal domain in the transferase. We conclude that crucial mutations in the globoside synthase gene cause the P(k) phenotype. >110.NF00099314 PMID:12111645 TI - A novel polymorphism in exon 11 of the WKL1 gene, shows no association with schizophrenia. AB - A missense mutation in exon 11 of the WKL1 gene on chromosome 22 was found to be associated with cases of catatonic schizophrenia in a single large pedigree. We have screened exon 11 of the WKL1 gene in 174 cases of schizophrenia, including cases of 22 cases of catatonic schizophrenia, but could not detect the previously reported mis-sense mutation. However in exon 11, we observed an insertion/deletion polymorphism, one-missense substitution and two synonymous substitutions. In addition, we also identified a nucleotide substitution in intron 11. All these polymorphisms appeared to be in complete linkage disequilibrium with one another. The polymorphisms were also identified in a UK pedigree with schizophrenia, however the polymorphisms did not segregate with the disease. To test for potential association between these polymorphisms and schizophrenia we sequenced an equal number of UK control individuals who were free of all psychiatric symptoms and had negative family histories for mental illness; the frequency of the insertion/deletion polymorphism was not significantly different in schizophrenia cases (42 out of 348 chromosomes, allele frequency 12%) compared to normal controls (40 out of 356 chromosomes, allele frequency 11%). The insertion/deletion was found to be in Hardy Weinberg equilibrium in both the schizophrenic and control groups. The insertion/deletion is composed of repeated sequence from exon 11 and intron 11 and is predicted to affect WKL1 protein structure. >111.NF00088546 PMID:11531333 TI - Structure of the C-terminal RNA-binding domain of hnRNP D0 (AUF1), its interactions with RNA and DNA, and change in backbone dynamics upon complex formation with DNA. AB - Heterogeneous nuclear ribonucleoprotein (hnRNP) D0 has two ribonucleoprotein (RNP) -type RNA-binding domains (RBDs), each of which can specifically bind to the UUAG-sequence. hnRNP D0 also binds specifically to single-stranded d(TTAGGG)(n), the human telomeric DNA repeat. We have already reported the structure and interactions with RNA of the N-terminal RBD (RBD1). Here, the structure of the C-terminal RBD (RBD2) determined by NMR is presented. It folds into a compact alpha beta structure comprising an antiparallel beta-sheet packed against two alpha-helices, which is characteristic of RNP-type RBDs. In addition to the four beta-strands commonly found in RNP-type RBDs, an extra beta-strand, termed beta 4(-), was found just before the fourth beta-strand, yielding a five-stranded beta-sheet. Candidate residues of RBD2 involved in the interactions with RNA were identified by chemical shift perturbation analysis. Perturbation was detected on the beta-sheet side, not on the opposite alpha-helix side, as observed for RBD1. It is notable that the beta 4(-) to beta 4 region of RBD2 is involved in the interactions in contrast to the case of RBD1. The chemical shift perturbation analysis also showed that RBD2 interacts with DNA in essentially the same way as with RNA. Changes in the backbone dynamics upon complex formation with DNA were examined by means of model free analysis of relaxation data. In free RBD2, the beta 4(-) to beta 4 region exhibits slow conformational exchange on the milli- to microsecond time scale. The exchange is quenched upon complex formation. The flexibility of free RBD2 may be utilized in the recognition process by allowing different conformational states to be accessed and facilitating induced fit. Additionally, faster flexibility on the nano- to picosecond time scale was observed for loop 3 located between beta 2 and beta 3 in free RBD2, which is retained by the complex as well. >112.NF01235993 PMID:11827176 TI - Consequences of CK2 signaling to the nuclear matrix. AB - Protein kinase CK2 is recognized as one of the key cellular signals for cell growth and proliferation. Its nuclear targeting appears to be critical to its role in these functions. In the nucleus, nuclear matrix (NM) which plays a major role in growth-related activities is a primary locus for CK2 signaling. A variety of growth stimuli evoke a rapid translocation of the CK2 to the NM whereas removal of these factors has the opposite effect. These studies, employing various experimental models of cell growth (involving different growth-stimulatory factors), have suggested that rapid shuttling of CK2 to the NM is a key feature of early growth control. By contrast, removal of growth-stimulatory factors leading to the loss of cell viability is associated with early loss of CK2 from the NM (and chromatin). This indicates that absence of CK2 from the nuclear compartment is contributory to induction of cell death via apoptosis, implying a protective role for CK2 against cell death. Here, we review the evidence that suggests that CK2 signaling in the NM is not only involved in cell growth but also in cell survival. >113.NF00990692 PMID:12122014 TI - Csk homologous kinase (CHK) and ErbB-2 interactions are directly coupled with CHK negative growth regulatory function in breast cancer. AB - Our previous studies demonstrated that Csk homologous kinase (CHK) acts as a negative growth regulator of human breast cancer through inhibition of ErbB-2/neu-mediated Src family kinase activity (Bougeret, C., Jiang, S., Keydar, I., and Avraham, H. (2001) J. Biol. Chem. 276, 33711-33720. The interaction between the CHK SH2 domain and Tyr(P)(1248) of the ErbB-2 receptor has been shown to be specific and critical for CHK function. In this report, we investigated whether the interaction of the CHK SH2 domain and ErbB-2 is directly related to the inhibition of heregulin-stimulated Src kinase activity. We constructed three CHK SH2 domain binding mutants: G129R (enhanced binding), R147K (inhibited binding), and R147A (disrupted binding). NMR spectra for the domains of each construct were used to evaluate their interaction with a Tyr(P)(1248)-containing ErbB-2 peptide. G129R showed enhanced binding to ErbB-2, whereas binding was completely disrupted by R147A. The enhanced binding mutant showed chemical shift changes at the same residues as wild-type CHK, indicating that this mutant has the same binding characteristics as the wild-type protein. Furthermore, inhibition of heregulin-stimulated Src kinase activity was markedly diminished by R147A, whereas G129R-mediated inhibition was stronger as compared with wild-type CHK. These results indicate that the specific interaction of CHK and ErbB-2 via the SH2 domain of CHK is directly related to the growth inhibitory effects of CHK. These new CHK high affinity binding constructs may serve as good candidates for inhibition of the ErbB-2/Src transduction pathway in gene therapy studies in breast cancer. >114.NF00076969 PMID:12015308 TI - Extracellular and cytoplasmic domains of endoglin interact with the transforming growth factor-beta receptors I and II. AB - Endoglin is an auxiliary component of the transforming growth factor-beta (TGF-beta) receptor system, able to associate with the signaling receptor types I (TbetaRI) and II (TbetaRII) in the presence of ligand and to modulate the cellular responses to TGF-beta1. Endoglin cannot bind ligand on its own but requires the presence of the signaling receptors, supporting a critical role for the interaction between endoglin and TbetaRI or TbetaRII. This study shows that full-length endoglin interacts with both TbetaRI and TbetaRII, independently of their kinase activation state or the presence of exogenous TGF-beta1. Truncated constructs encoding either the extracellular or the cytoplasmic domains of endoglin demonstrated that the association with the signaling receptors occurs through both extracellular and cytoplasmic domains. However, a more specific mapping revealed that the endoglin/TbetaRI interaction was different from that of endoglin/TbetaRII. TbetaRII interacts with the amino acid region 437-558 of the extracellular domain of endoglin, whereas TbetaRI interacts not only with the region 437-558 but also with the protein region located between amino acid 437 and the N terminus. Both TbetaRI and TbetaRII interact with the cytoplasmic domain of endoglin, but TbetaRI only interacts when the kinase domain is inactive, whereas TbetaRII remains associated in its active and inactive forms. Upon association, TbetaRI and TbetaRII phosphorylate the endoglin cytoplasmic domain, and then TbetaRI, but not TbetaRII, kinase dissociates from the complex. Conversely, endoglin expression results in an altered phosphorylation state of TbetaRII, TbetaRI, and downstream Smad proteins as well as a modulation of TGF-beta signaling, as measured by the reporter gene expression. These results suggest that by interacting through its extracellular and cytoplasmic domains with the signaling receptors, endoglin might affect TGF-beta responses. >115.NF00776855 PMID:11894222 TI - FOXP2 is not a major susceptibility gene for autism or specific language impairment. AB - The FOXP2 gene, located on human 7q31 (at the SPCH1 locus), encodes a transcription factor containing a polyglutamine tract and a forkhead domain. FOXP2 is mutated in a severe monogenic form of speech and language impairment, segregating within a single large pedigree, and is also disrupted by a translocation in an isolated case. Several studies of autistic disorder have demonstrated linkage to a similar region of 7q (the AUTS1 locus), leading to the proposal that a single genetic factor on 7q31 contributes to both autism and language disorders. In the present study, we directly evaluate the impact of the FOXP2 gene with regard to both complex language impairments and autism, through use of association and mutation screening analyses. We conclude that coding-region variants in FOXP2 do not underlie the AUTS1 linkage and that the gene is unlikely to play a role in autism or more common forms of language impairment. >116.NF00136200 PMID:12021920 TI - Expression of somatostatin receptor types 1-5 in 81 cases of gastrointestinal and pancreatic endocrine tumors. A correlative immunohistochemical and reverse-transcriptase polymerase chain reaction analysis. AB - Somatostatin receptors (SSTRs) have been extensively mapped in human tumors by means of autoradiography, reverse-transcriptase polymerase chain reaction (RT-PCR), in situ hybridization (ISH) and immunohistochemistry (IHC). We analyzed the SSTR type 1-5 expression by means of RT-PCR and/or IHC in a series of 81 functioning and non-functioning gastroenteropancreatic (GEP) endocrine tumors and related normal tissues. Moreover, we compared the results with clinical, pathological and hormonal features. Forty-six cases (13 intestinal and 33 pancreatic) were studied for SSTR 1-5 expression using RT-PCR, IHC with antibodies to SSTR types 2, 3, 5 and ISH for SSTR2 mRNA. The vast majority of tumors expressed SSTR types 1, 2, 3 and 5, while SSTR4 was detected in a small minority. Due to the good correlation between RT-PCR and IHC data on SSTR types 2, 3, and 5, thirty-five additional GEP endocrine tumors were studied with IHC alone. Pancreatic insulinomas had an heterogeneous SSTR expression, while 100% of somatostatinomas expressed SSTR5 and 100% gastrinomas and glucagonomas expressed SSTR2. Pre-operative biopsy material showed an overlapping immunoreactivity with that of surgical specimens, suggesting that the SSTR status can be detected in the diagnostic work-up. It is concluded that SSTRs 1-5 are heterogeneously expressed in GEP endocrine tumors and that IHC is a reliable tool to detect SSTR types 2, 3 and 5 in surgical and biopsy specimens. >117.NF00109313 PMID:12135940 TI - Regulation of Kv4.3 current by KChIP2 splice variants: a component of native cardiac I(to)? AB - BACKGROUND: The transient outward potassium current (I(to)) encoded by the Kv4 family of potassium channels is important in the repolarization of cardiac myocytes. KChIPs are a recently identified group of Ca2+-binding accessory subunits that modulate Kv4-encoded currents. KChIP2 is the only family member expressed in the heart. METHODS AND RESULTS: We previously cloned 2 novel splice variants of KChIP2 from human heart, named KChIP2S and KChIP2T. The transmural distribution of KChIP2 mRNA and protein in human and canine left ventricle was examined using kinetic RT-PCR and Western blots in the same tissues. A steep gradient of mRNA with greater KChIP2 expression in the epicardium was observed. However, no gradient of immunoreactive protein was observed. Immunocytochemistry reveals KChIP2 expression in the t-tubules and the nucleus. The predominant effects of all 3 KChIP2 splice variants on hKv4.3-encoded current are to increase the density, slow the current decay in a Ca2+-dependent manner, and hasten recovery from inactivation in a splice variant-specific fashion. CONCLUSIONS: A family of KChIP2 proteins is expressed in human hearts that exhibits differential modulation of hKv4.3 current in a Ca2+-dependent fashion. The effect of KChIP2 on the biophysical properties of expressed Kv4.3 current and the absence of a gradient of protein across the ventricular wall suggest that KChIP2 is either not a requisite component of human or canine ventricular I(to) or that its functional effect is being affected or additionally modified by other factors present in myocardial cells. >118.NF00140660 PMID:12393884 TI - Apocytochrome c blocks caspase-9 activation and Bax-induced apoptosis. AB - Complex networks of signaling pathways control the apoptotic response and, therefore, cell survival. However, these networks converge on a common machinery, of which the caspase cysteine proteases are key components. Diverse apoptotic stimuli release holocytochrome c from mitochondria, allowing holocytochrome c to bind apoptotic protease activating factor-1 (Apaf-1), which in turn binds caspase-9 both activating this caspase and forming an Apaf-1/caspase-9 holoenzyme. Cytochrome c lacking heme (the apo form) cannot support caspase activation, although the reason for this has not been studied. Here we show that apocytochrome c still binds Apaf-1 and that it can block holo-dependent caspase activation in a cell-free system. In addition we show that overexpression of apocytochrome c blocks Bax-induced apoptosis in cells. Thus it is possible to modulate cell survival by interfering with the Apaf-1/cytochrome c interaction. Given the key role played by Apaf-1/cytochrome c in the apoptotic process, and the role of apoptosis in degenerative disease, this interaction may serve as a novel therapeutic target. >119.NF00141471 PMID:12226101 TI - Expression of calcineurin B homologous protein 2 protects serum deprivation-induced cell death by serum-independent activation of Na+/H+ exchanger. AB - The calcineurin B homologous protein (designated CHP1) has been shown to be a common essential cofactor for the plasma membrane Na(+)/H(+) exchangers (NHEs) (Pang, T., Su, X., Wakabayashi, S., and Shigekawa, M. (2001) J. Biol. Chem. 276, 17367-17372). In this study, we characterized the function of another isoform of CHP (designated CHP2) that has a 61% amino acid identity with CHP1. CHP2, like CHP1, conferred the ability to NHEs 1-3 to express a high exchange activity by binding to the juxtamembrane region of the cytoplasmic domain of the exchanger, but it interacts more strongly (approximately 5-fold) with NHE1 than does CHP1. Although CHP1 is expressed ubiquitously at relatively high levels, CHP2 expression was extremely low in most human tissues but was higher in tumor cells. We produced stable cell clones overexpressing either CHP1 or CHP2 in which one of them is predominantly bound to NHE1. Serum (10%) induced a significant cytoplasmic alkalinization (0.1-0.2 pH unit) in cells co-expressing CHP1 and NHE1 but not in cells co-expressing CHP2 and NHE1. In the latter, pH(i) was high (7.4-7.5) even in the absence of serum, suggesting that NHE1 was already activated. Surprisingly, most (>80%) of CHP2/NHE1 cells unlike CHP1/NHE1 cells were viable even after long serum starvation (>7 days). Thus, the expression of CHP2 appears to protect cells from serum deprivation-induced death by increasing pH(i). These properties of CHP2/NHE1 cells are similar to those of malignantly transformed cells. We propose that serum-independent activation of NHE1 by bound CHP2 is one of the key mechanisms for the maintenance of high pH(i) and the resistance to serum deprivation-induced cell death in malignantly transformed cells. >120.NF00122681 PMID:12068077 TI - Further characterization of the molecular interaction between PSD-95 and NMDA receptors: the effect of the NR1 splice variant and evidence for modulation of channel gating. AB - Coexpression of PSD-95(c-Myc) with NR1-1a/NR2A NMDA receptors in human embryonic kidney (HEK) 293 cells resulted in a decrease in efficacy for the glycine stimulation of [3 H]MK801 binding similar to that previously described for l-glutamate. The inhibition constants (K (I) s) for the binding of l-glutamate and glycine to NR1-1a/NR2A determined by [3 H]CGP 39653 and [3 H]MDL 105 519 displacement assays, respectively, were not significantly different between NR1-1a/NR2A receptors coexpressed +/- PSD-95(c-Myc). The increased EC(50) for l-glutamate enhancement of [3 H]MK801 binding was also found for NR1-2a/NR2A and NR1-4b/NRA receptors thus the altered EC(50) is not dependent on the N1, C1 or C2 exon of the NR1 subunit. The NR1-4b but not the NR1-1a subunit was expressed efficiently at the cell surface in the absence of NR2 subunits. Total NR1-4b and NR1-4b/NR2A expression was enhanced by PSD-95(c-Myc) but whole cell enzyme-linked immunoadsorbent assays (ELISAs) showed that this increase was not due to increased expression at the cell surface. It is suggested that PSD-95(c-Myc) has a dual effect on NMDA receptors expressed in mammalian cells, a reduction in channel gating and an enhanced expression of NMDA receptor subunits containing C-terminal E(T/S)XV PSD-95 binding motifs. >121.NF00116445 PMID:12082094 TI - Functions of transforming growth factor-beta family type I receptors and Smad proteins in the hypertrophic maturation and osteoblastic differentiation of chondrocytes. AB - We investigated the effects of bone morphogenetic protein (BMP)-2, a member of the transforming growth factor-beta superfamily, on the regulation of the chondrocyte phenotype, and we identified signaling molecules involved in this regulation. BMP-2 triggers three concomitant responses in mouse primary chondrocytes and chondrocytic MC615 cells. First, BMP-2 stimulates expression or synthesis of type II collagen. Second, BMP-2 induces expression of molecular markers characteristic of pre- and hypertrophic chondrocytes, such as Indian hedgehog, parathyroid hormone/parathyroid hormone-related peptide receptor, type X collagen, and alkaline phosphatase. Third, BMP-2 induces osteocalcin expression, a specific trait of osteoblasts. Constitutively active forms of transforming growth factor-beta family type I receptors and Smad proteins were overexpressed to address their role in this process. Activin receptor-like kinase (ALK)-1, ALK-2, ALK-3, and ALK-6 were able to reproduce the hypertrophic maturation of chondrocytes induced by BMP-2. In addition, ALK-2 mimicked further the osteoblastic differentiation of chondrocytes induced by BMP-2. In the presence of BMP-2, Smad1, Smad5, and Smad8 potentiated the hypertrophic maturation of chondrocytes, but failed to induce osteocalcin expression. Smad6 and Smad7 impaired chondrocytic expression and osteoblastic differentiation induced by BMP-2. Thus, our results indicate that Smad-mediated pathways are essential for the regulation of the different steps of chondrocyte and osteoblast differentiation and suggest that additional Smad-independent pathways might be activated by ALK-2. >122.NF00126408 PMID:12540637 TI - Large-scale association studies of variants in genes encoding the pancreatic beta-cell KATP channel subunits Kir6.2 (KCNJ11) and SUR1 (ABCC8) confirm that the KCNJ11 E23K variant is associated with type 2 diabetes. AB - The genes ABCC8 and KCNJ11, which encode the subunits sulfonylurea receptor 1 (SUR1) and inwardly rectifying potassium channel (Kir6.2) of the beta-cell ATP-sensitive potassium (K(ATP)) channel, control insulin secretion. Common polymorphisms in these genes (ABCC8 exon 16-3t/c, exon 18 T/C, KCNJ11 E23K) have been variably associated with type 2 diabetes, but no large ( approximately 2,000 subjects) case-control studies have been performed. We evaluated the role of these three variants by studying 2,486 U.K. subjects: 854 with type 2 diabetes, 1,182 population control subjects, and 150 parent-offspring type 2 diabetic trios. The E23K allele was associated with diabetes in the case-control study (odds ratio [OR] 1.18 [95% CI 1.04-1.34], P = 0.01) but did not show familial association with diabetes. Neither the exon 16 nor the exon 18 ABCC8 variants were associated with diabetes (1.04 [0.91-1.18], P = 0.57; 0.93 [0.71-1.23], P = 0.63, respectively). Meta-analysis of all case-control data showed that the E23K allele was associated with type 2 diabetes (K allele OR 1.23 [1.12-1.36], P = 0.000015; KK genotype 1.65 [1.34-2.02], P = 0.000002); but the ABCC8 variants were not associated. Our results confirm that E23K increases risk of type 2 diabetes and show that large-scale association studies are important for the identification of diabetes susceptibility alleles. >123.NF00105835 PMID:12367750 TI - Regulation of HIV-1 transcription by NF-IL6 in activated Jurkat T cells. AB - To examine the mechanism of HIV-1 regulation by NF-IL6 in activated human cells, we selected a Jurkat cell line that did not contain endogenous NF-IL6. In this cellular environment, we evaluated the effect of exogenous NF-IL6 on transcription mediated by native and deleted LTR sequences. In Jurkat cells stimulated with LPS and PMA, LTR-mediated transcription was enhanced by NF-IL6. The results of deletion studies revealed a central role for the basal LTR region and the TATA element in the LTR, in upregulation of reporter gene expression by NF-IL6 in activated cells. In the selected cellular environment, regulation of transcription by NF-IL6 was not evident in studies of promoter regions of other genes. The results implied that the basal region of HIV-1 LTR includes molecular properties that support activation of HIV-1 by NF-IL6 in stimulated cells. >124.NF00028075 PMID:12110845 TI - Synaptotagmins I and IV promote transmitter release independently of Ca(2+) binding in the C(2)A domain. AB - At nerve terminals, a focal and transient increase in intracellular Ca(2+) triggers the fusion of neurotransmitter-filled vesicles with the plasma membrane. The most extensively studied candidate for the Ca(2+)-sensing trigger is synaptotagmin I, whose Ca(2+)-dependent interactions with acidic phospholipids and syntaxin have largely been ascribed to its C(2)A domain, although the C(2)B domain also binds Ca(2+) (refs 7, 8). Genetic tests of synaptotagmin I have been equivocal as to whether it is the Ca(2+)-sensing trigger of fusion. Synaptotagmin IV, a related isoform that does not bind Ca(2+) in the C(2)A domain, might be an inhibitor of release. We mutated an essential aspartate of the Ca(2+)-binding site of the synaptotagmin I C(2)A domain and expressed it in Drosophila lacking synaptotagmin I. Here we show that, despite the disruption of the binding site, the Ca(2+)-dependent properties of transmission were not altered. Similarly, we found that synaptotagmin IV could substitute for synaptotagmin I. We conclude that the C(2)A domain of synaptotagmin is not required for Ca(2+)-dependent synaptic transmission, and that synaptotagmin IV promotes rather than inhibits transmission. >125.NF00943016 PMID:12445816 TI - ABCC13, an unusual truncated ABC transporter, is highly expressed in fetal human liver. AB - In the present study, we have cloned the cDNA of ABCC13, a novel ABC transporter, from the cDNA library of adult human placenta. The ABCC13 gene spans approximately 70kb on human chromosome 21q11.2 and consists of 14 exons. The open reading frame of the ABCC13 cDNA encodes a peptide consisting of 325 amino acid residues. The amino acid sequence corresponding to putative membrane-spanning domains was remarkably similar to ABCC1, ABCC2, ABCC3, and ABCC6. The ABCC13 gene was expressed in the fetal liver at the highest level among the organs studied. While ABCC13 was expressed in the bone marrow, its expression in peripheral blood leukocytes of adult humans was much lower and no detectable levels were observed in differentiated hematopoietic cells. The expression of ABCC13 in K562 cells decreased during cell differentiation induced by TPA. These results suggest that the expression of human ABCC13 is related with hematopoiesis. >126.NF00115637 PMID:11933207 TI - Five novel mutations in the C1 inhibitor gene (C1NH) leading to a premature stop codon in patients with type I hereditary angioedema. AB - Hereditary angioedema (HAE) is a disorder characterised by recurrent attacks of localized subcutaneous or submucosal edema. It is inherited in an autosomal dominant fashion and caused by a deficiency of C1 inhibitor (C1 inh, or C1NH). Most patients with HAE have an absolute deficiency of C1 inh (type I HAE) while the rest (15% of kindreds) synthetize a dysfunctional C1 inh protein (type II HAE). In this report a novel use of denaturing gradient gel electrophoresis (DGGE) followed by direct sequencing of the C1 inhibitor gene is presented. Five novel mutations, one nonsense (p.S48X) and four small deletions resulting in frameshifts (g.2264-2265delAG, g.2304delC, g.8493-8494delCC and g.16676-16677delTG) have been identified in the C1 inhibitor gene in five families with type I HAE. All of these mutations lead to premature termination of translation and thus can be considered causative of the C1 inh deficiency. Moreover, two previously described mutations in the reactive center of C1 inh, p.R444C and p.R444H, have been detected in four unrelated patients with type II HAE. >127.NF00097702 PMID:11991731 TI - Cardiac expression and subcellular localization of the p38 mitogen-activated protein kinase member, stress-activated protein kinase-3 (SAPK3). AB - Despite the interest in the roles that mitogen-activated protein kinases (MAPKs) play in the heart, the role of the different MAPK isoforms has been relatively poorly defined. A third isoform of p38 MAPK, known variously as stress-activated protein kinase-3 (SAPK3), p38- gamma or ERK6, has been previously shown to differ from p38- alpha/ beta both in its molecular weight and its lack of inhibition by the compound SB203580. We have generated monoclonal antibodies with specificity for SAPK3 demonstrated by immunoblot analysis, immunofluorescence studies, and cloning of SAPK3 from a rat heart cDNA expression library. By immunoblotting, we confirmed high expression of SAPK3 in fast, slow and mixed fibre types of murine skeletal muscle and observed significant expression restricted to heart, lung, thymus and testes. In addition to expression in normal heart (human, mouse, rat, dog and pig), we observed constant expression in diseased human heart, as well as control and hypertrophic cultured neonatal rat cardiac myocytes. Immunolocalization in cultured cardiac myocytes followed by confocal microscopy showed punctate, non-nuclear SAPK3 staining. In contrast, p38- alpha/ beta staining was non-punctate and distributed throughout the cytosol and nucleus. Whereas treatment with Leptomycin B to prevent nuclear export processes promoted higher levels of p38- alpha/ beta staining in cardiac myocyte nuclei, there was no apparent change in SAPK3 localization under these conditions. These differences between p38- alpha/ beta and SAPK3 probably reflect the specialized functions of SAPK3 and emphasize the need to evaluate SAPK3 upstream activators and downstream targets in the heart. >128.NF00115397 PMID:11994747 TI - Closing in on the biological functions of Fps/Fes and Fer. AB - Fps/Fes and Fer are the only known members of a distinct subfamily of the non-receptor protein-tyrosine kinase family. Recent studies indicate that these kinases have roles in regulating cytoskeletal rearrangements and inside out signalling that accompany receptor ligand, cell matrix and cell cell interactions. Genetic analysis using transgenic mouse models also implicates these kinases in the regulation of inflammation and innate immunity. >129.NF00088551 PMID:12054582 TI - Chromosomal organization and localization of the human histone deacetylase 9 gene (HDAC9). AB - Epigenetically mediated modulation of gene promoter function through histone acetylation modifying enzymes, which regulate the acetylation state of histone proteins and other promoter-bound transcription factors, is increasingly appreciated as a key component in the regulation of reversible gene expression. While histone acetyltransferases (HATs), which are frequently part of multisubunit coactivator complexes, lead to the relaxation of chromatin structure and transcriptional activation, histone deacetylases (HDACs) tend to associate with multisubunit corepressor complexes, which result in chromatin condensation and transcriptional repression of specific target genes. We have isolated and characterized the human HDAC9 genomic sequence, which spans a region of 458 kb and which has one single chromosomal locus. Determination of the exon-intron splice-junctions established that HDAC9 is encoded by 23 exons ranging in size from 22 bp (exon 1) to 264 bp (exon 11). Characterization of the 5' flanking genomic region revealed that the human HDAC9 promoter lacks both the canonical TATA and CCAAT boxes; CpG elements are missing. The human HDAC9 open reading frame is 3036 bp long and encodes a 1011 aa protein with a predictive molecular weight of 111.3 kDa and an isoelectric point of 6.41. Fluorescence in situ hybridization analysis localized the human HDAC9 gene to chromosome 7p21, a region which has been associated particularly with the pathogenesis of gynecological tumors. >130.NF01099109 PMID:11840501 TI - Lack of association between HoxA1 and HoxB1 gene variants and autism in 110 multiplex families. AB - A recent report suggested that the HoxA1 and/or HoxB1 genes play a role in susceptibility to autism. To determine whether these findings could be confirmed, we screened these genes for DNA polymorphisms by sequencing all exons in 24 individuals with autism. We identified the same sequence variants in the genes that appeared in this report, which include one single-base substitution variant in HoxA1 and a common haplotype in HoxB1. We performed an association study by applying the transmission disequilibrium test to detect possible association of these variants to autism in 110 multiplex families. Our results demonstrated no deviation from the null hypothesis of no association. We have also separately examined transmissions within individual mating types, for paternal versus maternal alleles, to affected versus unaffected children, and for transmission to affected boys versus girls. None of these subsets revealed significant deviation from the null expectation. Our interpretation