Methylation of tumor associated genes in tissue and plasma samples from liver disease patients

To investigate whether aberrant hypermethylation in plasma DNA could be used as diagnosis makers for hepatocellular carcinoma (HCC), we performed methylation-specific PCR (MSP) to check the methylation status of five tumor associated genes in 36 cases of

Analysis of the effect of copper on the virulence of a pathogenic Edwardsiella tarda strain

Aim: To investigate the effect of copper on the virulence of Edwardsiella tarda. Methods and Results: The pathogenic Edw. tarda strain TX5 was cultured under copper-stressed conditions and examined for any potential alteration in capacities that are associated with pathogenicity. The results showed that compared to untreated TX5, Cu-treated TX5 exhibits reduced planktonic and biofilm growth, an impaired ability to adhere to host mucus, modulation of host immune response, and dissemination in host blood and liver. Consistent with these observations, the overall bacterial virulence of Cu-treated TX5 is significantly attenuated. SDS-PAGE analyses of whole cell protein production showed that Cu-treated TX5 differs from the untreated TX5 in its production of at least one protein. Quantitative real time reverse transcriptase PCR analyses showed that copper treatment decreased the expression of virulence-associated genes encoding components of the type III and type VI secretion systems, the Eth haemolysin system, and the LuxS/AI-2 quorum-sensing system. Conclusions: Prolonged exposure to copper has multiple effects on TX5 and results in significant attenuation of bacterial virulence. Significance and Impact of the Study: The results of this study demonstrate that copper treatment has a broad and profound effect on the virulence-associated capacities of TX5, which is exerted at least in part at the transcription level. These findings provide new insights to the antimicrobial mechanism of copper.

Attenuation of Edwardsiella tarda Virulence by Small Peptides That Interfere with LuxS/Autoinducer Type 2 Quorum Sensing

Edwardsiella tarda is a gram-negative pathogen with a broad host range that includes humans, animals, and fish. Recent studies have shown that the LuxS/autoinducer type 2 (AI-2) quorum sensing system is involved in the virulence of E. tarda. In the present study, it was found that the E. tarda LuxS mutants bearing deletions of the catalytic site (C site) and the tyrosine kinase phosphorylation site, respectively, are functionally inactive and that these dysfunctional mutants can interfere with the activity of the wild-type LuxS. Two small peptides, 5411 and 5906, which share sequence identities with the C site of LuxS, were identified. 5411 and 5906 proved to be inhibitors of AI-2 activity and could vitiate the infectivity of the pathogenic E. tarda strain TX1. The inhibitory effect of 5411 and 5906 on AI-2 activity is exerted on LuxS, with which these peptides specifically interact. The expression of 5411 and 5906 in TX1 has multiple effects (altering biofilm production and the expression of certain virulence-associated genes), which are similar to those caused by interruption of luxS expression. Further study found that it is very likely that 5411 and 5906 can be released from the strains expressing them and, should TX1 be in the vicinity, captured by TX1. Based on this observation, a constitutive 5411 producer (Pseudomonas sp. strain FP3/pT5411) was constructed in the form of a fish commensal isolate that expresses 5411 from a plasmid source. The presence of FP3/pT5411 in fish attenuates the virulence of TX1. Finally, it was demonstrated that fish expressing 5411 directly from tissues exhibit enhanced resistance against TX1 infection.

Attenuation of Edwardsiella tarda Virulence by Small Peptides That Interfere with LuxS/Autoinducer Type 2 Quorum Sensing

Edwardsiella tarda is a gram-negative pathogen with a broad host range that includes humans, animals, and fish. Recent studies have shown that the LuxS/autoinducer type 2 (AI-2) quorum sensing system is involved in the virulence of E. tarda. In the present study, it was found that the E. tarda LuxS mutants bearing deletions of the catalytic site (C site) and the tyrosine kinase phosphorylation site, respectively, are functionally inactive and that these dysfunctional mutants can interfere with the activity of the wild-type LuxS. Two small peptides, 5411 and 5906, which share sequence identities with the C site of LuxS, were identified. 5411 and 5906 proved to be inhibitors of AI-2 activity and could vitiate the infectivity of the pathogenic E. tarda strain TX1. The inhibitory effect of 5411 and 5906 on AI-2 activity is exerted on LuxS, with which these peptides specifically interact. The expression of 5411 and 5906 in TX1 has multiple effects (altering biofilm production and the expression of certain virulence-associated genes), which are similar to those caused by interruption of luxS expression. Further study found that it is very likely that 5411 and 5906 can be released from the strains expressing them and, should TX1 be in the vicinity, captured by TX1. Based on this observation, a constitutive 5411 producer (Pseudomonas sp. strain FP3/pT5411) was constructed in the form of a fish commensal isolate that expresses 5411 from a plasmid source. The presence of FP3/pT5411 in fish attenuates the virulence of TX1. Finally, it was demonstrated that fish expressing 5411 directly from tissues exhibit enhanced resistance against TX1 infection.

