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Query: UMLS:C0272170 (SDS)
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Antibodies reacting with human spermatozoa have been detected by various immunological techniques in the sera of subfertile men. Different patterns of sperm agglutination are observed with different sera, either head-to-head, tail-to-tail, or tail-tip-to-tail-tip. Differences have been detected between the clinically relevant antibodies in spontaneously infertile males and the less important antibodies in males who have undergone reversal of vasectomy. It has been suggested that the variations in agglutination patterns are due either to different classes of antibody or to binding of antibody to different antigens. In the present study immunoblotting techniques were used to characterize the reactivity of solubilized sperm proteins with serum samples exhibiting different modes of sperm agglutination. This involved the electrophoretic transfer of proteins from SDS gels to nitrocellulose sheets followed by overlay with serum antibody. Using these techniques we have attempted to characterize the antigens of spermatozoa which react with sera from both spontaneously infertile and vasovasostomized men. The results showed that although antisperm antibodies bind to discrete and sperm-associated antigens, there is no substantial difference between the antigenic patterns observed with antibodies producing different types of sperm agglutination. Neither the antigens detected, nor the intensity of reaction showed significant differences although there was a tendency for head-to-head agglutinating antibodies to react more strongly with the higher molecular weight antigens. Moreover, although with sequential serum samples the patterns of agglutination may change, the antigenic pattern remains unchanged.
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PMID:Characterization of sperm antigens reacting with human antisperm antibodies. 244 89

Washed ejaculated human spermatozoa were surface labelled with 125I, using solid phase (iodogen) or enzymic (lactoperoxidase) methods, while membrane components possessing terminal galactose or galactosamine residues were labelled with the galactose oxidase-sodium [3H]borohydride technique. All three procedures revealed the presence of 2 major labelled surface components. The first comprised a broad band of radioactivity migrating just behind the ion front on SDS-PAGE, which could be extracted with chloroform and methanol, suggesting a lipid-like composition. The second fraction exhibited properties consistent with a major glycoprotein component of the human sperm plasma membrane, giving a peak of radioactivity with Mr = 20,000, within which a discrete doublet of bands (Mr = 17,000 and 19,000) could be resolved by autoradiography. A more detailed analysis of the labelled protein fraction after TCA precipitation revealed a number of other surface components, the major ones of which exhibited Mr values of 30,000, 45,000, 66,000, 115,000 and 160,000. Western blot analysis was then used to determine whether any of the surface components described above interacted with the gamma-globulin fraction of antisera obtained from patients exhibiting idiopathic autoimmunity against sperm antigens. Using a purified membrane preparation as the target, antibodies were detected against numerous high molecular weight bands with Mr values similar to the major components of the human sperm surface (35,000, 45,000, 66,000, 90,000 and 150,000). The nature of the antigens targeted by these antisera did not correlate with the ability of the latter to stimulate or suppress sperm-oocyte fusion.
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PMID:Analysis of the surface labelling characteristics of human spermatozoa and the interaction with anti-sperm antibodies. 244 96

An antiserum to the purified porcine outer acrosomal membrane (OAM) was raised in rabbits and the IgG fraction isolated by ammonium sulphate precipitation and ion exchange chromatography. The antibodies reacted exclusively with the acrosomal cap of the sperm head as revealed by indirect immunofluorescence. In addition they cross-reacted not only with the acrosomal part of the spermatozoa of all mammalian species tested (bull, horse, rabbit, rat, mouse, hamster, mole, antelope, monkey, man) but also with the spermatozoa of the cock (Class: birds) and the rainbow trout (Class: fish). All the species exhibited similar development of the acrosomal cap during spermatogenesis, with the appearance of the immunofluorescent stain in early round spermatids. In the mole the localization of the acrosome in elongated testicular spermatids differed from that in all other species: Instead of prominent fluorescence over the apical part of the sperm an equatorial belt was formed. The cross-reactivity of the anti-boar OAM antibody with the acrosomes of different vertebrate species at the morphological level was supported by the results of Western blotting experiments with purified boar OAM proteins and the SDS-extractable proteins of bull and human spermatozoa, respectively. Using anti-OAM antibodies and antibodies against the acrosin inhibitors I and II described recently by Tschesche et al. (1982), in absorption and Western blotting experiments, it was demonstrated that the acrosin inhibitor proteins are integrated in the outer acrosomal membrane.
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PMID:Evolution and development of the outer acrosomal membrane (OAM) and evidence that acrosin-inhibitors are proteins of the OAM. 244 33

