Expression of angiotensin type 1 and 2 receptors in brain after transient middle cerebral artery occlusion in rats

Angiotensin II (Ang II) type 2 receptors (AT2Rs) have been associated with apoptosis. We hypothesized that AT2Rs are increased in stroke and may contribute effects of stroke to the brain. To test this, we have examined the expression of Ang II type 1 receptor (AT1R), AT2R and Ang II levels in the brain 24 h after transient middle cerebral artery occlusion (MCAO). The densities of AT1R and AT2R were measured by quantitative autoradiography (n=6). The levels of Ang II were measured by radioimmunoassay (RIA) (n=6) and by immunohistochemistry (n=3). AT1R levels on autoradiography showed a significant decrease (0.87±0.06 to 1.39±0.07 fmol/mg, p<0.01) in the ventral cortex of the stroke side compared to the cortices of non-stroke (NS) rats (n=4). There was no significant difference on ATIR in the contralateral verbal cortex of the stroke rats compared to NS control. In contrast, levels of AT2R in the ventral cortex of both the stroke and the contralateral sides were significantly increased (0.77±0.06, p<0.05 and 0.91±0.05, p<0.01 compared to 0.60±0.03 fmol/mg tissue, respectively). RIA showed that Ang II in the ventral cortex of both the stroke and the contralateral sides were significantly increased (241.63±47.72, p<0.01 and 165.51±42.59, p<0.05 compared to 76.80±4.10 pg/g tissue, respectively). Also, Ang II in the hypothalamus was significantly increased (179.50±17.49 to 118.50±6.65 pg/g tissue, p<0.05). Immunohistochemistry confirmed the increase of Ang II. These results demonstrate that brain Ang II and AT2Rs are increased whereas AT1Rs are decreased after transient MCAO in rats. We conclude that in stroke, Ang II and AT2R are activated and may contribute neural effects to brain ischemia.     

Effect of polyethylene glycol and dimethyl sulfoxide on the fusion of bovine nuclear transfer using mammary gland epithelial cells

The effects of polyethylene glycol and dimethyl sulfoxide (PEG/DMSO) treatment of donor cells on the fusion and subsequent development of bovine nuclear transfer embryos using mammary gland epithelial (MGE) cells before electrofusion (fresh MGE cells) was studied. The same study was conducted on those cells that were frozen and stored in liquid nitrogen, and then thawed (frozen-thawed MGE cells). Experiment 1 showed that the exposure time and pH of PEG/DMSO solution affected the fusion of nuclear transfer, and that a higher fusion rate was obtained when fresh MGE cells were exposed to PEG/DMSO solution at pH 8.0 for 5 min. In Experiment 2, the proportion of fused oocytes with fresh PEG/DMSO-treated cells (70 +/- 6%) was significantly higher than that with non-treated cells (50 +/- 13%, p < 0.05). The same tendency was observed when frozen-thawed cells as donor nuclei were used (48 +/- 6% vs. 34 +/- 12%, p < 0.05). In addition, PEG/DMSO treatment has neither harmful nor beneficial effects on the cleavage and development of the blastocyst stage of reconstructed embryos (p > 0.05). The fusion and cleavage rates of frozen-thawed cells were significantly lower than those of fresh cells (p < 0.05). After 10 blastocysts, derived from fresh PEG/DMSO-treated cells, were transferred to five recipient heifers, one live female calf was obtained. Experiment 3 showed that PEG/DMSO treatment reduced the viability of both fresh and frozen-thawed MGE cells (p < 0.05). We conclude that the PEG/DMSO treatment of fresh MGE cells, as well as the frozen-thawed cells, before electrofusion has a positive effect on the fusion of nuclear transfer without decreasing the in vitro development of reconstructed embryos.     

Possible role of cell surface H+ -ATP synthase in the extracellular ATP synthesis and proliferation of human umbilical vein endothelial cells

Extracellular ATP synthesis on human umbilical vein endothelial cells (HUVECs) was examined, and it was found that HUVECs possess high ATP synthesis activity on the cell surface. Extracellular ATP generation was detected within 5 s after addition of ADP and inorganic phosphate and reached a maximal level at 15 s. This type of ATP synthesis was almost completely inhibited by mitochondrial H(+)-ATP synthase inhibitors (e.g., efrapeptins, resveratrol, and piceatannol), which target the F(1) catalytic domain. Oligomycin and carbonyl cyanide m-chlorophenylhydrazone, but not potassium cyanide, also inhibited extracellular ATP synthesis on HUVECs, suggesting that cell surface ATP synthase employs the transmembrane electrochemical potential difference of protons to synthesize ATP as well as mitochondrial H(+)-ATP synthase. The F(1)-targeting H(+)-ATP synthase inhibitors markedly inhibited the proliferation of HUVECs, but intracellular ATP levels in HUVECs treated with these inhibitors were only slightly affected, as shown by comparison with the control cells. Interestingly, piceatannol inhibited only partially the activation of Syk (a nonreceptor tyrosine kinase), which has been shown to play a role in a number of endothelial cell functions, including cell growth and migration. These findings suggest that H(+)-ATP synthase-like molecules on the surface of HUVECs play an important role not only in extracellular ATP synthesis but also in the proliferation of HUVECs. The present results demonstrate that the use of small molecular H(+)-ATP synthase inhibitors targeting the F(1) catalytic domain may lead to significant advances in potential antiangiogenic cancer therapies.     

