The production of foot-and-mouth disease virus from BHK 21 C 13 cells grown on the surface of glass spheres.

In view of the advantages which are associated with the use of the BHK monolayer cell for the production of foot-and-mouth disease (FMD) virus, a unit system using glass spheres was developed to grow BHK monolayer cells and to test the susceptibility of such cells to FMD virus. The yield of cells and their susceptibility compares favorably with GREEN BHK monolayer cells which have been grown in Roux bottles.     

Altered glycoprotein synthesis in mouse epidermal cells treated with retinyl acetate in vitro

Retinyl acetate alters glycoprotein synthesis in mouse epidermal cells in culture. Epidermal glycoproteins were enzymatically digested to glycopeptides and separated on DEAE Sephadex A50 columns using different concentrations of LiCl. There was a two-fold increase in incorporation of fucose and glucosamine in the 0.2 M LiCl fraction from cells treated for 3 weeks with 12.5 μg/ml retinyl acetate and 1.25% DMSO as compared with DMSO controls. No changes were noted in other fractions. The glycopeptide from A treated cells isolated on 0.2 M LiCl had a higher molecular weight than glycopeptides from that same fraction eluted by control cells. This isolated newly synthesized glycopeptide from vitamin A treated cells appears to be a single product by rechromatography on DEAE Sephadex A50 and Sephadex G100 columns.     

Preparation of FMD type A87/IRN inactivated vaccine by gamma irradiation and the immune response on guinea pig

FMD is one of the most economically damaging diseases that affect livestock animals. In this study FMD Virus type A87/IRN was multiplied on BHK21 cells. The virus was titrated by TCID50 method, it was 10^7.5/ml. The FMD virus samples were inactivated by gamma ray from 60Co source at −20°C. Safety test was done by IBRS2 monolayer cell culture method, also antigenicity of irradiated and un-irradiated virus samples were studied by Complement Fixation Test. The Dose/Survival curve for irradiated FMD Virus was drawn, the optimum dose range for inactivation of FMDV type A87/IRN and unaltered antigenicity was obtained 40–44 kGy. The inactivated virus samples by irradiation and ethyleneimine (EI) were formulated respectively as vaccine with Al(OH)3 gel and other substances. The vaccines were inoculated to Guinea pigs and the results of Serum Neutralization Test for the normal vaccine and radio-vaccine showed protective titer after 8 months. The potency test of the inactivated vaccines was done, PD50 Value of the vaccines were calculated 7.06 and 5.6 for inactivated vaccine by EI and gamma irradiation respectively.     

Typing foot-and-mouth disease virus by fluorescent antibody technique.

Typing of foot-and-mouth disease (FMD) virus was performed by the direct fluorescent antibody (FA) technique. Type-specific FA was prepared from the following two sorts of procedures: (1) FA against live virus (FA-live) was prepared from hyperimmune serum taken from guinea pigs having received live FMD virus. Then it was adsorbed with concentrated heterotype antigen. (2) FA against inactivated virus (FA-Inact) was prepared from antiserum taken from guinea pigs immunized with purified FMD virus inactivated with acetylethyleneimine. Seventeen strains of FMD virus (seven strains of type A, seven strains of type O, and three strains of thpe C) were used. Type-specific FMD virus antigen was detected distinctly from the monolayer of BHK cells infected with each type of virus and fixed in acetone, in spite of negative results obtained from the cells fixed in methyl alcohol. All the 17 strains were typed successfully by the implementation of these two FA methods.     

Beta-glucanases of the yeast Pichia polymorpha.

Fractionation of proteins secreted into the culture medium by intact cells and protoplasts of Pichia polymorpha showing enzyme activity against laminarin, pustulan or p-nitrophenyl-beta-D-glucopyranoside has been performed, and the results compared with those obtained with cell-free extracts and lysed protoplasts. Fractionation with DEAE Sephadex A50 has proved to be the best method, yielding at least three fractions which hydrolyse laminarin. One of these fractions was active on both laminarin and pustulan. Filtration on Sephadex G-100 column only yielded one active preparation. Evidence supporting the conclusion that there are three different beta-glucanases located in the periplasmic space is presented.     

Isolation and purification of NADP-dependent "L-malate"-enzyme from corn leaves]

Isolation and purification of "malic-enzyme" NADP was done using fractionation by ammonium sulfate, anion-exchange chromatography on DEAE cellulose, gel-filtration through Sephadex G-200 and purification on DEAE Sephadex A-50. The isoenzyme isolated had a specific activity of 40-50 mkM/mg protein per min (approximately 80-fold purification) and contained negligible admixtures.     

