Precipitation of animal plasma proteins by puff-adder venom.

1. Plasma and serum samples obtained from various animals never previously exposed to snakes or snake venom were diffused against different concentrations of puff-adder, Bitis arietans, venom using the double immunodiffusion technique. 2. Depending upon venom concentration, two precipitin arcs could be produced in the case of all plasma samples used. No serum samples showed any arcs except pigeon serum, where one precipitin line was observed. 3. By altering the concentration of snake venom between 1% and 10% when immunodiffusing against plasma a change in position of the precipitin lines was observed and also the disappearance of one or both of the two bands at higher concentrations. This indicates that the arcs observed are in all probability due to precipitation of plasma protein fractions. 4. Previous results indicated that one of the two bands observed when diffusing venom against plasma was due to the precipitation of fibrinogen. By diffusing snake venom against heparin we have now shown that the second band involves this molecule and is not due to another coagulation factor as was suggested previously.     

Partial resolution of the enzymes catalyzing photophosphorylation. XV. Approaches to the active site of coupling factor I.

1. Prolonged treatment of coupling factor I (CF1) from spinach chloroplasts with trypsin free of chymotrypsin yielded an active ATPase. The isolated preparation showed only two polypeptide chains (mol wt 55,000 to 60,000) on acrylamide gels run in the presence of sodium dodecyl sulfate. The three smaller subunits of CF1 were not detectable. The preparation no longer served as a coupling factor for photophosphorylation in either EDTA- or silicotungstate-treated chloroplasts. 2. An antiserum prepared against coupling factor I from chloroplasts inhibited the ATPase activity of the trypsin-treated CF1. In contrast, antisera prepared against the two individual (denatured) subunits did not inhibit the ATPase activity when tested either alone or together, although each interacted with the trypsin-treated protein, forming precipitin lines in Ouchterlony plates. 3. The trypsin-treated enzyme was still cold-labile, showing that the three smaller subunits are not required for this property. However, the enzyme was no longer sensitive to the natural inhibitor protein which is one of its subunits (subunit epislon), but was still sensitive to inhibition by the flavonoid quercetin. 4. Two equivalents of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole were sufficient to inhibit about 80% of the ATPase activity of the coupling factor, irrespective of whether it contained two of five subunits. The inhibition was completely reversed by dithiothreitol. 5. Triated 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole was prepared. Treatment of the coupling factor with this tritium-labeled inhibitor followed by electrophoresis on acrylamide gels revealed that most of the radioactivity was incorporated into the beta subunit of the enzyme (molecular weight 56,000).     

Structural studies on a carbohydrate chain isolated from the lipopolysaccharide of Shigella boydii type 8

The lipopolysaccharide isolated from the cells of Shigella boydii type 8 bacteria gave precipitin bands against homologous antisera on Ouchterlony plates, whereas the carbohydrate-containing fractions obtained from it did not. One of the fractions was obtained in major proportion and contained 23.5% of sugars. A structure was assigned to the carbohydrate chain in this material by using the results of methylation, periodate oxidation, and deamination studies.     

蛇毒丝氨酸蛋白酶多样性──底物专一性免疫化学及序列比较研究

本文报道烙铁头（Ｔｒｉｍｅｒｅｓｕｒｕｓｍｕｃｒｏｓｑｕａｍａｔｕｓ）蛇毒纤维蛋白原溶酶（ＴＭＶＦｇ），眼镜王蛇（Ｏｐｈｉｏｐｈａｇｕｓｈａｎｎａｈ）蛇毒纤维蛋白原溶酶（ｏｈＳ１），竹叶青（Ｔｒｉｍｅｒｅｓｕｒｕｓｓｔｅｊｎｅｇｅｒｉ）蛇毒专一纤溶酶原激活剂（ｓｖ－ｐＡ）对５种小分子多肽底物的底物专一性，及这些蛇毒丝氨酸蛋白酶对各种凝血因子（第Ｘ因子、凝血酶原、纤溶酶原、蛋白Ｃ）的作用，并和其它蛇毒丝氨酸蛋白酶如矛头蝮（Ｂｏｔｈｒｏｐｓａｔｒｏｘ）蛇毒凝血酶样酶（Ｂａｔｒｏｘｏｂｉｎ）、铜头蝮（Ａｇｋｉｓｔｒｏｄｏｎｃｏｎｔｏｒｔｒｉｘｃｏｎｔｏｒｔｒｉｘ）蛇毒蛋白Ｃ激活剂ＡＣＣ－Ｃ、蝰蛇（Ｖｉｐｅｒａｒｕｓｓｅｌｌｉ）毒第Ⅴ因子激活剂ＲＶＶ－Ｖ进行比较研究。通过酶标偶联免疫反应研究了抗ｓｖ－ＰＡ抗体与各种丝氨酸蛋白酶的免疫交叉反应，并对蛇毒丝氨酸蛋白酶及相应功能的哺乳动物蛋白酶进行了序列比较分析。从底物专一性多样性及已知序列结构分化上对这一类蛇毒丝氨酸蛋白酶的结构与功能进行了探讨和研究。     

