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Nectin-like molecule 1 (NECL1)/CADM3/IGSF4B/TSLL1/SynCAM3 is a neural tissue-specific immunoglobulin-like cell–cell adhesion molecule downregulated at the mRNA level in 12 human glioma cell lines. Here we found that the expression of NECL1 was lost in six glioma cell lines and 15 primary glioma tissues at both RNA and protein levels. Re-expression of NECL1 into glioma cell line U251 would repress cell proliferation in vitro by inducing cell cycle arrest. And also NECL1 could decrease the growth rate of tumors in nude mice in vivo. To further investigate the mechanism why NECL1 was silenced in glioma, the basic promoter region located at −271 to +81 in NECL1 genomic sequence was determined. DNA bisulfite sequencing was performed to study the methylation status of CpG islands in NECL1 promoter; however, no hypermethylated CpG site was found. Additionally, the activity of histone deacetylase (HDACs) in glioma was higher than that in normal brain tissues, and the expression of NECL1 in glioma cell lines could be reactivated by HDACs inhibitor-Trichostatin A (TSA). So the loss of NECL1 in glioma was at least partly caused by histone deacetylation. Luciferase reporter assays, chromatin immunoprecipitation and co-immunoprecipitation (co-IP) assays indicated that Sp1 played an important role in this process by binding to either HDAC1 in untreated glioma cells or p300/CBP in TSA treated cells. Our finding suggests that NECL1 may act as a tumor suppressor in glioma and loss of it in glioma may be caused by histone deacetylation. © 2008 Wiley-Liss, Inc.

Here we report the negative correlation of phosphorylation of Smad1 and BMPR-IB expression with the development of human glioma. Western blot analysis showed that expression of both phospho-Smad1/5/8 and BMPR-IB were decreased in malignant glioma tissues compared with normal brain tissues. Kaplan-Meier survival curves revealed that lower expression ratio of phospho-Smad1/5/8 to Smad1 expression significantly correlates with poor patient survival. Transient transfection of BMPR-IB activates Smad1 signaling and induces differentiation and apoptosis of U251 and U87 glioblastoma cells. The effects could be blocked by cotransfection of Smad6. These results might provide new molecular marker and target for glioma diagnosis and therapy.

Nectin-like 1 (Necl-1)is a neural-specific cell adhesion molecule that is expressed in both the CNS and PNS. Previousin vitrostudies suggested thatNecl-1expression is essential for the axon-glial interaction and myelin sheath formation in the PNS. To investigate thein vivorole ofNecl-1in axonal myelination of the developing nervous system, we generated theNecl-1mutant mice by replacing axons 2–5 with the LacZ reporter gene. Expression studies revealed thatNecl-1is exclusively expressed by neurons in the CNS. Disruption ofNecl-1resulted in developmental delay of axonal myelination in the optic nerve and spinal cord, suggesting thatNecl-1plays an important role in the initial axon-oligodendrocyte recognition and adhesion in CNS myelination.

The TH (tyrosine hydroxylase) gene encodes the rate-limiting enzyme of catecholamine biosynthesis, and is involved in the pathogenesis of hypertension, but the relationship of its variants with hypertension has not been extensively studied. We designed a case-controlled study consisting of 503 HT (hypertensive) individuals and 490 NT (normotensive) individuals matched by region, age and gender to systematically investigate the association between the TH gene and hypertension. Based on the HapMap and dbSNP (where SNP is single nucleotide polymorphism) data, four SNPs, rs6356 A>G, rs6357 G>A, rs2070762 T>C and rs1800033 A>G in the TH gene were selected for genotyping. Rs1800033 was not polymorphic in our study population. No significant differences were observed for distributions of rs6356 and rs6357 between the HT and NT groups. However, both the genotype and allele frequencies of rs2070762 showed significant differences between cases and controls (P

Human DIXDC1 is a member of Dishevelled-Axin (DIX) domain containing gene family which plays important roles in Wnt signaling and neural development. In this report, we first confirmed that expression of Ccd1, a mouse homologous gene of DIXDC1, was up-regulated in embryonic developing nervous system. Further studies showed that Ccd1 was expressed specifically in neurons and colocalized with early neuronal marker Tuj1. During the aggregation induced by RA and neuronal differentiation of embryonic carcinoma P19 cells, expressions of Ccd1 as well as Wnt-1 and N-cadherin were dramatically increased. Stable overexpression of DIXDC1 in P19 cells promoted the neuronal differentiation. P19 cells overexpressing DIXDC1 but not the control P19 cells could differentiate into Tuj1 positive cells with RA induction for only 2 days. Meanwhile, we also found that overexpression of DIXDC1 facilitated the expression of Wnt1 and bHLHs during aggregation and differentiation, respectively, while inhibited gliogenesis by down-regulating the expression of GFAP in P19 cells. Thus, our finding suggested that DIXDC1 might play an important role during neurogenesis, overexpression of DIXDC1 in embryonic carcinoma P19 cells promoted neuronal differentiation, and inhibited gliogenesis induced by retinoic acid.

Post-translational modification by ubiquitin (Ub) and Ub-like modifiers is one of the most important mechanisms regulating a wide range of cellular processes in eukaryotes. Through mediating 26S proteasome-dependent degradation of substrates, the covalent modification of proteins by multiple Ub (ubiquitination) can regulate many different cellular functions such as transcription, antigen processing, signal transduction and cell cycle. To better understand ubiquitination and its functions, proteomic approaches have been developed to purify and identify more protein substrates. The S5a subunit of the 26S proteasome binds to poly-Ub chains containing four or more Ub. In this study, immobilized GST-S5a fusion protein was used to affinity-purify ubiquitinated proteins from Chang liver cells. The purified proteins were then identified with multi-dimensional LC combined with MS/MS. Eighty-three potential ubiquitination substrates were identified. From these proteins, 19 potential ubiquitination sites on 17 potential substrates were determined. These potential ubiquitination substrates are mainly related to important cellular functions including metabolism, translation and transcription. Our results provide helpful information for further understanding of the relationship between ubiquitination machinery and different cell functions.

