Pyrolysis gas-liquid chromatography applied to a study of variation in Arthroderma tuberculatum

Replicates of whole colonies of four species of closely related dermatophytes were analyzed by pyrolysis gas-liquid chromatography (PGLC). The four species included fifteen strains of Arthroderma tuberculatum, and two strains each of A. benhamiae, Nannizzia gypsea and N. incurvata.Individual/peaks on different pyrograms were identified as homologous with the aid of internal markers by the superimposition of pyrograms. The peak height data extracted from the pyrograms of the fungal samples were analyzed to compute average similarities between pairs of pyrograms. The average was calculated with each peak weighted equally, and log weighted for its information content. The results of the cluster analyses of proximities were generally similar.Most, but not all, replicates of each strain were similar enough to be clustered together. Some strains belonging to the same species were also similar enough to be grouped in one cluster. Other strains of a single species varied sufficiently to be put in separate clusters. The nearest neighbour to each OTU (pyrogram) was always a replicate of the same strain.     

流感病毒的裂解气相色谱分析

裂解气相色谱法(Pyrolysis Gas Chromatography,PGC)在微生物学的应用中曾多侧重于细菌的鉴定,Mayer最早用PGC做植物病毒的快速鉴定,80年代国内开始开展了病毒的PGC研究。本文报道用PGC分析流感病毒和新城疫病毒的初步结果。     

Purification and biochemical characterization of a basic superantigen (SPEX/SMEZ3) from Streptococcus pyogenes

A potent basic superantigen (designated streptococcal pyrogenic exotoxin X, SPEX/SMEZ3) was purified to homogeneity from culture supernatants of a Streptococcus pyogenes scarlatina strain of type 12 (genotype speA(-), speC(-)) and characterized. Sequence alignments revealed SPEX to be an allele of the streptococcal mitogens type Z (SMEZ). The N-terminal amino acid sequence of SPEX was found with LEVDNNSLLR to be identical to the recently described acidic superantigen SMEZ. Although SPEX/SMEZ genes were present in all of the streptococcal strains tested, a toxin production could only be detected in a small number of strains. The produced toxin concentration in the culture supernatants of positive strains differed between 0 and 20 ng ml(-1). The purified SPEX stimulated human T-lymphocytes with Vbeta8 specificity at extremely low concentrations (lower than 100 pg ml(-1)).     

Virulence prediction of Yersinia enterocolitica by pyrolysis gas-liquid chromatography.

Pyrolysis gas-liquid chromatography (PGLC) was used to differentiate between HeLa cell-invasive and noninvasive strains of Yersinia enterocolitica and between Sereny-positive and -negative strains. A temperature-programmed gas-liquid chromatograph, equipped with a high-resolution Carbowax 20M coated capillary column, separated the volatiles from pyrolyzed whole cells preparations and cell wall fractions. The resulting pyrolysis elution patterns (pyrograms) were divided into 313 30-s time interval areas. The time interval areas were normalized in relation to the entire pyrogram area and were evaluated by stepwise linear discriminant analysis. The results of the PGLC-statistical analyses showed good correlation in prediction of the HeLa cell invasivity test. The technique of PGLC coupled with statistical analyses is objective, in contrast to traditional methods of determining pathogenicity of Y. enterocolitica.     

Transformation of type polysaccharide antigen synthesis and hemolysin synthesis in streptococci

Transformation of the ability to synthesize type polysaccharide antigen and beta-hemolysin has been obtained in group F streptococci. Colonies possessing cells transformed to antigen synthesis were detected on the agar surface with fluorescein-labeled anti-type serum. This selection method, in contrast to those with antibiotics, allowed both transformed and nontransformed cells to grow, resulting in sectored colonies. These colonies could be subcultured to further establish the synthesis of antigen. Group F, group A, and group-like z deoxyribonucleic acid (DNA) labeled with type II antigen and hemolysin, and streptomycin resistance transferred each marker to a group F strain lacking a type antigen. DNA from group F and z3 strains labeled with type III antigen, and streptomycin resistance transferred both markers to group F and z3 strains lacking type antigen. A second F strain without type antigen was not transformed with any of these markers. A group H strain was transformed to streptomycin resistance only by the same types of DNA. Transformation to type II antigen synthesis always resulted in the formation of beta-hemolysin. All strains isolated from natural sources contained both markers. A mutant, obtained by nitrosoguanidine treatment of an FII(sr) strain, did not synthesize either the hemolysin or the antigen. This mutant still possessed the group antigen and streptomycin resistance. A close linkage of type II antigen and beta-hemolysin is indicated. The fluorescent-antibody staining of cells containing both group and type antigens showed a more intense ultraviolet adsorption for type than group antigen. A surface location (microcapsular) for the type antigen appeared likely. These results are of interest for studies on antigen biosynthesis, genetics, and classification of the streptococci.     