Purification of ornithine decarboxylase from regenerating rat liver

Ornithine decarboxylase was purified 175-fold over the crude 100 000 × g supernatant from homogenates of regenerating rat liver. It exhibited a single major band on acrylamide gels and a minor contaminant which may represent partially degraded enzyme. Antibody prepared against this enzyme gave a single precipitin line on Ouchterlony plates. The enzyme was precipitated by the antibody and substantial activity could be recovered from the immune precipitate. Several properties of the enzyme are described including differential effects of mercaptoethanol and dithiothreitol on enzyme activity.     

Proteomic analysis of Latrodectus tredecimguttatus venom for uncovering potential latrodectism-related proteins

Black widow spider is one of the most poisonous spiders in the world. Up to now, there have been few systematic analyses of the spider venom components, and the mechanism of action of the venom has not been completely understood. In this work, we employed combinative proteomic strategy to analyze the venom collected from living adult spider Latrodectus tredecimguttatus by electrical stimulation. The experiments demonstrated that the venom is primarily composed of high molecular weight proteins and has high abundance proteins around 100 kDa. The content of peptides and proteins with low molecular weight is low. A total of 75 nonredundant venom proteins with distinct function were unambiguously identified. Besides the known black widow spider venom proteins including latrotoxins, a variety of hydrolases and other proteins with special activity were found in the venom, such as proteinase, phospholipase, phosphatase, nuclease, fucolectin, venom allergen antigen 5-like protein and trypsin inhibitor, and so on. Their possible biological actions and relationship with latrodectism were discussed. The results help to understand the complexity and action mechanism of L. tredecimguttatus venom.     

黄腹潜蝇茧蜂寄生因子的特性及其对寄主的生理效应

初步研究了黄腹潜蝇茧蜂Opius caricivorae Fischer寄生因子的特性及其对寄主美洲斑潜蝇Liriomyza sativae Blanchard幼虫的生理效应。黄腹潜蝇茧蜂携带的主要因子是毒液。毒液器官是由一个土黄色的锥形毒囊和7个透明的椭圆形的毒腺及导管构成的;毒液的电泳图谱显示约有12条蛋白带,其中绝大多数低于100 kD,含量最高的3条蛋白带为43.5、25.9和20.1 kD;杜氏腺约有15条左右蛋白质条带,其中有5条含量很高（121.4、77.0、51.5、42.7和36.5 kD）。通过透射电镜观察,在黄腹潜蝇茧蜂毒腺分泌细胞和卵巢表皮细胞中新发现存在一种类病毒颗粒,这些球状颗粒直径大约为50 nm。雌蜂经Co60辐射处理后再寄生（即假寄生）3龄寄主幼虫,被寄生后的寄主依然能正常化蛹,但不能羽化;7 h后寄生体壁开始出现红斑;脂肪体形态结构无显著变化;绝大多数的蜂卵没有被包囊。推测在正常寄生的情况下可能是毒液抑制了寄主的包囊作用,而新发现的类病毒颗粒是否参与了这一过程目前还不清楚。     

Isolation of trypsin isozymes from the mosquito Aedes aegypti (L.)

Trypsin has been isolated from midgut homogenates of blood-fed females of Aedes aegypti by a simple two-step purification procedure: ion-exchange chromatography followed by affinity chromatography. The resulting mosquito trypsin contains a number of isozymes, among which 5 major SDS-PAGE bands are recognized with molecular weights of 26.7, 28.5, 29.7, 31.0 and 32.0 kdaltons, as are some minor bands above and below this range. The isozymic pattern is comparable to that in crude homogenates. Isoelectric focussing of purified trypsin however, revealed over 20 tryptic isozymes, demonstrating that several isozymes segregate into subforms. A high correlation between TAME-active fractions and their DFP equivalent was demonstrated by using ³H-labelled DFP as a marker for trypsin on native acrylamide gels.The purification factor and the specific activities are discussed with respect to the unusual amounts of protein of dietary origin present in the midgut homogenates. Interference of blood-borne coagulation factors of a tryptic nature is unlikely.     

