Determination of Staphylococcus species-specific antigens by using erythrocyte reagents

Species-specific (Staphylococcus aureus and S. epidermidis) antigenic and antibody erythrocyte diagnosticums have been prepared. Various tests made with the use of these diagnosticums have demonstrated that the activity of specific antigens is higher in hydrochloric acid extracts than in trichloroacetic and phenolic ones; this activity is linked with undialyzed components and depends on the peculiar features of the strain.     

Erythrocyte diagnostic agents for detecting bacterial antigens of the genus Citrobacter

Citrobacter antigenic and antibody erythrocyte diagnosticums, serogroups 1, 2, 3, 4, 5, 6, 7, 8, 11, 12, 13, and 22, have been developed. Tests with the use of these diagnosticums have proved to be highly sensitive and mainly group-specific. The antigen in the cellular form is best detected by means of the passive hemagglutination test and in the molecular form, by means of the neutralization test. The antibody-binding and agglutinating activities of strictly group-specific and cross-reacting O-antigenic determinants differ in their sensitivity to heating and to treatment with phenol. In the study of fecal samples taken from patients the above method for the detection of Citrobacter antigens has been shown to have high resolution.     

Escherichia-erythrocyte diagnostic agents

The conditions of a simple and practicable method for the preparation of effective antigenic nonprotein diagnosticums on the basis of water-phenol extracts of 23 Escherichia species have been developed. The method consists in heating the mixture of erythrocytes and the antigen in a boiling water bath for 60 minutes. The diagnosticums thus obtained are 16-30 times more sensitive in the passive hemagglutination test and 4-6 times more sensitive in the passive hemagglutination inhibition test than diagnosticums prepared with the use of tannin, rivanol, as well as by the common method for the preparation of nonprotein antigens. The minimum concentration of Escherichia cells detected in the passive hemagglutination inhibition test is 0.8-1.2 million cells/ml.     

Characteristics of erythrocyte diagnostic agents studied using optical methods

The quantitative characterization of erythrocyte diagnosticums (ED) has been made by optical methods (light microscopy with the use of an image analyzer, model Magiscan 2, and the opacity spectrum technique). The following parameters of ED have been determined: the average of the major axis (5.25 +/- 0.57 micron for ED from Shigella sonnei and 5.53 +/- 0.50 micron for ED from Shigella flexneri), the ratio of semiaxes (p approximately equal to 3), the major axis length distribution, the refractive index (1.076 +/- 0.002). For controlling the concentration of ED the use of the opacity spectrum technique is recommended.     

Increased sensitivity of the parapertussis diagnostic agent

The adsorption activity of formolated sheep red blood cells can be increased by their combined treatment with low-frequency ultrasound, tannin and Tween-40. The diagnosticums thus obtained have proved to be highly sensitive and specific and can be recommended for the detection of antibodies to Bordetella parapertussis in the sera of sick children.     

Obtaining erythrocyte diagnosticum for the detection of chlamydial antigen

It is shown possible to obtain erythrocyte diagnosticum for detection of chlamydial antigen, whose cell basis consists of a formalinized suspension of ram erythrocytes, sensibilized with hyperimmune antichlamydial sera by means of the amydol. High sensitivity and specificity of the diagnosticum, absolute correlation with the data obtained in the complex examination of patients with the urogenital tract pathology of the chlamydial etiology by other methods were determined in the course of investigation in the indirect hemagglutination test with diagnosticums of scrape specimens from these patients.     

Comparative study of the sensitivity and specificity of dried and liquid meningococcal erythrocyte diagnostic agents A, C and Y in a controlled trial

The comparative study of the sensitivity and specificity of dried and liquid meningococcal erythrocyte diagnosticums A, C and Y has been carried out in the indirect hemagglutination test with the sera of persons immunized with different doses of dried chemical meningococcal (group A) polysaccharide vaccine and persons receiving placebo under the conditions of a controlled epidemiological trial. The possibility of using, on principle, both liquid (A, C) and dried (A, C, Y) preparations in clinico-epidemiological studies has been established. The continuation of the research work aimed at the improvement of meningococcal diagnosticums and, in particular, at the development of polyvalent preparations seems to be justified.     

