Down-regulation of αGal epitopes by co-transfection of α1,3-galactosidase gene and α1,2–fucosyltransferase gene

The polycarbohydrate structure of Galα1- 3Ga1β1-4GluNAc-R （known as αGal epitopes of xenoantigen）, produced by α1-3-galactosyltransferase （α1,3-GT） in the course of animal development, is the major xenoantigen on the cell surface of porcine which causes hyperacute rejection in pig-to-human xenotransplantation. Alpha-1,3-galactosidase （AGL）, a hydrolytic enzyme, can remove the terminal α1,3-galactosyi from the Galα1-3Galβ1-4GluNAc-R structure resulting in cleaning αGai epitopes from the porcine cells. Aipha-1,2-fucosyitransferase （HT） can modify the surface carbohydrate phenotype of porcine cells, bringing about reduction of αGai epitopes expression. In this study, human AGL and HT gene were co-transfected to porcine fetal fibroblast （PFFb） in equimolar concentration to reduce the xenoantigen. Gene and protein of hAGL and HT were both detected to express at high level by RT-PCR and Western blot, respectively. There was an 84% reduction in αGai xenoantigen and an 82% increase in H antigen as assayed by flow cytometry in the AGL and HT gene co-transfected PFFb. The number and morphology of transgenic PFFb chromosome were normal. Findings indicate that Galα1-3Gal epitopes of PFFb could be down regulated by AGL and HT co-transfection without deleterious effects on the chromosomal profile of the transgenic ceil.     

Adhesion of subsets of human blood mononuclear cells to porcine endothelial cells

Cellular immune response is a major barrier to xenotransplantation, and cell adhesion is the first step in intercellular recognition. Flow-cytometric adhesion assay has been used to investigate the differential adhesions of monocyte (Mo), natural killer cell (NK) and T lymphocyte (T) present within human peripheral blood mononuclear cells (PBMC) to porcine aortic endothelial cells (PAEC), and to demonstrate the effect of human interferon-γ (hIFN-γ) or/and tumor necrosis factor-α (hTNF-α) pretreatment of PAEC on their adhesiveness for different PBMC subsets. The preferential sequence for PBMC subset binding to resting PAEC is Mo, NK and T cells, among which T cells show the slightest adherence; hTNF-α can act across the species, and augment Mo, NK and T cell adhesion ratios by 40%, 110% and 3 times, respectively. These results confirm at the cell level that host Mo and NK cells are major participants in the cellular xenograft rejection, thereby, providing a prerequisite for further studying the human Mo/NK-PAEC interactive mechanisms.     

Genistein inhibits human TNF-α-induced porcine endothelial cell adhesiveness for human monocytes and natural killer cells

Cellular immune response is a major barrier to xenotransplantation. Human tumor necrosis factor-α (hTNF-α) possesses cross-species activity and directly amplifies the immune rejection via the upregulation of adhesion molecules on porcine endothelium. We investigated the role of protein tyrosine phosphorylation in the induction of expression of E-sclectin and vascular cell adhesion molecule-1 (VCAM-1), and the augmentation of adhesion of human peripheral blood monocytes (PBMo) and natural killer cells (PBNK), after rhTNF-α-stimulation of porcine aortic endothelial cells (PAEC) in vitro, rhTNF-α-increased adhesiveness of PAEC for both PBMo and PBNK was dose-dependently reduced by pretreatment of PAEC with the selective protein tyrosine kinase (PTK) inhibitor genistein. The inhibitory effect occurred at the early time of PAEC activation triggered by rhTNF-α, and was completely reversible. PTK activity assay indicated that genistein also suppressed rhTNF-α stimulated activation of protein tyrosine kinases (PTKs) in PAEC in a dose-dependent manner. Flow cytometric analysis showed that genistein inhibited the upregulation of E-selectin and VCAM-1 by rhTNF-α. These results suggest that PTKs may regulate the expression of E-selectin and VCAM-1 on PAEC and the adherence of PBMo and PBNK induced by rhTNF-α. Moreover, dietary genistein, used as an adhesion antagonist, may contribute to managing the cell-mediated rejection in the clinical application.     

HLA－G inhibits xenogenetic cytotoxicity mediated by human NK cells and T lymphocytes against PECs

In order to investigate whether the non-classical HLA-G class I molecule protects the prcine endothelial cells(PECs)from the lysis mediated by human immune cells in pig to human discordant xenotransplantation,we have cloned HLA-G cDNA from a human placents by RT-PCR.Mammalian expression vector,pEFG-neo,was constructed by insertion of HLA-G cDNA in pEF-neo.We obtained efficiently expressed PECs by stable transfection.Cytotoxicity assay showed that overexpression of HLA-G on PECs was sufficient to inhibit human NK-92 cell lysis.The level of lysis was equal to or less than that of the lysis of human umbilical vein endothelial cells mediated by human NK-92 cells.It also indicated that HLA-G inhibited the lysis of PECs mediated by xeno-antigen specific T lymphocytes.The reduction of lysis ranged between 59.1% and 88.9A%.These findings suggest that the transgenic approach to overexpress HLA-G is believed to be a new immunotherapy in overconing the immune rejections in xenotransplantion,including delayed xenograft rejection and cell-mediated rejection.     