Nectin-like molecule 1 (NECL1)/CADM3/IGSF4B/TSLL1/SynCAM3, from now on referred to as NECL1, is a neural tissue-specific immunoglobulin-like cell–cell adhesion molecule which has Ca2+-independent homo- or heterophilic cell–cell adhesion activity and plays an important role in the formation of synapses, axon bundles and myelinated axons. Here we first detected the expression of NECL1 in human fetal and adult brains, and mouse brains at different developmental stages. The results indicated that two bands with molecular weights of about 62 kDa and 48 kDa were found in human fetal brain, while only one band with a molecular weight of about 48 kDa was found in human adult brain; two bands with molecular weights of about 62 kDa and 48 kDa whose expression level gradually increased were also found from mouse E16 to P14, while only one band with a molecular weight of about 48 kDa was found from P14. Bioinformatics analysis showed there were two putative N-glycosylation sites within human NECL1 at positions N25LS and N290KS and within mouse Necl1 at positions N23LS and N288KS, respectively. There was no O-glycosylation site in either human NECL1 or mouse Necl1. Based on the results of N-Glycosidase F treatment with human fetal brain tissue and lysates from transient transfection with human wild-type or glycosylation site mutant NECL1 in 293ET cells, we demonstrated that human NECL1 is an N-linked glycoprotein with a single glycosylation site at position N290KS. Cell aggregation assay further showed there was an increased adhesion activity after the glycosylation site mutation of NECL1 molecule.

Tumor necrosis factor alpha (TNFalpha) triggers a signaling pathway converging on the activation of NF-kappaB, which forms the basis for many physiological and pathological processes. In a kinase gene screen using a NF-kappaB reporter, we observed that overexpression of casein kinase 1alpha (CK1alpha) enhanced TNFalpha-induced NF-kappaB activation, and a CK1alpha kinase dead mutant, CK1alpha (K46A), reduced NF-kappaB activation induced by TNFalpha. We subsequently demonstrated that CK1alpha interacted with receptor interacting protein 1 (RIP1) but not with TRADD, TRAF2, MEKK3, IKKalpha, IKKbeta, or IKKgamma in mammalian cells. RIP1 is an indispensable molecule in TNFalpha/NF-kappaB signaling. We demonstrated that CK1alpha interacted with and phosphorylated RIP1 at the intermediate domain. Finally, we showed that CK1alpha enhanced RIP1-mediated NF-kappaB activation. Taken together, our studies suggest that CK1alpha is another kinase that regulates RIP1 function in NF-kappaB activation.

MicroRNAs are a class of small endogenous noncoding RNAs which play important regulatory roles mainly by post-transcriptional depression. Finding miRNA target genes will help a lot to understand their biological functions. We developed an ensemble machine learning algorithm which helps to improve the prediction of miRNA targets. The performance was evaluated in the training set and in FMRP associated mRNAs. Moreover, using human mir-9 as a test case, our classification was validated in 9 of 15 transcripts tested. Finally, we applied our algorithm on the whole prediction data set provided by miRanda website. The results are available at http://www.biosino.org/~kanghu/mRTP/mRTP.html.

Renalase, a novel flavin adenine dinucleotide-dependent amine oxidase, is secreted by the kidney, degrades circulating catecholamines, and modulates cardiac function and systemic blood pressure (BP). Its discovery may provide novel insights into the mechanisms of BP regulation and the pathogenesis of essential hypertension (EH). We designed a two-stage case-control study to investigate whether the renalase gene harbored any genetic variants associated with EH in the northern Han Chinese population. From the International Collaborative Study of Cardiovascular Disease in Asia (InterASIA in China), 1,317 hypertensive cases and 1,269 normotensive controls were recruited. These total 2,586 subjects were taken as the main study population in this study. In stage 1, all the eight selected single nucleotide polymorphisms (SNPs) of the renalase gene were genotyped and tested within a subsample (503 cases and 490 controls) of the main study population. By single locus analyses, three SNPs, rs2576178, rs2296545, and rs2114406, showed significant associations with EH (P < 0.05). In stage 2, these three SNPs were genotyped on the remaining individuals and analyzed using all the individuals. After Bonferroni correction for multiple comparisons, the associations of rs2576178 and rs2296545 with EH were still significant in stage 2. The cases had higher frequencies of rs2576178 G allele and rs2296545 C allele than the controls (0.55 versus 0.49, P < 0.0001; 0.61 versus 0.55, P < 0.0001). Particularly, under the codominant model, the adjusted odds ratios for rs2576178 GG genotype and rs2296545 CC genotype were 1.58 (95% CI, 1.25 to 2.00; P = 0.0002) and 1.61 (95% CI, 1.26 to 2.04; P = 0.0002), respectively. We also found risk-associated haplotypes and diplotypes, which further confirmed the significant association between the renalase gene and EH. These findings may provide novel genetic susceptibility markers for EH and lead to a better understanding of EH pathophysiology. In addition, further replications in other populations and functional studies would be warranted.

Accumulating evidence has suggested that the basic helix-loop-helix transcription factors play important roles in controlling neuronal fate specification and differentiation in the developing central nervous system. In this study, we report a detailed immunological study on the expression of Bhlhb5 in embryonic mouse spinal cord with a newly developed antibody. At the early stage of neural development, Bhlhb5 is specifically expressed in dI6 dorsal interneurons and in V1 and V2 ventral interneurons. At late stages of development, Bhlhb5 expression is detected in a subset of late-born dorsal association interneurons that migrate into the uppermost layer of the dorsal horn. Developmental Dynamics 236:829–835, 2007. © 2007 Wiley-Liss, Inc.

RNA-RNA and protein-RNA interactions are essential for post-transcriptional regulation in normal development and may be deregulated in cancer initiation and progression. The RNA-binding protein PCBP2, an oncogenic protein in human malignant gliomas, is an essential regulator of mRNA and miRNA biogenesis, stability and activity. Here, we identified Rho GDP dissociation inhibitor α (ARHGDIA) as a target mRNA that binds to PCBP2, and we uncovered the role of ARHGDIA as a putative metastasis suppressor through analyses of in vitro and in vivo models of EMT and metastasis. Furthermore, we demonstrated that ARHGDIA is a potential target of miR-151-5p and miR-16 in gliomas. The interaction between PCBP2 and the 3'UTR of the ARHGDIA mRNA may induce a local change in RNA structure that favors subsequent binding of miR-151-5p and miR-16, thus leading to the suppression of ARHGDIA expression. PCBP2 may facilitate miR-151-5p and miR-16 promotion of glioma cell migration and invasion through mitigating the function of ARHGDIA.

