纤维蛋白单体D区与E区聚合后E区结构的变化

应用纤维蛋白单克隆抗体IF 5 3,观察当纤维蛋白的“A”位点与另一纤维蛋白D区域的“a”位点结合后纤维蛋白E区的变化 .纤维蛋白原Aα链经赖氨酰肽链内切酶消化后 ,应用反相HPLC分离纯化 ;通过ELISA法检测单克隆抗体IF 5 3与纤维蛋白原及其衍生物的反应情况 ;应用放射免疫法检测RGD合成肽抑制纤维蛋白单体与IF 5 3反应的情况 .发现IF 5 3能与纤维蛋白原Aα链的一个片段反应 ,该片段经氨基酸序列分析显示为纤维蛋白原Aα链氨基末端 (1～ 2 9) .该抗体能与酸溶解的纤维蛋白单体和可溶性纤维蛋白及XDP反应 ,但不能与酸化纤维蛋白原或GPRP反应 ,因此IF 5 3的抗原决定簇在Aα 2 0～ 2 9,与凝血酶作用于纤维蛋白肽A ,暴露出的聚合位点“A”(Aα17～19)紧邻 .当GPRP存在于纤维蛋白原溶液时 ,经凝血酶作用产生这种纤维蛋白单体不能与IF 5 3反应 .Aα(93～ 99) (ILRGDFS)合成肽部分抑制纤维蛋白单体与IF 5 3的反应 .实验结果提示 ,当纤维蛋白单体相互聚合 ,或纤维蛋白单体与纤维蛋白原聚合时 ,纤维蛋白单体结构会发生变化 ,其中Aα2 0～ 2 9片段成为新抗原暴露于E区表面 ,并且Aα2 0～ 2 9与纤维蛋白原细胞粘附区域RGD1片段邻近     

Characterization of a novel human anti-HIV-1 gp41 IgM monoclonal antibody designated clone 37

Human monoclonal antibodies (HuMAbs) demonstrate great potential for passive immunotherapy against HIV-1. The gp41 transmembrane envelope glycoprotein of HIV has an important role in the pathogenicity of AIDS and importantly displays considerably less hypervariability than the gp120 surface envelope HIV glycoprotein, which makes it particularly a better candidate for the development of passive and active immunotherapies. The general aim of this study was to develop HuMAbs to HIV surface glycoproteins and particularly gp41. Peripheral blood mononuclear cells (PBMCs) were isolated from an HIV-seropositive long-term nondisease progressing patient. B-cells from this individual were then immortalized by Epstein-Barr virus (EBV) transformation, and antibody production was stabilized by fusion of transformed cells with a heteromyeloma. Subsets of the human heterohybridomas so generated were analyzed by ELISA. The hybridoma with the highest binding by immunoassay against gp160 was further analyzed. This hybridoma, designated as clone 37 (C37), was determined to be an IgM Kappa antibody and overlapping peptides of HIV envelope proteins (derived from the MN tissue culture line adapted HIV isolate) were used to map the specific binding domain of this HuMAb. Overlapping peptides designated 2026 (SWSNKSLDDIWNN, AA614-626), and 2027 (DDIWNNMTWMQWEREIDNYT, AA621-640) within the HIV-1 gp41 transmembrane glycoprotein were demonstrated to bind to C37 indicating that the specific binding domain for the antibody was DDIWNN. High affinity binding of C37 by ELISA to recombinant gp41 was demonstrated as well. Few IgM HuMAbs against HIV have been generated and characterized. Theoretically, because of the pentameric binding nature of IgM antibodies as well as their very efficient ability to activate complement, such reagents could have potential as anti-HIV agents.     

Megator, an essential coiled-coil protein that localizes to the putative spindle matrix during mitosis in Drosophila

We have used immunocytochemistry and cross-immunoprecipitation analysis to demonstrate that Megator (Bx34 antigen), a Tpr ortholog in Drosophila with an extended coiled-coil domain, colocalizes with the putative spindle matrix proteins Skeletor and Chromator during mitosis. Analysis of P-element mutations in the Megator locus showed that Megator is an essential protein. During interphase Megator is localized to the nuclear rim and occupies the intranuclear space surrounding the chromosomes. However, during mitosis Megator reorganizes and aligns together with Skeletor and Chromator into a fusiform spindle structure. The Megator metaphase spindle persists in the absence of microtubule spindles, strongly implying that the existence of the Megator-defined spindle does not require polymerized microtubules. Deletion construct analysis in S2 cells indicates that the COOH-terminal part of Megator without the coiled-coil region was sufficient for both nuclear as well as spindle localization. In contrast, the NH2-terminal coiled-coil region remains in the cytoplasm; however, we show that it is capable of assembling into spherical structures. On the basis of these findings we propose that the COOH-terminal domain of Megator functions as a targeting and localization domain, whereas the NH2-terminal domain is responsible for forming polymers that may serve as a structural basis for the putative spindle matrix complex.     

