Sadahiko Ishibashi

Neuroprotective effect of an antioxidant, ebselen, in patients with delayed neurological deficits after aneurysmal subarachnoid hemorrhage.

OBJECTIVE The effect of ebselen, a seleno-organic compound with antioxidant activity through a glutathione peroxidase-like action, on the outcome of subarachnoid hemorrhage was evaluated in a multicenter placebo-controlled double-blind clinical trial. METHODS Patients who suffered aneurysmal subarachnoid hemorrhages of Hunt and Kosnik Grades II through IV at admission and were able to start drug treatment within 96 hours of the ictus were enrolled. Early surgery was performed whenever possible. Oral administration of ebselen granules suspended in water (150 mg, twice a day) or placebo was started immediately after admission and continued for 2 weeks. The major end points were the Glasgow Outcome Scale at 2 weeks, 1 month, and 3 months after the start of treatment. The incidence of delayed ischemic neurological deficits clinically diagnosed as resulting from vasospasm and the incidence and extent of low-density areas on postoperative computed tomographic scans were also studied as secondary outcome measures. RESULTS Intent-to-treat analysis of the 286 patients enrolled in the trial (145 patients administered ebselen and 141 administered placebo) revealed that the incidence of clinically diagnosed delayed ischemic neurological deficits was unaltered. There were 52 (receiving ebselen) and 58 (receiving placebo) patients with delayed deficits; however, a significantly better outcome was observed after ebselen treatment than after placebo (P = 0.005, chi2 test). There was a corresponding decrease in the incidence and extent of low-density areas (P = 0.032, Wilcoxon rank sum test). CONCLUSION Ebselen reduced brain damage in patients with delayed neurological deficits after subarachnoid hemorrhage and may be a promising neuroprotective agent.

Progress of aging in human diploid cells transformed with a tsA mutant of simian virus 40

Normal human diploid cells, TIG-1, ceased to proliferate at about the 62 population doubling level (PDL). Transformed clones isolated from TIG-1 cells infected with wtSV40 and those with tsA900 SV40 cultured at 34 degrees C were subcultured up to about 80 PDL. When the culture temperature of tsA SV40-transformed cells was shifted from 34 to 39.5 degrees C at 51 PDL, the growth curve of these transformed cells changed to that of normal young cells. When shifted to 39.5 degrees C after 62 PDL, cells immediately reached the end of their proliferative lifespan even under such favourable conditions for growth as low cell density in fresh medium. Growth of wtSV40-transformed cells did not change markedly at either temperature. These findings suggest that the clock of aging progresses in transformed cells as in normal cells, around 62 PDL being the senescent state in both cases, and that T-antigen of the tsA mutant of SV40 supports the extension of the lifespan of human cells only at the permissive temperature.

Reinitiation of host DNA synthesis in senescent human diploid cells by infection with simian virus 40

Human diploid fibroblasts, TIG-1, cease to proliferate at about 60-62 population doubling level. In their senescent state used in this study, the percentage of nuclei labeled by [3H]thymidine for 48 h was around 1-2% in fresh medium containing 5-40% fetal bovine serum. The percentage of labelled nuclei increased up to 10-fold after infection with SV40. This increase reflects stimulation of cell DNA synthesis because: 1. The increase also occurred when ts A900 was used for infection at the non-permissive temperature, under these conditions viral DNA synthesis is inhibited; 2, the increase paralleled the stimulation of [3H]thymidine incorporation into DNA in a Hirt-precipitate fraction from SV40-infected cells. UV-irradiated SV40 had reduced ability to induce DNA synthesis. A viable deletion mutant of SV40, d1940, had almost the same activity to induce cell DNA synthesis as did wild-type SV40. Equilibrium density gradient centrifugation analysis of DNA labelled with 5-bromodeoxyuridine (BrdU) supported semiconservative replication rather than repair synthesis. We conclude that a considerable fraction of human diploid cells in a senescent population initiate host DNA replication by infection with SV40, although these cells cannot be stimulated with fetal bovine serum.

