Investigation of the antigenic composition of the cells and the exotoxin of Sh. dysenteriae (Shiga) by agar immunoelectrophoresis and polyacrylamide gel disc electrophoresis.

Using the method of immunoelectrophoresis in agar, we established the presence of 12 soluble antigens in Sh. dysenteriae (Shiga). We differentiated among them 3 specific antigens situated in the cathodic region. One of them is the somatic O antigen while the other two are thermolabile surface K antigens of these bacteria. K antigens of Grigoryev-Shiga bacteria differ from the earlier described K antigens of the Newcastle and Boyd Shigellae in their positive electric charge, in greater lability in relation to temperature and chemical effects and evidently also in their chemical character. The remaining protein antigens are common for either the genus of Shigella or for the family of enterobacteria. The exotoxin preparation isolated from the R strain of Sh. dysenteriae (Voile 30) by precipitation with trichloroacetic acid is represented in the immunophoreogram by five precipitation lines. Disc electrophoresis in polyacrylamide gel made possible the investigation of the composition of the exotoxin and isolation of the two fractions responsible for its toxic effect. The method of preparative electrophoresis in polyacrylamide gel can evidently be used for obtaining purified Sh. dysenteriae exotoxin preparations.     

Pulsed-field gel electrophoresis analysis of Bordetella pertussis populations in various European countries with different vaccine policies

The increasing incidence of pertussis in a number of countries, despite good vaccination coverage, is a cause for concern. We used pulsed-field gel electrophoresis (PFGE) typing to examine the genetic diversity of 101 clinical isolates of Bordetella pertussis, recovered during 1999-2001, and circulating in five different European countries to evaluate temporal and geographical distribution. This DNA fingerprinting approach seems to be a more discriminative epidemiological tool than sequencing of individual genes. Despite differences in vaccination policies in the five countries, these European isolates were found to be very similar and fell into the same major PFGE profile groups, with a predominance of one profile group. There was no evidence of geographic clustering, except that one new profile subgroup was predominantly found in one country. This study provides a baseline for continued surveillance of the B. pertussis population in Europe.     

Characterization of the filamentous hemagglutin from Bordetella pertussis by gel electrophoresis

Summary A highly purified preparation of filamentous hemagglutinin (FHA) from Bordetella pertussis was analyzed for its protein composition by gel electrophoretic methods. In this preparation of FHA the following native species could be detected by polyacrylamide gel electrophoresis (PAGE) at pH 3.2: S, and S₂ (inactive subunits or fragments); two monomers, a major form designated Ia (144K), and a minor form lb, differing only in net charge; and three oligomeric forms, designated II (213K), III (595K) and IV (1064K). Hemagglutinating activity was associated predominantly with component Ia. PAGE of FHA after derivatization with sodium dodecyl sulfate (SDS) showed there to be three major species, designated A, C and D. According to estimated molecular weight values, A, C and D are likely to correspond to S₂, Ia and II respectively. Isolated components II, III and IV yield all three SDS-species upon derivatization with SDS. Both moving boundary electrophoresis and gel electrofocusing showed hemagglutinating FHA to be a basic protein. Its apparent pI is 8.1.     

盘状聚丙烯酰胺凝胶电泳法分离血清蛋白实验的几点改进

对盘状聚丙烯酰胺凝胶电泳法分离血清蛋白实验提出了几点改进,以满足本科生实验的要求。实验主要比较和分析了两种封胶方法（原胶布封胶与改进的琼脂糖封胶）和两种染色方法（原考马斯亮蓝染色法与改进的考马斯亮蓝染色法）对凝胶分离血清蛋白实验的影响。结果显示,改进的盘状聚丙烯酰胺凝胶电泳法是一种灵敏、快速、简便、安全、分辨率高的实验方法。结论：改进的盘状聚丙烯酰胺凝胶电泳法分离血清蛋白实验非常适合本科生实验。     

Glutamine synthetase content and immunochemical analysis of different strains of Bordetella pertussis

In this investigation 3 groups of strains isolated from pertussis patients have been studied: typical (group 1), atypical in their cultural properties (group 2), unidentified Gram-negative bacilli agglutinated by pertussis and parapertussis antitoxins (group 3). Besides, B. pertussis cultures, obtained by subculturing 2 museum strains and 2 newly isolated strains on synthetic casein-charcoal agar with subinhibiting doses of antibiotics or specific immune sera added, have been studied. As indicated by the results of this study, strains belonging to groups 1 and 2 contain glutamine synthetase, while in strains of group 3 this enzyme is absent. In immunoelectrophoresis strains of group 3 have been found to contain not a single antigen similar to the antigens of strains belonging to groups 1 and 2. Electrophoresis in polyacrylamide gel has revealed to differences in the protein spectrum of the strains of these 3 groups. The investigation has shown that the determination of glutamine synthetase and immunoelectrophoresis can be used for the differentiation of B. pertussis from similar Gram-negative bacilli. B. pertussis strains, changed as the result of experiments with antibiotics and specific immune sera, have also been shown to retain their antigenic composition and protein spectrum and to have no essential difference in the content of glutamine synthetase.     

