Possible mechanism for the decrease of mitochondrial aspartate aminotransferase activity in ischemic and hypoxic rat retinas

AbstractGlutamate is believed to be an excitatory amino acid neurotransmitter in the retina. Enzymes for glutamate metabolism, such as glutamate dehydrogenase, ornithine aminotransferase, glutaminase, and aspartate aminotransferase (AAT), exist mainly in the mitochondria. The abnormal increase of intracellular calcium ions in ischemic retinal cells may cause an influx of calcium ions into the mitochondria, subsequently affecting various mitochondrial enzyme activities through the activity of mitochondrial calpain. As AAT has the highest level of activity among enzymes involved in glutamate metabolism, we investigated the change of AAT activity in ischemic and hypoxic rat retinas and the protection against such activity by calpain inhibitors. We used normal RCS (rdy+/rdy+) rats. For the in vivo studies, we clamped the optic nerve of anesthetized rats to induce ischemia. In the in vitro studies, the eye cups were incubated with Locke’s solution saturated with 95% N2/5% CO2. The activity of cytosolic AAT (cAAT) was about 20% of total activity, whereas mitochondrial AAT (mAAT) was about 75% in rat retina. Ninety minutes of ischemia or hypoxia caused a 20% decrease in mAAT activity, whereas cAAT activity remained unchanged. To examine the contribution of intracellular calcium ions to the degradation of mAAT, we used Ca2+-free Locke’s solution containing 1 mM EGTA, ryanodine (Ca2+ channel blocker), and thapsigargin (Ca2+-ATPase inhibitor). In the present study, thapsigargin in Ca2+-free Locke’s solution, but not ryanodine in this solution, was found to prevent AAT degradation. AAT degradation was also prevented by calpain inhibitors (Ca2+-dependent protease inhibitor) such as calpeptin at 1 nM, 10 nM, 0.1 μM, 1 μM and 10 μM, and by calpain inhibitor peptide, but not by other protease inhibitors (10 μM leupeptin, pepstatin, chymostatin). Additionally, we determined the subcellular localization of calpain activity and examined the change of calpain activity in ischemic rat retinas. Our results suggest that decreased activity of mAAT in ischemic and hypoxic rat retinas might be evoked by the degradation by calpain-catalyzed proteolysis in mitochondria

Essential Role of Thioredoxin 2 in Mitigating Oxidative Stress in Retinal Epithelial Cells

The retina is constantly subjected to oxidative stress, which is countered by potent antioxidative systems present in retinal pigment epithelial (RPE) cells. Disruption of these systems leads to the development of age-related macular degeneration. Thioredoxin 2 (Trx2) is a potent antioxidant, which acts directly on mitochondria. In the present study, oxidative stress was induced in the human RPE cell line (ARPE-19) using 4-hydroxynonenal (4-HNE) or C2-ceramide. The protective effect of Trx2 against oxidative stress was investigated by assessing cell viability, the kinetics of cell death, mitochondrial metabolic activity, and expression of heat shock proteins (Hsps) in Trx2-overexpressing cell lines generated by transfecting ARPE cells with an adeno-associated virus vector encoding Trx2. We show that overexpression of Trx2 reduced cell death induced by both agents when they were present in low concentrations. Moreover, early after the induction of oxidative stress Trx2 played a key role in the maintenance of the cell viability through upregulation of mitochondrial metabolic activity and inhibition of Hsp70 expression

福岡縣新宮濱に於ける砂丘植物群落とその土壌要素との關係

1) 新宮濱の砂丘植物群落は高潮線より僅々200米内外の間に明かに異つた三つの帶状群落を區別することが出來る。2) 第一は前砂丘植物群落であり, 第二は梢中生的傾向のある好砂砂丘植物群落で, 第三はクロマツを主とする砂丘林である。3) 砂層内の鹽分はその採集地點が内方に向ふにつれて急減し, 高潮線よりの距離と含鹽量との關係は雙曲線型曲線で表はすことが出來るものの如くである。4) それぞれの群落の生育地砂層内の鹽分分布には特有の型があり, 前砂丘植物群落では地表・地下共に鹽分濃度高く, 梢中生的傾向を持つ好砂砂丘植物群落では地表は鹽分濃度は地表下含度に比し割合に大である。砂丘林の部分では地表・地下共に低濃度で植物群落の區分と土壤に於ける含鹽状態區分とは大體一致を認めることが出來た。5) 土壤の酸度によつても大體鹽分によつたと同樣の三區分が出來る樣である。6) 隨つて土壤の鹽分と酸度とは本砂丘の植物群落の成立上重要なる役割を演じ居るものの如くである

Total syntheses of disulphated glycosphingolipid SB1a and the related monosulphated SM1a

Total syntheses of two natural sulphoglycolipids, disulphated glycosphingolipid SB1a and the structurally related monosulphated SM1a, are described. They have common glycan sequences and ceramide moieties and are associated with human epithelial carcinomas. The syntheses featured efficient glycan assembly and the glucosyl ceramide cassette as a versatile building block. The binding of the synthetic sulphoglycolipids by the carcinoma-specific monoclonal antibody AE3 was investigated using carbohydrate microarray technology

Active intestinal absorption of fluoroquinolone antibacterial agent ciprofloxacin by organic anion transporting polypeptide, Oatp1a5

Fluoroquinolone antimicrobial drugs are absorbed efficiently after oral administration despite of their hydrophilic nature, implying an involvement of carrier-mediated transport in their membrane transport process. It has been that several fluoroquinolones are substrates of organic anion transporter polypeptides OATP1A2 expressed in human intestine derived Caco-2 cells. In the present study, to clarify the involvement of OATP in intestinal absorption of ciprofloxacin, the contribution of Oatp1a5, which is expressed at the apical membranes of rat enterocytes, to intestinal absorption of ciprofloxacin was investigated in rats. The intestinal membrane permeability of ciprofloxacin was measured by in situ and the vascular perfused closed loop methods. The disappeared and absorbed amount of ciprofloxacin from the intestinal lumen were increased markedly in the presence of 7,8-benzoflavone, a breast cancer resistance protein inhibitor, and ivermectin, a P-glycoprotein inhibitor, while it was decreased significantly in the presence of these inhibitors in combination with naringin, an Oatp1a5 inhibitor. Furthermore, the Oatp1a5-mediated uptake of ciprofloxacin was saturable with a K mvalue of 140 μm, and naringin inhibited the uptake with an IC 50value of 18 μm by Xenopus oocytes expressing Oatp1a5. Naringin reduced the permeation of ciprofloxacin from the mucosal-to-serosal side, with an IC 50 value of 7.5 μm by the Ussing-type chamber method. The estimated IC 50 values were comparable to that of Oatp1a5. These data suggest that Oatp1a5 is partially responsible for the intestinal absorption of ciprofloxacin. In conclusion, the intestinal absorption of ciprofloxacin could be affected by influx transporters such as Oatp1a5 as well as the efflux transporters such as P-gp and Bcrp. Copyright © 2012 John Wiley & Sons, Ltd