Search

Changes in amount and activity of enzyme protein are critical factors in regulating intracellular metabolisms. However, since the metabolisms are proceeding in environment with complex architecture consisted of various membranes, spatial factors should be taken into consideration for the regulation. In this review, involvement of interaction between cytosolic and membrane proteins in metabolic regulation are discussed. It had been reported that hexokinase activity was found in mitochondrial fraction in spite of almost exclusive distribution of other glycolytic enzymes to soluble fraction, the tendency being marked in the brain and many types of tumor cells whereas mitochondrial hexokinase activity was quite low in the liver. Interested in such enzyme and tissue specificities, we investigated the significance and mechanism of the unique intracellular distribution of hexokinase. We found that mitochondria-bound hexokinase was more active than the cytosolic type in producing glucose 6-phosphate (G6P), probably due to the advantage in utilizing ATP produced in mitochondria. In addition, we also found that the binding stabilized hexokinase against G6P inhibition. As to the binding, it was reported that G6P released hexokinase from mitochondria while Mg2+ promoted the binding. In this respect, we found that polyamines promoted the binding at much lower concentration than that of Mg2+, and mitochondria-bound form had small hydrophobic domain at terminal region for the binding to porin on the outer membrane. Then, we found a protease which specifically cleaved the domain with little effect on catalytic activity and molecular size of the bindable form. Such a modifying protease was purified and identified as lysosomal cathepsin L. The protease activity was high in the liver and low in the brain, suggesting that the difference in the activity was responsible for the afore-mentioned tissue specificity. On the other hand, we examined regulatory mechanism for active oxygen production in neutrophils, since the production of superoxide anion (O2-) by NADPH oxidase was very low at the resting state while markedly increased on phagocytosis and chemical stimulation. Since the stimulants for the activation were so various in chemical nature, we postulated mechanism to converge the stimulation to the activation. Incidentally, we found increase in phosphorylation of 46-47 K protein, irrespective of the type of stimulation. Use of inhibitors and examination on the phosphorylation condition indicated protein kinase C (PKC) as the phosphorylating enzyme. In addition, we observed the 46-47 K protein existed in cytosol at resting state, while it was translocated to cell membranes in concurrence with the phosphorylation. Similar findings were obtained in many laboratories and those proteins were named cytosolic activating factors (and then p47-phox, etc.). These proteins associate with membrane proteins to constitutes the active from of NADPH oxidase. Next, we examined mechanism to shut off the O2- production, and found that the inactivation through disassembly of the constituents was attained by dephosphorylation of phosphorylated p47-phox by cytosolic protein phosphatase. Then we have also found that protein kinases other than PKC were involved in regulation of NADPH oxidase activity. Though phosphorylation of p47-phox etc. is deeply involved in the activation of NADPH oxidase, membrane perturbation, so-called priming, is required for the activation. We also reported some possible indications for the priming, and possible involvement of cytoskeletons in O2- production. Apart from protein phosphorylation, it has been reported that amphiphilic acidic compounds are potent activator for NADPH oxidase. We also have examined their effects to find that these compounds also caused the assembly of the NADPH oxidase constituents. Reversely, amphiphilic basic compounds suppressed suggesting significance of introduction of negative charge in NADPH oxidase activat

We investigated the effect of two isomers of retinoic acid (RA), all-trans RA and 9-cis RA, on the proliferation of Y79 human retinoblastoma cells. The two isomers inhibited the cell proliferation in a concentration-dependent manner. The IC50 for this inhibition by all-trans RA and 9-cis RA was 1.50 and 0.15 microM, respectively. The inhibitory effect of 9-cis RA on Y79 cell growth was observed within 24 hr, thereafter the cell number was gradually decreased. In contrast, no inhibition by all-trans RA of Y79 cell growth was observed within 24 hr, thereafter the cell number was slightly increased. In these cases, the cell viability at 4 days after the addition of 9-cis RA and all-trans RA was more than 90% and 95%, respectively. These results indicate that the two RA inhibit the proliferation of Y79 human retinoblastoma cells without inducing the cell death and that the effect of 9-cis RA on the inhibition of Y79 cell growth is much greater than that of all-trans RA.

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.

