Antigenic structure of saprophytic leptospirae and their classification]

The authors present the results of study of the serological properties of 46 strains of saprophytic leptospirae of different origin. On the basis of the affinity of the antigenic structure detected in the cross microagglutination reaction (MAR) 38 strains under study were united into 8 serological groups; the rest 8 strains were serologically independent in this reaction. The fact that 9 strains of water leptospirae isolated in the Armenian SSR belonged to one serological group was proved in the cross MAR and the test of aglutinin adsorption. This serological group was new and was named L. armenica. Five individual serological types of saprophytic leptospirae were differentiated in its composition. Comparative study of the serological interrelations between the group of strains isolated in Armenia and the strains of some serological groups and serological types the closest serological connections were noted in the K-1030 (serological group Armenica) and Bovedo (serological group Andamana) strains. It is believed that the existing division of the saprophytic leptospirae into two serological groups (Semaranga and Andamana) required widening and supplement by new serological groups and serological types.     

汞与硒在不同汞暴露水平地区猪肝脏和肾脏蛋白质中的分布

通过对贵州万山汞污染地区及北京地区猪肝脏和肾脏组织上清液进行凝胶过滤色谱分离(SephadexG 10 0 ) ,随后用原子荧光法测定它们蛋白质组分中汞和硒的含量 ,研究在汞暴露水平不同状态下微量元素汞和硒在动物体蛋白质分子水平上的分布 .发现这两个地区猪肝脏和肾脏组织上清液蛋白质组分中汞和硒的分布模式有明显差异 .贵州万山汞污染地区猪肝脏上清液中汞浓度比北京地区高 ,硒浓度也相应高 ,且前者与高分子量和低分子量蛋白结合的硒均明显高于后者 ;而北京地区猪肝脏上清液中的硒主要以与高分子量蛋白结合的形式存在 .贵州汞污染地区猪肝脏上清液中汞主要与高分子量蛋白结合 ,而北京地区猪肝脏上清液中汞则分布较为均匀 .贵州万山地区猪肾脏上清液中 ,含硒峰在高分子量蛋白区和低分子量区都有分布 ;而北京地区猪肾脏上清液中 ,硒则主要集中分布于高分子量蛋白范围 .这两个地区猪肾脏上清液中都有分子量约为 11kD的金属硫蛋白 (MT)存在 ,北京地区猪肾脏上清液中汞主要以与金属硫蛋白结合的形式出现 ,而贵州万山地区猪肾脏上清液中的汞除与金属硫蛋白结合外 ,尚有相当大部分是以与高分子量蛋白结合的形式存在 .研究结果表明 ,由于这两个地区汞暴露水平的差异 ,不仅使这两地区猪肝、肾上清液中的汞与硒含量     

Purification and characterisation of alpha-L-fucosidase from human placenta. pH-dependent changes in molecular size.

alpha-L-Fucosidase has been purified 12 000 fold from human placenta. The enzyme is a glycoprotein containing, by weight: 0.9% galactose; 1.9% mannose, 1.9% N-acetylglucosamine and 1.9% N-acetylneuraminic acid. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate separated proteins with molecular weights ot 55 000, 51 400 and 25 000. Resolution of the two larger protein bands varied with the gel system and these proteins may differ only in carbohydrate content. Gel filtration of te purified enzyme failed to separate the three proteins. Treatments with the cross-linking reagent dimethyl suberimidate prior to electrophoresis, resulted in a diminution of the original protein bands and the formation of oligomers with molecular weights of 80 000, 100 000, 130 000, and 144 000. These results suggest that the heavy (55 000 and 51 400) and light (25 000) proteins are structurally associated. The molecular weight of the native enzyme, measured by gel filtration, was dependent on the pH of the eluting buffer. At pH 5.0 or 6.0 a catalytically active peak was observed, with a molecular weight of 305 000. At pH 7.5 this peak was completely absent and the enzyme eluted as an asymmetrical peak with an apparent molecular weight of about 60 000. The reduction in apparent molecular weight at pH 7.5 was reversible by dialysis of isolated fractions at pH 6.0. In agreement with these findings the sedimentation coefficient was 8.5 S at pH 5.0 but only 3.6 S at pH 7.5. The results can be accounted for by the existence of a pH-dependent equilibrium between aggregated and dissociated forms of the enzyme or by pH-depedent conformational changes.     

