Screening of thermostable leucine and alanine dehydrogenases in thermophilicBacillus strains

Summary Screening of leucine and alanine dehydrogenases in thermophilicBacillus strains was carried out to develop their utilization for industrial and analytical catalysts. Out of the 28 thermophilic strains tested, four strains,Bacillus sp. DSM 405, 730 and 1521, andB. sphaericus DSM 462, abundantly produce both the enzymes. Both the enzyme activities in these thermophiles are enhanced by addition of the substrates to a polypeptone medium.     

Restriction fragment length polymorphism detected by human salivary amylase cDNA

Summary The plasmid clone which contains human salivary amylase cDNA was used to detect restriction fragment length polymorphisms (RFLPs). After double digestion with Pst 1 and Bam H1, a polymorphism with two alleles was observed. In Japanese, frequencies of these alleles, tentatively called 5.7kb and 6.5kb fragment alleles, are 0.55 and 0.45, respectively.     

Kinetics of formation of maltose and isomaltose through condensation of glucose by glucoamylase

A kinetic model was devised for the hydrolysis and synthesis of maltose and isomaltose by two glucoamylases from Rhizopus niveus and Aspergillus niger, and the validity of the model was verified experimentally at 313 K and pH 5.0. For both enzymes, the formations of maltose and isomaltose from glucose were parallel reversible reactions, and glucosyl transfer between maltose and isomaltose was not observed. The enzymes catalyzed rapid hydrolysis and synthesis of maltose. Isomaltose was hydrolyzed and synthesized more slowly, but the level produced from glucose was much higher than that of maltose. These hydrolysis and condensation reactions were expressed well by the model.     

Influence of predation on species coexistence in Volterra models

The possibility of predator-mediated coexistence of all species in model ecosystems of the Volterra type is discussed, that is, asymptotic behaviors of systems of two competing species are analyzed when one or two predators are added. All species in the communities can coexist in two distinct ways mathematically, that is, the species may coexist at equilibrium or may coexist in persistent oscillations. The stability of all species at equilibrium increases when one or two predators are added. The conditions for oscillatory coexistence in limit cycles or in chaotic behaviors of two-predator systems are more complicated than in those of one-predator systems. It is concluded that predator-mediated coexistence can be promoted by an intimate relationship between the competitive ability of the prey and the diet preference of the predators.     

Characterization of the env gene and long terminal repeat of molecularly cloned Friend mink cell focus-inducing virus DNA

The highly oncogenic erythroleukemia-inducing Friend mink cell focus-inducing (MCF) virus was molecularly cloned in phage lambda gtWES.lambda B, and the DNA sequences of the env gene and the long terminal repeat were determined. The nucleotide sequences of Friend MCF virus and Friend spleen focus-forming virus were quite homologous, supporting the hypothesis that Friend spleen focus-forming virus might be generated via Friend MCF virus from an ecotropic Friend virus mainly by some deletions. Despite their different pathogenicity, the nucleotide sequences of the env gene of Friend MCF virus and Moloney MCF virus were quite homologous, suggesting that the putative parent sequence for the generation of both MCF viruses and the recombinational mechanism for their generation might be the same. We compare the amino acid sequences in lymphoid leukemia-inducing ecotropic Moloney virus and Moloney MCF virus, and erythroblastic leukemia-inducing ecotropic Friend virus, Friend-MCF virus, and Friend spleen focus-forming virus. The Friend MCF virus long terminal repeat was found to be 550 base pairs long. This contained two copies of the 39-base-pair tandem repeat, whereas the spleen focus-forming virus genome contained a single copy of the same sequence.     

