Cytoplasmic Ca2+ release induced by microinjection of Ca2+ and effects of microinjected divalent cations on Ca2+ sequestration and exocytosis of cortical alveoli in the medaka egg

Intracellular release of Ca2+ by microinjection of Ca2+ was analyzed by measuring the luminescence of aequorin loaded in eggs of the medaka (Oryzias latipes). Microinjection of Ca2+ into the cortical cytoplasm induced propagative waves of cytoplasmic Ca2+ release and exocytosis of cortical alveoli initiated at the injection point. The Ca2+ wave was initiated with a time lag after some was sequestered at the region of the microinjection. Microinjection of Mg2+ or Mn2+ failed to trigger Ca2+ release and exocytosis. When the aequorin-loaded eggs were inseminated after microinjection of Mg2+, Mn2+, or Co2+ into a restricted region of the vegetal hemisphere, the wave of Ca release was propagated through the injected region toward the vegetal pole, but neither Ca sequestration (fall in Ca-aequorin luminescence) nor exocytosis occurred at the area of cortex where the eggs were injected with these divalent cations. These results suggest that a significant period is required to induce Ca2+ release from cytoplasmic stores by the increased Ca2+ concentration and that both the phenomena of Ca2+ release and Ca sequestration are involved in the process of exocytosis.     

Pioglitazone ameliorates endothelial dysfunction in obese rats with nephropathy

Endothelial dysfunction is a key event in the development of renovascular complications in the metabolic syndrome. The aim of this study was to elucidate the pathogenetic mechanisms involved in renovascular injuries in the Zucker obese rat, a model of the metabolic syndrome, and to examine the therapeutic effects of pioglitazone, a thiazolidinedione. Obese rats fed high-protein diet (OHP) for 12 weeks exhibited nephropathy and endothelial dysfunction, which were improved by pioglitazone. Accumulation of nitrotyrosine, a tracer of nitrative stress, was increased in aorta of the OHP group. The mRNA expressions of NADPH oxidase components and inducible nitric oxide synthase in the aorta were enhanced in the OHP group. Pioglitazone reduced nitrotyrosine in the aorta of the OHP group, inhibiting the augmented expression levels of both. These results suggest that nitrative stress could cause endothelial dysfunction in the rat model of metabolic syndrome with nephropathy, and that pioglitazone ameliorates these injuries, presumably by reducing nitrative stress.     

Synthetic study of VLA-4/VCAM-1 inhibitors: synthesis and structure-activity relationship of piperazinylphenylalanine derivatives

To improve the poor pharmacokinetic characteristics of VLA-4 inhibitors, novel piperazinylphenylalanine derivatives were designed. This structure is expected to improve physicochemical properties by increasing overall basicity. By changing components at the 4-position of piperazine and the terminal group of the amido bond, 12t was found to be the most potent of this series of compounds. In addition, dichlorobenzoyl derivative 12aa exhibited better oral availability and showed efficacy in an in vivo model after oral administration.     

emm Typing of group A streptococcus clinical isolates: identification of dominant types for throat and skin isolates

T and emm types were determined for group A streptococci isolated from patients with various infections during 1990-1999 in Toyama Prefecture, Japan. Out of 906 isolates, 872 isolates were divided into 20 T serotypes, and 34 isoltes were T nontypeable (TNT). T12, T1, and T4 were dominant among 699 throat isolates; on the other hand, T11, T28, TB3264, and TNT were dominant among 80 skin isolates. The emm types of 190 isolates were determined following specific PCR amplification and sequencing of the products. Twenty T serotypes were divided into 34 T type/emm type combinations. Thirty-four TNT isolates were divided into 14 emm types, in which emm58 was the most common (38%). Among 82 throat isolates randomly selected, predominant T types T12, T1, and T4 isolates were of the respective same numbers in emm type. T11/emm89, T28/emm28, TB3264/emm13w, and TNT/emm58 were predominant among 80 skin isolates. emm-type distribution observed in the present study was that usually reported in the western world. To our knowledge, 3 T/emm is a novel combination. These results show that emm typing allows the characterization of group A streptococci from various sources.     

Site-specific mechanisms of initiation by chelated iron and inhibition by alpha-tocopherol of lipid peroxide-dependent lipid peroxidation in charged micelles.