蝶蛹金小蜂毒液蛋白不同提取分离方法的比较

为了建立蝶蛹金小蜂Pteromalus puparum毒液抑制寄主血细胞免疫活性组分合适的分离纯化方法,就等电点沉淀法、乙醇沉淀法、75%硫酸铵沉淀法、75%硫酸铵沉淀法+40℃加热处理法,以及75%硫酸铵沉淀法分别与3种不同滤膜的分子大小截留法的组合等7种方法对毒液蛋白分离效果及活性的影响进行了比较。结果表明：等电点沉淀法获得的组分抑制寄主菜粉蝶Pieris rapae离体血细胞延展和包囊的活性最强,乙醇沉淀法次之,75%硫酸铵沉淀法最弱。从蛋白组分的SDS-PAGE图谱来看,等电点沉淀法获得毒液组分相对最纯,仅有3条主要谱带,分子量大小在45～116.2 kDa范围内;乙醇沉淀法次之,有5条主要谱带,分子量大小在24～116.2 kDa范围内;硫酸铵沉淀法的谱带组成与毒液蛋白粗提液相似。3种分子大小截留法获得的毒液组分的活性分析表明,强活性组分分子量大小可能都大于100 kDa。综合认为,7种方法中以等电点沉淀法提取分离蝶蛹金小蜂毒液蛋白相对为最适。     

IMMUNOCHEMICAL PROPERTIES OF S-100 PROTEINS AND THEIR SUBUNITS

Abstract— The immunological activities of two populations of bovine S-100 proteins with anti-S-100 serum were studied by complement fixation and rocket immunoelectrophoresis. The reactivities of subunits of these two populations were studied by crossed immunoelectrophoresis and rocket immunoelectrophoresis. Although the two populations conformed in all respects to the properties of S-100 protein, the immunological reactivity of one, III-IVa-1, was significantly lower than that of the other, III-IVb-1. The difference was much larger when the S-100 protein fractions were isolated in the absence of aids (mercaptoethanol, EDTA, EGTA, protease inhibitors). With bovine S-100 fractions, the three subunits separated by differences in charge as well as the four subunits separated by differences in molecular weight all reacted with the same antibody molecules in the antiserum. The reactivities of the subunits showed large quantitative differences.
Two populations of S-100 proteins from rat brain also showed differences in reactivity with anti-S-100 serum. The two subunits in each of these fractions reacted with anti-S-100 serum but with quantitative differences, the larger having almost double the activity of the smaller. These results provide firm evidence for the heterogeneity of S-100 proteins based on immunological activity of their subunit components. Different molecular species of S-100 proteins probably differ considerably in their reactivity with antibodies to S-100 protein. Some of the more reactive molecular species also appear to be much more labile, since the reactivity of some S-100 protein fractions was considerably reduced when they were isolated in the absence of aids.     

Genetic polymorphism and close linkage of two alpha 1-protease inhibitors in horse serum.

Two-dimensional electrophoretic analysis of horse serum proteins was done by a first-dimension separation in agarose gel (pH 5.4) followed by a second-dimension separation in horizontal polyacrylamide gel (pH 9.0). This method resulted in improved and reproducible separation of many alpha-globulins. Two groups of alpha 1-globulins, designated Pi1 and Pi2, were found to be protease inhibitors. Preliminary studies indicated that Pi1 and Pi2 proteins differed from each other in molecular weight and in protease inhibiting spectra. Extensive polymorphism was observed for both these proteins. Family data supported the hypothesis that Pi1 and Pi2 types were controlled by autosomal codominant alleles. For both Pi1 and Pi2 systems, most of the homozygous types showed two fractions each while the heterozygous types had 4 fractions. Six Pi1 and five Pi2 alleles were observed in two breeds of Swedish horses. Complete genetic linkage was observed for Pi1 and Pi2 loci as no recombinant type was observed in 40 informative matings studied.     