Early and transient increase in oxidative stress in the cerebral cortex of senescence-accelerated mouse

Age-related changes in oxidative stress in the cerebral cortex of SAMP8, a substrain of senescence-accelerated mouse (SAM), were investigated in comparison with those in SAMR1, which were used as a control. The lipid peroxide and protein carbonyl contents were transiently increased in SAMP8 from 4- to 8-weeks of age. The increases in lipid peroxide were seen only in the cerebral cortex and not in other regions of the cerebrum. Furthermore, the net generation of reactive oxygen species in cerebral cells was also increased in SAMP8. In addition, the activity of glutamine synthetase, which is known as an enzyme-highly sensitive to reactive oxygen, was decreased in the cerebral cortex of SAMP8 from 4- to 8-weeks of age. These results suggest that oxidative stress may be induced in the cerebral cortex of SAMP8 from 4- to 8-weeks of age, preceding the appearance of distinctive deficits in the brain of SAMP8.

ß-Subunit of Escherichia coli F1-ATPase: An amino acid replacement within a conserved sequence (G-X-X-X-X-G-K-T/S) of nucleotide-binding proteins

A mutant strain KF87 of E. coli with a defective ß‐subunit (Ala‐151 → Val) of F1‐ATPase was isolated. The mutation is within the conserved sequence (G‐X‐X‐X‐X‐G‐K‐T/S) of nucleotide‐binding proteins. The mutant F1‐ATPase had a much higher rate of uni‐site hydrolysis of ATP than the wild type, and about 6% of the wild‐type multi‐site activity. The mutant enzyme showed defective transmission of conformational change(s) between the ligand‐ and aurovertin‐binding sites.A mutant strain KF87 of E. coli with a defective beta-subunit (Ala-151----Val) of F1-ATPase was isolated. The mutation is within the conserved sequence (G-X-X-X-X-G-K-T/S) of nucleotide-binding proteins. The mutant F1-ATPase had a much higher rate of uni-site hydrolysis of ATP than the wild type, and about 6% of the wild-type multi-site activity. The mutant enzyme showed defective transmission of conformational change(s) between the ligand- and aurovertin-binding sites.

Changes in protein phosphorylation in guinea pig polymorphonuclear leukocytes by treatment with membrane-perturbing agents which stimulate superoxide anion production

Phosphorylation of proteins was examined in guinea pig polymorphonuclear leukocytes in relation to the effects of membrane-perturbing agents, which stimulate superoxide anion production, and their inhibitors. The phosphorylation was detected by 32P autoradiography after separation by two-dimensional electrophoresis of proteins phosphorylated in 32P-preloaded cells. Though phosphorylation of various proteins was stimulated by each of the membrane-perturbing agents, the stimulation was especially marked in six proteins. Phorbol myristate acetate and digitonin enhanced the phosphorylation of the six proteins, while myristate and concanavalin A increased the phosphorylation of five and three proteins, respectively, out of the six proteins. p-Bromophenacyl bromide, an inhibitor of phospholipase A2, inhibited the stimulatory effect of phorbol myristate acetate on both superoxide anion production and protein phosphorylation. Trifluoperazine, a calmodulin inhibitor, also inhibited the effect of phorbol myristate acetate on both, except for an increase in the phosphorylation of one out of the six proteins. alpha-Methylmannoside, an inhibitor of concanavalin A binding, inhibited the stimulation of the phosphorylation of the three proteins by concanavalin A. The results indicate that the activation of superoxide anion production by the membrane-perturbing agents in guinea pig polymorphonuclear leukocytes is accompanied by the phosphorylation of, at least some of, these six proteins.

Early appearance of abnormality of microperoxisomal enzymes in the cerebral cortex of senescence-accelerated mouse.

