Specificity of the protective action of a ribosomal Shigella vaccine and the absence of activity in the ribosomes from R mutants

The ribosomal preparations of S. sonnei and some other bacterial species were obtained by the method of differential centrifugation, and the specificity of their protective action was studied in the keratoconjunctivitis test on guinea pigs. The ribosomal preparations were introduced parenterally in a single injection, and their protective action was determined two weeks later by the challenge of the animals with S. sonnei virulent strain and the subsequent calculation of the efficiency index (EI) by the formula: EI = C-V/C X 100, where C and V are the percentage of resistant eyes in the control and vaccinated groups of the animals respectively. For the ribosomal preparation obtained from a homologous avirulent strain this index was equal to 58%, while for the heterologous ribosomes obtained from Escherichia coli, Salmonella minnesota and S. flexneri in was close to zero. The ribosomal preparations obtained from S. sonnei R-strain which had no surface or cytoplasmic O-antigen also proved to be ineffective in rendering protection against local Shigella infection. The results of this investigation are compared with the data obtained by other authors, and the analysis of these results leads to the conclusion that the O-specific component is the indispensable factor of the protective activity of many ribosomal vaccines and its molecular properties require further study. The possible role of other components of the ribosomal vaccine is also discussed.     

The action of ribosomal preparations on nonspecific resistance to bacterial infection and on early tolerance to endotoxic shock

Ribosomal preparations from Shigella flexneri and Shigella sonnei, introduced parenterally into mice, enhance their resistance to infection with the causative agents of typhoid fever and staphylococci. This effect is considerably less pronounced than that produced by the preparation of homologous lipopolysaccharide isolated by Boivin's method. After the administration of ribosomes nonspecific resistance to bacterial infective agents lasts for a short time. Ribosomal preparations do not enhance the resistance of mice to the lethal action of endotoxin.     

A protective factor of ribosomal shigellosis vaccine

Shigella ribosomal vaccine was shown previously to possess protective properties in the keratoconjunctival test on guinea pigs and to be capable of preventing experimental infection in 90% of challenged monkeys. The presence of the O-specific component (OSC) constituting about 0.5% of the ribosomal preparation by serological activity suggested its importance for the protective effect. This was studied in experiments with two O-specific immunosorbents prepared by coupling anti-O rabbit antibodies with Staphylococcus aureus cells or with CNBr-Sepharose. Ribosomes treated with immunosorbents proved to be lacking the serologically active OSC and lost their ability to induce O-antibody response in rabbits and mice. After the removal of this component ribosomal preparations were incapable of ensuring protection from Shigella kerato-conjunctival infection. The isolated OSC was also inactive in this test. The data obtained in this investigation confirm the hypothesis stating that the protective activity of Shigella ribosomal vaccine is based on the combined action of ribosomes and O-specific factor whose nature and properties require further study.     

Induction of murine B cell proliferation and immunoglobulin synthesis by some bacterial ribosomes

Ribosomal preparations from Klebsiella pneumoniae, Haemophilus influenzae, Streptococcus pyogenes, and Streptococcus pneumoniae were investigated with respect to their activating capacity towards murine lymphoid cells. The proliferation of BALB/c spleen cells was induced in a dose-dependent fashion (from 1 to 100 micrograms/ml) by ribosomes of K. pneumoniae, H. influenzae, and S. pyogenes with a peak activity at 48 or 72 hr of culture. The majority of the blast cells induced by these ribosomal preparations were positive for surface-immunoglobulin (S-Ig) and negative for Thy 1.2. Furthermore, K. pneumoniae, H. influenzae, and S. pyogenes ribosomes induced the synthesis of IgM and some IgA. Cell proliferation and induction of IgM production were also demonstrated with the 3 ribosomal preparations using spleen cells from athymic nude (nu+/nu+) mice, Lyb-5-defective CBA/N spleen cells, B cell-enriched and T cell-depleted BALB/c spleen cell suspensions, as well as spleen cells from the Ips gene-deficient C3H/HeJ strain. Cell culture supernatants contained specific anti-ribosome IgM antibodies. Antibodies of other specificities (anti-sheep erythrocytes) were also demonstrated in supernatants from K. pneumoniae-stimulated cultures. Evidence against a possible role of contamination of K. pneumoniae and H. influenzae ribosomes by lipopolysaccharide- or lipid A-associated proteins in this effect is discussed. Ribosomes from S. pneumoniae did not induce 3H-thymidine incorporation nor Ig production. None of the 4 ribosomal preparations was found to stimulate T cell blastogenesis or to induce interleukin-2 production by naive BALB/c spleen cells. Finally, ribosomes from H. influenzae, S. pyogenes, S. pneumoniae but not those of K. pneumoniae stimulated interleukin-1 production by adherent spleen cells, from BALB/c mice.     