Tropomyosin-related kinase (Trk) family receptors are a group of high affinity receptors for neurotrophin growth factors, which have pivotal functions in many physiological processes of nervous system. Trk receptors can dimerize and autophosphorylate upon neurotrophin stimulation, then recruit multiple adaptor proteins to transduct signal. In this report, we identified Dok5, a member of Dok family, as a new substrate of TrkB/C receptors. In yeast two-hybrid assay, Dok5 can interact with intracellular domain of TrkB and TrkC receptor through its PTB domain, but not with that of TrkA receptor. The interaction was then confirmed by GST pull-down assay and Co-IP experiment. Dok5 co-localized with TrkB and TrkC in differentiated PC12 cells, providing another evidence for their interaction. By using mutational analysis, we characterized that Dok5 PTB domain bound to Trk receptor NPQY motif in a kinase-activity-dependent manner. Furthermore, competition experiment indicated that Dok5 competed with N-shc for binding to the receptors at the same site. Finally, we showed that Dok5 was involved in the activation of MAPK pathway induced by neurotrophin stimulation. Taken together, these results suggest that Dok5 acts as substrate of TrkB/C receptors and is involved in neurotrophin induced MAPK signal pathway activation.

SARS‐CoV is a newly identified coronavirus (CoV) that causes severe acute respiratory syndrome (SARS). The SARS‐CoV nucleocapsid (N) protein is an important structural and functional protein. To identify cellular proteins that interact with the SARS‐CoV N protein and to elucidate the possible involvement of N protein in SARS‐CoV pathogenesis, a human lymphocyte cDNA library was screened using a yeast two‐hybrid system assay. hUbc9, a ubiquitin conjugating enzyme of sumoylation system, was found to interact specifically with the N protein, implying the post‐translational sumoylation of the N protein. Mapping studies localized the critical N sequences for this interaction to amino acids 170–210, which includes the SR‐rich motif. However, the consensus motif of sumoylation GK62EE in the N protein is not responsible for binding to hUbc9. Mutations of hUbc9 at the enzyme active site C93A or C93S severely impair the interaction with the N protein. The two proteins were also shown to colocalize in the cytoplasm of the transfected 293T cells. This is the first report demonstrating the interaction of hUbc9 with a structural protein of plus‐strand RNA viruses, indicating a new drug target for SARS‐CoV. J. Med. Virol. 78:1365–1373, 2006. © 2006 Wiley‐Liss, Inc.

We previously reported a significant linkage between human chromosome 8p22 with essential hypertension and systolic blood pressure levels. On the basis of this, we used an efficient age, sex and area-matched case-control scheme to test the association of the polymorphisms in the human alpha1A adrenergic receptor (ADRA1A) gene, located on chromosome 8p21-p11.2, with essential hypertension in a northern Han Chinese population.Seven polymorphisms were identified by direct sequencing of genomic DNA derived from 48 randomly recruited hypertensive and 48 healthy subjects. They were also examined for association with essential hypertension in 480 stage 2 hypertensive individuals and their individually matched controls.We observed significantly higher frequencies of the 347Arg allele and 2547G alleles in the cases compared with their controls (P = 0.04 and 0.007, respectively). McNemar's test revealed that carriers of 2547G alleles were at a greater risk of essential hypertension with an odds ratio of 3.00 [95% confidence interval (CI) 1.23-8.35]. We then performed a conditional logistic regression to adjust the effects of conventional risk factors, revealing an odds ratio of 2.84 for carriers of the 2547G allele (95% CI 1.15-6.99). With the haplotypic probabilities estimated using PHASE software, we performed haplotype trend regression analysis, showing a significant association between haplotype 7 and essential hypertension (P = 0.02), after adjustment for conventional risk factors.Our findings suggest that the genetic variations in the ADRA1A gene are significantly associated with essential hypertension, and may play an important role in the development of essential hypertension in this Chinese population.

Udgivelsesdato: 2006-Apr-27 After the completion of a draft human genome sequence, the International Human Genome Sequencing Consortium has proceeded to finish and annotate each of the 24 chromosomes comprising the human genome. Here we describe the sequencing and analysis of human chromosome 3, one of the largest human chromosomes. Chromosome 3 comprises just four contigs, one of which currently represents the longest unbroken stretch of finished DNA sequence known so far. The chromosome is remarkable in having the lowest rate of segmental duplication in the genome. It also includes a chemokine receptor gene cluster as well as numerous loci involved in multiple human cancers such as the gene encoding FHIT, which contains the most common constitutive fragile site in the genome, FRA3B. Using genomic sequence from chimpanzee and rhesus macaque, we were able to characterize the breakpoints defining a large pericentric inversion that occurred some time after the split of Homininae from Ponginae, and propose an evolutionary history of the inversion.

Nectins are Ca(2+)-independent immunoglobulin (Ig) superfamily proteins that participate in the organization of epithelial and endothelial junctions. Nectins have three Ig-like domains in the extracellular region, and the first one is essential in cell-cell adhesion and plays a central role in the interaction with the envelope glycoprotein D of several viruses. Five Nectin-like molecules (Necl-1 through -5) with similar domain structures to those of Nectins have been identified. Necl-1 is specifically expressed in neural tissue, has Ca(2+)-independent homophilic and heterophilic cell-cell adhesion activity, and plays an important role in the formation of synapses, axon bundles, and myelinated axons. Here we report the first crystal structure of its N-terminal Ig-like V domain at 2.4 A, providing insight into trans-cellular recognition mediated by Necl-1. The protein crystallized as a dimer, and the dimeric form was confirmed by size-exclusion chromatography and chemical cross-linking experiments, indicating this V domain is sufficient for homophilic interaction. Mutagenesis work demonstrated that Phe(82) is a key residue for the adhesion activity of Necl-1. A model for homophilic adhesion of Necl-1 at synapses is proposed based on its structure and previous studies.

