Method for isolating single cardiac cells in the guinea pig using pronase. The role of calcium ions

A method for isolation of guinea-pig cardiomyocytes with pronase has been developed. The method has been assessed in hearts perfused with solutions containing pronase (1 U/ml) and 200 microM Ca2+. Eighty per cent of the cells released were rod-shaped and 1.2 mM Ca2+ tolerant. Enriched medium 199 was used for all solutions. Sodium and slow inward currents recorded from cells dispersed with pronase were similar to those recorded from cells isolated after prolonged exposure to collagenase. Two principal factors are to be marked: (a) presence of high enough amounts of Ca2+ in enzyme solution (up to 200 microM); (b) use of the enriched medium in all the stages of the procedure.     

The role of molecular conformation in ion capture by carboxylic ionophores: a circular dichroism study of narasin A in single-phase solvents and liposomes

Conformational and thermodynamic aspects of cation binding by the carboxylic ionophore narasin A were studied by circular dichroism (CD). In single-phase solvents, dramatic increases in the maximum differential absorption (delta epsilon) of the C-11 carbonyl were observed upon the binding of K+, Na+ and protons to the free anionic form. These changes were associated with major shifts in the conformation equilibrium between extended and pseudocyclic conformers of narasin. Similar CD changes observed upon the binding of K+ to narasin A in dimyristoylphosphatidylcholine vesicles provided evidence that in the membrane environment, comparable conformation changes were associated with ion binding. Variation of the polar and protic properties of single-phase solvents was also found to influence the delta epsilon of the cation bound species of narasin A, supporting previous evidence for polarity-mediated modulation of conformation. Comparison of cation binding affinities indicated that in both single-phase solvents and liposomes, narasin had a marked equilibrium selectivity for K+ over Na+.     

Potential of Agrotis ipsilon nucleopolyhedrovirus for suppression of the black cutworm (Lepidoptera: Noctuidae) and effect of an optical brightener on virus efficacy.

Studies were performed in the laboratory, greenhouse and field to assess the potential of Agrotis ipsilon multicapsid nucleopolyhedrovirus (AgipMNPV) and a viral enhancing agent, M2R, for suppression of Agrotis ipsilon (Hufnagel). In laboratory droplet feeding bioassays, AgipMNPV was shown to be highly active against third-instar A. ipsilon. The optical brightener M2R significantly reduced LD50 estimates by approximately 160-fold, but had no direct effect on survival time estimates. In greenhouse trials, spray and bait formulations of AgipMNPV significantly reduced feeding damage to corn seedlings caused by third-instar A. ipsilon. In two sets of replicated field trials, bait formulations of AgipMNPV significantly reduced feeding damage to corn seedlings by third-instar A. ipsilon. However, there were no beneficial effects attributable to the inclusion of M2R in AgipMNPV formulations under greenhouse or field conditions. It seems likely that in an appropriately designed pest management program AgipMNPV could be used to suppress field populations of early and mid-instar A. ipsilon.     

The metabolism of leukotrienes in blood plasma studied by high-performance liquid chromatography

The metabolism of leukotrienes (B4, C4, D4, and E4) within human plasma was studied and a simple sample preparation is presented. It was demonstrated that leukotriene E4 and leukotriene B4 were stable during incubation at 37 degrees C using the in vitro system. In contrast, leukotriene C4 was metabolized by gamma-glutamyl transpeptidase activities into leukotriene D4 which was further metabolized by dipeptidase activities of plasma into leukotriene E4. The transition state inhibitor of gamma-glutamyl transpeptidase L-serine-borate decreased the metabolism of leukotriene C4 in plasma. Dilution of plasma demonstrated that the dipeptidase was more active compared to the gamma-glutamyl transpeptidase. The metabolizing activities of plasma were functionally characterized by fractionating the plasma proteins.     

Demonstration of the O-demethylation reaction catalyzed by a peroxidase: application to 9-methoxyellipticine

9-methoxy ellipticine, an antitumor compound, is O-demethylated in presence of the system peroxidase-H2O2; this reaction yields the corresponding electrophilic quinone-imine and methanol. This O-demethylation reaction is reported for the first time and might be possibly extended to some other antitumor drugs.     