SAMP8, a substrain of senescence-accelerated mouse (SAM), has been characterized by several age-related deficits in the brain. Previously, we reported that the contents of lipid peroxides and protein carbonyl, and net generation of H2O2 were increased in the cerebral cortex of SAMP8 at 4-8 weeks of age in comparison with those in age-matched SAMR1 substrain used as a control. To study the cause of these increases, we compared the activities of antioxidative enzymes in the cerebral cortex between the two substrains. The catalase activity was decreased by 75% in SAMP8 at 4-8 weeks of age, whereas neither superoxide dismutase nor glutathione peroxidase activities were changed. The change in the catalase activity was seen only in the cerebral cortex where oxidative stress was increased in SAMP8. On the contrary, the activity of acyl-CoA oxidase, a microperoxisomal H2O2-producing enzyme, in the cerebral cortex of SAMP8 was increased 1.6 fold in comparison with that in age-matched SAMR1 without change in the activity of D-amino acid oxidase. Furthermore, the changes in the activities of catalase and acyl-CoA oxidase with age were paralleled with those observed in oxidative stress in SAMP8. These results suggest that the abnormality of activities in two microperoxisomal enzymes, catalase and acyl-CoA oxidase, may be one of the cause of the early increase in oxidative stress observed in the cerebral cortex of 4-8 weeks old SAMP8.

Decreased d-glucose transport across renal brush-border membrane vesicles from streptozotocin-induced diabetic rats

The uptake of Na(+)-dependent D-glucose by renal brush-border membrane vesicles (BBMV) isolated from streptozotocin-induced diabetic rats was decreased as compared with controls. Since a Vmax of 4.8 nmol/mg protein per 30 s in diabetic BBMV was significantly decreased as compared with that of controls (Vmax = 7.0 nmol/mg protein per 30 s) without changing an apparent affinity for D-glucose, the decrease in the Na(+)-dependent D-glucose uptake in diabetic rats is likely to be due to the reduction in the number of the transporter. These results are also confirmed by the binding study of [3H]phlorizin to diabetic BBMV. When the blood glucose level is lowered in diabetic rats by both the treatment with insulin and starvation, the decreased Na(+)-dependent D-glucose uptake is returned to control level. These results suggest that Na(+)-dependent D-glucose reabsorption through the apical membrane in proximal tubular kidney cells is dynamically regulated by the change in blood glucose level.

Early changes in glucose metabolism in the cerebrum of senescence accelerated mouse: involvement of glucose transporter.

The rate of 6-[14C]D-glucose oxidation in cerebral cells of SAMP8, a substrain of senescence accelerated mouse, was investigated in vitro. The production of 14CO2 in dissociated intact brain cells prepared from the cerebrum of 4-8-week-old SAMP8 was higher than that of age-matched SAMR2 as a control mouse, while no difference between these two strains was observed in the production of 14CO2 in the cerebral homogenates. These results indicated that the increased metabolism of glucose in SAMP8 might be associated with the glucose transport system across the cell membrane. Therefore, 2-deoxy-D-glucose (2-DG) uptake into the brain cells and cytochalasin B (CB) binding to cerebral crude membranes were examined. Both the 2-DG uptake and the CB binding in SAMP8 were much greater than in SAMR2. Furthermore, the increased CB binding in SAMP8 was seen only in the cerebral cortex of 4- to 8-week-old mice, and neither in other regions of the cerebrum nor in other aged mice (2-week- and 40- to 48-week-old mice). These results suggest that the transient overproduction of the glucose transporter protein in the cerebral cortex is involved in the increased glucose metabolism in 4- to 8-week-old SAMP8.

Correlation between the presence of T-antigen and the reinitiation of host DNA synthesis in senescent human diploid fibroblasts after SV40 infection

Senescent human diploid fibroblasts, TIG-1, had labelling indices of about 0.5-3% when labelled with [3H]thymidine for 3 days in fresh medium containing 10% fetal bovine serum. When these cells were infected with SV40, the percentage of nuclei incorporating [3H]thymidine increased by about 10-fold. The frequency of T-antigen-positive cells and that of [3H]thymidine-incorporating cells were almost the same. About 80% of T-antigen-positive cells were also positive to incorporation of [3H]thymidine, and the same result was obtained in infected young cells. These results indicated that senescent human diploid cells which are brought to synthesize T-antigen always initiate DNA synthesis as young cells do. The characteristics of senescent cells as compared with younger cells was low incidence of T-antigen-positive cells after infection. The basis of low susceptibility of senescent cells to initiate DNA synthesis by SV40 infection thus seems to be concerned with an event after the adsorption of virus, but before the synthesis of a detectable amount of T-antigen.