Cotiarinase is a novel prothrombin activator from the venom of Bothrops cotiara

Snake venom serine proteinases (SVSPs) may affect hemostatic pathways by specifically activating components involved in coagulation, fibrinolysis and platelet aggregation or by unspecific proteolytic degradation. In this study, we purified and characterized an SVSP from Bothrops cotiara venom, named cotiarinase, which generated thrombin upon incubation with prothrombin. Cotiarinase was isolated by a two-step procedure including gel-filtration and cation-exchange chromatographies and showed a single protein band with a molecular mass of 29 kDa by SDS-polyacrylamide gel electrophoresis under reducing conditions. Identification of cotiarinase by mass spectrometric analysis revealed peptides that matched sequences of viperid SVSPs. Cotiarinase did not show fibrinogen-clotting, platelet-aggregating, fibrinogenolytic and factor X activating activities. Upon incubation with prothrombin the generation of thrombin was detected using the peptide substrate d-Phe-Pip-Arg-pNA. Moreover, mass spectrometric identification of prothrombin fragments generated by cotiarinase in the absence of co-factors (phospholipids, factor Va, factor Xa and Ca²⁺ ions), indicated the limited proteolysis of this protein to release prothrombin 1, fragment 1 and thrombin. Cotiarinase is a novel SVSP that acts on prothrombin to release active thrombin that does not match any group of the current classification of snake venom prothrombin activators.     

Carbohydrate Composition of Variant-Specific Surface Antigen Glycoproteins from Trypanosoma brucei

SYNOPSIS. The carbohydrate of variant-specific surface antigen glycoproteins from bloodstream forms of 13 cloned variants of Trypanosoma brucei was analyzed by gas-liquid chromatography. The glycoproteins contained from 6 to 17% carbohydrate by weight, and all contained the same 4 sugars: mannose, galactose, glucose, and glucosamine (probably as N-acetyl-glucosamine). The glycoprotein from variant 048, strain 427 contained (±20%) 11 mannose, 4 galactose, 4 glucose, and 5 glucosamine residues/mole of glycoprotein (molecular weight 65,000). Glucose was an integral component of the glycoproteins, not dissociable by sodium dodecyl sulphate, 8 M urea, or 1 M acetic acid. Some of the glucose was dissociated by trichloroacetic acid. Most of the glycoproteins formed precipitin bands with concanavalin A in Ouchterlony double diffusion, but none formed such bands with wheat germ agglutinin or Ricinus communis lectin (molecular weight 120,000).     

Immunological Homology Between Crystal and Spore Protein of Bacillus thuringiensis

Spore suspensions containing about 0.3% crystals and crystal suspensions containing about 0.1% spores were obtained from cultures of Bacillus thuringiensis by extraction with a two-phase system. Both preparations were tested for the presence of contaminating material from vegetative cells and were judged to be clean. Solutions of spore protein were obtained by extracting broken spores with phosphate buffer followed by extraction with either alkali- or urea-mercaptoethanol. The alkali spore or urea spore extracts had the same isoelectric point as crystal protein solubilized with these reagents. An antiserum prepared against alkali crystal solution precipitated alkali or urea spore extracts and crystal solutions but not phosphate spore extracts or extracts of whole cells. Lines of identity between spore and crystal precipitates were observed by using the Ouchterlony double-diffusion technique. Absorption of the antiserum with an excess of urea spore extract caused a disappearance of the precipitin bands originating from the spore protein and the homologous bands from the crystal protein. The results suggest that the crystal and endospore contain one or more common proteins.     

An improved staining procedure for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate polyacrylamide gels

The use of 3,3′,5,5′-tetramethylbenzidine-H₂O₂ as a stain for the peroxidase activity of cytochrome P-450 (or cytochrome P-450 in sodium dodecyl sulfate polyacrylamide gels is described in this report. This reagent can be used to detect very low levels of heme-associated peroxidase activity. The blue-stained bands on polyacrylamide gels are distinet, and the color is stable. The stained gels can be photographed or scanned at 690 nm because the gel background remains clear. The stain is easily removed from the gels to permit subsequent protein staining. Staining first for peroxidase activity has no effect on the subsequent protein staining profile. The peroxidase activity of cytochrome P-450 (or cytochrome P-420) in immunoprecipitates in Ouchterlony double diffusion plates can also be detected using this reagent.     