HLA-G inhibits xenogenetic cytotoxicity mediated by human NK cells and T lymphocytes against PECs

The worldwide shortage in the supply of human do-nor organs is becoming more and more pronounced. Xenotransplantation may probably give the hope to over-come the problem ultimately. Because it has a great pros-pect of clinical application, xenotransplantation has drawn great attention[1]. The pig appears to be an ideal source for human transplantation. But the xenograft has to face the challenge of three severe rejections (the hyperacute rejec-tion, the delayed xenograft rejection and acute ce…     

Selective activation of human beta-but not gamma-globin gene in human fibroblast x mouse erythroleukaemia cell hybrids.

The human alpha- and beta-globin genes have been activated in MEL X human fibroblast cell hybrids. However, even though the human gamma- and beta-globin genes are closely linked and were shown in these hybrid clones to be present in approximately equal numbers, no human gamma-globin mRNA was produced. Thus, the human beta- and gamma-globin genes in these cells are differentially regulated apparently by a positive regulatory factor(s) specific for individual globin genes.     

Differential expression analysis and regulatory network reconstruction for genes associated with muscle growth and adipose deposition in obese and lean pigs

During the growth and development of skeletal muscle cells and adipose cells, the regulatory mechanism of micro-effect polygenes determines porcine meat quality, carcass characteristics and other relative quantitative traits. Obese and lean type pig breeds show obvious differences in muscle growth and adipose deposition; however, the molecular mechanism underlying this phenotypic variation remains unknown. We used pathway-focused oligo microarray studies to examine the expression changes of 140 genes associated with muscle growth and adipose deposition in longissimus dorsi muscle at six growth stages （birth, 1, 2, 3, 4 and 5 months） of Landrace （a leaner, Western breed） and Taihu pigs （a fatty, indigenous, Chinese breed）. Variance analysis （ANOVA） revealed that differences in the expression of 18 genes in Landrace pigs and three genes in Taihu pigs were very significant （FDR-adjusted permutation, P 〈 0.01） and differences for 22 genes in Landrace pigs and seven genes in Taihu pigs were significant （FDR-adjusted permutation, P 〈 0.05） among six growth stages. Clustering analysis revealed a high level of significance （FDR-adjusted, P 〈 0.01） for four gene expression patterns, in which genes that strongly up-regulated were mainly associated with the positive regulation of myofiber formation and fatty acid biogenesis and genes that strongly down-regulated were mainly associated with the inhibition of cell proliferation and positive regulation of fatty acid β-oxidation. Based on a dynamic Bayesian network （DBN） model, gene regulatory networks （GRNs） were reconstructed from time-series data for each pig breed. These two GRNs initially revealed the distinct differences in physiological and biochemical aspects of muscle growth and adipose deposition between the two pig breeds; from these results, some potential key genes could be identified. Quantitative real-time RT-PCR （QRT-PCR） was used to verify the microarray data for five modulated genes, and a good correlation between     

ras-Related gene sequences identified and isolated from Saccharomyces cerevisiae

The oncogenes of Harvey and Kirsten murine sarcoma viruses (v-rasH and v-rasK) and their cellular homologues (c-rasH and c-rasK) constitute two members of the ras gene family. Each functional member of the ras gene family encodes a 21,000 molecular weight protein (p21ras). ras genes have been detected in a wide variety of vertebrate species, including Xenopus laevis (R. E. Steele, personal communication), and in Drosophila melanogaster. We report here the detection of ras-related genes in the yeast Saccharomyces cerevisiae, and the isolation of two ras-related molecular clones, c-rassc-1 and c-rassc-2, from the DNA of Saccharomyces. Both c-rassc-1 and c-rassc-2 hybridize specifically to probes prepared from mammalian ras DNA. Sequencing of c-rassc-1 reveals extensive amino acid homology between the protein encoded by c-rassc-1 and the p21 encoded by c-rasH. Our studies suggest that these clones can be used to elucidate the normal cellular functions of ras-related genes in this relatively simple eukaryotic organism.     

