Oligomerization and polymerization of the filovirus matrix protein VP40

The matrix protein VP40 from Ebola virus plays an important role in the assembly process of virus particles by interacting with cellular factors, cellular membranes, and the ribonuclearprotein particle complex. Here we show that the N-terminal domain of VP40 folds into a mixture of two different oligomeric states in vitro, namely hexameric and octameric ringlike structures, as detected by gel filtration chromatography, chemical cross-linking, and electron microscopy. Octamer formation depends largely on the interaction with nucleic acids, which in turn confers in vitro SDS resistance. Refolding experiments with a nucleic acid free N-terminal domain preparation reveal a mostly dimeric form of VP40, which is transformed into an SDS resistant octamer upon incubation with E. coli nucleic acids. In addition, we demonstrate that the N-terminal domain of Marburg virus VP40 also folds into ringlike structures, similar to Ebola virus VP40. Interestingly, Marburg virus VP40 rings reveal a high tendency to polymerize into rods composed of stacked rings. These results may suggest distinct roles for different oligomeric forms of VP40 in the filovirus life cycle.     

EB病毒对新生儿脐带血单核细胞来源的树突状细胞表型的影响

从脐带血单核细胞来源树突状细胞(DC)表型变化的角度来探讨EB病毒对DC表型的影响,以阐明其逃逸宿主免疫的机制。将GM-CSF和IL-4、EB病毒和LPS不同组合对单核细胞来源DC进行刺激培养,利用单染色和三染色免疫荧光抗体标记—流式细胞术检测单核细胞来源DC表面CD14、CD11c、CD1a、HLA-DR、CD86、MR和MHCⅠ类分子。加入EB病毒后,DC表面CD14分子表达的下调不明显,CD1a的表达则随病毒加入时细胞的分化阶段有关;同时EB病毒还影响了加入LPS后HLA-DR和CD86的升高和MR的降低;EB病毒还影响了细胞表面MHCⅠ类分子的表达。因此EB病毒可抑制单核细胞来源DC的分化和成熟,这可能是其逃逸宿主免疫的机制之一。     

原发性高血压患者血清NO、NOS及其亚型水平的分析

目的:测定原发性高血压患者外周血中血清一氧化氮(NO)、一氧化氮合酶(NOS)及其亚型水平,探讨NO/NOS系统参与血压调节的可能机制.方法:原发性高血压患者135例,正常对照组35例.采用化学法检测所有病例外周血的一氧化氮(NO)、总NOS、诱导型一氧化氮合酶(iNOS)和结构型一氧化氮合酶(cNOS)水平并作统计学分析.结果:高血压组NO、NOS、iNOS和cNOS水平均低于正常对照组,且差异具有显著性意义(分别为:P＜0.05,P＜0.01,P＜0.01,P＜0.01);iNOS/cNOS比值与正常对照组相比无显著差异(P＞0.05);对照组和高血压组的NOS浓度与iNOS和cNOS浓度均呈显著正相关;高血压组的cNOS水平与iNOS水平呈显著负相关.结论:高血压患者NO、NOS及其亚型浓度值可以作为临床评估高血压的参考指标;iNOS在正常人体内有表达;在高血压情况下,机体有通过增加iNOS的表达来弥补cNOS水平的病理性降低、调控NOS总体水平从而调节和平衡血压的趋势.     

Toll样受体介导的抗病毒感染效应及其机制

Toll样受体(Toll Like Receptors,TLRs)在天然免疫中发挥着重要的作用。近年,越来越多的证据表明TLRs也参与了天然免疫系统对病毒的识别,同时它在病毒感染及其转归中也扮演了重要的角色。TLR3、TLR4、TLR7、TLR8、TLR9等TLRs可识别特异性蛋白、DNA和RNA等病毒源性分子,启动胞内抗病毒信号传导机制,通过一系列转接蛋白和转录因子,介导了以干扰素反应为主要机制的固有免疫防御机制,同时也参与调控针对病毒的特异性免疫应答。此外,某些病毒具有针对TLRs的免疫逃逸机制。     

Chemoreceptor responses to substance P, physalaemin and eledoisin: Evidence for neurokinin-1 receptors in the cat carotid body

Substance P (SP) belongs to a group of peptides called tachykinins. Biological effects of SP are mediated by tachykinin receptors that have been classified as neurokinin-1 (NK-1), NK-2 and NK-3 subtypes. The aim of the present study is to elucidate the tachykinin receptor subtype(s) that mediate the excitatory effects of SP in the carotid body. For this purpose, we compared the carotid body responses elicited by SP with that of physalaemin and eledoisin. In other tissues, physalaemin exhibits equi or greater potency at NK-1 receptors and eledoisin exerts its effects more on NK-2 and NK-3 subtypes compared to SP. Experiments were performed on eight cats that were anaesthetized, paralyzed and artificially ventilated with room air. Close carotid body administration of SP and physalaemin produced dose-dependent augmentation of the chemoreceptor afferent activity. Chemoreceptor discharge, however, was unaffected by eledoisin. Compared to that by SP, the magnitude of excitation produced by physalaemin was the same at lower doses but significantly greater with the highest dose (100 nmol). The time course of the response induced by physalaemin, however, was the same as that by SP. The present results demonstrate that in the carotid body physalaemin is also either equi or relatively more potent than SP, whereas eledoisin has no effect on the chemoreceptor discharge. It is suggested that stimulation of the carotid body by SP is mediated by NK-1 but not NK-2 or NK-3 receptors.     