中国汉族人群若干疾病相关基因的定位研究

1. 中国一个轴前多指 II/III 型家系候选基因的定位 轴前多指（PPD）是人类众多肢体异常症状中比较常见的一种。前人的研 究把它定位到染色体7q36 上450Kb 的区段内。在这个候选区段内，不同的遗传 背景的几个多指家系的致病突变已被发现，它们位于SHH 基因的顺式调控元件 （ZRS）上，这个调控元件调控SHH 正常的时空表达，对指/趾的正常发育起着 重要的作用。在本研究之中，我们分析了我国一个大的多指家系，这个家系的多 指疾病的遗传方式是常染色体显性遗传并有接近100%的疾病外显率。通过连锁 分析和单倍型构建，我们把这个家系的致病基因定位到染色体7q36 上微卫星标 记D7S2465 和 D7S2423 之间1.7cM 的区域内，这个区域包含了上述的450Kb。 为了确定这个家系的致病突变，我们采用了直接测序的方法，筛查了我们定位的 这个区段内的5 个基因（HLXB9, C7orf2, NOM1, RNF32 和C7orf4）的编码区， SHH 基因的ZRS，C7orf2 基因的第5 内含子，和18 个在多物种中保守的非编码 区段（CNS）。我们的结果表明，在这个中国多指家系中，不是上述的5 个基因 和18 个多物种中保守非编码区段（CNS）的突变导致了多指的表型，也不是其 他研究小组报道的SHH 基因的ZRS 突变导致了多指表型。是否在我们界定的这 1.7cM 的候选区段内还存在着SHH 基因的其他调控元件，它的突变也一样会引 起SHH 基因异常的时空表达，最终导致多指表型的产生还需要进一步的研究来 证实。 2. 中国一个单纯性小眼球家系致病基因的定位 先天性小眼球是一种在临床上具有异质性的眼球发育异常疾病，表型从单 侧眼球体积变小到两侧眼球组织的完全缺失。单纯性小眼球疾病指的是除了眼球 的异常不伴随身体其他组织的异常，其遗传学上的致病机理到目前为止还未完全 清楚。之前的研究表明，不同遗传背景的小眼球家系的致病基因被定位到完全不 同的染色体区段上。本研究中的这个中国单纯性小眼球家系，具有常染色体显性的遗传方式，而且是第一个在分子水平上被研究的中国小眼球家系。为了研究这 个家系的致病基因，我们使用了382 个微卫星标记对这个家系进行了全基因组的 扫描，结果表明这个中国小眼球家系的致病基因定位于2 号染色体长臂上，这个 结果不同于以往报道的任何其他小眼球家系。两点连锁分析在重组率为0 时在微 卫星标记D2S2265 上得到最大值3.290，单倍型分析表明所有的受累个体在染色 体2q11-14 上微卫星标记D2S1890 和 D2S347 之间共享相同的单倍型，所以将 这个家系的致病基因定位到染色体2q11-14 上15 cM 的区段内。我们的结果进一 步提示了小眼球疾病的遗传异质性，并且定位了一个新的关于眼球发育的相关基 因。 3. 19 号染色体C 型凝集素家族与中国南方汉族人群的SARS 易感性研究 2003 年SARS 在全球爆发时，不同的个体在感染后表现出不同的病程和 最终的治疗结果，这被认为是个体本身的遗传因素在这个过程中起了一定的作 用，即不同个体在一些基因上的多态影响了他们对一些疾病的易感性。CD209L 基因是19 号染色体上C 型凝集素家族中的一员，它被证明是SARS 病毒的受体， 并且它的第4 外显子的VNTR 多态被认为和宿主对病毒的易感性有关。但是这 个结论在其后其他两个研究小组的工作未被得到证实。可能的原因有：(1) CD209L 基因本身的多态和宿主对SARS 病毒的易感性无关，而是和它紧密连锁 的其它基因的多态发挥真正的作用；(2)可能是由于以前未检测到的群体分层现 象的存在，使最初的研究得到了一个假阳性的结果。为了探讨这个问题，我们对 19 号染色体上C 型凝集素家族的4 个成员(FCER2, CLEC4G, CD209 和 CD209L) 作了全面的研究，我们采用了检测tagSNPs 的方法，在C 型凝集素家族基因簇上 选取了23 个tagSNPs 位点来代表这个基因簇的多态。我们检测了来自中国香港 的181 个SARS 病人和172 个匹配的正常对照，结果表明C 型凝集素家族的4 个基因跟中国南方汉族群体对SARS 病毒的易感之间没有相关性。同时我们检测 了来自中国不同地区的1145 个汉族样本CD209L 基因VNTR 的多态分布情况， 结果表明这个位点在中国人群中存在群体分层现象，这也解释了之前的研究小组 得到的假阳性结果可能正是由于他们忽略了这个问题而导致的