Streptococcus mutans ribosomal preparations: purification and properties

Ribosomal preparations were obtained from Streptococcus mutans. Sucrose density gradient analyses showed the ribosomes to be 70S and dissociated subunits to be 56S and 34S. The ribosomal preparation contained 57.4% RNA and 42.6% protein and gave an absorption maximum at 260 nm and a minimum at 235 nm and ribosomal particles were approximately 150-180 X 190-220 A as determined by electron microscopy. Immunodiffusion analysis of pooled antiserum raised by injecting the ribosomal preparation into rabbits disclosed precipitin lines with glucosyltransferase and lipoteichoic acid preparations from S. mutans. Gas chromatography showed rhamnose and glucose to be present in the ribosomal preparation indicating the presence of nonribosomal carbohydrate materials. The ribosomes were able to synthesize precipitable polypeptides when exogenous mRNA and tRNA were added and anti-ribosomal antibodies reduced this activity. Protease treatment rendered the ribosomal preparation less immunogenic in rats and less antigenic when the ribosomal preparation was used to coat erythrocytes for passive haemagglutination assays, while RNase treatment of the ribosomal preparation had no effect, suggesting that a protein(s) is the principal immunogenic moiety of the ribosomal antigen. Polyacrylamide gel electrophoresis of the ribosomal preparation revealed 27 protein bands of which five were found to react with hyperimmune rabbit antisera to the S. mutans ribosomal preparation by Western blot analysis. Washing the ribosomal preparation with 1 M NH4Cl did not remove any of the five immunogenic ribosomal protein antigens indicating that these were innate ribosomal proteins.     

Characterization of the mitogenic activity elicited by Neisseria gonorrhoeae ribosomal fractions.

Ribosomal preparations from Neisseria gonorrhoeae types 1 and 4 were examined for their in vitro stimulation of mouse splenocytes to determine the ribosomal moiety or contaminant responsible for the immunoproliferative activity. In immunodiffusion tests with homologous rabbit antiserum, crude 70S ribosomes formed four precipitin bands while the purified 30S and 50S subunits showed one major line. The same antiserum reacted with lysed N. gonorrhoeae and Neisseria meningitidis A cells but no precipitation occurred with Escherichia coli cells purified N. gonorrhoeae lipopolysaccharide (LPS). No membrane or LPS contaminant was detected in the purified 30S and 50S preparations. All the ribosomal preparations from virulent and non-virulent N. gonorrhoeae consistently stimulated the murine splenocytes. The mitogenic activity of the 30S and 50S ribosomal preparation was destroyed by treatment with trypsin but only slightly decreased by ribonuclease. It is suggested that the lymphoproliferative response elicited by gonococcal ribosomes is triggered by the protein moiety of the 30S or 50S subunits.     

痢疾菌苗研究进展

痢疾是全世界范围内流行的肠道传染病。随着分子生物学技术的发展,迄今已构建了新一代预防痢疾的疫苗,其中包括:具有侵袭力和不具侵袭力的单价口服活菌苗候选株;它们与大肠杆菌、伤寒沙门氏菌和血清型不同的痢疾杆菌组成的双价和三价菌苗候选株;以及以脂多糖和核糖体为基础的不经胃肠的组分菌。猴体和人体试验证明它们安全有效。预计在未来的10年中,将会有一个或几个安全有效的痢疾疫苗投放市场。     

Binding of erythromycin to the 50S ribosomal subunit is affected by alterations in the 30S ribosomal subunit

