Immunohistochemical investigation of different cytokeratins and vimentin in the human epididymis from the fetal period up to adulthood

Summary The anatomical distribution of cytokeratins and vimentin was investigated by means of immunohistochemistry in the human epididymis. Epithelial cells of the ductuli efferentes and the corpus epididymidis were positive for cytokeratins and vimentin. The expression of epithelial vimentin decreased toward the cauda epididymidis, whereas cytokeratins remained unchanged. The epithelium of the ductus deferens was negative when antibodies against vimentin were used. With monoclonal antibodies to individual cytokeratins, the presence of cytokeratins 7, 8, 18, and 19 was demonstrated histochemically throughout the epithelium of the epididymis. Monoclonal antibodies specific for cytokeratin 17 allowed immunohistochemical differentiation between the ductuli efferentes and the ductus epididymidis.     

Effects of nucleotides and divalent cations on phospholipase activity in Saccharomyces cerevisiae

Divalent cations activate the lysophospholipase and transacylase reactions catalyzed by the same enzymes in the yeast Saccharomyces cerevisiae. The activation was observed at neutral pH, but not at the pH optimum of lysophospholipase/transacylase, near 3.5. Adenine nucleotides, especially AMP and ADP, are strong inhibitors of the same group of enzymes. Half maximal inhibition by AMP was found at a concentration of about 20 M. The inhibition by nucleotides in low concentrations is enhanced by divalent cations.     

Heparin induces changes in the synthesis of porcine aortic endothelial cell heparan sulfate proteoglycans

Heparin is known to bind to cultured endothelial cells. This report documents that addition of heparin to endothelial cells results in an alteration of the heparan sulfate proteoglycan synthetic pattern. Specifically, the addition of saturating amounts of heparin to confluent cultures of porcine aortic endothelial cells results in an increase in the amount of radiolabeled heparan sulfate proteoglycan secreted into the growth medium. The increase is apparent as early as 8 h after heparin administration. Although there is often a decrease in the amount of cell surface heparan sulfate proteoglycan produced, it is not sufficient to account for the increase in the secreted form. Of the other glycosaminoglycans tested, only dextran sulfate and commercial heparan sulfate induce changes in heparan sulfate proteoglycan synthesis and secretion. Chondroitin sulfate glycosaminoglycans do not elicit this synthetic change. These data indicate that endothelial cells can alter the synthesis of heparan sulfate proteoglycans in response to extracellular signals including heparin and related glycosaminoglycans.     

Novel Pathway of Toluene Catabolism in the Trichloroethylene-Degrading Bacterium G4

o-Cresol and 3-methylcatechol were identified as successive transitory intermediates of toluene catabolism by the trichloroethylene-degrading bacterium G4. The absence of a toluene dihydrodiol intermediate or toluene dioxygenase and toluene dihydrodiol dehydrogenase activities suggested that G4 catabolizes toluene by a unique pathway. Formation of a hybrid species of ¹⁸O- and ¹⁶O-labeled 3-methylcatechol from toluene in an atmosphere of ¹⁸O₂ and ¹⁶O₂ established that G4 catabolizes toluene by successive monooxygenations at the ortho and meta positions. Detection of trace amounts of 4-methylcatechol from toluene catabolism suggested that the initial hydroxylation of toluene was not exclusively at the ortho position. Further catabolism of 3-methylcatechol was found to proceed via catechol-2,3-dioxygenase and hydroxymuconic semialdehyde hydrolase activities.     

Influence of cyanobacterial diet on Asplanchna predation risk in Brachionus calyciflorus

Asplanchna sylvestrii does not discriminate between groups of Brachionus calyciflorus fed either the cyanobacterium Anabaena flos-aquae or a control diet of Euglena gracilis. We based our analysis on the observed probabilities of attack, capture and ingestion during encounters between predator and prey. While A. sylvestrii was very sensitive to brachionid size, we found no significant affects of prey diet on predatory behavior. Thus, cyanobacterial diet did not influence the short-term predation risk of B. calyciflorus exposed to an effective predator. On the other hand, matched cohorts of A. sylvestrii fed B. calyciflorus cultured on the cyanobacterium reproduced more slowly than those fed the same prey cultured on the control food. With prolonged sympatry, therefore, the long-term risk of Asplanchna predation may be reduced for Brachionus by the latter's consumption of cyanobacteria.     

