Progesterone promotes the acrosome reaction in capacitated human spermatozoa as judged by flow cytometry and CD46 staining.
(17/2363)
The acrosome reaction is a necessary prerequisite for spermatozoa to acquire fertilizing ability. Several different moieties appear to promote the acrosome reaction through different pathways, including solubilized zona pellucidae, recombinant zona protein ZP3, follicular fluid, calcium ionophores, and mannosylated bovine serum albumin (BSA). Although many investigators have presented evidence that progesterone also promotes the acrosome reaction through the mediation of a non-genomic cell membrane receptor, this concept has been challenged. Other workers have suggested that progesterone does not promote an acrosome reaction in human spermatozoa, as judged by the detection of CD46, a complement regulatory protein present on the inner acrosome membrane, through flow cytometric analysis of large numbers of spermatozoa. Prior investigations were criticized by the limited numbers of spermatozoa enumerated visually, the use of non-specific staining techniques, and the failure to eliminate dead spermatozoa during the scoring of the acrosome reaction. We have repeated these experiments, using both a supravital dye to eliminate dead spermatozoa from flow cytometric analysis, and anti-CD46 monoclonal antibody to score acrosome-reacted spermatozoa. Care was taken to validate the adequacy of capacitation conditions, which were proven by the ability of spermatozoa to acrosome react in response to mannosylated BSA and to penetrate zona-free hamster eggs. Confocal microscopy was used to confirm that CD46 immunostaining was limited to the acrosomal region of the spermatozoon head. Our results indicate that progesterone does promote an acrosome reaction within capacitated spermatozoa. (+info)
Interference of nucleoside diphosphate derivatives of 2-deoxy-D-glucose with the glycosylation of virus-specific glycoproteins in vivo.
(18/2363)
The predominant effect of 2-deoxy-D-glucose on chick embryo cells infected with Semliki Forest virus is an interference with glycosylation of virus-specific glycoproteins; this results in a block of synthesis of infectious virus. Incorporation of radioactive mannose is blocked severely in the presence of 2-deoxyglucose in the cultural medium although it is readily phosphorylated and subsequently activated by GTP to yield GDP-mannose, which accumulates under these conditions. The intracellular concentrations of GDP-mannose and UDP-N-acetyl-D-hexosamine are not reduced in the presence of the inhibitor. An equimolar concentration of mannose in the cultural medium competes with the inhibitory effect of the deoxysugar and drops the cellular pool of GDP-2-deoxy-D-glucose below the level of detection, at the same time restoring the synthesis of infectious virus. When the intracellular concentration of UDP-2-deoxyglucose is reduced by addition of glucose into the cultural medium the inhibition of virus synthesis by the deoxysugar and the concentration of GDP-2-deoxyglucose within the cells remain near to the values when the inhibitor is present alone. It is concluded that among the metabolites of 2-deoxyglucose which occur in vivo after addition of 2-deoxyglucose to the culture medium, GDP-2-deoxyglucose is the agent responsible for inhibition of glycosylation of viral glycoproteins. (+info)
Role of wall phosphomannan in flocculation of Saccharomyces cerevisiae.
(19/2363)
Treatment with 60% hydrofluoric acid (HF) removed most of the phosphorus and small amounts of mannan, glucan and protein from walls of two non-flocculent strains (NCYC366 and NCYC1004) and two flocculent strains (NCYC1005 and NCYC1063) of Saccharomyces cerevisiae. Organisms of all strains showed increased flocculating ability following HF treatment. Flocculation of untreated organisms of NCYC1005 and NCYC1063, and of HF-treated organisms of all four strains, declined appreciably when they were washed in deionized water, with or without EDTA, and the flocculation was measured in deionized water instead of in 0-05 M-sodium acetate containing Ca2+. Treatment with 1,2-epoxypropane also caused a decrease in the flocculating ability of these organisms. Extracting the lipids from organisms of strains NCYC366 and NCYC1004 had no effect on their flocculating ability, but decreased the flocculating ability of organisms of strains NCYC1005 and NCYC1063. pH-electrophoretic mobility curves of untreated and HF-treated organisms confirmed the loss of wall phosphate by HF treatment, and indicated that HF treatment had little effect on the content of protein carboxyl groups in the outer wall layers. Mannose at 0-22 M completely prevented floc formation by organisms of strain NCYC1063; but, even at 0-33 M, it had very little effect on floc formation by HF-treated organisms of strains NCYC366 and NCYC1063. Organisms of all four strains bound fluorescein-conjugated concanavalin A to the same extent after treatment with HF as before, but this treatment led to a greatly diminished binding of of fluorescein-conjugated antiserum raised against organisms of strain NCYC366. The results indicate that phosphodiester linkages in yeast-wall mannan are not involved in bride formation through Ca2+ during floc formation and that this arises principally through carboxyl groups. (+info)
A pyruvated mannose-specific xanthan lyase involved in xanthan degradation by Paenibacillus alginolyticus XL-1.
