Complete replication of human sperm genome in egg extracts from Xenopus laevis.

To examine the ability of Xenopus egg extracts to support a complete replication cycle of human sperm genome, demembranated human spermatozoa were incubated with the extract from activated Xenopus laevis eggs. Most sperm heads were decondensed within 15 min. The heads became round within 30 min with diameters of 10-30 microns. The process of DNA replication in the pronuclei was monitored by two methods, bromodeoxyuridine incorporation and flow cytometry. The results indicate that DNA replication was initiated approximately 1.5 h after membrane structure formation and that it lasted up to 9 h. The amounts of DNA in most pronuclei were doubled by 4-9 h, depending on which donor toad was the source of the egg extract. Inclusion of the protein synthesis inhibitor, cycloheximide (100 micrograms/ml), had no obvious effect on human sperm DNA replication but appeared to prevent the pronuclei from degradation after a prolonged period (> 6 h) of incubation. After storage in liquid nitrogen for > 1.5 mo, the efficiency of the egg extracts in supporting sperm head decondensation and DNA replication was reduced for human sperm but not for Xenopus sperm. Possible applications of the use of Xenopus egg extract for human sperm activation and DNA replication are discussed.

Mycoplasma hyorhinis molecules that induce tumor necrosis factor alpha secretion by human monocytes.

Mycoplasma hyorhinis has been shown to induce the secretion of tumor necrosis factor alpha (TNF-alpha) from monocytes. To identify the molecules responsible for this activity, we separated sonicated M. hyorhinis lysate material by centrifugation at 100,000 x g into soluble (S) and particulate (P) fractions. The fractions were assayed for TNF-alpha-inducing activity by the L929 bioassay. Both the soluble and particulate fractions were able to induce TNF-alpha in roughly equal amounts. The optimum dose for both fractions was 1 micrograms/ml. Proteinase K treatment of either fraction eliminated the activity, suggesting that a protein component is involved in induction. Phase partitioning into Triton X-114 aqueous (A) and detergent (D) phases showed that the soluble fraction was composed of 80% aqueous-phase proteins, while the particulate fraction was > 75% detergent-phase proteins. All four fractions (SA, SD, PA, and PD) were able to induce TNF-alpha release. Treatment with NaIO4 to remove carbohydrate reduced the inducing activity of the SA phase by 80%, whereas that of the other fractions was unaffected by this treatment. The M(r)S of the inducing activity were determined by the monocyte Western (immunoblot) technique. The SA phase activity was associated with a single periodate-sensitive peak of 69 to 75 kDa. The two detergent phases had similar profiles of inducing activity, containing four peaks of activity. These peaks corresponded to 48 to 52, 43 to 45, 39 to 40, and 31 to 32 kDa. The PA fraction also contained four peaks of activity, 69 to 75, 55 to 57, 48 to 52, and 39 to 40 kDa. Thus, both a protein and glycan moiety from M. hyorhinis are capable of inducing TNF-alpha release from human monocytes.

A 48-kilodalton Mycoplasma fermentans membrane protein induces cytokine secretion by human monocytes.

Mycoplasma fermentans is one of several Mycoplasma species that have been reported to stimulate tumor necrosis factor (TNF) secretion from monocytes. This activity has been associated primarily with the mycoplasma membrane fraction. In this article, we have characterized a membrane protein that stimulates TNF and interleukin 1 beta secretion. The TNF-releasing activity partitioned into the Triton X-114 detergent phase, suggesting that the molecules is hydrophobic. The secretion of TNF is elevated in the presence of serum, which suggests that a serum component may play a role in the interaction between this mycoplasma protein and monocytes. Treatment of monocytes with monoclonal anti-CD14 antibody had no effect on the levels of TNF-releasing activity. By using the monocyte Western blot (immunoblot) technique, we have determined the molecular mass of the active molecule to be 48 kDa. This molecule appears to be distinct from the recently described family of variable lipoproteins of M. fermentans. Mycoplasma particulate material treated with proteinase K lost all inducing activity, whereas lipoprotein lipase-treated samples retained some level of activity.

Identification and characterization of proteinase K-resistant proteins in members of the class Mollicutes.

Proteins resistant to proteinase K are rare because of the potency, wide pH optimum, and low peptide bond specificity of this enzyme. Previously, only the prion proteins associated with transmissible spongiform encephalopathies, possibly related proteins in the mollicute Spiroplasma mirum, and proteinase K itself have been reported. We identified a new proteinase K-resistant protein, p40-pr, in two strains of Mycoplasma hyorhinis and in extracts of these organisms. p40-pr's are similar to prion proteins in their resistance to high doses of proteinase K and in the reversal of this resistance by strong denaturing conditions. However, p40-pr's were distinct immunologically, in relative molecular mass, and in their method of extraction. Two immunologically related forms of p40-pr were identified on sodium dodecyl sulfate (SDS) gels and Western immunoblots, a 40-kDa species in boiled samples and a 120-kDa species dissociable by boiling in SDS. Reduction with 2-mercaptoethanol did not affect the mass of p40-pr's or the 120-kDa forms. The development of proteinase K resistance of p40-pr correlated to age-dependent increases in organism protein-lipid ratios. p40-pr-like proteinase K-resistant proteins of 46 to 50 kDa were identified in four of eight additional species of the class Mollicutes but not in S. mirum. However, these mycoplasmal proteins did not react with antibody to the denatured 40-kDa form of M. hyorhinis p40-pr purified by electroelution. The chromatographically purified 46-kDa proteinase K-resistant protein of Mycoplasma orale was an arginine deiminase.

Microbiological cultivation of Mycoplasma hyorhinis from cell cultures.

