Role of nitric oxide and protein kinase C in the tachyphylaxis to vasopressin in rat aortic rings

The contribution of endothelium-derived mediators and protein kinase C in the tachyphylaxis to arginine vasopressin (AVP) was assessed in the rat aorta. Endothelium-intact (E+) and denuded rings (E-) obtained from the rat thoracic aorta were exposed to three administrations of a supramaximal concentration of AVP (100 nM), lasting 20 min and 45 min apart. N-Omega-nitro-L-arginine (NNLA), a non-selective inhibitor of all isoforms of NO synthase, and AMT, a selective inhibitor for the inducible (iNOS) and neuronal (nNOS) isoforms, diminished the tachyphylaxis to AVP significantly in both E+ and in E- rings. No iNOS could be detected by Western blots in freshly isolated rings or in rings exposed to AVP, despite a strong signal in rings isolated from LPS-treated rats, while nNOS could be constitutively detected. Inhibition of prostaglandins or epoxyeicosatrienoic acids (EETs) synthesis by diclofenac or clotrimazole, respectively, had no effect on tachyphylaxis while combination of these agents diminished tachyphylaxis in E+ only. Combination of NNLA, diclofenac and clotrimazole blocked completely the tachyphylaxis. Inhibition of PKC by either chelerythrine or bisindolylmaleimide I-HCl (BisI) led to a significant diminution of AVP tachyphylaxis only in E-. Activation of PKC with phorbol-12-myristate-13-acetate (PMA) simulated tachyphylaxis to AVP in E- only, effect blocked by the NO donor, SNP. In conclusion, NO produced from constitutive nNOS present in vascular smooth muscle cells participates in tachyphylaxis to AVP. PKC is involved in this tachyphylaxis only in E- rings, the presence of NO probably diminishing the effects of this kinase.     

KRP3A and KRP3B: candidate motors in spermatid maturation in the seminiferous epithelium

We have identified KRP3, a novel kinesin-related protein expressed in the mammalian testis, and have examined the tissue distribution and subcellular localization of isoforms of this protein. Isolation of KRP3 clones, using the head domain identified in a previous PCR screen as probe, identified at least two KRP3 isoforms in the rat. We have isolated coding sequences of two highly related cDNAs from the rat testis that we have termed KRP3A and KRP3B (kinesin-related protein 3, A and B). Both cDNAs code for predicted polypeptides with the three-domain structure typical of kinesin superfamily members; namely a conserved motor domain, a region capable of forming a limited coiled-coil secondary structure, and a globular tail domain. Although almost identical in their head and stalk domains, these motors diverge in their tail domains. This group of motors is found in many tissues and cell types. The KRP3B motor contains DNA-binding motifs and an RCC1 (regulator of chromosome condensation 1) consensus sequence in its tail domain. Despite this similarity, KRP3B is not associated with the same structures as RCC1. Instead, KRP3 isoforms localize with the nuclei of developing spermatids, and their immunolocalization in the testis overlaps with that of the small GTPase Ran. Like Ran, KRP3 motors are associated in a polarized fashion with the nucleus of maturing spermatids at various stages of elongation. Our findings suggest a possible role for KRP3 motor isoforms in spermatid maturation mediated by possible interaction with the Ran GTPase.     

L-type voltage-operated Ca(+2) channels modulate transient Ca(+2) influx triggered by activation of Sertoli cell surface L-selectin