Summary Expression of resistance to erythromycin in Escherichia coli, caused by an altered L4 protein in the 50S ribosomal subunit, can be masked when two additional ribosomal mutations affecting the 30S proteins S5 and S12 are introduced into the strain (Saltzman, Brown, and Apirion, 1974). Ribosomes from such strains bind erythromycin to the same extent as ribosomes from erythromycin sensitive parental strains (Apirion and Saltzman, 1974).Among mutants isolated for the reappearance of erythromycin resistance, kasugamycin resistant mutants were found. One such mutant was analysed and found to be due to undermethylation of the rRNA. The ribosomes of this strain do not bind erythromycin, thus there is a complete correlation between phenotype of cells with respect to erythromycin resistance and binding of erythromycin to ribosomes.Furthermore, by separating the ribosomal subunits we showed that 50S ribosomes bind or do not bind erythromycin according to their L4 protein; 50S with normal L4 bind and 50S with altered L4 do not bind erythromycin. However, the 30s ribosomes with altered S5 and S12 can restore binding in resistant 50S ribosomes while the 30S ribosomes in which the rRNA also became undermethylated did not allow erythromycin binding to occur.Thus, evidence for an intimate functional relationship between 30S and 50S ribosomal elements in the function of the ribosome could be demonstrated. These functional interrelationships concerns four ribosomal components, two proteins from the 30S ribosomal subunit, S5, and S12, one protein from the 50S subunit L4, and 16S rRNA.     

Stoichiometry of GTP hydrolysis during peptide synthesis on the ribosome. I. Factor-independent GTPase and ATPase of ribosomal preparations

It has been found that preparations of Escherichia coli (MRE-600) ribosomes can display GTPase and ATPase activities independent of elongation factors EF-Tu and EF-G. The GTPase and ATPase are localized on ribosomal 50S subparticles, whereas 30S subparticles are free of the activities and do not stimulate them upon association with the 50S subparticles to form complete ribosomes. The GTPase and ATPase can be removed from the ribosomes and their 50S subparticles by treatment with 1 M NH4Cl or 50% ethanol in the cold. Ribosomal preparations freed from the factor-independent GTPase and ATPase retain their basic functional features. The data obtained do not permit to solve finally whether the factor-independent GTPase and ATPase revealed are components of ribosomes or represent a contamination rather firmly bound to the ribosomes. However, in any case this finding can contribute to an uncoupled hydrolysis of GTP and should be considered when studying the stoichiometry of triphosphate expenditure in the process of ribosomal protein synthesis.     

Preparation of Escherichia coli 70S ribosomes labeled with 35S

[35S]--70S ribosomes (150 Ci/mmol) were isolated from E. coli MRE-600 cells grown on glucose-mineral media in the presence of [35S] ammonium sulfate. The labeled 30S and 50S subunits were obtained from [35S] ribosomes by centrifugation in a sucrose density gradient of 10--30% under dissociating conditions (0.5 mM Mg2+). The activity of [35S]--70S ribosomes obtained by reassociation of the labeled subunits during poly(U)-dependent diphenylalanine synthesis was not less than 70%. The activity of [35S]--70S ribosomes during poly(U)-directed polyphenylalanine synthesis was nearly the same as that of the standard preparation of unlabeled ribosomes. The 23S, 16S and 5S RNAs isolated from labeled ribosomes as total rRNA contained no detectable amounts of their fragments as revealed by polyacrylamide gel electrophoresis. The [35S] ribosomal proteins isolated from labeled ribosomes were analyzed by two-dimensional gel electrophoresis. The [35S] label was found in all proteins, with the exception of L20, L24 and L33 which did not contain methionine or cysteine residues.     

Ribosomal dysentery vaccine. I. Method of production, physical and chemical properties]

Studies of A. and G. Youmans on the experimental tuberculosis led to discovery of a fundamentally new type of vaccines (ribosomal vaccines) which proved to be highly effective in the prophylaxis of many experimental infections. Therefore it seems reasonable to prepare analogous vaccine from Shigellae for the study of its efficiency in experimental shigellosis. Ribosomal preparations from Shigella sonnei were prepared by sonic disruption of microbial cells followed by differential ultracentrifugation according to A. and G. Youmans' method with slight modifications. The yeild of ribosomal fraction was about 2 per cent by weight; all the series had an UV adsorption maximum at 260 nm, the ratio OD260:OD280 being approximately 2. They contained about 55% of RNA, 35% of protein and no more than 8% of saccharides. As shown by centrifugation in sucrose gradient and by analytical ultracentrifugation the preparations were homogeneous. The presence of undissociated ribosomes was confirmed by electron microscopy. Thus, the ribosomal preparations obtained proved to be sufficiently purified for carrying out experimental investigations of their biological activity.     

Characterization of Ribosomes from Dormant Spores of Bacillus cereus

Characterization of ribosomes from dormant spores and vegetative cells of Bacillus cereus strain T has been carried out. Polyuridylic acid binding activity, ribonuclease activity associated with ribosomes, thermal denaturation profile, and sedimentation coefficients are essentially identical for both ribosomal preparations. However, ribosomal protein content of dormant spore ribosomes is about 70% of that of vegetative ribosomes. Polyacrylamide gel electrophoresis of ribosomal proteins shows that some ribosomal proteins are missing from dormant spore ribosomes. Sucrose density gradient centrifugation of ribosomes shows the existence of defective ribosomal subunits, in addition to 30S and 50S subunits, in dormant spore ribosomes. These results indicate that the ribosomes from dormant spores are distinctively different from those of vegetative cells.     