The Shigella bacteria cause bacillary dysentery, which remains a significant threat to public health. The genus status and species classification appear no longer valid, as compelling evidence indicates that Shigella, as well as enteroinvasive Escherichia coli, are derived from multiple origins of E.coli and form a single pathovar. Nevertheless, Shigella dysenteriae serotype 1 causes deadly epidemics but Shigella boydii is restricted to the Indian subcontinent, while Shigella flexneri and Shigella sonnei are prevalent in developing and developed countries respectively. To begin to explain these distinctive epidemiological and pathological features at the genome level, we have carried out comparative genomics on four representative strains. Each of the Shigella genomes includes a virulence plasmid that encodes conserved primary virulence determinants. The Shigella chromosomes share most of their genes with that of E.coli K12 strain MG1655, but each has over 200 pseudogenes, 300 approximately 700 copies of insertion sequence (IS) elements, and numerous deletions, insertions, translocations and inversions. There is extensive diversity of putative virulence genes, mostly acquired via bacteriophage-mediated lateral gene transfer. Hence, via convergent evolution involving gain and loss of functions, through bacteriophage-mediated gene acquisition, IS-mediated DNA rearrangements and formation of pseudogenes, the Shigella spp. became highly specific human pathogens with variable epidemiological and pathological features.

Background & Aims: Overexpression of cyclooxygenase-2 (COX-2) is implicated in many steps of cancer development. Single nucleotide polymorphisms (SNPs) in the COX-2 promoter might contribute to differential COX-2 expression and subsequent interindividual variability in susceptibility to cancer. This study sought to identify functional SNPs in the COX-2 promoter and evaluated their effects on the risk of developing esophageal squamous cell carcinoma (ESCC). Methods: Thirty individual DNA samples were sequenced to search for SNPs, and the function of the SNPs was examined by a set of biochemical assays. Genotypes and haplotypes were analyzed in 1026 patients and 1270 controls, and odds ratios and 95% confidence intervals (CIs) were estimated by logistic regression. Results: Three SNPs, −1290A→G, −1195G→A, and −765G→C, were identified; the frequencies of variant alleles were 0.04, 0.51, and 0.02, respectively. The −1195G→A change creates a c-MYB binding site and displays a higher promoter activity. The −1195A-containing haplotypes had significantly increased luciferase expression and COX-2 messenger RNA levels in esophageal tissues compared with the −1195G-containing counterparts. A case-control analysis showed a 1.72-fold (95% CI, 1.35–2.20) and 2.24-fold (95% CI, 1.59–3.16) excess risk of developing ESCC for the −1195AA or −765CC genotype carriers compared with noncarriers. A greater risk of developing ESCC was observed for A−1195-C−765-containing haplotypes compared with G−1195-G−765-containing haplotypes, suggesting an interaction between the −1195G→A and −765G→C polymorphisms in the context of haplotype. Conclusions: These findings indicate that genetic variants in COX-2 may play a role in mediating susceptibility to esophageal cancer.

The SARS-nsp13 protein was identified as an mRNA cap1 methyltransferase. In this study, the nsp13 gene was cloned from the SARS-CoV PUMC02 strain viral RNA by RT-PCR, and inserted into the expression plasmid pET30a(+). The recombinant plasmid pET30a(+)-nsp13 was confirmed by restriction enzymes and sequencing analysis, and transformed into Escherichia coli BL21(DE3). The His-tag-fused protein was expressed by induction of 0.5mM IPTG and purified by a single Ni(2+) affinity chromatography. The protein was validated by western blot and MS analysis. A large quantity of the nsp13 protein obtained with this method may be useful for further study of its structure and function.

Summary Several recent studies have linked human chromosome 1p to essential hypertension (EH) or blood pressure (BP) levels. In an independent population of 148 hypertensive families from China we tested these findings. Thirty highly informative microsatellite markers spanning about 284 cM were genotyped. Qualitative linkage analysis was conducted using non-parametric linkage analysis implemented within the GENEHUNTER 2.0 software, whereas quantitative analysis was performed with the variance-component method integrated in the S.O.L.A.R. 1.7.4. software with an additional Haseman-Elston method using the SAGE/SIBPAL2 program. We observed suggestive linkage between D1S2890 (1p31, 80.9 cM) and hypertension using the multipoint non-parametric linkage analysis (NPL = 2.19, P = 0.01). In the quantitative analysis we didn't observe a significant excess of identity-by-descent allele sharing between the systolic blood pressure levels and the markers. However, the D1S207 microsatellite marker (1p21) which is located about 107 cM from the telomere of 1p showed weak linkage evidence with the diastolic blood pressure levels (LOD = 1.42). These findings suggest linkage of 1p31 with essential hypertension in the ethnic Chinese, and provide a potential clue for future studies involving candidate genes for hypertension.

Nervous system polycomb 1 (NSPc1) shares high homology with vertebrate PcG proteins Mel-18 and Bmi-1. The mRNA of NSPc1 is highly expressed in the developmental nervous system [Mech. Dev. 102 (2001) 219–222]. However, the functional characterization of NSPc1 protein is not clear. In the present study, using Western blotting technique, we aimed to describe the distributions of NSPc1 protein in rat tissues and cell lines. The subcellular localization of NSPc1 was examined in HeLa and SH-SY5Ycell lines, and its transcriptional repression activity was examined in COS-7 cell line. We found that the NSPc1 protein was localized mainly in the nucleus. NSPc1 remarkably repressed the transcription. Most interestingly, both the C-terminal of NSPc1 and two phosphorylation sites in the C-terminal, especially the PKC phosphorylation site at S183, were important in mediating transcription repression. Taken together, results from our study suggest that NSPc1, as a typical PcG family member, has powerful transcriptional repression ability, which may be related to the PKC signaling pathway.

We report the results of a genome-wide scan conducted in 219 individuals from 34 large multiplex nuclear pedigrees from the northern Han Chinese population at an average resolution of about 10 cM. Nonparametric two-point and multipoint linkage analyses were performed to detect evidence of linkage with type 2 diabetes in this study. On chromosome 1 four regions showed evidence of linkage with type 2 diabetes in northern Han Chinese. Of these regions a marker D1S193 (73 cM) showed evidence of linkage (two-point nonparametric linkage 2.409), and another region (around 190 cM) was a replication of several other studies performed in different ethnic populations. Evidences of linkage have been confirmed by typing additional markers (average distance 1-5 cM) flanking these two positive regions on chromosome 1. We also found indication of linkage with type 2 diabetes on chromosomes 2, 10, 12, 18, 20, and 22 by two-point linkage analyses.

