Molybdate transport by Bradyrhizobium japonicum bacteroids.

Bacteroid suspensions of Bradyrhizobium japonicum USDA 136 isolated from soybeans grown in Mo-deficient conditions were able to transport molybdate at a nearly constant rate for up to 1 min. The apparent Km for molybdate was 0.1 microM, and the Vmax was about 5 pmol/min per mg (dry weight) of bacteroid. Supplementation of bacteroid suspensions with oxidizable carbon sources did not markedly increase molybdate uptake rates. Anaerobically isolated bacteroids accumulated twice as much Mo in 1 h as aerobically isolated cells did, but the first 5 min of molybdate uptake was not dependent on the isolation condition with respect to O2. Respiratory inhibitors such as cyanide, azide, and hydroxylamine did not appreciably affect molybdate uptake, even at concentrations that inhibited O2 uptake. The uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and the ionophores nigericin and monensin significantly inhibited molybdate uptake. The electrogenic ionophores valinomycin and gramicidin stimulated molybdate uptake. Rapid pH shift experiments indicated that molybdate transport depends on a transmembrane proton gradient (delta pH), and it is probably transported electroneutrally as H2MoO4. Most of the 99MoO4(2-) taken up was not exchangeable with a 100-fold excess of unlabeled MoO4(2-). Tungstate was a competitive inhibitor of molybdate uptake, with a Ki of 0.034 microM, and vanadate inhibited molybdate uptake slightly.     

The effects of nonpenetrating cryoprotectants added to TEST-yolk-glycerol extender on the post-thaw motility of ram spermatozoa

The effects of adding five different concentrations of 17 polymeric compounds to TEST-yolk-glycerol extender on ram spermatozoa survival was studied. These were Aquacide (I, II, and III); dextran (0.8-1.6, 1.9, 15-20, 70, and 200-300 kDa); three types of Dri-Sweet; hydroxyethyl starch; methylcellulose, polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, and Supercol 912. All the compounds tested except the Dri-Sweet compounds and hydroxyethyl starch significantly (P less than 0.05) decreased percentages of motile cells in unfrozen samples. The use of dextran (0.8-1.6 kDa; hydrolyzed dextran separated by ethanol) and Aquacide II significantly (P less than 0.05) increased post-thaw motility of spermatozoa frozen in pellets. Dextran (15-20 kDa), dextran (0.8-1.6 kDa), Aquacide II, and hydroxyethyl starch significantly (P less than 0.05) increased the percentages of post-thaw motility of ram spermatozoa frozen in the presence of glycerol and egg yolk.     

Relationships between the human pepsinogen DNA and protein polymorphisms.

Pepsinogens (PGA) are the inactive precursors of pepsin, the major acid protease found in the stomach. The PGA gene family exhibits polymorphic variation in human populations that can either be demonstrated by electrophoretic analysis of the proteins or by analysis of the respective genes with cDNA probes. Here, we describe the interrelationships between the most common pepsinogen protein phenotypes and the corresponding pepsinogen haplotypes (A, B, and C) containing different combinations of the PGA3, PGA4, and PGA5 genes. We propose that this unusual genetic variation involving haplotypes that contain three, two, and one genes, respectively, is the result of molecular evolution by gene duplication.     

Growth hormone regulates the abundance of insulin-like growth factor I RNA in adult rat liver

Insulin-like growth factor I (IGF-I) is a mitogenic polypeptide present in the plasma of man and rat that is thought to mediate the actions of pituitary growth hormone on cartilage to promote skeletal elongation. In the rat, plasma levels of IGF-I show both developmental and hormonal regulation: levels are low at birth, increase with age, and are decreased in growth hormone-deficient adult animals. The present study demonstrates that these changes in plasma IGF-I reflect the abundance of IGF-I RNA in rat liver. A human IGF-I cDNA probe hybridized to multiple RNA species in adult rat liver with sizes 8.6, 4.6, 3.2, 2.1, and 1.0-1.4 kilobases. These RNA species were decreased by greater than 80% in neonatal (2- and 12-day-old) rat liver and by greater than 90% in liver from adult rats made growth hormone-deficient by hypophysectomy. Treatment of hypophysectomized rats with growth hormone increased the abundance of all species of IGF-I RNA. These results suggest that growth hormone regulates the expression of its physiological mediator by altering the synthesis, stability, or both of IGF-I RNA in rat liver.     

Fermentation by a new daunomycin-producing organism, Streptomyces insignis ATCC 31913.

A new organism belonging to the grey series of streptomycetes is described which produces 55 to 75 micrograms of daunomycin per ml in a sparged fermentor. This organism is not taxonomically related to other known daunomycin producers. Its proposed name in Streptomyces insignis ATCC 31913.     

Wound-induced proteinase inhibitors from tomato leaves. I. The cDNA-deduced primary structure of pre-inhibitor I and its post-translational processing

A cDNA containing the coding region for the complete amino acid sequence of wound-induced proteinase Inhibitor I from tomato leaves was constructed in the plasmid pUC9 and characterized. The open reading frame codes for a protein of 111 amino acids. This deduced amino acid sequence revealed the presence of a 42-amino acid N-terminal sequence that is not found in the native protein. This sequence appears to contain a 23-amino acid segment typical of a signal sequence followed by a 19-amino acid sequence containing 9 charged amino acids. The 42-amino acid sequence is apparently lost during maturation to the native Inhibitor I and represents 38% of the translated protein. The Inhibitor I amino acid sequence contains 71% identity with potato tuber Inhibitor I sequence and 35% identity with an inhibitor from the leech.     