Age-related changes in glycogen concentration in the heart of SAMP8, a substrain of senescence-accelerated prone mouse, were compared with that in SAMR1, a substrain of senescence-accelerated resistant mouse. The decrease by about 50% in the glycogen concentration in the hearts of 4-8 weeks old SAMP8 was observed in comparison with that in age-matched SAMR1, whereas there was no difference in adult hearts between the two substrains. Additionally, glycogen phosphorylase activity was significantly increased (1.3-fold) in the heart of 4–8 weeks old SAMP8 without the change in glycogen synthase activity between the two substrains. At the same age, the lipid peroxide concentration was also increased (1.4-fold) in the hearts of SAMP8, suggesting that oxidative stress was involved in the early damage to the hearts of SAMP8. Furthermore, the present findings that the cardiac glycogen concentration in SAMP8 decreases more quickly than that in SAMR1 may indicate the possibility that an accelerated aging of SAMP8 is related to the early changes in the energy sources in the heart.

We investigated the expression of glucose transporter (GLUT) protein isoforms in two human retinoblastoma cell lines, Y79 and WERI-Rb1, by Western blotting analysis with anti-GLUT1, 2, 3, and 4 antibodies. GLUT1 and GLUT4 proteins were detected in Y79, whereas GLUT1 and GLUT3 proteins were found in WERI-Rb1. GLUT2 protein was not detected in Y79 or WERI-Rb1. Our findings are of interest because (1) the expression of GLUT protein isoforms in the two retinoblastoma cell lines was different, and (2) GLUT4 protein, the insulin-sensitive GLUT isoform, was detected in Y79. This suggests that these cell lines have different mechanisms of glucose transport.

The expression of rho family along the crypt-villus axis of rat small intestine was investigated. The immunoblot analysis showed that the content of rac1 protein gradually increased from tip-villus to crypt-villus enterocytes without changes in the contents of rhoA and rac2 proteins. Furthermore, the contents of rac1 protein in the entire enterocytes were markedly decreased in streptozotocin-induced diabetic rats and restored by short-term treatment with insulin. These results suggest that differentiation and proliferation in the enterocytes of rat small intestine are possibly related to the expression of rac1 protein through insulin action.

The effects of sphingoid bases, sphingosine and dihydrosphingosine, which are protein kinase C (PKC) inhibitors, on NADPH oxidase were examined in a cell-free system. The bases inhibited cell-free activation of NADPH oxidase by arachidonic acid at lower concentration than N-acetylsphingosine. Thus, positive charge in the molecules may play a critical role in inhibition of the oxidase. Sphingosine did not change the Km value for NADPH, but shifted the optimum concentration of arachidonic acid for activation of the oxidase. Moreover, sphingosine suppressed the translocation of p47-phox, one of the cytosolic components of the oxidase, to the membrane fraction, suggesting that the base inhibits the assembly of the components.

Mechanisms for the cell-free activation of NADPH oxidase by sodium dodecyl sulfate (SDS) and arachidonate were compared in relation to their responsiveness to short chain diacylglycerols. The plasma membrane and cytosol prepared from guinea pig neutrophils were used for the cell-free system. The activation of NADPH oxidase by SDS was enhanced about 5- to 10-fold by 1,2-dioctanoylglycerol (diC8), but not by either 1,2-dihexanoylglycerol (diC6) or 1,2-didecanoylglycerol (diC10). However, none of these diacylglycerols potentiated the NADPH oxidase activation by arachidonate. The maximal extent of activation by the combination of SDS and diC8 was similar to that by arachidonate alone. In the presence of sufficient amounts of diC8 and SDS, GTP gamma S potentiated the activation of NADPH oxidase. The potentiating activity of diC8 was preserved in the membrane fraction, not in the cytosol fraction. These results suggest that arachidonate may possess the functions of both SDS and diC8 in the activation. In addition, diC8 and GTP gamma S seem to independently enhance the NADPH oxidase activation.

p47-phox is one of the cytosolic activation factors of NADPH oxidase in neutrophils and known to translocate to plasma membranes and function by protein kinase C-phosphorylation. In cytosol fraction, prepared from calyculin A-treated neutrophils, the activity of cytosolic factor to activate NADPH oxidase was more reduced than that from PMA-treated cells. But, p47-phox did not translocate to the membranes, even if p47-phox was hyperphosphorylated in the calyculin A-treated neutrophils. Such hyperphosphorylated p47-phox seemed to lose the activity to constitute NADPH oxidase complex.

