Permeable collagen-glycosaminoglycan cross-linked copolymers for the study of biological responses of coculturede Sertoli and spermatogenic cells

A novel collagen-glycosaminoglycan (C-GAG) substrate was developed to overcome the optical opacity of a HATF nitrocellulose substrate and to provide a more physiological permeable substrate for cocultured Sertoli and spermatogenic cells. Cocultures were prepared on optically transparent C-GAG discs attached to a polyester mesh to facilitate handling. Sertoli cells displayed a cuboidal-to-columnar shape; a large number of spermatogonia and primary spermatocytes connected by intercellular bridges were associated with basolateral and apical surfaces of Sertoli cells up to 12 days after plating. Rat Sertoli-spermatogenic cell cocultures have been used for testing the effect of toxicants on rat spermatogenesis in vitro. In our initial studies, we tested the effects of the toxicant gossypol on spermatogenic cells cocultured with Sertoli cells on nonpermeable (plastic) and permeable substrates (HATF nitrocellulose) under both standard culture conditions and during perifusion after achieving a continuous electrical-resistant cell monolayer. A selective mitochondrial structural damage was observed in spermatogenic cells (spermatogonia and spermatocytes) but not in the coexisting Sertoli cells. This damage was time- (15–60 min) and dose-dependent (0.1–10µM) and developed more rapidly under perifusion conditions. Similar mitochondrial damage was reported in the intact animal but required higher concentrations (mg) and longer administration time (months) for detection. Studies are in progress to evaluate the effect of additional toxic chemical agents on functional properties of Sertoli and spermatogenic cells in cocultures prepared on various classes of C-GAG substrates.Abbreviations C-GAG, collagen type I-glycosaminoglycans - C-C6S, collagen type I-chondroitin-6-sulfate - C-H, collagen type I-heparin     

Regulation of T cell receptor signaling by a src family protein-tyrosine kinase (p59fyn)

Engagement of the clonotypic antigen receptor (TCR) on T lymphocytes provokes an activation response leading to cell proliferation and lymphokine secretion. To examine the molecular basis of T cell signaling, we generated transgenic animals in which a lymphocyte-specific nonreceptor protein-tyrosine kinase p59fyn(T) is 20-fold overexpressed in developing T lineage cells. Thymocytes from these mice, analyzed using both cellular and biochemical assays, were remarkably hyperstimulable. Moreover, the responsiveness of normal thymocytes to TCR-derived signals correlated well with the extent to which p59fyn was expressed in these cells. Overexpression of a catalytically inactive form of p59fyn substantially inhibited TCR-mediated activation in otherwise normal thymocytes. These effects are unique to p59fyn; overexpression of a closely related T cell-specific tyrosine kinase, p56lck, elicits dramatically different phenotypes. Our results suggest that p59fyn is a critically important component of the TCR signal transduction apparatus.     

Dirofilaria immitis: surface properties of third- and fourth-stage larvae

The objective of this study was to analyze surface properties of larval Dirofilaria immitis with potential relevance to protective immunity. Comparisons were made between third (L3)- and fourth-stage larvae (L4) based on their net surface charge, surface carbohydrate and antigen composition, ability to nonspecifically absorb host proteins, complement activation, and nonspecific cellular adherence. It was determined that L3 had a net negative surface charge, whereas L4 had either a neutral or weakly positive surface charge. The lectin Con A, but not any of the other lectins tested, bound only to the surface of L4, and not to that of L3. Monoclonal antibodies were prepared which reacted with the surface of L3 or with the surface of L4, but never both. L4 were found to nonspecifically adsorb host protein to their surfaces, whereas L3 did not. Both L3 and L4 were found to activate complement through the alternate pathway. Finally, nonspecific cellular adherence was found on L3 both in vitro and in vivo but not on L4. The surfaces of L3 and L4 were thus shown to be significantly different and, potentially, in ways which would have great impact in the generation and effectiveness of a protective immune response.     

Forms and intracellular distribution of alpha-D-mannosidases in murine liver and spleen

1. The intracellular distribution of alpha-D-mannosidase in homogenates of murine liver and spleen was investigated by differential and gradient density centrifugation. 2. In both tissues an enzyme with a neutral pH optimum was found in the cytosol together with an alpha-D-mannosidase with optimal activity between pH 5.5 and 6.0 which was also partially membrane-bound. 3. In liver the acidic alpha-D-mannosidase was obtained almost entirely in a particulate form distributed equally between a heterogeneous low density region and heavy density lysosomes. 4. The lysosomal form of the liver enzyme was purified to electrophoretic homogeneity and shown to be a glycoprotein composed of four identical subunits of molecular weight 65 kDa. 5. Antibody raised against the purified liver alpha-D-mannosidase immunoprecipitated a polypeptide from spleen which had the same molecular size. This acidic enzyme was the predominant type of alpha-D-mannosidase in spleen, but in contrast to liver, it was obtained mainly in a cytosoluble form, the remaining activity being present in the heterogeneous light density compartment. 6. Although both tissues contain the same molecular form of the acidic alpha-D-mannosidase, in murine spleen this enzyme does not appear to be associated with stable heavy density lysosomes.     

