新型金属蛋白酶ADAMTS1在急性主动脉夹层中表达增加

急性主动脉夹层是在主动脉中膜变性基础上发生的一种致死性心血管疾病.含Ⅰ型血小板结合蛋白基序的解聚蛋白样金属蛋白酶ADAMTS1是一种新型细胞外金属蛋白酶,它参与动脉粥样硬化等多种血管疾病的发病过程.本研究发现,急性主动脉夹层病人血浆ADAMTS1水平明显高于急性心梗病人和正常对照.通过给老龄小鼠埋泵缓释血管紧张素Ⅱ建立了急性主动脉夹层动物模型.在血管紧张素Ⅱ埋泵后14天,小鼠主动脉夹层的发生率达到42%.血管紧张素Ⅱ给药组小鼠主动脉有大量巨噬细胞和中性粒细胞浸润,免疫荧光显示夹层部位过表达的ADAMTS1与巨噬细胞及中性粒细胞有共定位.此外,与对照组相比,夹层组织部位versican(ADAMTS1降解底物)的降解明显增加,其与ADAMTS1表达增加相一致.结果提示,巨噬细胞和中性粒细胞来源的过表达的ADAMTS1可能通过增加versican的降解促进了急性主动脉夹层的发生发展.     

KEAP1基因及其突变位点对非小细胞肺癌细胞株作用的实验初步研究

摘要 目的：研究KEAP1基因及KEAP1基因突变位点对肺癌细胞株的作用。方法：通过Western blot 方法,比较携带KEAP1 基因突变的肺癌细胞株（A549,NCI-H460,NCI-H838）与KEAP1 基因野生型的肺癌细胞株（NCI-H292, NCI-H1299, 95D, SPC-A1）之间,NRF2基因与NRF2下游基因HO-1的蛋白表达水平,检测并比较两组细胞的活性氧（ROS）含量;以新发现的非小细胞肺癌（NSCLC）病人的 KEAP1 体细胞突变作为模板构建 KEAP1 突变质粒,对自身存在 KEAP1 突变的肺癌细胞株A549,通过改造的 pMSCV 逆病毒转染体系,分别构建过表达空载,野生型及突变型 KEAP1 的 A549 稳定细胞株,比较过表达不同质粒的细胞间丙二醛（MDA）含量及 NRF2 下游抗氧化等相关基因的表达水平;通过克隆形成实验检测细胞增殖情况。结果：KEAP1基因突变组肺癌细胞株与KEAP1基因无突变的对照组细胞株相比,NRF2和HO-1蛋白表达显著增高,活性氧水平显著降低（P<0.01）;过表达野生型KEAP1 与过表达空载的 A549细胞相比,NRF2 及其下游基因转录水平表达显著下降（P<0.01）,蛋白水平表达下降,细胞内丙二醛水平显著增高（P<0.01）,克隆形成率显著降低（P<0.01）,而过表达突变型 KEAP1 与过表达空载的 A549细胞相比,NRF2 及其下游基因表达、细胞内丙二醛水平、克隆形成率均无显著差异（P>0.05）。结论：KEAP1基因具有抑癌作用,其突变为失活型突变,突变后KEAP1/NRF2通路激活,KEAP1基因突变可能通过改变细胞的氧化应激水平,促进肺癌的发生发展。     

白念珠菌唑类药物耐药相关转录因子研究进展

近年来白念珠菌的感染率呈逐年上升趋势,随着唑类药物的广泛应用,耐药菌株不断增多,已成为临床治疗的一大难题.白念珠菌的耐药机制主要与ERG 11基因的突变和过表达、药物外排泵相关基因表达增多及生物膜的形成等有关,由于转录因子是耐药基因表达的关键调节因子,关于锌簇转录因子与耐药关系的研究越来越多,如TAC 1、MRR 1、MRR 2、UPC 2、NDT 80等,其点突变可引起某些耐药基因的过表达而介导耐药,该领域研究已成为热点,该文就此研究进展做一概述.     