Isolation of a 70 000 molecular weight antigen of the Novikoff hepatoma.

Previous reports from this laboratory have indicated that a number of cytosol and nuclear proteins of Novikoff hepatoma cells were immunologically related [Yeoman, L. C., Jordan, J. J., Busch, R. K., Taylor, C. W., Savage, H., & Busch, H. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 3258; Busch, R. K., & Busch, H. (1977) Tumori 63, 347]. In preparation for analysis of their structure and function, studies were undertaken to purify nuclear antigen 2 from the cytosol of Novikoff hepatoma cells in high yield and purity. It was shown on Ouchterlony gels that cytosol nuclear antigen 2 formed a single immunoprecipitin band of identity with one of the bands extracted from Novikoff nuclear chromatin. In this study, a 70 000 molecular weight antigen was isolated from the cytosol of Novikoff hepatoma cells by ammonium sulfate fractionation, ion-exchange chromatography, and isoelectric focusing in a granulated gel bed. This protein which focused at a pI of 6.3 was labeled with 125I-labeled Bolton-Hunter reagent and purified on an Ultrogel AcA-44 column. As shown by electrophoresis on NaDodSO4-polyacrylamide gels, the antigen in the excluded volume migrated as a single protein with a molecular weight of 70 000. The overall purification over the starting material was 2890-fold.     

Primary culture of antvenom gland cells

Summary Venom from the antPseudomyrmex triplarinus reduces the symptoms and swelling of rheumatoid arthritis. The cells that produce the venom were dissected from larval and pupal ants and culture conditions studied. Cell dissociation, with minimal amount of damage, was done with 0.25% trypsin at 4°C with subsequent use of soybean trypsin inhibitor. A new medium was formulated and epidermal growth factor, fibroblast growth factor, insulin, cAMP, cGMP, and isoproterenol were beneficial. The optimum osmotic pressure was a relatively high 500 mOsm. Conditioning the medium with an established insect cell line was essential for long-term cell survival. Under these culture conditions the structural and metabolic integrity of the cells were maintained for up 12 mo.     

Immunological studies with an m-deficient histoplasmin skin-test antigen

Previous studies have shown that a single skin test to histoplasmin may induce complement-fixing antibodies or M precipitins (or both) to histoplasmin in histoplasmin-sensitive, but serologically negative, individuals. Ideally a skin-test antigen should be one which detects hypersensitivity without stimulating humoral antibodies. Histoplasmin skin-test antigens presently used contain both H and M antigens. The present study was undertaken to evaluate an histoplasmin skin-test antigen deficient in the M component but containing the H antigen. Thirty histoplasmin-hypersensitive subjects were bled prior to administration of the experimental skin-test antigen and at various time intervals thereafter. Only six of the thirty hypersensitive subjects showed serological responses. The sera of the six, however, only showed weak precipitin reactions, five showed M bands, and only one showed an M and an H band. None showed complement-fixation titers with either the yeast or mycelial antigens of Histoplasma capsulatum. Our data suggest that the use of a skin-test antigen purified to contain only H component would detect histoplasmin hypersensitivity without inducing antibodies and would eliminate false-positive serological reactions caused by the M component.     

Immunoaffinity purification of Schistosoma mansoni soluble egg antigens

Schistosoma mansoni egg antigens were purified from a heterogeneous mixture of soluble egg antigens (crude SEA) with an immunoaffinity column that consisted of the specific anti-SEA antibodies contained in 16-week S. mansoni-infected mouse serum bound to Sepharose 4B. On sodium dodecyl sulfate (SDS) gel electrophoresis, the purified antigen fraction yielded at least eight bands staining with Coomassie blue and at least five bands staining with Coomaisse blue and at least five bands reacting with periodic acid-Schiff (PAS). All of the proteins in the antigenic fraction appear to contain carbohydrate residues. Upon immunoelectrophoresis the antigen yielded four precipitin arcs. The antigenic fraction isolated by means of the immunoaffinity column was then compared to various fractions obtained from concanavalin A (Con A) chromatography of SEA. The results of Ouchterlony immunodiffusion and immunoelectrophoresis indicate that the antigenic fraction isolated by immunoaffinity purification of SEA contains the major antigens found in the fractions obtained from Con A chromatography of SEA. The results of SDS gel electrophoresis indicate that the major PAS-reacting bands of the antigenic fraction isolated by immunoaffinity purification are found in the 3rd peak (bound fraction) resulting from Con A chromatography of SEA, whereas the major Coomaisse blue-staining band in the isolated antigenic fraction is found in the 2nd peak (unbound fraction) from Con A chromatography of SEA.     