We studied involvement of tyrosine-phosphorylated proteins in activation of NADPH oxidase in guinea pig neutrophils. Pervanadate, which is the oxidized form of orthovanadate, induced[formula]production and protein tyrosine phosphorylation in neutrophils.[formula]production induced by pervanadate was more sensitive to the tyrosine kinase-specific inhibitor, ST-638, as compared with the production induced by PMA. On the other hand, staurosporine more selectively inhibited PMA-induced[formula]production than pervanadate-induced production. These results indicate that tyrosine kinase, not protein kinase C, is involved in pervanadate-induced[formula]production. The tyrosine-phosphorylated proteins were detected in both the cytosol and membrane fractions prepared from pervanadate-induced neutrophils. In order to examine if tyrosine residues of some components of NADPH oxidase were directly phosphorylated, tyrosine-phosphorylated proteins were removed from solubilized membranes prepared from the pervanadate-stimulated neutrophils by immunoprecipitation with an anti-phosphotyrosine antibody. NADPH oxidase activity in the solubilized membranes was not decreased by the treatment. These findings suggest that the components of NADPH oxidase are not tyrosine-phosphorylated by pervanadate treatment, that tyrosine phosphorylation may be involved in the signal transduction pathway of NADPH oxidase activation by pervanadate, and that this pathway is independent of the activation by protein kinase C.

It is well known that sodium dodecyl sulfate (SDS) activates NADPH oxidase in a cell-free system independently of protein kinase C (PKC). However, in intact neutrophils, direct evidence has never been presented to show that O − 2 production by SDS is actually due to the NADPH oxidase activation observed in the cell-free system. So, in this paper, we investigated the activation mechanism by SDS in intact guinea pig neutrophils. We previously reported that hypotonic treatment reversibly enhanced O − 2 production stimulated by PKC activators in intact neutrophils (M. Hiura et al. , 1991, Arch. Biochem. Biophys. 291, 31-37). In this paper, SDS also significantly stimulated O − 2 production in the intact cells under the hypotonic condition. This enhancement was gradual and was PKC inhibitor resistant. Furthermore, phosphorylation of the 46-kDa protein, one of cytosolic activation factors, was not detected by autoradiography of two-dimensional electrophoresis. Translocation of cytosolic activation factors was demonstrated by a decrease in the activity of the factors remained in the cytosol. In the presence of SDS, addition of 1-oleoyl-2-acetylglycerol, a PKC activator, further enhanced O − 2 production and translocation of the cytosolic activation factors. On the other hand, SDS remarkably increased membrane fluidity in intact neutrophils as well as in the cell-free system. These results indicate that activation of NADPH oxidase by SDS in intact neutrophils seems to be partly due to the same mechanism observed in cell-free activation, and that SDS alone slightly activates the oxidase and other stimulation, such as hypotonic and/or PKC activator treatments, is required for significant activation. The increase in the membrane fluidity may be one of the activation mechanisms of NADPH oxidase by SDS.

The effects of Panax ginseng extract, ginseng saponins, and some other triterpenoid saponins on glucose uptake were examined by using sheep erythrocytes. Initial rates of glucose transport were determined by measurements of 2-deoxy-D-glucose (2-DG) uptake. From kinetic analysis apparent Km and Vmax values of facilitated glucose transport in sheep erythrocytes were calculated as 2.3 +/- 0.08 mM and 1.4 +/- 0.05 nmol/min/10(9) cells. The results showed that ginseng extract stimulated glucose uptake in sheep erythrocytes dose-dependently. Ginseng saponins, in general, also stimulated glucose transport. The maximum effect was observed at 1 microM of ginsenoside Rb1 showing an increase of 24 +/- 5% above basal activity. However, ginsenoside Rg3, chikusetsusaponin Ia, and glycyrrhetic acid induced significant inhibitory effects on glucose transport in sheep erythrocytes.

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.