病毒性心肌疾病的基因时空表达特征

目的 研究病毒性心肌炎到扩张型心肌病全病程基因时空表达谱动力学特征.方法 用含有8 000个基因克隆的基因芯片检测CVB3反复感染Balb/c小鼠后第0,7,21天及第3,6,9个月心肌组织基因表达,并用SOM对差异性表达的基因进行聚类分析.结果（1）第10,14,15,19,20类基因在第7天时表达上调,第21,22类基因在第21天表达上调,而第23,24类基因在第7、21天表达均上调;（2）第6,7,11,12类基因在第7天时表达下调,第4,5类基因在第21天表达下调,而第2,3,8类基因则在第7,21天表达均下调;（3）第1,16类基因在第7天时表达下调,而第21天后则表现为上调;（4）有些基因如转移生长因子β结合蛋白、β-2微球蛋白,CD 53抗原却表现为持续性表达上调,而α-1微球蛋,ninjurin 1,舒血管素27,cytokine inducible SH2-containing protein 2则出现持续性表达下调.结论 建立了小鼠CVB病毒性心肌炎及扩张型心肌病全病程基因表达谱动力学模式,为进一步了解其分子发病机制提供全新的视野.     

临床异种移植百年进展(述评)

美国最近连续报道4例基因工程猪供给人的异种器官移植，这些进展打破了异种移植临床研究领域近30年的沉静。尤其是实验性的猪心脏移植取得初步成功，引发了广泛的关注。异种移植面向未来的临床应用迈出了关键性一步。这4个案例，无论是创新精神、技术进步、审批流程，还是伦理学的同步跟进，都值得我国同行学习和借鉴。30年来，基因工程（GE）动物器官已经能在非人类灵长类动物（NHPs）模型中长时间存活。这些实验中，预存抗体、补体激活、凝血状态等机制参与的超急性排异反应基本上得到有效控制。异种移植已经达到临床应用的起跑点。然而，未来仍然有四大问题需要解决：免疫排斥、生物安全、跨种适配和伦理心理。本文简要概括临床异种移植百年来的主要进展，其困难程度可见一斑，但希望和探索从未终止。     

王锦蛇(Elaphe carinata)Sox基因保守区的克隆及测序

参照人SRY基因HMG-box保守区序列设计一对兼并引物,PCR扩增了王锦蛇的Sox基因,采用SSCP技术筛选阳性克隆,并对其进行了测序.结果在雌雄个体中共筛选出4个Sox基因,其中一个为雌性独有,显示出性别差异性;4个Sox基因DNA序列及编码的氨基酸序列与人相应SOX基因的相似性分别为91%、91%、92%、91%和96%、98%、96%、96%,显示出高度的保守性.实验结果为王锦蛇的性别决定机制研究提供了分子资料.     

hASB-8相互作用蛋白的初步研究

hASB-8基因是对肿瘤细胞生长具有明显抑制作用的人类新基因．其编码蛋白属于人ASB蛋白家族中的一个成员,与小鼠中的ASB-8蛋白同源性达96％．保守结构域分析显示hASB-8在N端包含4个Ankyrin repeats,在C端包含了一个SOCS box．利用酵母双杂交技术,筛选了人的胎盘(Placenta)cDNA文库,获得了与KASB-8相互作用的2个蛋白,Elongin C和CDK4 binding protein;并在二倍体酵母体内进行了验证．这些试验提示hASB-8蛋白可能介导肿瘤细胞中靶蛋白和泛素复合体之间的相互作用,并与肿瘤细胞靶蛋白转录调节有关．     

Complexity of human T-cell antigen receptor beta-chain constant- and variable-region genes

Immune systems of vertebrates function via two types of effector cells, B and T cells, which are capable of antigen-specific recognition. The immunoglobulins, which serve as antigen receptors on B cells, have been well characterized with respect to gene structure, unlike the T-cell receptors. Recently, cDNA clones thought to correspond to the beta-chain locus of the human and mouse T-cell receptor have been described. The presumptive beta-chain clones detect gene rearrangement specifically in T-cell DNA and show homology with immunoglobulin light chains. The similarity of the T-cell beta-chain gene system to the immunoglobulin genes has been further demonstrated by the recent observation of variable- and constant-region gene segments as well as joining segments and putative diversity segments. We report here the characterization of cDNA and genomic clones encoding human T-cell receptor beta-chain genes. There are two constant-region genes (C beta 1 and C beta 2), each capable of rearrangement and expression as RNA. The gene arrangement, analogous to that of mouse beta-chain genes, shows strong evolutionary conservation of the dual C beta gene system in these two species.     

Xenozoonoses in xenotransplantation

Pig to human xenotransplantation is one of the possible ways to solve the problem of organ shortage, but the potential risk of xenozoonoses hinders the progress of xenotransplantation. Among pathogens that might cause xenozoonoses, porcine endogenous retrovirus (PERV) is undoubtedly the most noticeable. Current researches show that PERV exists in pigs' genomes and is able to express in multiple tissues and organs in pigs. Other experiments also show that PERV could successfully infect several human cell lines in vitro and could infect SCID mice through pig to mouse islet transplantation. The discussion and research on the risk of PERV is one of the hottest topics in current xenotransplantation, but the question of whether PERV will transmit to human through xenotransplantation has not been answered due to the lack of suitable animal models. More work should be done to evaluate the risk of xenozoonoses caused by PERV after pig to human xenotransplantation.     

