Penetration,adhesion, and fusion in mammalian sperm-egg interaction

Fertilization is the sum of the cellular mechanisms that pass the genome from one generation to the next and initiate development of a new organism. A typical, ovulated mammalian egg is enclosed by two layers: an outer layer of approximately 5000 cumulus cells and an inner, thick extracellular matrix, the zona pellucida. To reach the egg plasma membrane, sperm must penetrate both layers in steps requiring sperm motility, sperm surface enzymes, and probably sperm-secreted enzymes. Sperm also bind transiently to the egg zona pellucida and the egg plasma membrane and then fuse. Signaling in the sperm is induced by sperm adhesion to the zona pellucida, and signaling in the egg by gamete fusion. The gamete molecules and molecular interactions with essential roles in these events are gradually being discovered.     

Inhibition of a class C beta-lactamase by a specific phosphonate monoester

A phosphonate monoester, m-carboxyphenyl phenylacetamidomethylphosphonate, has been found to be a specific inhibitor of the class C beta-lactamase of Enterobacter cloacae P99. Inactivation is rapid (10(3) per second per molar concentration) and reactivation very slow (2.2 X 10(-6) per second). Apparently concerted with the inactivation, one equivalent (with respect to the enzyme) of m-hydroxybenzoate is released. Reactivation is accelerated by hydroxylamine and benzohydroxamate. This suggests that the loss of enzyme activity is due to phosphonylation of an active site functional group. This discovery holds the promise of a new general class of beta-lactamase inhibitors and, perhaps, antibiotics.     

Extracellular potassium ions mediate specific neuronal interaction

The giant interneurons from the nerve system of the cockroach Periplaneta americana exhibit a peculiar reciprocal synaptic interaction. The synaptic potentials are not blocked by addition of 5 millimolar cobalt chloride and have an extrapolated reversal potential close to 0 millivolt. Hyperpolarizing current injected into one cell does not spread to the other. Intracellular injection of tetraethylammonium ions into one giant interneuron increases the duration of the action potential of the injected cell to 30 milliseconds and reduces the rise time and amplitude of the postsynaptic response recorded in the other giant interneuron. These results indicate that the interaction between the interneurons is not mediated by conventional chemical or electrotonic synapses.. All evidence points to generation of the potentials by localized increases in extracellular potassium concentrations as a consequence of firing of one neuron.     

Regulation of myosin phosphatase by a specific interaction with cGMP- dependent protein kinase Ialpha

Contraction and relaxation of smooth muscle are regulated by myosin light-chain kinase and myosin phosphatase through phosphorylation and dephosphorylation of myosin light chains. Cyclic guanosine monophosphate (cGMP)-dependent protein kinase Ialpha (cGKIalpha) mediates physiologic relaxation of vascular smooth muscle in response to nitric oxide and cGMP. It is shown here that cGKIalpha is targeted to the smooth muscle cell contractile apparatus by a leucine zipper interaction with the myosin-binding subunit (MBS) of myosin phosphatase. Uncoupling of the cGKIalpha-MBS interaction prevents cGMP-dependent dephosphorylation of myosin light chain, demonstrating that this interaction is essential to the regulation of vascular smooth muscle cell tone.     

Inhibition of platelet aggregation by monoclonal antibody reactive with beta 2-microglobulin chain of HLA complex

A mouse monoclonal antibody that reacts with beta 2-microglobulin, the light chain of class I major histocompatibility antigens, inhibited the second wave of human platelet aggregation induced by adenosine diphosphate and epinephrine and blocked aggregation and platelet protein phosphorylation induced by sodium arachidonate. Thrombin-induced platelet aggregation was inhibited at threshold concentrations but not at higher concentrations. The antibody also inhibited aggregation and secretion in response to thromboxane A2 or the stable endoperoxide analog, U46619. These results suggest that beta 2-microglobulin in the histocompatibility complex is intimately associated with transmission of the endoperoxide-thromboxane signal at the platelet membrane.     

口服重组酵母(ApoB100肽段)对小鼠产生特异性抗体的诱导作用

【目的】开发一种针对低密度脂蛋白成分ApoB100的新型重组酵母疫苗,为动脉粥样硬化(Atherosclerosis,AS)的防治提供一种思路。【方法】利用PCR得到包含编码ApoB100的3 136-3 155位氨基酸残基的目的片段,将其克隆到酵母表达载体JMB88-HA-OVA-MCS上,构建JMB88-HA-OVA-hApoB载体,用硫酸铜诱导表达后获得HA-OVA-hApoB融合蛋白。采用口服的方法对昆明小白鼠免疫成功表达HA-OVA-hApoB蛋白的酵母细胞12周,每周1次,每次1×108个酵母细胞,免疫结束后4周采血,用Western blot检测小鼠是否产生特异性抗体。【结果】PCR扩增获得了859bp的人ApoB100基因。成功构建了JMB88-HA-OVA-hApoB载体。口服免疫12周后,HA-OVA-hApoB免疫组小鼠的血清中含有特异性ApoB100抗体。【结论】灌服表达人ApoB100肽段融合蛋白的重组酿酒酵母能诱导小鼠的免疫反应,使其产生特异性抗体。     

猪圆环病毒Ⅱ型特异抗体检测间接ELISA法的建立

利用巴斯德毕赤酵母表达的猪圆环病毒2型株Cap蛋白为包被抗原,成功地建立了一种检测血清中PCV-2特异抗体的间接ELISA方法.确定了血清样品阴、阳性判定标准.临床检测结果表明,自建ELISA试剂盒具有较高的敏感性和特异性.     