Expression and secretion of biologically active human atrial natriuretic peptide in Saccharomyces cerevisiae

A hybrid gene was constructed containing a fusion between the DNA sequences encoding the secretory precursor of the yeast mating pheromone alpha-factor and a synthetic sequence encoding a biologically active 24-amino acid carboxyl-terminal portion of the human atrial natriuretic peptide (hANP) precursor. Transformation of Saccharomyces cerevisiae with the hybrid gene resulted in the yeast cells secreting biologically active hANP into the extracellular medium. The secreted hANP was purified and found to be accurately processed at the junction in the chimeric alpha-factor/hANP protein, producing the desired mature hANP amino terminus. The secreted product was also folded correctly with respect to the single disulfide bond. However, the carboxyl terminus of the secreted hANP material was heterogeneous such that the major form lacked the last two amino acids of the peptide while the minor form was the full length material. The observed processing at the carboxyl terminus of the secreted hANP may reflect a normal processing event involved in alpha-factor peptide maturation.     

Differences in airway responsiveness to leukotriene D4 in allergic sheep with and without late bronchial responses

Allergic sheep respond to inhaled antigen with either acute and late bronchial obstructions (dual responders) or only acute bronchoconstriction (acute responders). In this study we tested the hypothesis that one factor which may distinguish between these two populations is the difference in sensitivity to a specific mediator of airway anaphylaxis, leukotriene (LT) D₄ (a major component of slow reacting substance of anaphylaxis). We postulated that if the hypothesis was correct than dual responders should demonstrate increased airway responses to inhaled LTD₄ and that this increased responsiveness should also be reflected by a more severe response to inhaled antigen. To test this we used animals from both groups with the same degree of non-specific airway responsiveness to carbachol and determined their airway responses to controlled inhlation challenges with synthetic LTD₄ and antigen. Airway responsiveness to carbachol was determined by measuring the change in specific lung resistance (SR_L) to increasing concentrations of carbachol aerosol, and then identifying, by linear interpolation, the provocative carbachol concentration which produced a 150% increase (PC₁₅₀) in SR_L. Airway responses to LTD₄, and antigen were determined by measuring the percentage change in SR_L after a controlled inhlation challenge with either aerosol. Airway responsiveness to carbachol was not different between the two groups. There was, however, a difference (p<0.05) in the airway response to the same dose of LTD₄ in the two groups. Dual responders showed a 297±72% increase in SR_L as compared to a 90±13% increase in SR_L in the acute responders. Dual responders also showed a greater immediate and more prolonged response to antigen than did acute responders. These results suggest that increased responsiveness to LTD₄ may be one factor which may distinguish dual responders from acute responders.     

Suppression of defective-sporulation phenotypes by mutations in the major sigma factor gene (rpoD) of Bacillus subtilis

Summary Mutations (crsA47 and crsA4) in the major sigma factor gene (rpoD) of Bacillus subtilis RNA polymerase have been found to be powerful intergenic suppressors of spoOB, spoOE, spoOF, spoOK and spoIIG mutations. The crsA47 suppressor restores sporulation of spoOE, spoOF, spoOK and spoIIG mutants to levels near those of wild type bacteria and substantially improves the sporulation of a spoOB strain. The crsA mutations are shown to prevent the induction by aliphatic alcohols of SpoO phenocopies in wild type B. subtilis cells.     

Molecular analysis of transferable tetracycline resistance plasmids from Clostridium perfringens.

Conjugative tetracycline resistance plasmids from 15 Clostridium perfringens isolates from piggeries were analyzed by restriction endonuclease digestion and agarose gel electrophoresis. Seven isolates from one farm were found to carry a 47-kilobase pair (kb) plasmid, pJIR5, which had EcoRI, XbaI, and ClaI profiles that were identical to those of a previously characterized plasmid, pCW3. An isolate from a second farm was found to carry a plasmid, pJIR6, which also was indistinguishable from pCW3. Five additional isolates from a third farm carried a 67-kb plasmid, pJIR2, which had at least 29 kb of DNA in common with pCW3. Finally, two isolates from a fourth farm were found to carry a 50-kb plasmid pJIR4, which appeared to consist of an entire pCW3 molecule with a 3-kb insertion. Comparative restriction maps of pCW3, pJIR2, and pJIR4 that identified the regions of homology among these plasmids were constructed. We suggest that many conjugative tetracycline resistance plasmids in C. perfringens may contain a pCW3-like core.