Studies on Two Colonial Types of Leptospira icterohaemorrhagiae with Special Reference to the Bottle Culture Method

A bottle culture method is described which favours the colony formation of Leptospira icterohaemorrhagiae. The Kitakata strain formed two morphological types of colonies when grown in rubber-stoppered 200 ml-square bottle cultures. One was compact and small (C type), while the other diffuse and large (D type). The C and D type sub-strains showed the following differences: The C type leptospirae were longer in body length, but less virulent for suckling mice, young hamsters, guinea pigs, or young rabbits. The C type leptospirae lost 90% of the colony forming units (CFU) after being heated at 45 C for 20 minutes. On the other hand, the D type leptospirae were shorter in body length, virulent in those animals examined and resistant to heating. No serological differences were observed between these two types of sub-strains by agglutination test and in agglutinin absorption test.     

Genetic polymorphism and close linkage of two α1-protease inhibitors in horse serum

Two-dimensional electrophoretic analysis of horse serum proteins was done by a first-dimension separation in agarose gel (pH 5.4) followed by a second-dimension separation in horizontal polyacrylamide gel (pH 9.0). This method resulted in improved and reproducible separation of many α-globulins. Two groups of aj-globulins, designated Pi1 and Pi2, were found to be protease inhibitors. Preliminary studies indicated that Pi1 and Pi2 proteins differed from each other in molecular weight and in protease inhibiting spectra. Extensive polymorphism was observed for both these proteins. Family data supported the hypothesis that Pi1 and Pi2 types were controlled by autosomal codominant alleles. For both Pi1 and Pi2 systems, most of the homozygous types showed two fractions each while the heterozygous types had 4 fractions. Six Pi1 and five Pi2 alleles were observed in two breeds of Swedish horses. Complete genetic linkage was observed for Pi1 and Pi2 loci as no recombinant type was observed in 40 informative matings studied.     

Bulk Preparation of CNS Cytoskeleton and the Separation of Individual Neurofilament Proteins by Gel Filtration: Dye-Binding Characteristics and Amino Acid Compositions

Abstract: The three major proteins of mammalian neurofilaments, of molecular weight 70,000, 160,000, and 210,000, have been resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel eJectrophoresis, and more recently, by ion-exchange chromatography in urea solution. We describe here a method to separate the neurofilament proteins by gel filtration without the use of SDS. A bulk preparation of cytoskeleton from rat spinal cord was first characterized. This preparation was then solubilized in a buffer containing 8 M urea and subjected to gel filtration. Individual neurofilament proteins, in milligram quantities, were harvested following the pooling of appropriate fractions. Gel electrophoresis showed a high degree of homogeneity in each of the three pooled fractions. Dye binding studies demonstrated that the protein of molecular weight 210,000 was relatively underrepresented when stained with Coomassie Blue, while all three neurofilament proteins showed similar dye binding properties with Fast Green. Amino acid analysis indicated that (1) all three neurofilament proteins contained a high content of acidic residues; (2) the molecular weight 210.000 protein contained >8 mol% proline; and (3) no simple oligomeric relationship existed among the neurofilament triplets.     

Partial purification of a boar sperm membrane protein inducing sperm agglutinating antibody

For the development of immunological contraception, attention is being concentrated on the possibility of using a sperm membrane antigen. Boar sperm membrane was extracted with triton-X 100 and fractionated by Sephadex G-150 column chromatography. The glycosylated and nonglycosylated portions of protein peaks from the gel filtration were obtained by fractionating on concanavalin A-Sepharose and eluting the bound protein with 0.3 M methyl mannoside. A glycosylated fraction was found to induce sperm agglutinating antibodies in rabbit. The partially purified protein has a molecular weight of 30 kilodaltons, as determined by sodium dodecyl polyaccyrlamide gel electrophoresis. Further work is planned on the histochemical determination of the origin of this protein and species cross-activity of the antibody.     