间接ELISA检测抗犬瘟热病毒抗体方法的建立

以大肠杆菌表达的犬瘟热病毒(CDV)重组核蛋白(GST-NP)经纯化后作为包被抗原,通过方阵ELISA滴定确定GST-NP的适宜包被浓度为1.31 μg·mL-1,并由此建立检测抗CDV抗体的间接ELISA方法.通过对已知抗CDV阳性血清及抗其他病毒血清的试验,表明所建立的间接ELISA可特异检测动物血清中的抗CDV抗体.另外,通过不同样品的重复试验以及不同批次的检测试验,证明该方法在用于检测CDV抗体时具有很好的重复性和稳定性.     

基于特异性对硫磷单克隆抗体的间接竞争酶联免疫吸附分析方法建立

[目的]制备特异性识别对硫磷(PA)的单克隆抗体,建立其间接竞争酶联免疫吸附分析方法(ic-ELISA),为实现对硫磷的农残快检提供技术支持.[方法]以化合物4-(二乙氧基硫代磷酸酯基)苯甲酸为半抗原,分别与牛血清白蛋白(BSA)和卵清白蛋白(OVA)偶联制备人工抗原,免疫7周龄Balb/c雌性小鼠,取抗血清效价最高、特异性最优小鼠的脾脏细胞与SP2/0骨髓瘤细胞进行细胞融合,采用间接酶联免疫方法(i-ELISA)及ic-ELISA分别评价融合细胞株的阳性和特异性,通过有限稀释法筛选稳定分泌对硫磷抗体的单克隆细胞株.再通过棋盘格试验筛选出抗原抗体最佳工作浓度组合建立对硫磷ic-ELISA标准曲线,并基于对硫磷单克隆抗体与各结构类似物的交叉反应率,评估ic-ELISA的特异性.采购西红柿样品进行对硫磷的添加回收试验,运用气相色谱法(GC)验证ic-ELISA的准确性.[结果]成功合成对硫磷人工抗原PA-BSA和PA-OVA,经5次免疫后融合小鼠的血清效价为8×103;通过有限稀释法筛选获得灵敏度高、特异性最强的对硫磷单克隆细胞株4F11A10.以2μg/mL包被抗原PA-OVA、0.25μg/mL细胞株4F11A10分泌抗体为最佳抗原抗体工作浓度组合,建立对硫磷ic-ELISA及其标准曲线,其半抑制浓度(IC50)为50 ng/mL,检测范围(IC20~IC80)为2.34~300 ng/mL;所建立的对硫磷ic-ELISA与5种有机磷农药(丙溴磷、杀扑磷、倍硫磷、水胺硫磷和甲拌磷)均无交叉反应.对比ic-ELISA和GC测定西红柿样品中对硫磷的添加回收试验结果,发现ic-ELISA的添加回收率为84.86%~100.27%,GC的添加回收率为97.60%~106.40%,两种方法的检测结果一致性较好.[结论]建立的ic-ELISA可用于快速检测蔬菜、水果等农产品中的对硫磷残留.     

抗A型禽流感病毒核蛋白特异性单克隆抗体研究

利用禽流感H9亚型病毒(AIV-H9)免疫Balb/c小鼠,取其脾细胞与骨髓瘤细胞进行融合,经免疫荧光试验(IFA)检测,以研制抗禽流感病毒(AIV)单克隆抗体。结果获得了5株特异性抗AIV核蛋白(NP)的单克隆抗体细胞株,分别命名为AIV-NP-2C3、AIV-NP-6A5、AIV-NP-3H9、AIV-NP-7B4、AIV-NP-2H4。这5株单克隆抗体能与所有试验的AIV-H9病毒反应,Western blotting方法鉴定结果表明,单克隆抗体仅识别60 ku的蛋白抗原,而不与新城疫病毒、禽网状内皮组织增殖症病毒、传染性法氏囊病毒等反应。初步应用结果显示,以这些单克隆抗体建立的间接免疫荧光试验或ELISA方法可迅速检测出禽流感病毒,这些单克隆抗体在禽流感的预防监测中将发挥重要的作用。     