Effect of different growth conditions on the discrimination of three bacteria by pyrolysis gas-liquid chromatography

High-resolution pyrolysis gas-liquid chromatography was applied to three bacteria (Escherichia coli NCTC 9001, Pseudomonas putida (NCIB 9494, and Staphylococcus aureus NCTC 8532) grown under a variety of conditions. Changing the culture medium drastically altered the quantitative aspects of the pyrograms of all three organisms, but the effects of culture time and incubation temperature were less severe. Mathematical analysis of the relative peak heights showed that four peaks could be used to discriminate the three bacteria however they were cultured.     

Effect of different growth conditions on the discrimination of three bacteria by pyrolysis gas-liquid chromatography.

High-resolution pyrolysis gas-liquid chromatography was applied to three bacteria (Escherichia coli NCTC 9001, Pseudomonas putida (NCIB 9494, and Staphylococcus aureus NCTC 8532) grown under a variety of conditions. Changing the culture medium drastically altered the quantitative aspects of the pyrograms of all three organisms, but the effects of culture time and incubation temperature were less severe. Mathematical analysis of the relative peak heights showed that four peaks could be used to discriminate the three bacteria however they were cultured.     

A Pyrolysis Gas-liquid Chromatography Study of Clostridium botulinum and Related Organisms

Low resolution pyrolysis gas-liquid chromatography could differentiate the following groups of Clostridium botulinum and related organisms: (1) Cl. botulinum type A. proteolytic types B and F and Cl. sporogenes ; (2) Cl. botulinum types C and D. and (3) Cl. botulinum type E and non-proteolytic types B and F. Toxin types A and B could be distinguished from type E and from type F.     

Purification and partial characterization of neuraminidase from type III group B streptococci

Extracellular neuraminidase from a type III fresh clinical isolate of a group B streptococcus was purified by a combination of salt fractionation, affinity chromatography of Affi-Gel blue, ion-exchange chromatography on diethylaminoethylcellulose, and gel filtration on Sephacryl S-200. These procedures yielded enzyme which was purified approximately 1,000-fold compared with the enzyme found in the original supernatant fluid. This type III streptococcal neuraminidase had a molecular weight of approximately 125,000 as estimated by filtration on Sephacryl S-200 and approximately 106,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In contrast to the majority of other bacterial neuraminidases, the type III group B streptococcal enzyme had no effect on colominic acid or N-acetylneuramin-lactose; however, it was quite active on bovine submaxillary mucin.     

Pyrolysis Gas Chromatography of Pseudomonas and Acinetobacter Species

Low resolution pyrolysis gas chromatography was performed on five species of Pseudomonas and two species of Acinetobacter. Conventional species groups could be differentiated by canonical variates analysis, and visual examination suggested that subspeciation would have been possible with more data.
Carbowax 20M has been used as the stationary phase for the majority of published work on pyrolysis gas chromatography of micro-organisms. However, with this material baseline resolution was poor, analysis times were long and column deterioration was relatively rapid. Peak area reproducibility on a single column averaged 10%, but it proved impossible to achieve quantitatively similar pyrograms on a new column. These serious drawbacks of Carbowax 20M limit the usefulness of this stationary phase for pyrolysis gas chromatography of micro-organisms.     

