Effect of synthetic physalaemin on splanchnic circulation in dogs

Physalaemin has been reported as one of the most potent vasodilator and hypotensive peptides (1-4). In spite of these studies, however, the effect of the peptide on splanchnic circulation is not known precisely. In the present study, the effect of synthetic physalaemin on superior mesenteric arterial blood flow, portal venous blood flow and pancreatic capillary blood flow was investigated in dogs. Dose dependent increases of superior mesenteric arterial blood flow and portal venous blood flow were induced in response to physalaemin (0.1-10.0 ng/kg). Superior mesenteric arterial blood flow and portal venous blood flow attained maximal increases of 77 +/- 8.9% and 70 +/- 8.6%, respectively, at a dose of 5 ng/kg. Physalaemin caused a dose-related decrease in systemic arterial blood pressure. Pancreatic capillary blood flow did not show significant change with the administration of physalaemin. These data suggest that physalaemin may play some physiological roles in the regulation of splanchnic circulation.     

Two types of cytochrome cd1 in the aerobic photosynthetic bacterium, Erythrobacter sp. OCh 114

Components I and II of cytochrome cd1 which had different spectral features were purified from the aerobic photosynthetic bacterium, Erythrobacter sp. strain OCh 114. Component I showed an absorption maxima at 700 and 406 nm in the oxidized form, and at 621, 552.5, 548 and 416 nm in the reduced form. Component II showed an absorption maxima at 635 and 410 nm in the oxidized form and at 628, 552.5, 548 and 417 nm in the reduced form. The relative molecular mass, Mr, of both cytochromes was determined to be 135,000 with two identical subunits. Components I and II showed pI values of 7.6 and 6.8, respectively. The redox potential of hemes ranged from +234 mV to +242 mV, except for the heme d1 of component I (Em7 = +134 mV). Components I and II showed both cytochrome c oxidase and nitrite reductase activities. Cytochrome c oxidase activity was strongly inhibited by a low concentration of nitrite and cyanide. Erythrobacter cytochromes c-551 and c-552 were utilized as electron donors for the cytochrome c oxidase reaction. The high affinity of cytochrome c-552 to component II (Km = 1.27 microM) suggested a physiological significance for this cytochrome. Erythrobacter cytochromes cd1 are unique in their presence in cells grown under aerobic conditions as compared to other bacterial cytochromes cd1 which are formed only under denitrifying conditions.     

Role of endocrine pancreas in temperature acclimation

Role of endocrine pancreas in temperature acclimation in rats was investigated. Plasma glucagon level increased and insulin level decreased in cold-acclimated rats (CA). The reverse was observed in heat-acclimated rats (HA). In the pancreas there were no changes in glucagon and insulin in CA, but a decrease in glucagon and an increase in insulin were found in HA. Plasma insulin/glucagon molar ratio (I/G) declined in CA and rose in HA. Pancreatic I/G rose in HA. Acute cold exposure elevated plasma glucagon, but did not affect plasma insulin. Pancreatic glucagon, insulin and I/G were not influenced by acute cold exposure, while plasma I/G decreased. Plasma I/G was inversely correlated with both blood free fatty acids and glucose levels. These results suggest that endocrine pancreas is closely associated with metabolic acclimation to cold and heat through its regulation of the metabolic direction to catabolic phase in cold acclimation and to anabolic phase in heat acclimation.     