Biochemical and molecular characterization of cowpea landraces using seed storage proteins and SRAP marker patterns

Seven landraces of cowpea [Vigna unguiculata (L.) Walp.] were assessed for genetic variability in total proteins, protein fractions viz. albumins, globulins, prolamins, and glutelins by SDS-polyacrylamide gel electrophoresis and DNA polymorphism using sequence-related amplified polymorphisms (SRAP) markers. The solubility-based protein fractionation data indicated that the salt soluble fraction (globulin) and water-soluble fraction (albumin) proteins were the predominant fractions in cowpea seeds comprising 45–50.3% and 31.2–35.5% of total soluble proteins, respectively. The electrophoretic pattern revealed the molecular heterogeneity among total proteins as well as different protein fractions. The molecular weights of protein bands obtained by SDS-PAGE varied between 10 to 250, 15 to 110, 15 to 150, and 15 to 130?kDa for total proteins, albumins, globulins, and glutelins, respectively. A large number of bands were found common to the various landraces, indicative of their close relationship with one another. However, a few bands distinctive to some specific landraces were also detected, indicating varietal differences. A 34 SRAP primer pair combination generated a total of 1003 amplicons (loci) showed 100% polymorphism with an average of 0.93 polymorphism information content (PIC) value. Landraces displayed an average 0.50 similarity coefficient which clustered the landraces corresponding to their growth habit in main clusters and to their geographical origin in subcultures. Molecular and biochemical analysis were correlated with a medium level (Mantel test, r?=?0.56, P?<?0.02). These findings revealed that seed proteins and DNA polymorphism provide valuable information regarding the variability among landraces and this information could be utilized for breeding purposes in the enhancement of protein quality and quantity in grain legumes.     

NEUROSECRETORY CELL PROTEIN METABOLISM IN THE LAND SNAIL,OTALA LACTEA

—Protein synthesis in an identified molluscan neurosecretory cell of the land snail, Otala lactea was examined using three different types of polyacrylamide gel electrophoresis. Cells taken from active snails synthesized specific low molecular weight proteins while those from aestivated snails did not. Most of the newly synthesized low molecular weight proteins in the active snails were lost from the cell body when the preparations was chased for 19 h in label-free enriched medium in the presence of anisomycin, an inhibitor of protein synthesis. If colchicine, a blocker of axonal transport, was included in the chase medium, the proteins present following a pulse were largely replaced by smaller molecular weight species. The results suggest that specific low molecular weight proteins are converted to smaller species and then transported from the cell body.     

Fractionation of Soluble Copper- and Zinc-Binding Proteins From Cattle Liver

The distribution of copper and zinc among soluble proteins in liver from normal slaughter cattle was examined after gel filtration of the proteins. Gopper- and zinc-binding proteins were mainly separated into three fractions. Varying amounts of zinc were eluted in a fourth fraction of molecular weight less than 2,000. A clear relationship was noted between the amount of copper bound to the low molecular weight fraction (m.w. ~ 10,000) and the total liver zinc concentration. The high molecular weight protein fraction (m.w. > 65,000) dominated in liver with zinc concentrations below 40 µg/g wet weight and total copper concentrations from 16 to 240 µg/g, while in liver with zinc concentrations above 40 µg/g and copper concentrations ranging from 20 to 107 µg/g, the low molecular weight metallothionein-like fraction dominated.     

Different induction of 70,000-Da heat shock protein and metallothionein in HeLa cells by copper.

