Role of kallikrein-kininogen system in insulin-stimulated glucose transport after muscle contractions.

Serum proteins [molecular weight (MW) > 10,000] are essential for increased insulin-stimulated glucose transport after in vitro muscle contractions. We investigated the role of the kallikrein-kininogen system, including bradykinin, which is derived from kallikrein (MW > 10,000)-catalyzed degradation of serum protein kininogen (MW > 10,000), on this contraction effect. In vitro electrical stimulation of rat epitrochlearis muscles was performed in 1) rat serum +/- kallikrein inhibitors; 2) human plasma (normal or kallikrein-deficient); 3) rat serum +/- bradykinin receptor-2 inhibitors; or 4) serum-free buffer +/- bradykinin. 3-O-methylglucose transport (3-MGT) was measured 3.5 h later. Serum +/- kallikrein inhibitors tended (P = 0.08) to diminish postcontraction insulin-stimulated 3-MGT. Contractions in normal plasma enhanced insulin-stimulated 3-MGT vs. controls, but contractions in kallikrein-deficient plasma did not. Supplementing rat serum with bradykinin receptor antagonist HOE-140 during contraction did not alter insulin-stimulated 3-MGT. Muscles stimulated to contract in serum-free buffer plus bradykinin did not have enhanced insulin-stimulated 3-MGT. Bradykinin was insufficient for postcontraction-enhanced insulin sensitivity. However, results with kallikrein inhibitors and kallikrein-deficient plasma suggest kallikrein plays a role in this improved insulin action.     

Transmitter release in hippocampal slices from rats with limbic seizures produced by systemic administration of kainic acid

The systemic injection of kainic acid (KA) has been shown to destroy neurons in the hippocampus and to induce limbic-type seizure activity. However, little is known on the neurochemical events that are associated with this convulsant effect. In the present work we studied the spontaneous and the K⁺-stimulated release of labeled -aminobutyric acid (GABA), glutamate, serotonin and dopamine, in hippocampal slices of KA-treated rats, at the moment of clinical seizures (2 h) and 72 h later. At the onset of convulsions we found a 40–45% decrease in the K⁺-stimulated release of GABA. The release of the other neurotransmitters was not significantly affected by KA treatment. After 72 h GABA release was still reduced by 30–40%. It is concluded that the epileptogenic effect of KA in the hippocampus is probably related to a diminished inhibitory GABAergic neurotransmission.     

The amino-terminal half of rotavirus SA114fM VP4 protein contains a hemagglutination domain and primes for neutralizing antibodies to the virus.

We have previously reported the synthesis in Escherichia coli of polypeptide MS2-VP8', which contains the amino-terminal half of the SA114fM VP4 protein fused to MS2 bacteriophage polymerase sequences (C. F. Arias, M. Lizano, and S. López, J. Gen. Virol. 68:633-642, 1987). In this work we have synthesized the carboxy-terminal half of the VP4 protein also fused to the MS2 polymerase. This protein, designated MS2-VP5', was recognized by sera to the complete virion and was able to induce antibodies to the virus when administered to mice; however, these antibodies had no neutralizing activity. The two chimeric polypeptides were tested for their ability to agglutinate erythrocytes and to prime the immune system of mice. Bacterial lysates enriched for the MS2-VP8' hybrid polypeptide, but not those enriched for the MS2-VP5' protein or those containing proteins from the host E. coli strain, had hemagglutinating activity. This hemagglutination was inhibited by sera to SA114fM rotavirus. In addition, a single dose of the MS2-VP8' polypeptide was able to prime the immune system of mice for an augmented neutralizing antibody response when the animals were subsequently immunized with purified SA114fM virus.     

Distinctions between the multiple cationic forms of rat liver glutathione S-transferase

Three cationic glutathione S-transferase forms isolated from rat liver were characterized as dimers that originated from different combinations of two subunit types, Ya and Yc. The cationic forms were purified using lysyl glutathione affinity matrices and were chromatographically resolved from anionic glutathione S-transferases that contain Yb subunits. The three classes of cationic transferase exhibited similar specific activities with 1-chloro-2,4-dinitrobenzene as a substrate, all forms cross-reacted with antibodies to glutathione S-transferase B, and all had comparable secondary structures and tryptophan fluorescence properties. In spite of those similarities, the Yc-containing forms were clearly distinguishable from Ya forms on the basis of characteristic differences in circular dichroic patterns associated with their aromatic side chains. All cationic transferases bound bilirubin with stoichiometric ratios of 1 mol/dimeric protein molecule, but discrete differences in mode of binding were ascribed to forms containing Ya subunits as compared to Yc dimers. Binding to Yc forms was of lower affinity and may be associated with the catalytic region of the protein since glutathione effectively displaced bilirubin from the Yc component.     