肿瘤发生与相关基因突变的关联

肿瘤的发生常与其相关基因的突变有关,这些突变会引起编码基因的蛋白质的结构或数量的改变,从而导致其相关基因功能的丧失。突变在基因上发生的位置及类型的不同也会对基因的表达造成不同的影响。本文介绍了存在于肿瘤相关基因编码区及非编码区的突变与肿瘤发生发展的相互关系。本文还就肿瘤相关基因的突变类型阐述肿瘤的靶向治疗,提供了癌症治疗及预防的新思路。     

常染色体非特异性精神发育迟滞相关基因研究进展

常染色体上一些基因与神经系统的发育和功能密切相关, 突变后可导致非特异性精神发育迟滞。文章从基因定位、表达、生物学功能与突变后致病机理等方面, 对常染色体非特异性精神发育迟滞相关基因的研究现状进行了综述, 并展望了今后这一领域的研究前景。     

限制性酶介导整合技术在盘基网柄菌的应用

1前言突变分析技术在许多功能基因的分离和表达中起着关键的作用,前人已经利用电离辐射、紫外诱变、PCR和转座子等技术对许多模式生物进行了随机突变研究。分离具有特殊细胞分化功能的基因需要建立大群体的随机突变体库,但是用传统方法产生的随机突变体的鉴定与突变表型相关基因的     

慢病毒载体沉默ADAMTS6人非小细胞肺癌稳转株的构建

目的:通过数据库预测ADAMTS6在非小细胞肺癌(Non-small-cell lung cancer,NSCLC)组织中的表达及其与NSCLC患者临床预后的关系,构建ADAMTS6的shRNA干扰载体并建立ADMATS6的NSCLC稳定敲减细胞株。方法:通过Oncomine数据库分析ADAMTS6在NSCLC组织和肺正常组织的表达差异,通过Kaplan-Meier Plotter数据库分析ADAMTS6的表达水平与临床NSCLC患者预后关系,设计合成ADAMTS6的shRNA干扰序列,shRNA模板退火并与双酶切pGLV3-GFP线性化载体连接,转化挑取阳性菌落后送测序。干扰质粒进行病毒包装并感染人NSCLC细胞株NCI-H358,使用嘌呤霉素进行稳定敲减细胞株筛选。荧光观察慢病毒感染细胞密度,通过qRT-PCR和Western blot检测ADAMTS6的mRNA和蛋白水平的敲减效果。结果:Oncomine数据库分析结果显示NSCLC组织中ADAMTS6 mRNA表达较正常肺组织显著升高(P0.001);Kaplan-Meier Plotter数据库分析结果显示高表达ADAMTS6的NSCLC患者预后较低表达ADAMTS6的NSCLC患者差(P0.05);pGLV3-GFP载体双酶切线性化后与shRNA退火模板连接成功,测序结果正确。荧光观察显示慢病毒感染细胞密度在95%左右,通过qRT-PCR和Western blot检测ADAMTS6慢病毒干扰质粒已成功敲减ADAMTS6的m RNA和蛋白水平。结论:ADAMTS6的高表达可能与NSCLC患者的不良临床预后密切相关。本研究构建了ADAMTS6的慢病毒感染质粒,并成功建立NCI-H358稳定敲减细胞株,为进一步研究ADAMTS6在NSCLC中的作用及机制奠定了基础。     

小鼠白内障基因突变方式、分布及基因定位

哺乳动物发育相关的功能基因的鉴定多来源于自发或诱发突变的小鼠。小鼠白内障作为较易鉴定的性状,目前业已明确的突变多达140余个。自发突变的小鼠主要来源于大规模饲养,诱发突变主要通过X射线与ENU处理获得,基因敲除与转基因小鼠亦可获得白内障性状。已知的白内障相关基因分布于小鼠20条染色体,其中以1号染色体最多,并常于小鼠胚胎时期起始表达。小鼠白内障表型多由单基因突变引起,突变的确定主要通过F2代家系全基因组扫描、精细定位、单倍型分型与测序验证等程序。本文从小鼠白内障基因突变来源、基因分布、基因定位与基因表达等角度较为全面的阐述了小鼠先天性白内障的研究进展。     