Hepatic adenylate cyclase. Development-dependent coupling to the beta-adrenergic receptor in the neonate

Guanine nucleotide-dependent modulation of agonist binding to the beta-receptor reflects coupling of the receptor to the nucleotide regulatory protein. Similarly, guanine nucleotide-dependent stimulation of adenylate cyclase can be used as an index of coupling between the regulatory protein and the catalytic unit of the cyclase. Using both approaches we have studied coupling in the beta-adrenergic receptor-adenylate cyclase system in rabbit liver during neonatal development. With [3H]dihydroalprenolol as ligand, the Bmax was relatively unchanged (200-300 fmol/mg of protein) between birth and end of day 1 and was similar to adult values. Guanyl-5'-yl imidodiphosphate-dependent shift in agonist (l-isoproterenol) competition curves was biphasic, decreasing from 10-fold in membranes isolated from animals at term to about 6-fold in membranes from 6-h-old neonates, and increasing progressively in older animals to a maximal measurable value of 42-fold in the adult. The ability of guanyl-5'-yl imidodiphosphate, GTP, GTP plus isoproterenol, NaF, or forskolin to activate adenylate cyclase was also biphasic and age-dependent. With Mn2+ the measured activity was not at any time greater than the activity at term. Pretreatment of membranes with cholera toxin resulted in differential levels of enhancement of adenylate cyclase activity wherein much lower enhancement was observed in membranes from neonatal animals. With [32P]NAD as substrate, cholera toxin-catalyzed ADP-ribosylation of membranes indicated development-dependent accumulation of Ns peptides. From these results we suggest that there is a decreased efficiency in the coupling of the beta-adrenergic receptor to hepatic adenylate cyclase in early neonatal life. The molecular basis for the biphasic nature of the coupling is presently unclear.     

Isolation of enamelinlike proteins from blue shark (Prionace glauca) enameloid

A sequential dissociative extraction scheme was used to extract proteins from developing Blue Shark enameloid. The first extraction solution (4 M guanidine HC1) solubilized the polypeptides, mainly collagenous, not closely associated with the hydroxyapatite. The next extraction solution (4 M guanidine HC1, 0.5 M ethylenediaminetetraacedic acid (EDTA] solubilized the proteins more closely associated with the tooth mineral component. After extraction, the proteins were separated and isolated with gel electrophoresis. Protein molecular weights were determined and selected proteins were isolated for amino acid composition analysis. The two proteins isolated were tested for mammalian enamel protein antigenic determinants by a "Dot" immunobinding assay. The isolated proteins were enamelinlike by extraction criteria and amino acid composition. Further, the two proteins share antigenic determinants with mammalian enamel proteins.     

Genetic reconstruction and functional analysis of the repeating lipoyl domains in the pyruvate dehydrogenase multienzyme complex of Escherichia coli

The dihydrolipoamide acetyltransferase component (E2p) of the pyruvate dehydrogenase complex of Escherichia coli contains three highly homologous sequences of about 100 residues that are tandemly repeated to form the N-terminal half of the polypeptide chain. All three sequences include a lysine residue that is a site for lipoylation and they appear to form independently folded functional domains. These lipoyl domains are in turn linked to a much larger (about 300 residues) subunit-binding domain of the E2p chain that aggregates to form the octahedral inner core of the complex and also contains the acetyltransferase active site. In order to investigate whether individual lipoyl domains play different parts in the enzymic mechanism, selective deletions were made in vitro in the dihydrolipoamide acetyltransferase gene (aceF) so as to excise one or two of the repeating sequences. This was facilitated by the high degree of homology in these sequences, which allowed the creation of hybrid lipoyl domains that closely resemble the originals. Pyruvate dehydrogenase complexes incorporating these genetically reconstructed E2p components were purified and their structures were confirmed. It was found that the overall catalytic activity, the system of active site coupling, and the ability to complement pyruvate dehydrogenase complex mutants, were not significantly affected by the loss of one or even two lipoyl domains per E2p chain. No special role can be attached thus far to individual lipoyl domains. On the other hand, certain genetic deletions affecting the acetyltransferase domain caused inactivation of the complex, highlighting particularly sensitive areas of that part of the E2p chain.     

Transposon donor plasmids, based on ColIb-P9, for use in Pseudomonas putida and a variety of other gram negative bacteria

Summary The properties of pLG221, a derivative of the ColIb plasmid carrying the transposon Tn5 are described. This plasmid can be used to introduce Tn5 by conjugation from Escherichia coli into a variety of Gram negative bacteria outside the host range for maintenance of ColIb. Plasmid pLG221, and a similar plasmid pLG223 carrying Tn10 may be of general utility as vectors for transposon-mediated mutagenesis in a variety of Gram negative bacteria.