(20/2363)
The xanthan-degrading bacterium Paenibacillus alginolyticus XL-1, isolated from soil, degrades approximately 28% of the xanthan molecule and appears to leave the backbone intact. Several xanthan-degrading enzymes were excreted during growth on xanthan, including xanthan lyase. Xanthan lyase production was induced by xanthan and inhibited by glucose and low-molecular-weight enzymatic degradation products from xanthan. A xanthan lyase with a molecular mass of 85 kDa and a pI of 7.9 was purified and characterized. The enzyme is specific for pyruvated mannosyl side chain residues and optimally active at pH 6.0 and 55 degrees C. (+info)
A role for the mannose-sensitive hemagglutinin in biofilm formation by Vibrio cholerae El Tor.
(21/2363)
While much has been learned regarding the genetic basis of host-pathogen interactions, less is known about the molecular basis of a pathogen's survival in the environment. Biofilm formation on abiotic surfaces represents a survival strategy utilized by many microbes. Here it is shown that Vibrio cholerae El Tor does not use the virulence-associated toxin-coregulated pilus to form biofilms on borosilicate but rather uses the mannose-sensitive hemagglutinin (MSHA) pilus, which plays no role in pathogenicity. In contrast, attachment of V. cholerae to chitin is shown to be independent of the MSHA pilus, suggesting divergent pathways for biofilm formation on nutritive and nonnutritive abiotic surfaces. (+info)
Physicochemical characterization of Cajanus cajan lectin: effect of pH and metal ions on lectin carbohydrate interaction.
(22/2363)
The association constant of Cajanus cajan lectin for methyl alpha-D-mannopyranoside was studied by equilibrium dialysis method. An attempt was also made to understand the metal ion requirements and to establish that ionizable groups are responsible for lectin-carbohydrate interaction. The N-terminal sequence up to 27 amino acid residues was found to be more than 80% homologous with other mannose-specific legume lectins of the tribe Viceae. Like concanavalin A and pea lectin it also exhibits high affinity for the sugar alpha-methyl mannose and at 37 degrees C the association constant was found to be 1.4x104 M-1. The lectin required one Ca2+ and one Mg2+ per mole and during the lectin sugar interaction two ionizable groups with pK of 3.75 and 8.3 are ionized. Whether the secondary structure is similarly affected with pH changes and presence or absence of metal ion was investigated by circular dichroism studies. Results suggested that changes in carbohydrate binding properties of the Cajanus cajan lectin due to change in pH and addition of metal ions are not accompanied by any significant change in secondary structure. (+info)
Glucocorticoids affect human dendritic cell differentiation and maturation.
(23/2363)
Because dendritic cells (DC) play a major role in the initiation of T cell-mediated immunity, we studied the effects of glucocorticoids, well-known inhibitors of the immune and inflammatory response, on the differentiation and maturation of human DC. DC were differentiated from human monocytes by culture with GM-CSF and IL-4 for 7 days with and without dexamethasone (Dex). Cells treated with Dex (10-8 M) (Dex-DC) developed a characteristic dendritic morphology; however, membrane phenotype analysis demonstrated that they were not fully differentiated. Dex-DC expressed low levels of CD1a and, unlike untreated cells, high levels of CD14 and CD16. Molecules involved in Ag presentation (CD40, CD86, CD54) were also impaired. In contrast, molecules involved in Ag uptake (mannose receptor, CD32) and cell adhesion (CD11/CD18, CD54) were up-regulated. After exposure to TNF-alpha or CD40 ligand, Dex-DC expressed lower levels of CD83 and CD86 than untreated cells. Dex-DC showed a higher endocytic activity, a lower APC function, and a lower capacity to secrete cytokines than untreated cells. Overall, these results indicate that DC differentiated in the presence of Dex are at a more immature stage. Moreover, Dex also partially blocked terminal maturation of already differentiated DC. In conclusion, our data suggest that glucocorticoids may act at the very first step of the immune response by modulating DC differentiation, maturation, and function. (+info)
Phosphorylation of arylsulphatase A occurs through multiple interactions with the UDP-N-acetylglucosamine-1-phosphotransferase proximal and distal to its retrieval site by the KDEL receptor.
(24/2363)
Phosphorylation of oligosaccharides of the lysosomal enzyme arylsulphatase A (ASA), which accumulate in the secretions of cells that mis-sort most of the newly synthesized lysosomal enzymes due to a deficiency of mannose 6-phosphate receptors, was found to be site specific. ASA residing within the secretory route of these cells contains about one third of the incorporated [2-3H]mannose in phosphorylated oligosaccharides. Oligosaccharides carrying two phosphate groups are almost 2-fold less frequent than those with one phosphate group and only a few of the phosphate groups are uncovered. Addition of a KDEL (Lys-Asp-Glu-Leu) retention signal prolongs the residence time of ASA within the secretory route 6-fold, but does not result in more efficient phosphorylation. In contrast, more than 90% of the [2-3H]mannose incorporated into secreted ASA (with or without a KDEL retention signal) is present in phosphorylated oligosaccharides. Those with two phosphate groups are almost twice as frequent as those with one phosphate group and most of the phosphate groups are uncovered. Thus, ASA receives N-acetylglucosamine 1-phosphate groups in a sequential manner at two or more sites located within the secretory route proximal and distal to the site where ASA is retrieved by the KDEL receptor, i.e. proximal to the trans-Golgi. At each of these sites up to two N-acetylglucosamine 1-phosphate groups can be added to a single oligosaccharide. Of several drugs known to inhibit transit of ASA through the secretory route only the ionophore monensin had a major inhibitory effect on phosphorylation, uncovering and sialylation. (+info)