Modification of commerical and custom-built slab gel electrophoresis units for two-dimensional polyacrylamide gel electrophoresis

Many commercial and custom-built slab gel electrophoresis units can be modified to function as two-dimensional polyacrylamide gel electrophoresis units with the insertion of Plexiglas adapters. These adapters can be made for about $50 a pair and can be used for either temporary or permanent modification of the slab gel units. The physical dimensions of the adapters can be varied to permit great flexibility in the diameter of cylinder gels and the thickness of slab gels that can be run together. For example, proteins from 6-mm cylinder gels can be easily separated on 1-mm slab gels, which can then be dried for autoradiography.     

Apparatus for discontinuous electrophoresis in polyacrylamide gel slabs

Construction of an inexpensive slab discontinuous electrophoresis apparatus is described. Using this apparatus 23 human serum proteins were resolved and the gel could be scanned with a standard densitometer to yield a trace with discrete peaks or pronounced shoulders for each protein band. The advantages of a single homogeneous slab for comparative studies are indicated.     

Use of disc electrophoresis in polyacrylamide gel method for the separation of soluble proteins of vibrios]

The authors present the results of a comparative study of the protein spectra of 80 strains of various vibrios. Proteinograms were elaborated by the method of disc-electrophoresis in polyacrylamide gel for each strain and species. There were revealed 25 protein peaks with definite mobility coefficients. The number of peaks and their areas varied in different species. The qualitative and the quantitative differences between the vibrio species were established. Results of investigations demonstrated a possibliity of using the protein spectra for the differentiation of microorganisms.     

Dissociation constants of glucan phosphorylases of rabbit tissues studied by polyacrylamide gel disc electrophoresis

Using polyacrylamide gel disc electrophoresis, a simple and sensitive stain method for glucan phosphorylase (EC 2.4.1.1) was developed. With this method 0.3–1.5 μg or 1–5 units of phosphorylase could be demonstrated as a sharp band within a few hours. Mobility of phosphorylase fraction was retarded in gels containing glycogen. From the change of mobility as a function of glycogen concentrations, the dissociation constants of phosphorylases of rabbit skeletal muscle, liver, and brain with rabbit liver glycogen was calculated. They were 6.1 × 10⁻⁴, 22 × 10⁻⁴, and 13 × 10⁻⁴m, respectively. From the electrophoretic mobility, rabbit tissue phosphorylases could be classified into two: those of brain and kidney, and those of skeletal muscle and liver. When the electrophoresis gel, however, contained glycogen in a considerable concentration, their mobilities were retarded, and the retardation was more marked with those of skeletal muscle and brain than with those of liver and kidney. Hence, all four tissue phosphorylases could be distinguished only by the disc gel containing glycogen.     

A method for horizontal polyacrylamide slab gel electrophoresis

We present a simplified method of preparation of polyacrylamide gels which is totally analogous to the procedure now widely used to pour and run horizontal agarose gels. The acrylamide is poured into an open air gel mold consisting of a glass plate with a masking tape border and a comb. It is subsequently run in a submarine horizontal electrophoresis apparatus. The electrophoretic mobility and resolution of DNA fragments obtained in such gels are identical to results obtained with gels poured and run in the vertical configuration. Numerous advantages of horizontal polyacrylamide gel electrophoresis are discussed.     

Ultrastructure of freshly isolated strains of Bordetella pertussis

The study of the electronograms of B. pertussis strains isolated in the foci of pertussis revealed the existence of the morphological variants of these cells, differing in the character of the cell wall, the state of the cytoplasm, the presence of amorphous inclusions of medium electron-optical density. The morphological variants did not correlate with the character of B. pertussis colonies isolated from blood-charcoal agar. The ultrastructure of the cells belonging to the second morphological variant was similar to that of the cells from the museum strain, altered by tetracycline treatment in the course of the experiment.