湖南烙铁头蛇毒蛋白组分的双向电泳分析

烙铁头蛇是世界上剧毒的蛇种之一,其所携带的毒素能够导致严重的机体损伤。应用蛋白质双向电泳技术,对湖南烙铁头蛇蛇毒蛋白的蛋白质组分进行分析。通过等电聚焦和SDS-PAGE凝胶电泳分析获得完整的烙铁头蛇毒全蛋白质的图谱,经胶体考马斯亮蓝染色后,应用PDQuest软件对蛋白表达谱进行分析。通过等电聚焦和SDS-PAGE凝胶电泳有83个蛋白质组分被检测出来。其中大约90.00%的蛋白质的相对分子质量(Mr)分布在15～45 kDa之间,大约72.29%的蛋白质等电点(pI)在4.0～7.0之间。通过对烙铁头蛇毒的蛋白组学研究,获得其蛇毒蛋白质组分的表征特点,为后续进一步研究各组分的身份和潜在功能奠定基础,既可以提出新的治疗方案又可以为新的药理应用提供宝贵资源。     

蜈蚣(Scolopendra subspinipes mutilans L.Koch)毒的化学组成和生物活性

系统分析少棘蜈蚣粗毒的化学组成,蛋白质含量86.23％、水不溶物质0.24％、还原糖0.23％、水份2.1％,仅含Ser、Pro、Arg等三种游离氨基酸,鉴定了11种微量元素,碱性凝胶电泳显示20条蛋白质谱带。分析了10种酶的活性、出血毒性及对血小板聚集的影响,其中精氨酸酯酶活力最高,不存在类凝血酶、淀粉酶活性及出血毒性,蜈蚣毒的浓度为0.3μg/μL时强烈诱导血小板的聚集。     

Antigenic relatedness of immunoglobulins toLegionella pneumophila serogroups 1–6 from humans and the nonhuman primateMacaca fascicularis

The purpose of this study was to determine whether cynomolgus monkey antisera toLegionella pneumophila serogroups 1–6 antigens could be used as positive controls in the indirect immunofluorescence assay (IFA) for legionellosis. Immunoelectrophoretic mobilities and Ouchterlony analyses with heavy chain-specific antisera and IFA titers with immunoglobulin class-specific conjugates were used to show antigenic relatedness of immunized monkey immunoglobulins to those produced as a result of infection in humans. Identical immunoelectrophoretic precipitation patterns were obtained for human and monkey sera with antihuman gamma, mu, and alpha heavy-chain-specific antisera. Ouchterlony analyses showed precipitin bands of partial identity between human and monkey IgG, IgM, and IgA classes. IFA titers in the monkey hyperimmune antisera were >16,000 with antihuman conjugate. These data suggest that hyperimmune cynomolgus monkey antisera are suitable alternatives to human sera for IFA-positive controls.     

竹叶青蛇毒凝血酶样酶氨基酸序列报道

蛇毒凝血酶样酶可作为蛋白酶结构与功能研究的良好模型,并已广泛用于各种血栓疾病的诊断和治疗,因而测定其一级结构具有重要意义．利用逆转录与聚合酶链反应相结合的ＲＴ－ＰＣＲ法,扩增出竹叶青蛇毒凝血酶样酶（ＴＳＶ－ＴＥＬ１）的ｃＤＮＡ;将扩增的ｃＤＮＡ片段克隆入ｐＧＥＭ－Ｔ载体中,经末端终止法测定核苷酸序列,推导出竹叶青蛇毒凝血酶样酶的全序列．竹叶青蛇毒凝血酶样酶由２３４个氨基酸组成并含有１个位于Ａｓｎ２０的Ｎ－型糖基结合位点．竹叶青蛇毒凝血酶样酶序列与其它蛇种来源凝血酶样酶具有较大相似性,其与黄绿烙铁头蛇毒凝血酶样酶序列相似度为８４％,与美洲矛头蝮蛇毒凝血酶样酶序列相似度为６８％,而与牛凝血酶Ｂ链序列相似度仅为２５％．