Does enhanced expression of the Na+-CA2+ exchanger increase myocardial vulnerability to ischemia/reperfusion injury in rabbit hearts?

The present study focused on examining the efficacy of feeding a rutin-glucose derivative (G-rutin) to inhibit glycation reactions that can occur in muscle, kidney and plasma proteins of diabetic rats. Both thiobarbituric acid-reactive substance levels and protein carbonyl contents in muscle and kidney were significantly (p < 0.05) reduced in streptozotocin-induced diabetic rats fed G-rutin supplemented diet, compared to diabetic rats fed control diet. The N -fructoselysine content in muscle and kidney, a biomarker of early glycation reaction, was markedly (p < 0.05) increased by diabetes, but significantly (p < 0.05) reduced in diabetic rats fed G-rutin. Advanced glycation end-products (AGEs) in serum and kidney protein were measured by immunoblot using anti-AGE antibody, and were also reduced in diabetic rats fed dietary G-rutin. Feeding G-rutin also slightly inhibited aldose reductase activity in these animals. These results demonstrate for the first time that dietary G-rutin consumption can provide potential health benefits that are related to the inhibition of tissue glycation reactions common to diabetes.     

Cold-active esterase from Psychrobacter sp. Ant300: gene cloning, characterization, and the effects of Gly-->Pro substitution near the active site on its catalytic activity and stability

The gene encoding an esterase (PsyEst) of Psychrobacter sp. Ant300, a psychrophilic bacterium isolated from Antarctic soil, was cloned, sequenced, and expressed in Escherichia coli. PsyEst, which is a member of hormone-sensitive lipase (HSL) group of the lipase/esterase family, is a cold-active, themolabile enzyme with high catalytic activity at low temperatures (5-25 degrees C), low activation energy (e.g., 4.6 kcal/mol for hydrolysis of p-nitrophenyl butyrate), and a t(1/2) value of 16 min for thermal inactivation during incubation at 40 degrees C and pH 7.9. A three-dimensional structural model of PsyEst predicted that Gly(244) was located in the loop near the active site of PsyEst and that substitution of this amino-acid residue by proline should potentially rigidify the active-site environment of the enzyme. Thus, we introduced the Gly(244)-->Pro substitution into the enzyme. Stability studies showed that the t(1/2) value for thermal inactivation of the mutant during incubation at 40 degrees C and pH 7.9 was 11.6 h, which was significantly greater than that of the wild-type enzyme. The k(cat)/K(m) value of the mutant was lower for all substrates examined than the value of the wild type. Moreover, this amino-acid substitution caused a shift of the acyl-chain length specificity of the enzyme toward higher preference for short-chain fatty acid esters. All of these observations could be explained in terms of a decrease in active-site flexibility brought about by the mutation and were consistent with the hypothesis that cold activity and thermolability arise from local flexibility around the active site of the enzyme.     

Hypocretin/dopamine interaction in sleep-related motor function

Sleep and Biological Rhythms -     

Glutathione S‐transferase π complexes with and stimulates Na+,K+‐ATPase

Glutathione S‐transferase (GST) was found to complex with the Na⁺,K⁺‐ATPase as shown by binding assay using quartz crystal microbalance. The complexation was obstructed by the addition of antiserum to the α‐subunit of the Na⁺,K⁺‐ATPase, suggesting the specificity of complexation between GST and the Na⁺,K⁺‐ATPase. Co‐immunoprecipitation experiments, using the anti‐α‐subunit antiserum to precipitate the GST‐Na⁺,K⁺‐ATPase complex and then using antibodies specific to an isoform of GST to identify the co‐precipitated proteins, revealed that GSTπ was complexed with the Na⁺,K⁺‐ATPase. GST stimulated the Na⁺,K⁺‐ATPase activity up to 1.4‐fold. The level of stimulation exhibited a saturable dose–response relationship with the amount of GST added, although the level of stimulation varied depending on the content of GSTπ in the lots of GST received from supplier. The stimulation was also obtained when recombinant GSTπ was used, confirming the results. When GST was treated with reduced glutathione, GST activity was greatly stimulated, whereas the level of stimulation of the Na⁺,K⁺‐ATPase activity was similar to that when untreated GST was added. When GST was treated with H₂O₂, GST activity was greatly diminished while the stimulation of the Na⁺,K⁺‐ATPase activity was preserved. The results suggest that GSTπ complexes with the Na⁺,K⁺‐ATPase and stimulates the latter independent of its GST activity. Copyright © 2012 John Wiley & Sons, Ltd.     