Modified colorimetric ninhydrin methods for peptidase assay

Four colorimetric procedures suitable for the determination of peptidase activity on peptides having a free α- or ε-amino group are described. Two of the methods (A and B) are modifications of the conventional ninhydrin method described by S. Moore and W. H. Stein ((1948) J. Biol. Chem.176, 367–388; (1954) Ibid.211, 907–913); the heating time is shortened to 5 min at 100°C and the pH of the buffer in the reagent is lowered to 4.0. Method A differs from method B in buffer concentration. The other two methods (C and D) are modifications of the Cd-ninhydrin method described by A. P. Tsarichenko ((1966) Nauch. Tr. Krasnodar. Gos. Pedagog. Inst.70, 86–88, as cited in Chem. Abs.67, 79479c); the water content in the reagent is reduced to $\text{1}\text{20}$ of the original reagent and the sample is heated for 5 min at 84°C. Method C differs from method D in the ratio of sample to reagent. In contrast to the free amino acids which are sufficiently colored, various peptides and amino acid derivatives except for the glycylpeptides give only a faint color with these methods. These four methods are not only useful for the determination of peptidase activity on peptides (e.g., Leu-Gly and tert-butyloxycarbonyl-glycyl-lysyl-leucine), but are also useful for the determinations of amidase activity on amino acid amides (e.g., Leu-NH₃) and esterase activity on amino acid esters (e.g., tyrosine ethyl ester).     

NADH2- and succinate-dependent O2 uptake in chromatophores from Chromatium vinosum

The O₂ uptake linked to NADH₂ and succinate oxidation was observedin chromatophores from photosynthetically grown Chromatium vinosum. The maximal rate was 60–120 nmoles of O₂ uptake per minper {diaeresis}mole of bacterio-chlorophyll. The rate of O₂uptake linked to NADH₂ oxidation was higher in the neutral-to-acidicpH range than in the alkaline range, whereas that linked tosuccinate oxidation was higher in the alkaline range. The O₂ uptake linked to NADH₂ oxidation was inhibited by rotenone,amytal, antimycin A, KCN and NaN₃, while that linked to succinateoxidation was inhibited by antimycin A, KCN and NaN₃. Malate,citrate, pyruvate, acetate, -ketoglutarate, NADPH₂ and thiosulfatedid not serve as substrates for the O₂ uptake of isolated chromatophores. The rates of the O₂ uptake linked to both NADH₂ and succinateoxidation were not stimulated by adding uncouplers or underphosphorylating conditions. Little or no ATP was synthesizedin the dark, coupled to either NADH₂ or succinate oxidation,in spite of a high activity of photophosphorylation in Chromatiumchromatophores. (Received February 26, 1980; )     

Template-independent poly(A) x poly(U) synthesizing activity of different forms of Bacillus subtilis RNA polymerase

The steady-state kinetic parameters of the tripeptides D-Val-Leu-Lys-, Ala-Phe-Lys-, and < Glu-Phe-Lys- in which the free carboxyl group was substituted with p-nitroaniline (substrate) or chloromethane (inhibitor), towards the serine proteinases plasmin (EC 3.4.21.7), thrombin (EC 3.4.21.5), urokinase, factor Xa, and trypsin (EC 3.4.21.4) were investigated. The p-nitroanilide derives were found to be very good substrates for plasmin, 2.5--40-times less efficient towards trypsin and very poor (100--10 000-times less efficient) substrates for thrombin, factor Xa and urokinase. The chloromethyl ketone derivatives were comparably efficient inhibitors of plasmin and trypsin and in general very poor (100--10 000-times weaker) inhibitors of thrombin, factor Xa and urokinase. D-Val-Leu-Lys-pNA however was a very poor substrate but D-Val-Leu-Lys-CH2Cl a very efficient inhibitor for thrombin. The variability in susceptibility of the substrates towards the enzymes was due to differences in their Michaelis constant, in their deacylation rate constant or both. the variable efficiency of the inhibitors was mostly due to differences in their dissociation constant and much less to differences in their alkylation rate constant. Only a poor correlation (r = 0.25) was found between the efficiency of the p-nitroanilides as substrate and their homologous chloromethyl ketones as inhibitor. The most notable discrepancy was observed with the D-Val-Leu-Lys derivatives towards thrombin.     

Induction of G1 cell cycle arrest in human umbilical vein endothelial cells by flavone's inhibition of the extracellular signal regulated kinase cascade.