Characterization of two membrane-associated protease genes obtained from screening out-membrane protein genes of Flavobacterium columnare G(4)

In order to identify genes encoding the outer membrane proteins (OMPs) of the myxobacter Flavobacterium columnare G(4), the expression library of the bacterium was screened by using rabbit antisera developed against its OMPs. Positive colonies of Escherichia coli M15 containing fragments encoding the bacterial OMPs were selected for cloning the relevant genes by genomic walking methods. Two genes encoding a membrane-associated zinc metalloprotease and prolyl oligopeptidase are reported in this paper. The membrane-associated zinc metalloprotease gene (map) is 1800 bp in length, coding for 449 amino acids (aa). Despite the presence of a conserved motif HEXXH for all metalloproteases, the special HEXXH similar to 32 aa similar to E motif of the F. columnare G(4) Map and its low level of identity with other reported zinc-containing metalloproteases may imply that the membrane-associated zinc metalloprotease of F. columnare G(4) represents a new family of zincins. The gene encoding prolyl oligopeptidase (Pop), a serine proteinase, is 2352 bp in length, coding for 649 aa. Sequence homology analysis revealed that the Pop is also novel as it has <50% identity with other reported prolyl oligopeptidase family proteins. The present study represents the first to employ anti-fish bacterial OMP sera to screen genes of membrane-associated proteases of fish pathogenic bacteria, and to provide necessary information for the examination of the role of the two genes in the infection and pathogenesis of F. columnare.

Features and trend of loss of promoter-associated CpG islands in the human and mouse genomes

CpG islands (CGIs) are often considered as gene markers, but the number of CGIs varies among mammalian genomes that have similar numbers of genes. In this study, we investigated the distribution of CGIs in the promoter regions of 3,197 human-mouse ortholo

A common SNP of MCPH1 is associated with cranial volume variation in Chinese population

Microcephaly (MCPH) genes are informative in understanding the genetics and evolution of human brain volume. MCPH1 and abnormal spindle-like MCPH associated (ASPM) are the two known MCPH causing genes that were suggested undergone recent positive selectio

Functional consequences of new exon acquisition in mammalian chromodomain Y-like (CDYL) genes

The origin of new exons is an important mechanism for proteome diversity. Here, we report the recurrent origination of new exons in mammalian chromodomain Y-like (CDYL) genes and the functional consequences associated with the acquisition of the new exons

Polymorphisms in the C-type lectin genes cluster in chromosome 19 and predisposition to severe acute respiratory syndrome coronavirus (SARS-CoV) infection

Background: Polymorphisms of CLEC4M have been associated with predisposition for infection by the severe acute respiratory syndrome coronavirus (SARS-CoV). DC-SIGNR, a C-type lectin encoded by CLEC4M, is a receptor for the virus. A variable number tandem

Molecular evolution of the keratin associated protein gene family in mammals, role in the evolution of mammalian hair

Background: Hair is unique to mammals. Keratin associated proteins (KRTAPs), which contain two major groups: high/ultrahigh cysteine and high glycine-tyrosine, are one of the major components of hair and play essential roles in the formation of rigid and

Positive selection drives population differentiation in the skeletal genes in modern humans

During the course of evolution, the human skeletal system has evolved rapidly leading to an incredible array of phenotypic diversity, including variations in height and bone mineral density. However, the genetic basis of this phenotypic diversity and the relatively rapid tempo of evolution have remained largely undocumented. Here, we discover that skeletal genes exhibit a significantly greater level of population differentiation among humans compared with other genes in the genome. The pattern is exceptionally evident at amino acid-altering sites within these genes. Divergence is greater between Africans and both Europeans and East Asians. In contrast, relatively weak differentiation is observed between Europeans and East Asians. SNPs with higher levels of differentiation have correspondingly higher derived allele frequencies in Europeans and East Asians. Thus, it appears that positive selection has operated on skeletal genes in the non-African populations and this may have been initiated with the human colonization of Eurasia. In conclusion, we provide genetic evidence supporting the rapid evolution of the human skeletal system and the associated diversity of phenotypes.