Resistance against cycloheximide in cell lines from Chinese hamster and human cells is conferred by the large subunit of cytoplasmic ribosomes

Summary Cell lines from Chinese hamster ovary [CHO-K1-D3] and human fibroblast cells [46, XX, 18p-] were mutagenized with N-nitrosomethylurea followed by a selection for cycloheximide resistance. Two mutants resistant against the durg were selected from either wildtype. 80S ribosomes and their ribosomal subunits were isolated from all mutant and wildtype cells. 80S ribosomes reassociated from the isolated subunits were as active as isolated 80S couples in the poly (U) dependent poly (Phe) synthesis. Hybrid 80S ribosomes constructed from subunits of the various cell lines of the same species were fully active, whereas the interspecies 80S hybrids were not active at all in poly (Phe) synthesis.Hybrid 80S ribosomes from subunits of mutant and the ocrresponding wildtype cells were tested in the poly (U) assay in the presence and absence of cycloheximide. The results strikingly indicate that in all four mutant cell lines the resistance against cycloheximide is conferred by the large subunit of cytoplasmic ribosomes.Abbreviations CHM Cycloheximide - CHO Chinese hamster ovarien - FBS foetal bovine serum - Eagle MEM Eagle minimal essential medium - EMS Ethyl-metansulfonate - NMU N-nitrosomethylurea     

Isolation of ribosomal subparticles from human placenta containing intact rRNA and determination of the functional activity of the 80S ribosome

The method for isolation of human placenta ribosomal subunits containing intact rRNA has been determined. The method uses fresh unfrozen placenta. Activity of 80S ribosomes obtained via reassociation of 40S and 60S subunits in non-enzymatic poly(U)-mediated Phe-tRNAPhe binding, was near 75% (maximal [14C]Phe-tRNA(Phe) binding was 1.5 mol Phe-tRNA(Phe) per mol of 80S ribosomes). Activity of 80S ribosomes with damaged rRNA isolated from frozen placenta was 2 times lower (the maximum level of poly(U)-dependent Phe-tRNA(Phe) binding was 0.7 mol per mol of ribosomes). The activity 80S ribosomes in poly(U)-mediated synthesis of polyphenylalanine was determined by using fractionated ("ribosomeless") protein synthesising system from rabbit reticulocytes. In this system up to the 50 mol of Phe residues per mol of 80S ribosomes are incorporated in acid insoluble fraction in 1 hour, at 37 degrees C. The obtained level of [14C]phenylalanine incorporation is three times as much as the amount of Phe residues observed for the ribosomal subunits, isolated from frozen placenta.     

Requirement of the conformational stability of a Salmonella ribosomal vaccine for its mouse protection

The 43-kDa non-O antigenic component isolated from the crude ribosomal fraction of Salmonella typhimurium [9] was further purified by affinity chromatography (43-kDa protein: 43-kDp). Immunization with 43-kDp did not induce complete mouse protection in CF1 mice to 500 LD50 of S. typhimurium, although it elicited a substantial IgG antibody response. The 43-kDp exhibited the mitogenicity to splenocytes (CF1 and C3H/HeJ) and B cell-rich populations (CF1). Complexing 43-kDp with the compact ribosomes of Streptococcus pyogenes by formaldehyde (complex vaccine: CV) elicited both IgM and IgG antibodies to 43-kDp. CV induced a boosting effect to enhance IgG antibody response. Moreover, CV generated delayed-type hypersensitivity to salmonella antigens and also conferred complete protection against 500 LD50 challenge of S. typhimurium to CF1 mice. These abilities of CV were reduced or impaired by RNase digestion. CV was able to induce partial or complete protection in inbred mouse strains (C3H/HeN, C3H/HeJ, DBA/2 and A/J). These data, in addition to other reports, suggest that conformational stability between ribosomes and contaminating substances such as 43-kDp or O-antigens might be required for the overall effects of the ribosomal vaccine.     

