Crude vesicles in which prodigiosin is localized were separated from pigmented Serratia marcescens. The bacteria were grown on peptone-glycerol agar plate, suspended in saline, and fractionated into cells, vesicles, and supernatant by differential centrifugation. Electron microscopic observations showed that the fractionation was conducted properly and the separated vesicles were lysed in distilled water. The vesicles suspended in saline retained 100 kilodalton protein of which amount is correlated with prodigiosin level, but the 100kDa protein was found in the supernatant when the vesicles were lysed in distilled water. The vesicle fraction retained few colony-forming units and little detectable activity of NADH oxidase, but showed much higher activities of protease and nuclease than the cell fraction. The profiles of the activities of the protease and the nuclease in the fractions were different from each other, that is, the protease activity in the vesicle fraction was lower than that in the supernatant fraction, whereas the nuclease activity in the vesicle fraction was higher than that in the supernatant fraction, suggesting that the two extracellular enzymes were released from the pigmented bacteria by different mechanisms.

View full abstract

We have studied the host defense factors that operate during the course of chronic respiratory tract infection caused by Klebsiella pneumoniae 27 in CBA/J mice. A large number of polymorphonuclear leukocytes (PMNs) rapidly infiltrated the alveolar spaces after infection. Treatment with cyclophosphamide (CY) before infection greatly reduced the infiltration of PMNs and caused an increase in bacterial counts. CY treatment of mice in the chronic phase also caused bacterial proliferation in the lungs. The administration of a high titer immune serum efficiently reduced the bacterial counts in the lungs during the early phase but not during the chronic phase. The proliferation of bacteria induced by CY treatment was not suppressed by the administration of the immune serum in either phase. When the mice were exposed to an aerosol containing Pseudomonas aeruginosa P9 in the chronic phase, the organisms from the secondary infection were eliminated from the lungs in the same manner as in the case of primary infection with P. aeruginosa. Thus, PMNs seem to play an important role in the suppression of bacterial proliferation in the early and chronic phases, and the specific antibody might have a supplementary effect on the defensive action of PMNs in the chronic phase. It is also presumed that the bacteria in the chronic phase of infection are sequestered at sites hardly accessible to PMNs.

View full abstract

Rickettsia was isolated from a patient with Japanese spotted fever, and its proliferation in cultured green monkey kidney cells was observed by electron microscopy. In the course of this study, we observed fusion of infected cells to uninfected cells which may be a way of spreading the rickettsiae from a cell to another. On the other hand, whirlpool-like, multilayer membranous structures, similar to the mesosomes of gram-negative bacteria, were sometimes seen in the rickettsial cells. The other profiles common to the other rickettsiae in spotted fever group were observed, such as the electron-lucent halo zone around the rickettsiae, and external fibrous materials on their surface, but intranuclear multiplication was rarely observed.

View full abstract

Gene transfer in seven pathogenic enteric bacteria was studied using an RP4::mini-Mu element, the plasmid pULB113. From the E. coli K-12 host strain the plasmid could be efficiently transferred to these enteric bacteria, but its transfer back to E. coli K-12 was not as efficient, being detected only in Shigella dysenteriae 1, S. flexneri and the ‘smooth’ variant of S. sonnei. In these three species, transposition of chromosomal fragments into the plasmid to produce R-prime plasmid was also detected at a frequency of approximately 10^-5. Transposition was random as suggested by the recovery at approximately the same frequency (10^-5 to 10^-6) of R-primes involving 20 different auxotrophic markers from widely separated chromosomal locations. Formation of R-prime plasmids expressing toxicity in the E. coli K-12 recipient strain was also efficient in S. dysenteriae 1 but the toxin-activity was rapidly lost from these R-primes. In our experiments, the plasmid pULB113 incorporated relatively small amounts of chromosomal DNA as determined by restriction endonuclease digestion. For a Thy⁺ R-prime that we analyzed, the amount of cloned DNA was approximately 15kb.

