Dual energy metabolism-dependent effect of Ureaplasma urealyticum infection on sperm activity.

Genital Ureaplasma urealyticum infection is considered a sexually transmitted infection. It has long been debated whether the presence of U. urealyticum in semen may be a possible cause of infertility. Long-term incubation (4 hours or overnight) of sperm cells with U. urealyticum in vitro resulted in a significant inhibition of sperm motility and membrane alteration whereas a short incubation (45 minutes) of sperm cells with ureaplasmas resulted in an acceleration of sperm velocity. The aim of this study was to understand these contradictory reports of U. urealyticum infection on sperm motility. Spermatozoa from fresh ejaculates of normozoospermic semen of men who were referred to the university Male Fertility Laboratory for semen analysis, with no history of genital tract infection, and from normal Assaf breed rams were infected in vitro with U. urealyticum serotype 8, at different pHs and O2 concentrations. Sperm viability and motility and changes in extracellular pH were evaluated. A significant (16%-43%) increase in sperm activity was observed upon infection at alkaline pH (7.8) under aerobic or hypoxic conditions, and a 58% increase was observed under anaerobic conditions and pH 7.2. When the infection was conducted under aerobic conditions and acidic pH (6.3), or under hypoxic conditions at neutral pH (7.2), an 8%-25% inhibition of sperm activity was observed. These results indicate that when sperm activity depends on mitochondrial oxidative phosphorylation, usually at low pHs, U. urealyticum competes with mitochondrial energy production and therefore reduces sperm motility and viability. However, when sperm energy metabolism depends on glycolysis, usually at higher pHs, U. urealyticum stimulates glycolysis and sperm activity.

In vivo and in vitro impairment of human and ram sperm nuclear chromatin integrity by sexually transmitted Ureaplasma urealyticum infection.

The incidence of Ureaplasma urealyticum infection in the semen of infertile men is variable (7%-42%). Evidence has accumulated through routine semen analysis to suggest that this infection can cause embryo loss without necessarily affecting sperm quality. The aim of this study was to specifically investigate the effects of U. urealyticum infection on sperm chromatin stability and DNA integrity, which are known to be correlated to pregnancy outcome. Sperm cells isolated from human semen infected in vivo with U. urealyticum exhibited a low percentage of stable chromatin as determined by nuclear chromatin decondensation assay (42% +/- 4.8%, n = 8) and a high percent of denatured DNA as determined by sperm chromatin structure assay (60.9% +/- 9.1%, n = 7). After doxycyclin treatment, a significant improvement in both parameters was observed (73.7% +/- 3.6%, P: < 0.001 and 30.1% +/- 3.5%, P: < 0.008, respectively). Sperm cells infected in vitro exhibited higher rates of viability and motility than uninfected cells. In contradistinction, U. urealyticum caused significant dose- and time-dependent chromatin decondensation and DNA damage. The percentage of human sperm cells with denatured DNA increased significantly by 54.9% +/- 23.9% and 47. 9% +/- 12.1%, after 30 min infection with serotypes 8 and 3, respectively, at a multiplicity of infection of 100 ureaplasmas per sperm compared with uninfected control cells. The damage to DNA was significantly more pronounced in infected ram sperm (180.9% +/- 21. 5%). These results indicate that preserved sperm activity post U. urealyticum infection resulted in damage to paternal DNA, although a high fertilization rate was maintained, and embryonic development may, therefore, be impaired.

Meloidogyne javanica surface proteins: characterization and lability.