Ejaculated human spermatozoa were studied to assess their nuclear maturity. After SDS or SDS-EDTA treatment, asthenozoospermic semen had a lower resistance to decondensation than normozoospermic semen and contained more stained immature nuclei after aniline blue staining. It showed a higher uptake of ethidium bromide, specific for DNA. There was no difference in the binding of 14C iodoacetamide in the two groups. Therefore, asthenozoospermic semen could be characterized by its relative nuclear immaturity.
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PMID:Human spermatozoal nuclear maturity in normozoospermia and asthenozoospermia. 246 2

We investigated the effect of salt on the fluorescence staining procedure for quantification of the amount of DNA in cell nuclei in situ. For this, NaCl was added at various concentrations to the Hoechst 33258 fluorochrome (Hoe) medium for staining DNA. The fluorescence intensity of free DNA-Hoe solution was not changed by the addition of NaCl, but that of the nuclei-Hoe complex in situ increased 4-fold on increasing the NaCl concentration up to 1 M. SDS polyacrylamide gel electrophoresis showed that histones H1, H2A, and H2B dissociated from cell nuclei in the presence of 1 M NaCl, resulting in increasing accessibility of DNA to the fluorochrome. The applicability of the NaCl-aided fluorescence staining method was evaluated by measuring the ploidy classes of various cells. The amount of DNA in spermatozoa is half that in 2 n hepatocytes, but by the conventional Hoe staining procedure the fluorescence intensity of spermatozoa is higher than that of 2 n hepatocytes, due to differences in accessibility of the dye to DNA. In contrast, by the NaCl-aided procedure, the fluorescence intensity of 2 n hepatocytes was twice that of spermatozoa. The effectiveness of the NaCl-aided Hoe staining method was checked using cultivated human gingival cells and hepatocytes of LEC rats with hereditary hepatitis. In all cases, reasonable proportionality between the fluorescence intensity and the amount of DNA was observed.
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PMID:NaCl-aided Hoechst 33258 staining method for DNA quantification and its application. 247 69

Spleen cells from female mice of the C57BL/6 strain iso-immunized with an homogenate from 3-week-old mice testis were fused with P3U1 cells. After cloning, two hybridomas, producing IgM, were obtained. Tissue specificity of the monoclonal antibody (Moab) in ascitic fluid was investigated by indirect immunofluorescence. Moab 1A1 reacted specifically with the cytoplasm of spermatogonia, spermatocytes and spermatids, but not with spermatozoa. Testicular antigen recognized by Moab 1A1 (Moab1A1-TA) was prepared by tissue sonication and then subjected to gel filtration. Moab1A1-TA detected in the void volume by ELISA was analyzed by SDS-PAGE and Western blotting. Immuno-staining of membrane filters revealed a broad area within the 45-205 kD range. Moab1A1-TA was treated with proteolytic enzymes, but no changes were observed after Western blotting. Thus, Moab1A1-TA was further digested by peptidases and glycolytic enzymes, electrophoresed using cellulose acetate membranes and immuno-stained with Moab 1A1. Evidence obtained from these experiments strongly suggests that Moab1A1-TA consists of an acidic peptide and a carbohydrate molecule. The antigenicity would be included in the carbohydrate epitope. Moreover, partial digestion of Moab1A1-TA by keratanase indicates that the lacto-series structure is included in the antigenic carbohydrate moiety.
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PMID:A testis-specific antigen of the C57BL/6 mouse. 247 26