Changes in amount and activity of enzyme protein are critical factors in regulating intracellular metabolisms. However, since the metabolisms are proceeding in environment with complex architecture consisted of various membranes, spatial factors should be taken into consideration for the regulation. In this review, involvement of interaction between cytosolic and membrane proteins in metabolic regulation are discussed. It had been reported that hexokinase activity was found in mitochondrial fraction in spite of almost exclusive distribution of other glycolytic enzymes to soluble fraction, the tendency being marked in the brain and many types of tumor cells whereas mitochondrial hexokinase activity was quite low in the liver. Interested in such enzyme and tissue specificities, we investigated the significance and mechanism of the unique intracellular distribution of hexokinase. We found that mitochondria-bound hexokinase was more active than the cytosolic type in producing glucose 6-phosphate (G6P), probably due to the advantage in utilizing ATP produced in mitochondria. In addition, we also found that the binding stabilized hexokinase against G6P inhibition. As to the binding, it was reported that G6P released hexokinase from mitochondria while Mg2+ promoted the binding. In this respect, we found that polyamines promoted the binding at much lower concentration than that of Mg2+, and mitochondria-bound form had small hydrophobic domain at terminal region for the binding to porin on the outer membrane. Then, we found a protease which specifically cleaved the domain with little effect on catalytic activity and molecular size of the bindable form. Such a modifying protease was purified and identified as lysosomal cathepsin L. The protease activity was high in the liver and low in the brain, suggesting that the difference in the activity was responsible for the afore-mentioned tissue specificity. On the other hand, we examined regulatory mechanism for active oxygen production in neutrophils, since the production of superoxide anion (O2-) by NADPH oxidase was very low at the resting state while markedly increased on phagocytosis and chemical stimulation. Since the stimulants for the activation were so various in chemical nature, we postulated mechanism to converge the stimulation to the activation. Incidentally, we found increase in phosphorylation of 46-47 K protein, irrespective of the type of stimulation. Use of inhibitors and examination on the phosphorylation condition indicated protein kinase C (PKC) as the phosphorylating enzyme. In addition, we observed the 46-47 K protein existed in cytosol at resting state, while it was translocated to cell membranes in concurrence with the phosphorylation. Similar findings were obtained in many laboratories and those proteins were named cytosolic activating factors (and then p47-phox, etc.). These proteins associate with membrane proteins to constitutes the active from of NADPH oxidase. Next, we examined mechanism to shut off the O2- production, and found that the inactivation through disassembly of the constituents was attained by dephosphorylation of phosphorylated p47-phox by cytosolic protein phosphatase. Then we have also found that protein kinases other than PKC were involved in regulation of NADPH oxidase activity. Though phosphorylation of p47-phox etc. is deeply involved in the activation of NADPH oxidase, membrane perturbation, so-called priming, is required for the activation. We also reported some possible indications for the priming, and possible involvement of cytoskeletons in O2- production. Apart from protein phosphorylation, it has been reported that amphiphilic acidic compounds are potent activator for NADPH oxidase. We also have examined their effects to find that these compounds also caused the assembly of the NADPH oxidase constituents. Reversely, amphiphilic basic compounds suppressed suggesting significance of introduction of negative charge in NADPH oxidase activat

A previously found proteinase possibly involved in the modification of hexokinase to eliminate the mitochondria-binding ability without appreciable change in the catalytic activity (called hexokinase-processing enzyme hereafter), was purified by sequential chromatographies from rat liver and its properties were examined. The hexokinase-processing enzyme had carbohydrate moieties as evidenced by adsorption on immobilized concanavalin A, and had a molecular weight of about 23,000 as estimated by SDS-PAGE and gel filtration chromatography. Benzyloxycarbonyl-phenylalanyl-L-arginine-4-methylcoumaryl-7-amide (Z-Phe-Arg-MCA)-hydrolyzing activity was co-purified with this processing activity throughout the purification, while the hydrolyzing activity for benzyloxycarbonyl-L-arginyl-L-arginine-4-methylcoumaryl-7-amide (Z-Arg-Arg-MCA) was not. The processing activity, as well as Z-Phe-Arg-MCA hydrolyzing activity, was highly sensitive to cysteine proteinase inhibition, for example, by leupeptin and N-[N-3-(trans-carboxirane-2-carbonyl)-L-leucyl]agmatine (E-64). Furthermore, the enzyme preparation reacted with an antibody against cathepsin L purified from rat kidney. These results indicated that cathepsin L may be involved in the above-mentioned processing of hexokinase.

Exposure to the pentobarbital potently inhibited the 2-deoxy glucose uptake in cultured neuroblastoma cells. The inhibition was assumed to be due to saturation of the uptake in the early stage where the incorporation was linear in the nontreated cells. On the contrary, the incorporation of 3-O-methyl glucose, another glucose analog which is not phosphorylated by hexokinase, was not altered by the treatment with pentobarbital. These results suggest that the suppression of hexokinase is involved in the above-mentioned effect of pentobarbital.

