Molecular cloning of bovine leukemia virus DNA integrated into the bovine tumor cell genome

The bovine leukemia virus (BLV) DNA harbored in the bovine tumor cell genome was cloned in lambda Charon 4A phage. Using either representative or 3' half-enriched BLV cDNA as a blot hybridization probe, clone lambda BLV-1 was shown to carry 9 kb of the BLV genome, flanked by cellular sequences at both ends. Restriction mapping with twelve endonucleases and hybridization of the DNA fragments to BLV cDNA representing a 3'-end portion of the viral genome revealed the presence and precise location of two long terminal repeats (LTRs) and virus-cell junctions. Thus, lambda BLV-1 appears to contain the complete BLV genome and flanking tumor cellular sequences. The restriction map of the cloned BLV proviral DNA closely resembles that previously reported for unintegrated linear proviral DNA, but differs significantly from that of the integrated provirus of another BLV isolate, the difference occurring preferentially in the putative gag and pol genes.     

Effect of the sequences upstream from the ribosome-binding site on the yield of protein from the cloned gene for phage MS2 coat protein

The translational efficiency of the coat protein gene of phage MS2 has been examined in vivo with respect to neighbouring sequences. The cloned MS2 DNA has been gradually shortened starting at the 5' or 3' terminus, and its effect on coat protein synthesis monitored. Removal of the 3'-terminal sequences had little influence. In contrast, the gradual removal of the 5'-terminal region profoundly reduces translation. Long before the ribosomal binding site (RBS) of the coat protein (CP) gene is reached, the yield of CP has dropped by one order of magnitude. Functional half-lives of the various messengers were found not to be significantly different. Available evidence indicates that the secondary structure of the RBS in native and shortened MS2 RNA is identical. We infer that important determinants for ribosome recognition lie 5' to the RBS region of the MS2 RNA coat gene.     

Immunosuppressive properties of leukotriene D4 and E4 in vitro

Leukotrienes D₄ and E₄ whose physiological function has been associated with smooth muscle contraction, are demonstrated to be potent suppressors of immunocompetent cell function. In concentrations as low as 10^?12 M both leukotriene D₄ and E₄ can inhibit mitogen-induced lymphocyte transformation. Higher concentrations of leukotrienes E₄ and D₄ will inhibit appearance of antibody-forming cells in tissue culture. These data suggest another role for the leukotrienes in addition to their function as slow-reacting substances.     

Amino group modification inhibits ristocetin cofactor activity of human von Willebrand factor

Purified von Willebrand factor rapidly loses activity when treated under mild conditions with the highly specific amino group reagent trinitrobenzenesulfonic acid. Greater than 90 percent inhibition of activity is achieved by modification of only 7 percent of the amino groups. Other modifications such as acetylation and succinylation also abolish activity. It is unlikely that the essential rapidly reacting amino groups function simply in an electrostatic manner since modifications such as amidination and methylation which produce derivatives which retain positive charge are also inactive or nearly so.     

Binding, internalization and intracellular processing of 125I-epidermal growth factor purified by isoelectric focusing

When epidermal growth factor (EGF) which had been extensively purified by HPLC was subjected to iodination with sodium ¹²⁵iodide, 5 major species of differing isoelectric points were produced. Some of these species bound to rat fibroblasts with different affinities but were internalized with equal efficiency. Examination of the internalized ¹²⁵I-labelled molecules revealed processing of all the ¹²⁵I-EGF species to macromolecules with more acidic isoelectric points. The ¹²⁵I-EGF species with a pI of 4.5 corresponded in electrofocusing behavior with intact non-iodinated EGF. Other EGF species probably represented molecules which were covalently modified as a result of the iodination procedure.     

