Purification and characterization of the vascular permeability factor produced by Bacillus cereus

Purification of an extracellular protein exhibiting the vascular permeability activity produced by Bacillus cereus was performed by ammonium sulfate precipitation followed by chromatography on DE-32 cellulose, Sephadex G-100, and Sephadex G-75. The purified protein was found to be electrophoretically and antigenically almost homogeneous although it contained a trace of contaminant. The molecular weight of the protein was calculated to be 45,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified protein showed vascular permeability activity and mouse lethal toxicity, and caused fluid accumulation in ligated mouse intestinal loops, whereas it did not show any hemolytic and lecithinase activities. From these findings, the purified protein is suggested to be an enterotoxin (or a diarrheagenic toxin) responsible for diarrhea caused by B. cereus in a diarrheal-type food poisoning.     

Purification of a heat labile dermonecrotic toxin from culture fluid of Pasteurella multocida

A highly pure heat-labile dermonecrotic toxin (DNT) of Pasteurella multocida was isolated from bacterium-free broth culture fluid. The protocol for the isolation included the following steps: ammonium sulphate precipitation, gel filtration, ion exchange chromatography and preparative polyacrylamide gel electrophoresis (PAGE). About 1 mg of purified DNT was recovered from 3 l of broth culture fluid. The final product was toxic for embryonic bovine lung (EBL) cells, lethal for mice, dermonecrotic in the guinea pig skin test and inactivated by heating at 56 degrees c. The recovery of biological activity was about 5% that of the original culture fluid and the specific activity had increased about 4000 times. After sodium dodecyl sulphate (SDS)-PAGE and silver staining a single band appeared, indicating that the purified DNT was free from contaminating proteins. The molecular weight of the toxin was approximately 125,000 daltons. The minimal toxic dose of DNT protein for embryonic bovine lung cells was about 2 ng, the minimal dermonecrotic dose in the guinea pig skin test was about 80 ng and the 50% lethal dose for mice about 300 ng.     

Isolation of exfoliative toxin from Staphylococcus intermedius and its local toxicity in dogs

A rounding effect was demonstrated in cultured cells inoculated with the culture filtrates (CFs) of 60 strains of Staphylococcus intermedius derived from dogs affected with pyoderma. Exfoliative toxin (ET)-like toxin (ETLT) was isolated from the CF of S. intermedius strain D-52, which exhibited strong rounding activity and then was purified by gel filtration on a Sephadex G-75 column, and by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The ETLT caused exfoliation in 1-day-old chickens, suckling Syrian hamsters, and dogs, but not in suckling mice. The ETLT was serologically different from exfoliative toxin A (ETA), exfoliative toxin B (ETB), exfoliative toxin C (ETC), S. hyicus exfoliative toxin A (SHETA), and SHETB, as shown by Western blot analysis. The molecular weight of the ETLT was estimated at 30 kDa by SDS-PAGE. In the present study, we propose the ETLT was a novel type of ET, S. intermedius exfoliative toxin (SIET).     

家兔胚源性特异蛋白－1的分离纯化及其理化性质

经ＳｅｐｈａｄｅｘＧ７５和PＡＧＥ从家兔早孕期子宫液中分离纯化了一种胚源性特异蛋白质。分析表明，该蛋白质分子量为５３８００，ｐＩ为５．８，Ｎ－末端为赖氨酸，分子中不含糖、脂成分，但富含异亮氨酸、亮氨酸，缺乏半胱氨酸。     

Induction of pneumonia in rabbits by use of a purified protein toxin from Pasteurella multocida.

Heat-labile toxin from a cell sonicate of a virulent type-D strain of Pasteurella multocida was purified by ammonium sulfate precipitation followed by ion exchange chromatography, gel filtration chromatography, and polyacrylamide gel electrophoresis. Toxic activity was assayed during toxin purification by cytopathic effect in Vero or bovine embryonic lung cell cultures. Toxicity for cells correlated with dermonecrosis in guinea pig skin. Toxicity was accounted for by a single protein with a molecular weight of 149,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Rabbits were inoculated intranasally with purified toxin to determine whether toxin had a role in the induction of pneumonia in rabbits infected with P multocida. Pneumonia, pleuritis, acute hepatic necrosis, and splenic lymphoid atrophy were found in 4 of 5 rabbits. One of 5 rabbits had bilateral turbinate atrophy. Western blotting with monoclonal antibodies to toxin from a P multocida isolate causing atrophic rhinitis in pigs revealed the toxin that induces pleuritis and pneumonia in rabbits to be the same or a closely related toxin.     

Purification and characterisation of chicken liver monomeric glutathione peroxidase

1. A novel glutathione peroxidase, which is distinct from tetrameric glutathione peroxidase, was purified to homogeneity from a broiler chick liver cytosolic fraction using 5 different column chromatographic methods.