The failure of many cell culture isolates of Mycoplasma hyorhinis to grow on microbiological media has stressed the need for alternate assays to detect these organisms. The use of freshly prepared yeast extract in mycoplasmal media together with incubation in 5% CO2/air successfully detected M. hyorhinis in 12 of 12 infected cultures. These were not detected by the use of conventional mycoplasmal media using aerobic or anaerobic incubation. This assay may also be helpful in detection of other mycoplasmal species commonly isolated from cell cultures.

Benzo[a]pyrene-induced immunotoxicity: comparison to DNA adduct formation in vivo, in cultured splenocytes, and in microsomal systems.

Benzo[a]pyrene (BaP)/DNA adduct formation appears to be involved in carcinogenesis, but the relationship between adduct formation and BaP-induced immunotoxicity is unknown. We compared DNA adduct formation (32P-postlabeling analysis) to suppression of polyclonal immune responses (3H-TdR incorporation and IgM secretion) and decreases in cell viability in B6C3F1 female mouse splenic leukocytes (SPL). BaP administration (200 mg/kg, ip) resulted in suppression of polyclonal responses and substantial DNA adduct formation in mouse SPL. SPL adduct levels were similar to those in liver, lung, kidney, and stomach. In vitro exposure of SPL to BaP without rat liver activation enzymes (S9) caused decreases in SPL viability and immune responses that were dependent on dose and exposure period. However, DNA adduct formation in SPL was very low between 1 and 200 microM BaP. S9 enhanced the toxicity of BaP for SPL cultures. Adduct formation was rapid and dose related in +S9 incubates. The low level of BaP activation by SPL was confirmed in microsomal incubations in which splenic microsomes exhibited much lower aryl hydrocarbon hydroxylase (AAH) activity and ability to form DNA-adducting metabolites than did microsomes from liver or lung. Results indicate that immunosuppression produced by BaP in these systems was due to cytotoxic effects. It appears that these effects were caused by two separate mechanisms, one dependent on and one independent of DNA adduct formation. Since SPL had high levels of DNA adducts after ip injection of BaP, reactive metabolites of BaP may be involved in the immunotoxicity seen in vivo.

Comparative analysis of mycoplasma ribosomal RNA operons.

Using differential probes derived from the Escherichia coli rrnB operon, we were able to map the ribosomal operons of Mycoplasma pneumoniae, M. hyorhinis, and M. arthritidis. All organisms contained only one ribosomal RNA operon, and the genes were linked in the eubacterial order, 16S-23S-5S. The HindIII sites in M. hyorhinis and M. pneumoniae were highly conserved.

Blast transformation of mouse splenic B-lymphocytes with extracts of Mycoplasma hyorhinis.

A soluble fraction of Mycoplasma hyorhinis containing three major protein components with Mrs of 53, 43, and 35 kDa was developed. This fraction caused blast transformation of mouse B-lymphocytes from the spleen and peritoneum.

Pleomorphism among murine tissue-associated gp70s.

The gp70s isolated from normal mouse tissues by radioimmune precipitation and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) were shown to be highly pleomorphic. Their apparent molecular weights calculated from SDS-PAGE ranged from 75000 for thymus gp70 to 65000 for epididymal secretion gp70. Differences in the Mr of tissue-associated gp70s were confirmed by double-label co-electrophoresis studies. In addition, a high degree of primary structural pleomorphism among tissue-associated gp70s was demonstrated using two-dimensional tryptic peptide fingerprint analysis. These studies showed that most of the conserved peptides of tissue-associated gp70s were also common to xenotropic murine leukaemia virus (MuLV) gp70s. Thus, tissue-associated gp70s are probably encoded by endogenous xenotropic MuLV env genes or gene fragments. Tissue-associated gp70s also showed a very high level of primary structural pleomorphism. These phenomena were observed for gp70s derived from the tissues of several strains of mice. Tissue-associated gp70 pleomorphism may arise as a consequence of at least two simultaneously operating mechanisms. First, the expression of pleomorphic forms of gp70s on murine tissues may be regulated by mechanisms that also determine the differentiated state of the tissues. Second, endogenous xenotropic env genes may be modified by recombinational or mutational events among these genes, or among cellular genes that regulate the expression of endogenous proviral genes.

Infection of mouse lymphoblastoid cell lines with Mycoplasma hyorhinis: complex nature of mycoplasma-host cell interactions.

Mycoplasma hyorhinis infection of lymphoid cells is a complex process. Mycoplasmas adsorb to cell surface receptors and undergo lateral redistribution on the cell membrane. This process culminates in the formation of co-caps of mycoplasmas and specific cell surface antigens. One or more of these antigens may be a M. hyorhinis receptor(s) or may bear a receptor moiety(s). We show that the cell surface antigens Thy-1.2 and Thy-1.1, and to a lesser extent H-2 and gp70, but not T200, are co-capped with M. hyorhinis on the membranes of acutely infected mouse thymic lymphoblastoid cell lines. These antigens may represent multiple receptor(s) for M. hyorhinis since there is no correlation between the expression of any individual antigen and the susceptibility of these cell lines to infection.

Characterization of the serum protein inhibitor of the V.D.R.L. test.

The V.D.R.L. test for syphilis was used to evaluate the ability of various serum protein fractions to inhibit cardiolipin flocculation tests for syphilis. Serum protein fractions obtained by modifications of the method of Ecker et al. (1) were chromatographed by DEAE or gel filtration and characterized by immunodiffusion and electrophoresis. These experiments indicated that the V.D.R.L. inhibitor substance was in the IgM fraction of serum, although it has not yet been determined whether the inhibitor is an IgM antibody or another component which cofractionates with IgM.