Serum C-reactive protein (CRP) has been strongly implicated in the pathogenesis of coronary heart disease (CHD). We report here on the association between gene coding for CRP and CHD in the ethnic Han population of China. For this purpose two polymorphisms of -717A>G and +2147A>G of the CRP gene were identified by direct sequencing of genomic DNA derived from 48 randomly selected patients and these were further investigated for associations with CHD in 619 male patients and 615 age-matched male normal controls. The frequency of A allele carriers of -717A>G polymorphism was significantly higher in patients than in controls by univariate analysis. After controlling for other risk factors the association between this polymorphism and CHD remained significant by multivariate logistic regression analysis. Individuals carrying the -717A allele had an approx. 6.8-fold higher risk of developing CHD (odds ratio 6.84 compared with those not carrying this allele). Haplotype analysis confirmed the results of individual polymorphism analyses. However, the resolution of effect size is poor, which may be due to the deficiency in sample size of this study. Neither polymorphism was observed to have an influence on serum CRP level. Since the frequency difference between CHD cases and controls for the -717A allele carriers is only 2.28%, and homozygosity for -717G occurs in only 1.78% of subjects, -717 A>G polymorphism is not a major determinant of population genetic risk of CHD in the Chinese population. The association of this polymorphism with CHD supports the belief that carriers of -717A allele of the CRP gene are genetically predisposed to CHD in the Chinese Han population, and it remains possible that this polymorphism is in disequilibrium with one as yet unidentified functional polymorphism in the vicinity.

Neuroendocrine-specific protein (NSP) -reticulons are endoplasmic reticulum-associated protein complexes, which are localized in the endoplasmic reticulum (ER) and identified as markers for neuroendocrine differentiation. In the present study, human reticulon 3 gene (hRTN3) was cloned and its expression pattern in a variety of tissues was investigated. Truncated hRTN3s corresponding to the C-terminal (hRTN3-C) domain were expressed and purified. hRTN3 mRNA was down-regulated during the differentiation of human neuroblastoma cell line SH-SY5Y induced by all-trans-retinoic acid (RA), which suggests that, like other members of the reticulon family, hRTN3 is a potential marker for neuroendocrine differentiation.

Objective— An extensive association analysis of PON gene cluster (PONs) with coronary heart disease (CHD) was performed in Chinese Han population. Methods and Results— Thirty polymorphisms of PON1, PON2, and PON3 gene were identified by direct sequencing of genomic DNA derived from 48 randomly selected patients. Twelve polymorphisms were additionally investigated for association with CHD in 474 male patients and 475 controls. Univariate analyses showed the cases had significantly higher frequencies of PON1 192Q allele, 160R allele, −162A allele, and PON2 311C allele than were seen in the controls. Logistic regression analyses revealed only the PON1 R160G and −162G/A polymorphisms remained significantly associated with CHD ( P =0.0054 and P =0.0002). Haplotype analyses for various polymorphism combinations additionally confirmed the results of individual polymorphism analyses. Only the frequencies of haplotypes containing −162A allele were significantly higher, whereas only the frequencies of haplotypes containing 160G allele were significantly lower in cases than in controls in various polymorphism combinations. Conclusions— This extensive association study has identified the PON1 −162G/A and R160G polymorphisms to be independently associated with CHD in Chinese Han population and warrants additional study to elucidate the biological mechanism.

Objectives Several genome-wide scans recently accomplished in the ethnic Chinese revealed a number of candidate loci possibly contributing to essential hypertension, and some appeared to be replicable in 2q14-q23 and 5q32. The current study aimed to examine the linkage of qualitative and blood pressure quantitative traits in essential hypertension with these genomic regions in a large sample of Chinese hypertensive families. Methods We performed a genetic analysis on 148 randomly ascertained families containing 328 affected sib pairs, grouped into two geographically distinct subsets. Five highly informative microsatellite markers (D2S151, D2S142, D5S2090, D5S413 and D5S2013) were genotyped, and linkage analyses were performed with different genetic models. Results We did not observe consistent evidence for excess allele sharing identity by descent in either of the qualitative or the quantitative test. However, higher LOD scores were found at D5S2013 in North Group subset with Haseman-Elston and maximum likelihood (ML) variance (no dominance variance, NDV) algorithms. With the ML (NDV) algorithm, the LOD was 1.410 for diastolic blood pressure at this locus, although this was not statistically significant. Conclusions These findings provide no evidence to support a significant linkage of 2q14-q23 or 5q32 with essential hypertension or blood pressure quantitative traits in the ethnic Chinese, and indicate the aetiologic diversity and complexity of hypertension. Previous reports implied 2q14-q23 or beta 2- adrenergic receptor gene potentially linked to essential hypertension in the ethnic Chinese. To replicate these results and perform quantitative linkage analysis, we genotyped members of 148 hypertensive families with five highly informative microsatellite markers. We observed no evidence of excess allele sharing identity by descent in sib pairs, revealing a lack of linkage between 2q14-q23 or 5q32 (chromosome region harboring the gene encoding beta 2 adrenergic receptor) and hypertension in our study sample.

We have sequenced the genome of Shigella flexneri serotype 2a, the most prevalent species and serotype that causes bacillary dysentery or shigellosis in man. The whole genome is composed of a 4 607 203 bp chromosome and a 221 618 bp virulence plasmid, designated pCP301. While the plasmid shows minor divergence from that sequenced in serotype 5a, striking characteristics of the chromosome have been revealed. The S.flexneri chromosome has, astonishingly, 314 IS elements, more than 7-fold over those possessed by its close relatives, the non-pathogenic K12 strain and enterohemorrhagic O157:H7 strain of Escherichia coli. There are 13 translocations and inversions compared with the E.coli sequences, all involve a segment larger than 5 kb, and most are associated with deletions or acquired DNA sequences, of which several are likely to be bacteriophage-transmitted pathogenicity islands. Furthermore, S.flexneri, resembling another human-restricted enteric pathogen, Salmonella typhi, also has hundreds of pseudogenes compared with the E.coli strains. All of these could be subjected to investigations towards novel preventative and treatment strategies against shigellosis.