2. The enzyme in cytosol was separated from ‘classic’ tetrameric glutathione peroxidase and glutathione S‐transferases by DEAE‐Sephacel and Sephadex G‐100 chromatographies and further purified by Mono Q, hydroxylapatite and sulphobro‐mophthalein‐S‐glutathione‐agarose chromatographies.

3. The molecular weight of the purified enzyme determined by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis was 19,500 and that found by gel filtration chromatography was comparable. This indicates that the enzyme protein is a single polypeptide. The isoelectric point of the enzyme was determined as 7.0 by polyacrylamide gel isoelectric focusing.

4. The purified enzyme catalysed the reduction of hydrogen peroxide, cumene hydroperoxide, tert‐butyl hydroperoxide and linoleic acid hydroperoxide. Furthermore, it reduced phosphatidylcholine hydroperoxide in the absence of phospholi‐pase A2. The optimum pH for the enzyme reaction was 7.0. The antiserum against the purified enzyme reacted with the 19.5 kDa polypeptide in the liver cytosol of duck and quail.     

The purification and characterisation of cervine IgM and IgG

A procedure is described for the isolation of immunoglobulin G (IgG) and immunoglobulin M (IgM) from hyperimmune cervine serum. Hybrids of red deer (Cervus elaphus) and wapiti (Cervus canadensis) were immunised with keyhole limpet hemocyanin (KLH). An immunoglobulin-containing fraction was precipitated from the hyperimmune serum using ammonium sulphate. The antigen-specific immunoglobulins were purified by KLH-conjugated sepharose affinity chromatography and further separated into IgM and IgG by gel-filtration chromatography. Purified immunoglobulin was analysed by polyacrylamide gel electrophoresis and isoelectric focusing. The molecular weights and isoelectric points of the composite chains of cervine IgG and IgM are presented.     

家蚕铜锌超氧化物歧化酶的分离纯化及其部分性质的研究

家蚕体液经热变性、DEAE 琼脂糖柱层析、SephacrylS 2 0 0凝胶过滤和等电点聚焦电泳纯化 ,获得达到均一程度的高活性的铜锌超氧化物歧化酶 (Cu .Zn SOD)。活性回收率为 35 % ,纯化倍数为 1911倍。测得该酶的分子量约为 32 0 0 0D ,亚基分子量约为 16 0 0 0D。该酶在紫外光区与可见光区的吸收峰分别为 2 70nm和 6 76nm。测得该酶的等电点为pH 7.15。     

桑叶中抗凝血活性成分的初步分离与纯化

采用乙醇分级沉淀和Sephadex G100凝胶层析的方法,结合凝血指标检测,对桑叶中的抗凝血活性成分进行了分离纯化。结果表明:桑叶的抗凝血活性主要集中在桑叶的水提取液中,能够显著延长人体血浆的活化部分凝血活酶时间(APTT)值。桑叶水提取液经30%乙醇沉淀、凝胶层析和醋酸纤维素薄膜电泳表明其抗凝血活性成分是一种多糖组分。试验还表明,高浓度乙醇处理对桑叶多糖的抗凝血活性没有影响。     

Purification of three fragments of the dermonecrotic toxin from Pasteurella multocida

Dermonecrotic toxin purified from sonicates of Pasteurella multocida was mildly trypsinized. The trypsinized preparations were reversibly dissociated into three polypeptides, with molecular weights of about 23,000 (fragment a), about 64,000 (fragment b), and about 74,000 (fragment c) by treatment with 100 mM dithiothreitol and 6 M urea. Upon removal of dithiothreitol and urea from the dissociated toxin by dialysis, the fragments reassociated and formed dermonecrotic toxin indistinguishable from the native toxin. The three fragments were separated from the dissociated toxin by gel filtration on a Sephadex G-200 column equilibrated with buffer containing 4 M urea and 1 mM dithiothreitol. The purified fragments a, b, and c did not show dermonecrotic activity for guinea pigs. Immunodiffusion and immunoelectrophoretic analysis with rabbit anti-dermonecrotic antiserum showed that the three purified fragments were antigenically distinct but had partial identity with the native toxin.     