of these findings is that it is unlikely that HoxA1 and HoxB1 play a significant role in the genetic predisposition to autism. >131.NF00089606 PMID:11831556 TI - CSF total tau, Abeta42 and phosphorylated tau protein as biomarkers for Alzheimer's disease. AB - With the arrival of effective symptomatic treatments and the promise of drugs that may delay progression, we now need to identify Alzheimer's disease (AD) at an early stage of the disease. To diagnose AD earlier and more accurately, attention has been directed toward peripheral biochemical markers. This article reviews promising potential cerebrospinal fluid (CSF) biomarkers for AD focussing on their role in clinical diagnosis. In particular, two biochemical markers, CSF total tau (t-tau) protein and the 42 amino acid form of beta-amyloid (Abeta42), perform satisfactorily enough to achieve a role in the clinical diagnostic settings of patients with dementia together with the cumulative information from basic clinical work-up, genetic screening, and brain imaging. These CSF markers are particularly useful to discriminate early or incipient AD from age-associated memory impairment, depression, and some secondary dementias. In order to discriminate AD from other primary dementia disorders, however, more accurate and specific markers are needed. Preliminary evidence strongly suggests that quantification of tau phosphorylated at specific sites in CSF improves early detection, differential diagnosis, and tracking of disease progression in AD. >132.NF00095768 PMID:12112824 TI - Cytological grading, apoptosis, and Bcl-2 protein expression in breast cancer. AB - A prospective study was conducted on 27 cases of infiltrating duct carcinoma of breast diagnosed by fine-needle aspirate cytology (FNAC), subjected to mastectomy following the FNA diagnosis. Cytological grading, Bcl-2 score, and quantification of apoptotic cell count were done on FNA material. Next, the carcinomas were graded on the corresponding histopathological sections. The overall concordance between cytological and histological gradings was 77.7%, with maximum concordance in Grade I tumors and minimum in Grade II. The mean apoptotic rates were 0.59 +/- 0.722, 2.11 +/- 0.707, and 2.95 +/- 0.854 in Grades I, II, and III, respectively. Similarly Bcl-2 scores were 1.36 +/- 0.82, 0.22 +/- 0.13, and 0.14 +/- 0.116, in Grades I, II, and III lesions, respectively. When the cytological grade was correlated with histological grade alone, and subsequently along with Bcl-2 scores and apoptotic rates, there was a significant improvement from 0.662-0.713 (P value, 0.001). >133.NF01222501 PMID:12386808 TI - Cdk9, a member of the cdc2-like family of kinases, binds to gp130, the receptor of the IL-6 family of cytokines. AB - Cdk9 is a member of the Cdc2-like family of kinases. It binds to members of the family of cyclin T (T1, T2a and T2b) and to cyclin K. The Cdk9/cyclin T complex appears to be involved in regulating several physiological processes. In fact Cdk9 is the kinase of the P-TEFb complex, involved in basal transcription. Cdk9 has also been described as the kinase of the TAK complex, homologous to P-TEFb and involved in HIV replication. Here we show that Cdk9 interacts with gp130, the receptor of the Interleukin-6 (IL-6) family of cytokines, which includes Leukemia Inhibitory Factor (LIF), Oncostatin M (OSM), Ciliary Neurotrophic Factor (CNTF), Interleukin-11 (IL-11) and Cardiotrophin (CT-1). IL-6 is a key regulator of hematopoiesis, immunological responses and inflammation. In addition, IL-6 plays a major role in the endocrine and nervous systems. Signal transduction by gp130 is mediated by physical interaction of the cytoplasmic region of gp130 with cellular kinases and results in the transcriptional activation of cellular and viral genes. We found that Cdk9 interacts in vitro with the cytoplasmic region of gp130 and we succeded in reproducing this interaction in vivo. Cdk9 expression was found both in the nucleus and in the cytoplasm. The binding occurring between Cdk9 and gp130 increased upon IL-6 stimulation. We also observed that Cdk9 synergized with IL-6 in inducing the activation of an IL-6-responsive reporter plasmid. In summary, these results point to a previously undisclosed role for Cdk9 in signal transduction. >134.NF01070140 PMID:12531696 TI - Notch2 protein distribution in human teeth under normal and pathological conditions. AB - Notch signaling is essential for the appropriate differentiation of many cell types during development and, furthermore, is implicated in a variety of human diseases. Previous studies have shown that although the Notch1, -2, and -3 receptors are expressed in developing and injured rodent teeth, Notch2 expression was predominant after a lesion. To pursue the role of the Notch pathway in tooth development and disease, we have analyzed the expression of the Notch2 protein in embryonic and adult wounded human teeth. During the earlier stages of tooth development, the Notch2 protein was expressed in the epithelium, but was absent from proliferating cells of the inner enamel epithelium. At more advanced stages, Notch2 was expressed in the enamel-producing ameloblasts, while it was absent in mesenchyme-derived odontoblasts that synthesize the dentin matrix. Although Notch2 was not expressed in the pulp of adult intact teeth, it was reexpressed during dentin repair processes in odontoblasts and subodontoblastic cells. Transforming growth factor beta-1, which stimulates odontoblast differentiation and hard tissue formation after dental injury, downregulated Notch2 expression in cultured human dental slices, in vitro. These observations are consistent with the notion that Notch signaling is an important element in dental physiological and pathogenic conditions. >135.NF01070224 PMID:12124628 TI - Adenovirus oncoproteins inactivate the Mre11-Rad50-NBS1 DNA repair complex. AB - In mammalian cells, a conserved multiprotein complex of Mre11, Rad50 and NBS1 (also known as nibrin and p95) is important for double-strand break repair, meiotic recombination and telomere maintenance. This complex forms nuclear foci and may be a sensor of double-strand breaks. In the absence of the early region E4, the double-stranded DNA genome of adenovirus is joined into concatemers too large to be packaged. We have investigated the cellular proteins involved in this concatemer formation and how they are inactivated by E4 products during a wild-type infection. Here we show that concatemerization requires functional Mre11 and NBS1, and that these proteins are found at foci adjacent to viral replication centres. Infection with wild-type virus results in both reorganization and degradation of members of the Mre11-Rad50-NBS1 complex. These activities are mediated by three viral oncoproteins that prevent concatemerization. This targeting of cellular proteins involved in genomic stability suggests a mechanism for 'hit-and-run' transformation observed for these viral oncoproteins. >136.NF00137104 PMID:12107724 TI - Impaired glucose tolerance is associated with increased serum concentrations of interleukin 6 and co-regulated acute-phase proteins but not TNF-alpha or its receptors. AB - AIMS/HYPOTHESIS: A population-based sample was studied to define immune abnormalities in individuals at risk of Type II (non-insulin-dependent) diabetes mellitus because of impaired glucose tolerance. METHODS: A total of 1653 individuals aged 55 to 74 years participated in a population based survey in Southern Germany (KORA Survey 2000). Those without a history of diabetes were subjected to an OGTT. Randomly selected subjects with IGT ( n=80) were compared with non-diabetic control subjects ( n=77) and patients with Type II diabetes ( n=152) of the same population-based sample after matching for age and sex. Immune parameters were analysed in serum with rigidly evaluated ELISA. RESULTS: Serum pro-inflammatory cytokine interleukin 6 (IL-6) concentrations were higher in subjects with IGT and Type II diabetes than in the control subjects (median 1.8 and 2.5 vs 0.8 pg/ml, p<0.0001). Soluble IL-6 receptors potentiate IL-6 bioactivity and their concentrations were mildly increased in Type II diabetes ( p<0.05). These immune changes seem relevant because IL-6 dependent acute-phase proteins C-reactive protein, serum amyloid A protein and fibrinogen were also increased in IGT and Type II diabetes. Circulating concentrations of TNF-alpha and its two receptors sTNF-R60 and sTNF-R80 were not increased in IGT subjects compared with the control subjects. CONCLUSION/INTERPRETATION: Our study shows systemic up-regulation of selected inflammatory mediators in patients with Type II diabetes and IGT. The pattern observed is non-random and fits with an IL-6 associated rather than TNF-alpha associated response. >137.NF00140831 PMID:11911837 TI - Association of dopamine D(3) receptors with actin-binding protein 280 (ABP-280). AB - Proteins that bind to G protein-coupled receptors have been identified as regulators of receptor localization and signaling. In our previous studies, a cytoskeletal protein, actin-binding protein 280 (ABP-280), was found to associate with the third cytoplasmic loop of dopamine D(2) receptors. In this study, we demonstrate that ABP-280 also interacts with dopamine D(3) receptors, but not with D(4) receptors. Similar to the dopamine D(2) receptor, the D(3)/ABP-280 association is of signaling importance. In human melanoma M2 cells lacking ABP-280, D(3) receptors were unable to inhibit forskolin-stimulated cyclic AMP (cAMP) production significantly. D(4) receptors, however, exhibited a similar degree of inhibition of forskolin-stimulated cAMP production in ABP-280-deficient M2 cells and ABP-280-replent M2 subclones (A7 cells). Further experiments revealed that the D(3)/ABP-280 interaction was critically dependent upon a 36 amino acid carboxyl domain of the D(3) receptor third loop, which is conserved in the D(2) receptor but not in the D(4) receptor. Our results demonstrate a subtype-specific regulation of dopamine D(2)-family receptor signaling by the cytoskeletal protein ABP-280. >138.NF00090206 PMID:12141441 TI - PAX genes in development and disease: the role of PAX2 in urogenital tract development. AB - PAX genes play an important role in fetal development. Moreover, heterozygous mutations in several PAX genes cause human disease. Here we review the role of PAX2 in kidney development, focusing on the morphological effects of PAX2 mutations. We discuss the role of PAX2 in the context of an inhibitory field model of kidney branching morphogenesis and summarize recent progress in this area. >139.NF00081216 PMID:12547701 TI - CC chemokine receptor 8 in the central nervous system is associated with phagocytic macrophages. AB - CC chemokine receptor 8 (CCR8) has been detected in vitro on type 2 helper and regulatory lymphocytes, which might exert beneficial functions in multiple sclerosis (MS) and on macrophages and microglia, possibly promoting tissue injury in MS lesions. To discriminate the relevant expression pattern in vivo, we defined the cell types that expressed CCR8 in MS lesions and determined the relationship of CCR8 expression and demyelinating activity. CCR8 was not expressed on T cells but was associated with phagocytic macrophages and activated microglia in MS lesions and directly correlated with demyelinating activity. To identify factors associated with CCR8 expression, the study was extended to other central nervous system (CNS) pathologies. CCR8 was consistently expressed on phagocytic macrophages and activated microglia in stroke and progressive multifocal leukoencephalopathy, but not expressed on microglia in pathologies that lacked phagocytic macrophages such as senile change of the Alzheimer's type. CCR8 was up-regulated by macrophage differentiation and activating stimuli in vitro. In summary CNS CCR8 expression was associated with phagocytic macrophages and activated microglial cells in human CNS diseases, suggesting that CCR8 may be a feasible target for therapeutic intervention in MS. CCR8 expression may also indicate a selective program of mononuclear phagocyte gene expression. >140.NF00126254 PMID:12297502 TI - Inhibition of silencing and accelerated aging by nicotinamide, a putative negative regulator of yeast sir2 and human SIRT1. AB - The Saccharomyces cerevisiae Sir2 protein is an NAD(+)-dependent histone deacetylase that plays a critical role in transcriptional silencing, genome stability, and longevity. A human homologue of Sir2, SIRT1, regulates the activity of the p53 tumor suppressor and inhibits apoptosis. The Sir2 deacetylation reaction generates two products: O-acetyl-ADP-ribose and nicotinamide, a precursor of nicotinic acid and a form of niacin/vitamin B(3). We show here that nicotinamide strongly inhibits yeast silencing, increases rDNA recombination, and shortens replicative life span to that of a sir2 mutant. Nicotinamide abolishes silencing and leads to an eventual delocalization of Sir2 even in G(1)-arrested cells, demonstrating that silent heterochromatin requires continual Sir2 activity. We show that physiological concentrations of nicotinamide noncompetitively inhibit both Sir2 and SIRT1 in vitro. The degree of inhibition by nicotinamide (IC(50) < 50 microm) is equal to or better than the most effective known synthetic inhibitors of this class of proteins. We propose a model whereby nicotinamide inhibits deacetylation by binding to a conserved pocket adjacent to NAD(+), thereby blocking NAD(+) hydrolysis. We discuss the possibility that nicotinamide is a physiologically relevant regulator of Sir2 enzymes. >141.NF01222508 PMID:11927593 TI - The newly identified human nuclear protein NXP-2 possesses three distinct domains, the nuclear matrix-binding, RNA-binding, and coiled-coil domains. AB - Using a monoclonal antibody that recognizes a nuclear matrix protein, we selected a cDNA clone from a lambdagt11 human placenta cDNA library. This cDNA encoded a 939-amino acid protein designated nuclear matrix protein NXP-2. Northern blot analysis indicated that NXP-2 was expressed in various tissues at different levels. Forcibly expressed green fluorescent protein-tagged NXP-2 as well as endogenous NXP-2 was localized in the nucleus and distributed to the nuclear matrix. NXP-2 was released from the nuclear matrix when RNase A was included in the buffer for nuclear matrix preparation. Mapping of functional domains was carried out using green fluorescent protein-tagged truncated mutants of NXP-2. The region of amino acids 326-353 was responsible for nuclear matrix binding and contained a cluster of hydrophobic amino acids that was similar to the nuclear matrix targeting signal of acute myeloleukemia protein. The central region (amino acids 500-591) was demonstrated to be required for RNA binding by Northwestern analysis, although NXP-2 lacked a known RNA binding motif. The region of amino acid residues 682-876 was predicted to have a coiled-coil structure. The RNA-binding, nuclear matrix-binding, and coiled-coil domains are structurally separated, suggesting that NXP-2 plays important roles in diverse nuclear functions, including RNA metabolism and maintenance of nuclear architecture. >142.NF01070309 PMID:12481029 TI - The DNA methyltransferase-like protein DNMT3L stimulates de novo methylation by Dnmt3a. AB - Dnmt3L is required for the establishment of maternal methylation imprints at imprinting centers (ICs). Dnmt3L, however, lacks the conserved catalytic domain common to DNA methyltransferases. In an attempt to define its function, we coexpressed DNMT3L with each of the two known de novo methyltransferases, Dnmt3a and DNMT3B, in human cells and monitored de novo methylation by using replicating minichromosomes carrying various ICs as targets. Coexpression of DNMT3L with DNMT3B led to little or no change in target methylation. However, coexpression of DNMT3L with Dnmt3a resulted in a striking stimulation of de novo methylation by Dnmt3a. Stimulation was observed at maternally methylated ICs such as small nuclear ribonucleoprotein polypeptide N (SNRPN), Snrpn, and Igf2rAir, as well as at various nonimprinted sequences present on the episomes. Stimulation of Dnmt3a by DNMT3L was also observed at endogenous sequences in the genome. Therefore, DNMT3L acts as a general stimulatory factor for de novo methylation by Dnmt3a. The implications of these findings for the function of DNMT3L and Dnmt3a in DNA methylation and genomic imprinting are discussed. >143.NF00108436 PMID:11980634 TI - Platelet-derived growth factor D: tumorigenicity in mice and dysregulated expression in human cancer. AB - Platelet-derived growth factor (PDGF) has been directly implicated in developmental and physiological processes, as well as in human cancer and other proliferative disorders. We have recently isolated and characterized a novel protease-activated member of the PDGF family, PDGF D. PDGF D has been shown to be proliferative for cells of mesenchymal origin, signaling through PDGF receptors. Comprehensive and systematic PDGF D transcript analysis revealed expression in many cell lines derived from ovarian, renal, and lung cancers, as well as from astrocytomas and medulloblastomas. beta PDGF receptor profiling further suggested autocrine signaling in several brain tumor cell lines. PDGF D transforming ability and tumor formation in SCID mice was further demonstrated. Exploiting a sensitive PDGF D sandwich ELISA using fully human monoclonal antibodies, PDGF D was detected at elevated levels in the sera of ovarian, renal, lung, and brain cancer patients. Immunohistochemical analysis confirmed PDGF D localization to ovarian and lung tumor tissues. Together, these data demonstrate that PDGF D plays a role in certain human cancers. >144.NF00911921 PMID:11810296 TI - A major marker for normal tension glaucoma: association with polymorphisms in the OPA1 gene. AB - Normal tension glaucoma (NTG) is a major form of glaucoma, associated with intraocular pressures that are within the statistically normal range of the population. OPA1, the gene responsible for autosomal dominant optic atrophy represents an excellent candidate gene for NTG, as the clinical phenotypes are similar and OPA1 is expressed in the retina and optic nerve. Eighty-three well-characterized NTG patients were screened for mutations in OPA1 by heteroduplex analysis and bi-directional sequencing. Sequences found to be altered in NTG subjects were examined for variations in 100 population controls. A second cohort of 80 NTG patients and 86 population controls was subsequently screened to determine whether the initial findings could be replicated. A single nucleotide polymorphism (SNP) on intervening sequence (IVS) 8 (IVS8 + 4 C/T) was found to be strongly associated with the occurrence of NTG in both cohorts (chi(2)=7.97, P=0.005 in the first cohort, chi(2)=9.93, P=0.002 in the second cohort; odds ratio 3.1 (95% CI: 1.8-5.6). A second SNP (IVS8 + 32 T/C) appeared to be associated with disease in the first cohort (chi(2)=4.71, P=0.030), but this finding could not be replicated in the second cohort. In the combined cohort, the compound at-risk genotype IVS8 + 4 C/T, + 32 T/C was strongly associated with the occurrence of NTG (chi(2)=22.04, P=0.00001 after correcting for testing four genotypes). These results indicate that polymorphisms in the OPA1 gene are associated with NTG and may be a marker for the disease. >145.NF00114756 PMID:12095614 TI - Direct addition of BimL to mitochondria does not lead to cytochrome c release. AB - Pro-apoptotic members of the Bcl-2 family can be subdivided in two classes according to their structure: a group including Bax, Bak, and Bok that display Bcl-2 homology (BH) 1, BH2 and BH3 domains and a second group including Bid (BH3 interacting domain death agonist), Bad, Bim (Bcl-2 interacting mediator of cell death) and several others that contain only a BH3 domain, the BH3-only proteins. The BH3-only proteins have been proposed to activate pro-apoptotic members of the Bax subfamily to trigger a mitochondrial pathway that leads to the release of cytochrome c and other apoptogenic factors. Here we report that the mechanism of action of Bim is different from that of Bid. Although overexpression of Bid or Bim in cells leads to cytochrome c release, only Bid is able to trigger the release of cytochrome c through Bax activation when added directly to isolated mitochondria. Bim(L), although unable to activate Bax, can directly inhibit Bcl-2 or Bcl-x(L). Our data suggest two functional classes of BH3-only proteins: those such as Bid which directly activate Bax-like proteins leading to mitochondrial membrane permeability and apoptosis and those such as Bim which inhibit anti-apoptotic proteins and render the cells more susceptible to apoptogenic stimuli. >146.NF00097631 PMID:11991808 TI - Human CCS gene: genomic organization and exclusion as a candidate for amyotrophic lateral sclerosis (ALS). AB - BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive lethal disorder of large motor neurons of the spinal cord and brain. In approximately 20% of the familial and 2% of sporadic cases the disease is due to a defect in the gene encoding the cytosolic antioxidant enzyme Cu, Zn-superoxide dismutase (SOD1). The underlying molecular defect is known only in a very small portion of the remaining cases and therefore involvement of other genes is likely. As SOD1 receives copper, essential for its normal function, by the copper chaperone, CCS (Copper Chaperone for SOD), we considered CCS as a potential candidate gene for ALS. RESULTS: We have characterized the genomic organization of CCS and determined exon-intron boundaries. The 823 bp coding region of the CCS is organized in 8 exons. We have evaluated involvement of the CCS in ALS by sequencing the entire coding region for mutations in 20 sporadic ALS patients. CONCLUSIONS: No causative mutations for the ALS have been detected in the CCS gene in 20 sporadic ALS patients analyzed, but an intragenic single nucleotide polymorphism has been identified. >147.NF00097555 PMID:12147251 TI - Both tissue inhibitors of metalloproteinases-1 (TIMP-1) and TIMP-2 activate Ras but through different pathways. AB - Tissue inhibitors of metalloproteinases-1 (TIMP-1) and TIMP-2 have growth-stimulating activity for a wide range of cell types. Ras, which comprises a family of three members, i.e, Ha-Ras, Ki-Ras, and H-Ras, is known to participate in growth control in all its facets, including cell proliferation, transformation, differentiation, and apoptosis. In this study, we tested the hypothesis that Ras might be involved in the cell growth-promoting activity of TIMPs. Using MG-63 human osteosarcoma cells, we demonstrated that both TIMP-1 and TIMP-2 caused an increase in the Ras-GTP level in a dose-dependent manner. Our previous results indicated that TIMP-1 activity is mediated through the tyrosine kinase (TYK)/mitogen-activated protein kinase (MAPK) pathway. Here, we demonstrated that Ras activation by TIMP-1 was inhibited by a specific TYK inhibitor, herbimycin A, suggesting that the TYK/MAPK signaling pathway was involved in Ras activation by TIMP-1. However, the activation of Ras by TIMP-2 was inhibited by an inhibitor specific for cyclic AMP-dependent protein kinase (PKA), H89, suggesting the involvement of the PKA-mediated pathway. Furthermore, TIMP-2 promoted the formation of a complex between Ras-GTP and phosphoinositide 3-kinase. >148.NF00079574 PMID:12213900 TI - Human fallopian tubes express prostacyclin (PGI) synthase and cyclooxygenases and synthesize abundant PGI. AB - Animal studies unequivocally support the indispensable role of prostaglandin (PG) and cyclooxygenase (COX) in ovulation and implantation. Available data also suggest that PG and COX may be important in the transport of embryos. The effects of PGE(2) and PGF(2alpha) on the contractility of human tubal muscle have been studied extensively; the expression of COX in human fallopian tubes was also reported. Despite all these, two fundamentally important questions remained to be answered: 1) which PGs are produced by human fallopian tubes; and 2) which COX isoform(s) is expressed by the fallopian tubes. We used reverse-phase HPLC to study the metabolism of [1-(14)C] arachidonic acid by the fallopian tubes. We found that 6 keto-PGF(1alpha), a stable metabolite of prostacyclin (PGI), and PGE(2) constituted 56% +/- 10% and 35% +/- 10% (mean +/- SEM, four samples), respectively, of total eicosanoids synthesized. Western blot analysis revealed the expression of both COX isoforms. Immunohistochemistry study showed that both COX-1 and -2 were localized to nonciliated epithelia and tubal smooth muscle. In addition, COX-2 was also expressed in ciliated epithelial cells. Western blot analysis revealed the expression of PGI synthase (PGIS) and PGI receptor by fallopian tubes. Immunohistochemistry confirmed the expression of PGIS by luminal epithelia, tubal smooth muscle, vascular endothelial cells, and vascular smooth muscle cells. Iloprost, a PGI analog, inhibited the activities of circular and longitudinal muscles of the fallopian tube. Thus, the fallopian tube expresses both COX isoforms and PGIS. Furthermore, it is a source and a target of PGI. PGI and COX may be important to gamete function, embryo transport, and embryo development. >149.NF00079494 PMID:11847269 TI - Alternative type I and I' turn conformations in the beta8/beta9 beta-hairpin of human acidic fibroblast growth factor. AB - Human acidic fibroblast growth factor (FGF-1) has a beta-trefoil structure, one of the fundamental protein superfolds. The X-ray crystal structures of wild-type and various mutant forms of FGF-1 have been solved in five different space groups: C2, C222(1), P2(1) (four molecules/asu), P2(1) (three molecules/asu), and P2(1)2(1)2(1). These structures reveal two characteristically different conformations for the beta8/beta9 beta-hairpin comprising residue positions 90-94. This region in the wild-type FGF-1 structure (P2(1), four molecules/asu), a his-tagged His93-->Gly mutant (P2(1), three molecules/asu) and a his-tagged Asn106-->Gly mutant (P2(1)2(1)2(1)) adopts a 3:5 beta-hairpin known as a type I (1-4) G1 beta-bulge (containing a type I turn). However, a his-tagged form of wild-type FGF-1 (C222(1)) and a his-tagged Leu44-->Phe mutant (C2) adopt a 3:3 beta-hairpin (containing a type I' turn) for this same region. A feature that distinguishes these two types of beta-hairpin structures is the number and location of side chain positions with eclipsed C(beta) and main-chain carbonyl oxygen groups (Psi is equivalent to +60 degrees). The effects of glycine mutations upon stability, at positions within the hairpin, have been used to identify the most likely structure in solution. Type I' turns in the structural data bank are quite rare, and a survey of these turns reveals that a large percentage exhibit crystal contacts within 3.0 A. This suggests that many of the type I' turns in X-ray structures may be adopted due to crystal packing effects. >150.NF00329866 PMID:12114499 TI - HIV-1 Tat interaction with RNA polymerase II C-terminal domain (CTD) and a dynamic association with CDK2 induce CTD phosphorylation and transcription from HIV-1 promoter. AB - Human immunodeficiency virus, type 1 (HIV-1), Tat protein activates viral gene expression through promoting transcriptional elongation by RNA polymerase II (RNAPII). In this process Tat enhances phosphorylation of the C-terminal domain (CTD) of RNAPII by activating cell cycle-dependent kinases (CDKs) associated with general transcription factors of the promoter complex, specifically CDK7 and CDK9. We reported a Tat-associated T-cell-derived kinase, which contained CDK2. Here, we provide further evidence that CDK2 is involved in Tat-mediated CTD phosphorylation and in HIV-1 transcription in vitro. Tat-mediated CTD phosphorylation by CDK2 required cysteine 22 in the activation domain of Tat and amino acids 42-72 of Tat. CDK2 phosphorylated Tat itself, apparently by forming dynamic contacts with amino acids 15-24 and 36-49 of Tat. Also, amino acids 24-36 and 45-72 of Tat interacted with CTD. CDK2 associated with RNAPII and was found in elongation complexes assembled on HIV-1 long-terminal repeat template. Recombinant CDK2/cyclin E stimulated Tat-dependent HIV-1 transcription in reconstituted transcription assay. Immunodepletion of CDK2/cyclin E in HeLa nuclear extract blocked Tat-dependent transcription. We suggest that CDK2 is part of a transcription complex that is required for Tat-dependent transcription and that interaction of Tat with CTD and a dynamic association of Tat with CDK2/cyclin E stimulated CTD phosphorylation by CDK2. >151.NF00777929 PMID:12101186 TI - The transcriptional regulating protein of 132 kDa (TReP-132) enhances P450scc gene transcription through interaction with steroidogenic factor-1 in human adrenal cells. AB - The human P450scc gene is regulated by the tissue-specific orphan nuclear receptor, steroidogenic factor-1 (SF-1), which plays a key role in several physiologic processes including steroid synthesis, adrenal and gonadal development, and sexual differentiation. Several studies have demonstrated the interaction of SF-1 with different proteins. However, it is clear that additional factors not yet identified are involved with SF-1 to regulate different target genes. Recently, it was demonstrated that a novel transcriptional regulating protein of 132 kDa (TReP-132) regulates expression of the human P450scc gene. The overexpression of TReP-132 in adrenal cells increases the production of pregnenolone, which is associated with the activation of P450scc gene expression. Considering the colocalization of TReP-132 and SF-1 in steroidogenic tissues such as the adrenal and testis, and the presence of two putative LXXLL motifs in TReP-132 that can potentially interact with SF-1, the relationship between these two factors on the P450scc gene promoter was determined. The coexpression of SF-1 and TReP-132 in adrenal NCI-H295 cells cooperates to increase promoter activity. Pull-down experiments demonstrated the interaction between TReP-132 and SF-1, and this was further confirmed in intact cells by coimmunoprecipitation/Western blot and two-hybrid analyses. Deletions and mutations of the TReP-132 cDNA sequence demonstrate that SF-1 interaction requires the LXXLL motif found at the amino-terminal region of the protein. Also, the "proximal activation domain" and the "AF-2 hexamer" motif of SF-1 are involved in interaction with TReP-132. Consistent with previous studies showing interaction between CBP/p300 and SF-1 or TReP-132, the coexpression of these three proteins results in a synergistic effect on P450scc gene promoter activity. Taken together the results in this study identify a novel function of TReP-132 as a partner in a complex with SF-1 and CBP/p300 to regulate gene transcription involved in steroidogenesis. >152.NF00091024 PMID:11931760 TI - The dissociation of cohesin from chromosomes in prophase is regulated by Polo-like kinase. AB - The separation of sister chromatids in anaphase depends on the dissociation of cohesin from chromosomes. In vertebrates, some cohesin is removed from chromosomes at the onset of anaphase by proteolytic cleavage. In contrast, the bulk of cohesin is removed