Near the base of mammalian seminiferous epithelium, Sertoli cells are joined by tight junctions, which constitute the blood-testis barrier. Differentiating germ cells are completely enveloped by Sertoli cells and must traverse the tight junctions during spermatogenic cycle. Following the specific ligand activation of L-selectin, the up-regulated Rho family small G-proteins have been implicated as important modulators of tight junctional dynamics. Although the activation of L-selectin transmits subsequent intracellular signals in a Ca(+2)-dependent fashion in various cell types, little is understood regarding the signaling pathways utilized by L-selectin in Sertoli cells. Therefore, we have examined the possible resultant calcium influx triggered by specific ligand-activation of cell surface L-selectin receptors or by cross-linking of L-selectin with anti-L-selectin. Spectrofluorimetric studies demonstrate increase of intracellular Ca(+2) levels immediately after the treatment of the L-selectin ligands, fucoidan and sialyl Lewis-a, or after treatment with anti-L-selectin antibody. We then determined the mechanism of Ca(+2) influx by investigating L- and T-type voltage-operated Ca(+2) channels, which have been suggested to present in the membranes of Sertoli cells. Data demonstrate that Sertoli cells treated with L-type voltage-operated Ca(+2) channel antagonists, nifedipine, diltiazem, or verapamil, lead to dose-dependent blockage of L-selectin-induced Ca(+2) influx. Cells treated with mibedradil, a T-type voltage-operated Ca(+2) channel antagonist, results in little or no blocking effect. Therefore, we conclude that activation of Sertoli cell L-selectin induces Ca(+2) influx, which is at least partially regulated by L-type voltage-operated Ca(+2) channels.     

In vitro growth control of selected pathogens by Lactobacillus acidophilus- and Lactobacillus casei-fermented milk

AIMS: Food-borne pathogen inhibition was tested in the presence of a mixture of Lactobacillus acidophilus and Lactobacillus casei during fermentation under controlled pH conditions. METHODS AND RESULTS: The growth of Escherichia coli O157:H7, Salmonella serotype Typhimurium, Staphylococcus aureus, Listeria innocua, Enterococcus faecium and Enterococcus faecalis was evaluated for 48 h at 37 degrees C. In the presence of the lactic acid bacteria (LAB), an increase of the generation time was observed for all the gram-positive bacteria evaluated. Staphylococcus aureus was the most sensitive strain showing an increase of the generation time by 210%. However, for all the gram-negative bacteria evaluated, no inhibition occurred after 8 h of fermentation. The soluble portion of Lact. acidophilus- and Lact. casei-fermented milk was recuperated and tested for its antimicrobial activity. Listeria innocua and Staph. aureus were the most sensitive to the presence of fermented milk supernatant showing an inhibition of 85.9% and 84.7%, respectively. This soluble fraction was neutralized to eliminate the antimicrobial effect of the organic acids produced; the most sensitive strains were L. innocua and E. coli O157:H7 showing an inhibition of 65.9% and 61.9%, respectively. Finally, the soluble fraction was neutralized and irradiated at 45 kGy using a (60)Co source to eliminate the possible antimicrobial effect of both organic acids and bacteriocin-like substances. Enterococcus faecalis, E. coli O157:H7 and Staph. aureus were the most affected bacteria by this fraction, showing 39.1, 32 and 31.2% inhibition, respectively. CONCLUSIONS: The results obtained in this study suggest the implication of both organic acids and bacteriocin-like inhibitory substances in the antimicrobial activity observed in the soluble fraction of the probiotic preparation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed the antimicrobial mechanisms of action of Lact. acidophilus- and Lact. casei-fermented milk used to prevent antibiotic-associated diarrhoea.     

Temporal expression of membrane antigens during mouse spermatogenesis.

The temporal expression of cell surface antigens during mammalian spermatogenesis has been investigated using isolated populations of mouse germ cells. Spermatogenic cells at advanced stages of differentiation, including pachytene primary spermatocytes, round spermatids, and residual bodies of Regaud and mature spermatozoa, contain common antigenic membrane components which are not detected before the pachytene stage of the first meiotic prophase. These surface constituents are not detected on isolated populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, or leptotene and zygotene primary spermatocytes. These results have been demonstrated by immunofluorescence microscopy, by complement-mediated cytotoxicity, and by quantitative measurements of immunoglobulin (Ig) receptors on the plasma membrane of all cell populations examined. The cell surface antigens detected on germ cells are not found on mouse thymocytes, erythrocytes, or peripheral blood lymphocytes as determined by immunofluorescence and by cytotoxicity assays. Furthermore, absorption of antisera with kidney and liver tissue does not reduce the reactivity of the antibody preparations with spermatogenic cells, indicating that these antigenic determinants are specific to germ cells. This represents the first direct evidence for the ordered temporal appearance of plasma membrane antigens specific to particular classes of mouse spermatogenic cells. It appears that at late meiotic prophase, coincident with the production of pachytene primary spermatocytes, a variety of new components are inserted into the surface membranes of developing germ cells. The further identification and biochemical characterization of these constituents should facilitate an understanding of mammalian spermatogenesis at the molecular level.     