Nuclear factor III, a novel sequence-specific DNA-binding protein from HeLa cells stimulating adenovirus DNA replication

Dissection and reconstitution of the adenovirus DNA replication machinery has led to the discovery of two HeLa nuclear proteins which are required in conjunction with three viral proteins. One of these, nuclear factor I (NF-I), recognizes an internal region of the origin between nucleotides 25 and 40 and by binding to one side of the helix stimulates the initiation reaction up to 30-fold. NFI-binding sites have been observed upstream of several cellular genes, such as chicken lysozyme, human IgM and human c-myc, and coincide in most cases with DNase I hypersensitive regions. Here we report the identification of a novel DNA-binding protein from HeLa nuclei, designated NF-III, that recognizes a sequence in the adenovirus origin very close to the NFI-binding site, between nucleotides 36 and 54. This sequence includes the partially conserved nucleotides TATGATAATGAG. NF-III stimulates DNA replication four- to sixfold by increasing the initiation efficiency. Potential cellular binding sites include promoter elements of the histone H2B gene, the human interferon beta gene, the human and mouse immunoglobulin VK and VH genes and the mammal/chicken/Xenopus laevis U1 and U2 small nuclear RNA genes. Furthermore, a subset of the herpes simplex virus immediate early promoter specific TAATGARAT elements is homologous with the adenovirus 2 (Ad-2) NFIII-binding site.     

A diverse range of gene products are effectors of the type I interferon antiviral response

The type I interferon response protects cells against invading viral pathogens. The cellular factors that mediate this defence are the products of interferon-stimulated genes (ISGs). Although hundreds of ISGs have been identified since their discovery more than 25 years ago, only a few have been characterized with respect to antiviral activity. For most ISG products, little is known about their antiviral potential, their target specificity and their mechanisms of action. Using an overexpression screening approach, here we show that different viruses are targeted by unique sets of ISGs. We find that each viral species is susceptible to multiple antiviral genes, which together encompass a range of inhibitory activities. To conduct the screen, more than 380 human ISGs were tested for their ability to inhibit the replication of several important human and animal viruses, including hepatitis C virus, yellow fever virus, West Nile virus, chikungunya virus, Venezuelan equine encephalitis virus and human immunodeficiency virus type-1. Broadly acting effectors included IRF1, C6orf150 (also known as MB21D1), HPSE, RIG-I (also known as DDX58), MDA5 (also known as IFIH1) and IFITM3, whereas more targeted antiviral specificity was observed with DDX60, IFI44L, IFI6, IFITM2, MAP3K14, MOV10, NAMPT (also known as PBEF1), OASL, RTP4, TREX1 and UNC84B (also known as SUN2). Combined expression of pairs of ISGs showed additive antiviral effects similar to those of moderate type I interferon doses. Mechanistic studies uncovered a common theme of translational inhibition for numerous effectors. Several ISGs, including ADAR, FAM46C, LY6E and MCOLN2, enhanced the replication of certain viruses, highlighting another layer of complexity in the highly pleiotropic type I interferon system.     

用抑制差减杂交技术分离烯丙异噻唑诱导水稻特异表达的基因

烯丙异噻唑(PBZ)处理水稻根能使其产生对稻瘟病的系统获得性抗性,因此在东南亚稻区被广泛用于防治稻瘟病,然而关于其作用的分子机理还知之甚少．运用抑制差减杂交技术,试图通过分离鉴定受PBZ诱导调控的关键基因,探索其作用的分子机理．以PBZ处理后的水稻叶片cDNA为目标群体(tester),以未处理水稻叶片cDNA为对照群体(driver),用经过对照cDNA差减的、烯丙异噻唑处理的cDNA群体构建了一个含260个重组子的差减文库．通过差示筛选鉴定出了26个。PBZ诱导水稻特异表达和增强表达的候选克隆．对26个cDNA克隆进行了双向测序和同源性比较,发现其中3个克隆：rJAB1,rTAB2和蛋白磷酸酯酶2Aδ调节亚基同型物基因,位于抗病相关信号转导途径上,它们与哺乳动物和人类免疫途径上的信号因子有明显相似之处,因此推断可能与诱导抗性有关．另外8个克隆与已知基因同源性为70％～99％．经Northern杂交分析,其中rJAB1(编码c-jun激活区结合蛋白1)受烯丙异噻唑和稻瘟菌诱导表达;膜糖蛋白同源基因及肌动蛋白(actin)α1受烯丙异噻唑诱导表达,部分克隆为低丰度转录本．