Regulation of Endothelial Nitric-oxide Synthase (NOS) S-Glutathionylation by Neuronal NOS: EVIDENCE OF A FUNCTIONAL INTERACTION BETWEEN MYOCARDIAL CONSTITUTIVE NOS ISOFORMS*

Myocardial constitutive No production depends on the activity of both endothelial and neuronal NOS (eNOS and nNOS, respectively). Stimulation of myocardial β₃-adrenergic receptor (β₃-AR) produces a negative inotropic effect that is dependent on eNOS. We evaluated whether nNOS also plays a role in β₃-AR signaling and found that the β₃-AR-mediated reduction in cell shortening and [Ca²⁺]_i transient amplitude was abolished both in eNOS^−/− and nNOS^−/− left ventricular (LV) myocytes and in wild type LV myocytes after nNOS inhibition with S-methyl-l-thiocitrulline. LV superoxide (O₂^˙̄) production was increased in nNOS^−/− mice and reduced by l-N^ω-nitroarginine methyl ester (l-NAME), indicating uncoupling of eNOS activity. eNOS S-glutathionylation and Ser-1177 phosphorylation were significantly increased in nNOS^−/− myocytes, whereas myocardial tetrahydrobiopterin, eNOS Thr-495 phosphorylation, and arginase activity did not differ between genotypes. Although inhibitors of xanthine oxidoreductase (XOR) or NOX2 NADPH oxidase caused a similar reduction in myocardial O₂^˙̄, only XOR inhibition reduced eNOS S-glutathionylation and Ser-1177 phosphorylation and restored both eNOS coupled activity and the negative inotropic and [Ca²⁺]_i transient response to β₃-AR stimulation in nNOS^−/− mice. In summary, our data show that increased O₂^˙̄ production by XOR selectively uncouples eNOS activity and abolishes the negative inotropic effect of β₃-AR stimulation in nNOS^−/− myocytes. These findings provide unequivocal evidence of a functional interaction between the myocardial constitutive NOS isoforms and indicate that aspects of the myocardial phenotype of nNOS^−/− mice result from disruption of eNOS signaling.     

Improved yield and stability of amylase by multipoint covalent binding on polyglutaraldehyde activated chitosan beads: Activation of denatured enzyme molecules by calcium ions

In this study raw starch digesting amylase (RSDA) from Aspergillus carbonarius (Bainier) Thom IMI 366159 was stabilized by covalent binding on polyglutaraldehyde (PG), glutaraldehyde (G) activated chitosan beads or post immobilization cross linking of enzyme adsorbed on chitosan. Presence of Ca²⁺ ions (0.5–1.5 mM) activated the PG and G derivatives but repressed the crosslinked enzyme. Optimum pH for cross linked derivative increased by 2 units but was unaltered for PG and G derivatives. Immobilized amylase exhibited improved thermal and storage stability. Immobilized derivatives had no loss of activity after 1 month storage and retained above 90% activity after 10 batch reactions of 60 min each. Immobilization successfully stabilized RSDA and immobilized enzyme from A. carbonarius can be applied in numerous industries for cheap, cost effective and environmentally friendly starch hydrolytic processes to simple sugars.     

Enhancement of the Anti-inflammatory Effect of Bromelain by Its Immobilization on Probiotic Spore of Bacillus cereus

The therapeutic application of bromelain is limited due to its sensitivity to operating conditions such as high acidity, gastric proteases in the stomach juice, chemicals, organic solvents and elevated temperature. We hypothesized that bromelain immobilized on probiotic bacterial spores would show enhanced therapeutic activity through possible synergistic or additive effects. In this study, the oedema inhibition potential of bromelain immobilized on probiotic Bacillus spores was compared to the free enzyme using the carrageenan paw oedema model with Wistar rats. In batch A rats (carrageenan-induced inflammation 30 min after receiving oral treatments), group 7 rats treated with a lower dose of spore-immobilized bromelain suspension showed the highest oedema inhibition, 89.20 ± 15.30%, while group 4 treated with a lower dose of free bromelain had oedema inhibition of 60.25 ± 13.00%. For batch B rats (carrageenan-induced inflammation after receiving oral treatment for three days), group 7 rats treated with a lower dose of spore-immobilized bromelain suspension showed higher inhibition percentage (81.94 ± 8.86) than group 4 treated with a lower dose of free bromelain (78.45 ± 4.46) after 24 h. Our results showed that used alone, the enzyme and the spores produced oedema inhibition and improved the motility of the rats. The spore-immobilized bromelain formulation performed approximately 0.9-fold better than the free bromelain and the free spores at the lower evaluated dose.