甲壳类和贝类原肌球蛋白的广谱单克隆抗体制备与鉴定

原肌球蛋白（Tropomyosin, TM）被认为是甲壳类和贝类的主要过敏原,为了应对与日俱增的甲壳类和贝类过敏风险,本研究以南美白对虾（Litopenaeus vannamei）的TM富集液为抗原免疫BALB/c小鼠,通过杂交瘤细胞融合技术筛选获得1株南美白对虾过敏原TM的单克隆抗体细胞株2F8,并利用ELISA、Western Blot、生物信息学等方法对2F8单抗进行免疫学特性分析,确定其效价、特异性、抗体亚型、结合区域,以TM的特异性单抗2F8和兔多抗构建双抗夹心胶体金试纸条分析单抗2F8的应用可行性。结果显示,单抗2F8的抗体亚型为IgG1型、效价为1.28×105,抗体与TM抗原的IC50值为20.21,亲和力高;纯化的单抗2F8可以特异性识别甲壳类、贝类和鱿鱼的主要过敏原TM,存在3个潜在的TM抗原结合区域AA0-31,AA38-43和AA58-67。制备的试纸条检测结果显示,该试纸条可在10 min内目测5 ng/mL的南美白对虾TM（相当于12.5 μg/kg）,且可用于甲壳类和贝类肌肉中TM 的检测,研究表明所制备的2F8单抗亲和力高,可用于虾蟹贝和鱿鱼中的过敏原TM的鉴定与检测,具有较好的应用价值。本研究为过敏原TM的鉴定和检测提供了重要生物材料,也为今后TM的快速检测试剂盒开发提供了技术支持。     

Stimulation of cells by antibody

Tumor cell lines exposed to immunoglobulins specific for cell surface antigens developed increased cellular incorporation of [(125)I]iododeoxyuridine and [(3)H]thymidine (up to 200-fold increases over cells treated with normal rabbit immunoglobulins). Antibody-stimulated cells multiplied more rapidly and lived longer than control cells in tissue culture. These observations were made both with cells substituted with 2,4,6-trinitrophenol and purified antibody against 2,4,6-trinitrophenol, and with several cell lines and their respective whole-cell antibodies. Antibodies that were stimulatory at low concentrations were cytotoxic at high concentrations. These observations may have significance in regard to enhancing effects of antibodies on tumor cell growth in vivo.     

Inhibition of idiotype--anti-idiotype interaction for detection of a parasite antigen: a new immunoassay

Described in this report is an immunoradiometric assay of general applicability that is based on a new principle: the inhibition of the interaction between monoclonal antibodies by an antigen. The advantages of this assay are that it measures concentrations of single epitopes, purified antigen is not required, and the reagents can be obtained in unlimited amounts and are homogeneous. Its features are particularly attractive when the antigen has not been purified and is a minor component of a complex mixture of molecule.     

Specific inhibition of plaque formation to phosphorylcholine by antibody against antibody

Spleen cells from BALB/c mice immunized with heat-killed rough pneumococci (strain R36A) or spleen cells from normal mice immunized in vitro with the same antigen produce direct hemolytic plaques against sheep erythrocytes coated with pneumococcal C polysaccharide or conjugated with phosphorylcholine. Formation of plaques is specifically inhibited by phosphorylcholine or by antiserum to mouse immunoglobulin A myeloma protein which binds phosphorylcholine. Thus, the myeloma proteins and normal BALB/c antibodies share similar idiotypic determinants. This experimental system is suitable for probing the role of the antigen receptor in the immune response.     

外源重组蛋白制备鹅免疫球蛋白轻链特异性多克隆抗体

采用RT-PCR方法扩增鹅免疫球蛋白轻链恒定区编码序列(GoIgCL),构建原核表达载体pET30a-IgCL,在RosettaTM(DE3)pLysS宿主菌中表达鹅免疫球蛋白轻链恒定区重组蛋白rGoCL,以纯化后rGoCL作为免疫原制备兔抗GoIgL多克隆抗体,对多抗进行鉴定分析.结果表明,rGoCL在大肠杆菌中获得可溶性表达,可代替天然分离的轻链作为免疫原制备轻链特异性抗体,多抗效价为1 204800.研究为鹅免疫球蛋白的蛋白结构、功能分析以及鹅免疫诊断试剂研发奠定基础.     

Inhibition of protein synthesis by spectinomycin

Spectinomycin selectively inhibits protein synthesis in cells and in extracts of Escherichia coli. Mutations to high-level resistance to this antibiotic map close to the streptomycin locus, and the site of action of spectinomycin, like that of streptomycin, is the 30S ribosomal subunit, as shown by experiments with reconstituted 70S ribosomes containing subunits from sensitive and from resistant ribosomes. In contrast to streptomycin, however, spectinomycin is not bactericidal and causes no detectable misreading of polyribonucleotides.     

Inhibition of cell motility by interferon

Interferon derived from human leukocytes, human fibroblasts, and mouse fibroblasts was found to inhibit the motility of cultured cells. It inhibits the tumor-induced motility of capillary endothelial cells as well as the spontaneous migration of other cell types. The ability of a given preparation of interferon to inhibit the motility of a given cell type is proportional to its antiviral activity in that particular cell type. Antiserum to human leukocyte interferon neutralizes both the motility-inhibitory activity and the antiviral activity of this preparation.