Erythrocyte reagents in the study of Erysipelothrix antigens

The activity of species-specific and type-specific antigens in various preparations isolated from the bacterial mass of standard strains of Erysipelothrix, and also in bacterial cells was studied by means of prepared erysipeloid erythrocyte antigen (species-specific and with general type- and species-specificity) and antibody (species-specific, with general type- and species-specificity as also with type-specificity only) diagnosticums. It has been demonstrated that the activity of these antigens differs in preparations from different strains, depending on the method of extraction. An efficient method of serotyping of Erysipelothrix, based on agglutination of erythrocyte antibody diagnosticums, was proposed.     

The Effects of Conjugation in Stentor coeruleus

SYNOPSIS. Culture methods favorable for conjugation in Stentor coeruleus are described and the effects of conjugation on over 12,000 conjugating pairs of S. coeruleus are presented.     

Determination of Shigella antigens in the feces of people, immunized with enteral vaccine prepared from S. flexneri antigen

The dynamic determination of the presence of the specific antigen and its activity in the excreta of humans subjected to enteral immunization with vaccine prepared from S. flexneri antigen was made in the agglutination test and neutralization test with the use of, respectively, antibody and antigenic erythrocyte diagnosticums. In the feces and urine of the vaccinees antibodies occurred less commonly and, as a rule, they were less active than those detected in dysentery patients at the corresponding time from the beginning of the disease. The occurrence of Shigella antigens in the feces of the vaccinees was greater than in their urine at the corresponding time. Similarities and differences in the dynamics of the isolation of Shigella antigen from dysentery patients and from the vaccinees receiving enteral vaccine prepared from S. flexneri antigens were established.     

Effect of the species origin of erythrocytes and of the methods for their subsequent handling on their electrophoretic mobility in producing diagnostic agents

Changes in electrophoretic motility correlate with the effect produced by different reagents used in the production of erythrocyte reagents. This characteristic may be one of the criteria for the evaluation of the quality of erythrocytes in the development of diagnosticums.     

Circulating immune complexes in the diagnosis of Mycoplasma pneumoniae infection

The possibility of detecting M. pneumoniae antigen and antibodies to it, incorporated into immune complexes, in the sera of patients with acute pneumonia by means of erythrocyte diagnosticums was studied, and the immunological characterization of these complexes was made. In patients with mycoplasmal pneumonia M. pneumoniae antigen and specific antibodies, both free and incorporated into immune complexes, were found to circulate in the blood. In children, antigenemia was detected twice as frequently as in adults. Dissociated M. pneumoniae antigens had different molecular weight, their location on the gel chromatogram of the serum being in fractions 7S and 19S. The dissociation of immune complexes permits the detection of M. pneumoniae antigen and antibodies to it in a bound state by means of the passive hemagglutination test, thus increasing the frequency of positive results in the diagnosis of M. pneumoniae infection.     

O:2-CRM197 Conjugates against Salmonella Paratyphi A

Enteric fevers remain a common and serious disease, affecting mainly children and adolescents in developing countries. Salmonella enterica serovar Typhi was believed to cause most enteric fever episodes, but several recent reports have shown an increasing incidence of S. Paratyphi A, encouraging the development of a bivalent vaccine to protect against both serovars, especially considering that at present there is no vaccine against S. Paratyphi A. The O-specific polysaccharide (O:2) of S. Paratyphi A is a protective antigen and clinical data have previously demonstrated the potential of using O:2 conjugate vaccines. Here we describe a new conjugation chemistry to link O:2 and the carrier protein CRM₁₉₇, using the terminus 3-deoxy-D-manno-octulosonic acid (KDO), thus leaving the O:2 chain unmodified. The new conjugates were tested in mice and compared with other O:2-antigen conjugates, synthesized adopting previously described methods that use CRM₁₉₇ as carrier protein. The newly developed conjugation chemistry yielded immunogenic conjugates with strong serum bactericidal activity against S. Paratyphi A.     

Disulfide bridge as a linker in nucleic acids’ bioconjugation. Part I: An overview of synthetic strategies

This 2-part article reviews methods of oligonucleotides functionalization with thiol tethers and their consecutive use in conjugation with other (macro)molecules via a disulfide bridge. This relatively inexpensive, robust and reversible method of conjugation of DNAs, RNAs and their analogs holds a prominent position in a modern biochemistry toolbox and therefore there is a wealth of literature on the subject. In part I methods of thiol/disulfide groups introduction into oligonucleotide strands have been systematized and discussed. A digest of conjugation methods is presented as well.     

