Experimental Salmonellosis VIII. Postinfective Immunity and Its Significance for Conferring Cellular Immunity

In the process of live-vaccine immunization of Salmonella enteritidis infection in mice, the relation between the number of bacteria in the organs of mice and their protecting effect was studied. Treatment with antibiotics was used to control the number of immunizing bacteria in the tissues. Mice, which were infected with 10(-5) mg (1,000 mouse MLD) of virulent S. enteritidis and treated with kanamycin simultaneously, acquired high antilethal resistance against infection with the same organisms. However, the administration of large amounts of kanamycin, which caused a rapid decrease in bacterial numbers in the organs of infected mice, was incapable of conferring immunity. This indicated the necessity of persistence of live bacteria in the host for the production of immunity. A large number of microorganisms were maintained for 53 weeks in a diffusion chamber inserted into the mouse abdominal cavity. The mice implanted with diffusion chambers containing large numbers of virulent S. enteritidis did not acquire antilethal resistance against infection with the same organisms, although agglutinins against S. enteritidis were observed in these mice. Agglutinin was also found in the fluid contained in diffusion chambers inserted into mice immunized with a killed vaccine of S. enteritidis. This indicated that antibody penetrated the membrane filter of diffusion chambers from outside to inside and vice versa. From these results, it is suggested that contact of live microorganisms with the host cell is necessary for conferring postinfective immunity in salmonellosis.     

Experimental Salmonellosis X. Cellular Immunity and Its Antibody in Mouse Mononuclear Phagocytes

A cell-associated antibody was detected in the peritoneal mononuclear phagocytes (referred to as monocytes) of mice hyperimmunized with live vaccine of Salmonella enteritidis, by use of immune transfer and immune adherence hemagglutination techniques. The cellular antibody inhibited the growth of a virulent strain of S. enteritidis with the aid of complement and lysozyme on nutrient agar plates. This type of bactericidal antibody could not be detected in the monocytes of mice immunized with killed vaccine of S. enteritidis. The antibody extracted from the peritoneal monocytes of mice hyperimmunized with live vaccine was identified as a macroglobulin by ultracentrifugal analysis.     

The actin domain of Gardner-Rasheed feline sarcoma virus inhibits kinase and transforming activities.

The transforming gene product, P70gag-actin-fgr, of Gardner-Rasheed feline sarcoma virus (GR-FeSV) is a single polypeptide composed of regions derived from cellular and viral genes. Gamma actin and c-fgr genes are the two known cellular components of the GR-FeSV genome. In the present study, sequences representing each cell-derived gene were deleted and the resulting constructs were tested for transforming activity by transfection of NIH 3T3 cells. Constructs lacking a portion of the c-fgr proto-oncogene failed to induce focus formation, demonstrating the essential nature of this component for GR-FeSV oncogenic activity. In contrast, the construct lacking the actin domain was more active than GR-FeSV DNA in transformation assays. Protein specified by the actin deletion mutant possessed a 2.4-fold greater specific protein-tyrosine kinase activity compared with that of the wild-type gene product. Furthermore, the actin domain had no detectable effect on the ability of the fgr kinase to associate with cytoskeleton or to phosphorylate unique cellular proteins on tyrosine. Our findings demonstrate that the actin domain inhibits focus formation and impairs protein-tyrosine kinase activity.     

Structure, function and expression of a murine homeobox protein AREC3, a homologue of Drosophila sine oculis gene product, and implication in development.

The cDNA clones encoding ARE(Na,K-ATPase alpha1 subunit gene regulatory element) binding protein AREC3 were isolated from myoblast C2C12 cells and mouse skeletal muscle cDNA library. At least four alternatively spliced forms of AREC3 cDNA were identified. Sequence analysis indicates that AREC3 has an extensive homology with the Drosophila sine oculis gene product required for development of the entire visual system [Cheyette et al.(1994) Neuron 12, 977-996]. The homologous region including a homeodomain is required for specific DNA binding to ARE. A transactivation domain was identified in the C-terminal part of the AREC3 by reporter gene assays using GAL4-AREC3 fusion protein constructs. Immunohistochemistry revealed that AREC3 localized to the nucleus and cytoplasm of myoblast C2C12 cells, and the production of AREC3 is augmented during muscle differentiation. Western blot analysis indicated that the 115 kDa form of AREC3 protein is increased in the cytoplasmic extract, and the 67kDa form is increased both in nuclear and cytoplasmic extracts of C2C12 cells during muscle differentiation.     

Molecular cloning of a chloroplastic proteinassociated with both the envelope and thylakoid membranes

Chloroplasts consist of six morphologically distinct compartments. Each compartment has a specific set of polypeptides that perform distinct biochemical functions. We report here the identification of a membrane-associated protein with a novel localization. This protein was synthesized as a 37 kDa precursor and was processed to a mature protein of 30 kDa after being imported into isolated pea chloroplasts. Fractionation of chloroplasts showed that the 30 kDa mature protein was associated with both of the envelope membranes as well as with thylakoid membranes. Immunocyto-chemical localization of the 30 kDa protein revealed that the protein occurred in clusters in the vicinity of both the envelope and the thylakoid. Possible functions of this 30 kDa protein, inferred from its novel localization pattern, are discussed.Abbreviations CAB light-harvesting chlorophyll a/b-binding protein of photosystem II - prCAB precursor protein to CAB - SS small subunit of ribulose-1,5-bisphosphate carboxylase - prSS precursor protein to SS - RCF relative centrifugation force     

Anti-bacterial antibodies in Epstein-Barr virus (EBV)-transformed oligoclonal B-cell lines established from normal persons and autoimmune disease patients

We have established 950 and 430 oligoclonal B-lymphoblastoid cell lines (LCL) from two normal persons and eight autoimmune disease patients, respectively by using Epstein-Barr virus (EBV)-induced transformation. To re-evaluate the EBV technique for production of human monoclonal antibodies (mAb) related to infectious disease, we screened these oligoclonal LCLs for antibodies against 31 bacterial strains systematically. A total of 74 cultures out of 1380 were reactive to a total of 18 strains out of 31. Among these, eight cultures showed 10^-3 antibody (Ab) titers to Pseudomonas aeruginosa serotypes C, E, F and I, Staphylococcus aureus, Serratia marcescens and Bacillus cereus. Ten cultures showed 10^-2 Ab titers to Ps. aeruginosa serotypes D, E, F and I, Ps. maltophilia, Staph, epidermidis, Klebsiella ozaenae, Ser. marcescens and B. subtilis. The results reveal the further possibilities for the EBV technique to produce various infectious disease-related human mAbs.