鸡和猪分泌型免疫球蛋白A结构蛋白的比较

通过SephadexG 2 0 0凝胶过滤和DEAE纤维素柱 ,分别从胆汁和初乳中提纯鸡和猪的分泌型免疫球蛋白A (SIgA)。SDS PAGE结果显示 ,鸡SIgA的轻链分子量约为 2 6 0 0 0～ 2 80 0 0 ,与鸡IgG的轻链分子量相似 ,而重链分子量约为 670 0 0～ 70 0 0 0 ,比鸡IgG的分子量要大。猪的SIgA与猪IgG的轻链和重链的分子量均相同。轻链的分子量约为 2 6 0 0 0～ 2 80 0 0 ;重链的分子量约为 53 0 0 0～ 570 0 0。猪SIgA中J链的分子量为 1 6 0 0 0～ 1 70 0 0。本实验证明鸡SIgA轻链的分子量与猪的SIgA的轻链相似 ,而鸡SIgA重链的分子量则高于猪的SIgA的重链     

Purification of IgG from serum with caprylic acid and ammonium sulphate precipitation is not superior to ammonium sulphate precipitation alone

Immunoglobulin G (IgG) from bovine serum raised against Aeromonas Salmonicida was purified by ammonium sulphate precipitation (ASP) or caprylic acid treatment followed by ammonium sulphate precipitation (CAAS). Purity of IgG samples prepared by both methods were examined by High Performance Gel Permeation Chromatography, electrophoresis and antibody activity assay. Results suggest that IgG prepared by ASP is better than that obtained by CAAS method in terms of the yield of the IgG monomers and the recovery of the antibody activity.     

莆田黑猪精液中NAGase酶学特性研究

试验对以莆田黑猪精液为材料分离纯化得到的N-乙酰-β-D-氨基葡萄糖苷酶(NAGase)进行了理化特性研究。经硫酸铵分级沉淀、DEAE Sepharose Fast Flow离子交换层析和Sephadex G100分子筛层析获得PAGE电泳纯化的NAGase酶制剂。以对硝基苯-N-乙酰-β-D-氨基葡萄糖苷(pNP-GlcNAc)为底物,研究酶催化水解的相关性质。分离纯化获得的酶制剂比活力为1561.42 U/mg,分子质量为58 ku,只有1个亚基,等电点pI为9.13。酶的最适pH为5.6,最适温度为45 ℃,酶在pH 3.6～7.8之间稳定,当pH>8时迅速失活,在50 ℃以下处理30 min酶活力保存稳定,高于50 ℃时,酶活力迅速降低。酶促反应动力学符合米氏双曲线方程,米氏常数K_m为0.82 mmol/L,最大反应速度V_m为39.23 μmol/(L·min)。催化pNP-GlcNAc反应的活化能为27.30 kJ/mol。金属离子中Na⁺、K⁺、Mg²⁺、Ca²⁺对酶活力无明显影响,Zn²⁺、Cu²⁺、Pb²⁺对酶有抑制作用。     

Purification and properties of cholesterol oxidase and choline phosphohydrolase from Rhodococcus equi.

Cholesterol oxidase (CO) and choline phosphohydrolase (CPH) exoenzymes were isolated from culture supernatants of Rhodococcus equi ATCC 33701 and their hemolytic and cytotoxic activities examined. The purifications involved differential ammonium sulphate precipitation, ion exchange and gel filtration chromatography. A purification of 32.8-fold and a yield of 0.3% of CO were determined by synergistic hemolysis of sheep red blood cells (SRBC) presensitized with Staphylococcus aureus beta toxin. The enzymatic activity of CO was also demonstrated by oxidation of aqueous cholesterol suspensions. The activity of CO was reversibly inhibited by concentration. A purification of 412.4-fold and a yield of 1.7% of CPH were determined by hydrolysis of p-nitrophenyphosphorylcholine. Purity of both exoenzymes was confirmed by immunoblotting. On sodium dodecyl sulphate polyacrylamide gel electrophoresis, the CO had a molecular mass (Mr) of 60 kd and the CPH a Mr of 65 kd. Choline phosphohydrolase did not hydrolyse sphingomyelin. Sphingomyelinase C (SMC) activity was however demonstrated in concentrated culture supernatants. This dissociation of SMC from CPH activity indicates that R. equi produces two distinct phospholipase C exoenzymes, a CPH and a SMC. Both CO and CPH combined, or individually, did not lyse native SRBC even with subsequent chilling of the cells at 4 degrees C ("hot-cold" treatment). Purified CO lysed beta toxin-sensitized SRBC. The CPH showed only minor hemolytic activity against such sensitized SRBC even at high concentrations. Combination of CO and CPH in lysis of beta toxin sensitized SRBC showed only minor additive rather than synergistic effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serum concentration and properties of alpha-fetoprotein and serum level of albumin in sucking piglets

Porcine alpha-fetoprotein (AFP) was purified from the sera of four-day-old piglets using an immunoadsorbent column. The molecular weight of the AFP was 80,000 when determined by gel filtration on Sephadex G-200 and 75,000 when determined by sodium dodecylsulphate polyacrylamide gel electrophoresis. The isoelectric point of the AFP was pH 4.85 at 0 degree C. The maximum concentration of serum AFP was reached on the fourth day after birth (mean value 1.1 mg ml-1), and it then decreased to 10 micrograms ml-1 when the piglets were 35 days old. The concentration of serum albumin increased rapidly between birth and seven days old, reaching 16 to 17 mg ml-1 at seven days old. Between birth and seven days old, the serum concentrations of AFP and albumin were approximately inversely proportional.     