from chromosomes already in prophase and prometaphase by an unknown mechanism that does not involve cohesin cleavage. We show that Polo-like kinase is required for the cleavage-independent dissociation of cohesin from chromosomes in Xenopus. Cohesin phosphorylation depends on Polo-like kinase and reduces the ability of cohesin to bind to chromatin. These results suggest that Polo-like kinase regulates the dissociation of cohesin from chromosomes early in mitosis. >153.NF00135250 PMID:11716065 TI - Hyaluronate receptors mediating glioma cell migration and proliferation. AB - The extracellular matrix (ECM) of the central nervous system (CNS) is enriched in hyaluronate (HA). Ubiquitous receptors for HA are CD44 and the Receptor for HA-Mediated Motility known as RHAMM. In the present study, we have investigated the potential role of CD44 and RHAMM in the migration and proliferation of human astrocytoma cells. HA-receptor expression in brain tumor cell lines and surgical specimens was determined by immunocytochemistry and western blot analyses. The ability of RHAMM to bind ligand was determined through cetylpyridinium chloride (CPC) precipitations of brain tumor lysates in HA-binding assays. The effects of HA, CD44 blocking antibodies, and RHAMM soluble peptide on astrocytoma cell growth and migration was determined using MTT and migration assays. Our results show that the expression of the HA-receptors, CD44, and RHAMM, is virtually ubiquitous amongst glioma cell lines, and glioma tumor specimens. There was a gradient of expression amongst gliomas with high grade gliomas expressing more RHAMM and CD44 than did lower grade lesions or did normal human astrocytes or non-neoplastic specimens of human brain. Specific RHAMM variants of 85- and 58-kDa size were shown to bind avidly to HA following CPC precipitations. RHAMM soluble peptide inhibited glioma cell line proliferation in a dose-dependent fashion. Finally, while anti-CD44 antibodies did not inhibit the migration of human glioma cells, soluble peptides directed at the HA-binding domain of RHAMM inhibited glioma migration both on and off an HA-based ECM. These data support the notion that HA-receptors contribute to brain tumor adhesion, proliferation, and migration, biological features which must be better understood before more effective treatment strategies for these tumors can be found. >154.NF01222518 PMID:11690642 TI - Expression pattern of human P2Y receptor subtypes: a quantitative reverse transcription-polymerase chain reaction study. AB - The diverse biological actions of extracellular nucleotides in tissues and cells are mediated by two distinct classes of P2 receptor, P2X and P2Y. The G protein-coupled P2Y receptors comprise at least six mammalian subtypes (P2Y(1,2,4,6,11,12)), all of which have been cloned from human tissues, as well as other species. The P2Y receptor subtypes differ in their pharmacological selectivity for various adenosine and uridine nucleotides, which overlap in some cases. Data concerning the mRNA expression patterns of five P2Y receptors (P2Y(1,2,4,6,11)) in different human tissues and cells are currently quite limited, while P2Y mRNA distribution in the human brain has not previously been studied. In this study, we have addressed this deficiency in receptor expression data by using a quantitative reverse transcription-polymerase chain reaction approach to measure the precise mRNA expression pattern of each P2Y receptor subtype in a number of human peripheral tissues and brain regions, from multiple individuals, as well as numerous human cell lines and primary cells. All five P2Y receptors exhibited widespread yet subtype-selective mRNA expression profiles throughout the human tissues, brain regions and cells used. Our extensive expression data indicate the many potentially important roles of P2Y receptors throughout the human body, and will help in elucidating the physiological function of each receptor subtype in a wide variety of human systems. >155.NF00109417 PMID:12151397 TI - Induction of homologue of Slimb ubiquitin ligase receptor by mitogen signaling. AB - Homologue of Slimb (HOS) is the substrate-recognizing component of the SCF(HOS)-Roc1 E3 ubiquitin protein ligase. This ligase mediates ubiquitination of the inhibitor of NF-kappaB transcription factor (IkappaB). We have found that HOS is highly expressed in a number of human cancer cell lines. The rates of the HOS gene transcription as well as HOS mRNA and protein levels were up-regulated in cells treated with mitogens or transfected with the inducers of mitogen-activated protein kinase pathway. Conversely, mitogen withdrawal strikingly reduced HOS levels during differentiation of mouse myoblasts. Activators of mitogen-activated protein kinase accelerated IkappaBalpha degradation and increased NF-kappaB transcriptional activity. Inhibition of HOS function via expression of dominant negative HOS (HOS(DeltaF)) initiated mouse myoblast differentiation and prevented Ras-mediated acceleration of IkappaBalpha degradation as well as NF-kappaB trans-activation and transformation of NIH3T3 cells. These data link the induction of HOS in proliferating cells with mitogen-signaling-dependent inhibition of cell differentiation and promotion of cell transformation. >156.NF00114683 PMID:12478664 TI - Hodgkin's lymphoma cell lines are characterized by frequent aberrations on chromosomes 2p and 9p including REL and JAK2. AB - Four Hodgkin's lymphoma cell lines (KM-H2, HDLM-2, L428, L1236) were analyzed for cytogenetic aberrations, applying multiplex fluorescence in situ hybridization, chromosome banding and comparative genomic hybridization. Each line was characterized by a highly heterogeneous pattern of karyotypic changes with a large spectrum of different translocated chromosomes (range 22-57). A recurrent finding in all cell lines was the presence of chromosomal rearrangements of the short arm of chromosome 2 involving the REL oncogene locus. Furthermore, multiple translocated copies of telomeric chromosomal segments were frequently detected. This resulted in a copy number increase of putative oncogenes, e.g., JAK2 (9p24) in 3 cell lines, FGFR3 (4p16) and CCND2 (12p13) in 2 cell lines as well as MYC (8q24) in 1 cell line. Our data confirm previous cytogenetic results from primary Hodgkin's tumors suggesting an important pathogenic role of REL and JAK2 in this disease. In addition, they provide evidence for a novel cytogenetic pathomechanism leading to increased copy numbers of putative oncogenes from terminal chromosomal regions, most probably in the course of chromosomal stabilization by telomeric capture. >157.NF01223249 PMID:12359436 TI - Vpr- and Nef-dependent induction of RANTES/CCL5 in microglial cells. AB - Microglia are pivotal in the pathogenesis of AIDS dementia, as they serve as the major target of HIV infection in the CNS. In addition, activation of microglia correlates best with clinical dementia. Although the beta-chemokine RANTES/CCL5 is important in modulating HIV infection as well as cellular activation, no information is available regarding how its expression is regulated in microglia by HIV-1. Here we report that RANTES/CCL5 expression is induced in microglia by HIV-1, but that this requires infection by HIV-1. This conclusion was supported by (1) the delayed kinetics coinciding with viral replication; (2) the lack of effect of X4 viruses; (3) inhibition by the reverse transcriptase inhibitor AZT, and (4) the lack of effect of cytokine antagonists or antibodies. Interestingly, RANTES/CCL5 production was dependent on the viral accessory protein Vpr, in addition to Nef, demonstrating a novel role for Vpr in chemokine induction in primary macrophage-type cells. Furthermore, the specific p38 MAP kinase inhibitor SB203580 augmented chemokine expression in microglia, indicating a negative role played by p38. These data suggest unique features of RANTES/CCL5 regulation by HIV-1 in human microglial cells. >158.NF01196322 PMID:12207029 TI - Dissection of the functional differences between sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 1 and 3 isoforms by steady-state and transient kinetic analyses. AB - Steady-state and transient-kinetic studies were conducted to characterize the overall and partial reactions of the Ca(2+)-transport cycle mediated by the human sarco(endo)plasmic reticulum Ca(2+)-ATPase 3 (SERCA3) isoforms: SERCA3a, SERCA3b, and SERCA3c. Relative to SERCA1a, all three human SERCA3 enzymes displayed a reduced apparent affinity for cytosolic Ca(2+) in activation of the overall reaction due to a decreased E(2) to E(1)Ca(2) transition rate and an increased rate of Ca(2+) dissociation from E(1)Ca(2). At neutral pH, the ATPase activity of the SERCA3 enzymes was not significantly enhanced upon permeabilization of the microsomal vesicles with calcium ionophore, indicating a difference from SERCA1a with respect to regulation of the lumenal Ca(2+) level (either an enhanced efflux of lumenal Ca(2+) through the pump in E(2) form or insensitivity to inhibition by lumenal Ca(2+)). Other differences from SERCA1a with respect to the overall ATPase reaction were an alkaline shift of the pH optimum, increased catalytic turnover rate at pH optimum (highest for SERCA3b, the isoform with the longest C terminus), and an increased sensitivity to inhibition by vanadate that disappeared under equilibrium conditions in the absence of Ca(2+) and ATP. The transient-kinetic analysis traced several of the differences from SERCA1a to an enhancement of the rate of dephosphorylation of the E(2)P phosphoenzyme intermediate, which was most pronounced at alkaline pH and increased with the length of the alternatively spliced C terminus. >159.NF00080178 PMID:12200142 TI - Interaction of oncogenic papillomavirus E6 proteins with fibulin-1. AB - Human papillomavirus (HPV) infection is the primary risk factor for the development of cervical cancer. The papillomavirus E6 gene is essential for virus-induced cellular transformation and the viral life cycle. Important insight into the mechanism of E6 function came from the discovery that cancer-related HPV E6 proteins promote the degradation of the tumor suppressor p53. However, mounting evidence indicates that interaction with p53 does not mediate all E6 activities. To explore the p53-independent functions of E6, we performed a yeast two-hybrid screen and identified fibulin-1 as an E6 binding protein. Fibulin-1 is a calcium-binding plasma and extracellular matrix protein that has been implicated in cellular transformation and tumor invasion. The interaction between E6 and fibulin-1 was demonstrated by both in vitro and in vivo assays. Fibulin-1 is associated specifically with cancer-related HPV E6s and the transforming bovine papillomavirus type 1 E6. Significantly, overexpression of fibulin-1 specifically inhibited E6-mediated transformation. These results suggest that fibulin-1 plays an important role in the biological activities of E6. >160.NF00116385 PMID:12134158 TI - Unconventional Rac-GEF activity is mediated through the Dock180-ELMO complex. AB - Mammalian Dock180 and ELMO proteins, and their homologues in Caenorhabditis elegans and Drosophila melanogaster, function as critical upstream regulators of Rac during development and cell migration. The mechanism by which Dock180 or ELMO mediates Rac activation is not understood. Here, we identify a domain within Dock180 (denoted Docker) that specifically recognizes nucleotide-free Rac and can mediate GTP loading of Rac in vitro. The Docker domain is conserved among known Dock180 family members in metazoans and in a yeast protein. In cells, binding of Dock180 to Rac alone is insufficient for GTP loading, and a Dock180 ELMO1 interaction is required. We can also detect a trimeric ELMO1 Dock180 Rac1 complex and ELMO augments the interaction between Dock180 and Rac. We propose that the Dock180 ELMO complex functions as an unconventional two-part exchange factor for Rac. >161.NF00943036 PMID:12096920 TI - Interleukin-1F7B (IL-1H4/IL-1F7) is processed by caspase-1 and mature IL-1F7B binds to the IL-18 receptor but does not induce IFN-gamma production. AB - We have recently reported the identification of four novel members of the interleukin-1 (IL-1) family which we designated as IL-1 homologue 1-4 (IL-1H1-4). These proteins exhibit significant sequence homology to other members of the IL-1 family. Of these homologues, only IL-1H4 (renamed IL-1F7b) was predicted to contain a propeptide domain and a caspase cleavage site. We now report that caspase-1 cleaves IL-1F7b at the predicted site to generate mature IL-1F7b. Caspase-4 was also able to process IL-1F7b, albeit inefficiently. Other caspases and Granzyme-B did not cleave IL-1F7b. Furthermore, adenovirus-mediated expression of IL-1F7b in HEK 293 cells led to in situ processing and secretion of mature IL-1F7b. In a screen to identify a potential receptor, both pro and mature IL-1F7b bound to the soluble IL-18 receptor alpha-Fc (IL-18Ralpha-Fc) but not to the soluble IL-1R-Fc or ST2R-Fc fusion proteins. Mature IL-1F7b bound to the IL-18Ralpha-Fc protein with higher affinity than the pro form, although the affinities for both proteins were significantly lower than that observed for IL-18. Consistent with this observation, only IL-18 and not IL-1F7b induced IFN-gamma production by KG1a cells. We also report that pro and mature IL-1F7b form homodimers with association constants of 4 microM and 5 nM, respectively, suggesting biological relevance to IL-1F7b processing. Finally, we have localized the expression of IL-1F7b protein in discrete cell populations including plasma cells and tumor cells. These data suggest that IL-1F7b may be involved in immune response, inflammatory diseases and/or cancer. >162.NF00113796 PMID:12070015 TI - Novel transcription factors in human CD34 antigen-positive hematopoietic cells. AB - Transcription factors (TFs) and the regulatory proteins that control them play key roles in hematopoiesis, controlling basic processes of cell growth and differentiation; disruption of these processes may lead to leukemogenesis. Here we attempt to identify functionally novel and partially characterized TFs/regulatory proteins that are expressed in undifferentiated hematopoietic tissue. We surveyed our database of 15 970 genes/expressed sequence tags (ESTs) representing the normal human CD34(+) cells transcriptosome (http://westsun.hema.uic.edu/cd34.html), using the UniGene annotation text descriptor, to identify genes with motifs consistent with transcriptional regulators; 285 genes were identified. We also extracted the human homologues of the TFs reported in the murine stem cell database (SCdb; http://stemcell.princeton.edu/), selecting an additional 45 genes/ESTs. An exhaustive literature search of each of these 330 unique genes was performed to determine if any had been previously reported and to obtain additional characterizing information. Of the resulting gene list, 106 were considered to be potential TFs. Overall, the transcriptional regulator dataset consists of 165 novel or poorly characterized genes, including 25 that appeared to be TFs. Among these novel and poorly characterized genes are a cell growth regulatory with ring finger domain protein (CGR19, Hs.59106), an RB-associated CRAB repressor (RBAK, Hs.7222), a death-associated transcription factor 1 (DATF1, Hs.155313), and a p38-interacting protein (P38IP, Hs. 171185). The identification of these novel and partially characterized potential transcriptional regulators adds a wealth of information to understanding the molecular aspects of hematopoiesis and hematopoietic disorders. >163.NF00122868 PMID:11712085 TI - Expression and function of adrenomedullin and its receptors in Conn's adenoma cells. AB - Adrenomedullin (ADM) is a hypotensive peptide, that derives from the proteolytic cleavage of pro(p)ADM and acts through two subtypes of receptors, called L1-receptor (L1-R) and calcitonin receptor-like receptor (CRLR). CRLR may function as a calcitonin gene-related peptide or a selective ADM receptor depending on the expression of the subtype 1 or the subtypes 2 and 3 of a family of proteins, named receptor-activity modifying proteins (RAMPs). Reverse transcription (RT)-polymerase chain reaction (PCR) allowed the detection of pADM mRNA in dispersed cells of eight Conn's adenomas (aldosteronomas). These cells also expressed peptidyl-glycine alpha-amidating monooxigenase, the enzyme converting immature ADM to the mature form, and contained sizeable amounts of ADM-immunoreactivity as measured by radioimmunoassay. RT-PCR also demonstrated the presence in aldosteronoma cells of the specific mRNAs of L1-R, CRLR and RAMPs 1-3. ADM (10(-8) M) inhibited angiotensin-II (10(-9) M)-simulated aldosterone secretion from cultured aldosteronoma cells, without affecting basal production. ADM (10(-8) M) also enhanced basal proliferation rate of cultured cells, as estimated by the 5-bromo-2'-deoxyuridine immunocytochemical technique. Both effects of ADM were annulled by the ADM-receptor selective antagonist ADM22-52 (10(-7) M). In conclusion, our study provides evidence that aldosteronoma cells express both ADM and ADM22-52-sensitive receptors. These findings, coupled with the demonstration that ADM exerts an aldosterone antisecretagogue action and a proliferogenic effect on cultured aldosteronoma cells, make it likely that endogenous ADM system plays a potentially important role in the paracrine or autocrine functional control of Conn's adenomas. >164.NF01030797 PMID:11739281 TI - Modulation of endothelial cell growth arrest and apoptosis by vascular endothelial growth inhibitor. AB - Vascular endothelial growth inhibitor (VEGI), a new member of the tumor necrosis factor family, is an endothelial cell-specific gene and a potent inhibitor of endothelial cell proliferation, angiogenesis, and tumor growth. We report here that VEGI mediates the following two activities in endothelial cells: early G(1) arrest in G(0)/G(1) cells responding to growth stimuli, and programmed death in proliferating cells. G(0)/G(1)-synchronized bovine aortic endothelial cells were treated with VEGI before and after the onset of the growth cycle. When the cells were stimulated with growth conditions but treated simultaneously with VEGI, a reversible, early-G(1) growth arrest occurred, evidenced by the lack of late G(1) markers such as hyperphosphorylation of the retinoblastoma gene product and upregulation of the c-myc gene. Additionally, VEGI treatment led to inhibition of the activities of cyclin-dependent kinases CDK2, CDK4, and CDK6. In contrast, VEGI treatment of cells that had entered the growth cycle resulted in apoptotic cell death, as evidenced by terminal deoxytransferase labeling of fragmented DNA, caspase 3 activation, and annexin V staining, all of which were lacking in nonproliferating cells treated with VEGI. Additionally, stress-signaling proteins p38 and JNK were not as fully activated by VEGI in quiescent as compared with proliferating populations. These findings suggest a dual role for VEGI, the maintenance of growth arrest and induction of apoptosis, in the modulation of the endothelial cell cycle. >165.NF00015868 PMID:11867536 TI - The roX genes encode redundant male-specific lethal transcripts required for targeting of the MSL complex. AB - The roX1 and roX2 genes of Drosophila produce male-specific non-coding RNAs that co-localize with the Male-Specific Lethal (MSL) protein complex. This complex mediates up-regulation of the male X chromosome by increasing histone H4 acetylation, thus contributing to the equalization of X-linked gene expression between the sexes. Both roX genes overlap two of approximately 35 chromatin entry sites, DNA sequences proposed to act in cis to direct the MSL complex to the X chromosome. Although dosage compensation is essential in males, an intact roX1 gene is not required by either sex. We have generated flies lacking roX2 and find that this gene is also non-essential. However, simultaneous removal of both roX RNAs causes a striking male-specific reduction in viability accompanied by relocation of the MSL proteins and acetylated histone H4 from the X chromosome to autosomal sites and heterochromatin. Males can be rescued by roX cDNAs from autosomal transgenes, demonstrating the genetic separation of the chromatin entry and RNA-encoding functions. Therefore, the roX1 and roX2 genes produce redundant, male-specific lethal transcripts required for targeting the MSL complex. >166.NF00079829 PMID:11901358 TI - Fine mapping of the chromosome 2p12-16 dyslexia susceptibility locus: quantitative association analysis and positional candidate genes SEMA4F and OTX1. AB - A locus on chromosome 2p12-16 has been implicated in dyslexia susceptibility by two independent linkage studies, including our own study of 119 nuclear twin-based families, each with at least one reading-disabled child. Nonetheless, no variant of any gene has been reported to show association with dyslexia, and no consistent clinical evidence exists to identify candidate genes with any strong a priori logic. We used 21 microsatellite markers spanning 2p12-16 to refine our 1-LOD unit linkage support interval to 12cM between D2S337 and D2S286. Then, in quantitative association analysis, two microsatellites yielded P values<0.05 across a range of reading-related measures (D2S2378 and D2S2114). The exon/intron borders of two positional candidate genes within the region were characterized, and the exons were screened for polymorphisms. The genes were Semaphorin4F (SEMA4F), which encodes a protein involved in axonal growth cone guidance, and OTX1, encoding a homeodomain transcription factor involved in forebrain development. Two non-synonymous single nucleotide polymorphisms were found in SEMA4F, each with a heterozygosity of 0.03. One intronic single nucleotide polymorphism between exons 12 and 13 of SEMA4F was tested for quantitative association, but no significant association was found. Only one single nucleotide polymorphism was found in OTX1, which was exonic but silent. Our data therefore suggest that linkage with reading disability at 2p12-16 is not caused by coding variants of SEMA4F or OTX1. Our study outlines the approach necessary for the identification of genetic variants causing dyslexia susceptibility in an epidemiological population of dyslexics. >167.NF00088497 PMID:12393185 TI - Genomic organization of an intron-containing sperm protein 17 gene (Sp17-1) and an intronless pseudogene (Sp17-2) in humans: a new model. AB - Sp17 was initially thought to be a sperm specific protein involved in the interaction of the spermatozoon with the oocyte's surrounding extracellular glycoprotein matrix. Recent reports, however, indicate that Sp17 expression is neither testis-specific nor is it exclusively used for binding to the zona pellucida of the oocyte. In this study, we provide comprehensive characterization of the genomic structure of Sp17. We identified an intron-containing gene (Sp17-1) containing five exonic and four intronic sequences. Analysis of Sp17 transcripts using rapid amplification of DNA complementary to RNA (cDNA) ends (RACE) and polymerase chain reaction (PCR) techniques showed the presence of alternative polyadenylation resulting in the production of varying lengths of mRNAs as well as the usage of different transcriptional start sites. Moreover, an earlier description of the human Sp17 mRNA describing a splice variant could not be confirmed. Comparison to mouse Sp17 gene organization demonstrated a high degree of conservation, suggesting selective evolutionary pressure for this protein to retain a conserved gene architecture. Additionally, we identified a second gene (Sp17-2), whose most striking characteristic was the complete absence of introns. This Sp17-2 gene has likely arisen by reverse transcription (RT) of a spliced Sp17-1 mRNA with subsequent integration into the human genome. Its open reading frame (ORF) is interrupted by stop codons, giving rise to a pseudogene. Furthermore, Southern blot analysis of human genomic DNA indicated the possibility of additional Sp17 species within the human genome. >168.NF00100215 PMID:12208868 TI - Overexpression of ABCG5 and ABCG8 promotes biliary cholesterol secretion and reduces fractional absorption of dietary cholesterol. AB - Two ATP-binding cassette (ABC) transporters, ABCG5 and ABCG8, have been proposed to limit sterol absorption and to promote biliary sterol excretion in humans. To test this hypothesis, a P1 clone containing the human ABCG5 and ABCG8 genes was used to generate transgenic mice. The transgenes were expressed primarily in the liver and small intestine, mirroring the expression pattern of the endogenous genes. Transgene expression only modestly affected plasma and liver cholesterol levels but profoundly altered cholesterol transport. The fractional absorption of dietary cholesterol was reduced by about 50%, and biliary cholesterol levels were increased more than fivefold. Fecal neutral sterol excretion was increased three- to sixfold and hepatic cholesterol synthesis increased two- to fourfold in the transgenic mice. No significant changes in the pool size, composition, and fecal excretion of bile acids were observed in the transgenic mice. Transgene expression attenuated the increase in hepatic cholesterol content induced by consumption of a high cholesterol diet. These results demonstrate that increased expression of ABCG5 and ABCG8 selectively drives biliary neutral sterol secretion and reduces intestinal cholesterol absorption, leading to a selective increase in neutral sterol excretion and a compensatory increase in cholesterol synthesis. >169.NF00074103 PMID:12054167 TI - Localization of the gene causing autosomal dominant osteopetrosis type I to chromosome 11q12-13. AB - The osteopetroses are a heterogeneous group of genetic conditions characterized by increased bone density due to impaired bone resorption by osteoclasts. Within the autosomal dominant form of osteopetrosis, the radiological type I (ADOI) is characterized by a generalized osteosclerosis, most pronounced at the cranial vault. The patients are often asymptomatic but some suffer from pain and hearing loss. ADOI is the only type of osteopetrosis not associated with an increased fracture rate. Linkage analysis in two families with ADOI from Danish origin enabled us to assign the disease-causing gene to chromosome 11q12-13. A summated maximum lod score of +6.54 was obtained with marker D11S1889 and key recombinants allowed delineation of a candidate region of 6.6 cM between markers D11S1765 and D11S4113. Previously, genes causing other conditions with abnormal bone density have been identified from this chromosomal region. The TCIRG1 gene was shown to underly autosomal recessive osteopetrosis (ARO), and, recently, mutations in the LRP5 gene were found both in the osteoporosis-pseudoglioma syndrome and the high bone mass trait. Because both genes map within the candidate region for ADOI, it can not be excluded that ADOI is caused by mutations in either the TCIRG1 or the LRP5 gene. >170.NF00137122 PMID:11846562 TI - Crystal structure of a ternary SAP-1/SRF/c-fos SRE DNA complex. AB - Combinatorial DNA binding by proteins for promoter-specific gene activation is a common mode of DNA regulation in eukaryotic organisms, and occurs at the promoter of the c-fos proto-oncogene. The c-fos promoter contains a serum response element (SRE) that mediates ternary complex formation with the Ets proteins SAP-1 or Elk-1 and the MADS-box protein, serum response factor (SRF). Here, we report the crystal structure of a ternary SAP-1/SRF/c-fos SRE DNA complex containing the minimal DNA-binding domains of each protein. The structure of the complex reveals that the SAP-1 monomer and SRF dimer are bound on opposite faces of the DNA, and that the DNA recognition helix of SAP-1 makes direct contact with the DNA recognition helix of one of the two SRF subunits. These interactions facilitate an 82 degrees DNA bend around SRF and a modulation of protein-DNA contacts by each protein when compared to each of the binary DNA complexes. A comparison with a recently determined complex containing SRF, an idealized DNA site, and a SAP-1 fragment containing a SRF-interacting B-box region, shows a similar overall architecture but also shows important differences. Specifically, the comparison suggests that the B-box region of the Ets protein does not significantly influence DNA recognition by either of the proteins, and that the sequence of the DNA target effects the way in which the two proteins cooperate for DNA recognition. These studies have implications for how DNA-bound SRF may modulate the DNA-binding properties of other Ets proteins such as Elk-1, and for how other Ets proteins may modulate the DNA-binding properties of other DNA-bound accessory factors to facilitate promoter-specific transcriptional responses. >171.NF00107800 PMID:10512872 TI - GERp95, a membrane-associated protein that belongs to a family of proteins involved in stem cell differentiation. AB - A panel of mAbs was elicited against intracellular membrane fractions from rat pancreas. One of the antibodies reacted with a 95-kDa protein that localizes primarily to the Golgi complex or the endoplasmic reticulum (ER), depending on cell type. The corresponding cDNA was cloned and sequenced and found to encode a protein of 97.6 kDa that we call GERp95 (Golgi ER protein 95 kDa). The protein copurifies with intracellular membranes but does not contain hydrophobic regions that could function as signal peptides or transmembrane domains. Biochemical analysis suggests that GERp95 is a cytoplasmically exposed peripheral membrane protein that exists in a protease-resistant complex. GERp95 belongs to a family of highly conserved proteins in metazoans and Schizosaccharomyces pombe. It has recently been determined that plant and Drosophila homologues of GERp95 are important for controlling the differentiation of stem cells (Bohmert et al., 1998; Cox et al., 1998; Moussian et al., 1998). In Caenorhabditis elegans, there are at least 20 members of this protein family. To this end, we have used RNA interference to show that the GERp95 orthologue in C. elegans is important for maturation of germ-line stem cells in the gonad. GERp95 and related proteins are an emerging new family of proteins that have important roles in metazoan development. The present study suggests that these proteins may exert their effects on cell differentiation from the level of intracellular membranes. >172.NF00119061 PMID:12482858 TI - Evidence for a role of p38 kinase in hypoxia-inducible factor 1-independent induction of vascular endothelial growth factor expression by sodium arsenite. AB - Recently we have demonstrated that sodium arsenite induces the expression of hypoxia-inducible factor 1alpha (HIF-1alpha) protein and vascular endothelial growth factor (VEGF) in OVCAR-3 human ovarian cancer cells. We now show that arsenic trioxide, an experimental anticancer drug, exerts the same effects. The involvement of phosphatidylinositol 3-kinase and mitogen-activated protein kinase (MAPK) pathways in the effects of sodium arsenite was investigated. By using kinase inhibitors in OVCAR-3 cells, both effects of sodium arsenite were found to be independent of