To clarify the physiological roles of heat shock proteins induced by copper, we studied the synthesis of these proteins and metallothionein, as well as the level and nature of copper incorporated into HeLa cells. Incubation in medium containing 200 microM cupric sulfate and above induced the synthesis of 70,000-Da heat shock protein (hsp70) in these cells. However, the synthesis of hsp70 did not increase in the presence of less than 200 microM cupric sulfate. On the other hand, the synthesis of metallothionein increased due to 100 microM cupric sulfate. The uptake of copper into the cells depended on the cupric sulfate concentration in the medium. To analyze the nature of the intracellular copper, cell extracts were separated by gel filtration chromatography into three fractions: the high molecular weight, metallothionein, and low molecular weight fractions. No copper was found in the low molecular weight fraction of control cells, but appeared distinctly at 200 microM cupric sulfate and above. Copper in the high molecular weight fraction also began to increase at 200 microM cupric sulfate and above, whereas in the metallothionein fraction it began to increase even at 50 to 100 microM cupric sulfate. Furthermore, inhibition of cell growth was also observed at 200 microM cupric sulfate and above but not at 100 microM and below.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of lectins for detection of electrophoretically separated glycoproteins transferred onto nitrocellulose sheets

A method of rapidly identifying lectin-binding glycoproteins separated by polyacrylamide gel electrophoresis is described. The method is particularly useful for comparing the glycoprotein content of different cell types and fractions. Normal rat liver, Novikoff hepatoma, and rat mammary tumor cell line 13762 MAT-B were fractionated to give purified nuclei and other fractions defined by their sedimentation properties in low ionic strength buffer. The subcellular fractions were separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, transferred to nitrocellulose sheets, and localized by an immunochemical method to identify lectin-binding activities. The localization pattern of concanavalin A and wheat germ agglutinin-binding activities in the fractions from the three cell types showed the greatest similarities between the glycoprotein contents of normal liver and Novikoff hepatoma fractions. On a per-cell basis the purified nuclei from each of the cell types contained less activity overall than did other particulate cell fractions. Washing the nuclei from normal liver and Novikoff hepatoma, but not MAT-B cells, in nonionic detergent removed or depressed most of the lectin-binding activities. However, two major bands were unaffected by the detergent. One of these localized with wheat germ agglutinin at an apparent molecular weight of 62,000 in the nuclei of all three cell types. The other localized with concanavalin A at an apparent molecular weight of 200,000 in normal liver and Novikoff hepatoma nuclei.     

New data on the molecular heterogeneity of brain-specific protein S100

A comparative study of distribution of labeled products of endogenous limited proteolysis of the "major" (MF) and "minor" (S100-26) fractions of proteins S100 from rat brain by ion-exchange chromatography was carried out with a view of testing the hypothesis on the formation of proteins S100 molecular associates as a possible cause of molecular weight heterogeneity of proteins S100. There is evidence that proteins S100-MF and S100-26 are different species of brain-specific S100 proteins. The experimental results also suggest that the brain-specific proteins S100-MF and S100-26 are adsorbed both by glial cells and by neurons. Some physico-chemical properties of peptide proteolytic products of various species of proteins S100 were investigated.     

A novel 115-kD peripheral membrane protein is required for intercisternal transport in the Golgi stack

We have used an in vitro Golgi protein transport assay dependent on high molecular weight (greater than 100 kD) cytosolic and/or peripheral membrane proteins to study the requirements for transport from the cis- to the medial-compartment. Fractionation of this system indicates that, besides the NEM-sensitive fusion protein (NSF) and the soluble NSF attachment protein (SNAP), at least three high molecular weight protein fractions from bovine liver cytosol are required. The activity from one of these fractions was purified using an assay that included the second and third fractions in a crude state. The result is a protein of 115-kD subunit molecular mass, which we term p115. Immunodepletion of the 115-kD protein from a purified preparation with mAbs removes activity. Peptide sequence analysis of tryptic peptides indicates that p115 is a "novel" protein that has not been described previously. Gel filtration and sedimentation analysis indicate that, in its native state, p115 is a nonglobular homo-oligomer. p115 is present on purified Golgi membranes and can be extracted with high salt concentration or alkaline pH, indicating that it is peripherally associated with the membrane. Indirect immunofluorescence indicates that p115 is associated with the Golgi apparatus in situ.     