Experimental Staphyloenterotoxicosis in Mink

Experimental staphyloenterotoxicosis was produced in minks by oral administration of mink feed containing 5 or 200 µg of purified enterotoxin A per test animal. The animals became very exhausted after the ingestion of toxin. Vomiting was observed in two of seven minks of the lower toxin group with a latent period of 2.5 to 4.0 h. The higher toxin concentration caused vomiting in four of seven test animals with a latent period of 2.0 to 2.5 h. Vomitus was accompanied by strong salivation. Poor appetite was observed in four of seven minks having ingested 5 µg of SEA, and 200 µg caused total loss of appetite in all the test animals. After a test period of 22 h all the animals but one had normal appetite. Diarrhoea was prominent in three of seven minks with the low toxin concentration and in all with the high toxin concentration. Statistically significant haemato-logical changes compared to the control group were an increase in neutrophil count and a decrease in lymphocyte count in the high toxin group. Significant changes in the blood chemical data were an increase in blood urea nitrogen with 200 µg of SEA and a decline in the cholesterol level in both toxin groups.     

Attempts to separate female Ascaris suum antigen and to investigate its partial characterization

The location and separation of Ascaris suum antigen for serological testing was investigated. The antigenic constituent was rich in the ovary of the adult worm and was obtained by dialysis with 50% ammonium sulphate saturated solution. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting analysis demonstrated that the heat labile antigenic preparation showed one major and seven faint bands. The major band seemed also to be a glycoprotein. The sera from pigs with/without hepatic milk spot showed relatively high precipitation titres, while, those from the specific pathogen free pigs manifested low titres.     

Purification and some of the properties of alkaline phosphatase in guinea fowls (Numida meleagris galeata)

1. Alkaline phosphatase activity in the plasma of different strains of guinea fowls showed considerable variation both within and between sexes as well as within and between strains. 2. The enzymes from different strains of wild guinea fowls had different mobilities on disc polyacrylamide electrophoresis but each was characterised by a single band. 3. When the enzyme was purified 163-fold from the plasma of a domesticated grey breasted strain, both ion-exchange chromatography and gel-filtration purification steps yielded a single band of enzyme. 4. The purified enzyme had a molecular weight of 79,400 +/- 3,000 and was stable up to 60 degrees C at the optimum pH of 9.6. 5. Evidence is provided that guinea fowl alkaline phosphatase is a metalloenzyme.     

Immunoglobulin G responses in 21 dogs with skin diseases to antigens from different isolates of Staphylococcus intermedius

The aim of this study was to characterise the immunoglobulin G (IgG) response in 21 dogs with or without pyoderma to antigens from six isolates of Staphylococcus intermedius. The staphylococcal proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis, transferred electrophoretically on to a membrane and subjected to immunoblotting with the dogs' serum. Gels containing separated proteins from the six isolates revealed 29 to 33 distinct bands with molecular weights ranging from 20 to 230 kDa. All the dogs' sera contained IgG that recognised 12 to 24 bands (mean 17), regardless of whether the dogs had pyoderma. The recognised proteins had molecular weights ranging from 20 to 198 kDa but the majority had molecular weights below 75 kDa. The most intense band in all six isolates had a molecular weight of 28 to 29 kDa. The antibody responses to the six isolates were essentially similar except that there were significantly more bands in the response to isolate 2 than to isolate 6, and occasional differences in the intensity of individual bands. All 21 dogs mounted an IgG response to multiple antigens in S intermedius, which differed only marginally between the six isolates. This lack of variation provides evidence that the host's response to different isolates of S intermedius is not a major factor in canine pyoderma.     

The protective antigens of equine herpesvirus type 1.

Equine herpesvirus type 1 was cultivated in swine testis cell cultures and partially purified by differential centrifugation and centrifugation in a linear sucrose density gradient. The viral envelope was separated from the nucleocapsid by treatment with Rexol 25J followed by sucrose gradient centrifugation. The envelope and nucleocapsid preparations were then electrophoresed in polyacrylamide gel after solubilization with sodium dodecyl sulphate. Hamsters were immunized with various preparations of the partially purified virus, including live or inactivated equine herpesvirus type 1 and viral envelope and nucleocapsid, all derived from the Kentucky D strain of the virus. Challenge of the immunized hamsters, with a hamster-adapted strain of equine herpesvirus type 1 demonstrated protection only in those animals which had been vaccinated with envelope-containing materials. When vaccination was carried out with fractions of electrophoresed envelope or nucleocapsid, protection was induced only by polypeptides of high molecular weight containing a glycoprotein component of the envelope of equine herpesvirus type 1.