Drug residue formation from ronidazole, a 5-nitroimidazole. II. Involvement of microsomal NADPH-cytochrome P-450 reductase in protein alkylation in vitro

Purified liver microsomal NADPH-cytochrome P-450 reductase is able to catalyze the activation of [14C]ronidazole to metabolite(s) which bind covalently to protein. Like the reaction catalyzed by microsomes, protein alkylation catalyzed by the reductase is (1) sensitive to oxygen, (2) requires reducing equivalents, (3) is inhibited by sulfhydryl-containing compounds and (4) is stimulated several fold by either flavin mononucleotide (FMN) or methytlviologen. A cytochrome P-450 dependent pathway of ronidazole activation can be demonstrated as judged by the inhibition of the reaction by carbon monoxide, metyrapone and 2,4-dichloro-6-phenylphenoxyethylamine but the involvement of specific microsomal cytochrome P-450 isozymes has not been definitively established. Milk xanthine oxidase is also capable of catalyzing ronidazole activation. Polyacrylamide sodium dodecyl sulfate (SDS)-gel electrophoresis reveals that the reactive intermediate(s) of ronidazole does not alkylate proteins selectively.     

Drug residue formation from ronidazole, a 5-nitroimidazole. III. Studies on the mechanism of protein alkylation in vitro

Ronidazole (1-methyl-5-nitroimidazole-2-methanol carbamate) is reductively metabolized by liver microsomal and purified NADPH-cytochrome P-450 reductase preparations to reactive metabolites that covalently bind to tissue proteins. Kinetic experiments and studies employing immobilized cysteine or blocked cysteine thiols have shown that the principal targets of protein alkylation ara cysteine thiols. Furthermore, ronidazole specifically radiolabelled with 14C in the 4,5-ring, N-methyl or 2-methylene positions give rise to equivalent apparent covalent binding suggesting that the imidazole nucleus is retained in the bound residue. In contrast, the carbonyl-14C-labeled ronidazole gives approx. 6--15-fold less apparent covalent binding indicating that the carbamoyl group is lost during the reaction leading to the covalently bound metabolite. The conversion of ronidazole to reactive metabolite(s) is quantitative and reflects the amazing efficiency by which this compound is activated by microsomal enzymes. However, only about 5% of this metabolite can be accounted for as protein-bound products under the conditions employed in these studies. Consequently, approx. 95% of the reactive ronidazole metabolite(s) can react with other constituents in the reaction media such as other thiols or water. Based on these results, a mechanism is proposed for the metabolic activation of ronidazole.     

Structural changes in bovine oocytes during final maturation in vivo

On the basis of structural observations bovine oocytes were grouped into four successive classed: 0, those before the luteinizing hormone (LH) surge; 1, those up to 8 h following the LH peak level; 2, those between 8 and 19 h after the LH peak level; and 3, those between 19 h after the LH peak level and ovulation. Oocytes in class 0 had mitochondria located in a generally peripheral position. Interior to the mitochondria were elements of rough endoplasmic reticulum (RER) and numerous membrane-bound vesicles which bore ribosome-like particles on their outer surface. The first visible changesater the LH peak level as seen in class 1 were the formation of the periviteline space with loss of contact between the cumulus cells and the oocyte, and ruffing of the nuclear envelope. These changes were followed b the resumption of meiosis as defined by germinal-vesicle breakdown (GVBD), the disappearance of RER, and the formation fo clusters of mitochondria in association with lipid droplets and elementrs of smooth endolasmic reticulum (SER). The period between 8 and 19 h following LH peak level (class 2) was characterized by intensive clustering of mitochoncria in association with lipid droplets and elements of SER, conversion of lipid, fusion of vesicles, and the appearance of ribosomes in the cytoplasm. During the final stage (class 3), the polar body was extruded, the mitochondria dispersed, and the majority of the organelles became located toward the center of the cell. The relatively organelle-free cortical region contained cortical granules immediately adjacent to the plasma membrane together with aggregates of tubular SER. The structural changes are discussed in the context of follicular steroidogenesis and oocyte developmental competence.