We presented a procedure to identify novel protein-coding genes from human fetal hippocampus ESTs database generated in our previous study. The protocol is involved in searching the GenBank non-redundant (nr) protein database with ESTs coding regions query. We used ESTScan program to detect and reconstruct coding regions in the ESTs sequence data. With almost 10,000 raw ESTs analyzed, 4,061 coding regions were extracted. The corresponding amino acid sequences were then searched against the GenBank nr-protein databases; all the similarity hits were parsed in a homology analysis workbench. 22 ESTs were identified as the “seed” ESTs, which might be the fragment of the novel protein-coding genes. Indeed, further analysis of these ESTs in full-length cDNA cloning in silico, as well as the mapping information in UCSC and the added experimental proof, verified the effectiveness of the strategy. Taking advantage of rare ESTs resources, and the effective strategy, we would explore more novel genes in the human genome. [The ESTs sequence data described in this paper have been submitted to the GenBank data library (http://www.ncbi.nlm.nih.gov/dbEST) under accession nos. BM174886-958, BM259607-9; and the full length cDNA sequence can be obtained in http://www.chgb.org.cn/hippocampus.htm]

Thioredoxin is a ubiquitous dithiol oxidoreductase found in many organisms and involved in numerous biochemical processes. Human thioredoxin-like protein (hTRXL) is differentially expressed at different development stages of human fetal cerebrum and belongs to an expanding family of thioredoxins. We have solved the crystal structure of the recombinant N-terminal catalytic domain (hTRXL-N) of hTRXL in its oxidized form at 2.2-A resolution. Although this domain shares a similar three-dimensional structure with human thioredoxin (hTRX), a unique feature of hTRXL-N is the large number of positively charged residues distributed around the active site, which has been implicated in substrate specificity. Furthermore, the hTRXL-N crystal structure is monomeric while hTRX is dimeric in its four crystal structures (reduced, oxidized, C73S and C32S/C35S mutants) reported to date. As dimerization is the key regulatory factor in hTRX, the positive charge and lack of dimer formation of hTRXL-N suggest that it could interact with the acidic amino-acid rich C-terminal region, thereby suggesting a novel regulation mechanism.

LHX4 gene is a member of the LIM-homeobox gene family and plays a critical role in the development of motor neurons. We have isolated a cDNA of human LHX4 from a library of the adult human spinal cord. Its sequence is 92% homologous to that of the mouse Lhx4. The genomic structure of the LHX4 gene and its chromosomal localization were determined. The gene was mapped on human chromosome 1q 24.1-1q 24.3 and composed of six exons. The homeodomain was encoded by two exons, exons 4 and 5. The first LIM domain was coded by exon 2, and the second by exon 3. Human MTE Array was used to study the expression profile of LHX4 in 72 human tissues. The expression was specific in the CNS including the fetal brain, the spinal cord, and the cerebral cortex. In situ hybridization of the adult rodent CNS showed the abundant expression of LHX4 in the cerebral cortex and motor neurons of the spinal cord. Our results suggest that LHX4 may play a role in the CNS, especially the neocortex and the spinal cord, and provide a basis to investigate potential involvement of the LHX4 gene in human diseases.

Internal ribosomal entry site (IRES)-mediated translation initiation is constitutively activated during stress conditions such as tumorigenesis and hypoxia. The RNA editing enzyme ADAR1 plays an important role in physiology and pathology. Initially, we found that the ADAR1 p150 or p110 transcript levels were decreased in glioma cells compared with normal astrocyte cells. In contrast, protein levels of ADAR1 p110 were significantly upregulated in glioma tissues and cells. This expression pattern indicated translationally controlled regulation. We identified an 885-nt sequence that was located between AUG1 and AUG2 within the ADAR1 mRNA that exhibited IRES-like activity. Furthermore, we confirmed that the translational mode of ADAR1 p110 was mediated by PTBP1 in glioma cells. The protein levels of PTBP1 and ADAR1 were cooperatively expressed in glioma tissues and cells. Knocking down ADAR1 p110 significantly decreased cell proliferation in three types of glioma cells (T98G, U87MG and A172). The removal of a minimal IRES-like sequence in a p150-overexpression construct could effectively abolish p110 induction and resulted in the slight suppression of cell proliferation compared with ADAR1-p150 overexpression in siPTBP1-treated T98G cells. In summary, our study revealed a mechanism whereby ADAR1 p110 can be activated by PTBP1 through an IRES-like element in glioma cells, and ADAR1 is essential for the maintenance of gliomagenesis.

Brain-enriched miR-128 is repressed in glioma cells, and could inhibit the proliferation of gliomas by targeting genes such as E2F3a and BMI1. To identify more targets of miR-128 in glioblastoma multiforme, the pulse stable isotope labeling with amino acids in cell culture (pSILAC) technique was used to test its impact on whole protein synthesis in T98G glioma cells. We successfully identified 1897 proteins, of which 1459 proteins were quantified. Among them, 133 proteins were downregulated after the overexpression of miR-128. Through predictions using various bioinformatics tools, 13 candidate target genes were chosen. A luciferase assay validated that 11 of 13 selected genes were potential targets of miR-128, and a mutagenesis experiment confirmed CBFB, CORO1C, GLTP, HnRNPF, and TROVE2 as the target genes. Moreover, we observed that the expression of CORO1C, TROVE2, and HnRNPF were higher in glioma cell lines compared to normal brain tissues and presented a tendency toward downregulation after overexpression of miR-128 in T98G cells. Furthermore, we have validated that CORO1C, TROVE2, and HnRNPF could inhibit glioma cell proliferation. In sum, our data showed that the integration of pSILAC and bioinformatics analysis was an efficient method for seeking the targets of miRNAs, and plentiful targets of miR-128 were screened and laid the foundation for research into the miR-128 regulation network.