Characterization of surface coat (SC) proteins including carbohydrate-binding proteins and glycoproteins of the plant-parasitic nematode Meloidogyne javanica 2nd-stage juvenile (J2) is reported. Extraction of surface proteins with sodium dodecyl sulfate (SDS) and separation by denaturing polyacrylamide gel electrophoresis (SDS-PAGE) results with bands at 6, 9, 14, 22, 26, 31, 46, 49, 58, 66, 80, 205 and 250 kDa. On Western blots, the neoglycoprotein, fucosylated-, mannosylated- and glucosylated-bovine serum albumin, reacted with the 14, 22, 26, 58 and 66 kDa bands. The lectins, Concanavalin A and wheat-germ agglutinin (WGA) labelled surface protein bands of 6, 9, 14, 31, 58 and 66 kDa; WGA also labelled the 22 and 26 kDA bands. Biotin reagents were used to specifically trace surface proteins on live J2. SDS-PAGE of biotinylated J2 extracts revealed only 2 specific biotin-protein bands at 46 and 49 kDa. The labile and transitory nature of Meloidogyne javanica SC was demonstrated by the dynamics of human red blood cells (HRBC) adherence to J2 of different ages. HRBC adherence was also used to demonstrate the SC recovery of detergent-treated J2, which was further exhibited in the SDS-PAGE profiles.

Adherence of Mycoplasma gallisepticum involves variable surface membrane proteins.

Adherence of Mycoplasma gallisepticum to erythrocytes was examined by colony immunoblotting, detergent phase fractionation, trypsin treatment, comparison of protein profiles, and comparison of erythrocyte-bound mycoplasma protein fractions of hemadsorption-positive and -negative mutants. The binding of M. gallisepticum to chicken or human erythrocytes was found to be mediated via surface-exposed membrane proteins undergoing high-frequency phase variation.

Adherence of Mycoplasma pneumoniae to human alveolar macrophages.

The human pathogen Mycoplasma pneumoniac causes primary atypical-cold agglutinin-positive pneumonia. Since alveolar macrophages internalize mycoplasma as part of their immune defense, we studied characteristics of the human macrophage receptor for opsonized and nonopsonized M. pneumoniae. The glass-adhering subpopulation of M. pneumoniae attached more than the non-adherent subpopulation. The attachment was dose-dependent and enhanced by opsonization in the presence of human serum. It is inhibited by sulfated compounds such as dextran-sulfate and polyanetholsulfonic acid, but not by dextran or several monosaccharides, suggesting that sulfated glycolipids on the macrophage surface may act as receptors for M. pneumoniae binding. In addition, sialylated compounds, such as fetuin and alpha 1-acid glycoprotein, were found to be potent inhibitors of the attachment, also indicating the role of sialic acid residue in recognition and attachment of M. pneumoniae to human alveolar macrophages.

Mycoplasmas regulate HIV-LTR-dependent gene expression.

Mycoplasmas have been incriminated in setting the stage for HIV infection and full-blown AIDS. We tested the possible involvement of mycoplasmas in activation of HIV. Two cell lines, 293 fibroblasts and Jurkat CD4+ T-cells, transfected with plasmids harboring a transcription fusion construct between HIV-long terminal repeat (HIV-LTR) and either luc or cat genes, were infected with several mycoplasmas (M. fermentans; M. penetrans, M. pirum and Ureaplasma urealyticum) and the reporter gene expression was monitored. The data presented here suggest that mycoplasmas, and specifically their membranes, play a role in the activation of HIV-LTR mediated transcription.

Carbohydrate-recognition domains on the surface of phytophagous nematodes.

Human red blood cells (HRBC) adhered to preparasitic second-stage juveniles (J2) of Heterodera avenae, Heterodera schachtii, Meloidogyne javanica, Pratylenchus mediterraneus, Rotylenchulus reniformis, and Tylenchulus semipenetrans over the entire nematode body. Binding was conspicuously confined to the head and tail of Longidorus cohni, Xiphinema brevicolle, and Xiphinema index. Binding was Ca2+ and Mg2+ dependent. In contrast, HRBC did not adhere to Anguina tritici, Aphelenchoides subtenius, Ditylenchus dipsaci, M. javanica females, and Panagrellus redivivus, even in the presence of these cations. Incubation of M. javanica J2 with fucose, glucose, N-acetylglucosamine, mannose, or trypsin decreased the intensity of subsequent HRBC binding, while galactose and N-acetylgalactosamine increased binding intensity. HRBC binding was diminished when nematodes were pretreated with trypsin and eliminated when pretreatments with detergents removed the surface coat. HRBC adhered to nylon fibers coated with surface coat extracted from M. javanica J2; binding was Ca2+ and Mg2+ dependent and diminished when the nylon fibers were coated with bovine serum albumin or preincubated with fucose and mannose. These results demonstrate that HRBC adhesion involves carbohydrate moieties of HRBC and corresponding carbohydrate-recognition domains (CRD) distributed in the nematode surface coat. To our knowledge this is the first report of a surface CRD in the phylum Nematoda.