View full abstract

The latent MDV (Marek's disease virus) genomes are folded into nucleosomal structures in both virus-productive and -nonproductive lymphoblastoid cell lines, MDCC-MSB1 (MSB-1) and -RP1 (RP-1), respectively. There was no difference between transcriptionally active and inactive regions of MDV genome with regard to nucleosomal patterns. In order to investigate whether nucleosomal structure is correlated with the repression of the transcription of MDV genome in lymphoblastoid cells, we examined the DNaseI sensitivity of nucleosomal MDV DNA in lymphoblastoid cell lines, MSB-1 and RP-1. No difference in the DNaseI sensitivity between transcriptionally active and inactive MDV DNA regions in lymphoblastoid cells was observed.

View full abstract

The proliferative response of peripheral blood mononuclear cells (PBMC) from healthy adults to human herpesvirus-6 (HHV-6) was examined. It was found that PBMC from 13 of 14 HHV-6-seropositive adults apparently proliferated in response to stimulation with HHV-6 antigen in contrast to the lack of response of cord blood mononuclear cells. In order to confirm the presence of HHV-6-specific memory T cells in the peripheral blood of healthy adults, we established HHV-6-specific T-cell clones from an HHV-6-seropositive individual. CD4⁺ T-cell clones generated from HHV-6-stimulated PBMC were found to proliferate upon stimulation with HHV-6 in the presence of autologous antigen-presenting cells, but not in response to herpes simplex virus type 1 antigen or mock-infected control antigen. These results indicate that a T-cell immune response against HHV-6 infection is generally present in healthy adult populations.

View full abstract

Relative ability of distinct isotypes of human major histocompatibility complex class II molecules to bind staphylococcal enterotoxin A (SEA) was investigated. SEA-binding was observed in L cells transfected with DR2 and DQw1 genes. By contrast, it was not detected in L cells transfected with DPw4 and DP (Cp63) genes. All the transfectants supported SEA-induced IL-2 production by human T cells. Levels of the accessory activity were low in the DPw4 and DP (Cp63) transfectants compared with the DR2 and DQw1 transfectants. In view of the observation that all the transfectants express well the transfected gene products on their surface, the results indicate that DR and DQ molecules bind SEA with high affinity, while DP molecules bind it with fairly low affinity.

View full abstract

The chemical structure of the 2-keto-3-deoxyoctonate (KDO) region of the lipopolysaccharide (LPS) isolated from O1 V. cholerae NIH 41R (Ogawa) was elucidated by dephosphorylation, periodate oxidation and methylation analysis. Methylation analysis of KDO in the dephosphorylated LPS revealed the presence of 5-O-acetyl-1, 2, 4, 6, 7, 8-hexa-O-methyl-3-deoxy-octitol and 2-keto-3-deoxy-heptulosonic acid was detected in the methanolysate of the periodate-oxidized and dephosphorylated LPS. These results indicated that the site of binding of KDO to the core oligosaccharide is position C5 as in enteric gram-negative bacterial LPS, while only one molecule of the KDO residue carrying phosphate on position C4 is present in the inner core region of the LPS in contrast to enteric gram-negative bacterial LPS in which one molecule of KDO carrying KDO or KDO2→4KDO disaccharide instead of the phosphate group at position C4 is present in its main chain.

View full abstract

A herpes simplex virus ribonucleotide reductase (RR) null mutant, ICP6Δ, exhibited hypersensitivity to hydroxyurea, and to the precursors of allosteric inhibitors of cellular RR. The mutant was also much more sensitive than the parental KOS to all of antiviral nucleoside analogs tested, including acyclovir (ACV), ganciclovir (DHPG) and BVaraU. Our data indicate that cellular RR is essential for the growth of ICP6 Δ, and suggest that inhibitors of viral RR could act as potentiators of all of anti-herpetic nucleoside analogs whose targets are viral DNA polymerase.

View full abstract