To obtain information on the role of iron-catalyzed lipid peroxidation in the presence of the small amount of lipid peroxide in deterioration of biological membranes, we examined factors affecting peroxidation of fatty acids in charged micelles. Peroxidation of linoleic acid (LA) was catalyzed by Fe2+ via reductive cleavage of linoleic acid hydroperoxide (LOOH) in negatively charged sodium dodecyl sulfate micelles, but not in positively charged tetradecyltrimethylammonium bromide (TTAB) micelles. However, this Fe2(+)-induced, LOOH-dependent lipid peroxidation could be induced in TTAB micelles in the presence of a negatively charged iron chelator, nitrilotriacetic acid (NTA). The linoleic acid alkoxy radical (LO.) generated by the LOOH-dependent Fenton reaction was also trapped by N-t-butyl-alpha-phenylnitrone at the surface of TTAB micelles in the presence of NTA, but not in its absence. The degradation rates of two spin probes, N-oxyl-4,4'-dimethyloxazolidine derivatives of stearic acid (5-NS and 16-NS), were investigated to determine the site of production of radicals formed during LOOH-dependent lipid peroxidation. The rate of consumption of 16-NS during the LOOH-dependent Fenton-like reaction was higher in TTAB micelles containing LA than in those containing lauric acid (LauA), although the rates of formation of LO. in the two types of fatty acid micelles were similar. The rates of 5-NS consumption in LA and LauA micelles were almost the same and were as low as that of 16-NS consumption in LauA micelles. 16-NS was more inhibitory than 5-NS of LOOH-dependent lipid peroxidation, and this inhibition was associated with its higher consumption of 16-NS than of 5-NS. alpha-Tocopherol inhibited NTA-Fe2(+)-induced LOOH-dependent lipid peroxidation in TTAB micelles, and was oxidized during this inhibition process. The rate and amount of alpha-tocopherol oxidized by the LOOH-dependent Fenton reaction were higher in LA micelles than in LauA micelles. alpha-Tocopherol inhibited the consumption of 16-NS during NTA-Fe2(+)-induced LOOH-dependent lipid peroxidation more effectively than that of 5-NS. The distribution of the chromanol moiety of alpha-tocopherol was studied by the fluorescence quenching method. There was no difference between Stern-Volmer plots of the quenchings of alpha-tocopherol fluorescence by 5-NS and 16-NS. From these results, we discuss the mechanism of induction of LOOH-dependent peroxidation of LA and the mechanism of the antioxidant effects of alpha-tocopherol on it from the viewpoint of site-specific reaction.     

In vivo measurement of 31P nuclear magnetic resonance spectrum of aging mouse brain

31P nuclear magnetic resonance spectra of the brains of young, mature and old C57BL/6 mice were measured in vivo. Under normal conditions, no significant changes with age were observed in the spectra and there were no statistically significant age-related differences in the ratio of phosphocreatine to adenosine triphosphate (ATP). When the animals were stressed by subjecting them to oxygen restriction, the young and mature mice showed no change in the ratio, whereas it increased in the old mice. These results are consistent with the view that energy metabolism is unimpaired in the brains of unstressed old animals but that ATP synthesis may be decreased with age in a stressed animal.     

Change in Intracellular Calcium Ions upon Maturation in Starfish Oocytes

A transient increase in intracellular Ca²⁺ upon maturation in starfish oocyte was revealed by light emission of aequorin microinjected into the cell. One minute application of 1-methyladenine (1-MeAde) to a limited area of the oocyte surface was sufficient to induce the Ca²⁺ transient over the entire cell though it did not induce the germinal vesicle breakdown (GVBD). Ten minutes application of 1-MeAde induced a similar Ca²⁺ transient followed by GVBD. Even when the transient increase of Ca²⁺ was inhibited by injecting EGTA into the oocyte, 1-MeAde treatment for a long period induced GVBD. These facts indicate that the Ca²⁺ increase is neither necessary nor sufficient for maturation of the starfish oocyte.
When the oocyte, which had been treated with 1-MeAde for 1 min at a limited area around the animal pole, was treated again with 1-MeAde for 10 min starting about 15 min after the first treatment, a Ca²⁺ transient similar to the first one was induced and was followed by GVBD. By contrast, in the oocyte treated with 1-MeAde at an area around the vegetal pole, neither Ca²⁺ transient nor GVBD was induced by the second treatment with 1-MeAde. These results indicate a difference in responsiveness to the hormone between the animal hemisphere and the vegetal hemisphere of the oocyte.