A halocin acting on Na+/H+ exchanger of haloarchaea as a new type of inhibitor in NHE of mammals.

The capability of halocin H6 (a bacteriocin-like protein produced by haloarchaea Haloferax gibbonsii) to inhibit Na+/H+ exchanger (NHE) in mammalian cells and its cardio-protective efficacy on the ischemic and reperfused myocardium were evaluated in the present study. H6 inhibits NHE activity (measured by a flow cytometry method) in a dose-dependent form of cell lines of mammalian origin (HEK293, NIH3T3, Jurkat and HL-1) as well as in primary cell culture from human skeletal muscle (myocytes and fibroblasts). In vivo, an ischemia-reperfusion model in dogs by coronary arterial occlusion was used (two hours of regional ischemia and three hours of reperfusion). In animals treated with halocin H6 there was a significant reduction of premature ventricular ectopic beats and infarct size, whereas blood pressure and heart rate remained unchanged. Up to date, halocin H6 is the only described biological molecule that exerts a specific inhibitory activity in NHE of eukaryotic cells.

Crystal structure of beta-glucosidase A from Bacillus polymyxa: insights into the catalytic activity in family 1 glycosyl hydrolases.

Family 1 glycosyl hydrolases are a very relevant group of enzymes because of the diversity of biological roles in which they are involved, and their generalized occurrence in all sorts of living organisms. The biological plasticity of these enzymes is a consequence of the variety of beta-glycosidic substrates that they can hydrolyze: disaccharides such as cellobiose and lactose, phosphorylated disaccharides, cyanogenic glycosides, etc. The crystal structure of BglA, a member of the family, has been determined in the native state and complexed with gluconate ligand, at 2.4 A and 2.3 A resolution, respectively. The subunits of the octameric enzyme display the (alpha/beta)8 barrel structural fold previously reported for other family 1 enzymes. However, significant structural differences have been encountered in the loops surrounding the active-center cavity. These differences make a wide and extended cavity in BglA, which seems to be able to accommodate substrates longer than cellobiose, its natural substrate. Furthermore, a third sub-site is encountered, which might have some connection with the transglycosylating activity associated to this enzyme and its certain activity against beta-1,4 oligosaccharides composed of more than two units of glucose. The particular geometry of the cavity which contains the active center of BglA must therefore account for both, hydrolytic and transglycosylating activities. A potent and well known inhibitor of different glycosidases, D-glucono-1,5-lactone, was used in an attempt to define interactions of the substrate with specific protein residues. Although the lactone has transformed into gluconate under crystallizing conditions, the open species still binds the enzyme, the conformation of its chain mimicking the true inhibitor. From the analysis of the enzyme-ligand hydrogen bonding interactions, a detailed picture of the active center can be drawn, for a family 1 enzyme. In this way, Gln20, His121, Tyr296, Glu405 and Trp406 are identified as determinant residues in the recognition of the substrate. In particular, two bidentate hydrogen bonds made by Gln20 and Glu405, could conform the structural explanation for the ability of most members of the family for displaying both, glucosidase and galactosidase activity.

Lactose utilization by Saccharomyces cerevisiae strains expressing Kluyveromyces lactis LAC genes.

Whey generated in cheese manufacture continues being an industrial problem without a satisfactory solution. Genetic modification of the yeast S. cerevisiae to obtain strains able to utilize lactose, is a prerequisite for the utilization of this yeast to convert cheese whey into useful fermentation products (i.e. biomass, heterologous protein and other recombinant products). Although the construction of S. cerevisiae Lac(+) strains has been achieved by different strategies, most of these strains have unsuitable characteristics, such as genetic instability of the Lac phenotype or diauxic growth. In previous communications we have described the construction of genetically stable strains of S. cerevisiae that assimilate lactose with a high efficiency. These strains carry multiple copies of Kluyveromyces lactis LAC4 and LAC12 genes, which code for a beta-galactosidase and a lactose permease, respectively. In this work we report additional results about the effect of gene dosage, and analyze the performance of a selected strain in the bioconversion of cheese whey. Additionally, we describe the construction of a new strain, which combines the Lac(+) phenotype with additional properties of biotechnological interest: flocculence, and the ability to hydrolyze starch.

In vitro proliferation, differentiation and immuno-magnetic bead purification of human myoblasts.

OBJECTIVES: In vitro culture of myoblasts and subsequent grafting into injured myocardium represents a new therapeutic approach for the treatment of myocardial infarct. A major limitation to developing enough myoblasts to engrafting purpose is the isolation and purification. In the present work we purified myoblast from primary culture using an immunomagnetic bead technique. METHODS: Primary culture was obtained by trypsin-EDTA digestion of human muscle biopsies. Cells were cultured in DMEM growth medium containing 10% FBS, 2 mM L-glutamine and antibiotics. Immunotechniques using both monoclonal anti-myosin heavy chain (skeletal fast) and 5.1.H11 antibody combining with flow cytometry did identification of myoblasts. Positive selection was on myoblasts bound to 5.1.H11 incubating with human antimouse IgG coated magnetic beads (Dynabead) and subsequent isolation by magnet, releasing cells from beads with DNAse. RESULTS: More than 59% of primary cell culture are positive to 5.1.H11 and decreasing with passage. The coating of culture dish surface increased specific growth rate of myoblast clones twice. Positive selection allows to increasing concentration of myoblasts from 8.4% in mixed culture to more than 90% without affecting neither viability nor platting efficiency. CONCLUSION: Purification procedure reported here is easy, efficient and requires small amount of sample, which will facilitate the purpose of autologous implant.