Analysis of Ribosomes from Viomycin-Sensitive and -Resistant Strains of Mycobacterium smegmatis

Viomycin-resistant strains were isolated from Mycobacterium smegmatis. Ribosomes were isolated and tested for drug resistance in subcellular systems containing poly(U) as messenger ribonucleic acid. Resistance to viomycin in these strains was due to altered ribosomes. Further analysis showed that viomycin resistance of two mutants with low level resistance (20 mug/ml) was due to altered 30S ribosomal subunits. Another mutant that was highly resistant to viomycin (1 mg/ml), however, had altered 50S ribosomal subunits.     

Effects of the ribosomal subunit association on the chemical modification of the 16S and 23S RNAs from Escherichia coli

70S ribosomes and 30S and 50S ribosomal subunits from Escherichia coli were modified under non-denaturing conditions with the chemical reagent dimethylsulfate. The ribosomal 23S and 16S RNAs were isolated after the reaction and the last 200 nucleotides from the 3' ends were analyzed for differences in the chemical modification. A number of accessibility changes could be detected for 23S and 16S RNA when 70S ribosomes as opposed to the isolated subunits were modified. In addition to a number of sites which were protected from modification several guanosines showed enhanced reactivities, indicating conformational changes in the ribosomal RNA structures when 30S and 50S subunits associate to a 70S particle. Most of the accessibility changes can be localized in double-helical regions within the secondary structures of the two RNAs. The results confirm the importance of the ribosomal RNAs for ribosomal functions and help to define the RNA domains which constitute the subunit interface of E. coli ribosomes.     

Interaction of the cytoplasmic membrane and ribosomes in Escherichia coli; altered ribosomal proteins in sucrose-dependent spectinomycin-resistant mutants.

Alterations in the ribosomes of sucrose-dependent spectinomycin-resistant (Sucd-Spcr) mutants of Escherichia coli were studied. Subunit exchange experiments showed that 30S subunits were responsible for the resistance of ribosomes to spectinomycin in all Sucd-Spcr mutants tested. Proteins of 30S ribosomes were analyzed by carboxymethyl cellulose column chromatography based on their elution positions. Mutants YM22 and YM93 had an altered 30S ribosomal protein component, S5, and mutant YM50 had an altered protein, S4. Although a shift of elution position was not detected for all the 30S ribosomal proteins from mutant YM101, the amount of protein S3 was appreciably lowered in the isolated 30S subunits. A partial reconstitution experiment with protein S3 prepared from both the wild-type strain and YM101 revealed that the mutant had altered protein S3 which is responsible for the spectinomycin resistance. These alterations in 30S subunits are discussed in relation to the interaction between ribosomes and the cytoplasmic membrane.     

Effect of immunization with an enterobacterial vaccine on the rate of plating out of Shigella from the intestinal mucosa of mice orally infected

The oral and subcutaneous immunization of mice with the vaccine prepared from S. minnesota strain R595, chemotype Re, known as enterobacterial vaccine, was found to significantly decrease the number of shigellae in cultures obtained by the inoculation of homogenized mucosal samples taken from the large intestine of mice, the vaccine prepared from S. minnesota isogeneous strain SF 1111 with the intact structure of lipopolysaccharide had no such activity. Antisera, obtained by the immunization of rabbits with enterobacterial vaccine, contained high titers of antibodies to Re-glycolipid and were capable of decreasing the isolation rate of shigellae from homogenized mucosal samples taken from the large intestine of mice; at the same time S. flexneri were found capable of binding with antibodies to glycolipid of Re-chemotype.     

Functional macrophage activity in infection with virulent and avirulent strains of the dysentery microbe

The effect of S. flexneri virulent and avirulent (vaccine) strains 2a on the cytoplasmic membrane of mouse macrophages has been studied by evaluating the action of these bacteria on the activity of 5-nucleotidase. The dynamics of the activity of 5-nucleotidase after the introduction of both virulent and avirulent strains has a phasic character with alternating rises and falls in the activity of this enzyme in comparison with the control. S. flexneri vaccine strain produces mainly a stimulating effect on the functional activity of peritoneal macrophages in mice, which is confirmed by a decrease in the activity of 5-nucleotidase; on the contrary, S. flexneri virulent strain- has mainly an inhibiting effect on the functional activity of peritoneal macrophages, which is confirmed by an increase in the activity of 5-nucleotidase in these cells. The comparative study of changes in the activity of 5-nucleotidase, following the introduction of S. flexneri, in mice, previously immunized with smallpox vaccine, and in intact mice has shown that the use of animals immunized with smallpox vaccine in the study of metabolic characteristics may lead to distortions in the results of the experiment.