Cyclodextrins as carriers of cholesterol and fatty acids in cultivation of mycoplasmas.

The design of fully or partly defined media for mycoplasma cultivation involves the need to provide the essential lipids, cholesterol and long-chain fatty acids, in an assimilable and nontoxic form. This study introduces cyclodextrins (CDs) as carriers of these lipids, thus suggesting alternatives to serum or bovine serum albumin (BSA). The effects of beta-CD and two forms of chemically modified beta-CD, dimethyl-beta-CD (Dimeb) and hydroxypropyl-beta-CD (Hyprob), on the growth of Mycoplasma capricolum and Acholeplasma laidlawii were investigated in a basal medium as well as in serum- and BSA-supplemented media. beta-CD was found to inhibit the growth of the sterol-requiring M. capricolum in both serum and BSA media, but it stimulated the growth of the sterol-independent A. laidlawii. Inhibition by beta-CD was explained by its capacity to form a water-insoluble CD-cholesterol complex, thus rendering it unavailable to the cells. Dimeb, despite its strong complexing ability for lipids, was found to be toxic to all mycoplasma species in both liquid cultures and agar diffusion susceptibility tests. In sharp contrast to beta-CD and Dimeb, Hyprob (with a degree of substitution of 4.2) added at 5 and 10 mM to a basal medium supplemented with lipids permitted growth of M. capricolum. Comparison of growth curves in the two conventional serum and BSA media with those in two Hyprob media revealed comparable growth and growth rates.

Adherence of Ureaplasma urealyticum to human erythrocytes.

Ureaplasma urealyticum (four serotypes and two clinical isolates) were metabolically labeled with radioactive methionine to a high specific activity. Labeling allowed the study of the mechanism of adherence to human erythrocytes. The adherence mechanism was complex and partially mediated by proteinaceous surface components. The binding sites on the erythrocytes were partially sensitive to neuraminidase treatment, and adherence was inhibited by glycophorin and dextran sulfate, indicating recognition of sialyl residues and sulfated compounds.

Wheat Germ Agglutinin Bound to the Outer Cuticle of the Seed Gall Nematodes Anguina agrostis and A. tritici.

The presence of wheat germ agglutinin (WGA) on the cuticular surface of the seed gall nematodes Anguina agrostis and Anguina tritici was demonstrated, and the nature of its binding was examined. Crude extracts from the cuticles of A. tritici agglutinated human red blood cells, and only N-acetylglucosamine (GlucNAc) inhibited the agglutination. Distribution of the lectin was visualized by treating live infective juveniles (J2) with rabbit anti-WGA antibody and staining with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG. The lectin bound to the outer cuticular surface of the whole body wall. Pretreatment with GlucNAc oligomers did not reduce the fluorescence created by the anti-WGA-WGA binding, indicating at least a partial nonspeciflc adhesion of the WGA to the nematode surface. Proteolytic enzyme pretreatments diminished the fluorescence, whereas lipase and periodate pretreatments increased the fluorescence. Adult females and males were labeled only on the head and tail, whereas eggs were not labeled at all. It was concluded that the WGA on the J2 cuticle originates from the host.

Panagrellus redivivus and Caenorhabditis elegans: evidence for the absence of sialic acids.

