Enzyme-Substrate Complex of p-Hydroxybenzoate Hydroxylase; One of the Activated Intermediates of the Overall Reaction

The transglucosidation reaction of brewer’s yeast α-glucosidase was examined under the co-existence of l-sorbose and phenyl-α-glucoside. As the transglucosidation products, three kinds of new disaccharide were chromatographically isolated. It was presumed that these disaccharides consisting of d-glucose and l-sorbose were 1-O-α-d-glucopyranosyl-l-sorbose ([α]_D+89.0), 3-O-α-d-glucopyranosyl-l-sorbose ([α]_D+69.1) and 4-O-α-d-glucopyranosyl-l-sorbose ([α]_D+81.0). The principal product formed in the enzyme reaction was 1-O-α-d-glucopyranosyl-l-sorbose.     

In vitro analysis of Bcl-2 proteins in mitochondria and endoplasmic reticulum: similarities in anti-apoptotic functions and differences in regulation

Anti-apoptotic Bcl-2 localizes in the membranes of mitochondria and endoplasmic reticulum (ER) and resists a broad range of apoptotic stimuli. However, the precise function of Bcl-2 in ER is still unclear. We herein examined the anti-apoptotic potencies of Bcl-2 in mitochondria and ER in vitro. The mitochondria isolated from HeLa cells, which have little or practically no Bcl-2, were apoptosis-competent. That is, membrane-bound Bax was activated and cytochrome c was released when the isolated mitochondria were incubated at 35 degrees C. Cytochrome c release from the apoptosis-competent mitochondria was suppressed by co-incubation with the mitochondria with overexpressed Bcl-2 (Bcl-2 mitochondria), suggesting that Bcl-2 anchored in one mitochondrion can suppress cytochrome c release from another mitochondrion. Similar results were obtained when microsomes with overexpressed Bcl-2 (Bcl-2 microsomes) were co-incubated with apoptosis-competent mitochondria. A quantitative titration analysis showed that Bcl-2 in the ER suppresses cytochrome c release as efficiently as that in the mitochondria. An immunoprecipitation assay showed that Bcl-2 in both mitochondria and ER binds to Bax at almost the same degree. However, in the presence of tBid, co-incubation of apoptosis-competent mitochondria with Bcl-2 microsomes, but not with Bcl-2 mitochondria, diminished the Bax-binding to Bcl-2 significantly, suggesting that Bcl-2 in ER is readily inactivated by tBid. Co-incubation assay further confirmed that Bcl-2 in the ER, but not Bcl-2 in the mitochondria, is potentially inactivated by tBid. Our quantitative in vitro studies indicate that Bcl-2 in mitochondria and ER are similarly potent in inhibiting Bax-associated apoptosis of other mitochondria, but are regulated by tBid differently.     

The antioxidative function of eicosapentaenoic acid in a marine bacterium, Shewanella marinintestina IK-1

When the eicosapentaenoic acid (EPA)-deficient mutant strain IK-1Delta8 of the marine EPA-producing Shewanella marinintestina IK-1 was treated with various concentrations of hydrogen peroxide (H(2)O(2)), its colony-forming ability decreased more than that of the wild type. Protein carbonylation, induced by treating cells with 0.01 mM H(2)O(2) under bacteriostatic conditions, was enhanced only in cells lacking EPA. The amount of cells recovered from the cultures was decreased more significantly by the presence of H(2)O(2) for cells lacking EPA than for those producing EPA. Treatment of the cells with 0.1 mM H(2)O(2) resulted in much lower intracellular concentrations of H(2)O(2) being consistently detected in cells with EPA than in those without EPA. These results suggest that cellular EPA can directly protect cells against oxidative damage by shielding the entry of exogenously added H(2)O(2) in S. marinintestina IK-1.     

A 350-S recovery period does not necessarily allow complete recovery of peak power output during repeated cycling sprints

The aim of this study was to determine whether a 350-s recovery period allows recovery of peak power output (PPO) to its initial value under the condition of a blood lactate (La) concentration higher than 10 mmol.L-1 during repeated cycling sprints (RCS). RCS (10x10-s cycling sprints) were performed under two conditions. Under one condition, the recovery period of RCS was fixed at 35 s (RCS35), and under the other condition, a 350-s recovery period was set before the 5th and 9th sets, and a 35-s recovery period was set before the other sets (RCScomb). In RCScomb, PPO in the 5th set recovered to that in the 1st set, but PPO in the 9th set did not. Under both conditions, blood La concentration progressively increased and reached approximately 14 mmol.L-1 at the end of the RCS. In RCScomb, VO2 immediately before the 5th set was not significantly different from that immediately before the 9th set. Mean power frequency (MPF) values estimated by a surface electromyogram from the vastus lateralis in the 5th and 9th sets were significantly higher in RCScomb than in RCS35. In conclusion, a 350-s recovery period does not allow recovery of PPO to its initial value under the condition of a blood La concentration of 14 mmol.L-1 during RCS.     