1. The distribution of phosphatidylinositol3, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate hydrolysis or phosphatidylinositol-specific phospholipase C (PI-PLC), activity in the bull reproductive system showed the highest specific activity in the isolated spermatozoa (SZ) followed by testis and different epididymal segments. Both the head and tail fractions of SZ were active. 2. The optimal solubilization of the enzyme from SZ was obtained with 0.2% Triton X-100 or at 0.05% detergent concentration when combined with a 60 sec sonication. The sucrose gradient centrifugation showed that PI-PLC was enriched in membrane fraction distinct from mitochondria and acrosomes. 3. The enzyme was purified by ammonium sulphate precipitation and fractionations by hydrophobic interaction chromatography, gel filtration, Con A-Sepharose affinity and chromatofocusing columns. The purified enzyme was able to hydrolyse all phosphatidylinositol substrates with optimum at pH 7.0 and activation by Ca2+, Cd2+ and Mn2+ but not phospholipids lacking the inositol residue. 4. In PAGE (8-25% gradient) the purified (aggregated) enzyme did not enter the gel. In SDS-PAGE two closely located bands were found with Mr-values of 15,000 and 18,000. Isoelectric focusing showed a wide band at pl 4.5-5.1. 5. Gel filtration resulted in a broad elution peak indicating multiple molecular forms (aggregates); the basic form had an apparent molecular weight of 100,000. The binding of the enzyme to Con A-Sepharose indicated that the enzyme is a glycoprotein.
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PMID:Purification and characterization of phosphatidylinositol-specific phospholipase C from bovine spermatozoa. 255 6

A 125I-labelled calmodulin gel overlay procedure in the presence and the absence of Ca2+ was used to evaluate bull spermatozoa calmodulin-binding proteins. Frozen spermatozoa were thawed, washed and incubated for 6 h before being processed for SDS polyacrylamide gel electrophoresis and the 125I-labelled calmodulin gel overlay procedure. In non-incubated spermatozoa, up to 14 binding proteins were detected. Some exhibited greater calmodulin binding in the presence of Ca2+ while others exhibited greater binding when Ca2+ was absent. When heparin (2 micrograms/ml) was present in the incubation medium, a decrease in the calmodulin binding to the proteins of Mr 28,000 and 30,000 was detected in the presence of Ca2+ and EGTA. This effect of heparin was time- and dose-dependent and was increased by the presence of the acrosin inhibitor benzamidine. Sperm capacitation could thus be related to a decrease in the binding of calmodulin to these proteins.
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PMID:Effect of heparin on the expression of calmodulin-binding proteins in bull spermatozoa. 270 98

Immunizing Balb-c mice with washed motile human spermatozoa enabled the production of 2 monoclonal spermatozoal antibodies designated Ki-Sp II-13 and Ki-Sp VI-2. Immunohistochemically Ki-Sp VI-2 reacts only with mature spermatozoa. The monoclonal antibody Ki-Sp II-13 recognizes besides spermatozoa also T- and B-lymphocytes. Electronmicroscopically both antibodies react with the surface membrane of spermatozoa in the area of the head, the neck and the proximal part of the tail. Functional tests show a high sperm-agglutinating and sperm-immobilizing activity, using different test systems for Ki-Sp II-13. Both antibodies proved to be IgG. On immunoprecipitation Ki-Sp II-13 was identified as IgG-2a and Ki-Sp VI-2 as IgG1. Immunoprecipitates of SDS-electrophoretically separated and radiolabeled sperm surface antigens revealed a molecular weight of 18 KD for Ki-Sp II-13 and of 24 KD for Ki-Sp VI-2.
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PMID:[Monoclonal antibodies Ki-Sp II-13 and VI-2 react with surface membrane of human spermatozoa]. 271 62

An anti-sticking factor (ASF-I) that showed high affinity for inhibiting adhesion of spermatozoa to glass was isolated from goat epididymal plasma and characterized. The factor was purified approx. 5600-fold and showed a single protein band when examined by non-denaturation and SDS-polyacrylamide gel electrophoresis. The molecular mass and S20w value of ASF-I were approx. 47 kDa and 4.25 S. ASF-I at a concentration of 1 nM showed nearly maximal anti-sticking activity when approx. 60% of the intact spermatozoa were prevented from adhesion to glass and it showed a high degree of protein specificity. Studies with trypsin and glycosidases demonstrated that both the sugar and protein parts of the molecule are essential for its anti-sticking activity. Evidence has been presented to support the view that the outer surface of sperm possesses specific ASF-I receptors that bind to 125I-labelled ASF and mediate cell adhesion to glass. ASF-I also showed high affinity for inhibiting agglutination of corpus-epididymal spermatozoa. The ASF activity was found to be distributed in all the tissues tested and its specific activity was markedly higher in blood plasma than in the tissues. The results suggest that ASF may play an important biological role by serving as a specific inhibitor of cell-substratum and cell-cell adhesions.
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PMID:Purification and characterization of an anti-sticking factor from goat epididymal plasma that inhibits sperm--glass and sperm--sperm adhesions. 271 14


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