Effect of detergents on sterol synthesis in a cell-free system of yeast

In order to obtain information about the reactivity of enzymes in sterol synthesis of yeast, the effects of some detergents were investigated. Among the detergents used, Triton X-100 was found to exert a unique action, and its effect on the incorporation of 14C-labeled acetate, mevalonate, farnesyl pyrophosphate, or S-adenosyl-L-methionine into squalene, 2,3-oxidosqualene, and sterols in a cell-free system was examined. Triton X-100 showed virtually no effect on the enzyme activities in the reactions from acetyl CoA to farnesyl pyrophosphate, but it had a marked effect on reactions from farnesyl pyrophosphate to ergosterol. Evidence was obtained suggesting that Triton X-100 apparently activated squalene synthetase (EC 2.5.1.21) but inhibited squalene epoxidase (EC 1.14.99.7) and delta 24-sterol methyltransferase (EC 2.1.1.41). The activity of epoxidase was protected from the inhibition by increasing the concentration of cell-free extracts or by the prior addition of lecithin liposomes to the reaction mixture. The inhibition of methyltransferase was partially reversed by treatment with Bio-heads SM-2, but that of epoxidase was not reversed by the treatment.     

Effects of dopamine on chemoreflexes in breathing

The effects of intravenous infusion of dopamine (20 microgram.min) on the steady-state ventilatory and carotid chemoreceptor responses to successive levels of isocapnic hypoxia and hyperoxic hypercapnia were investigated in cats anesthetized with alpha-chloralose. Dopamine infusion was followed by a maximal decrease in ventilation in about 20 s. Thereafter, the effect diminished and stabilized. Termination of dopamine infusion was promptly followed by an increase in ventilation. These ventilatory responses were smaller than the corresponding carotid chemoreceptor responses. The steady-state effect of dopamine infusion was to diminish ventilation at all levels of arterial O2 tension, the decrease being greater during hypoxia than that during hyperoxia. Bilateral section of the carotid sinus nerves significantly diminished but did not abolish the inhibitory effect of dopamine on ventilation during hyperoxia. Thus the ventilatory depression due to dopamine infusion is not entirely due to its effect on the carotid chemoreceptors. Dopamine decreased ventilatory responses to successive levels of hypercapnia by the same magnitude without changing the slope of the response curves. The steady-state relationship between chemoreceptor activity and ventilation shows that the ventilatory equivalent for carotid chemoreceptor activity is increased during dopamine infusion because of its greater inhibitory effect on carotid chemoreceptor activity than on ventilation with the decrease of arterial O2 tension.     

The antitumor potency of progesterone antagonists is due to their differentiation potential.

A new therapy for the progesterone receptor positive mammary carcinoma may be the treatment with progesterone antagonists. This new class of antihormones causes a strong inhibition of tumor growth comparable to the potency of ovariectomy in a panel of experimental mammary carcinomas. The mechanisms of the strong tumor-inhibiting action of progesterone antagonists on experimental mammary carcinomas mainly depends on a progesterone receptor mediated process leading to induction of terminal differentiation and a blockade of the cell cycle. To further characterize the antitumor mechanism of progesterone antagonists we analyzed the effects of Onapristone and ZK 112.993 on DMBA- and MNU-mammary tumors of the rat and MXT-tumors of the mouse after different therapy intervals. These hormone-dependent mammary tumors normally display intraductal growth in papillary, cribiform or solid formation, whereas after treatment periods of 2-6 weeks with progesterone antagonists they displayed dysplastic ductal and acinous formations, usually filled with secretory material. Whereas tumor size, mitotic index, and the grade of tumor malignancy decreased distinctly, the volume fraction of glandular structures in the tumors as well as the appearance of apoptosis increased 3-fold compared to the controls. In addition, the mammary glands of progesterone antagonist treated animals showed the morphological features of differentiation with the appearance of secretory activity. Interestingly, the staining pattern of some of the lectins used, especially UEA 1 binding pattern, fits to the concept of differentiation since recent studies revealed a higher degree of fucosylation only in benign lesions of human breast cancers. Therefore, these data underline the concept of a differentiation potential of progesterone antagonists on progesterone receptor positive mammary carcinomas.