ptsH基因过表达对乳酸乳球菌N8自身免疫耐受性及其生理代谢的影响

目的:通过基因工程方法提高HPr蛋白编码基因ptsH在乳链菌肽(nisin)高产野生乳酸乳球菌株N8中的表达,揭示ptsH基因与乳酸乳球菌乳链菌肽耐受性等相关生物学功能的关系。方法:构建ptsH过表达质粒pLEV16-ptsH并转化至N8,使其ptsH基因过量表达,进而对比分析ptsH过表达菌株与野生菌株在生长曲线、乳链菌肽耐受性、效价、Biolog等方面的差异。结果:N8-ptsH过表达菌株与N8菌株在菌落形态、大小、表面湿滑程度及生长曲线等方面没有明显差异;ptsH基因过表达使N8菌株的乳链菌肽耐受性提高了8.3%,2个乳链菌肽耐受性相关基因nisI和nisF的表达量分别提高了15.45倍和近45倍;ptsH基因过表达略微减缓了N8菌株中乳链菌肽的产生,但乳链菌肽的最终产量略有提高;ptsH基因过表达菌株中PTS系统糖苷和磷酸化糖类的利用率比原始菌株显著提高。结论:ptsH基因主要与乳酸乳球菌的乳链菌肽耐受性有关。     

水稻长穗颈eui突变体长选3S最上节间基因差异表达研究

利用水稻全基因组芯片和半定量PCR对水稻长穗颈eui突变体长选3S与对照培矮64S最上节间快速伸长期的基因差异表达分析,在检测到的56 983芯片杂交点只发现527个差异表达转录本,其中上调表达的362个,下调表达的165个.生物信息分析发现,差异表达的转录本包括信号传导相关基因、膜及运输基因、细胞生长与凋亡相关基因、复制与修复相关基因、碳代谢相关基因,次生代谢物合成相关基因和其它功能未知基因.研究结果为了解水稻最上节间伸长机制提供了非常有用的信息.     

Regulation of procollagen amino-propeptide processing during mouse embryogenesis by specialization of homologous ADAMTS proteases: insights on collagen biosynthesis and dermatosparaxis

Mutations in ADAMTS2, a procollagen amino-propeptidase, cause severe skin fragility, designated as dermatosparaxis in animals, and a subtype of the Ehlers-Danlos syndrome (dermatosparactic type or VIIC) in humans. Not all collagen-rich tissues are affected to the same degree, which suggests compensation by the ADAMTS2 homologs ADAMTS3 and ADAMTS14. In situ hybridization of Adamts2, Adamts3 and Adamts14, and of the genes encoding the major fibrillar collagens, Col1a1, Col2a1 and Col3a1, during mouse embryogenesis, demonstrated distinct tissue-specific, overlapping expression patterns of the protease and substrate genes. Adamts3, but not Adamts2 or Adamts14, was co-expressed with Col2a1 in cartilage throughout development, and with Col1a1 in bone and musculotendinous tissues. ADAMTS3 induced procollagen I processing in dermatosparactic fibroblasts, suggesting a role in procollagen I processing during musculoskeletal development. Adamts2, but not Adamts3 or Adamts14, was co-expressed with Col3a1 in many tissues including the lungs and aorta, and Adamts2(-/-) mice showed widespread defects in procollagen III processing. Adamts2(-/-) mice had abnormal lungs, characterized by a decreased parenchymal density. However, the aorta and collagen fibrils in the aortic wall appeared normal. Although Adamts14 lacked developmental tissue-specific expression, it was co-expressed with Adamts2 in mature dermis, which possibly explains the presence of some processed skin procollagen in dermatosparaxis. The data show how evolutionarily related proteases with similar substrate preferences may have distinct biological roles owing to tissue-specific gene expression, and provide insights into collagen biosynthesis and the pathobiology of dermatosparaxis.     