Hic-5 communicates between focal adhesions and the nucleus through oxidant-sensitive nuclear export signal

hic-5 was originally isolated as an H(2)O(2)-inducible cDNA clone whose product was normally found at focal adhesions. In this study, we found that Hic-5 accumulated in the nucleus in response to oxidants such as H(2)O(2). Other focal adhesion proteins including paxillin, the most homologous to Hic-5, remained in the cytoplasm. Mutation analyses revealed that the C- and N-terminal halves of Hic-5 contributed to its nuclear localization in a positive and negative manner, respectively. After the finding that leptomycin B (LMB), an inhibitor of nuclear export signal (NES), caused Hic-5 to be retained in the nucleus, Hic-5 was demonstrated to harbor NES in the N-terminal, which was sensitive to oxidants, thereby regulating the nuclear accumulation of Hic-5. NES consisted of a leucine-rich stretch and two cysteines with a limited similarity to Yap/Pap-type NES. In the nucleus, Hic-5 was suggested to participate in the gene expression of c-fos. Using dominant negative mutants, we found that Hic-5 was actually involved in endogenous c-fos gene expression upon H(2)O(2) treatment. Hic-5 was thus proposed as a focal adhesion protein with the novel aspect of shuttling between focal adhesions and the nucleus through an oxidant-sensitive NES, mediating the redox signaling directly to the nucleus.     

Enhancement of transpiration by ethylene is responsible for absence of internodal elongation in floating rice at low humidity

Internodal elongation in floating rice (Oryza sativa) is known to be enhanced by treatment with ethylene or gibberellic acid (GA3) at high relative humidity (RH). However, ethylene-induced internodal elongation is inhibited at low RH, while GA3-induced internodal elongation is hardly affected by humidity. We examined the effects of ethylene and GA3 on the rate of transpiration in stem segments incubated at 30% or 100% RH. Ethylene promoted the transpiration of stem segments at 30% RH, but not at 100% RH, while GA3 had little effect on transpiration at either 30% or 100% RH. We propose that the absence of ethylene-induced internodal elongation at low RH is due, at least in part, to ethylene-induced transpiration.     

Identification of the lantibiotic nisin Q,a new natural nisin variant produced by Lactococcus lactis 61-14 isolated from a river in Japan

Lactococcus lactis 61-14 isolated from river water produced a bacteriocin active against a wide range of Gram-positive bacteria. N-terminal amino acid sequencing, mass spectral analysis of the purified bacteriocin, and genetic analysis using nisin-specific primers showed that the bacteriocin was a new natural nisin variant, termed nisin Q. Nisin Q and nisin A differ in four amino acids in the mature peptide and two in the leader sequence.     

Pleiotropic phenotypes of fission yeast defective in ubiquinone-10 production. A study from the abc1Sp (coq8Sp) mutant

We previously constructed two Schizosaccahromyces pombe ubiquinone-10 (or Coenzyme Q10) less mutants, which are either defective for decaprenyl diphosphate synthase or p-hydroxybenzoate polyprenyl diphosphate transferase. To further confirm the roles of ubiquinone in S. pombe, we examined the phenotype of the abc1Sp (coq8Sp) mutant, which is highly speculated to be defective in ubiquinone biosynthesis. We show here that the abc1Sp defective strain did not produce UQ-10 and could not grow on minimal medium. The abc1Sp-deficient strain required supplementation with antioxidants such as cysteine or glutathione to grow on minimal medium. In support of the antioxidant function of ubiquinone, the abc1Sp-deficient strain is sensitive to H2O2 and Cu2+. In addition, expression of the stress inducible ctt1 gene was much induced in the ubiquinone less mutant than wild type. Interestingly, we also found that the abc1-deficient strain as well as other ubiquinone less mutants produced a significant amount of H2S, which suggests that oxidation of sulfide by ubiquinone may be an important pathway for sulfur metabolism in S. pombe. Thus, analysis of the phenotypes of S. pombe ubiquinone less mutants clearly demonstrate that ubiquinone has multiple functions in the cell apart from being an integral component of the electron transfer system.     

Detection of Chlamydia species-specific serum antibodies by prior adsorption of common genus-specific antibodies

To establish a method for the detection of Chlamydia species-specific antibodies to the three species of Chlamydia responsible for human disease, the author attempted to remove Chlamydia genus-specific antibodies by prior adsorption with heterologous Chlamydia antigen. The effects of adsorption with heterologous antigen were investigated by the microplate immunofluorescence antibody technique. The Chlamydia genus-specific antibodies in immune animal sera were significantly reduced by prior adsorption with heterologous Chlamydia antigen. Chlamydia pecorum which does not infect humans was found to be useful for the adsorption. A preliminary test using Chlamydia trachomatis-infected human sera showed that this adsorption method with C. pecorum is applicable to the serodiagnosis of human Chlamydia infections.     

Stimulatory effect of regucalcin on proteolytic activity is impaired in the kidney cortex cytosol of rats with saline ingestion

The effect of regucalcin (RC) on neutral proteolytic activity in the cytosol of rat kidney cortex was investigated. Proteolytic activity was significantly increased by the presence of RC (0.01 + 0.10 M) in the enzyme reaction mixture. This increase was completely abolished by the addition of anti-RC monoclonal antibody (150 ng/ml). When the renal cortex cytosol was incubated without RC addition, the degradation of globin of substrate was demonstrated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. This degradation was clearly inhibited by the addition of anti-RC antibody (150 ng/ml), indicating that protein degradation results partly from the cytosolic endogenous RC. Meanwhile, proteolytic activity was significantly decreased in the renal cortex cytosol of rats with saline ingestion for 2, 7, and 14 days. The effect of RC (0.1 M) in increasing proteolytic activity was weakened in the kidney cortex cytosol of saline-ingested rats. The present study suggests that endogenous RC plays a role in the activation of proteases in the renal cortex cytosol, and that the RC effect is impaired in saline-ingested rats.