Catecholamine-induced changes in transmembrane potential and temperature in normal and dystrophic hamster brown adipose tissue

Previous studies have shown that norepinephrine (NE) elicits trans-membrane potential changes in skeletal muscle cells from normal and dystrophic (BIO 14.6) hamsters, with the magnitude of these changes being significantly less in dystrophic cells. To determine if the decreased response of the dystrophic muscle cells reflects a more generalized phenomenon, the present study was designed to evaluate the effects of NE on membrane properties of brown adipocytes. $\text{In vivo}$ techniques using glass microelectrodes were similar to those used in the muscle studies. NE injection (2 to 5 μg/kg body wt, i.v.) into anesthetized hamsters was followed by membrane depolarization, the magnitude of which did not significantly differ in the dystrophic and normal adipocytes. For example, upon administration of 5 μg NE/kg body wt, the average depolarization was $\text{14.5 ± 1.3}\text{mV}\text{(}\text{X}\text{±}\text{S.E.}\text{)}$ for 20 dystrophic cells and 14.1 ± 1.8 mV for 18 normal cells. The depolarizations following i.v. infusion of isoproterenol and phenylephrine also had similar amplitudes in both normal and dystrophic cells. Despite this lack of difference in plasma membrane responses, NE induced a significantly smaller rise in interscapular brown fat temperature in the dystrophic (0.09°C) than in the normal hamsters (0.26°C) following administration of 5 μg NE/kg body wt. Thus, the decreased responsiveness to NE of dystrophic sarcolemma did not occur with the plasma membrane of brown adipocytes, although brown fat temperature changes in the dystrophic hamsters were decreased in amplitude.     

Intermediate sucrose octa-acetate sensitivity suggests a third allele at mouse bitter taste locus Soa and Soa-Rua identity

Mice have been characterized as either tasters or non-tastersof the bitter compound sucrose octa-acetate(SOA). However, 11of 17 supposedly non-taster inbred strains were found to avoid1 mM SOA. All 17 strains were indifferent to 0.1 mM SOA. Tasterstrains avoided both concentrations. The intermediate phenotypewas dubbed demitaster. A consistent phenotypic dominance orderwas found in crosses among both inbred and outbred strains (taster> non-taster > demitaster). Demitasters were found (withtasters) in an outbred strain showing monogenic segregationfor SOA avoidance. This, plus monogenic segregation in a back-crossof taster to demitaster inbred strains, suggested a third alleleat the Soa locus (Soa^c). Demitaster allelism was supported bythe strong associations found in 15 strains between the threeSOA phenotypes and HindIII restriction fragment patterns forthe closely linked Prp (proline rich protein) loci. SOA demitasterstrains were also intermediate in raffinose undeca-acetate (RUA)avoidance. Furthermore, B6.SW-Soa² congenic mice avoided notonly SOA, but RUA and eight other acetylated sugars. A previouslyproposed separate RUA-sensitivity gene (Rua) thus appeared tobe redundant.     

Ligand-induced association of rat lymphocyte membrane proteins with the detergent-insoluble lymphocyte cytoskeletal matrix

There are two classes of membrane protein capping on the basis of ligand requirements. Surface immunoglobulin (Slg), the prototype of the first class, requires a single ligand for cap induction. RT1 (rat histocompatibility proteins) requires two antibodies for cap induction. The lateral mobility of Slg is relatively restricted compared with RT1. These differences may be due to differential interaction with the cytoskeleton. After ligand binding 71% of Slg becomes detergent insoluble and is associated with the lymphocyte cytoskeletal matrix. The insolubilization occurs at 4 degrees C and is not inhibited by sodium azide or cytoskeleton-active drugs. The insolubilized ligand-receptor complex can be solubilized by a cytoskeleton destabilizing buffer. In contrast, only 20% of RT1 becomes associated with the lymphocytic cytoskeleton after ligand binding. The ligand-induced receptor-cytoskeleton interaction influences capping behavior and may play a role in cell activation.     

The effectiveness of constructed wetland for treatment of woodwaste leachate

Percolation of rainfall through woodwaste piles leaches natural chemicals from the wood residuals that can have adverse impacts on the environment. A study was conducted on a woodwaste storage site, adjacent to the Lower Fraser River, near Mission, BC, Canada. The objective of this research was to evaluate the effectiveness of constructed wetland for treatment of this woodwaste leachate. The leachate was characterized by high oxygen demand, tannin and lignin, and volatile fatty acids (VFAs), but low pH and nutrients. Diluted leachate passed through six pilot-scale wetland cells, four planted with cattail (Typha latifolia) and two unplanted controls, with a hydraulic retention time of 7 days and an average depth of 40 cm. Nutrient addition and pH adjustments were made to improve contaminant removal. Reductions in contaminants were consistently achieved, with average removals for BOD, COD, VFAs and tannin and lignin of 60, 50, 69 and 42%, respectively. Climatic conditions had an impact on the performance of the constructed wetland. Further operation of the system will help to elucidate the seasonal fluctuations. Aging of the constructed wetland system increased the treatment performance.