A novel type of autophagy occurs together with vacuole genesis in miniprotoplasts prepared from tobacco culture cells

Mature plant cells have large vacuoles. But how these vacuoles are formed has not been fully understood. It has been reported that autophagy is involved in the genesis of plant vacuoles. Thus we examined whether autophagy occurs in the vacuole genesis of a plant cell model called miniprotoplasts, in which preexisting large vacuoles have been removed. We prepared miniprotoplasts from tobacco culture cells (BY-2) and observed the formation of vacuoles by light and electron microscopy. The miniprotoplasts had few vacuoles immediately after preparation, but had large vacuoles after 1 to 2 d. When the cysteine protease inhibitor E-64c or E-64d was added to culture media, almost all vacuoles formed contained materials of cytoplasmic origin. This result suggests that autophagy occurs together with the genesis of the vacuoles in miniprotoplasts. 3-Methyladenine and phosphatidylinositol 3-kinase inhibitors such as wortmannin and LY294002, all of which block starvation?induced autophagy in tobacco culture cells and constitutive autophagy in Arabidopsis root cells, did not affect the autophagy in miniprotoplasts. Thus the form of autophagy in miniprotoplasts is probably different from the form of autophagy that arises as a result of sucrose starvation and constitutive autophagy in root tip cells. The causal connection between autophagy and vacuole genesis in miniprotoplasts was not clarified in this study.     

Circumnutational movement in rice coleoptiles involves the gravitropic response: analysis of an agravitropic mutant and space‐grown seedlings

Plants exhibit helical growth movements known as circumnutation in growing organs. Some studies indicate that circumnutation involves the gravitropic response, but this notion is a matter of debate. Here, using the agravitropic rice mutant lazy1 and space‐grown rice seedlings, we found that circumnutation was reduced or lost during agravitropic growth in coleoptiles. Coleoptiles of wild‐type rice exhibited circumnutation in the dark, with vigorous oscillatory movements during their growth. The gravitropic responses in lazy1 coleoptiles differed depending on the growth stage, with gravitropic responses detected during early growth and agravitropism during later growth. The nutation‐like movements observed in lazy1 coleoptiles at the early stage of growth were no longer detected with the disappearance of the gravitropic response. To verify the relationship between circumnutation and gravitropic responses in rice coleoptiles, we conducted spaceflight experiments in plants under microgravity conditions on the International Space Station. Wild‐type rice seeds were germinated, and the resulting seedlings were grown under microgravity or a centrifuge‐generated 1 g environment in space. We began filming the seedlings 2 days after seed imbibition and obtained images of seedling growth every 15 min. The seed germination rate in space was 92–100% under both microgravity and 1 g conditions. LED‐synchronized flashlight photography induced an attenuation of coleoptile growth and circumnutational movement due to cumulative light exposure. Nevertheless, wild‐type rice coleoptiles still showed circumnutational oscillations under 1 g but not microgravity conditions. These results support the idea that the gravitropic response is involved in plant circumnutation.     

Analysis of responses to endothelins in isolated porcine blood vessels by using a novel endothelin antagonist, BQ-153.

We examined the effects of a novel ETA-selective endothelin (ET) antagonist, BQ-153, on vascular responses to ET-1 and ET-3 in isolated porcine coronary and pulmonary blood vessels, to clarify the roles of ET receptor subtypes in the regulation of vascular smooth muscle tension. With endothelium-denuded vascular tissues, the concentration-contraction curve (CCC) for ET-1 appeared as a single sigmoidal shape for all types of tissue. The CCC for ET-1 was antagonized by BQ-153 (2 and 10 microM) in all tissues, but part of the contraction was resistant. The CCC for ET-3 usually consisted of two different phases with higher (first phase) and lower (second phase) sensitivities to the peptide. Only the second phase of CCC for ET-3 was completely inhibited by BQ-153 (2 microM) in all tissues, while the first phase was resistant. The BQ-153-resistant contractile phases of ET-1 and ET-3-induced vasoconstriction appeared to have similar sensitivity in all tissues, and the contractile activity varied with each type of tissue. With endothelium-intact materials, the potencies of ET-1 and ET-3 for endothelium-dependent vasorelaxation in pulmonary artery were almost equivalent. BQ-153 (10 microM) did not inhibit ET-induced vasorelaxation. These results indicate that ET-induced vasoconstriction is mediated not only through ETA but also through ETnonA (probably ETB), and that the relative proportions of the ET-receptor subtypes mediating contractions vary in different vascular areas. In addition, results showed that ET-induced endothelium-dependent vasorelaxation is mediated through ETB.     

Molecular monitoring of bleomycin-induced pulmonary fibrosis by cDNA microarray-based gene expression profiling.

Pulmonary fibrosis is a progressive disorder whose molecular pathology is poorly understood. Here we developed an in-house cDNA microarray ("lung chip") originating from a lung-normalized cDNA library. By using this lung chip, we analyzed global gene expression in a murine model of bleomycin-induced fibrosis and selected 82 genes that differed by more than twofold intensity in at least one pairwise comparison with controls. Cluster analysis of these selected genes showed that the expression of genes associated with inflammation reached maximum levels at 5 days after bleomycin administration, while genes involved in the development of fibrosis increased gradually up to 14 days after bleomycin treatment. These changes in gene expression signature were well correlated with observed histopathological changes. The results show that microarray analysis of animal disease models is a powerful approach to understanding the gene expression programs that underlie these disorders.