Drosophila ash2 is a member of the trxG gene super family, some human homologues of which are involved in hematopoiesis and leukemia. We report here the identification of the human homologue of Drosophila ash2 and its alternative splicing isoform, ASH2L1 and ASH2L2. ASH2L proteins are 60% homologous to Drosophila ash2. ASH2L also has a zinc finger motif (C2C2) although it is not identical to that in ASH2. Expression profile analysis showed that the amount of ASH2L transcripts is extremely high in fetal liver, testis, and leukemia cell lines with erythroid and megakaryocytic potential such as K562, Hel, and Dami. We treated these cells with differentiation inducers phorbol ester and hemin. We found that ASH2L is downregulated rapidly and dramatically in K562, Hel, and Dami cells during phorbol ester induced differentiation with megakaryocytic features. However, its expression is maintained at a high level during erythroid differentiation of K562 cells induced with hemin. These results suggest that ASH2L plays a role in hematopoiesis and is associated with some special kinds of leukemia.

Copper is one of the most important trace elements to life. HumanHCUTA is a novel cDNA encoding a 156aa protein, which may participate in human copper tolerance system. The HCUTA protein is highly similar to protein CUT A1 ofE. coli. The whole opening reading frame ofHCUTA cDNA was amplified by PCR and cloned into pET28a + express vector, and the HCUTA protein was effectively expressed inE. coli BL21 (DE3).

In order to identify the genes associated with glioblastoma differentiation, some ESTs, expressed differentially in the control cell and the differentiated human glioblastoma cell line BT-325 induced by the all-trans retinoid acid, have been isolated by the method of DDRT-PCR. Of the 46 ESTs sequenced, 19 are from new genes. A full-length 1 535-bp cDNA, termed geneGDR1, has been isolated from the human cDNA library using the probe designed according to one of the novel ESTs, HGBB098. The open reading frame ofGDR1 gene encodes a putative protein containing 334 amino acid residues. Blast against the current GenBank DNA and protein sequence database did not reveal significant homology with any known proteins. RT-PCR shows thatGDR1 mRNA level increased in the differentiated BT-325 cells after being treated with RA. The different expression patterns ofGDR1 mRNA in human tissues have been detected through the multiple tissue Northern blot hybridization.

Thembl (muscleblind) gene ofDrosophila encodes a nuclear protein which contains two Cys3His motifs. The mutation ofmbl gene will disturb the differentiation of all theDrosophila’s photoreceptors. Primers have been designed according to human EST086139, which is highly homologous tombl gene. Human fetal brain cDNA library has been screened and a novel cDNA clone has been obtained. The 2595 bp cDNA, designatedMBLL (muscleblind-like), contains an open reading frame which encodes 255 amino acids and has 4 Cys3His motifs (GenBank Acc. AF061261). The amino acids sequence shares high homology toDrosophila’s mbl. The Northern blot and RNA dot blot hybridization of 43 human adult tissues and 7 fetal tissues show thatMBLL is a widely expressed gene, but the expression amounts differ in these tissues.

After screening human fetal brain cDNA library, a novel human cDNA encoding an RNA-binding protein was cloned and namedGRY-RBP. HumanGRY-RBP cDNA is 2 932 bp in length, and encodes a protein of 623 amino acids with a deduced molecular mass of 69.6 ku. Human GRY-RBP contains three RNA recognition motifs (RRMs) and a glycine (G), tyrosin (Y)-rich C-terminus. Computer searches with sequence database revealed its moderate homology with RRMs of many RRM RNA-binding proteins. In this study, the full coding region and partial non-coding region of mouseGRY-RBP cDNA were amplified by PCR using mouse brain cDNA library as template. Three transcripts ofGry-rbp are expressed in all tissues as shown by Northern blot, but the amounts in heart, planceta and skeletal muscle are considerably higher than those in other tissues.

mRNA differential display (DDRT-PCR) has been used to analyze different human fetal brain tissues of different developmental stages (13- and 33-week). According to the sequence of one EST obtained in this assay, a pair of primers have been designed to screen the arrayed human fetal brain cDNA library. A 1.2-kb cDNA clone has been found. This cDNA consists of an 867 bp open reading frame, a 132 bp 5′ untranslated sequence and a 209 bp 3′ untranslated sequence with a typical polyadenylation signal. The coding region predicts a protein of 289 amino acids. Its N-terminal of 105 residues is highly homologous to human thioredoxin, while no homology has been found in the databases with its C-terminal of 184 residues. Its N-terminal region also contains the conserved active site sequence CGPC (Cys-Gly-Pro-Cys) of thioredoxin. It was named human Thioredoxin-like gene (hTRXL).

Genes encoding proteins with PHD (plant homeodomain) finger motif (C4HC3) are highly conserved fromArabidopis toHomo sapiens. One of the major functions of these genes is regulating the expression of homeotic genes during the stage of embryonic development. They play a role in cell-cycle and cell differentiation and seem to be related with some human malignant diseases, such as leukemia. A human placenta cDNA library has been screened with cDNA probe amplified by PCR. The PCR primers have been designed according to theM96 (a mouse gene encoding a protein with PHD domain) homologous data in dbEST. A 2.1 kb insert fragment in a positive cDNA clone has been isolated and sequenced. This new full-length cDNA is namedPHF2 (GenBank Acc: AF052205). The putative protein composed of 567 aa has two typical PHD fingers at its N-terminus. Meanwhile it is identified that there are several alternatively spliced transcripts ofPHF2 in different human tissues through the PCR amplification, Northern blot hybridization and analysis of genomic DNA structure.

Primers for screening cDNA library have been designed according to EST AA453734 which is corresponding to the mouse LIM domain binding protein Ldbl. Arrayed human fetal brain cDNA library has been screened by PCR and routine hybridization method. A 2398 bp-cD-NA clone has been obtained. The cDNA encodes a 347 amino acids protein highly homologous to the mouse Ldbl,Xenopus Xldbl andDrosophila Chip. It also contains an LIM binding domain and a nuclear localization signal. It has been namedLDB1 ( LIM domain binding protein 1), GenBank accession number is AF052389. Northern blot showed a 2.4 kb band, and the expression amounts ofLDBI in heart, brain and lung were considerably higher than those in other tissues.

In order to learn the mechanism of brain development and differentiation, 90 expressed sequence tags (ESTs) were isolated by differential display from the cerebrum and cerebellum of 13-week and 33-week fetal brains. After searching database, 74 of them represented novel genes, some of them were homologous to the brain development related genes. Using total cDNA probes, 79 of the ESTs and their expression differences in fetal brain were further characterized.