Novel mutations in ADAMTS13 CUB domains cause abnormal pre-mRNA splicing and defective secretion of ADAMTS13

Hereditary thrombotic thrombocytopenic purpura (TTP) is an autosomal recessive thrombosis disorder, caused by loss-of-function mutations in ADAMTS13. Mutations in the CUB domains of ADAMTS13 are rare, and the exact mechanisms through which these mutations result in the development of TTP have not yet been fully elucidated. In this study, we identified two novel mutations in the CUB domains in a TTP family with an acceptor splice-site mutation (c.3569−1, G>A, intron 25) and a point missense mutation (c.3923, G>A, exon 28), resulting in a glycine to aspartic acid substitution (p.G1308D). In vitro splicing analysis revealed that the intronic mutation resulted in abnormal pre-mRNA splicing, and an in vitro expression assay revealed that the missense mutation significantly impaired ADAMTS13 secretion. Although both the patient and her brother displayed significantly reduced ADAMTS13 activity and increased levels of ultra-large VWF (ULVWF) multimers in plasma, only the female developed acute episodes of TTP. Our findings indicate the importance of the CUB domains for the protein stability and extracellular secretion of ADAMTS13.     

Dynamic induction of ADAMTS1 gene in the early phase of acute myocardial infarction

Extracellular matrix (ECM)-degrading enzymes such as matrix metalloproteases (MMPs) play an essential role in the repair of infarcted tissue, which affects ventricular remodeling after myocardial infarction. ADAMTS1 (A disintegrin and metalloprotease with thrombospondin motifs), a newly discovered metalloprotease, was originally cloned from a cancer cell line, but little is known about its contribution to disease. To test the hypothesis that ADAMTS1 appears in infarcted myocardial tissue, we examined ADAMTS1 mRNA expression in a rat myocardial infarction model by Northern blotting, real-time RT-PCR and in situ hybridization. Normal endothelium expressed little ADAMTS1 mRNA, while normal myocardium expressed no detectable ADAMTS1 mRNA. Up-regulation of ADAMTS1 was demonstrated by Northern blot analysis and real-time RT-PCR at 3 h after coronary artery ligation. In situ hybridization revealed strong ADAMTS1 mRNA signals in the endothelium and myocardium in the infarcted heart, mainly in the infarct zone, at 3 h after myocardial infarction. The rapid and transient up-regulation of the ADAMTS1 gene in the ischemic heart was distinct from the regulatory patterns of other MMPs. Our study demonstrated that the ADAMTS1 gene is a new early immediate gene expressed in the ischemic endothelium and myocardium.     

Mutational and functional analysis reveals ADAMTS18 metalloproteinase as a novel driver in melanoma

The disintegrin-metalloproteinases with thrombospondin domains (ADAMTS) genes have been suggested to function as tumor suppressors as several have been found to be epigenetically silenced in various cancers. We performed a mutational analysis of the ADAMTS gene family in human melanoma and identified a large fraction of melanomas to harbor somatic mutations. To evaluate the functional consequences of the most commonly mutated gene, ADAMTS18, six of its mutations were biologically examined. ADAMTS18 mutations had little effect on melanoma cell growth under standard conditions, but reduced cell dependence on growth factors. ADAMTS18 mutations also reduced adhesion to laminin and increased migration in vitro and metastasis in vivo. Melanoma cells expressing mutant ADAMTS18 had reduced cell migration after short hairpin RNA-mediated knockdown of ADAMTS18, suggesting that ADAMTS18 mutations promote growth, migration, and metastasis in melanoma.     

ADAMTS1 inhibits lymphangiogenesis by attenuating phosphorylation of the lymphatic endothelial cell-specific VEGF receptor

Angiogenesis and lymphangiogenesis play roles in malignant tumor progression, dissemination, and metastasis. ADAMTS1, a member of the matrix metalloproteinase family, is known to inhibit angiogenesis. Recombinant ADAMTS1 was shown to strongly inhibit angiogenesis. We investigated whether ADAMTS1 inhibited lymphangiogenesis in the present study. We examined cell proliferation and cell migration in normal human dermal lymphatic microvascular endothelial cells (HMVEC-dLy) transduced with or without adenoviral human ADAMTS1 gene therapy. We then examined the VEGFC/VEGFR3 signal transduction pathway in ADAMTS1-transduced HMVEC-dLy. Cell proliferation and tube formation in Matrigel were significantly lower with transduced ADAMTS1 than with control (non-transduced HMVEC-dLy). The phosphorylation of VEGFR3 was also attenuated by ADAMTS1 gene therapy in HMVEC-dLy. Immunoprecipitation assays revealed that ADAMTS1 formed a complex with VEGFC. Our results demonstrated that ADAMTS1 inhibited lymphangiogenesis in vitro. The data highlight the new function of ADAMTS1 in the regulation of lymphangiogenesis and the therapeutic potential of ADAMTS1 in cancer therapy.     