phosphatidylinositol 3-kinase and p44/p42 MAPKS but were attenuated by inhibition of p38 MAPK. A role for p38 in the regulation of HIF-1alpha and VEGF expression was supported further by analysis of activation kinetics. Experiments in mouse fibroblast cell lines, lacking expression of c-Jun N-terminal kinases 1 and 2, suggested that these kinases are not required for induction of HIF-1alpha protein and VEGF mRNA. Unexpectedly, sodium arsenite did not activate a HIF-1-dependent reporter gene in OVCAR-3 cells, indicating that functional HIF-1 was not induced. In agreement with this hypothesis, up-regulation of VEGF mRNA was not reduced in HIF-1alpha(-/-) mouse fibroblast cell lines. Altogether, these data suggest that not HIF-1, but rather p38, mediates induction of VEGF mRNA expression by sodium arsenite. >173.NF00118414 PMID:12444553 TI - Expression of hpttg proto-oncogene in lymphoid neoplasias. AB - Pituitary tumor-transforming gene (pttg) is a distinct proto-oncogene which is expressed in certain normal tissues with high proliferation rate and in a variety of tumors. PTTG is the vertebrate analog of yeast securins Pds1 and Cut2 with a key role in the regulation of sister chromatid separation during mitosis. Impairment of PTTG regulated functions is expected to lead to chromosomal instability and aneuploidy. Human pttg (hpttg) is abundantly expressed in Jurkat T lymphoblastic lymphoma cells but not in normal peripheral blood leukocytes. To obtain additional data on the potential role of hpttg in lymphomagenesis we selected 150 cases of lymphoid tumors for the assessment of hpttg expression in tumor tissues. Immunohistochemical studies on formalin-fixed, paraffin-embedded tissues revealed hPTTG in 38.8% of B-cell lymphomas, 70.2% of T-cell lymphomas, and 73.1% of Hodgkin's lymphomas. Among B-cell lymphomas, the most frequently immunostained tumors were plasma cell tumors, diffuse large cell lymphomas, and follicle center cell lymphomas. In Hodgkin's disease, immunoreactivity was mainly noted in Reed-Sternberg cells. In conclusion, the frequent overexpression of hpttg in many histological subtypes of lymphoma suggests the involvement of this proto-oncogene in lymphomagenesis. >174.NF00117443 PMID:12411432 TI - Identification and characterization of a novel human sphingosine-1-phosphate phosphohydrolase, hSPP2. AB - Sphingosine 1-phosphate (S1P) is a bioactive lipid molecule that acts as both an extracellular signaling mediator and an intracellular second messenger. S1P is synthesized from sphingosine by sphingosine kinase and is degraded either by S1P lyase or by S1P phosphohydrolase. Recently, mammalian S1P phosphohydrolase (SPP1) was identified and shown to constitute a novel lipid phosphohydrolase family, the SPP family. In this study we have identified a second human S1P phosphohydrolase, SPP2, based on sequence homology to human SPP1. SPP2 exhibited high phosphohydrolase activity against S1P and dihydrosphingosine 1-phosphate. The dihydrosphingosine-1-phosphate phosphohydrolase activity was efficiently inhibited by excess S1P but not by lysophosphatidic acid, phosphatidic acid, or glycerol 3-phosphate, indicating that SPP2 is highly specific to sphingoid base 1-phosphates. Immunofluorescence microscopic analysis demonstrated that SPP2 is localized to the endoplasmic reticulum. Although the enzymatic properties and localization of SPP2 were similar to those of SPP1, the tissue-specific expression pattern of SPP2 was different from that of SPP1. Thus, SPP2 is another member of the SPP family that may play a role in attenuating intracellular S1P signaling. >175.NF00114852 PMID:12496348 TI - Up-regulation of neuronal calcium sensor-1 (NCS-1) in the prefrontal cortex of schizophrenic and bipolar patients. AB - The delineation of dopamine dysfunction in the mentally ill has been a long-standing quest of biological psychiatry. The present study focuses on a recently recognized group of dopamine receptor-interacting proteins as possible novel sites of dysfunction in schizophrenic and bipolar patients. We demonstrate that the dorsolateral prefrontal cortex in schizophrenia and bipolar cases from the Stanley Foundation Neuropathology Consortium display significantly elevated levels of the D2 dopamine receptor desensitization regulatory protein, neuronal calcium sensor-1. These levels of neuronal calcium sensor-1 were not influenced by age, gender, hemisphere, cause of death, postmortem period, alcohol consumption, or antipsychotic and mood stabilizing medications. The present study supports the hypothesis that schizophrenia and bipolar disorder may be associated with abnormalities in dopamine receptor-interacting proteins. >176.NF01222528 PMID:11574907 TI - Mutations in the human orthologue of the mouse underwhite gene (uw) underlie a new form of oculocutaneous albinism, OCA4. AB - Oculocutaneous albinism (OCA) affects approximately 1/20,000 people worldwide. All forms of OCA exhibit generalized hypopigmentation. Reduced pigmentation during eye development results in misrouting of the optic nerves, nystagmus, alternating strabismus, and reduced visual acuity. Loss of pigmentation in the skin leads to an increased risk for skin cancer. Two common forms and one infrequent form of OCA have been described. OCA1 (MIM 203100) is associated with mutations of the TYR gene encoding tyrosinase (the rate-limiting enzyme in the production of melanin pigment) and accounts for approximately 40% of OCA worldwide. OCA2 (MIM 203200), the most common form of OCA, is associated with mutations of the P gene and accounts for approximately 50% of OCA worldwide. OCA3 (MIM 203290), a rare form of OCA and also known as "rufous/red albinism," is associated with mutations in TYRP1 (encoding tyrosinase-related protein 1). Analysis of the TYR and P genes in patients with OCA suggests that other genes may be associated with OCA. We have identified the mouse underwhite gene (uw) and its human orthologue, which underlies a new form of human OCA, termed "OCA4." The encoded protein, MATP (for "membrane-associated transporter protein") is predicted to span the membrane 12 times and likely functions as a transporter. >177.NF00019355 PMID:12446795 TI - The Drosophila atypical protein kinase C-ref(2)p complex constitutes a conserved module for signaling in the toll pathway. AB - Recent results showed the critical role of the mammalian p62-atypical protein kinase C (aPKC) complex in the activation of NF-kappaB in response to different stimuli. Here we demonstrate using the RNA interference technique on Schneider cells that the Drosophila aPKC (DaPKC) is required for the stimulation of the Toll-signaling pathway, which activates the NF-kappaB homologues Dif and Dorsal. However, DaPKC does not appear to be important for the other Drosophila NF-kappaB signaling cascade, which activates the NF-kappaB homologue Relish in response to lipopolysaccharides. Interestingly, DaPKC functions downstream of the nuclear translocation of Dorsal or Dif, controlling the transcriptional activity of the Drosomycin promoter. We also show that the Drosophila Ref(2)P protein is the homologue of mammalian p62 as it binds to DaPKC, its overexpression is sufficient to activate the Drosomycin but not the Attacin promoter, and its depletion severely impairs Toll signaling. Collectively, these results demonstrate the conservation of the p62-aPKC complex for the control of innate immunity signal transduction in Drosophila melanogaster. >178.NF00127248 PMID:11857550 TI - Hydrocephalus and intestinal aganglionosis: is L1CAM a modifier gene in Hirschsprung disease? AB - Congenital hydrocephalus associated with aqueductal stenosis and/or agenesis of the corpus callosum has been described in newborn males with mutations in L1CAM, a gene that encodes a neural cell adhesion molecule. These males usually have severe mental retardation and may have spastic paraplegia and adducted thumbs. In contrast, Hirschsprung disease, or absence of ganglion cells in the distal gut, has rarely been described in such individuals. We report a male infant who had severe hydrocephalus identified in the prenatal period with evidence of aqueductal stenosis and adducted thumbs at birth. He developed chronic constipation, and rectal biopsy confirmed the diagnosis of Hirschsprung disease. Molecular testing of the L1CAM gene revealed a G2254A mutation, resulting in a V752M amino acid substitution. A common polymorphism in RET, but no mutation, was identified. Our patient represents the third example of coincident hydrocephalus and Hirschsprung disease in an individual with an identified L1CAM mutation. We hypothesize that L1CAM-mediated cell adhesion may be important for the ability of ganglion cell precursors to populate the gut, and that L1CAM may modify the effects of a Hirschsprung disease-associated gene to cause intestinal aganglionosis. >179.NF00123684 PMID:11741957 TI - A lipid-regulated docking site on vinculin for protein kinase C. AB - During cell spreading, binding of actin-organizing proteins to acidic phospholipids and phosphorylation are important for localization and activity of these proteins at nascent cell-matrix adhesion sites. Here, we report on a transient interaction between the lipid-dependent protein kinase Calpha and vinculin, an early component of these sites, during spreading of HeLa cells on collagen. In vitro binding of protein kinase Calpha to vinculin tail was found dependent on free calcium and acidic phospholipids but independent of a functional kinase domain. The interaction was enhanced by conditions that favor the oligomerization of vinculin. Phosphorylation by protein kinase Calpha reached 1.5 mol of phosphate/mol of vinculin tail and required the C-terminal hydrophobic hairpin, a putative phosphatidylinositol 4,5-bisphosphate-binding site. Mass spectroscopy of peptides derived from in vitro phosphorylated vinculin tail identified phosphorylation of serines 1033 and 1045. Inhibition of C-terminal phospholipid binding at the vinculin tail by mutagenesis or deletion reduced the rate of phosphorylation to < or =50%. We suggest a possible mechanism whereby phospholipid-regulated conformational changes in vinculin may lead to exposure of a docking site for protein kinase Calpha and subsequent phosphorylation of vinculin and/or vinculin interaction partners, thereby affecting the formation of cell adhesion complexes. >180.NF00132596 PMID:12016134 TI - Analysis of human cellular retinol-binding protein II promoter during enterocyte differentiation. AB - Cellular retinol binding protein II (CRBP II) is a vitamin A-binding protein that is expressed specifically in small intestinal villus absorptive cells. Previous studies have shown that retinoic acid upregulates endogenous human CRBP II gene expression in differentiated Caco-2 cells. To better characterize the regulation of human CRBP II expression, we analyzed the ability of receptor-selective agonists to enhance transcription from the 5'-upstream flanking region of the human CRBP II gene. Stable transfection experiments showed that the proximal 2.8-kb region of the human CRBP II gene is sufficient for retinoic acid inducibility in differentiated Caco-2 cells. However, direct sequence analysis and transient transfection experiments indicate that, unlike the rat CRBP II promoter, the human CRBP II promoter is not a direct retinoid X receptor target. The results indicate that the retinoic acid responsiveness of the human CRBP II promoter is mediated by an indirect mechanism and that this mechanism is associated with enterocyte differentiation. >181.NF00106676 PMID:12084720 TI - Identification of human male germ cell-associated kinase, a kinase transcriptionally activated by androgen in prostate cancer cells. AB - Androgen is involved in both normal development and malignant transformation of prostate cells. The signal transduction pathways associated with these processes are not well understood. Using a novel kinase display approach, we have identified a protein kinase, human male germ cell-associated kinase (hMAK), which is transcriptionally induced by the androgenic hormone 5alpha-dihydrotestosterone (DHT). The kinetics of induction is rapid and dose-dependent, and the induction is not blocked by cycloheximide treatment. Real time reverse transcription-PCR studies demonstrated a 9-fold induction of hMAK by 10 nm DHT at 24 h post-stimulation. The expression levels of hMAK in prostate cancer cell lines are in general higher than those of normal prostate epithelial cells. A reverse transcription-PCR product encompassing the entire hMAK open reading frame was isolated. The results from sequencing analysis showed that the hMAK protein is 623 amino acids in length and contains a kinase catalytic domain at its N terminus, followed by a proline/glutamine-rich domain. The catalytic domain of this kinase contains sequence motifs related to both the cyclin-dependent kinase and the mitogen-activated protein kinase families. When expressed in COS1 cells, hMAK is kinase-active as demonstrated by autophosphorylation and phosphorylation of exogenous substrate and is localized in the nucleus. A 3.7-kilobase pair promoter of the hMAK locus was isolated from a human genomic DNA bacterial artificial chromosome clone and was shown to be activated by DHT. This activation can be blocked by an anti-androgen drug bicalutamide (Casodex), implicating the involvement of androgen receptor in this process. Taken together, these data suggest that hMAK is a protein kinase targeted by androgen that may participate in androgen-mediated signaling in prostate cancer cells. >182.NF00124497 PMID:12368450 TI - Mycobacterium tuberculosis-infected human macrophages exhibit enhanced cellular adhesion with increased expression of LFA-1 and ICAM-1 and reduced expression and/or function of complement receptors, FcgammaRII and the mannose receptor. AB - The entry of Mycobacterium tuberculosis (Mtb) into the host macrophage and its survival in this environment are key components of tuberculosis pathogenesis. Following intracellular replication of the bacterium within alveolar macrophages, there is spread of bacilli to regional lymph nodes in the lungs and subsequent presentation of antigens to the host immune system. How this process occurs remains poorly understood, but one mechanism may involve the migration of macrophages containing Mtb across the alveoli to lymph nodes, where there is development of a protective host response with formation of granulomas composed in part of aggregated and fused, apoptotic, infected macrophages. Leukocyte integrins, including lymphocyte function-associated antigen-1 (LFA-1) and complement receptors CR3 and CR4, and their counter receptors play a major role in macrophage adhesion processes and phagocytosis. In this study, the appearance of Mtb-infected macrophages over time was examined, using inverted-phase microscopy and an in vitro culture model of human monocyte-derived macrophages (MDMs). Prior to and immediately following infection of the MDMs with Mtb, the macrophages appeared as individual cells in monolayer culture; however, within 24 h of infection with Mtb, the MDMs began to migrate and adhere to each other. The kinetics of this response were dependent on both the m.o.i. and the length of infection. Quantitative transmission electron microscopy studies revealed that macrophage adhesion was accompanied by increases in levels of LFA-1 and its counter receptor (ICAM-1), decreases in surface levels of the phagocytic receptors CR3, CR4 and FcgammaRII, and an increase in major histocompatibility complex Class II (MHC-II) molecules at 72 h post-infection. Decreases in surface levels of CR3 and CR4 had a functional correlate, with macrophages containing live bacilli showing a diminished phagocytic capacity for complement-opsonized sheep erythrocytes; macrophages containing heat-killed bacilli did not show this diminished capacity. The modulation of macrophage adhesion and phagocytic proteins may influence the trafficking of Mtb-infected macrophages within the host, with increases in levels of LFA-1 and ICAM-1 enhancing the adhesive properties of the macrophage and decreases in phagocytic receptors diminishing the phagocytic capacity of an already-infected cell, potentially allowing for maintenance of the intracellular niche of Mtb. >183.NF00118098 PMID:12010812 TI - Inactivating mutations of CASP10 gene in non-Hodgkin lymphomas. AB - Caspase 10 (Mch4/FLICE2) is a caspase homologous to caspase 8. A recent report described that inherited CASP10 gene mutations underlie defective lymphocyte and dendritic cell apoptosis in autoimmune lymphoproliferative syndrome (ALPS). In this study, to explore the possibility that mutation of this gene might be involved in the development of non-Hodgkin lymphoma (NHL), we have analyzed the entire coding region and all splice sites of the CASP10 gene for the detection of somatic mutations in 117 human NHLs. Overall, 17 NHLs (14.5%) were found to have CASP10 mutations, which were identified in the coding regions of the prodomain (n = 3), the p17 large protease subunit (n = 11), and the p12 small protease subunit (n = 3). We expressed the tumor-derived caspase 10 mutants in 293 cells and found that apoptosis was suppressed. These data suggest that the inactivating mutations of the CASP10 gene might lead to the loss of its apoptotic function and contribute to the pathogenesis of some human NHLs. >184.NF00125389 PMID:11793467 TI - New insights into ocular albinism type 1 (OA1): Mutations and polymorphisms of the OA1 gene. AB - Albinism ocular type 1 (OA1) is an X-linked type of albinism that mainly effects pigment production in the eye, resulting in hypopigmentation of the retina, nystagmus, strabismus, foveal hypoplasia, abnormal crossing of the optic fibers, and reduced visual acuity. The OA1 gene is located on chromosome Xp22.32 and the coding sequence is divided into nine exons. The protein is an integral transmembrane protein that has weak similarities to G protein-coupled receptors. A total of 25 missense, two nonsense, nine frameshift, and five splicing mutations have been reported in the OA1 gene associated with OA1. There are also several deletions of some or all exons of the OA1 gene with deletions of exon 2 resulting from unequal crossing-over, due to flanking Alu repeats. Mutation and polymorphism data on this gene is available from the International Albinism Center - Albinism Database web site (http://www.cbc.umn.edu/tad). >185.NF00079670 PMID:12376704 TI - Subthalamic GAD gene therapy in a Parkinson's disease rat model. AB - The motor abnormalities of Parkinson's disease (PD) are caused by alterations in basal ganglia network activity, including disinhibition of the subthalamic nucleus (STN), and excessive activity of the major output nuclei. Using adeno-associated viral vector-mediated somatic cell gene transfer, we expressed glutamic acid decarboxylase (GAD), the enzyme that catalyzes synthesis of the neurotransmitter GABA, in excitatory glutamatergic neurons of the STN in rats. The transduced neurons, when driven by electrical stimulation, produced mixed inhibitory responses associated with GABA release. This phenotypic shift resulted in strong neuroprotection of nigral dopamine neurons and rescue of the parkinsonian behavioral phenotype. This strategy suggests that there is plasticity between excitatory and inhibitory neurotransmission in the mammalian brain that could be exploited for therapeutic benefit. >186.NF00888340 PMID:11929878 TI - Sec34 is implicated in traffic from the endoplasmic reticulum to the Golgi and exists in a complex with GTC-90 and ldlBp. AB - Sec34p/Grd20p has been implicated in endoplasmic reticulum (ER)-to-Golgi transport and/or post-Golgi trafficking events and exists in a protein complex consisting of at least eight subunits in yeast. Although the mammalian counterpart (Sec34) of Sec34p has been molecularly identified, its role and interacting partners remain undefined. In this study, we have prepared antibodies specifically against the recombinant N-terminal fragment of Sec34 that recognize a polypeptide of about 93 kDa and label the Golgi apparatus. In a well-characterized semi-intact cell assay that reconstitutes transport of the envelope glycoprotein (VSVG) of vesicular stomatitis virus from the ER to the Golgi apparatus, anti-Sec34 antibodies inhibited the transport in a dose-dependent manner. The inhibition by anti-Sec34 antibodies could be neutralized by a noninhibitory amount of the antigen. Large-scale immunoprecipitation of rat liver cytosol with immobilized anti-Sec34 antibodies has co-immunoprecipitated GTC-90 and ldlBp, two peripheral Golgi proteins previously shown to exist in separate protein complexes. Two mammalian homologues (Dor1 and Cod1) of the yeast Sec34 complex were similarly recovered in the Sec34 immunoprecipitates. When expressed in transfected cells, epitope-tagged ldlCp and Cod2 were co-immunoprecipitated with anti-Sec34 antibodies with efficiencies comparable to that observed for tagged ldlBp, Dor1, and Cod1. Direct interactions of Sec34 with ldlBp and ldlCp were further demonstrated in vitro. These results suggest that Sec34, GTC-90, and ldlBp/ldlCp are part of the same protein complex(es) that regulates diverse aspects of Golgi function, including transport from the ER to the Golgi apparatus. >187.NF00099193 PMID:11801662 TI - Fc alpha RI/CD89 circulates in human serum covalently linked to IgA in a polymeric state. AB - The FcR for IgA CD89/FcalphaRI, is a type I receptor glycoprotein, expressed on myeloid cells, with important immune effector functions. In vitro CD89 can be released from CD89-expressing cells upon activation. Little information is available on the existence of this soluble molecule in vivo. Using specific and sensitive ELISA techniques (detection limit 50 pg/ml), we were not able to detect circulating CD89 in human sera. However, using Western blotting, a 30-kDa soluble CD89 molecule was demonstrated in both serum and plasma. Moreover, using a specific semiquantitative dot-blot system, we found CD89 in all human sera tested (mean concentration 1900 ng/ml). Size fractionation of human serum using gel filtration chromatography showed that the CD89 molecule was predominantly present in larger molecular mass fractions. Direct complexes between IgA and CD89 were demonstrated by anti-IgA affinity purification, and when analyzed under nonreducing conditions appeared to be covalently linked. Size fractionation of affinity-purified IgA showed the presence of soluble CD89 only in the high molecular mass fractions of IgA, but not in monomeric IgA. High molecular mass complexes of CD89-IgA could be distinguished from J chain containing dimeric IgA. These data show that CD89 circulates in complex with IgA, and suggest that CD89 might contribute to the formation of polymeric serum IgA. >188.NF01099219 PMID:12596048 TI - Influence of interleukin-12 receptor beta1 polymorphisms on tuberculosis. AB - Host genetic factors may be important determinants of susceptibility to tuberculosis, and several candidate gene polymorphisms have been shown to date. A series of recent reports concerning rare human deficiencies in the type-1 cytokine pathway suggest that more subtle variants of relevant genes may also contribute to susceptibility to tuberculosis at the general population level. To investigate whether polymorphisms in the interleukin-12 receptor (IL-12R) gene predispose individuals to tuberculosis, we studied these genes by single-strand conformational polymorphism analysis and direct sequencing. Although no common polymorphisms could be identified in the IL-12R beta 2 gene ( IL-12RB2), we confirmed four single nucleotide polymorphisms (SNPs; 641A-->G, 684C-->T, 1094T-->C, and 1132G-->C) causing three missense variants (Q214R, M365T, G378R) and one synonymous substitution in the extracellular domain of the IL-12R beta 1 gene ( IL12RB1). All SNPs were in almost perfect linkage disequilibrium (D'=0.98), and two common haplotypes of IL12RB1(allele 1: Q214-M365-G378; allele 2: R214-T365-R378) were revealed. Polymerase chain reaction/restriction fragment length polymorphism and sequence analyses were used to type IL12RB1polymorphisms in 98 patients with tuberculosis and 197 healthy controls in Japanese populations. In our case-control association study of tuberculosis, the R214-T365-R378 allele (allele 2) was over-represented in patients with tuberculosis, and homozygosity for R214-T365-R378 (the 2/2 genotype) was significantly associated with tuberculosis (odds ratio: 2.45; 95% CI: 1.20-4.99; P=0.013). In healthy subjects, homozygotes for R214-T365-R378 had lower levels of IL-12-induced signaling, according to differences in cellular responses to IL-12 between two haplotypes. These data suggest that the R214-T365-R378 allele, i.e., variation in IL12RB1, contribute to tuberculosis susceptibility in the Japanese population. This genetic variation may predispose individuals to tuberculosis infection by diminishing receptor responsiveness to IL-12 and to IL-23, leading to partial dysfunction of interferon-gamma-mediated immunity. >189.NF00095955 PMID:12439719 TI - Quantitative analysis of human kallikrein gene 14 expression in breast tumours indicates association with poor prognosis. AB - KLK14 (formerly known as KLK-L6) is a recently identified member of the human kallikrein gene family. This family harbours several genes aberrantly expressed in various cancers as well as established (PSA/hK3, hK2) and potential (hK6, hK10) cancer markers. Similar to other kallikrein genes, KLK14 was found to be regulated by steroid hormones, particularly androgens and progestins, in breast and ovarian cancer cell lines. Preliminary studies indicated that KLK14 is differentially expressed in breast, ovarian, prostatic and testicular tumours. Given the above, we determined the prognostic significance of KLK14 expression in breast cancer. We studied KLK14 expression in 178 histologically confirmed epithelial breast carcinomas by quantitative reverse transcription-polymerase chain reaction and correlated with clinicopathological variables (tumour stage, grade, histotype etc.) and with outcome (disease-free survival and overall survival), monitored over a median of 76 months. KLK14 mRNA levels ranged from 0 to 1,219 arbitrary units in breast cancer tissues, with a mean+/-s.e. of 136+/-22. An optimal cutoff value of 40.5 arbitrary units was selected, to categorise tumours as KLK14-positive or negative. Higher concentrations of KLK14 mRNA were more frequently found in patients with advanced stage (III) disease (P=0.032). No statistically significant association was found between KLK14 and the other clinicopathological variables. KLK14 overexpression was found to be a significant predictor of decreased disease-free survival (hazard ratio of 2.31, P=0.001) and overall survival (hazard ratio of 2.21, P=0.005). Cox multivariate analysis indicated that KLK14 was an independent prognostic indicator of disease-free survival and overall survival. KLK14 also has independent prognostic value in subgroups of patients with a tumour size </=2 cm and positive nodal, oestrogen receptor and progestin receptor status. We conclude that KLK14 expression, as assessed by quantitative reverse transcription-polymerase chain reaction, is an independent marker of unfavourable prognosis for breast cancer. >190.NF00097656 PMID:11815625 TI - Cytokine signaling: STATS in plasma membrane rafts. AB - STAT transcription factors signal from the plasma membrane to the nucleus in response to growth factors and cytokines. We have investigated whether plasma membrane "rafts" are involved in cytokine-activated STAT signaling. Cytokine-free human hepatoma Hep3B cells or cells treated with interleukin-6 (IL-6) or orthovanadate (a general activator of STATs) were fractionated, and plasma membrane raft fractions were obtained by equilibrium sedimentation or flotation through discontinuous sucrose gradients using either non-detergent or detergent-based (saponin or Triton X-100) methods. By Western blotting the plasma membrane raft fractions obtained using either non-detergent or detergent-based methods contained significant amounts of STAT1 and STAT3 (up to approximately 10% of the total cytoplasmic amount) as well as the integral raft proteins caveolin-1 and flotillin-1, the IL-6-receptor signal transducing chain gp130, the interferon-gamma receptor alpha chain (IFN-gammaRalpha), and the chaperone glucose-regulated protein 58 (GRP58/ER-60/ERp57). Upon activation of signaling by IL-6 or orthovanadate the respective Tyr-phosphorylated STAT species were now also observed in the membrane raft fraction but in a form deficient in DNA binding. The data show pre-association of STATs with plasma membrane rafts in flotation fractions, which also contained caveolin-1 and flotillin-1, and suggest that Tyr phosphorylation may not in itself be sufficient to cause the departure of PY-STATs from plasma membrane rafts. Methyl-beta-cyclodextrin, which sequesters cholesterol and disrupts plasma membrane rafts, markedly inhibited IL-6- and IFN-gamma-induced STAT signaling. Signaling through specialized raft microdomains may be a general mechanism operating at the level of the plasma membrane through which cytokines and growth factors activate STAT species (the "raft-STAT signaling hypothesis"). >191.NF00083100 PMID:11950878 TI - Human CAP1 is a key factor in the recycling of cofilin and actin for rapid actin turnover. AB - Cofilin-ADF (actin-depolymerizing factor) is an essential driver of actin-based motility. We discovered two proteins, p65 and p55, that are components of the actin-cofilin complex in a human HEK293 cell extract and identified p55 as CAP1/ASP56, a human homologue of yeast CAP/SRV2 (cyclase-associated protein). CAP is a bifunctional protein with an N-terminal domain that binds to Ras-responsive adenylyl cyclase and a C-terminal domain that inhibits actin polymerization. Surprisingly, we found that the N-terminal domain of CAP1, but not the C-terminal domain, is responsible for the interaction with the actin-cofilin complex. The N-terminal domain of CAP1 was also found to accelerate the depolymerization of F-actin at the pointed end, which was further enhanced in the presence of cofilin and/or the C-terminal domain of CAP1. Moreover, CAP1 and its C-terminal domain were observed to facilitate filament elongation at the barbed end and to stimulate ADP-ATP exchange on G-actin, a process that regenerates easily polymerizable G-actin. Although cofilin inhibited the nucleotide exchange on G-actin even in the presence of the C-terminal domain of CAP1, its N-terminal domain relieved this inhibition. Thus, CAP1 plays a key role in speeding up the turnover of actin filaments by effectively recycling cofilin and actin and through its effect on both ends of actin filament. >192.NF00137212 PMID:11854171 TI - KRIT1 association with the integrin-binding protein ICAP-1: a new direction in the elucidation of cerebral cavernous malformations (CCM1) pathogenesis. AB - Mutations in KRIT1, a protein initially identified based on a yeast two-hybrid interaction with the RAS-family GTPase RAP1A, are responsible for the development of the inherited vascular disorder cerebral cavernous malformations (CCM1). As the function of