Adenosine deaminase from pig thyroid gland purification and some properties

Adenosine deaminase was isolated from the pig thyroid gland and purified over 900-fold using DEAE Sephadex A-50 column chromatography, Sephadex G-100 gel filtration and DEAE Sephadex A-50 rechromatography. The enzyme was specific towards adenosine. The Michaelis constant based on the Lineweaver-Burk plot was 5 × 10^?5M. The optimum pH was about 7.0, and molecular weight 44 700.     

内切葡聚糖苷水解酶的分离纯化和内源荧光性质

利用离子交换和分子筛层析技术从拟康氏木霉Ｓ－３８固态发酵液中提纯了两个内切葡聚糖苷酶组分．测定了一个组分的内源荧光性质,结果表明：该酶分子的内源荧光几乎都来自色氨酸．Ｎ－溴代琥珀酰亚胺（ＮＢＳ）修饰导致了酶活力的完全丧失,但是酶荧光残留了２５％,抑制剂纤维二糖与酶结合可使部分酶荧光得到保护,同时这种结合也可以保护一定的色氨酸荧光不被外来淬灭剂淬灭．     

Detection of multiple forms of polyphosphate phosphohydrolase from Endomyces magnusii]

Labile polyphosphate phosphohydrolase from Endomyces magnusii is 27-fold purified by means of fractionation with ammonium sulphate, gel filtration on Sephadex G-75 and Biogel P-60 and chromatography on DEAE cellulose. Chromatography on DEAE Sephadex A-50, isoelelctric focusing and polyacrylamide gel electrophoresis of the enzyme preparation revealed 3 different fractions with polyphosphate phosphohydrolase activity (PPPH1, PPPH2 and PPPH3). Relative content of these fractions in E. magnusii cells is 30%, 55% and 15% respectively. Isoelectric points are: PPPH1--pH 5.1--5.2; PPPH2--pH 6.0--6.1; PPPH3--pH 6.3--6.4. PPPH1 and PPPH2 are found to be the most labile. PPPH3 is more stable under isolation procedure and storage. The fractions have similar molecular weight (48 000 +/- 3000).     

Purification, Properties, and Localization of Two Carbonic Anhydrases from Amaranthus cruentus Leaves

The method has been developed for obtaining two purified forms of carbonic anhydrase (CA, A and B forms) from amaranth (Amaranthus cruentus L.) leaves. The method includes precipitation with ammonium sulfate, fractionation by ion-exchange chromatography on DEAE Sephadex A-50, gel filtration on AcA-34 ultragel, and ion-exchange chromatography on DEAE cellulose. The molecular weights of A and B forms were different and equaled to 151 and 251 kD, respectively. The results suggest that SH groups and zinc play important roles in the catalytic activity of both CA forms. Both forms exhibited a high hydratase activity and did not represent allosteric enzymes. However, the catalytic properties of A form, evaluated from the pH dependence of kinetic parameters, differed from those of B form, which was apparently caused by dissimilar structures of these forms. Furthermore, the A form was localized in chloroplast membranes of bundle sheath cells, whereas B form was a soluble enzyme located in the cytoplasm of mesophyll cells.     

尖吻蝮蛇毒内一种新抗凝血因子ACF II 的纯化及特性

利用阴离子交换层析、凝胶过滤及阳离子交换层析三步方法, 从皖南尖吻蝮蛇毒中分离纯化到一个新的抗凝血因子ＡＣＦＩＩ（ａｎｔｉｃｏａｇｕｌａｔｉｏｎ ｆａｃｔｏｒＩＩ） 。纯化的ＡＣＦＩＩ在ＰＡＧＥ、ＳＤＳＰＡＧＥ和ＩＥＦＰＡＧＥ图谱上均呈单一区带。ＡＣＦＩＩ由两条分子量为１４．６ ｋＤ 的肽链通过二硫键连接在一起, 其等电点为７．０。ＡＣＦＩＩ具有显著的抗凝血活性, 在体外延长ＰＰＴ 时间的最终浓度为０ ．４ ｍｇ／Ｌ。ＡＣＦＩＩ不具有类凝血酶活性、磷脂酶Ａ２ 活性和纤溶活性,也没有出血活性和毒性, 是一种潜在的高效抗凝药物。     