ADAMTS7B, the full-length product of the ADAMTS7 gene, is a chondroitin sulfate proteoglycan containing a mucin domain

We have characterized ADAMTS7B, the authentic full-length protein product of the ADAMTS7 gene. ADAMTS7B has a domain organization similar to that of ADAMTS12, with a total of eight thrombospondin type 1 repeats in its ancillary domain. Of these, seven are arranged in two distinct clusters that are separated by a mucin domain. Unique to the ADAMTS family, ADAMTS7B is modified by attachment of the glycosaminoglycan chondroitin sulfate within the mucin domain, thus rendering it a proteoglycan. Glycosaminoglycan addition has potentially important implications for ADAMTS7B cellular localization and for substrate recognition. Although not an integral membrane protein, ADAMTS7B is retained near the cell surface of HEK293F cells via interactions involving both the ancillary domain and the prodomain. ADAMTS7B undergoes removal of the prodomain by a multistep furin-dependent mechanism. At least part of the final processing event, i.e. cleavage following Arg(220) (mouse sequence annotation), occurs at the cell surface. ADAMTS7B is an active metalloproteinase as shown by its ability to cleave alpha(2)-macroglobulin, but it does not cleave specific peptide bonds in versican and aggrecan attacked by ADAMTS proteases. Together with ADAMTS12, whose primary structure also predicts a mucin domain, ADAMTS7B constitutes a unique subgroup of the ADAMTS family.     

Two Independent Mutations in ADAMTS17 Are Associated with Primary Open Angle Glaucoma in the Basset Hound and Basset Fauve de Bretagne Breeds of Dog

PurposeMutations in ADAMTS10 (CFA20) have previously been associated with primary open angle glaucoma (POAG) in the Beagle and Norwegian Elkhound. The closely related gene, ADAMTS17, has also been associated with several different ocular phenotypes in multiple breeds of dog, including primary lens luxation and POAG. We investigated ADAMTS17 as a candidate gene for POAG in the Basset Hound and Basset Fauve de Bretagne dog breeds.MethodsWe performed ADAMTS17 exon resequencing in three Basset Hounds and three Basset Fauve de Bretagne dogs with POAG. Identified variants were genotyped in additional sample cohorts of both breeds and dogs of other breeds to confirm their association with disease.ResultsAll affected Basset Hounds were homozygous for a 19 bp deletion in exon 2 that alters the reading frame and is predicted to lead to a truncated protein. Fifty clinically unaffected Basset Hounds were genotyped for this mutation and all were either heterozygous or homozygous for the wild type allele. Genotyping of 223 Basset Hounds recruited for a different study revealed a mutation frequency of 0.081 and predicted frequency of affected dogs in the population to be 0.007. Based on the entire genotyping dataset the association statistic for the POAG-associated deletion was p = 1.26 x 10⁻¹⁰. All affected Basset Fauve de Bretagne dogs were homozygous for a missense mutation in exon 11 causing a glycine to serine amino acid substitution (G519S) in the disintegrin-like domain of ADAMTS17 which is predicted to alter protein function. Unaffected Basset Fauve de Bretagne dogs were either heterozygous for the mutation (5/24) or homozygous for the wild type allele (19/24). Based on the entire genotyping dataset the association statistic for the POAG-associated deletion was p = 2.80 x 10⁻⁷. Genotyping of 85 dogs of unrelated breeds and 90 dogs of related breeds for this variant was negative.ConclusionThis report documents strong associations between two independent ADAMTS17 mutations and POAG in two different dog breeds.