Surface-associated glycosyltransferase activities in rat sertoli cells in vitro

We have previously demonstrated fucosyltransferase (FT) activity on mouse germ cell surfaces at different stages of spermatogenesis. To complement these findings, here we report FT activity on the Sertoli cell (SC) surface. SC isolated and cultured from 20-day-old rat testes displayed FT activity with a V_max of 12.5 pmoles/mg protein/min and a K_m of 22 μM, while purified Sertoli cell plasma membranes (SCPM) showed FT activity with a V_max of 10 pmoles/mg protein/min and a K_m of 18.2 μM for GDP-[¹⁴C]-L-fucose. Fucosyltransferase activities were 16.7 and 2.6 pmoles/mg protein/min in SC and SCPM, respectively; 16% of FT activity is, therefore, on the cell surface. To test whether the expression of FT activity in SC was regulated by hormones and growth factors, SC were cultured in serum-free medium supplemented with insulin, transferrin, sodium selenite, and epidermal growth factor (medium 4F) or in 4F plus follicle-stimulating hormone, testosterone, hydrocortisone, and vitamin E (medium 8F). We found that FT activity in SC is not modulated by these hormones or growth factors (4F or 8F). For comparison with FT, galactosyltransferase (GalTase) activities in SC and SCPM were also determined. SC displayed GalTase activity with a V_max of 50 pmoles/mg protein/min and a K_m of 38.5 μM, while SCPM showed GalTase activity with a V_max of 25 pmoles/mg protein/min and a K_m of 20.8 μM for UDP-[³H]-galactose. Galactosyltransferase activities were 29.2 and 9.6 pmoles/mg protein/min in SC and SCPM, respectively. Therefore, ～33% of the total cell GalTase activity was detected on the surface membranes of rat Sertoli cells. These results suggest that cell surface glycosyltransferases may be involved in Sertoli cell function during mammalian spermatogenesis. © 1993 Wiley-Liss, Inc.     

Catabolic Gene Probe Analysis of an Aquifer Microbial Community Degrading Creosote-Related Polycyclic Aromatic and Heterocyclic Compounds

Abstract The biodegradation of a mixture of several creosote-related compounds, p-cresol, phenanthrene, fluorene, and carbazole was examined in columns containing aquifer sands. The aquifer material, itself, had an effect on the migration of the test compounds, with p-cresol being retarded the least, followed by carbazole, then fluorene, and finally phenanthrene. The biodegradation of all the compounds was greatly enhanced by the inclusion of p-cresol (10 ppm) in the substrate mixture. Associated with this enhanced degradation was a 100-fold increase in the total culturable bacterial population, and increases in the xylE- and ndoB-positive bacterial populations of more than three orders of magnitude. The products of these two genes are involved in the degradation of monocyclic and polycyclic aromatic compounds, respectively. In columns that did not receive p-cresol, there was no significant change in either the total culturable bacterial population density or the xylE-positive bacterial population, but there were significant increases of one to two orders of magnitude in the ndoB-positive bacterial populations. The results suggest that the ndoB gene probe can detect bacteria capable of utilizing phenanthrene, carbazole, and possibly fluorene. Received: 26 January 1996; Accepted: 20 June 1996     

Resistance of earthworms Lumbricus terrestris and Allolobophora turgida to the fungicide captan 50 W.P

Two species of earthworms were kept in Muck, Chicot and Ste-Sophie soils treated with captan 50 W.P. solutions of 700, 1 400 and 2 800 ppm. After a 42-day treatment period, L. terrestris had a 95% survival and A. turgida 100% survival. Using a gas chromatograph method, no captan was detected in tissue extracts of exposed earthworms. Based on the survival rate and the undetected presence of captan in earthworm tissues, we concluded that soil residues of this product are unlikely to be harmful to the animal's ecology.