Antigenic variation in foot-and-mouth disease: studies based on the virus neutralization reaction

The neutralization reaction is the most appropriate in vitro reference test system for assessing intratypic antigenic variation as it involves the antigenic determinants responsible for virus strain specificity and evoking protective antibody. Antigenic relationships determined in different neutralization test systems were independent of the system used and were assumed to truly reflect antigenic variation. The two-dimensional microneutralization test was found to be appropriate for foot and mouth disease (FMD) virus strain differentiation. To minimize test to test variation, comparisons are performed as matched pairs. The pooled variance of the test system is used to assess the significance of the relationships obtained. Antisera from convalescent animals were less specific than those from vaccinates. Serum quality seemed less critical for the virus neutralization than the complement fixation reaction. A system for FMD virus strain differentiation based on the use of the virus neutralization reaction taking into account the statistical and biological significance of observed r values is recommended.     

Characterization of gamma-glutamylamidase-glutaminase activity in Saccharomyces cerevisiae

In a foregoing paper we have shown the presence in the yeast Saccharomyces cerevisiae of an enzyme catalyzing the hydrolysis of L-gamma-glutamyl-p-nitroanilide, but apparently distinct from gamma-glutamyltranspeptidase. The cellular level of this enzyme was not regulated by the nature of the nitrogen source supplied to the yeast cell. Purification was attempted, using ion exchange chromatography on DEAE Sephadex A 50, salt precipitations and successive chromatographies on DEAE Sephadex 6B and Sephadex G 100. The apparent molecular weight of the purified enzyme was 14,800 as determined by gel filtration. As shown by kinetic studies and thin layer chromatography, the enzyme preparation exhibited only hydrolytic activity against gamma-glutamylarylamide and L-glutamine with an optimal pH of about seven. Various gamma-glutamylaminoacids, amides, dipeptides and glutathione were inactive as substrates and no transferase activity was detected. The yeast gamma-glutamylarylamidase was activated by SH protective agents, dithiothreitol and reduced glutathione. Oxidized glutathione, ophtalmic acid and various gamma-glutamylaminoacids inhibited competitively the enzyme. The activity was also inhibited by L-gamma-glutamyl-o-(carboxy)phenylhydrazide and the couple serine-borate, both transition-state analogs of gamma-glutamyltranspeptidase. Diazooxonorleucine, reactive analog of glutamine, inactivated the enzyme. The physiological role of yeast gamma-glutamylarylamidase-glutaminase is still undefined but is most probably unrelated to the bulk assimilation of glutamine by yeast cells.     

Combined Use of Clofibrate and Cholestyramine or DEAE Sephadex in Hypercholesterolaemia

A comparison was made of the effect of DEAE (diethylaminoethyl) Sephadex (an anion exchange resin) and cholestyramine (Questran) with and without the addition of clofibrate in normal and hypercholesterolaemic patients. DEAE Sephadex (12-15 g/day) alone appeared to be as effective as cholestyramine in lowering the plasma cholesterol by 12-15%. Clofibrate acted synergistically with DEAE Sephadex and increased the activity of the latter by over twofold. This combination proved superior to that of clofibrate and cholestyramine and has the greatest potential use in the treatment of type II pattern hyperlipoproteinaemia.     

Isolation of a non-thionein copper-binding protein from liver of copper-injected rats

1. The characterization of a low molecular weight, non-thionein, Cubinding protein isolated from rat liver is reported. The protein was isolated following chronic administration of Cu(NO₃)₂ using a combination of Sephadex G-75 and Sephadex DEAE A-25 chromatography. The protein did not bind to fully equilibrated Sephadex DEAE which formed the basis of the isolation procedure. 2. The final protein pereparation was found to be homogeneous by a variety of electrophoretic techniques and was distinguished from metallothionein on the basis of its behaviour on ion exchange and electrophoretic systems, spectral properties, and amino acid composition and metal content. It contains 6.8% cysteine and was found to bind Cu in a ratio of 1.5:1 based on a molecular weight of 11 000. 3. These results confirm the necessity to use techniques other than gel filtration alone to obtain adequate separation of low molecular weight metal-binding protein fractions.     