the KRIT1 protein and its role in CCM pathogenesis remain unknown, we performed yeast two-hybrid screens to identify additional protein binding partners. A fragment containing the N-terminal 272 amino acid residues of KRIT1, a region lacking similarity to any known protein upon database searches, was used as bait. From parallel screens of human fetal brain and HeLa cDNA libraries, we obtained multiple independent isolates of human integrin cytoplasmic domain-associated protein-1 (ICAP-1) as interacting clones. The interaction of KRIT1 and ICAP-1 was confirmed by GST-KRIT1 trapping of endogenous ICAP-1 from 293T cells. The alpha isoform of ICAP-1 is a 200 amino acid serine/threonine-rich phosphoprotein which binds the cytoplasmic tail of beta1 integrins. We show that mutagenesis of the N-terminal KRIT1 NPXY amino acid sequence, a motif critical for ICAP-1 binding to beta1 integrin molecules, completely abrogates the KRIT1/ICAP-1 interaction. The interaction between ICAP-1 and KRIT1, and the presence of a FERM domain in the latter, suggest that KRIT1 might be involved in the bidirectional signaling between integrin molecules and the cytoskeleton. Furthermore, these data suggest that KRIT1 might affect cell adhesion processes via integrin signaling in CCM1 pathogenesis. >193.NF00115830 PMID:12417738 TI - In vivo interference with Skp1 function leads to genetic instability and neoplastic transformation. AB - Skp1 is involved in a variety of crucial cellular functions, among which the best understood is the formation together with Cul1 of Skp1-cullin-F-box protein ubiquitin ligases. To investigate the role of Skp1, we generated transgenic (Tg) mice expressing a Cul1 deletion mutant (Cul1-N252) able to sequestrate and inactivate Skp1. In vivo interference with Skp1 function through expression of the Cul1-N252 mutant into the T-cell lineage results in lymphoid organ hypoplasia and reduced proliferation. Nonetheless, after a period of latency, Cul1-N252 Tg mice succumb to T-cell lymphomas with high penetrance (>80%). Both T-cell depletion and the neoplastic phenotype of Cul1-N252 Tg mice are largely rescued in Cul1-N252, Skp1 double-Tg mice, indicating that the effects of Cul1-N252 are due to a sequestration of the endogenous Skp1. Analysis of Cul1-N252 lymphomas demonstrates striking karyotype heterogeneity associated with c-myc amplification and c-Myc overexpression. We show that the in vitro expression of the Cul1-N252 mutant causes a pleiotrophic phenotype, which includes the formation of multinucleated cells, centrosome and mitotic spindle abnormalities, and impaired chromosome segregation. Our findings support a crucial role for Skp1 in proper chromosomal segregation, which is required for the maintenance of euploidy and suppression of transformation. >194.NF00082217 PMID:12086470 TI - Infertility of CD9-deficient mouse eggs is reversed by mouse CD9, human CD9, or mouse CD81; polyadenylated mRNA injection developed for molecular analysis of sperm-egg fusion. AB - CD9 is a membrane protein belonging to the tetraspanin family. Despite CD9's broad tissue distribution, the only abnormality observed in CD9-deficient mice was infertility of females, which was responsible for a defect in the sperm-egg fusion process. However, the function of CD9 in sperm-egg fusion is not clear at all because the technique to analyze the activity of molecules in sperm-egg fusion has not been established. We demonstrated that the exogenous mouse CD9, expressed by polyadenylated mRNA injection at the germinal-vesicle stage oocytes, was precisely localized to the egg plasma membrane, and the expression reversed the infertility of CD9(-/-) eggs. Then, two other tetraspanins, human CD9 and mouse CD81, overexpressed with this technique on CD9(-/-) eggs restored the fertilization rate up to approximately 90 and approximately 50% against that of wild type eggs, respectively. Moreover, in the presence of an anti-mouse CD9 mAb, which blocks sperm-egg fusion, expression of human CD9 or mouse CD81 on eggs also rescued the fusibility. These results suggested that human CD9 plays a crucial role in human fertilization, and mouse CD81 has the potential to compensate for CD9 function in sperm-egg fusion. In addition, the polyadenylated mRNA injection is effective for molecular analysis of sperm-egg fusion. >195.NF00126362 PMID:11904153 TI - Conformational change and destabilization of cataract gammaC-crystallin T5P mutant. AB - Human lens gammaC-crystallin and T5P mutant were cloned, and their biophysical properties and thermodynamic stability were studied. CRYGC (T5P) is one of the many gamma-crystallin mutant genes for autosomal dominant congenital cataracts. This mutation is associated with Coppock-like cataract, and has the phenotype of a dust-like opacity of the fetal lens nucleus. During cloning and overexpression, the majority of T5P mutant was found in the inclusion body. This property is unique among the many cataract gamma-crystallin mutant genes. It is thus worthwhile to study what factors contribute to this unique property of gammaC-crystallin. One possibility is changes in conformation and stability, which can be studied using spectroscopic measurements. In this study, conformational change was studied by circular dichroism and fluorescence measurements, and conformational stability was determined by thermal unfolding probed by Trp fluorescence and time-dependent light scattering. The T5P mutation obviously changes conformation and decreases conformational stability. >196.NF00119154 PMID:12121992 TI - SNAP-23 is a target for calpain cleavage in activated platelets. AB - The role of calpain in platelet function is generally associated with aggregation and clot retraction. In this report, data are presented to show that one component of the platelet secretory machinery, SNAP-23, is specifically cleaved by calpain in activated cells. Other proteins of the membrane fusion machinery, e.g. syntaxins 2 and 4 and alpha-SNAP, are not affected. In vitro studies, using permeabilized platelets, demonstrate that cleavage is time- and calcium-dependent. Analysis of SNAP-23 cleavage products suggests that the calpain cleavage site(s) is in the C-terminal third of the molecule potentially between the cysteine-rich acyl attachment sites and the C-terminal coiled-coil domain. The time course of cleavage is most consistent with late calpain-mediated events such as pp60(c-src) cleavage, but not early events such as protein-tyrosine phosphatase-1B activation. SNAP-23 cleavage is inhibited by calpeptin, calpastatin, calpain inhibitor IV, and E-64d, but not by caspase 3 inhibitor III or cathepsin inhibitor I. When tested for their effect on secretion, none of the calpain-specific inhibitors significantly affected release of soluble components from any of the three platelet granule storage pools. These results indicate that SNAP-23 cleavage occurs after granule release and therefore may play a role in affecting granule membrane exteriorization. This is consistent with the ultrastructural morphology of calpeptin-treated platelets after activation. >197.NF00126282 PMID:12105219 TI - Mass spectrometric analysis of GAP-43/neuromodulin reveals the presence of a variety of fatty acylated species. AB - GAP-43 (neuromodulin) is a protein kinase C substrate that is abundant in developing and regenerating neurons. Thioester-linked palmitoylation at two cysteines near the GAP-43 N terminus has been implicated in directing membrane binding. Here, we use mass spectrometry to examine the stoichiometry of palmitoylation and the molecular identity of the fatty acid(s) attached to GAP-43 in vivo. GAP-43 expressed in either PC12 or COS-1 cells was acetylated at the N-terminal methionine. Approximately 35% of the N-terminal GAP-43 peptides were also modified by palmitate and/or stearate on Cys residues. Interestingly, a variety of acylated species was detected, in which one of the Cys residues was acylated by either palmitate or stearate, or both Cys residues were acylated by palmitates or stearates or a combination of palmitate and stearate. Depalmitoylation of membrane-bound GAP-43 did not release the protein from the membrane, implying that additional forces function to maintain membrane binding. Indeed, mutation of four basic residues within the N-terminal domain of GAP-43 dramatically reduced membrane localization of GAP-43 without affecting palmitoylation. These data reveal the heterogeneous nature of S-acylation in vivo and illustrate the power of mass spectrometry for identification of key regulatory protein modifications. >198.NF00080518 PMID:12089359 TI - The mRNA structure has potent regulatory effects on type 2 iodothyronine deiodinase expression. AB - Type 2 deiodinase (D2) is a selenoenzyme catalyzing the activation of T(4) to T(3). D2 activity/mRNA ratios are often low, suggesting that there is significant posttranscriptional regulation. The D2 mRNA in higher vertebrates is more than 6 kb, containing long 5' and 3' untranslated regions (UTRs). The D2 5'UTRs are greater than 600 nucleotides and contain 3-5 short open reading frames. These full-length 5'UTRs reduce the D2 translation efficiency approximately 5-fold. The inhibition by human D2 5'UTR is localized to a region containing the first short open reading frame encoding a tripeptide-MKG. This inhibition was abolished by mutating the AUG start codon and weakened by modification of the essential purine of the Kozak consensus. Deletion of the 3.7-kb 3'UTR of the chicken D2 mRNA increased D2 activity approximately 3.8-fold due to an increase in D2 mRNA half-life. In addition, alternatively spliced D2 mRNA transcripts similar in size to the major 6- to 7-kb D2 mRNAs but not encoding an active enzyme are present in both human and chicken tissues. Our results indicate that a number of factors reduce the D2 protein levels. These mechanisms, together with the short half-life of the protein, ensure limited expression of this key regulator of T(4) activation. >199.NF00113973 PMID:11577372 TI - Alpha-B crystallin gene (CRYAB) mutation causes dominant congenital posterior polar cataract in humans. AB - Congenital cataracts are an important cause of bilateral visual impairment in infants. In a four-generation family of English descent, we mapped dominant congenital posterior polar cataract to chromosome 11q22-q22.3. The maximum LOD score, 3.92 at recombination fraction 0, was obtained for marker D11S898, near the gene that encodes crystallin alpha-B protein (CRYAB). By sequencing the coding regions of CRYAB, we found in exon 3 a deletion mutation, 450delA, that is associated with cataract in this family. The mutation resulted in a frameshift in codon 150 and produced an aberrant protein consisting of 184 residues. This is the first report of a mutation, in this gene, resulting in isolated congenital cataract. >200.NF00122964 PMID:10898795 TI - BCoR, a novel corepressor involved in BCL-6 repression. AB - BCL-6 encodes a POZ/zinc finger transcriptional repressor that is required for germinal center formation and may influence apoptosis. Aberrant expression of BCL-6 due to chromosomal translocations is implicated in certain subtypes of non-Hodgkin's lymphoma. The POZ domains of BCL-6 and several other POZ proteins interact with corepressors N-CoR and SMRT. Here we identify and characterize a novel corepressor BCoR (BCL-6 interacting corepressor), which is expressed ubiquitously in human tissues. BCoR can function as a corepressor when tethered to DNA and, when overexpressed, can potentiate BCL-6 repression. Specific class I and II histone deacetylases (HDACs) interact in vivo with BCoR, suggesting that BCoR may functionally link these two classes of HDACs. Strikingly, BCoR interacts selectively with the POZ domain of BCL-6 but not with eight other POZ proteins tested, including PLZF. Additionally, interactions between the BCL-6 POZ domain and SMRT, N-CoR, and BCoR are mutually exclusive. The specificity of the BCL-6/BCoR interaction suggests that BCoR may have a role in BCL-6-associated lymphomas. >201.NF00564623 PMID:11856366 TI - Ornithine decarboxylase-antizyme is rapidly degraded through a mechanism that requires functional ubiquitin-dependent proteolytic activity. AB - Antizyme is a polyamine-induced cellular protein that binds to ornithine decarboxylase (ODC), and targets it to rapid ubiquitin-independent degradation by the 26S proteasome. However, the metabolic fate of antizyme is not clear. We have tested the stability of antizyme in mammalian cells. In contrast with previous studies demonstrating stability in vitro in a reticulocyte lysate-based degradation system, in cells antizyme is rapidly degraded and this degradation is inhibited by specific proteasome inhibitors. While the degradation of ODC is stimulated by the presence of cotransfected antizyme, degradation of antizyme seems to be independent of ODC, suggesting that antizyme degradation does not occur while presenting ODC to the 26S proteasome. Interestingly, both species of antizyme, which represent initiation at two in-frame initiation codons, are rapidly degraded. The degradation of both antizyme proteins is inhibited in ts20 cells containing a thermosensitive ubiquitin-activating enzyme, E1. Therefore we conclude that in contrast with ubiquitin-independent degradation of ODC, degradation of antizyme requires a functional ubiquitin system. >202.NF00927018 PMID:12133833 TI - Association of the adaptor TANK with the I kappa B kinase (IKK) regulator NEMO connects IKK complexes with IKK epsilon and TBK1 kinases. AB - Canonical activation of NF-kappa B is mediated via phosphorylation of the inhibitory I kappa B proteins by the I kappa B kinase complex (IKK). IKK is composed of a heterodimer of the catalytic IKK alpha and IKK beta subunits and a presumed regulatory protein termed NEMO (NF-kappa B essential modulator) or IKK gamma. NEMO/IKK gamma is indispensable for activation of the IKKs in response to many signals, but its mechanism of action remains unclear. Here we identify TANK (TRAF family member-associated NF-kappa B activator) as a NEMO/IKK gamma-interacting protein via yeast two-hybrid analyses. This interaction is confirmed in mammalian cells, and the domains required are mapped. TANK was previously shown to assist NF-kappa B activation in a complex with TANK-binding kinase 1 (TBK1) or IKK epsilon, two kinases distantly related to IKK alpha/beta, but the underlying mechanisms remained unknown. Here we show that TBK1 and IKK epsilon synergize with TANK to promote interaction with the IKKs. The TANK binding domain within NEMO/IKK gamma is required for proper functioning of this IKK subunit. These results indicate that TANK can synergize with IKK epsilon or TBK1 to link them to IKK complexes, where the two kinases may modulate aspects of NF-kappa B activation. >203.NF01196429 PMID:12504121 TI - Genomic structure, expression, and transcriptional regulation of human Gal beta 1,3 GalNAc alpha 2,3-sialyltransferase gene. AB - In this report, we describe transcriptional regulation of the human Gal beta 1,3 GalNAc alpha 2,3-sialyltransferase II (hST3Gal II) gene. The results of 5'-RACE showed that the forms of two mRNAs differed only in the 5'-untranslated region (Types 1 and 2). According to analysis of the genomic structure, the transcriptional regulation of Type 1 and Type 2 mRNA isoforms depended on the p1 and p2 promoters, respectively. Both the mRNA isoforms were detected in various human tissues except colon, skeletal muscle, and peripheral blood leukocytes by RT-PCR analysis. In colon tissue, the Type 2 mRNA was detected, however, Type 1 mRNA was not detected. To elucidate the molecular basis of hST3Gal II gene expression, we isolated and characterized the function of the genomic region of hST3Gal II containing the p1 and p2 promoters. The activity of p2 promoter is much higher than that of the p1 promoter in the colon adenocarcinoma cell line, COLO205. These results suggest that the hST3Gal II gene is expressed specifically by alternative promoter utilization and is regulated in a tissue-restricted fashion at the level of transcription. >204.NF00099608 PMID:12011764 TI - Is the CD14 receptor gene a marker for smoking dependence? AB - BACKGROUND: The CD14 receptor is a myeloid cell-specific receptor, which plays a role in host defense and cell stimulation. CD14 positive cells have been detected in lung, and also in brain, and the CD14 receptor is thought to play a role in asthma and allergy reactions. C-159-->T polymorphism in the promoter of the CD14 gene has been associated with myocardial infarction, but not in all studies. Our goal was to establish whether this polymorphism is associated with some of the risk factors of MI. MATERIAL/METHODS: With PCR and subsequent restriction analysis we evaluated C-159-->T polymorphism in the CD14 gene in 135 representative selected male Caucasians. RESULTS: We detected a significantly higher frequency of T/T homozygotes (p<0.025) in subjects who had never smoked (15 of 60, 25.0%) as compared to smokers and past smokers (6 of 75, 8.0%). CONCLUSIONS: The C-159-->T polymorphism in the CD14 gene could be a genetic marker associated with smoking dependence, but confirmation in a large population study is necessary. >205.NF00078954 PMID:11729309 TI - Delineation of mRNA export pathways by the use of cell-permeable peptides. AB - The transport of messenger RNAs (mRNAs) from the nucleus to the cytoplasm involves adapter proteins that bind the mRNA as well as receptor proteins that interact with the nuclear pore complex. We demonstrate the utility of cell-permeable peptides designed to interfere with interactions between potential adapter and receptor proteins to define the pathways accessed by particular mRNAs. We show that HuR, a protein implicated in the stabilization of short-lived mRNAs containing AU-rich elements (AREs), serves as an adapter for c-fos mRNA export through two pathways. One involves the HuR shuttling domain, HNS, which exhibits a heat shock-sensitive interaction with transportin 2 (Trn2); the other involves two protein ligands of HuR-pp32 and APRIL-which contain leucine-rich nuclear export signals (NES) recognized by the export receptor CRM1. Heterokaryon and in situ hybridization experiments reveal that the peptides selectively block the nucleocytoplasmic shuttling of their respective adapter proteins without perturbing the overall cellular distribution of polyadenylated mRNAs. >206.NF00086973 PMID:12225856 TI - Unique assignment of inter-subunit association in GABA(A) alpha 1 beta 3 gamma 2 receptors determined by molecular modeling. AB - Recent publications defined requirements for inter-subunit contacts in a benzodiazepine-sensitive GABA(A) receptor (GABA(A)R alpha 1 beta 3 gamma 2). There is strong evidence that the heteropentameric receptor contains two alpha 1, two beta 3, and one gamma 2 subunit. However, the available data do not distinguish two possibilities: When viewed clockwise from an extracellular viewpoint the subunits could be arranged in either gamma 2 beta 3 alpha 1 beta 3 alpha 1 or gamma 2 alpha 1 beta 3 alpha 1 beta 3 configurations. Here we use molecular modeling to thread the relevant GABA(A)R subunit sequences onto a template of homopentameric subunits in the crystal structure of the acetylcholine binding protein (AChBP). The GABA(A) sequences are known to have 15-18% identity with the acetylcholine binding protein and nearly all residues that are conserved within the nAChR family are present in AChBP. The correctly aligned GABA(A) sequences were threaded onto the AChBP template in the gamma 2 beta 3 alpha 1 beta 3 alpha 1 or gamma 2 alpha 1 beta 3 alpha 1 beta 3 arrangements. Only the gamma 2 alpha 1 beta 3 alpha 1 beta 3 arrangement satisfied three known criteria: (1) alpha 1 His(102) binds at the gamma 2 subunit interface in proximity to gamma 2 residues Thr(142), Phe(77), and Met(130); (2) alpha 1 residues 80-100 bind near gamma 2 residues 91-104; and (3) alpha 1 residues 58-67 bind near the beta 3 subunit interface. In addition to predicting the most likely inter-subunit arrangement, the model predicts which residues form the GABA and benzodiazepine binding sites. >207.NF00089567 PMID:12107172 TI - Nuclear localization of CDC25B1 and serine 146 integrity are required for induction of mitosis. AB - CDC25B phosphatases are essential regulators that control cyclin-dependent kinases activities at the entry into mitosis. In this study, we demonstrate that serine 146 is required for two crucial features of CDC25B1. It is essential for CDC25B1 to function as a mitotic inducer and to prevent CDC25B1 export from the nucleus. We also show that serine 146 is phosphorylated in vitro by CDK1-cyclin B. However, phosphorylation of CDC25B does not stimulate its phosphatase activity, and mutation of serine 146 had no effect on its catalytic activity. Serine 146 phosphorylation is proposed to be a key event in the regulation of the CDC25B function in the initiation of mammalian mitosis. >208.NF00083111 PMID:11781319 TI - The ankyrin-B C-terminal domain determines activity of ankyrin-B/G chimeras in rescue of abnormal inositol 1,4,5-trisphosphate and ryanodine receptor distribution in ankyrin-B (-/-) neonatal cardiomyocytes. AB - Ankyrins are a closely related family of membrane adaptor proteins that are believed to participate in targeting diverse membrane proteins to specialized domains in the plasma membrane and endoplasmic reticulum. This study addresses the question of how individual ankyrin isoforms achieve functional specificity when co-expressed in the same cell. Cardiomyocytes from ankyrin-B (-/-) mice display mis-localization of inositol 1,4,5-trisphosphate receptors and ryanodine receptors along with reduced contraction rates that can be rescued by expression of green fluorescent protein (GFP)-ankyrin-B but not GFP-ankyrin-G. We developed chimeric GFP expression constructs containing all combinations of the three major domains of ankyrin-B and ankyrin-G to determine which domain(s) of ankyrin-B are required for ankyrin-B-specific functions. The death/C-terminal domain of ankyrin-B determined activity of ankyrin-B/G chimeras in localization in a striated pattern in cardiomyocytes and in restoration of a normal striated distribution of both ryanodine and inositol 1,4,5-trisphosphate receptors as well as normal beat frequency of contracting cardiomyocytes. Further deletions within the death/C-terminal domain demonstrated that the C-terminal domain determines ankyrin-B activity, whereas deletion of the death domain had no effect. C-terminal domains are the most divergent between ankyrin isoforms and are candidates to encode the signal(s) that enable ankyrins to selectively target proteins to diverse cellular sites. >209.NF00074123 PMID:12154078 TI - Regulation of the CDK-related protein kinase PCTAIRE-1 and its possible role in neurite outgrowth in Neuro-2A cells. AB - PCTAIRE-1 is a CDK-related protein kinase found in terminally differentiated cells in brain and testis, and in many immortalised and transformed cell lines. Bacterially expressed PCTAIRE is completely inactive as a protein kinase, but is a very good substrate for protein kinase A (PKA), which phosphorylates a total of four sites in the N-terminus of PCTAIRE-1. Phosphorylation of one of these sites, Ser119, generates a 14-3-3 binding site, which is functional in vitro as well as in vivo. Mutation of another PKA site, Ser153, to an alanine residue generated an activated kinase in transfected mammalian cells. This activity was comparable to that of CDK5 activated by a bacterially expressed, truncated version of p35(nck), p21. Gel filtration analysis of a brain extract suggested that monomeric PCTAIRE-1 was the active species, implying that PCTAIRE-1 may not be a true CDK, in that it does not require a partner (cyclin-like) subunit for kinase activity. Finally, we found that various forms of PCTAIRE-1 transfected into neuroblastoma cell lines could either promote or inhibit neurite outgrowth, suggesting a potential role for the PCTAIRE-1 gene product in the control of neurite outgrowth. >210.NF00080523 PMID:11727264 TI - Expression of multidrug resistance 1 and glutathione-S-transferase-Pi protein in nasopharyngeal carcinoma. AB - Radiotherapy is the modality of choice for the treatment of nasopharyngeal carcinoma (NPC). However, systemic chemotherapy has recently been found to play an increasing role in the treatment of advanced or metastatic disease. The status of drug resistance gene expression that has crucial impact on chemotherapy has not been fully addressed for patients with NPC. In this study, we examined the expression of multidrug resistance 1 (MDR-1) and glutathione-S-transferase-Pi (GST-Pi) in primary, recurrent, and metastatic NPC using results of immunohistochemical examinations. The results were correlated with the expression of Epstein-Barr virus (EBV) latent protein, latent membrane protein 1 (LMP1), and clinicopathologic features, including stage, histopathologic types, and survival rates. MDR-1 protein expression was detected in 18 (12.6%) of 143 patients with primary NPC, 14 (32.6%) of 43 with recurrent NPC, and O (0%) of 20 with metastatic NPC, whereas 83 (58%) of 143 patients with primary NPC, 30 (69.8%) of 43 with recurrent NPC, and 13 (65%) of 20 with metastatic NPC expressed GST-Pi. EBV-LMP1 was expressed in 59 (41.3%) of 143 patients with primary NPC, 23 (53.5%) of 43 with recurrent NPC, and 9 (45%) of 20 with metastatic NPC. Simultaneous expression of MDR1 and GST-Pi was observed in 13 (72.2%) of 18 patients with primary NPC and 12 (85.7%) of 14 with recurrent NPC. The expression of LMP1 was detected in only 6 of the 13 patients with primary NPC and 6 of the 12 with recurrent NPC. We concluded that the expression of GST-Pi was more frequent in NPC tumor tissues than the expression of MDR-1. The expression of MDR-1 correlated with clinicopathologic features of primary NPC, including the histopathologic types and survival rates, but not with disease stage. The expression of GST-Pi did not correlate with clinicopathologic features. The expression of MDR-1 and GST-Pi did not correlate with expression of EBV-LMP1 for patients with NPC. >211.NF00081416 PMID:12606287 TI - Transcriptional activation of BMP-4 and regulation of mammalian organogenesis by GATA-4 and -6. AB - Transcription factors GATA-4, -5, and -6 constitute an evolutionary conserved subfamily of vertebrate zinc finger regulators highly expressed in the developing heart and gut. Genetic evidence suggests that each protein is essential for embryonic development, but their exact functions are not fully elucidated. Moreover, because all three proteins share similar transcriptional properties in vitro, and because transcripts for two or more GATA genes are present in similar tissues, the molecular basis underlying in vivo specificity of GATA factors remains undefined. Knowledge of the exact cell types expressing each protein and identification of downstream targets would greatly help define their function. We have used high-resolution immunohistochemistry to precisely determine the cellular distribution of the GATA-4, -5, and -6 proteins in murine embryogenesis. The results reveal novel sites of expression in mesodermal and ectodermal cells. In particular, GATA-4 and -6 expression was closely associated with yolk sac vasculogenesis and early endoderm-mesoderm signaling. Additionally, GATA-6 was strongly expressed in the embryonic ectoderm, neural tube, and neural crest-derived cells. This pattern of expression closely paralled that of BMP-4, and the BMP-4 gene was identified as a direct downstream target for GATA-4 and -6. These findings offer new insight into the function of GATA-4 and -6 during early stages of embryogenesis and reveal the existence of a positive cross-regulatory loop between BMP-4 and GATA-4. They also raise the possibility that part of the early defects in GATA-4 and/or GATA-6 null embryos may be due to impaired BMP-4 signaling. >212.NF00124913 PMID:11756564 TI - Isolation of the human testatin gene and analysis in patients with abnormal gonadal development. AB - We have previously isolated the testatin gene using a modified mRNA differential display method on RNA from developing male and female mouse gonads. This gene is specifically expressed during early testis development, immediately after the onset of the testis-determining gene SRY: The protein encoded by testatin has features that are characteristic for type 2 cystatins, a family of small inhibitors of cystein proteases such as the cathepsins. We have now isolated the human orthologue of this gene. We describe here the sequence, genomic structure, chromosomal location, and expression pattern of the human testatin gene. Like mouse testatin, human testatin is specifically expressed in the testis, suggesting that it has a function in reproduction. We have therefore also investigated whether the human testatin gene plays a role in disorders of gonadal development, by sequencing the gene in patients with gonadal dysgenesis, with true hermaphroditism, and in children with less well-defined intersex conditions. We found no sequence aberrations in these patients apart from an H109P polymorphism which was also found in fertile controls. This is the first genetic analysis of testatin in humans. >213.NF00081335 PMID:12064938 TI - Cryo-electron microscopy of trichocyte (hard alpha-keratin) intermediate filaments reveals a low-density core. AB - Trichocyte intermediate filaments (IF) are the principal components of epidermal appendages such as hair and nail. Based on studies by a variety of techniques, it has been inferred that trichocyte IF are structurally similar to other kinds of IF. However, some basic structural attributes have yet to be established: in particular, it has remained unclear whether IF are hollow. We have examined trichocyte IF isolated from rat vibrissae and human hair follicles by electron microscopy. Scanning transmission electron microscopy of freeze-dried specimens yielded mass-per-unit-length values of approximately 32 kDa/nm, with the human preparations also containing filaments at half this density, corresponding to two rather than four protofibrils. Radial density profiles calculated from cryo-electron micrographs of vitrified specimens preserved in a near-native state revealed a low-density region of approximately 3 nm diameter around the filament axis. A minor species of filament with the same internal structure was surface-decorated with material arranged with a helical pitch length of 9.3 nm. These filaments appear to represent IF coated with associated proteins-perhaps, "high-sulfur" proteins-readied for incorporation into the filament-matrix biocomposite of the mature hair. >214.NF00091136 PMID:12482935 TI - The promoter of IL-18 binding protein: activation by an IFN-gamma -induced complex of IFN regulatory factor 1 and CCAAT/enhancer binding protein beta. AB - The IL-18 binding protein (IL-18BP) is a circulating inhibitor of the proinflammatory cytokine IL-18. It is constitutively expressed in mononuclear cells, and elevated expression is induced by IFN-gamma. In this study, we characterized the IL-18BP promoter. We first showed that induction is at the transcriptional level and requires de novo protein synthesis. The