Migration of mouse antibody-secreting hybridoma cells from blood to genital tract and its regulation by sex hormones are associated with the differential expression patterns of adhesion molecules and chemokines in the tract rather than in the antibody-secreting cells

To understand better the molecular mechanisms of differential migration of antibody-secreting cells (ASCs) into mouse genital tracts, and regulation by sex hormones, surface markers, hormone receptors and adhesion molecules in mouse SG2 and PA4 hybridoma cells, respectively, secreting IgG2b and polymeric IgA antibody were detected by flow cytometry or RT-PCR. Semiquantitative RT-PCR was also used for measuring mRNA expression of adhesion molecules and chemokines (VCAM-1, ICAM-1, P-selectin, JAM-1 and CXCL12) in genital tracts of various adult mouse groups. The mRNAs of androgen receptor, estrogen receptor beta and CXCR4 were expressed in the ASCs. Sex hormones had no effect on expression of these molecules in ASCs. Except for VCAM-1, mRNA of all examined genes was expressed in normal mouse genital tracts. The mean of relative amounts of ICAM-1 and CXCL12 mRNA in all examined organs of females were higher (2.1- and 1.9-fold) than those in males. After orchiectomy or ovariectomy, the expression of ICAM-1, CXCL12 and P-selectin mRNA in the examined organs increased, except JAM-1 in male and CXCL12 in female. Sex hormone treatment recovered the changes to normal levels of mRNA expression in many examined genital tissues. In combination with our previous work, preferential migration of ASCs into female genital tract and regulation of migration by sex hormones are associated with expression patterns of adhesion molecules and chemokines in genital tract rather than in ASCs. (C) 2006 Elsevier Ireland Ltd. All rights reserved.

Genome-wide identification and characterization of cytochrome P450 monooxygenase genes in the ciliate Tetrahymena thermophila

Background: Cytochrome P450 monooxygenases play key roles in the metabolism of a wide variety of substrates and they are closely associated with endocellular physiological processes or detoxification metabolism under environmental exposure. To date, however, none has been systematically characterized in the phylum Ciliophora. T. thermophila possess many advantages as a eukaryotic model organism and it exhibits rapid and sensitive responses to xenobiotics, making it an ideal model system to study the evolutionary and functional diversity of the P450 monooxygenase gene family. Results: A total of 44 putative functional cytochrome P450 genes were identified and could be classified into 13 families and 21 sub-families according to standard nomenclature. The characteristics of both the conserved intron-exon organization and scaffold localization of tandem repeats within each P450 family clade suggested that the enlargement of T. thermophila P450 families probably resulted from recent separate small duplication events. Gene expression patterns of all T. thermophila P450s during three important cell physiological stages (vegetative growth, starvation and conjugation) were analyzed based on EST and microarray data, and three main categories of expression patterns were postulated. Evolutionary analysis including codon usage preference, sit-especific selection and gene-expression evolution patterns were investigated and the results indicated remarkable divergences among the T. thermophila P450 genes. Conclusion: The characterization, expression and evolutionary analysis of T. thermophila P450 monooxygenase genes in the current study provides useful information for understanding the characteristics and diversities of the P450 genes in the Ciliophora, and provides the baseline for functional analyses of individual P450 isoforms in this model ciliate species.

Identification of immune genes in grass carp Ctenopharyngodon idella in response to infection of the parasitic copepod Sinergasilus major

The parasitic copepod Sinergasilus major is an important pathogen of grass carp Ctenopharyngodon idella. To understand the immune response of grass carp to the copepod infection, suppression subtractive hybridization method was employed to characterize genes up-regulation during the copepod infection in liver and gills of the fish. One hundred and twenty-two dot blot positive clones from infected subtracted library were sequenced. Searching available databases by using these nucleotide sequences revealed that 23 genes are immune-related, including known acute-phase reactants, and four novel genes encoding proteins such as source of immunodominant MHC-associated peptides (SIMP), TNF receptor-associated factor 2 binding protein (T2BP), poliovirus receptor-related protein 1 precursor, glycoprotein A repetitions predominant (GARP). The differential expression of seven immune genes, i.e. GARP, alpha-2-macroglobulin, MHC class I, C3, SIMP, T2BP, transferrin, as a result of infection was further confirmed by RT-PCR, with the up-regulation of alpha-2-macroglobulin, MHC class I, C3, SIMP and T2BP in the liver of infected fish, and down-regulation of SIMP in the gills of infected fish. The present study provides foundation for understanding grass carp immune response and candidate genes for further analysis.