Cleavage of proteins of reproductive secretions by extracellular proteinases of Tritrichomonas foetus

Cleavage of host defense proteins from reproductive secretions was investigated as a potential virulence mechanism for Tritrichomonas foetus extracellular proteinases. Three categories of susceptibility to digestion were found among the defense proteins tested. Cleavage of fibrinogen, fibronectin, and albumin occurred rapidly with more than 50% of these digested within 30 min. Lactoferrin, immunoglobulin G1, and immunoglobulin G2 were more than 50% digested after 4 h. Transferrin, immunoglobulin M, and immunoglobulin A were the most resistant to the Tritrichomonas foetus extracellular proteinases, since 50% or more of the parent molecule remained after 24 h. The responsible proteinases were classified as cysteine (thiol) proteinases because cleavage was inhibited by the cysteine proteinase specific inhibitor, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane and not by the serine proteinase specific inhibitor, phenylmethylsulfonyl fluoride. In addition, alpha 2-macroglobulin, but not alpha 1-antitrypsin, inhibits the action of the proteinases. The ratio of this naturally occurring inhibitor to the quantity of proteinases released may determine whether the above substrates are cleaved in vivo. Since these substrates are implicated in iron acquisition, cell adherence, and acquired immunity, Tritrichomonas foetus proteinases are likely to play a role in host-parasite interactions.     

Monoclonal anti-idiotype induces antibodies against bovine Q17 rotavirus.

This study describes, for the first time, the production and use of an "internal-image" anti-idiotypic monoclonal antibody (MAb) to elicit a rotavirus-specific antibody response. An immunoglobulin G2a MAb, designated RQ31 (MAb1), specific for the outer capsid protein VP4 of bovine Q17 rotavirus and capable of neutralizing viral infection in vitro was used to generate an anti-idiotypic MAb (MAb2). This MAb2, designated RQA2, was selected by enzyme-linked immunosorbent assay (ELISA) using F(ab')2 fragments of RQ31. RQA2 (MAb2) inhibited the binding of RQ31 (MAb1) to the virus but had no effect on the binding of other rotavirus-specific MAbs. The MAb2 also inhibited virus neutralization mediated by MAb1 in a dose-dependent fashion. Naive guinea pigs immunized with the MAb2 produced anti-anti-idiotypic antibodies (Ab3) that reacted with bovine Q17 rotavirus in an ELISA and neutralized rotavirus infection in vitro. The Ab3 response was characterized as MAb1-like because the Ab3 recognizes only the Q17 and neonatal calf diarrhea virus rotavirus strains in ELISA, as did RQ31 (MAb1). The Ab3 response also possessed two other characteristics of RQ31: the abilities to bind the 1.36 (double-capsid) but not the 1.38 (single-capsid) purified rotavirus fraction in ELISA and to immunoprecipitate the VP4 rotavirus protein.     

Factors affecting triple staining of human sperm

We have previously described a triple stain for evaluating normal acrosome reactions of human sperm. This procedure uses trypan blue to distinguish live and dead sperm, Bismarck brown to stain the sperm's postacrosomal region, and rose Bengal to stain the sperm's acrosome. We have recently found that batches of rose Bengal vary significantly in their ability to produce good staining of the acrosome in this procedure. This appears to be due to variations in the intrinsic pH of rose Bengal solutions and the presence of nondye contaminants in the stain. In this study, we have evaluated acrosomal staining using 6 batches of rose Bengal and report a method for achieving uniform staining quality with each batch. Solutions of rose Bengal (0.8%) are made up in 0.1 M Tris HCl (pH 2.3) buffer and adjusted to pH 5.3 if necessary. For most batches of rose Bengal this promotes precipitation of some of the dye and an unidentified contaminating crystal. The precipitate is removed by centrifugation, and the supernatants have been found to give good to excellent staining of the acrosomes for all batches tested. Solutions of both rose Bengal and Bismarck brown are stable for at least 5 days but their pH values should be monitored daily and adjusted to 5.3 and 1.8 respectively if drifting occurs. We have also observed some variation in the intensity of rose Bengal staining of the acrosome from donor to donor and recommend that staining times in rose Bengal be adjusted for each donor.     

Accumulation of cadmium and lead in the gills of Mytilus edulis: X-ray microanalysis and chemical analysis.

The accumulation of Cd and Pb in the gills of the lamellibranch mollusc Mytilus edulis has been studied by electron microscopy, X-ray microanalysis, atomic absorption spectroscopy and radionuclide monitoring. The patterns of accumulation of the two elements differ markedly as do the sites of deposition whithin the gills. Lead is found extracellularly as crystalline deposits in the basal lamina which forms the capillary walls of the gill lamellae. The Pb is found associated with Ca in equiatomic ratios and occurs either as a mixed or complex carbonate. Cadmium is always associated with S and frequently with P in membrane bound vesicles within the cells of the gill epithelium and in the amoebocytes. The S is probably attributable to the presence of cysteine residues in a metal binding protein which can be extracted from the gills. Analysis of the metal binding protein shows that it binds Ag, Cd, Cu, Fe, Hg, Sn and Zn. Its amino acid composition is similar to that reported for eels and limpets but has a lower cysteine content than mammalian metal binding protein.     

Ouabain sensitivity is linked to ras -transformation in human HOS cells

Mouse cells transformed by the retroviral oncogene v-Ki- ras are significantly more sensitive to the toxic effects of 1mM ouabain than are their nontransformed counterparts. We have extended these findings to a human cell line (HOS). HOS cells (ATCC CRL 1543) are relatively resistant to treatment with 1 microM ouabain while KHOS cells (transformed by Kirsten murine sarcoma virus) are extremely sensitive. Two flat revertant cell lines isolated from the KHOS line and lacking the v- ras gene sequences are resistant to ouabain. This effect may be observed morphologically and can also be demonstrated by dye exclusion and plating efficiency tests. In addition, the toxic effects of ouabain may be rapidly and efficiently quantitated in a 51Cr-release assay. This differential lethality may be used to enrich the proportion of non-transformed revertants in populations of mutagen-treated transformed cells.