IL-18BP promoter resides within 1.6 kb DNA upstream of the first exon and includes at least six regulatory elements. We identified in the basal promoter a gamma-activated sequence (GAS) proximal to the transcription start site (base 1), followed by an IFN regulatory factor 1 response element (IRF-E) and two CCAATenhancer binding protein beta (CEBPbeta) sites, all of which are essential for basal promoter activity. Furthermore, GAS and IRF-E were essential for IFN-gamma-induced transcription. Indeed, sera of IRF-1-deficient mice lacked basal and IFN-gamma-induced IL-18BP. We found that after induction of IRF-1 by IFN-gamma, it formed a complex with CEBPbeta, which bound to the IRF-E and GAS-containing proximal DNA. In contrast, the IFN-gamma-induced signal transducer and activator of transcription 1 dimer did not associate with this GAS. In addition, we identified a silencer element and a distal enhancer at bases -1081 to -1272, which was also physically associated with IRF-1. The IRF-1-CEBPbeta complex described here probably plays a fundamental role in regulating additional IFN-gamma-responsive genes. >215.NF00117705 PMID:12608515 TI - Autosomal dominant macular dystrophy in a large Canadian family. AB - BACKGROUND: We studied a large Canadian family (178 total family members) spanning seven generations with autosomal dominant macular dystrophy. We performed a study to identify the gene mutation responsible for the disease in the family. METHODS: Participating family members were evaluated clinically. Genetic linkage, genotyping, mutation screening and an extensive genealogic investigation were performed. RESULTS: The common clinical findings in affected family members included progressive early- to mid-onset visual loss and extensive areas of central chorioretinal atrophy. Two-point linkage analysis indicated linkage to chromosome 6p. Direct DNA sequencing showed a C/T transition in codon 172 of the retinal degeneration slow (RDS) gene creating an amino acid change to Arg172Trp. Haplotype analysis of affected family members using microsatellite markers distributed around the RDS gene locus revealed that the markers were not conserved when compared to members of British families with the Arg172Trp mutation. Genealogic studies indicated the family immigrated to Canada from Ireland in 1843. INTERPRETATION: A newly identified large family with autosomal dominant macular dystrophy is described. The phenotypic appearance of the fundus is similar to that of previously described patients with an Arg172Trp mutation in the RDS gene. Haplotype analysis of markers spanning the disease locus identified a new founder for this mutation. The identification of the disease-causing gene in this family allows for better genetic counselling for patients with this condition and provides a basis to distinguish clinically similar types of macular dystrophy based on the clinical phenotype. >216.NF00075018 PMID:12213191 TI - Intracellular signaling by growth hormone variant (GH-V). AB - Placental growth hormone, or growth hormone variant (GH-V), is a member of the growth hormone gene family. Its physiologic role during pregnancy remains undefined. Although extensive work has been done characterizing the signaling pathways activated by hGH, the signaling pathways which are activated by GH-V have not been characterized. We investigated cellular signaling by GH-V in model systems in which signaling by hGH has been well characterized. GH-V activates the STAT5b transcription factor in the IM-9 human lymphocyte and 3T3-F442A preadipocyte cell lines, and in primary porcine smooth muscle cells, which all homologously express GH receptors. In contrast, hCS does not activate signaling in these GH receptor-expressing cells. Like hGH, signaling by GH-V is inhibited by the GH antagonist (G120K). These results provide preliminary evidence that GH-V plays a major role in affecting target cells expressing the GH receptor, thus potentially exerting significant GH-like effects on maternal physiology during pregnancy. >217.NF00109445 PMID:11705854 TI - Hypoxia-regulated carbonic anhydrase-9 (CA9) relates to poor vascularization and resistance of squamous cell head and neck cancer to chemoradiotherapy. AB - PURPOSE: Carbonic anhydrases are proteins involved in the catalytic hydration of carbon dioxide to carbonic acid. Recent studies show that carbonic anhydrase 9 (CA9) is up-regulated by hypoxia and that its immunohistochemical tissue distribution follows the distribution of the radiosensitizer pimonidazole (C. C. Wykoff et al., Cancer Res. 60: 7075-7083, 2001). Therefore, CA9 expression may show hypoxia levels of clinical importance. EXPERIMENTAL DESIGN: We assessed the expression of CA9 and the microvessel density (MVD; CD31-positive) in 75 locally advanced squamous cell head and neck cancers treated with concurrent chemoradiotherapy with carboplatin. RESULTS: Strong membrane/cytoplasmic CA9 expression, noted in 20/75 (26.6%) tumors, mainly occurred in tumors with very poor vascularization (expression in 63% versus 14%; P < 0.0001), was located around areas of focal necrosis, and was related to poor complete response rate (40% versus 70%; P = 0.02). These observations suggested that CA9 might be a marker of clinically important hypoxia. Combining the CA9 staining and the tumor angiogenicity (MVD), we identified three groups of patients: (a) hypoxic tumors; (b) euoxic highly angiogenic tumors; and (c) euoxic non-highly angiogenic tumors. Groups (a) and (b) had a very poor local relapse-free survival (P < 0.0001). CONCLUSIONS: Stratification of patients undergoing radical radiotherapy using the CA9/MVD model may be useful for the individualization of therapeutic strategies combining antiangiogenesis and hypoxia targeting with radiotherapy. >218.NF00081257 PMID:12547389 TI - Specific stimulation of human apurinic/apyrimidinic endonuclease by heat shock protein 70. AB - We previously demonstrated the stimulation of human apurinic/apyrimidinic endonuclease 1 (HAP1) by heat shock protein 70 (HSP70). In this work, we further defined the functional interaction between these proteins. Digestion of HSP70 by trypsin released 48 and 43 kDa amino terminal fragments that retained the ability to stimulate HAP1. In agreement with this result, an HSP70 N-terminal deletion mutant protein containing amino acids 1-385 was comparable to the full-length protein in its ability to enhance HAP1 activity. HSP70 mutants containing carboxy terminal amino acids 386-640 stimulated HAP1 only slightly, as did unrelated proteins. These results implicate the amino terminal portion of HSP70 in stimulating the activity of HAP1. >219.NF00124837 PMID:11809883 TI - hMutSalpha forms an ATP-dependent complex with hMutLalpha and hMutLbeta on DNA. AB - The DNA binding properties of hMutSalpha and hMutLalpha and complex formation of hMutSalpha with hMutLalpha and hMutLbeta were investigated using binding experiments on magnetic bead-coupled DNA substrates with nuclear extracts as well as purified proteins. hMutSalpha binding to homoduplex DNA was disrupted by lower NaCl concentrations than hMutSalpha binding to a mismatch. ATP markedly reduced the salt resistance of hMutSalpha binding but hMutSalpha still retained affinity for heteroduplexes. hMutSalpha formed a complex with hMutLalpha and hMutLbeta on DNA in the presence of ATP. This complex only formed on 81mer and not 32mer DNA substrates. Complex formation was enhanced by a mismatch in the DNA substrate, and hMutLalpha and hMutLbeta were shown to enter the complex at different ATP concentrations. Purified hMutLalpha showed an intrinsic affinity for DNA, with a preference for single-stranded over double-stranded DNA. >220.NF00134639 PMID:12207887 TI - VCY2 protein interacts with the HECT domain of ubiquitin-protein ligase E3A. AB - VCY2 locates in the AZFc region on chromosome Yq and is frequently deleted in infertile men with severe oligozoospermia or azoospermia. VCY2 is a testis-specific protein with unknown function. This study was to identify the protein that interacts with VCY2. We used the full-length VCY2 as bait to screen the human testis cDNA library using yeast two-hybrid approach. We identified a number of positive-interacting clones that encode ubiquitin-protein ligase E3A (UBE3A). UBE3A contains a HECT domain that binds VCY2. The specificity of the interaction was confirmed by co-immunoprecipitation and yeast mating. Northern blot analyses revealed two UBE3A transcripts 1.4 and 2kb that were abundantly expressed in human testis. We also showed that both VCY2 and UBE3A mRNAs were expressed in ejaculated human spermatozoa, indicating that both genes localize in the germ cell compartment. These data suggest that UBE3A ubiquitination may be required for VCY2 function. >221.NF00932291 PMID:11727258 TI - MUC4 expression is increased in dysplastic cervical disorders. AB - The female uterine cervix has 2 characteristic populations of epithelial cells: the endocervix is composed by mucus-secreting cells that express several mucin genes, and the exocervix has a typical stratified squamous epithelium and does not express secreted mucins. Among human mucin genes, the MUC4 sequence has a transmembrane domain, and its molecular structure suggests that it has a protective role and also may be implicated in intracellular signalling. The aim of this study is to analyze whether changes in the expression of MUC4 can be detected associated with the squamous dysplastic transformation of exocervical epithelium. MUC4 expression has been analyzed by immunohistochemistry, Western blotting, and in situ hybridization. Using immunohistochemical techniques, MUC4 is found in normal endocervix (n = 11) and is absent or only focally detected in the normal stratified cervical epithelium (n = 18). In samples from squamous metaplasia (n = 9), MUC4 is variably expressed (10% to 50% positive cells), whereas MUC4 is strongly detected in dysplastic cervical epithelia. The greatest number of positive cells is found in samples with moderate and severe dysplasia in which MUC4 is detected in 100% of the analyzed samples (n = 16). These results have been confirmed by Western blotting and by detection of MUC4 transcripts using in situ hybridization. The present data suggest that MUC4 is activated during the process of squamous dysplastic transformation and may be used as a marker for this pathologic process. >222.NF00088920 PMID:11836615 TI - Expression of the genes of methyl-binding domain proteins in human gliomas. AB - DNA methylation is the most common epigenetic alteration in tumor genomes and might result in transcriptional repression of tumor suppressor genes. Moreover, recent results have demonstrated that both specific methylation patterns and functional components of the mismatch repair system are involved in the development of therapy resistance of tumor cells. Here we investigated the expression of the genes of methyl binding domain containing proteins (MBD) in human gliomas both in vivo and in vitro. We found expression of MBDs including MBD1, MBD2, MBD3 and MBD4/MED1 in all glioma cell lines and glioma biopsies. No differences existed in vitro with regard to individual MBDs and individual cell lines. In vivo, MBD1 and MBD2 were also expressed in all biopsies with only minor differences between individual tumors. MBD3 and MBD4/MED1, however, showed a correlation of expression with the grade of malignancy. Astrocytomas and anaplastic astrocytomas showed a weak expression compared with a high expression in glioblastoma multiforme. >223.NF00097597 PMID:11823470 TI - Caspase-2 can trigger cytochrome C release and apoptosis from the nucleus. AB - The cysteine proteases specific for aspartic residues, known as caspases, are localized in different subcellular compartments and play specific roles during the regulative and the executive phase of the cell death process. Here we investigated the subcellular localization of caspase-2 in healthy cells and during the execution of the apoptotic program. We have found that caspase-2 is a nuclear resident protein and that its import into the nucleus is regulated by two different nuclear localization signals. We have shown that in an early phase of apoptosis caspase-2 can trigger mitochondrial dysfunction from the nucleus without relocalizing into the cytoplasm. Release of cytochrome c occurs in the absence of overt alteration of the nuclear pores and changes of the nuclear/cytoplasmic barrier. Addition of leptomycin B, an inhibitor of nuclear export, did not interfere with the ability of caspase-2 to trigger cytochrome c release. Only during the late phase of the apoptotic process can caspase-2 relocalize in the cytoplasm, as consequence of an increase in the diffusion limits of the nuclear pores. Taken together these data indicate the existence of a nuclear/mitochondrial apoptotic pathway elicited by caspase-2. >224.NF00079777 PMID:11966773 TI - Expression, modulation and signalling of IL-17 receptor in fibroblast-like synoviocytes of patients with rheumatoid arthritis. AB - Interleukin-17 (IL-17) has been characterized as a proinflammatory cytokine produced by CD4+ CD45RO+ memory T cells. Overproduction of IL-17 was detected in the synovium of patients with rheumatoid arthritis (RA) compared to patients with osteoarthritis. In contrast to the restricted expression of IL-17, the IL-17 receptor (IL-17R/CDw217) is expressed ubiquitously. Using a real-time RT-PCR assay, we detected similar absolute levels of IL-17R mRNA expression in fibroblast-like synoviocytes (SFC) from patients with RA (mean 9 pg/microg total RNA; ranged from 0.1 pg to 96 pg IL-17R mRNA/microg total RNA) compared to synoviocytes of non-RA patients. Analysis of the IL-17R surface expression confirmed the results obtained for IL-17R mRNA expression. Exposure of SFC to IL-17 led to a mRNA induction of CXC chemokines IL-8, GRO-alpha and GRO-beta. An anti-IL-17 antibody blocked these effects of IL-17. The MAPK p38 appears necessary for the regulation of IL-8, GRO-alpha and GRO-beta expression as shown by inhibition with SB203580. The inhibitors genistein (tyrosine kinase inhibitor) and calphostin C (inhibitor of protein kinase C) reduced significantly the IL-17-stimulated mRNA expression of IL-8, GRO-alpha and GRO-beta in SFC, whereas PD98059 (inhibitor of MEK-1/2) was without effect. Pharmacological drugs used in therapy of RA, such as cyclosporin and methotrexate, induced a fourfold increase of IL-17R mRNA expression and augmented the IL-17-stimulated IL-8 expression. Our results support the hypothesis that IL-17/IL-17R may play a significant role in the pathogenesis of RA contributing to an unbalanced production of cytokines as well as participating in connective tissue remodelling. >225.NF00888526 PMID:12200131 TI - Hypoxia activates beta(1)-integrin via ERK 1/2 and p38 MAP kinase in human vascular smooth muscle cells. AB - Hypoxia plays an important role in vascular remodeling and directly affects vascular smooth muscle cell (VSMC) functions. VSMC adhesion participates in changes of vascular structure; however, little is known about VSMC adhesion under hypoxic conditions. It was the aim of the present study to investigate the effects of hypoxia on adhesion mechanisms in human VSMCs. Compared to normoxic cells, hypoxia (1% O(2), 24h) significantly increased adhesion of VSMCs to collagen I by 30.2% and fibronectin by 58.0%. This effect was completely inhibited in the presence of the pharmacological ERK 1/2 mitogen-activated protein kinase (MAPK) pathway inhibitor PD98059 (30 microM) or the p38 MAPK inhibitor SB203580 (1 microM). Basal adhesion of normoxic cells was not affected by pretreatment with PD98059 and SB203580. Hypoxia induced a time-dependent activation of ERK 1/2 and p38 MAPK activation in human VSMCs, which were completely abolished by PD98059 or SB203580, respectively. Since adhesion of VSMCs to fibronectin and collagen I involves beta(1)-integrin receptors, we used a blocking antibody against beta(1)-integrin (P5D2) to examine potential effects of hypoxia on beta(1)-integrins. P5D2 significantly reduced VSMC adhesion to fibronectin and collagen I in normoxia and hypoxia in a comparable manner; however, beta(1)-integrin protein or mRNA levels were not affected by hypoxia. As evidenced by flow cytometry, hypoxia induced a activation of beta(1)-integrins by exposing an conformationally sensitive epitope on the beta(1)-subunit. These results demonstrate that hypoxia enhances adhesion of VSMC on extracellular matrix proteins by activating beta(1)-integrin. >226.NF00111014 PMID:11988089 TI - Crystal structure of human dehydroepiandrosterone sulphotransferase in complex with substrate. AB - Dehydroepiandrosterone sulphotransferase (DHEA-ST) is an enzyme that converts dehydroepiandrosterone (DHEA), and some other steroids, into their sulphonated forms. The enzyme catalyses the sulphonation of DHEA on the 3alpha-oxygen, with 3'-phosphoadenosine-5'-phosphosulphate contributing the sulphate. The structure of human DHEA-ST in complex with its preferred substrate DHEA has been solved here to 1.99 A using molecular replacement with oestradiol sulphotransferase (37% sequence identity) as a model. Two alternative substrate-binding orientations have been identified. The primary, catalytic, orientation has the DHEA 3alpha-oxygen and the highly conserved catalytic histidine in nearly identical positions as are seen for the related oestradiol sulphotransferase. The substrate, however, shows rotations of up to 30 degrees, and there is a corresponding rearrangement of the protein loops contributing to the active site. This may also reflect the low identity between the two enzymes. The second orientation penetrates further into the active site and can form a potential hydrogen bond with the desulphonated cofactor 3',5'-phosphoadenosine (PAP). This second site contains more van der Waal interactions with hydrophobic residues than the catalytic site and may also reflect the substrate-inhibition site. The PAP position was obtained from the previously solved structure of DHEA-ST co-crystallized with PAP. This latter structure, due to the arrangement of loops within the active site and monomer interactions, cannot bind substrate. The results presented here describe details of substrate binding to DHEA-ST and the potential relationship to substrate inhibition. >227.NF00110043 PMID:12411321 TI - Furin gene (fur) regulation in differentiating human megakaryoblastic Dami cells: involvement of the proximal GATA recognition motif in the P1 promoter and impact on the maturation of furin substrates. AB - The convertase furin is involved in the maturation of key growth/aggregation mediators synthesized by the platelet producers, megakaryocytes, but the regulation of furin in these cells remains unknown. Computer-assisted search of the furin promoter sequence revealed multiple potential binding motifs for GATA-1, suggesting that furin is expressed and regulated in these cells. Using megakaryoblastic Dami cells, we observed that fur mRNA expression increased gradually on phorbol 12-myristate 13-acetate-induced differentiation, reaching maximum levels (8.3-fold increase) at 10 days. Transient transfections with P1, P1A, or P1B fur-LUC-promoter constructs revealed that in Dami cells, the P1 promoter is the strongest and the most sensitive to forced expression of GATA-1. Coexpression of GATA-1 and its comodulator, Friend of GATA-1 (FOG-1), resulted in a cooperative increase in P1 activity. Deletion analysis indicated that important GATA-1-regulated sequences are located in the most proximal region of the P1 promoter. Further analysis revealed 2 potential GATA-binding motifs at positions -66 and +62. Point mutation of each of the 2 motifs indicated that the intactness of the first GATA site is required for full basal and GATA-1-stimulated promoter activity. Finally, the inhibition of furin activity through gene transfer of the inhibitor alpha1-AT-PDX led to a block in maturation of the furin substrates transforming growth factor-beta1 and platelet-derived growth factor. Taken together, these results indicate that the most proximal GATA element in the P1 promoter is needed for fur gene expression in megakaryoblastic cells. They also suggest that proper regulation of the fur gene in megakaryocytes has an impact on the activation of furin substrates involved in megakaryocyte maturation and platelet functions. >228.NF00011064 PMID:12101100 TI - Critical evaluation of the role of the Toll-like receptor 18-Wheeler in the host defense of Drosophila. AB - Essential aspects of innate immune responses to microbial infections appear to be conserved between insects and mammals. In particular, in both groups, transmembrane receptors of the Toll superfamily play a crucial role in activating immune defenses. The Drosophila Toll family member 18-Wheeler had been proposed to sense Gram-negative infection and direct selective expression of peptides active against Gram-negative bacteria. Here we re-examine the role of 18-Wheeler and show that in adults it is dispensable for immune responses. In larvae, 18wheeler is required for normal fat body development, and in mutant larvae induction of all antimicrobial peptide genes, and not only of those directed against Gram-negative bacteria, is compromised. 18-Wheeler does not qualify as a pattern recognition receptor of Gram-negative bacteria. >229.NF00100248 PMID:12138090 TI - QM, a putative tumor suppressor, regulates proto-oncogene c-yes. AB - The QM gene encodes a 24.5 kDa ribosomal protein L10 known to be highly homologous to a Jun-binding protein (Jif-1), which inhibits the formation of Jun-Jun dimers. Here we have carried out screening with the c-Yes protein and found that a QM homologous protein showed interactions with c-Yes and other Src family members. We have found that two different regions of QM protein were associated with the SH3 domain of c-Yes. The QM protein does not contain canonical SH3 binding motifs or previously reported amino acid fragments showing interaction with SH3 domains. Several c-Yes kinase activity assays indicated that the QM protein reduced c-Yes kinase activity by 70% and that this suppression is related not only to the two SH3 binding regions but also to the C-terminal region of QM. Moreover, our autophosphorylation assays clarified that this regulation resulted from the inhibition of c-Yes autophosphorylation. Immunofluorescence studies showed that the QM proteins and c-Yes are able to interact in various tumor cell lines in vivo. The increases of the c-Yes protein and mRNA levels were detected when the QM was transfected. These results suggest that the QM protein might be a regulator for various signal transduction pathways involving SH3 domain-containing membrane proteins. >230.NF00081421 PMID:11920156 TI - Mutation screening and imprinting analysis of four candidate genes for autism in the 7q32 region. AB - Genetic studies indicate that chromosome 7q is likely to contain an autism susceptibility locus (AUTS1). We have followed a positional candidate gene approach to identify the relevant gene and report the analysis of four adjacent genes localised to a 800 kb region in 7q32 that contains an imprinted domain: PEG1/MEST, COPG2, CPA1 and CPA5-a previously uncharacterised member of the carboxypeptidase gene family. Screening these genes for DNA changes and association analysis using intragenic single nucleotide polymorphisms (SNPs) provided no evidence for an etiological role in IMGSAC families. We also searched for imprinting mutations potentially implicated in autism: analysis of both DNA methylation and replication timing indicated a normal imprinting regulation of the PEG1/COPG2 domain in blood lymphocytes of all patients tested. The analysis of these four genes strongly suggests that they do not play a major role in autism aetiology, and delineates our strategy to screen additional candidate genes in the AUTS1 locus. >231.NF00134722 PMID:11784720 TI - Calpain-mediated cleavage of the cyclin-dependent kinase-5 activator p39 to p29. AB - The activity of cyclin-dependent kinase-5 (Cdk5) is tightly regulated by binding of its neuronal activators p35 and p39. Upon neurotoxic insults, p35 is cleaved to p25 by the Ca(2+)-dependent protease calpain. p25 is accumulated in ischemic brains and in brains of patients with Alzheimer's disease. p25 deregulates Cdk5 activity by causing prolonged activation and mislocalization of Cdk5. It is unknown whether p39, which is expressed throughout the adult rat brain, is cleaved by calpain, and whether this contributes to deregulation of Cdk5. Here, we show that calpain cleaved p39 in vitro, resulting in generation of a C-terminal p29 fragment. In vivo, p29 was generated in ischemic brain concomitant with increased calpain activity. In fresh brain lysates, generation of p29 was Ca(2+)-dependent, and calpain inhibitors abolished p29 production. The Ca(2+) ionophore ionomycin and the excitotoxin glutamate induced production of p29 in cultures of cortical neurons in a calpain-dependent manner. Like p25, p29 was more stable than p39 and caused redistribution of Cdk5 in cortical neurons. Our data suggest that neurotoxic insults lead to calpain-mediated conversion of p39 to p29, which might contribute to deregulation of Cdk5. >232.NF00106860 PMID:12016153 TI - Polymorphism at GSTM1, GSTM3 and GSTT1 gene loci and susceptibility to oral cancer in an Indian population. AB - This study evaluates the influence of genetic polymorphism at GSTM1, GSTM3 and GSTT1 gene loci on oral cancer risk among Indians habituated to the use of, smokeless tobacco, bidi or cigarette. DNA extracted from white blood cells of 297 cancer patients and 450 healthy controls by the proteinase K phenol-chloroform extraction procedure were analyzed by the polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) analyses. Lifetime tobacco exposure was evaluated as a risk factor in relation to the polymorphism at the GST gene loci using logistic regression analysis. There was no significant difference in the distribution of the GSTM3 and GSTT1 genotypes between oral cancer patients and controls. In contrast, a significant 3-fold increase in risk was seen for patients with the GSTM1 null genotype (age adjusted OR = 3.2, 95% CI 2.4-4.3). The impact of the GSTM1 null genotype on oral cancer risk was also analyzed in separate groups of individuals with different tobacco habits. The odds ratio associated with the GSTM1 null genotype was 3.7 (95% CI 2.0-7.1) in tobacco chewers, 3.7 (5% CI 1.3-7.9) in bidi smokers and 5.7 (95% CI 2.0-16.3) in cigarette smokers. Furthermore, increased lifetime exposure to chewing tobacco appeared to be associated with a 2-fold increase in oral cancer risk in GSTM1 null individuals. The results suggest that the GSTM1 null genotype is a risk factor for development of oral cancer among Indian tobacco habitues. >233.NF00125815 PMID:11960377 TI - Overexpression of B-type cyclins alters chromosomal segregation. AB - To identify genes which overexpression results into chromosomal instability (CIN), we developed a biological approach based on a yeast indicator strain in which CIN can be detected by a sectoring phenotype. Screening in this strain of a yeast genomic library cloned into a high copy vector led us to identify, among the clones generating 100% of sectoring colonies, Clb5, one of the six B-type cyclins present in yeast. Overexpression of cyclin B2 and cyclin B1, the two human homologs of Clb5, in the CIN indicator strain resulted also into a sectoring phenotype and induced, like overexpression of Clb5, an abnormal sensitivity to benomyl, indicating that overexpression of B-type cyclins alters the spindle checkpoint. In a series of 38 primary colorectal cancers, we detected in five tumors (13%) an accumulation of cyclin B1, which was neither related to mRNA overexpression nor to mutation within the coding region, and in five other tumors (13%) a 2-10-fold increase of cyclin B2 mRNA which was not related to gene amplification. These results suggest that overexpression of cyclins B, resulting from different mechanisms, could contribute, through an alteration of the spindle checkpoint, to the chromosomal instability observed in cancer. >234.NF00123871 PMID:11846980 TI - Structure and function of IRF-7. AB - Interferon (IFN) regulatory factors (IRF) are a family of transcription factors with multiple functions. IRF-7 was initially cloned within the biologic context of Epstein-Barr virus (EBV) latency and discovered to have an intimate relation with the EBV primary oncogenic protein, latent membrane protein-1 (LMP-1). EBV regulates and uses IRF-7 as a secondary mediator for several target genes involved in latency and immune regulation. Other than its functions in EBV latency, IRF-7 has been identified as one of the major players in virally induced IFN production that is central to innate immunity. Thus, IRF-7 plays important roles in a variety of biologic systems. >235.NF00080537 PMID:12054577 TI - Expression of the Musashi1 gene encoding the RNA-binding protein in human hepatoma cell lines. AB - Musashi1, a neural RNA-binding protein, plays an important role in regulating cell differentiation in precursor cells. Recently, expression of Musashi1 has been detected in human tumor tissues such as gliomas and melanomas, suggesting its involvement in oncogenic development. To determine any association between Musashi1 and the development of liver cancer, we investigated its gene expression in seven human hepatoma cell lines: HuH6, HuH7, Hep3B, SK-Hep1, HepG2, HLE, and HLF. Musashi1 mRNA expression was analyzed using the reverse-transcription polymerase chain reaction (PCR), and the PCR products were sequenced using a subcloning procedure. Musashi1 protein expression was analyzed in HuH7 and HepG2 cells by Western blot and immunofluorescence staining. Musashi1 mRNA was detected in the HuH6, HuH7, and Hep3B hepatoma cell lines, but not in the others. Sequencing of the PCR-amplified Musashi1 cDNA in these three cell lines showed the expected sequence of the human Musashi1 gene. Musashi1 protein expression was confirmed in HuH7 cells, which were positive for Musashi1 mRNA expression, but not in HepG2 cells. These results suggest that Musashi1 expression may be an important factor in the development of several types of carcinoma such as human hepatoma, and may be a useful molecular marker for tumor detection. >236.NF00864016 PMID:12151362 TI - Mutations in the Drosophila glycoprotein hormone receptor, rickets, eliminate neuropeptide-induced tanning and selectively block a stereotyped behavioral program. AB - Adult insects achieve their final form shortly after adult eclosion by the combined effects of specialized behaviors that generate increased blood pressure, which causes cuticular expansion, and hormones, which plasticize and then tan the cuticle. We examined the molecular mechanisms contributing to these processes in Drosophila by analyzing mutants for the rickets gene. These flies fail to initiate the behavioral and tanning processes that normally follow ecdysis. Sequencing of rickets mutants and STS mapping of deficiencies confirmed that rickets encodes the glycoprotein hormone receptor DLGR2. Although rickets mutants produce and release the insect-tanning hormone bursicon, they do not