Alterations in mRNA expression of ribosomal protein S9 in hydrogen peroxide-treated neurotumor cells and in rat hippocampus after transient ischemia

This study was designed to isolate new genes related to apoptosis in rat pheochromocytoma (PC12) cells treated with hydrogen peroxide (H₂O₂), and to characterize the roles of the genes using both in vitro and in vivo models of oxidative injury. cDNA libraries were prepared from H₂O₂-treated and -untreated PC12 cells, and a ribosomal protein S9 (RPS9) clone was isolated by a differential screening method. Increase of RPS9 expression in both H₂O₂-treated PC12 and neuroblastoma (Neuro-2A) cells was shown by Northern blot analysis. Viability of the antisense-transfected Neuro-2A (RPS9-AS) cells following H₂O₂ treatment was significantly reduced in a dose-dependent manner. In an in vivo model of transient forebrain ischemia, an increase in RPS9 expression was prominent by 1 day postischemia in the granule cell layer neurons of the dentate gyrus. Both activation of caspase-3 and significant recovery of viability following pretreatment with cycloheximide were shown in RPS9-AS cells treated with H₂O₂. These data suggest that RPS9 plays a protective role in oxidative injury of neuronal cells.     

Hippocampal Long-Term Potentiation Attenuated by Lesions in the Marginal Division of Neostriatum

The marginal division (MrD) is a spindled-neurons consisted zone at the caudal border of the neostriatum in the mammalian brain and has been verified as contributing to associative learning and declarative memory in the rat and human with behavior and functional magnetic resonance imaging methods. It was proved to have functional connections with the limbic system. Whether the MrD has influence on the hippocampal long-term potentiation (LTP) was investigated in this study. LTP was induced from the dentate gyrus (DG) in the hippocampus by high-frequency stimulation (HFS) to the perforant path (PP). The amplitude of the population spike (PS) and the slope of the excitatory postsynaptic potential (EPSP) increased significantly to form LTP in the DG of the hippocampus after HFS of PP in normal and saline-injected control groups of rats. Lesions introduced in the MrD reduced significantly both the amplitude of PS and the slope of the EPSP following HFS of the PP. The results indicated that lesions in the MrD could attenuate LTP formation in the hippocampus. Our data suggest that the MrD might very possibly have excitatory functional influence on the hippocampus and therefore might influence the function of the hippocampus.     

不完全酶切去除载体多个相同酶切位点的简易方法

以去除真核表达载体pIGF3的3个EcoR Ⅰ位点为例,介绍一种通过不完全酶切去除载体多个相同酶切位点的方法。本方法步骤简单,经济实用。     

重要生物恐怖病原及其医学防护对策

生物恐怖问题由来已久,但直到美国“9.11”事件后的炭疽芽孢袭击才引起人们广泛关注。生物恐怖已成为21世纪全人类的威胁,防范生物恐怖病原袭击已成为各国政府的当务之急。本文主要介绍了对生物恐怖的定义、生物恐怖病原种类和致病特征,以及医学防护对策。     

甘薯品种营养成份与抗小象虫的相关性研究

通过田间和网室测定结果,抗小象虫较好的甘薯品种有抗虫1号、台农26、Tis2534、Ricin和鸡蛋黄;抗小象虫较弱的品种有新种花、惠红早、“329”、广薯15等。甘薯品种的营养成份与抗小象虫相关性分析结果表明,抗、感品种与粗纤维、粗脂肪含量无明显相关,与粗淀粉有显负相关,R1=-0．9935,而与粗蛋白和18种氨基酸总量有显正相关,R2=0．9741,R3=0．9621。表现粗淀粉含量高的品种,其虫害指敦较低,抗虫性强;而粗蛋白含量和18种氨基酸总量高的品种,其虫害指数较高,抗虫性则表现较弱。测定分析说明了甘薯品种营养成份与抗虫性存在一定的相关性。     

Crystallization and preliminary X-ray crystallographic analysis of orotate phosphoribosyltransferase from Helicobacter pylori

Orotic acid phosphoribosyltransferase (PyrE) (EC 2.4.2.10) is a key enzyme in de novo uridine monophosphate (UMP) biosynthesis. It catalyzes the reaction between orotic acid and 5-phosphoribosyl-1-pyrophosphate (PRPP) to yield orotidine monophosphate (OMP), which is transformed to uridine monophosphate by decarboxylation. H. pylori PyrE was crystallized at 294 +/- 1 K by the hanging drop vapor-diffusion method. The crystals belong to the space group P2(1)2(1)2(1) with unit-cell dimensions a = 95.8, b = 104.9, c = 281.1 A, alpha = beta = gamma = 90 degrees. A set of diffraction data was collected to 3.29 A resolution using synchrotron X-ray radiation.     

Ca2+: a stabilizing component of the transglutaminase activity of Galphah (transglutaminase II)

Galphah (transglutaminase type II; tissue transglutaminase) is a bifunctional enzyme with transglutaminase (TGase) and guanosine triphosphatase (GTPase) activities. The GTPase function of Galphah is involved in hormonal signaling and cell growth while the TGase function plays an important role in apoptosis and in cross-linking extracellular and intracellular proteins. To analyze the regulation of these dual enzymatic activities we examined their calcium-dependence and thermal stability in enzymes from several cardiac sources (mouse heart, and normal, ischemic and dilated cardiomyopathic human hearts). The GTP binding activity of Galphah was markedly inhibited by Ca2+ whereas the TGase activity was strongly stimulated, suggesting that Ca2+ acts as a regulator, switching Galphah from a GTPase to a TGase. The TGase function of Galphah of both mouse and human hearts was more thermostable in the presence of Ca2+.