Studies on the ribosomal RNA operons of Listeria monocytogenes

Mannose-resistant hemagglutinating fimbrial antigen F165 is produced by Escherichia coli strains associated with septicemia in piglets and calves. A fimbrial component with an M(r) of 17,200 as determined by SDS-PAGE was purified to homogeneity from F165-positive E. coli strain 4787 of serogroup O115. This fimbrial component of F165 antigen was named F165(2). Separation procedures included fast protein liquid chromatography with a Superose 12 column followed by ultracentrifugation and 0.15 M ethanolamine buffer (pH 10.5) dissociation. Upon removal of ethanolamine, the fimbrial component reassociated into fimbriae. Amino acid composition analysis indicated that the fimbrial component molecule comprised 158 amino acid residues of which 37.3% were hydrophobic. The amino acid composition and the isoelectric point (9.5) were readily distinguishable from those of F1 fimbriae. The amino acid sequence was determined for approximately 40% of the molecule. For the first 33 residues, the F165(2) sequence was identical to that of F1B fimbriae and very similar to that of F1C. Fimbriae F165(2) could nevertheless be differentiated antigenically from F1C fimbriae as demonstrated by the immunodot technique using cross-absorbed antisera.     

A new type of mitogenic factor produced by Streptococcus pyogenes.

A new type of mitogenic factor (protein) was purified from the culture supernatant of a strain of Streptococcus pyogenes by SP-Sephadex C-25 column chromatography, preparative isoelectric focusing and reversed-phase high-performance liquid chromatography. The purified factor, showing marked mitogenic activity in rabbit peripheral blood lymphocytes, gave a single-band staining for protein on SDS-PAGE. The molecular weight of the purified mitogenic factor was determined to be 25,370, which was different from those calculated from reported amino acid sequences deduced from 4 different nucleotide sequences of 3 kinds of streptococcal pyrogenic exotoxins (two SPEAs, SPEB and SPEC). The amino acid sequence of the N-terminal region of the purified mitogenic factor was determined to be Gln-Thr-Gln-Val-Ser-Asn-Asp-Val-Val-Leu-Asn-Asp-Gly-Ala-Ser-Lys-Tyr-Leu- Asn-Glu - Ala-, which was also different from the reported N-terminal sequences deduced from the 4 different nucleotide sequences. These data indicate that this mitogenic factor is distinct from the already described streptococcal pyrogenic exotoxins.     

Structural investigation on the lipopolysaccharide of Escherichia coli rough mutant F653 representing the R3 core type.

The chemical structure of the core oligosaccharide of the lipopolysaccharide isolated from Escherichia coli rough mutant strain F653, representing the enterobacterial R3 core type, was investigated by quantitative and methylation analyses, nuclear magnetic resonance spectroscopy, gas-liquid chromatography/mass spectrometry, and determined as [formula: see text] All sugars are present as alpha-pyranosides but the anomeric configurations of the 3-deoxy-D-manno-octulopyranosonic acid (Kdo) residues could not be determined. The third Kdo and the heptose-linked GlcN residue are present in nonstoichiometric amounts; the GlcN residues may be, at least partially, N-acetylated.     

Aggregated Form of Dextransucrases from Leuconostoc mesenteroides NRRL B-512F and Its Constitutive Mutant

Purified dextransucrases [EC 2.4.1.5], DSW-D and DSW-G, from Leuconostoc mesenteroides B-512F were obtained from affinity chromatography with DEAE-Sephadex A-50 by elution with clinical dextran and guanidine-HCl, respectively, DSM-G was purified from the B-512F mutant strain SH 3002, which produces dextransucrase constitutively. Although the sugar contents of the purified enzymes were different, their molecular masses by SDS–PAGE were all 170kDa. DSW-D and DSW-G were highly aggregated and the all the activities were eluted at the void volume (V₀) on Sepharose 6B, while the DSM-G was eluted at 1.2 × V₀ volume. On rechromatography, DSM-G was separated into three peaks corresponding to the aggregated form, monomeric form, and partially digested form, respectively. The aggregation of Leuconostoc dextransucrase was looser than that of streptococcal glucosyltransferases, but the structures of these enzymes had high homology with each other.     

Glycolipids from the cell walls of streptococci

A streptococcus, serologically defined as a Z₃III strain was compared with a mutant strain Z₃ lacking the type III polysaccharide antigen. The loss of type antigen represents a decrease in the carbohydrate content of the cell wall of the mutant and is accompanied by long-chain formation, increased sensitivity to streptomycin and agglutination in saline. Cell-wall preparations can be freed of membrane contamination by treatment with hot sodium dodecylsulfate (SDS). This resulted in a doubling of the ratio of muramic acid to lysine and in the disappearance of phospholipids. It could be shown that the membrane-free cell walls of these strains still contained appreciable amounts of glycolipids which could be identified as monoglucosyl glyceride and diglucosyl-diglyceride.     