酪蛋白水解物对雷帕霉素产生菌Streptomyces hygroscopicus ATCC29253 接合转移效率的影响

吸水链霉菌(Streptomyces hygroscopicus)ATCC29253能够产生非常丰富的次级代谢产物,除了雷帕霉素外,还可以分泌尼日利亚菌素、洋橄榄叶素、六烯类抗生素等多种具有生物活性的物质,具有重要的研究价值和应用前景。【目的】而建立高效的遗传操作系统是研究该链霉菌相关代谢产物合成机理和构建基因工程菌株的基础。【方法】我们测试了不同培养基及供体菌对吸水链霉菌及其它链霉菌接合转移效率的影响。【结果】我们发现酪蛋白水解物和镁离子能显著提高S.hygroscopicus ATCC29253接合转移效率。通过随机组合试验,筛选出最佳的MgCl2和酪蛋白水解物浓度组合,使得S.hygroscopicus ATCC29253接合转移效率达到1.5×10-4。同时我们还发现酪蛋白水解物可以明显改变S.lividans、S.albus、S.avermitilis的接合转移效率。【结论】本研究首次发现酪蛋白水解物不光对S.hygroscopicus ATCC29253接合转移有作用,对其它常用的链霉菌如S.lividans、S.albus、S.avermitilis等的接合转移效率都有显著的影响。     

Intergeneric conjugal gene transfer from Escherichia coli to the sweet potato pathogen Streptomyces ipomoeae

Aims:  To develop an effective gene transfer system for Streptomyces ipomoeae , the causative agent of soil rot disease of sweet potatoes.
Methods and Results:  Of the several methods investigated, introduction of genes into S. ipomoeae could only be achieved using intergeneric conjugation from Escherichia  coli . However, even results for that method varied greatly and were dependent on using particular media for S. ipomoeae spore preparation and conjugation. Transconjugant to recipient ratios as great as 4·1 × 10⁻⁵ were achieved when International Streptomyces Project Medium 4 was used for both sporulation and conjugation protocols. Both site-specifically integrating and autonomously replicating plasmids could be introduced and maintained in S. ipomoeae, and plasmids could be introduced with approximate equivalent frequencies from either methyl-proficient or methyl- deficient E. coli donors; the latter result indicates a likely absence of relevant methyl-specific restriction in S. ipomoeae .
Conclusions:  Efficient transfer of genes into S. ipomoeae was achieved here by using an optimized intergeneric mating procedure.
Significance and Impact of the Study:  The described protocol will facilitate further genetic manipulation of this agriculturally important pathogen.     

Relationships between Cell Cycle and Conjugation in 3 Hypotrichs*

SYNOPSIS. Relationships between the cell cycle and the beginning of conjugation were analyzed for 3 hypotrichs: Diophrys scutum, Oxytricha bifaria, and Euplotes crassus. The first 2 species enter conjugation with micronuclei in G₁; the latter species with a micronucleus in G₂. The 1st micronuclear division of conjugating E. crassus is mitotic. Thus meiotic DNA replication occurs when the cells of each species have already entered the mating process. Cells from asynchronous populations start conjugation with their macronuclei primarily in G₁ or more rarely at the beginning of the S stage in a percentage significantly different from that expected on the basis of random mating among all cells in the population. Also, macronuclear replication, when already begun, was blocked in cells undergoing conjugation. Therefore only the G₁ or the very early S stages of the cell cycle are compatible with conjugation in the 3 analyzed species.     

The utilization of alanine, glutamic acid, and serine as amino acid substrates for glycine N-acyltransferase

The conjugation of benzoyl-CoA with the aliphatic and acidic amino acids by glycine N-acyltransferase, as well as the amides of the latter group, was investigated. Bovine and human liver benzoyl-amino acid conjugation were investigated using electrospray ionization tandem mass spectrometry (ESI-MS-MS). Bovine glycine N-acyltransferase catalyzed conjugation of benzoyl-CoA with Gly (Km(Gly) = 6.2 mM), Asn (Km(Asn) = 129 mM), Gln (Km(Gln) = 353 mM), Ala (Km(Ala) = 1573 mM), Glu (Km(Glu) = 1148 mM) as well as Ser in a sequential mechanism. In the case of the human form, conjugation with Gly (Km(Gly) = 6.4 mM), Ala (Km(Ala) = 997 mM), and Glu was detected. The presence of these alternative conjugates did not inhibit bovine glycine N-acyltransferase activity significantly. Considering the relatively low levels at which these conjugates are formed, it is unlikely that they will have a significant contribution to acyl-amino acid conjugation under normal conditions in vivo. However, their cumulative contribution to acyl-amino acid conjugation under metabolic disease states may prove to have a useful contribution to detoxification of elevated acyl-CoAs.     