melanize when injected with extracts containing bursicon. In contrast, mutants do melanize in response to injection of an analog of cyclic AMP, the second messenger for bursicon. Hence, rickets appears to encode a component of the bursicon response pathway, probably the bursicon receptor itself. Mutants also have a behavioral deficit in that they fail to initiate the behavioral program for wing expansion. A set of decapitation experiments utilizing rickets mutants and flies that lack cells containing the neuropeptide eclosion hormone, reveals a multicomponent control to the activation of this behavioral program. >237.NF00125576 PMID:11836498 TI - Hermansky-Pudlak syndrome is caused by mutations in HPS4, the human homolog of the mouse light-ear gene. AB - Hermansky-Pudlak syndrome (HPS) is a disorder of organelle biogenesis in which oculocutaneous albinism, bleeding and pulmonary fibrosis result from defects of melanosomes, platelet dense granules and lysosomes. HPS is common in Puerto Rico, where it is caused by mutations in the genes HPS1 and, less often, HPS3 (ref. 8). In contrast, only half of non-Puerto Rican individuals with HPS have mutations in HPS1 (ref. 9), and very few in HPS3 (ref. 10). In the mouse, more than 15 loci manifest mutant phenotypes similar to human HPS, including pale ear (ep), the mouse homolog of HPS1 (refs 13,14). Mouse ep has a phenotype identical to another mutant, light ear (le), which suggests that the human homolog of le is a possible human HPS locus. We have identified and found mutations of the human le homolog, HPS4, in a number of non-Puerto Rican individuals with HPS, establishing HPS4 as an important HPS locus in humans. In addition to their identical phenotypes, le and ep mutant mice have identical abnormalities of melanosomes, and in transfected melanoma cells the HPS4 and HPS1 proteins partially co-localize in vesicles of the cell body. In addition, the HPS1 protein is absent in tissues of le mutant mice. These results suggest that the HPS4 and HPS1 proteins may function in the same pathway of organelle biogenesis. >238.NF00025785 PMID:12208494 TI - Towards a new T-fold protein?: the coproporphyrinogen III oxidase sequence matches many structural features from urate oxidase. AB - Urate oxidase (UOX) and coproporphyrinogen III oxidase (CPO) are two unusual oxidases as they accomplish their catalytic act with no co-factor nor metal ion. They both require molecular oxygen, and lead to hydrogen peroxide in addition to the product. UOX is composed of two contiguous Tunneling-fold domains and CPO appears to be also divided into two structurally equivalent domains. Moreover, each of these putative domains can be coherently aligned on UOX domains. Although their sequences are very distant, we therefore suggest that functional CPO dimer is built around a tunnel, with the substrate sitting above it, on the N- and C-terminal side. This overall model is supported by mutation data and is coherent with the chemical events expected for substrate processing by CPO. >239.NF00828217 PMID:11928817 TI - Distinct expression pattern of two related human proteins containing multiple types of protease-inhibitory modules. AB - We have recently identified a gene (the WFIKKN gene) on human chromosome 16 (16p13.3) that encodes a secreted protein containing WAP-type, Follistatin/ Kazal type, Kunitz-type and NTR-type protease-inhibitory modules and an Immunoglobulin domain [Trexler et al., Proc. Natl. Acad. Sci. USA 98 (2001), 3705 - 3709]. In the present work we show that a gene on chromosome 17 encodes a WFIKKN-related protein (WFIKKNRP) that has the same domain organization as the WFIKKN protein. The exon-intron structure of the two genes is also similar as both genes have a single phase 0 intron that splits their WAP domains in equivalent positions. In view of the presence of several protease inhibitory modules in these proteins it seems likely that they serve to control the action of multiple types of proteases. The tissue expression pattern of the two proteins, however, is markedly different suggesting that they have distinct biological roles. Whereas the WFIKKN gene is expressed primarily in adult pancreas, liver and thymus but not in brain and ovary, significant expression of the WFIKKNRP gene is observed in ovary, testis and brain, but not in liver. Pronounced differences could also be seen in the case of fetal tissues: expression of the WFIKKN gene was highest in the lung, skeletal muscle and liver, whereas the WFIKKNRP gene was expressed primarily in brain, skeletal muscle, thymus and kidney. >240.NF00141779 PMID:11972883 TI - Identification of a novel HLA-DPB1 allele, DPB1*02014, by sequence-based typing. AB - In this report we describe the identification of a novel DPB1 allele, DPB1*02014, found in an Italian Caucasian individual.The new allele was detected during routine HLA sequence-based typing (SBT) for an individual undergoing bone marrow transplantation. DPB1*02014 was identical to DPB1*02012 except for a single nucleotide substitution in codon 72 (GTG-->GTT). This nucleotide change represents a synonimous mutation, as both triplets code for a valine.This new allele has been submitted to GenBank and assigned the accession number AF326565. The WHO Nomenclature Committee has officially assigned the name DPB1*02014. >241.NF00132869 PMID:12421953 TI - Characterization of the interaction between L-ficolin/p35 and mannan-binding lectin-associated serine proteases-1 and -2. AB - Ficolins are oligomeric lectins comprising a collagen-like and a fibrinogen-like domain, with a binding specificity for N-acetylglucosamine. It has been reported recently that L-ficolin/P35 associates with mannan-binding lectin (MBL)-associated serine proteases (MASP-1 and -2) and MBL-associated protein 19 (MAp19) in serum and forms complexes able to activate complement. Using surface plasmon resonance spectroscopy we have shown that recombinant MASP-1 and -2, their N-terminal CUB1 (module originally found in complement proteins C1r/C1s, Uegf, and bone morphogenetic protein-1)-epidermal growth factor (EGF)-CUB2 and CUB1-EGF segments, and MAp19 bind to immobilized L-ficolin/P35 in the presence of Ca(2+) ions. Comparable K(d) values were obtained for the full-length proteases and their CUB1-EGF-CUB2 segments (9.2 and 10 nM for MASP-1 and 4.6 and 5.4 nM for MASP-2, respectively), whereas higher values were obtained for the CUB1-EGF segments (26.7, 15.6, and 14.3 nM for MASP-1, MASP-2, and MAp19). These values are in the same range as those determined for the interaction of these proteins with MBL. Binding was Ca(2+) dependent and was only partly sensitive to EDTA for MASP-1, MASP-2, and MASP-2 CUB1-EGF-CUB2. Half-maximal binding was obtained at comparable Ca(2+) concentrations for MASP-1 and MASP-2 (0.45 and 0.47 micro M, respectively), their CUB1-EGF-CUB2 segments (0.37 and 0.72 micro M), and their CUB1-EGF segments (0.31 and 0.79 micro M). These values are lower than those determined in the case of MBL, indicating a difference between MBL and L-ficolin/P35 with respect to the Ca(2+) dependence of their interaction with the MASPs. Preincubation of the MASPs with soluble MBL inhibited subsequent binding to immobilized L-ficolin/P35 and, conversely, suggesting that these lectins compete with each other for binding to the MASPs in vivo. >242.NF00911891 PMID:12095414 TI - Expression of the leucocyte common antigen-related (LAR) tyrosine phosphatase is regulated by cell density through functional E-cadherin complexes. AB - The leucocyte common antigen-related phosphatase (LAR) has been implicated in receptor tyrosine kinase signalling pathways while also displaying cell-density-dependency and localization to adherens junctions. Whereas physiological substrates for LAR have not been identified unequivocally, beta-catenin associates with LAR and is a substrate in vitro. With the implication that LAR may play a role in regulating E-cadherin-dependent cell-cell communication and contact inhibition, the relationship of LAR with E-cadherin was investigated. LAR expression increased with cell density in the human breast cancer cell line MCF-7 and in Ln 3 cells derived from the 13762NF rat mammary adenocarcinoma. LAR protein levels decreased rapidly when cells were replated at a low density after attaining high expression of LAR at high cell density. COS-7 cells displayed comparable density-dependent regulation of LAR expression when transiently expressing exogenous LAR under the control of a constitutively active promoter, indicating that the regulation of expression is not at the level of gene regulation. Disrupting homophilic E-cadherin complexes by chelating extracellular calcium caused a marked decrease in LAR protein levels. Similarly, blocking E-cadherin interactions with saturating amounts of E-cadherin antibody (HECD-1) also led to a rapid and pronounced loss of cellular LAR. In contrast, mimicking cell-surface E-cadherin engagement by plating cells at low density on to dishes coated with HECD-1 resulted in a 2-fold increase in LAR expression compared with controls. These results suggest that density-dependent regulation of LAR expression is mediated by functional E-cadherin and may play a role in density-dependent contact inhibition by regulating tyrosine phosphorylation in E-cadherin complexes. >243.NF00105899 PMID:11812157 TI - Determining the differences in actin binding by human ADF and cofilin. AB - The actin-depolymerizing factor (ADF)/cofilin family of proteins play an essential role in actin dynamics and cytoskeletal re-organization. Human tissues express two isoforms in the same cells, ADF and cofilin, and these two proteins are more than 70% identical in amino acid sequence. We show that ADF is a much more potent actin-depolymerizing agent than cofilin: the maximum level of depolymerization at pH 8 by ADF is about 20 microM compared to 5 microM for cofilin, but little depolymerization occurs at pH 6.5 with either protein. However, we find little difference between the two proteins in their binding to filaments, their severing activities or their activation of subunit release from the pointed ends of filaments. Likewise, they show no significant differences in their affinities for monomeric actin: both bind 15-fold more tightly to actin.ADP than to actin.ATP. Complexes between actin.ADP and ADF or cofilin associate with both barbed and pointed ends of filaments at similar rates (close to those of actin.ATP and much higher than those of actin.ADP). This explains why high concentrations of both proteins reverse the activation of subunit release at pointed ends. The major difference between the two proteins is that the nucleating activity of cofilin-actin.ADP complexes is twice that of ADF-actin.ADP complexes and this, in turn, is twice that of actin.ATP alone. It is this weaker nucleating potential of ADF-actin.ADP that accounts for the much higher steady-state depolymerizing activity. The pH-sensitivity is due to the nucleating activity of complexes being greater at pH 6.5 than at pH 8. Sequence analysis of mammalian and avian isoforms shows a consistent pattern of charge differences in regions of the protein associated with F-actin-binding that may account for the differences in activity between ADF and cofilin. >244.NF00097769 PMID:12393901 TI - LIGHT, a member of the tumor necrosis factor ligand superfamily, prevents tumor necrosis factor-alpha-mediated human primary hepatocyte apoptosis, but not Fas-mediated apoptosis. AB - LIGHT is a member of tumor necrosis factor (TNF) superfamily, and its receptors have been identified as lymphotoxin-beta receptor (LTbetaR) and the herpesvirus entry mediator (HVEM)/ATAR/TR2, both of which lack the cytoplasmic sequence termed the "death domain." The present study has demonstrated that LIGHT inhibits TNFalpha-mediated apoptosis of human primary hepatocytes sensitized by actinomycin D (ActD), but not Fas- or TRAIL-mediated apoptosis. Furthermore, LIGHT does not prevent some cell lines such as HepG2 or HeLa from undergoing ActD/TNFalpha-induced apoptosis. This protective effect requires LIGHT pretreatment at least 3 h prior to ActD sensitization. LIGHT stimulates nuclear factor-kappaB (NF-kappaB)-dependent transcriptional activity in human hepatocytes like TNFalpha. The time course of NF-kappaB activation after LIGHT administration is similar to that of the pretreatment required for the anti-apoptotic effect of LIGHT. LIGHT inhibits caspase-3 processing on the apoptotic protease cascade in TNFalpha-mediated apoptosis but not Fas-mediated apoptosis. In addition, increased caspase-3 and caspase-8 activities in ActD/TNFalpha-treated cells are effectively blocked by LIGHT pretreatment. However, LIGHT does not change the expression of TNFRp55, TNFRp75, and Fas. These results indicate that LIGHT may act as an anti-apoptotic agent against TNFalpha-mediated liver injury by blocking the activation of both caspase-3 and caspase-8. >245.NF00100413 PMID:12096904 TI - Nucleolin associates with a subset of the human Ro ribonucleoprotein complexes. AB - Ro RNPs are evolutionarily conserved, small cytoplasmic RNA-protein complexes with an unknown function. In human cells, Ro RNPs consist of one of the four hY RNAs and two core proteins: Ro60 and La. Recently, the association of hnRNP I and hnRNP K with particles containing hY1 and hY3 RNAs has been described. The association of three other proteins, namely calreticulin, Ro52 and RoBPI, with (subsets of) the Ro RNPs is still controversial. To gain more insight into the composition and function of the Ro RNPs, we have immunopurified these particles from HeLa cell extracts using monoclonal antibodies against Ro60 and La. Using this approach, we have identified the RNA-binding protein nucleolin as a novel subunit of Ro RNP particles containing hY1 or hY3 RNA, but not hY4 and hY5 RNA. Using an in vitro hY RNA-binding assay we established that the internal pyrimidine-rich loop of hY1 and hY3 RNA is essential for the association of nucleolin. The binding is critically dependent on the presence of all four RNP motifs of nucleolin, but not of the C-terminal RGG-box. Moreover, we demonstrate that, in contrast to nucleolin and hnRNP K, nucleolin and hnRNP I can bind simultaneously to the internal pyrimidine-rich loop of hY1 RNA. We postulate that nucleolin functions in the biogenesis and/or trafficking of hY1 and hY3 RNPs through the nucleolus and subsequent transport to the cytoplasm. >246.NF00082240 PMID:11896048 TI - Characterization of a brain-enriched chaperone, MRJ, that inhibits Huntingtin aggregation and toxicity independently. AB - Molecular chaperones are involved in a wide range of cellular events, such as protein folding and oligomeric protein complex assembly. DnaK- and DnaJ-like proteins are the two major classes of molecular chaperones in mammals. Recent studies have shown that DnaJ-like family proteins can inhibit polyglutamine aggregation, a hallmark of many neurodegenerative diseases, including Huntington's disease (HD). Although most DnaJ-like proteins studied are ubiquitously expressed, some have restricted expression, so it is possible that some specific chaperones may affect polyglutamine aggregation in specific neurons. In this report, we describe the isolation of a DnaJ-like protein MRJ and the characterization of its chaperone activity. Tissue distribution studies showed that MRJ is highly enriched in the central nervous system. In an in vitro cell model of HD, overexpressed MRJ effectively suppressed polyglutamine-dependent protein aggregation, caspase activity, and cellular toxicity. Collectively, these results suggest that MRJ has a relevant functional role in neurons. >247.NF01034720 PMID:12110693 TI - Identification, characterization, and functional study of the two novel human members of the semaphorin gene family. AB - We cloned two novel human transmembrane semaphorins, (HSA)SEMA6C and (HSA)SEMA6D, that belong to the class VI subgroup of the semaphorin family. The genes for SEMA6C and SEMA6D are mapped on chromosome 1q12-21.1 and 15q21.1, respectively. Among the adult tissues, SEMA6C is expressed only in skeletal muscle, whereas SEMA6D is expressed abundantly in kidney, brain, and placenta and moderately in the heart and skeletal muscles. During murine development, neither SEMA6C nor SEMA6D was expressed in embryonic day 10.5 (E10.5) embryos, but both were highly expressed in the areas of the lateral ventricle, the striatum, the wall of the midbrain, the pons/midbrain junction, and the choroid plexus of E13 embryos. Were neurons, neither axons nor astrocytes, highly expressed both semaphorins. Three isoforms of SEMA6C and five isoforms of SEMA6D derived from alternative splicing were identified, and their expression was regulated in a tissue- and development-dependent manner. Deletion analysis indicated that a sema domain and a PSI domain are integrally necessary for correct post-translation modification and subcellular localization. The extracellular domain of SEMA6C inhibited axonal extension of nerve growth factor-differentiated PC12 cells and induced the growth cone collapse of chicken dorsal root ganglion, rat hippocampal neurons, and rat cortical neurons in a dose-responsive manner. SEMA6D acted like SEMA6C except it had no significant effect on the growth cones of rat cortical neurons. >248.NF00126711 PMID:12296995 TI - TAK1-dependent activation of AP-1 and c-Jun N-terminal kinase by receptor activator of NF-kappaB. AB - The receptor activator of nuclear factor kappa B (RANK) is a member of the tumor necrosis factor (TNF) receptor superfamily. It plays a critical role in osteoclast differentiation, lymph node organogenesis, and mammary gland development. The stimulation of RANK causes the activation of transcription factors NF-kappaB and activator protein 1 (AP1), and the mitogen activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK). In the signal transduction of RANK, the recruitment of the adaptor molecules, TNF receptor-associated factors (TRAFs), is an initial cytoplasmic event. Recently, the association of the MAPK kinase kinase, transforming growth factor-beta-activated kinase 1 (TAK1), with TRAF6 was shown to mediate the IL-1 signaling to NF-kappaB and JNK. We investigated whether or not TAK1 plays a role in RANK signaling. A dominant-negative form of TAK1 was discovered to abolish the RANK-induced activation of AP1 and JNK. The AP1 activation by TRAF2, TRAF5, and TRAF6 was also greatly suppressed by the dominant negative TAK1. The inhibitory effect of the TAK1 mutant on RANK- and TRAF-induced NF-kappaB activation was also observed, but less efficiently. Our findings indicate that TAK1 is involved in the MAPK cascade and NF-kappaB pathway that is activated by RANK. >249.NF00118612 PMID:11891225 TI - Laminin-10/11 and fibronectin differentially prevent apoptosis induced by serum removal via phosphatidylinositol 3-kinase/Akt- and MEK1/ERK-dependent pathways. AB - Cell adhesion to the extracellular matrix inhibits apoptosis, but the molecular mechanisms underlying the signals transduced by different matrix components are not well understood. Here, we examined integrin-mediated antiapoptotic signals from laminin-10/11 in comparison with those from fibronectin, the best characterized extracellular adhesive ligand. We found that the activation of protein kinase B/Akt in cells adhering to laminin-10/11 can rescue cell apoptosis induced by serum removal. Consistent with this, wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, or ectopic expression of a dominant-negative mutant of Akt selectively accelerated cell death upon serum removal. In contrast to laminin-10/11, fibronectin rescued cells from serum depletion-induced apoptosis mainly through the extracellular signal-regulated kinase pathway. Cell survival on fibronectin but not laminin was significantly reduced by treatment with PD98059, a specific inhibitor of mitogen- or extracellular signal-regulated kinase kinase-1 (MEK1) and by expression of a dominant-negative mutant of MEK1. Laminin-10/11 was more potent than fibronectin in preventing apoptosis induced by serum depletion. These results, taken together, demonstrate laminin-10/11 potency as a survival factor and demonstrate that different extracellular matrix components can transduce distinct survival signals through preferential activation of subsets of multiple integrin-mediated signaling pathways. >250.NF00521426 PMID:12167609 TI - Generation and phenotypic analysis of CHIF knockout mice. AB - Corticosteroid hormone-induced factor (CHIF) is a short epithelial-specific protein that is independently induced by aldosterone and a high-K(+) diet. It is a member of the FXYD family of single-span transmembrane proteins that include phospholemman, Mat-8, and the gamma-subunit of Na(+)-K(+)-ATPase. A number of studies have suggested that these proteins are involved in the regulation of ion transport and, in particular, functionally interact with the Na(+)-K(+)-ATPase. The present study describes the characterization, targeted disruption, and phenotypic analysis of the mouse CHIF gene. The CHIF knockout mice are viable and not distinguishable from wild-type littermates under normal conditions. Under K(+) loading, they have a twofold higher urine volume and an increased glomerular filtration rate. Similar but smaller effects are observed in mice fed a low-Na(+) diet. Treating K(+)-loaded mice for 10 days with furosemide resulted in lethality in the knockout mice (17 of 39) but not in the wild-type group (1 of 39). The data are consistent with an effect of CHIF on the Na(+)-K(+)-ATPase that is specific to the outer and inner medullary duct, its major expression site. >251.NF00082407 PMID:12011083 TI - Differences in the regulation of the classical and the alternative pathway for bile acid synthesis in human liver. No coordinate regulation of CYP7A1 and CYP27A1. AB - It has been reported that there is a coordinate regulation of sterol 27-hydroxylase (CYP27A1) and cholesterol 7alpha-hydroxylase (CYP7A1) in rats. Thus, the levels of the mRNA corresponding to these two enzymes were found to change in the same direction in rat liver and in isolated rat hepatocytes. In contrast, other groups have not seen such regulation of CYP27A1 in rabbit liver or in rat liver when using an activity assay. In the present work, the effect of bile acid treatment on human CYP27A1/luciferase reporter activity was studied in a transient transfection assay in human liver-derived HepG2 cells. Neither the endogenous 27-hydroxylase activity nor the CYP27A1/luciferase reporter activity were down-regulated by treatment of HepG2 cells with chenodeoxycholic acid or taurochenodeoxycholic acid. We also measured CYP27A1 mRNA and CYP7A1 mRNA in liver of humans subjected to treatment with chenodeoxycholic acid, ursodeoxycholic acid, hydroxymethylglutaryl (HMG)-CoA reductase inhibitor and a combination of HMG-CoA reductase inhibitor and cholestyramine. There was a 60-fold variation in the levels of CYP7A1 mRNA but only a 5-fold variation in the levels of CYP27A1 mRNA. There was no correlation between the two mRNA species. It is concluded that, in humans, there is little or no coordinate regulation of CYP7A1 and CYP27A1 at the transcriptional level, and that CYP27A1 is not subject to a negative feedback control by bile acids. The results underline that marked species differences may exist in mechanisms for control of synthesis of bile acids and cholesterol homeostasis. >252.NF00083138 PMID:12135764 TI - Memapsin 2 (beta-secretase) cytosolic domain binds to the VHS domains of GGA1 and GGA2: implications on the endocytosis mechanism of memapsin 2. AB - Memapsin 2, or beta-secretase, is a membrane-anchored aspartic protease that initiates the cleavage of beta-amyloid precursor protein (APP) leading to the production of beta-amyloid peptide in the brain and the onset of Alzheimer's disease. Memapsin 2 and APP are both endocytosed into endosomes for cleavage. Here we show that the cytosolic domain of memapsin 2, but not that of memapsin 1, binds the VHS domains of GGA1 and GGA2. Gel-immobilized VHS domains of GGA1 and GGA2 also bound to full-length memapsin 2 from cell mammalian lysates. Mutagenesis studies established that Asp(496), Leu(499) and Leu(500) were essential for the binding. The spacing of these three residues in memapsin 2 is identical to those in the cytosolic domains of mannose-6-phosphate receptors, sortilin and low density lipoprotein receptor-related protein 3. These observations suggest that the endocytosis and intracellular transport of memapsin 2, mediated by its cytosolic domain, may involve the binding of GGA1 and GGA2. >253.NF00081197 PMID:12416719 TI - The expression of alpha-dystrobrevin and dystrophin during skeletal muscle regeneration. AB - The expression of alpha-dystrobrevin and dystrophin in rat tibialis anterior muscles was chronologically evaluated during a cycle of regeneration after myonecrosis induced by the injection of cardiotoxin. In immunohistochemical studies, alpha-dystrobrevin and dystrophin were first stained weakly at the sarcolemma of some regenerating muscle fibers on day 5. On day 7, alpha-dystrobrevin was still stained weakly, whereas dystrophin was stained conspicuously. After day 10, alpha-dystrobrevin and dystrophin were both stained conspicuously on almost all regenerating muscle fibers. In the Western blot analysis, alpha-dystrobrevin and dystrophin were first detected as visible bands on days 5 and 7, respectively. The bands of alpha-dystrobrevin and dystrophin both darkened sequentially up to day 10. The protein levels based on the densitometrical analysis of the bands on each day were converted to the percentage of the protein level on day 28, which was taken as 100%. The sequential line based on these data showed that alpha-dystrobrevin and dystrophin reached 50% of the protein level on day 28 by 6.6 and 5.3 days, respectively. These data provide evidence that alpha-dystrobrevin regenerates more slowly than dystrophin in skeletal muscle. >254.NF00137168 PMID:12084819 TI - Live-cell imaging reveals divergent intracellular dynamics of polyglutamine disease proteins and supports a sequestration model of pathogenesis. AB - Protein misfolding and aggregation are central features of the polyglutamine neurodegenerative disorders, but the dynamic properties of expanded polyglutamine proteins are poorly understood. Here, we use fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) with green fluorescent protein fusion proteins to study polyglutamine protein kinetics in living cells. Our results reveal markedly divergent mobility states for an expanded polyglutamine protein, ataxin-3, and establish that nuclear inclusions formed by this protein are aggregates. Additional studies of green fluorescent protein-tagged cAMP response element binding protein coexpressed with either of two mutant polyglutamine proteins, ataxin-3 and huntingtin, support a model of disease in which coaggregation of transcriptional components contributes to pathogenesis. Finally, studies of a third polyglutamine disease protein, ataxin-1, reveal unexpected heterogeneity in the dynamics of inclusions formed by different disease proteins, a finding which may help explain disease-specific elements of pathogenesis in these neurodegenerative disorders. >255.NF00135467 PMID:11948912 TI - Cooperative E-box regulation of human GLI1 by TWIST and USF. AB - Sonic hedgehog signaling plays a critical role in vertebrate patterning, and signaling defects are associated with severe birth defects and cancer in man. GLI1 encodes a critical transcription activator in this pathway. GLI1 is expressed in human basal cell carcinomas and sarcomas. Despite the significance of the GLI1 gene in human disease, few immediate upstream regulators of GLI1 expression are known. We previously demonstrated that a 5' region, including 5' flanking sequence, an untranslated exon, and 425 bp of the first intron, regulates the human GLI1 gene. Here we show that inactivating mutations in E-box, GC box, AP-2, GATA, GSG, PuF, and Zeste sites identified three critical regulatory elements, including a GC box that binds Sp1 and two intronic E-boxes that bind USF proteins or Twist. Expression of Twist but not a frame shift mutation of Twist activates the wild-type human GLI1 regulatory sequences but not with inactivating mutations of the E-boxes. Twist activates GLI1 reporter expression through E-box +482 but requires binding of USF proteins to E-box +157. Twist mutations cause human birth defects and Twist is overexpressed in many rhabdomyosarcomas, suggesting that one of Twist's primary roles is the regulation of GLI1. >256.NF00970219 PMID:12023963 TI - Involvement of TRAF4 in oxidative activation of c-Jun N-terminal kinase. AB - We previously found that the angiogenic factors TNFalpha and HIV-1 Tat activate an NAD(P)H oxidase in endothelial cells, which operates upstream of c-Jun N-terminal kinase (JNK), a MAPK involved in the determination of cell fate. To further understand oxidant-related signaling pathways, we screened lung and endothelial cell libraries for interaction partners of p47(phox) and recovered the orphan adapter TNF receptor-associated factor 4 (TRAF4). Domain analysis suggested a tail-to-tail interaction between the C terminus of p47(phox) and the conserved TRAF domain of TRAF4. In addition, TRAF4, like p47(phox), was recovered largely in the cytoskeleton/membrane fraction. Coexpression of p47(phox) and TRAF4 increased oxidant production and JNK activation, whereas each alone had minimal effect. In addition, a fusion between p47(phox) and the TRAF4 C terminus constitutively activated JNK, and this activation was decreased by the antioxidant N-acetyl cysteine. In contrast, overexpression of the p47(phox) binding domain of TRAF4 blocked endothelial cell JNK activation by TNFalpha and HIV-1 Tat, suggesting an uncoupling of p47(phox) from upstream signaling events. A secondary screen of endothelial cell proteins for TRAF4-interacting partners yielded a number of proteins known to control cell fate. We conclude that endothelial cell agonists such as TNFalpha and HIV-1 Tat initiate signals that enter basic signaling cassettes at the level of TRAF4 and an NAD(P)H oxidase. We speculate that endothelial cells may target endogenous oxidant production to specific sites critical to cytokine signaling as a mechanism for increasing signal specificity and decreasing toxicity of these reactive species. >257.NF00117649 PMID:11969340 TI - The distribution of activin and activin receptors in gestational tissues across human pregnancy and during labour. AB - The aim of this study was to investigate localization and content of activin beta A-subunit and activin receptors in gestational tissues throughout pregnancy and in association with term labour. Placenta and fetal membranes were collected from women undergoing first and second trimester terminations and from women before and after term labour. Activin beta A-subunit and activin receptors IA, IB, IIA and IIB were studied by immunohistochemistry. Term tissues were analysed for activin A and follistatin content by ELISA and the presence of receptor proteins was assessed by Western hybridization. Activin beta A-subunit was localized to the syncytiotrophoblast and cytotrophoblast in placentae from