Basic streptococcal superantigens (SPEX/SMEZ or SPEC) are responsible for the mitogenic activity of the so-called mitogenic factor (MF)

The mitogenic factor (MF) of group A streptococci has been reported to be a superantigen stimulating human T cells carrying Vbeta2, 4 and 8 and has been designated streptococcal pyrogenic exotoxin F (SPEF). MF was also shown to possess DNase activity. Here we have purified MF from culture supernatants of different Streptococcus pyogenes strains. Surprisingly, the MF preparations from different strains showed different Vbeta specificities depending on the expression of SPEC or SMEZ3 by the producing strain. Their mitogenic activity decreased upon further purification. In addition, the mitogenic activity could be only neutralized by antibodies against the basic streptococcal superantigens SPEC or SPEX (SMEZ3) but not by antibodies against MF itself although the latter were able to neutralize completely the DNase activity of MF. We found that streptodornase type B (SDB) was expressed in two molecular forms (SDBI and SDBII), differing only by one additional N-terminal arginine at SDBI. MF was found identical to the enzyme SDBII but is devoid of superantigenic properties and should no longer be called a superantigen or a pyrogenic exotoxin.     

重组人组织型纤溶酶原激活剂(rht-PA)及其突变体的纯化

稳定高效表达重组人组织型纤溶酶原激活剂 (rht PA)的CHO细胞株和表达组合突变体的细胞株进行了 3L转瓶培养 .将培养上清分别进行了Lys Sepharose 4B亲和层析和Zn2 + Sepharose 4B层析两步纯化 ,rht PA纯度提高了 5 34倍 ,比活达 2 5× 10 5IU mg ,产率为 73% ;突变体纯度提高了1119倍 ,比活达 5 9× 10 5IU mg ,产率为 6 9% .纯化产物SDS PAGE分析显示 ,rht PA和突变体基本都呈单一条带 ,扫描分析均达到 98%以上纯度 .rht PA和突变体在纯化系统中的行为作对照分析发现 ,突变体的构建思想在Lys Sepharose 4B亲和层析过程中有充分体现 .这两步层析组合是很好的纯化t PA及其突变体的方法 ,尤其是Lys Sepharose 4B纯化突变体效果更好     

Characterization of antigen purified from type 3 strains of Pasteurella multocida and its use for an enzyme-linked immunosorbent assay

Surface antigens were purified from a type 3, 4 rabbit isolate of Pasteurella multocida designated as R11146. Two protein peaks were obtained by gel filtration with Sephadex G-200 from crude saline extract. Major antigenic activity was detected in the first peak. The first peak was absorbed onto DEAE-cellulose and eluted by a linear gradient of NaCl. Four fractions eluted from the column contained a single antigen which was identical to an antigen purified from another type 3 strain, P-1059. Also, they uniformly contained two protein species of molecular weights of 44,000 and 25,500. Six Pasteurella-free rabbits were infected intranasally with R11146 isolate and antibody response was determined by an enzyme-linked immunosorbent assay (ELISA) with the use of an antigen purified from P-1059 strain. Serum samples from the infected rabbits showed ELISA titers at the plateau stage by 21 or 28 days post-inoculation. Highest titers ranged from 1:15,000 to 1:16,000, while all the preinoculation sera had titers lower than 1:10. The high titers generally persisted for longer than 98 days after the infection. These results indicate that ELISA using a purified type 3 antigen is useful to detect P. multocida infection in rabbits by a type 3-related strain.     

Metalloproteinase from Bacillus subtilis: - "intracellular" and extracellular enzymes

"Intracellular" metalloproteinase was purified to homogeneity from Bacillus subtilis 103 crude cell extract, using affinity chromatography on bacitracin-Sepharose 4B. The degree of purification and the yield of the enzyme were about 260-fold and 3%, respectively. In its physico-chemical properties and the amino acid composition the enzyme is very similar, if not identical, to the extracellular metalloproteinase isolated from the culture filtrate of the same strain. Extracellular metalloproteinase-deficient mutant strain Bacillus subtilis SMY-512 does not produce the "intracellular" enzyme either. THe activity of "intracellular" metalloproteinase in the periplasmic space of the cells is about 70% of that in the cytoplasm, thus being indicative of a rather regular distribution of the enzyme throughout the cell compartment.