Dietary flavonoids have demonstrated anti-carcinogenic activity in several animal models, but their mechanisms of action have not yet been clearly established. Here, we show that flavone, a parent compound of flavonoids, inhibits the proliferation, migration, and capillary tube formation of human umbilical vein endothelial cells (HUVECs). Flow cytometric analysis showed that flavone arrests the cell cycle progression at G(1) phase in HUVECs. We observed the down-regulation of the hyperphosphorylated form of retinoblastoma gene product and cyclin-dependent kinases 2 and 4 in flavone-treated cells, but it had no affect on the expression of p53 and cyclin-dependent kinase inhibitors p21(CIP/Waf1) and p27(Kip). Flavone almost completely inhibited the activation of extracellular signal regulated kinase 1. The present results suggest that the flavone moiety of flavonoids is required for anti-proliferative activity of flavonoids and that anti-carcinogenic action of flavonoids in vivo was mediated, at least in part, by inhibiting angiogenesis.     

Genetic Analysis of Comamonas acidovorans Polyhydroxyalkanoate Synthase and Factors Affecting the Incorporation of 4-Hydroxybutyrate Monomer

The polyhydroxyalkanoate (PHA) synthase gene of Comamonas acidovorans DS-17 (phaC_Ca) was cloned by using the synthase gene of Alcaligenes eutrophus as a heterologous hybridization probe. Complete sequencing of a 4.0-kbp SmaI-HindIII (SH40) subfragment revealed the presence of a 1,893-bp PHA synthase coding region which was followed by a 1,182-bp β-ketothiolase gene (phaA_Ca). Both the translated products of these genes showed significant identity, 51.1 and 74.2%, respectively, to the primary structures of the products of the corresponding genes in A. eutrophus. The arrangement of PHA biosynthesis genes in C. acidovorans was also similar to that in A. eutrophus except that the third gene, phaB, coding for acetoacetyl-coenzyme A reductase, was not found in the region downstream of phaA_Ca. The cloned fragment complemented a PHA-negative mutant of A. eutrophus, PHB⁻4, resulting in poly-3-hydroxybutyrate accumulation of up to 73% of the dry cell weight when fructose was the carbon source. The heterologous expression enabled the incorporation of 4-hydroxybutyrate (4HB) and 3-hydroxyvalerate monomers. The PHA synthase of C. acidovorans does not appear to show any preference for 4-hydroxybutyryl-coenzyme A as a substrate. This leads to the suggestion that in C. acidovorans, it is the metabolic pathway, and not the specificity of the organism’s PHA synthase, that drives the incorporation of 4HB monomers, resulting in the efficient accumulation of PHA with a high 4HB content.     

Expression and Characterization of (R)-Specific Enoyl Coenzyme A Hydratase Involved in Polyhydroxyalkanoate Biosynthesis by Aeromonas caviae

Complementation analysis of a polyhydroxyalkanoate (PHA)-negative mutant of Aeromonas caviae proved that ORF3 in the pha locus (a 402-bp gene located downstream of the PHA synthase gene) participates in PHA biosynthesis on alkanoic acids, and the ORF3 gene is here referred to as phaJ_Ac. Escherichia coli BL21(DE3) carrying phaJ_Ac under the control of the T7 promoter overexpressed enoyl coenzyme A (enoyl-CoA) hydratase, which was purified by one-step anion-exchange chromatography. The N-terminal amino acid sequence of the purified hydratase corresponded to the amino acid sequence deduced from the nucleotide sequence of phaJ_Ac except for the initial Met residue. The enoyl-CoA hydratase encoded by phaJ_Ac exhibited (R)-specific hydration activity toward trans-2-enoyl-CoA with four to six carbon atoms. These results have demonstrated that (R)-specific hydration of 2-enoyl-CoA catalyzed by the translated product of phaJ_Ac is a channeling pathway for supplying (R)-3-hydroxyacyl-CoA monomer units from fatty acid β-oxidation to poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) biosynthesis in A. caviae.