The sinusoidal domain of the plasma membrane of rat hepatocytes contains an amiloride-sensitive Na+/H+ antiport

ATP-dependent trapping of [14C]methylamine was demonstrated in vesicles selectively derived from the sinusoidal plasma membrane of rat hepatocytes; activity was lacking in vesicles prepared from the canalicular domain of the plasma membrane of rat hepatocytes. The proton movement was inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, strophanthidin, vanadate, amiloride, and absence of sodium. 22Na efflux from sinusoidal membrane vesicles increased inversely to extravesicular pH. The results indicate that the sinusoidal plasma membrane of rat hepatocytes contains a Na+/H+ antiport.     

The mechanism of biliary secretion of reduced glutathione. Analysis of transport process in isolated rat-liver canalicular membrane vesicles

Transport of reduced glutathione (GSH) was studied in isolated rat liver canalicular membrane vesicles by a rapid filtration technique. The membrane vesicles exhibit uptake of [2-3H]glycine--labeled GSH into an osmotically reactive intravesicular space. Although the canalicular membrane vesicles possess gamma-glutamyltransferase and aminopeptidase M, enzymes that hydrolyze glutathione into component amino acids, inactivation of the vesicle-associated transferase by affinity labeling with L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) had no effect on the initial rate of GSH transport. Chemical analysis revealed that intact GSH accounted for most of vesicle-associated radioactivity. The initial rate of transport followed saturation kinetics with respect to GSH concentration; an apparent Km of 0.33 mM and V of 1.47 nmol/mg protein in 20 s were calculated. These results indicate that transport of GSH across the canalicular membranes is a carrier-mediated process. Replacement of NaCl in the transport medium by KCl, LiCl or choline chloride had no effect on the transport activity of the vesicles. The rate of GSH uptake by the vesicles was enhanced by valinomycin-induced K+-diffusion potential (vesicle inside-positive) and was inhibited by probenecid, indicating that GSH transport across the canalicular membranes is electrogenic and involves the transfer of negative charge. The transport of GSH was inhibited by oxidized glutathione or S-benzyl-glutathione. This transport system in canalicular plasma membranes may function in biliary secretion of GSH and its derivatives which are synthesized in hepatocytes by oxidative processes or glutathione S-transferase.     

The effects of mating design on introgression between chromosomally divergent sunflower species

Population genetic theory suggests that mating designs employing one or more generations of sib-crossing or selfing prior to backcrossing are more effective than backcrossing alone for moving alleles across linkage groups where effective recombination rates are low (e.g., chromosomally divergent linkages). To test this hypothesis, we analyzed the effects of chromosomal structural differences and mating designs on the frequency and genomic distribution of introgressed markers using the domesticated sunflower, Helianthus annuus, and one of its wild relatives, H. petiolaris, as the experimental system. We surveyed 170 progeny, representing the end products of three different mating designs (design I, P-F₁-BC₁-BC₂-F₂-F₃; design II, P-F₁-F₂-BC₁-BC₂-F₃; and design III, P-F₁-F₂-F₃-BC₁-BC₂), for 197 parental RAPD markers of known genomic location. Comparison of observed patterns of introgression with expectations based on simulations of unrestricted introgression revealed that much of the genome was protected from introgression regardless of mating design or chromosomal structural differences. Although the simulations indicated that all markers should introgress into multiple individuals in each of the three mating designs, 20 of 58 (34%) markers from collinear linkage groups, and 112 of 139 (81%) markers from rearranged linkage groups did not introgress. In addition, the average size of introgressed fragments (12.2 cM) was less than half that predicted by theoretical models (26–33 cM). Both of these observations are consistent with strong selection against introgressed linkage blocks, particularly in chromosomally divergent linkages. Nonetheless, mating designs II and III, which employed one and two generations of sib-mating, respectively, prior to backcrossing, were significantly more effective at moving alleles across both collinear and rearranged linkages than mating design I, in which the backcross generations preceded sib-mating. Thus, breeding strategies that include sib-crossing, in combination with backcrossing, should significantly increase the effectiveness of gene transfer across complex genic or chromosomal sterility barriers.     

Short communication: Effect of water activity on cell growth and phenol oxidase production by Streptomyces cyaneus var. viridochromogenes in surface culture

In surface cultures of Streptomyces cyaneus var. viridochromogenes, NaCl depressed water activity (a _w) without supporting growth. Reducing a _w from 0.987 to 0.951 led to 3- and 4-fold increases in intracellular and extracellular phenol oxidase activities, respectively.