The metabolism of metals in rat placenta

The status and transfer of metals across the rat placenta were studied by subcellular and molecular fractionations of this organ at 2 and 24 h after iv injection of radiolabeled metals. The soluble and nuclear fractions showed higher contents of copper and zinc, whereas most of the nickel was associated with the soluble fraction. Cadmium was almost evenly distributed between the microsomal and nuclear fractions. Gel filtration of the soluble fractions showed nickel associated with an unknown low molecular weight form; zinc with high molecular weight proteins; copper with metallothionein, ceruloplasmin, and high molecular weight proteins; and cadmium with high molecular weight proteins and metallothionein.     

Effects of cold, starvation and immobilization on the composition of acid-soluble nuclear proteins in chicken liver

Nuclear proteins soluble in 0.2 M sulphuric acid were isolated from the liver of three groups of hens subjected for 60 hours to starvation, immobilization or cold exposure. The obtained proteins were separated by means of one-dimensional and two-dimensional electrophoresis on polyacrylamide gel. It was observed that this exposure of the birds to stress caused no qualitative changes in liver nuclear proteins. Histones, histone-like proteins - M1, M2, M3, uM1, HMG 1 and 2 proteins, and a large group of non-histone protein fractions gave nearly identical patterns. However, several components of nuclear proteins were found whose quantity changed in the liver of the birds subjected to stress. These changes were observed in a protein with molecular weight about 27 000 daltons and two proteins weighing over 100 000 daltons.     

Characterization of polyagglutinating and surface antigens in Pseudomonas aeruginosa

Mutants of Pseudomonas aeruginosa PAC1R (serotype O:3) which were resistant to bacteriophage D were isolated and shown to react with O:5d, O:9 and O:13 antisera as well as O:3. Antisera to the parent strain and to the three polyagglutinating (PA) mutants also showed cross-reactions. The mutants differed from the parent strain in their lipopolysaccharide (LPS) composition. The LPS from two of the three mutants yielded high molecular weight polysaccharide fractions. Although the high molecular weight fraction from one of the mutants contained the amino sugars and other components characteristic of the O:3 serotype strains, its mobility on Sephadex G75 was different from that of the parent strain. The high molecular weight material from the second mutant lacked the O-antigenic determinants but these were present in a semi-rough LPS fraction. The third mutant appeared rough and completely lacked the O-antigenic components. These three mutants were compared with the parent strain and with a non-agglutinating LPS-defective mutant which lacked both O-antigenic side chains and all neutral sugars in the outer core. Agglutination with absorbed sera and haemagglutination using purified LPS and ELISA detection suggested that wall components other than LPS were responsible for some of the cross-reactions observed. The components responsible were detected after SDS-PAGE of crude outer membrane fractions by a combination of Coomassie blue and silver-staining and antigenic components were detected by immunoelectrophoresis and ELISA-linked immunoblotting of the gels. The main antigenic determinants detected by antiserum to the parent strain were in the high molecular weight O-polysaccharide fractions and in the semirough fractions of the LPS, with some activity due to the H protein of the outer membrane. O:5d antisera reacted with unidentified high molecular weight polysaccharide fractions. Cross-reactions with the O:9 antiserum appeared to be due mainly to the F porin and, to a lesser extent, to the G and E proteins of the outer membrane. O:13 antiserum reacted with high molecular weight polysaccharide fractions but also with the rough core and F and H protein. Cross-reactivity of the other three mutant antisera could largely be interpreted in terms of the outer membrane components exposed in each strain. One reacted strongly with the F porin and the rough core, while the others reacted with a number of protein and LPS-derived fractions.(ABSTRACT TRUNCATED AT 400 WORDS)