Production of a new thermostable neutral alpha-galactosidase from a strain of Bacillus stearothermophilus

An intracellular, thermostable, neutral α-galactosidase (α-D -galactoside galactohydrolase EC 3.2.1.32) was produced in pilot plant quantities from a strain of Bacillus stearothermophilus. The organism was cultured at 50°C in a soluble neutral medium containing water extract of soybean meal (3%) and 0.5% yeast extract. The enzyme biosynthesis was inducible and sensitive to catabolite repression. After autolysis of the cells, the α-galactosidase was selectively and quantitatively complexed from clarified beer directly onto DEAE Sephadex; and enzyme-rich fractions were batchwise eluted with an increasing gradient of NaCl solutions. The eluates were given two consecutive isopropyl alcohol precipitations, and the aqueous solutions of the second precipitate were dialyzed and lyophilized. Final product activity recovery was 72% based on the crude fermentation beer. Best specific activity was 5.2 u/mg protein. Further laboratory purification (DEAE Sephadex and Bio-Gel P200) yielded a product with 14.2 u/mg protein.     

黑曲霉木聚糖酶的底物特异性和低聚木糖生产

对黑曲霉（Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ）木聚糖酶进行了纯化研究,结果表明,经Ｓｅｐｈａｄｅｘ Ｇ－１００和ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－５０分离后,获得三个组分,称为Ｘ１、Ｘ２、Ｘ３。它们经ＰＡＧＥ电泳分析均为单一组分。对Ｘ１、Ｘ２、Ｘ３的相关性质,特别是底物特异性也作了研究,纯酶Ｘ３组分或部分纯化的酶可用于生产低聚木糖,产品得率为１０％。     

Identification of polysaccharides from pericarp tissues of litchi (Litchi chinensis Sonn.) fruit in relation to their antioxidant activities

A large number of polysaccharides are present in the pericarp tissues of harvested litchi fruits. A DEAE Sepharose fast-flow anion-exchange column and a Sephadex G-50 gel-permeation column were used to isolate and purify the major polysaccharides from litchi fruit pericarp tissues. Antioxidant activities of these major polysaccharide components were also evaluated. An aqueous extract of the polysaccharides from litchi fruit pericarp tissues was chromatographed on a DEAE anion-exchange column to yield two fractions. The largest amount of the polysaccharide fraction was subjected to further purification by gel filtration on Sephadex G-50. The purified product was a neutral polysaccharide, with a molecular weight of 14 kDa, comprised mainly of 65.6% mannose, 33.0% galactose and 1.4% arabinose. Analysis by Smith degradation indicated that there were 8.7% of (1-->2)-glycosidic linkages, 83.3% of (1-->3)-glycosidic linkages and 8.0% of (1-->6)-glycosidic linkages in the polysaccharide. Furthermore, different polysaccharide fractions extracted and purified from litchi fruit pericarp tissues exhibited strong antioxidant activities. Among these fractions, the purified polysaccharide had the highest antioxidant activity and should be explored as a novel potential antioxidant.     

蕲蛇蛇毒抗凝血因子的分离纯化与特性

目的 寻找蕲蛇蛇毒中的抗凝血因子。方法 利用硫酸铵沉降、阴离子交换层析、阳离子交换层析及高效液相色谱层析,从蕲蛇蛇毒中分离纯化到一个抗凝血因子。结果 纯化的这一组份在PAGE、SDS—PAGE上均呈单一区带,分子量约为25．4kD,由两条分子量分别为15．0kD和16．0kD的肽链通过二硫键连接在一起。这一组份在体外显著地延长血浆复钙时间和凝血酶原时间,但不延长牛凝血酶时间,也不具有磷脂酶A2活性、纤溶活性和出血活性。结论 蕲蛇蛇毒中舍右一种新的抗凝血因子。     

Purification and characterization of the larvicidal toxin of Bacillus sphaericus 1593M

We present here a procedure for purifying the larvicidal toxin from sporulating cells of Bacillus sphaericus 1593M and describe some of the biochemical and biophysical properties of this toxin. The procedure involves solubilization of the cell-wall/membrane bound toxin by sonication of cells followed by repeated rounds of freezing and thawing at 50 degrees C. Further purification involved Sephadex G-100 and DEAE Sephacel chromatography. We show by Sephadex G-100 chromatography that at pH 7.5 the smallest active form of the toxin has an Mr of 38,000 and that this toxin can reversibly aggregate to molecular forms of a size higher than 2 X 10(5) Mr. By shifting the pH from 7.5 to 8.5 only the aggregated forms can be observed.