Conjugation of isoprene monoepoxides with glutathione, catalyzed by alpha, mu, pi and theta-class glutathione S-transferases of rat and man

In the present study, the enzymatic conjugation of the isoprene monoepoxides 3,4 epoxy-3-methyl-1-butene (EPOX-I) and 3,4-epoxy-2-methyl-1-butene (EPOX-II) with glutathione was investigated, using purified glutathione S-transferases (GSTs) of the alpha, mu, pi and theta-class of rat and man. HPLC analysis of incubations of EPOX-I and EPOX-II with [35S]glutathione (GSH) showed the formation of two radioactive fractions for each isoprene monoepoxide. The structures of the EPOX-I and EPOX-II GSH conjugates were elucidated with 1H-NMR analysis. As expected, two sites of conjugation were found for both isoprene epoxides. EPOX-II was conjugated more efficiently than EPOX-I. In addition, the mu and theta class glutathione S-transferases were much more efficient than the alpha and pi class glutathione S-transferases, both for rat and man. Because the mu- and theta-class glutathione S-transferases are expressed in about 50 and 40-90% of the human population, respectively, this may have significant consequences for the detoxification of isoprene monoepoxides in individuals who lack these enzymes. Rat glutathione S-transferases were more efficient than human glu tathione S-transferases: rat GST T1-1 showed about 2.1-6.5-fold higher activities than human GST T1-1 for the conjugation of both EPOX-I and EPOX-II, while rat GST M1-1 and GST M2-2 showed about 5.2-14-fold higher activities than human GST M1a-1a. Most of the glutathione S-transferases showed first order kinetics at the concentration range used (50-2000 microM). In addition to differences in activities between GST-classes, differences between sites of conjugation were found. EPOX-I was almost exclusively conjugated with glutathione at the C4-position by all glutathione S-transferases, with exception of rat GST M1-1, which also showed significant conjugation at the C3-position. This selectivity was not observed for the conjugation of EPOX-II. Incubations with EPOX-I and EPOX-II and hepatic S9 fractions of mouse, rat and man, showed similar rates of GSH conjugation for mouse and rat. Compared to mouse and rat, human liver S9 showed a 25-50-fold lower rate of GSH conjugation.     

Proteolytic processing of micronuclear H3 and histone phosphorylation during conjugation in Tetrahymena thermophila

During vegetative growth, micronuclei of the ciliated protozoan Tetrahymena thermophila contain two electrophoretically distinct forms of H3, H3S and H3F [4, 5]. Of these two forms, H3F is unique to micronuclear chromatin and is derived from H3S by a physiologically regulated proteolytic processing event [5]. While the function of this processing event is not clear, several lines of evidence [2, 5] suggest that it may be related to chromatin condensation during mitosis. In this report pulse-chase experiments have been used to study the processing of H3S into H3F during the sexual phase of the life cycle, conjugation. Our results demonstrate that even though micronuclei divide mitotically (and meiotically) several times during the mating process, processing of H3S into H3F does not occur. Failure of H3S to be converted into H3F during these divisions causes a significant increase in the amount of H3S (relative to H3F) as conjugation proceeds. By 10 h of conjugation, essentially all of the micronuclear H3 is in the form of H3S (also see [3]). As long as mating cells are maintained under starvation conditions, processing of H3S into H3F does not occur. However, if exconjugants are returned to food and allowed to proceed through the first true cell division following exconjugation, processing of H3S into H3F occurs. Thus, the return of the processing of H3(3) into H3F following conjugation seems to be tightly coupled to a division which is part of a cell division cycle (as appears to be the case with vegetatively growing cells). The relevancy of these results to the differentiation of new macro- and micronuclei is discussed. H3F is specifically phosphorylated in growing cells, and it has been suggested that this phosphorylation event may be related to chromatin condensation during mitosis [2]. Since in mating cells H3S becomes the more predominant form of H3, the pattern of histone phosphorylation was examined during stages of conjugation where micronuclei are active in mitotic division (6-7 h). While a low level of phosphate label is observed over H3S in mating cells, more phosphate label is associated with the small amount of H3F which remains in micronuclei at this stage of conjugation. We also observe significant amounts of phosphate label associated with micronuclear H2A, H2B, and H4 and each of the micronuclear H1-like molecules, alpha, beta and gamma.