Radiofrequency hepatic ablation with internally cooled electrodes and hybrid applicators with distant saline infusion using an in vivo porcine model.

AIMS: Radiofrequency ablation (RFA) of tumors by means of internally cooled (ICE) or multitined expandable electrodes combined with infusion of saline into the tissue may improve results. Our aim was to determine the efficacy of a previously optimized hybrid ICE system (ICE combined with infusion of saline into the tissue at a distance of 2mm) in comparison with a conventional ICE cluster electrode in porcine liver in vivo. METHODS: A total of 32 RFA were performed on a total of 10 farm pigs using two RFA systems: Group A (n=16): Cluster electrode. Group B (n=16): Hybrid system (with continuous infusion of 100ml/h of 20% NaCl at 2mm distance from the electrode shaft by an independent isolated needle). Livers were removed for macroscopic and histological assessment after the procedure. Coagulation volume, coagulation diameters, coefficient of variability (CV) of coagulation volume, sphericity ratio (SR), deposited power (DP), deposited energy (DE), deposited energy per coagulation volume (DEV) and rise of animal temperature during the procedure were compared and correlated among groups. Additionally, linear regression analysis was modeled to study the relationship between deposited energy and either coagulation volume and rise of animal temperature during the procedure in both groups. RESULTS: Both coagulation volume and short diameter of coagulation were significantly greater (p<0.05) in group B compared to group A (22.7+/-11.0 cm(3) and 3.1+/-0.7 cm vs. 13.5+/-7.7 cm(3) and 2.5+/-0.5 cm, respectively). A similar CV and SR was observed among groups (57.1% and 1.4+/-0.3 vs. 48.6% and 1.3+/-0.2 for groups B and A, respectively). In group B, DE and DP were more than double group A, but DEV was nearly twice as high (9782 J/cm(3) vs. 5342 J/cm(3), for groups B and A, respectively). No significant relationship between DE and coagulation volume was encountered. CONCLUSION: Efficacy of a single ICE may be improved with continuous infusion of saline at around 2 mm from the electrode shaft. Coagulation volume obtained with this improved system may be even greater than that obtained with a cluster electrode.

Methanosarcina mutant unable to produce methane or assimilate carbon from acetate.

Mutants of Methanosarcina barkeri 227 resistant to monofluoroacetate were isolated from monofluoroacetate-treated cultures. Mutant strain FAr9 was 100 times more resistant to monofluoroacetate than the wild-type strain and was deficient in carbon uptake and CH4 and CO2 production from methyl-labeled acetate. Methanol was assimilated at increased levels. Strain FAr9 was unable to shift from using methanol to using acetate for growth and exhibited increased sensitivity to growth inhibition by NaCN in methanol-containing complex medium. Unlike parent strain 227, acetate addition to methanol-containing media did not prevent NaCN inhibition. The specific activities of enzymes of exogenous acetate assimilation, CO dehydrogenase, and enzymes of the tricarboxylic acid cycle were similar for mutant and parent strain cell extracts. Mutation to monofluoroacetate resistance did not confer simultaneous resistance to 2-bromoethanesulfonate or pyruvate or alter propionate uptake. We conclude that strain FAr9 is either an acetate permeability mutant or is defective in an activation step required for the catabolism and anabolism of acetate.

Inhibition of methanogenesis and carbon metabolism in Methanosarcina sp. by cyanide.

NaCN was tested for its inhibitory effects on growth of and metabolism by Methanosarcina barkeri 227. NaCN (10 microM) inhibited catabolism of acetate methyl groups to CH4 and CO2 but did not inhibit methanogenesis from methanol, CO2, methylamine, or trimethylamine. NaCN also inhibited the assimilation of methanol or CO2 (as the sole carbon source) into cell carbon and stimulated the assimilation of acetate. These results suggest that inhibition by NaCN was a result of its action as an inhibitor of in vivo CO dehydrogenase. The results also implicate CO dehydrogenase in the oxidation of acetate but not methanol methyl groups to CO2.

Amino acid substitutions enhancing thermostability of Bacillus polymyxa beta-glucosidase A.

Mutations enhancing the thermostability of beta-glucosidase A of Bacillus polymyxa, a family 1 glycosyl hydrolase, have been obtained after hydroxylamine mutagenesis of a plasmid containing the bglA gene, transformation of Escherichia coli with the mutagenized plasmid, and identification of transformant colonies that showed beta-glucosidase activity after a thermal treatment that inactivated the wild-type enzyme. Two additive mutations have been characterized that cause replacement of glutamate at position 96 by lysine and of methionine at position 416 by isoleucine respectively. The thermoresistant mutant enzymes showed increased resistance to other denaturing agents, such as pH and urea, while their kinetic parameters did not change. CD spectra indicated that the E96K replacement caused an increase in alpha-helix content. The observed effect of the M416I mutation is consistent with the lower content of cysteine and methionine found in family 1 enzymes of thermophilic species compared with similar ones from mesophilic organisms.