TrkC is a dependence receptor and many reports have shown that neurotrophin-3 promotes cell survival by inhibiting TrkC-induced apoptosis in many cell lines. However, the identity of the adaptor protein involved in the NT-3/TrkC signaling pathway regulating cell death and survival remains unclear. The downstream of tyrosine kinase/docking protein (Dok) adaptor protein 5 is one substrate of the TrkC receptor. Because NT-3 and its receptor, TrkC, are strongly expressed by sensory neurons, we measured the expression of Dok5 and TrkC in the developing mouse spinal cord and dorsal root ganglia (DRG). We found that the number of cells positive for both Dok5 and TrkC decreases with DRG development. Immunoprecipitation and immunofluorescence staining showed that Dok5 interacted with TrkC and partially colocalized with TrkC in DRG neurons. In HEK293T cells, TrkC triggered apoptosis, but NT-3 prevented TrkC-induced apoptosis. Interestingly, siRNA knockdown of Dok5 expression partially prevented the protection of NT-3 against TrkC-induced apoptosis by regulating the activity of caspase-3. Taken together, we concluded that Dok5 is necessary for NT-3 signaling to block TrkC-induced apoptosis.

The timing and progression of axonal myelination are precisely controlled by intercellular interactions between neurons and glia in development. Previous in vitro studies demonstrated that Nectin like 4 (Necl-4, also known as cell adhesion molecule Cadm-4 or SynCAM-4) plays an essential role in axonal myelination by Schwann cells in the peripheral nervous system (PNS). However, the role of Necl-4 protein in axonal myelination in the developing central nervous system (CNS) has remained unknown. In this study, we discovered upregulation of Necl-4 expression in mature oligodendrocytes at perinatal stages when axons undergo active myelination. We generated Necl4 gene knockout mice, but found that disruption of Necl-4 gene did not affect oligodendrocyte differentiation and myelin formation in the CNS. Surprisingly, disruption of Necl-4 had no significant effect on axonal myelination in the PNS either. Therefore, our results demonstrated that Necl-4 is dispensable for axonal myelination in the developing nervous system.

PCBP2 is a member of the poly(C)-binding protein (PCBP) family, which plays an important role in posttranscriptional and translational regulation by interacting with single-stranded poly(C) motifs in target mRNAs. Several PCBP family members have been reported to be involved in human malignancies. Here, we show that PCBP2 is upregulated in human glioma tissues and cell lines. Knockdown of PCBP2 inhibited glioma growth in vitro and in vivo through inhibition of cell-cycle progression and induction of caspase-3-mediated apoptosis. Thirty-five mRNAs were identified as putative PCBP2 targets/interactors using RIP-ChIP protein-RNA interaction arrays in a human glioma cell line, T98G. Four-and-a-half LIM domain 3 (FHL3) mRNA was downregulated in human gliomas and was identified as a PCBP2 target. Knockdown of PCBP2 enhanced the expression of FHL3 by stabilizing its mRNA. Overexpression of FHL3 attenuated cell growth and induced apoptosis. This study establishes a link between PCBP2 and FHL3 proteins and identifies a new pathway for regulating glioma progression.

Gliomas are the most common and deadly type of primary brain tumor. In this study, we showed that cAMP response element-binding protein (CREB), a proto-oncogenic transcription factor that is overexpressed in gliomas, can promote gliomagenesis by modulating the expression of oncogenic microRNA-23a (mir-23a). First, we found that CREB is highly expressed in glioma tissues and cell lines. CREB is also essential for glioma cell growth and cell survival in vitro and is critical for gliomagenesis in vivo. Second, microRNA microarray, ChIP-chip, ChIP-quantitative PCR, and luciferase reporter assays showed that CREB directly binds to the regulatory sequences of mir-23a and enhance the expression of mir-23a. Moreover, mir-23a was confirmed as a functional downstream target of CREB in glioma cell growth and cell survival. Finally, using computational prediction followed by experimental confirmation, we identified PTEN, which is frequently silenced in gliomas, as a downstream target of mir-23a. Taken together, we propose that CREB promotes gliomagenesis and acts as a modulator of oncogenic mir-23a, which represses the tumor suppressor PTEN.

We performed a meta-analysis of 2 genome-wide association studies of coronary artery disease comprising 1,515 cases with coronary artery disease and 5,019 controls, followed by de novo replication studies in 15,460 cases and 11,472 controls, all of Chinese Han descent. We successfully identified four new loci for coronary artery disease reaching genome-wide significance (P < 5 × 10−8), which mapped in or near TTC32-WDR35, GUCY1A3, C6orf10-BTNL2 and ATP2B1. We also replicated four loci previously identified in European populations (PHACTR1, TCF21, CDKN2A/B and C12orf51). These findings provide new insights into biological pathways for the susceptibility of coronary artery disease in Chinese Han population.

Migration-proliferation dichotomy is a common mechanism in gliomagenesis; however, an understanding of the exact molecular mechanism of this “go or grow” phenomenon remains largely incomplete. In the present study, we first found that microRNA-9 (miR-9) is highly expressed in glioma cells. MiR-9 inhibited the proliferation and promoted the migration of glioma cells by directly targeting cyclic AMP response element-binding protein (CREB) and neurofibromin 1 (NF1), respectively. Our data also suggested a migration-inhibitory role for CREB through directly regulating the transcription of NF1. Furthermore, we found that the transcription of miR-9-1 is under CREB's control, forming a negative feedback minicircuitry. Taken together, miR-9 inhibits proliferation but promotes migration, whereas CREB plays a pro-proliferative and anti-migratory role, suggesting that the CREB-miR-9 negative feedback minicircuitry plays a critical role in the determination of “go or grow” in glioma cells.

The Trx domain of human thioredoxin-like protein has been purified and crystallized using ammonium sulfate as precipitant. The crystal belongs to space group C2, with unit-cell parameters a = 87.5, b = 48.5, c = 29.8 A, beta = 99.59 degrees. It has one molecule per asymmetric unit and diffracts beyond 2.2 A under cryoconditions (100 K) using an in-house Cu rotating-anode X-ray generator.