Complementary experiments were performed to indicate the presence or absence of sialic acids in axenically cultured Panagrellus redivivus and Caenorhabditis elegans. Competitive displacement experiments with radiolabeled Limax flavus agglutinin demonstrated the presence of sialic acid in nematodes grown in medium which contained liver extract as a growth factor but the absence of sialic acid when heme was substituted for liver extract. This finding suggested that sialic acid present in the liver medium was responsible for conflicting results of other studies. Transmission electron microscopy of thin sections from nematodes labeled with an LFA-ferritin conjugate revealed no label to the surface area of the cephalic chemosensilla. Fluorometric analysis with a modification of the thiobarbituric acid assay was negative for sialic acid. Analyses by gas chromatography-mass spectrometry, sensitive to the high picomole range, were also negative for sialic acid. Taken together the results provide evidence for the absence of sialic acid in P. redivivus and C. elegans using the most sensitive and diagnostic technique currently available.

Glycosidase activities of mycoplasmas.

The activities of alpha- and beta-glucosidase, beta-galactosidase and beta-N acetylglucosaminidase were assessed at acidic pH by fluorimetry using the appropriate 4-methylumbelliferyl substrate in four Mycoplasma species (M. pneumoniae, M. gallisepticum, M. hominis and M. capricolum) and in Acholeplasma laidlawii. The glycosidase activities were in a low range (0.1-4.2 nmole per h per mg protein) with the exception of higher activities of beta-N-acetylglucosaminidase in A. laidlawii. The enzyme levels of a virulent and a nonvirulent strain of M. pneumoniae were comparable. Despite the very sensitive assay, neuraminidase activity was not detected in M. pneumoniae and M. gallisepticum. No induction of alpha-glucosidase could be demonstrated for M. pneumoniae or A. laidlawii. At least part of the glycosidase activities was localized in the membrane fraction of all mycoplasmas studied. This may support the hypothesis that pathogenic mycoplasmas, being membrane parasites, may modify, by their glycosidases, some host cell glycoconjugates. However, our study did not distinguish the pathogenic mycoplasmas to possess a characteristic glycosidase profile.

Characterization of monoclonal antibodies to human platelet myosin that recognize highly conserved epitopes within the 50 kDa fragment of myosin subfragment-1.

Three monoclonal antibodies directed against human platelet myosin heavy chains (MCH) that recognize homologous sequences contained within the functionally active subfragment-1, in platelet and rabbit skeletal muscle myosin were studied. These antibodies are distinguished by their affinities to different myosins and their differential effect on various ATPase activities. Epitope mapping was accomplished by analyzing antibody binding to proteolytic peptides of myosin head subfragment-1 under various experimental conditions. The epitopes recognized by these anti-human platelet MHC monoclonal antibodies reside within a small region of the 50 kDa fragment, beginning 9 kDa from its C-terminus and extending a stretch of 6 kDa towards the N-terminus. These epitopes lie between residues 535-586, and are contained within a highly conserved area of myosin heavy chain.

Structural and immunological properties of myosin from human platelet external and internal membranes.

Our previous studies indicate that platelets contain two myosin isoforms, one of them localized in the membrane while the other in the cytoplasmic compartment. Structural and functional differences of these myosins have been characterized. In this study two platelet membrane subfractions, the external and the internal membranes, were isolated simultaneously from a crude membrane fraction and their purity was characterized using specific marker enzymes. Myosin was shown to be present in both membrane fractions and its structural and immunological properties were investigated. The electrophoretic mobilities of myosin in both membrane preparations were identical to the mobility of its cytoplasmic counterpart. Two-dimensional peptide mapping of the iodinated tryptic peptides of the myosin heavy chains indicated that at least one peptide is missing in the maps of the myosins from the external and internal membranes as compared to their soluble counterpart. Our data suggest that myosin is located in three distinct platelet compartments: cytosol, external and internal membranes. The same myosin isoform is located in the two membrane compartments, while the isoform found in the cytosol is different. The observed variations in the structure of the two isoforms may reflect differences in their respective physiological functions.