Bioethics and academic freedom

The author describes the events surrounding his attempts to lecture on the subject of euthanasia in West Germany in June 1989. Singer, who defends the view that active euthanasia for some newborns with handicaps may be ethically permissible, had been invited to speak to professional and academic groups. Strong public protests against Singer and his topic led to the cancellation of some of his engagements, disruptions during others, and harrassment of the German academics who had invited him to speak. These incidents and the subject of euthanasia became matters of intense national debate in West Germany, but there was little public or academic support for Singer's right to be heard. Singer argues that bioethics and bioethicists must have the freedom to challenge conventional moral beliefs, and that the events in West Germany illustrate the grave danger to that freedom from religious and political intolerance.     

Preparation of polysaccharide-enzyme conjugates for competitive binding assays

A variety of bacterial O-polysaccharides were covalently linked to enzymes and it was demonstrated with three discrete monoclonal antibodies that enzyme-glycoconjugates function as convenient labelled antigens in direct enzyme immunoassays, particularly competitive assays that quantify bacterial O-antigens. Two strategies, each involving reductive amination, were used to couple O-polysaccharides to enzymes, while retaining high enzymic activity. Reduction of the Schiff base formed between, 1,3-diaminopropane and the terminal reducing ketodeoxyoctanoic acid (KDO) residue present in the majority of the lipopolysaccharide (LPS) core domains, following mild acid removal of Lipid A, offered the most direct route to mono-aminated polysaccharide. Alternatively, mild periodate oxidation of KDO and heptose residues generated multiple aldehyde targets for Schiff base formation, without affecting the O-antigenic determinant. Hetero- and homobifunctional coupling reagents, sulphosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate and discuccinimidyl suberate, activated polysaccharide for coupling to enzymes at amino and sulphydryl sites and produced conjugates that retained at least 95% of the original enzymic activity. The most suitable enzyme conjugates, especially for competitive inhibition EIA were those bearing one polysaccharide chain, and these were easily prepared from horse-radish peroxidase. Although the extent of conjugation of activated polysaccharide to -galactosidase and alkaline phosphatase could be controlled by reaction stoichiometry, the use of these enzymes was a less effective utilization of valuable antigen and enzyme.Issued as NRCC No. 31634     

Pertussis toxin inhibits CAMP-induced desensitization of adenylate cyclase in Dictyostelium discoideum

cAMP binds to surface receptors of Dictyostelium discoideum cells, transducing the signal to adenylate cyclase, guanylate cyclase and to chemotaxis. The activation of adenylate cyclase is maximal after 1 min and then declines to basal levels due to desensitization, which is composed of two components: a rapidly reversible adaptation process, and a slowly reversible down-regulation of cAMP receptors. Adaptation is correlated with receptor phosphorylation.The chemotactic response and the cAMP-induced cGMP response were not significantly altered in D. discoideum cells pretreated with pertussis toxin. The initial increase of cAMP levels was identical in control and toxin treated cells, suggesting that activation of adenylate cyclase was also not affected. However, cAMP synthesis continued in toxin treated cells, due to a strongly diminished desensitization. Pertussis toxin inhibited the adaptation of adenylate cyclase stimulation, but not the down-regulation or phosphorylation of the cAMP receptors. Adenylate cyclase in D. discoideum membranes can be stimulated or inhibited by GTP, depending on the conditions used. Pertussis toxin did not affect the stimulation of adenylate cyclase but nullified the inhibition. In membranes from desensitized control cells, stimulation of adenylate cyclase by GTP was lost, whereas inhibition was retained. Stimulation of adenylate cyclase in membranes from desensitized pertussis toxin treated cells was diminished but not absent. These results indicate that receptor phosphorylation is not sufficient for adaptation of adenylate cyclase, and that a pertussis toxin substrate, possibly Gi, is also involved in this process.Abbreviations used ATPS Adenosine 5-0-(3-Thiotriphosphate) - GTPS Guanosine 5-0-(3-thiotri-phosphate) - (Sp)-cAMPS Adenosine 3,5-monophosphorothioate-Sp-isomer - dcAMP 2-deoxyadenosine 3,5-monophosphate - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - DTT Dithiothreitol - buffer A 10 mM KH₂PO₄/Na2HPO₄, pH 6.5 - buffer B 40 mM Hepes/NaOH, 0.5 mM EDTA, 250 mM sucrose, pH 7.7