It was found that (-)rugulosin, an antibiotic isolated from Myrothecium verucaria, had a potent anti-phage effect on RNA phages (MS2, GA and Qβ) and DNA phages (IA and T4). The effect was almost independent of the host bacterial strains used. By a detailed investigation using MS2, it was revealed that the antibiotic did not affect the free phage or host bacterium alone but inhibited phage multiplication, and the degree of the inhibition depended on the multiplicity of infection. The inhibition was not mainly due to a drop in the burst size but rather was due to a decrease of the phageproducing cells during the early stages of phage infection and replication.

View full abstract

The mechanism of anti-phage action of (-)rugulosin was investigated mainly using phage MS2. In the presence of the antibiotic, phage adsorption was not inhibited and phage-bacterium complexes did not remain in an antiserum-sensitive state. However, phage-bacterium complexes temporarily remained in a shear-sensitive state during the time immediately following phage infection and phage RNA penetrating into the host bacteria was decreased. The antibiotic also acted on preformed antiserum-and shear-resistant infective centers and inhibited the in uitro RNA synthesis by Qβ replicase. These results indicate that the mechanim of action of the antibiotic consists of at least 2 parts, i.e., the inhibition of the penetration of phage RNA into the host bacteria and some early intracellular reaction(s) for phage multiplication.

View full abstract

The mode of action of the autolytic enzymes of Clostridiumt botulinumt type A strain 190L was investigated using a partially purified autolysin. The autolysin completely solubilized SDS-treated cell walls of the organism, liberating 1.2 moles of NH₂-terminalL-alanine and 0.6 moles of reducing groups per mole of glutamic acid. Neither the NH₂-termini of other amino acids nor COOH-termini of any amino acids were released, These results show that the autolysin contains an N-acetylmuramyl-L-alanine amidase and a hexosaminidase. A disaccharide and peptides were isolated from the wall lysate in a chromatographically homogeneous state. The reducing end of the disaccharide was elucidated to be N-acetylglucosamine by borohydride reduction. This fact indicates that the hexosaminidase is likely to be an endo-β-N-acetylglucosaminidase. A possible structure of the cell wall peptidoglycan is proposed.

View full abstract

A seroepidemiological study of rubella in northern Japan was made by testing for hemagglutinin-inhibitory (HAI) antibodies to rubella virus using an HAI test with our modification. An epidemic of exanthematous disease, which occurred in 1966-1967, was confirmed as rubella by the age specific distribution of rubella HAI antibody. However, one of the 14 areas tested in the region, a Piedmont village, was found to have had no rubella experience during the last 15 years. In general, a higher incidence of high titered HAI antibody was observed among the younger ages, suggesting a decrease of antibody titer with an increase in age after infection. Through these studies, it is evident that 100% of adults over 20 years of age possessed HAI antibody in all age groups and all areas so far examined. This fact may possibly explain the extremely rare incidence of congenital rubella syndrome in the northern part of Japan.

View full abstract

Adjuvant action of lipopolysaccharide (LPS) extracted from Salmonella typhiinuriumLT2 on anti-sheep red blood cell (sRBC) antibody response in mouse spleen was studied at the cellular level by the technique of localized hemolysis in agar. Injection of LPS caused significant increase in direct 19S and indirect 7S plaque-forming cells (PFC) in mouse spleen after the primary antigenic stimulation, while the adjuvant effect of LPS could hardly be assessed in the secondary PFC response. The adjuvant effect on the primary PFC response was most conspicuous when LPS was injected at the same time as the sRBC, and no effect could be observed when LPS was injected 1 or 5hr before PFC determination. The mice who had received LPS with the primary sRBC stimulation showed much higher PFC level after the secondary sRBC stimulation, indicating the increase in the memory cells. Cell population consisting of lymphoid cells and macrophages was separated into 'adherent' (macrophage-rich) and 'non-adherent' (lymphoid cell-rich) fractions by incubating in petri dishes, and effect of LPS on each fraction was examined. Transfer of non-adherent lymphoid cells treated with LPS in vitro and simultaneous sRBC injection resulted in a significant increase of PFC in the spleen of the syngeneic recipient mice, but transfer of LPS-treated macrophages did not show any effect, suggesting that LPS acts on lymphocytes but not on macrophages. This idea appeared also to be supported by the results that LPS exerted antagonistic effect on the immune suppression caused by heterologous anti-lymphocyte serum, anti-sRBC serum and cortisone which are currently supposed to suppress the antigen reactive cells.

View full abstract

An immune ribonucleic acid (RNA) was extractable from the spleen cells of mice hyperimmunized with live vaccine of Salmonella enteritidis. The RNA was capable of inducing cellular immunity and developing cellular antibody in the peritoneal macrophages of mice injected with this agent. It was found that cellular immunity was detectable even 90 days after injection in the peritoneal macrophages of mice which had received an intraperitoneal injection with this agent. Results of serial passive transfers of cellular immunity through immune RNA led us to the conclusion that this agent does not contain antigen or fragment thereof and may replicate actively in the recipient cells, although the mechanism still remains to be elucidated. The development of cellular immunity by immune RNA was inhibited by puromycin but not by actinomycin D. However, serial passive transfers of cellular immunity through immune RNA was inhibited by treatment of recipient mouse with actinomycin D, implying the role of DNA-dependent RNA polymerase in the processing of immune RNA in recipient cells. Using these results, the role of immune RNA and the possible mechanisms of immune RNA replication are discussed.

View full abstract

Ribonucleic acid fractions extracted from spleens of immunized mice were shown to convert non-immune spleen cells to antibody-forming cells. RNA fractions which possessed the converting activity appeared in the spleen 3 to 5 days after immunization. Sucrose density gradient separation of crude RNA revealed that most of the active materials were in the 8 to 12S fraction and not in other fractions. In methylated-albuminKieserguhr-column chromatography, the activity was also recovered in a limited molecular size range. Specific activity of the RNA fractions was increased by purification procedures.

View full abstract

Cynomolgus monkey kidney cells were found to be a highly sensitive medium for plaque formation of Sendai virus and provided a practical method for the infectivity assay of the virus. The method gave reproducible titers and was as sensitive as the egg inoculation method and was simple enough to be used routinely. Sendai virus formed no recognizable plaques on chick embryo cell monolayers. However, the incorporation of a small amount of trypsin into the overlay medium induced plaques. This method gave comparable titers to those obtained on monkey kidney monolayers. Sendai virus formed plaques on calf kidney cell monolayers, but difficulties in the application of the method to infectivity assay arose from the fact that the plaques formed in calf kidney cells were small and not homogeneous in size and were not clearly defined.

View full abstract

Sendai virus infection induced enhancement of ³²P incorporation into phospholipids in chick embryo, monkey kidney and bovine kidney cells, as previously observed in chorioallantoic membranes of chick embryos. These findings indicate that phospholipid synthesis is enhanced upon Sendai virus infection. Ultraviolet irradiation abolished the ability of the virus to induce the enhanced synthesis of phospholipids, a fact suggesting that the phenomenon depends upon infectivity of the virus. Gamma irradiation of host cells little affected the enhanced ³²P labeling of cellular phospholipids, suggesting that the function of host cell DNA may not be directly involved in the phenomenon.

View full abstract