all gestational ages, and to the amniotic epithelial and chorionic trophoblast layer at term. In placentae of first and second trimester, receptor proteins were localized to the syncytium, whereas at term, the distribution was confined predominantly to vascular endothelial cells of villous blood vessels. Receptor proteins in amnion were localized to some epithelial cells, mesenchyme and chorionic trophoblast. These findings suggest that activin A is secreted by and targets the placental syncytium and amniotic epithelium in early pregnancy, but at term targets the vascular endothelium of placenta and the fetal membranes. There were no differences with labour onset. >258.NF00089673 PMID:11883937 TI - A novel negative regulatory element in the human collagenase-3 proximal promoter region. AB - We have identified in the human collagenase-3 promoter a novel negative regulatory element, GAAAAGAAAAAG, designated AGRE (AG-Rich Element). The AGRE site functionality was characterized in human osteoarthritic (OA) chondrocytes as well as four cell lines. The cells were transfected with a plasmid consisting of the first 133 bp of the collagenase-3 promoter and its AGRE mutated or deleted derivatives. The absence of a functional AGRE site resulted in a statistically significant increase of the collagenase-3 basal transcription that was not affected by the collagenase-3 inducers IL-1beta and TGF-beta1. Two specific protein-AGRE binding complexes were detected by EMSA, and their presence depended on the physiological state of the cell. Indeed, normal chondrocytes and synovial fibroblasts and the four cell lines showed only a slower-migrating complex (complex 1). In OA chondrocytes, the type of complex discriminated two groups--the low-OA chondrocytes, showing low collagenase-3 basal levels and high inducibility of IL-1beta stimulation (complex 1), and the high-OA chondrocytes with high collagenase-3 basal levels and low IL-1beta inducibility (a faster-migrating complex, designated complex 2). UV cross-linking revealed the presence of 48 and 97 kDa proteins in complex 1 and 27, 35, and 73 kDa proteins in complex 2. These findings suggest that the AGRE site plays a rate-limiting role in human collagenase-3 production. >259.NF01099179 PMID:12620808 TI - The L1 major capsid protein of human papillomavirus type 11 interacts with Kap beta2 and Kap beta3 nuclear import receptors. AB - We have previously shown that the L1 major capsid protein of low-risk HPV11 binds to the Kap alpha2 adapter and enters the nucleus via a Kap alpha2beta1-mediated pathway. In this study, we discovered that HPV11 L1 capsomeres bind to Kap beta2 import receptor, known to mediate nuclear import of hnRNP A1 via interaction with its nuclear localization signal termed M9. Significantly, binding of HPV11 L1 capsomeres to Kap beta2 inhibited the nuclear import of Kap beta2, and its specific M9-containing cargo. Interestingly, HPV11 L1 capsomeres also interacted with Kap beta3 import receptor and inhibited Kap beta3 nuclear import. Moreover, the L1 capsomeres of high-risk HPV-16 shared these activities. These data suggest that HPV L1 major capsid proteins interact with Kap beta2 and Kap beta3, and they may inhibit the Kap beta2- and Kap beta3-mediated nuclear import pathways during the productive phase of the viral life cycle when the virions are assembled and released. >260.NF00089677 PMID:12049201 TI - BAG-1, an anti-apoptotic tumour marker. AB - BAG-1 is a multifunctional and anti-apoptotic or anti-cell death protein that interacts with a variety of cellular proteins and affects their functions. On the cell surface, it binds to the cytosolic domain of the growth factor receptors and enhances the protection from cell death triggered by growth factor receptors. In the cytosol, it binds to Bcl-2 and heat shock protein, and modulates their functions. In the nucleus, it binds to a variety of nuclear hormone receptors and inhibits hormone-induced apoptosis. BAG-1 is widely overexpressed in a variety of tumour cell lines and cancer tissues. In addition, differential expression of BAG-1 isoforms has been observed. Preclinical studies indicate that overexpression of BAG-1, especially its nuclear and cytoplasmic isoforms, may be useful as a prognostic and/or predictive biomarker. Pilot clinical studies have demonstrated that overexpression of nuclear BAG-1 may be associated with a shorter survival in breast and laryngeal carcinomas. Conversely, overexpression of cytoplasmic BAG-1 may be associated with a better clinical outcome in early stage breast cancer and in non-small cell lung cancer. Further large-scale clinical studies are warranted to establish the role of BAG-1 as a novel prognostic and/or predictive biomarker in the clinical management of these common malignancies. >261.NF00100423 PMID:11514614 TI - Dynamin-related protein Drp1 is required for mitochondrial division in mammalian cells. AB - Mutations in the human dynamin-related protein Drp1 cause mitochondria to form perinuclear clusters. We show here that these mitochondrial clusters consist of highly interconnected mitochondrial tubules. The increased connectivity between mitochondria indicates that the balance between mitochondrial division and fusion is shifted toward fusion. Such a shift is consistent with a block in mitochondrial division. Immunofluorescence and subcellular fractionation show that endogenous Drp1 is localized to mitochondria, which is also consistent with a role in mitochondrial division. A direct role in mitochondrial division is suggested by time-lapse photography of transfected cells, in which green fluorescent protein fused to Drp1 is concentrated in spots that mark actual mitochondrial division events. We find that purified human Drp1 can self-assemble into multimeric ring-like structures with dimensions similar to those of dynamin multimers. The structural and functional similarities between dynamin and Drp1 suggest that Drp1 wraps around the constriction points of dividing mitochondria, analogous to dynamin collars at the necks of budding vesicles. We conclude that Drp1 contributes to mitochondrial division in mammalian cells. >262.NF00012058 PMID:12154120 TI - RNAi is activated during Drosophila oocyte maturation in a manner dependent on aubergine and spindle-E. AB - Gene silencing by double-stranded RNA is a widespread phenomenon called RNAi, involving homology-dependent degradation of mRNAs. Here we show that RNAi is established in the Drosophila female germ line. mRNA transcripts are translationally quiescent at the arrested oocyte stage and are insensitive to RNAi. Upon oocyte maturation, transcripts that are translated become sensitive to degradation while untranslated transcripts remain resistant. Mutations in aubergine and spindle-E, members of the PIWI/PAZ and DE-H helicase gene families, respectively, block RNAi activation during egg maturation and perturb translation control during oogenesis, supporting a connection between gene silencing and translation in the oocyte. >263.NF00082330 PMID:11744098 TI - Estrogen neuroprotection: the involvement of the Bcl-2 binding protein BNIP2. AB - To identify genes selectively induced by estrogens in cells of neural origin we have treated with a low concentration of 17 beta-estradiol (E2) the estrogen-receptor positive SK-ER3 neuroblastoma cells and we have isolated messages modulated by the hormonal treatment at short (1 h) and longer (17 h) times. By using the ddPCR approach we identified numerous messages which content was significantly and reproducibly altered by the hormonal treatment. Among these messages we focused our attention on bnip2, which expression was inhibited by estradiol. bnip2 was found to be a member of the BNIP family of genes of unknown physiological activity at the time. Investigations carried out in our laboratory proved a strong correlation between the increased expression of bnip2 gene and cell death induced by toxic stimuli. Furthermore, we showed that transfection of the bnip2 cDNA results in massive cell death and Bcl-2 overexpression counteracts the toxic effect of bnip2. These findings suggest that the proteins encoded by these two genes either interact or act in an opposite manner on the same mechanisms triggering the apoptotic cascade of events. Time-course experiments carried out in different cell systems and with a variety of neurotoxic agents proved a strong correlation between estrogen-induced decrease in bnip2 expression and the time required for estrogen to exert its protective effect. These observations led us to hypothesize an involvement of bnip2 in estrogen effects on cell survival. The finding that bnip2 is developmentally regulated may suggest a role of this gene in those brain areas where the differentiation is orchestrated by estradiol. Investigations in non-neural cells show that bnip2 is the mediator of the anti-apoptotic activity of estrogens in a variety of cells and thus might represent an important target for the evaluation of the activity of novel synthetic ligands for the estrogen receptor. >264.NF00100349 PMID:12095712 TI - Microanatomical localization of PD-1 in human tonsils. AB - PD-1 is an immunoinhibitory receptor, which belongs structurally to the CD28 family. PD-1-deficient mice show breakdown of peripheral tolerance and manifest multiple autoimmune symptoms. We previously described expression of PD-1 on activated T and B lymphocytes and myeloid cells. However, little is known about the microanatomical distribution of PD-1 in lymphoid organs. In this study, we performed immunohistochemistry using monoclonal antibodies against human PD-1. In human tonsils, PD-1 was expressed on most of T cells and a small subset of centrocytes in the light zone of germinal centers (GCs), where clonal selection of centrocytes takes place. These results suggest that PD-1 may play an important role in GC reaction. >265.NF01017801 PMID:12219026 TI - Identification of mitogen-activated protein kinase kinase as a chemoresistant pathway in MCF-7 cells by using gene expression microarray. AB - BACKGROUND: Components of the mitogen-activated protein kinase (MAPK) cascade have been implicated in apoptotic regulation. This study used gene expression profiling analysis to identify and implicate mitogen-activated protein kinase kinase (MEK5)-BMK1 (big mitogen-activated kinase-1)/extracellular signal related protein kinase (ERK5) pathway as a novel target involved in chemoresistance. METHODS: Differential gene expression between apoptotically sensitive (APO+) and apoptotically resistant (APO-) MCF-7 cell variants was determined by using microarray and confirmed by reverse transcriptase- polymerase chain reaction (RT-PCR). An apoptotic/viability reporter gene assay was used to deter-mine the effects of the transfection of a dominant-negative mutant of BMK1 (BMK1/DN) in conjunction with apoptotic-inducing agents (etoposide, tumor necrosis factor-alpha [TNF], or TNF-related apoptosis-inducing ligand [TRAIL]), with or without phorbol ester (PMA). RESULTS: Of the 1186 genes detected through microarray analysis, MEK5 was increased 22-fold in APO- cells. Overexpression of MEK5 was confirmed by using RT-PCR analysis. Expression of BMK1/DN alone resulted in a dose-dependent increase in cell death versus control (P <.05). In addition, BMK1/DN enhanced the sensitivity of MCF-7 cells to treatment-induced cell death (P <.05). The ability of PMA to partially suppress TRAIL- and TNF-induced cell death was inhibited by BMK1/DN. However, only TRAIL-induced activity suppression reached statistical significance (P <.05). CONCLUSIONS: The overexpression of MEK5 in APO- MCF-7 breast carcinoma cells shows that this MAPK signaling protein represents a potent survival molecule. Molecular inhibition of MEK5 signaling may represent a mechanism for sensitizing cancer cells to chemotherapeutic regimens. >266.NF00074313 PMID:11761714 TI - Cloning, mapping and expression analysis of C15orf4, a novel human gene with homology to the yeast mitochondrial ribosomal protein Ym130 gene. AB - We have identified C15orf4, a novel human gene showing homology to the yeast mitochondrial ribosomal protein YmL30. C15orf4 encodes a transcript of 1,006 nt with an ORF of 279 amino acids and a predicted protein size of 31.7 kDa. Expression of C15orf4 is enriched in testis. C15orf4 was positioned to chromosome 15q24 by radiation hybrid mapping. We have identified the C15orf4 mouse orthologue as well as homologues in other species. >267.NF00082170 PMID:12055595 TI - Molecular misreading of the ubiquitin B gene and hepatic mallory body formation. AB - BACKGROUND & AIMS: Molecular misreading of the ubiquitin B gene has been documented in the cerebral cortex of patients with Alzheimer's disease and Down syndrome. This novel process consists of the unfaithful conversion of genomic information into aberrant transcripts and its subsequent translation into +1 proteins. METHODS: Because Mallory bodies (MBs) also contain ubiquitinated proteins, we stained 11 autopsied and 6 biopsied MB-containing livers from patients with steatohepatitis with an antibody to ubiquitin(+1) to look for the presence of mutant (ubiquitin(+1)) protein. Antibodies to wild-type ubiquitin were used to document the presence of MBs in all cases. RESULTS: Ubiquitin(+1) immunoreactivity was detected in all MB-containing livers with steatohepatitis; no ubiquitin(+1) immunoreactivity was found in 13 MB-free liver controls. A subpopulation (about one third of the MBs) of the MB-containing hepatocytes in autopsied livers showed ubiquitin(+1) immunoreactivity (i.e., ubiquitin and ubiquitin(+1) colocalized in MBs). MB-containing liver biopsy specimens showed colocalization of ubiquitin and ubiquitin(+1) in every MB. Western blot analysis showed an ubiquitin(+1) band of 11 kilodaltons. Molecular misreading of the ubiquitin B gene (DeltaGU) was shown in one of the livers, which contained numerous MBs using an expression cloning strategy. CONCLUSIONS: The results showed that molecular misreading of the ubiquitin B gene occurred in hepatocytes in virtually all of the MB-containing livers tested. Ubiquitin(+1) protein was only found within the MBs and therefore may act by interfering with the degradation of the MBs because ubiquitin(+1) may inhibit proteolytic function of the proteasome. >268.NF00080713 PMID:12032142 TI - Subunit H of the V-ATPase binds to the medium chain of adaptor protein complex 2 and connects Nef to the endocytic machinery. AB - Nef is an accessory protein of human and simian immunodeficiency viruses (HIV and SIV) that is required for efficient viral infectivity and pathogenicity. It decreases the expression of CD4 on the surface of infected cells. V1H is the regulatory subunit H of the vacuolar membrane ATPase (V-ATPase). Previously, the interaction between Nef and V1H has been found to facilitate the internalization of CD4, suggesting that V1H could connect Nef to the endocytic machinery. In this study, we demonstrate that V1H binds to the C-terminal flexible loop in Nef from HIV-1 and to the medium chain (mu2) of the adaptor protein complex 2 (AP-2) in vitro and in vivo. The interaction sites of V1H and mu2 were mapped to a central region in V1H from positions 133 to 363, which contains 4 armadillo repeats, and to the N-terminal adaptin-binding domain in mu2 from positions 1 to 145. Fusing Nef to V1H reproduced the appropriate trafficking of Nef. This chimera internalized CD4 even in the absence of the C-terminal flexible loop in Nef. Finally, blocking the expression of V1H decreased the enhancement of virion infectivity by Nef. Thus, V1H can function as an adaptor for interactions between Nef and AP-2. >269.NF00117732 PMID:11898002 TI - Expression and function of histamine receptors 1 and 2 on human monocyte-derived dendritic cells. AB - BACKGROUND: Histamine is a well-known mediator of inflammatory and allergic reactions and has immunomodulatory capacities. There is increasing evidence that dendritic cells as professional antigen-presenting cells play a major role in the development of allergic reactions. However, a possible link between histamine and dendritic cells has not been investigated thus far. OBJECTIVE: We investigated the effect of histamine on human monocyte-derived dendritic cells (MoDCs). METHODS: Expression of histamine H1 and H2 receptors (H1R and H2R) on MoDCs was assessed by means of RT-PCR and flow cytometry. Functional exploration of these receptors was performed by monitoring the increase of intracellular calcium levels (H1R), cyclic adenosine monophosphate formation (H2R), F-actin polymerization, and IL-12p70 production. RESULTS: MoDCs express both H1R and H2R. Stimulation of dendritic cells with histamine resulted in F-actin polymerization and cyclic adenosine monophosphate production through H2R. Influx of calcium could not be detected after stimulation of dendritic cells with histamine under conditions in which histamine induced calcium influx in monocytes. Histamine and H1R and H2R agonists downregulated IL-12p70 production of prestimulated MoDCs. CONCLUSION: MoDCs express histamine H1 and H2 receptors. Our results indicate chemotactic (F-actin polymerization) and immunomodulatory (inhibition of IL-12p70 production) effects of histamine on MoDCs. Therefore histamine might represent a link between immediate-type hypersensitivity reactions and cellular inflammation in allergic disease (eg, in atopic dermatitis). >270.NF00133698 PMID:12620798 TI - Binding of HTLV-1 tax oncoprotein to the precursor of interleukin-16, a T cell PDZ domain-containing protein. AB - HTLV-1 Tax oncoprotein interacts with various cellular factors and modulates transcription and the cell cycle. In that role it is sufficient to create T cell malignancies in the absence of HTLV-1 infection. HTLV-1 Tax protein has been reported to bind to cellular proteins containing PDZ domains in vitro. The precursor of human interleukin 16, pro-IL-16, is an abundant cellular protein present in human peripheral blood T cells. Pro-IL-16 contains three PDZ domains. It has been shown that expression of pro-IL-16 in pro-IL-16 negative cells induces a G(0)/G(1) arrest in the cell cycle. The current studies demonstrate that Tax binds to pro-IL-16 in HTLV-1 infected human T cells. We mapped the Tax binding site to the first PDZ domain of pro-IL-16. Over-expression of Tax in COS cells resulted in fewer cells in G(0)/G(1) consistent with its activity to induce G(1)- to S-phase progression in lymphocytes, while over-expression of pro-IL-16 in COS cells resulted in G(0)/G(1) arrest. Co-expression of wild type Tax with pro-IL-16 in COS cells negated the effects of pro-IL-16, an effect not observed with Tax mutated at its PDZ binding C-terminus. These results suggest that one of the effects of Tax on growth deregulation in HTLV-1 infected T cells might be mediated by its binding to pro-IL-16. >271.NF00026699 PMID:11872800 TI - Living with lethal PIP3 levels: viability of flies lacking PTEN restored by a PH domain mutation in Akt/PKB. AB - The phosphoinositide phosphatase PTEN is mutated in many human cancers. Although the role of PTEN has been studied extensively, the relative contributions of its numerous potential downstream effectors to deregulated growth and tumorigenesis remain uncertain. We provide genetic evidence in Drosophila melanogaster for the paramount importance of the protein kinase Akt [also called protein kinase B (PKB)] in mediating the effects of increased phosphatidylinositol 3,4,5-trisphosphate (PIP3) concentrations that are caused by the loss of PTEN function. A mutation in the pleckstrin homology (PH) domain of Akt that reduces its affinity for PIP3 sufficed to rescue the lethality of flies devoid of PTEN activity. Thus, Akt appears to be the only critical target activated by increased PIP3 concentrations in Drosophila. >272.NF01188288 PMID:12601022 TI - TGFbeta-induced factor: a candidate gene for high myopia. AB - PURPOSE: To investigate the coding exons of transforming growth factor (TGF)-beta-induced factor (TGIF) for mutations in Chinese patients with high myopia. METHODS: Seventy-one individuals with high myopia of -6.00 D or less and 105 control subjects were screened by DNA sequencing for sequence alterations. Univariate analysis and logistic regression were performed to identify single-nucleotide polymorphisms (SNPs) and their interactions in TGIF that may be associated with myopia. RESULTS: Six SNPs showed a significant difference (P < 0.05) between patient and control subject in univariate analysis. Four of them cause codon changes: G223R, G231S, P241T, and A262G. Among all the SNPs that entered multivariate analysis, only 657(T-->G) showed statistical significance in the logistic regression model (odds ratio 0.133; 95% confidence interval 0.037-0.488; P = 0.002). CONCLUSIONS: TGIF is a probable candidate gene for high myopia. Further studies are needed to identify the underlying mechanism. >273.NF00112011 PMID:11773079 TI - Functional interaction of STAT3 transcription factor with the coactivator NcoA/SRC1a. AB - Signal transducer and activator of transcription 3 (STAT3) transcription factors are cytoplasmic proteins that induce gene activation in response to cytokine receptor stimulation. Following tyrosine phosphorylation, STAT3 proteins dimerize, translocate to the nucleus, and activate specific target genes. This transcriptional activation by STAT3 proteins has been shown to require the recruitment of coactivators such as CREB-binding protein (CBP)/p300. In the present study, we show that steroid receptor coactivator 1, NcoA/SRC1a, originally identified as a nuclear receptor coactivator, also functions as a coactivator of STAT3 proteins. In coimmunoprecipitations, NcoA/SRC1a was found to associate with STAT3 following IL-6 stimulation of HepG2 hepatoma cells. Pull-down experiments indicated that the N-terminal part of NcoA/SRC1a associates with the activation domain of STAT3. Overexpression of NcoA/SRC1a or its SRC1e isoform enhanced transcriptional activation by STAT3 proteins in transient transfection experiments. This ability of NcoA/SRC1a to enhance STAT3 activity is dependent upon the presence of the CBP-interacting domain, activation domain 1. Using chromatin immunoprecipitation assays, we found that STAT3, NcoA/SRC1a, and CBP/p300 are simultaneously recruited to the p21(waf1) promoter following interleukin-6 stimulation. Taken together, these data suggest that CBP/p300 and NcoA/SRC1a may function in a common pathway to regulate STAT3 transcriptional activity. >274.NF00084043 PMID:12016223 TI - Assembly of human small nuclear RNA gene-specific transcription factor IIIB complex de novo on and off promoter. AB - In humans, transcription factor IIIB (TFIIIB)-alpha governs basal transcription from small nuclear RNA genes by RNA polymerase III (pol III). One of the components of this complex, BRFU/TFIIIB50, is specific for these promoters, whereas TATA-binding protein (TBP) and hB" are required for pol III transcription from both gene external and internal promoters. We show that hB" is specifically recruited to a promoter-bound TBP.BRFU complex, which we have previously demonstrated as forming on TATA-containing templates. The N-terminal region of BRFU, containing a zinc ribbon domain, acts as a damper of hB" binding. TBP deactivates this negative mechanism through protein-protein contacts with both BRFU and hB", which may then promote their cooperative binding to form TFIIIB-alpha. In addition, we have identified a GC-rich sequence downstream from the TATA box (the BURE) which, depending on the strength of TATA box, can either enhance BRFU binding to the TBP.DNA complex or hB" association with the BRFU.TBP.DNA complex, and subsequently stimulate pol III transcription. Moreover, mutation of the BURE reduces pol III transcription and induces transcription by RNA polymerase II from the U2 gene promoter carrying a minimal TATA box. >275.NF00100198 PMID:11912161 TI - Coamplification of DAD-R, SOX5, and EKI1 in human testicular seminomas, with specific overexpression of DAD-R, correlates with reduced levels of apoptosis and earlier clinical manifestation. AB - Seminomas and nonseminomas represent the invasive stages of testicular (TGCTs) of adolescents and adults. Although TGCTs are characterized by extra copies of the short arm of chromosome 12, the genetic basis for gain of 12p in the pathogenesis of this cancer is not yet understood. We have demonstrated that gain of 12p is related to invasive growth and that amplification of specific 12p sequences, i.e., 12p11.2-p12.1, correlates with reduced apoptosis in the tumors. Here we show that three known genes map within the newly determined shortest region of overlap of amplification (SROA): DAD-R, SOX5, and EKI1. Whereas EKI1 maps close to the telomeric region of the SROA, DAD-R is the first gene at the centromeric region within the 12p amplicon. Although all three genes are amplified to the same level within the SROA, expression of DAD-R is significantly up-regulated in seminomas with the restricted 12p amplification compared with seminomas without this amplicon. DAD-R is also highly expressed in nonseminomas of various histologies and derived cell lines, both lacking such amplification. This finding is of particular interest because seminomas with the restricted 12p amplification and nonseminomas are manifested clinically in the third decade of life and show a low degree of apoptosis. In contrast, seminomas lacking a restricted 12p amplification, showing significantly lower levels of DAD-R with pronounced apoptosis, manifest clinically in the fourth decade of life. A low level of DAD-R expression is also observed in normal testicular parenchyma and in parenchyma containing the precursor cells of this cancer, i.e., carcinoma in situ. Therefore, elevated DAD-R expression in seminomas and nonseminomas correlates with invasive growth and a reduced level of apoptosis associated with an earlier clinical presentation. These data implicate DAD-R as a candidate gene responsible in part for the pathological effects resulting from gain of 12p sequences in TGCTs. In addition, our results also imply differences in expression regulation of DAD-R between seminomas and nonseminomas. >276.NF00090362 PMID:11924557 TI - Mutational and haplotype analysis of AGL in patients with glycogen storage disease type III. AB - Glycogen storage disease type III (GSD III) is a rare autosomal recessive inherited disorder caused by a deficiency of the glycogen-debranching enzyme (AGL). We investigated two GSD III patients and identified four different mutations. Nucleotide sequence analysis revealed patient 1 of Chinese descent to be a compound heterozygote for a novel nonsense mutation, R34X, and the splicing mutation (IVS32-12A > G) reported in a Japanese patient. Patient 2 of Japanese origin was found to be compound heterozygous for a novel nonsense mutation, Y1148X, and the splicing mutation (IVS14+1G > T) that we had described previously. To determine whether splicing mutations occurred independently, we performed intense AGL haplotype analysis using 21 intragenic polymorphic markers plus a novel polymorphism IVS32-97 A/G in the vicinity of the IVS32 splicing mutation. Patient 1 of Chinese origin and the Japanese patient homozygous for the IVS32-12A > G were found to have different haplotypes, indicating the IVS32-12A > G mutation to be a recurrent mutation. This is the first recurrent mutation established by intense haplotyping in the AGL gene. >277.NF00090445 PMID:11831386 TI - Mrnp41 (Rae 1p) associates with microtubules in HeLa cells and in neurons. AB - Mrnp41 (hRae1p) is an evolutionarily highly conserved protein, which is a potential component of mRNP particles and plays a role in nuclear mRNA export. The protein is mainly localized at the nuclear pore complex, but is also associated with distinct nuclear domains and with a meshwork of numerous small particles in the cytoplasm (Kraemer and Blobel (1997): Proc. Natl. Acad. Sci. USA 91, 1519-1523). We show that the cytoplasmic pattern of mrnp41 is sensitive to treatment with the microtubule (MT)-depolymerizing drug nocodazole which causes disappearance of mrnp41 from the cell periphery and concentration around the nucleus. By immunofluorescence we demonstrate that mrnp41 colocalizes with MT in HeLa cells and displays an MT-like distribution in cultured neurons. Association of mrnp41 with MT is further demonstrated by copurification with MT from pig brain throughout several steps of polymerization and depolymerization. Separation of MT-associated proteins (MAPs) by phosphocellulose (PC) chromatography showed copurification of mrnp41 with MAPs. These data show an association of mrnp41 with MT and, moreover, demonstrate that an intact MT system is necessary for dispersion of mrnp41-containing particles to the cellular periphery. The essential role of mrnp41 in spindle pole separation and cell cycle progression may also be related to its ability to bind to MTs. >278.NF01155028 PMID:12050388 TI - Human herpesvirus 6 variant A but not variant B induces fusion from without in a variety of human cells through a human herpesvirus 6 entry receptor, CD46. AB - Human herpesvirus 6 (HHV-6) is a lymphotropic betaherpesvirus that productively infects T cells and monocytes. HHV-6 isolates can be differentiated into two groups, variants A and B (HHV-6A and HHV-6B). Here, we show a functional difference between HHV-6A and -6B in that HHV-6A induced syncytium formation of diverse human cells but HHV-6B did not. The syncytium formation induced by HHV-6A was observed 2 h after infection; moreover, it was found in the presence of cycloheximide, indicating that HHV-6A induced fusion from without (FFWO) in the target cells. Furthermore, the fusion event was dependent on the expression of the HHV-6 entry receptor, CD46, on the target cell membrane. In addition, we determined that short consensus repeat 2 (SCR2), -3, and -4 of the CD46 ectodomain were essential for the formation of the virus-induced syncytia. Monoclonal antibodies against glycoproteins B and H of HHV-6A inhibited the fusion event, indicating that the syncytium formation induced by HHV-6A required glycoproteins H and B. These findings suggest that FFWO, which HHV-6A induced in a variety of cell lines, may play an important role in the pathogenesis of HHV-6A, not only in lymphocytes but also in various tissues, because CD46 is expressed ubiquitously in human tissues. >279.NF00133862 PMID:11719522 TI - Molecular cloning and characterization of the human diacylglycerol kinase beta (DGKbeta) gene: alternative splicing generates DGKbeta isotypes with different properties. AB - Diacylglycerol kinases are key modulators of levels of diacylglycerol, a second messenger involved in a variety of cellular responses to extracellular stimuli. A number of diacylglycerol kinases encoded by separate genes are present in mammalian genomes. We have cloned cDNAs encoding several isoforms of the human homologue of the rat diacylglycerol kinase beta gene and characterized two such isoforms that differ at their carboxyl terminus through alternative splicing and the usage of different polyadenylation signals. Quantitative analysis of gene expression in a panel of human tissue cDNAs revealed that transcripts corresponding to both isoforms are co-expressed