Characterization of a 105-kDa plasma membrane associated glycoprotein that is involved in West Nile virus binding and infection

This study attempts to isolate and characterize West Nile virus-binding molecules on the plasma membrane of Vero and murine neuroblastoma cells that is responsible for virus entry. Pretreatment of Vero cells with proteases, glycosidases (endoglycosidase H, alpha-mannosidase), and sodium periodate strongly inhibited West Nile virus infection, whereas treatments with phospholipases and heparinases had no effect. The virus overlay protein blot detected a 105-kDa molecule on the plasma membrane extract of Vero and murine neuroblastoma cells that bind to WN virus. Treatment of the 105-kDa molecules with beta-mercaptoethanol resulted in the virus binding to a series of lower molecular weight bands ranging from 30 to 40 kDa. The disruption of disulfide-linked subunits did not affect virus binding. N-linked sugars with mannose residues on the 105-kDa membrane proteins were found to be important in virus binding. Specific antibodies against the 105-kDa glycoprotein were highly effective in blocking virus entry. These results strongly supported the possibility that the 105-kDa protease-sensitive glycoprotein with complex N-linked sugars could be the putative receptor for WN virus.     

HLA-B*07 allele frequencies and haplotypic associations in Koreans

We have investigated the frequencies of HLA-B*07 alleles and their haplotypic associations with HLA-A, -C and -DRB1 loci in 489 healthy unrelated Koreans, including 214 parents from 107 families. All of the 45 samples (9.2%) typed as B7 by serology were analyzed for B*07 alleles using polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) method. Two different B*07 alleles were detected: B*0702 (allele frequency 0.041) and B*0705 (0.005). Two characteristic haplotypes showing strong linkage disequilibrium in Koreans were A*2402-Cw*07-B*0702-DRB1*0101 (haplotype frequency 0.028) and A*2901-B*0705-DRB1*0803 (0.005). The characteristic haplotype A*2901-B*0705-DRB1*0803, found in 100% (5/5) of B*0705-positive individuals, has not been previously described in other ethnic groups. HLA-B7 alleles comprise distinctive extended haplotypes in the Korean population. The probability of HLA-B7 allele mismatches among ABDR-matched unrelated donor-recipient pairs is expected to be low in Koreans.     

A case of progression from type II cryoglobulinaemia to Waldenstrom’s macroglobulinaemia in a patient with chronic hepatitis C

In recent years, much interest has been generated over the potential of human embryonic stem cells in transplantation medicine. The ground-breaking study of Fraidenraich and colleagues conclusively demonstrated that rescue of lethal cardiac defects in Id knockout mutant mouse embryos was not due to transplanted embryonic stem cells giving rise to functional new tissues within the defective embryonic heart. Instead, there is indirect evidence that the observed therapeutic effect was due to various secreted factors emanating from the transplanted cells. This therefore introduces the exciting prospect of utilizing human embryonic stem cells as "catalysts" to promote biological repair and regeneration in transplantation therapy. Nevertheless, the immunological barrier against allogenic transplantation, as well as the teratogenic potential of human embryonic stem cells poses major technical challenges. A possible strategy to overcome the immunological barrier may be to impose a temporary regimen of immunosuppressive drugs followed by their gradual withdrawal, once adequate tissue regeneration has been achieved. Other more novel alternatives include the use of microencapsulation to block interaction with the transplant recipient's immune system, and co-transplantation with bone marrow-derived mesenchymal stem cells, which have been demonstrated to possess immuno-suppressive properties. The teratogenic potential of human embryonic stem cells could possibly be alleviated by directing the differentiation of these cells to specific lineages prior to transplantation, or through mitotic inactivation. Co-transplantation with autologous adult stem cells may represent a novel strategy to further enhance the "catalytic" effects of human embryonic stem cells. The various factors secreted by human embryonic stem cells could then have a concentrated localized effect on relatively large numbers of co-transplanted autologous adult stem cells, which may in turn lead to enhanced repair and regeneration of the damaged tissue or organ. This new therapeutic strategy needs to rigorously investigated, in view of its potentially important clinical applications.     

丁型肝炎病毒核酶反式切割HBV mRNA片段的实验研究

目的　探讨反式作用丁型肝炎病毒 (HDV)核酶体外切割乙型肝炎病毒 (HBV)mRNA片段的可行性。方法　将化学合成的核酶cDNA克隆到含有T7启动子的载体PGEM 4Z中。利用体外转录技术转录出核酶及底物 ,研究其体外切割活性。利用E H作图法进行核酶的酶促动力学研究。结果　在体外实验中显示两酶均能成功的将底物切割 ,37℃温浴 90min的切割百分率为 5 0 %和5 1%。利用E H作图法进行的酶促动力学研究中求得Rc1、Rc2的Km值分别为 0 6 1μmol L、0 5 8μmol L,Kcat值分别为 :0 6 4·min 1 、0 6 0·min 1 。结论　反式作用HDV核酶对非HDV底物 HBVmRNA片段的成功切割为寻找新的HBV的反义抑制手段开辟了途径。     

小发卡RNA抗呼吸道合胞病毒效应的研究

目的 为从基因水平抑制呼吸道合胞病毒（respiratory syncytial virus，RSV）的复制，针对RSV的M2-2基因构建小发卡结构状RNA（short hairpin RNA，shRNA）重组质粒，在细胞水平观察shRNA对RSV复制的影响。方法 将成功构建的针对RSV M2-2基因的重组质粒pshRNA8260转染HEp-2细胞，利用光镜观察pshRNA8260对RSV致HEp-2细胞病变效应（cytopathogenic effect，CPE）的影响并计算CPE抑制率，空斑形成实验检测RSV滴度变化。结果 成功构建了针对人RSV M2-2基因mRNA的pshRNA8260重组质粒，研究发现pshRNA8260能明显改善RSV所致的病变效应，降低RSV在细胞内复制的病毒滴度。结论 针对RSV M2-2基因的pshRNA82（g）重组质粒具有明显的特异性抗RSV效应的作用。