Demonstration of bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes by monoclonal antibodies

Calmodulin-dependent cyclic nucleotide phosphodiesterase from bovine brain is found to be composed of two distinct subunits, 60,000- and 63,000-dalton polypeptides. Peptide mapping of the subunits by partial proteolysis demonstrated that the 60-kDa polypeptide is not derived from the 63-kDa species. The interaction of the enzyme with three monoclonal antibodies, A6, C1, and A2, and the analysis of immunocomplexes by sucrose density gradient centrifugation revealed that calmodulin-dependent cyclic nucleotide phosphodiesterase exists in three different forms, i.e. (a) homodiamer of 60-kDa, (b) heterodimer of 60- and 63-kDa, and (c) homodimer of 63-kDa. A6 antibody reacts with both 60- and 63-kDa polypeptides indicating that they are immunologically related. C1 and A2 antibodies react with only 60-kDa polypeptide species. By using C1 Sepharose 4B affinity column chromatography, the 63-kDa homodimer which did not bind to the column (Fraction I) was separated from the 60-kDa polypeptide containing isozymes (the heterodimer and the 60-kDa homodimer) which were retained on the column and later eluted as a mixture (Fraction II). Fraction I, the 63-kDa homodimer enzyme, has higher Vmax toward cGMP as substrate than cAMP whereas the opposite was found with Fraction II. The specific activity of Fraction II enzyme toward cAMP was approximately 500 mumol/min/mg, the highest value ever reported for brain calmodulin-dependent cyclic nucleotide phosphodiesterase preparations.     

Probability of paternity exclusion and the number of loci needed to determine the fathers in a troop of macaques

We calculated the probability of paternity exclusion (P) in 6 troops of rhesus and Japanese macaques housed in open enclosures and 68 wild troops of Japanese, crab-eating, and toque macaques using 33 genetic loci which encoded the blood protein variations detected by electrophoretic techniques. In the open enclosures, especially of rhesus troops, we obtained a fairly high probability of paternity exclusion and succeeded in determining the fathers of offspring. However, we found significant differences between the observed and calculated probabilities in most of the troops. These differences were ascribed to a situation whereby the Hardy-Weinberg equilibrium had not been attained in the troops and/or the numbers of variable loci were too small. In the wild troops of Japanese, crab-eating, and toque macaques, the means ofP were 0.2274 (0.0192–0.5017), 0.4635 (0.1676–0.7151), and 0.7382 (0.6266–0.7954), respectively. We also estimated the number of loci needed to determine the fathers of offspring with a probability of 0.8 assuming that ten males were present in the troop. The estimated number was about 13.5 times, 5 times and 1.8 times the number of loci examined on average in the troops of Japanese, crab-eating and toque macaques, respectively. This means that determination of most of the fathers of offspring in wild troops of these macaques, even of toque macaques which have a rather high probability of paternity exclusion, is difficult so long as we employ only electrophoretic techniques.     

Identification of the native form of chicken gizzard myosin light chain kinase with the aid of monoclonal antibodies

Examination, by immunoblotting, of myosin light chain kinase-containing fractions obtained during purification of the enzyme from chicken gizzard has shown that a single species (Mr = 136,000) exists in the muscle and that this enzyme is degraded, primarily to a 130,000-dalton fragment, during purification. These conclusions were confirmed by phosphorylation of the different species of myosin light chain kinase by the isolated catalytic subunit of cyclic AMP-dependent protein kinase.     

Radiometric assays for mammalian epoxide hydrolases and glutathione S-transferase

A number of epoxides, including cis- and trans-stilbene oxides, were assayed as substrates for epoxide hydrolases (EHs) by gas-liquid chromatography. Radiolabeled stilbene oxides were prepared by sodium borotritide reduction of desyl chloride followed by ring closure with base treatment. Rapid radiometric assays for EHs were performed by differential partitioning of the epoxide into dodecane, while the product diol remained in the aqueous phase. Glutathione (GSH) transferase was similarly assayed by partitioning the epoxide and diol, if formed metabolically, into 1-hexanol, while the GSH conjugate was retained in the aqueous phase. The cytosolic EH rapidly hydrates the trans isomer while the cis is very poorly hydrated. In contrast, the cis is a better substrate for the microsomal EH than the trans. GSH transferase utilized both epoxides as substrates, but conjugation is faster with the cis isomer. Cytosolic EH activity is high in mouse but very low in rat and guinea pig. Microsomal EH activity, in contrast, is highest in guinea pig, intermediate in rat, and the lowest in mouse. GSH transferase activity, which is high in all three species, can be inhibited by chalcone, with an I₅₀ of 3.1 × 10^?5m. These assays facilitate the rapid evaluation and direct comparison of epoxide-metabolizing systems in cell homogenates used in short-term mutagenicity assays, cell or organ culture, and possibly in vivo.