Two lines of immortal human fibroblasts were isolated following transfection of TIG-3 cells with plasmid DNA, pMT-1ODtsA, that contained SV40 early gene with a deletion in replication origin and ts mutation in coding sequence for T-antigen. These cells continued proliferation at 34 degrees C, over 565 population doubling level (PDL) which is far over the limited division potential of untransformed normal TIG-3 of 70-80 PDL. When the culture temperature was shifted to 40 degrees C after 70 PDL, they ceased proliferation immediately. One of these immortal clones, SVts8, lost its ts phenotype after retransformation with wtT-antigen gene. These results indicated that the function of intact T-antigen is required for maintenance of immortal proliferation, at least in one of the SV40 transformed immortal clones.

Treatment of guinea pig polymorphonuclear leukocytes (PMNL) with phorbol 12-myristate 13-acetate (PMA) induced an increase in phosphorylation of 46 kDa protein(s) in parallel with activation of NADPH oxidase. In response to PMA stimulation, phosphorylated 46 kDa protein(s) increased markedly in the membrane fraction, accompanied by a decrease in the unphosphorylated form(s) in the cytosol. The results indicate that the 46 kDa protein(s) may be translocated concomitantly with its phosphorylation. On the other hand, in a cell-free activation system reconstituted from the cytosol and plasma membranes of unstimulated PMNL, arachidonic acid caused the translocation of the 46 kDa protein(s) from the cytosol to the plasma membranes concomitantly with an enhancement of NADPH oxidase activity. These results suggest that activation of NADPH oxidase is dependent on an association of 46 kDa protein(s) with the membranes both in intact PMNL and in the cell-free system.

The superoxide anion (O2−) production of guinea pig polymorphonuclear leukocytes (PMNL) by platelet-activating factor (PAF) was greatly enhanced by the addition of 1-oleoyl-2-acetylglycerol (OAG), even at low concentrations of OAG at which O2 production was little induced. The enhanced production was biphasic, depending on concentrations of PAF. One was saturable at a lower concentration range of PAF and probably mediated through a putative PAF receptor—islet-activating protein-sensitive GTP-binding protein (G protein) pathway. Another was increased in a concentration-dependent manner at a higher concentration range of PAF and apparently mediated through both receptor—G protein-dependent and -independent pathways. These results suggest that at least two types of action of PAF are involved in the synergistic stimulation of O2− production by PAF and OAG in PMNL.

tsJT16 is a temperature-sensitive (ts) mutant of rat fibroblasts that has a ts defect in a function operating soon after the growth stimulation from the G0 phase. After the growth stimulation, the cells express several cell-cycle-dependent genes at both temperatures while they fail at the nonpermissive temperature to synthesize a protein p70 identified on two-dimensional gel electrophoresis. Here we report that 1) synthesis of p70 began within 1 h of stimulation, continued up to the 7th hour and then decreased; 2) the half-life of p70 was shortened after 6 h after the stimulation; 3) p70 was localized in the nuclear fraction; 4) p70 was likely to be a primarily induced protein; 5) mRNA of p70 was supposed to be synthesized exclusively within 2 h of growth stimulation. These and the previous results suggest that p70 is a nuclear protein responsible for the early stage of transition of cells from the G0 toward the S phase and is induced via a different signal transduction sequence from that for the c-fos gene.

The effect of standardized Panax ginseng extract (G115 Pharmaton) on D-glucose uptake by Ehrlich ascites tumour cells from mouse was examined. Measurements were carried out using [ 3 H]-2-deoxy-D-glucose, a non-metabolizable glucose analogue. [ 3 H]-2-deoxy-D-glucose was transported from the medium to cells and phosphorylated but was neither further metabolized nor eliminated. Thus, the amount taken in the cells was a measure of D-glucose uptake. The results showed that G115 stimulated D-glucose transport and the maximum effect was obtained at a G115 concentration of 2.0 μg/mL representing an increase of about 35% above basal activity

Both the plasma membrane-rich fraction and specific granule-rich fraction prepared from human neutrophil lysate by Percoll centrifugation have been reported to contain cytochrome b558, a membrane activation factor for NADPH oxidase. In this study, the plasma membrane-rich fraction and specific granule-rich fraction of guinea pig neutrophils were prepared, and the abilities of both fractions to activate NADPH oxidase in a cell-free system consisting of either fraction, cytosol and arachidonate were compared. There was no difference in the Km value for NADPH between NADPH oxidase activated by specific granules or by plasma membranes. Optimum concentrations of arachidonate for the activation of NADPH oxidase in both the fractions were also the same. However, after freeze-thawing, the specific granules markedly lost the ability, compared to plasma membranes. Such instability of specific granules was also observed on hypotonic- or deoxycholate-treatment. The inactivation by freeze-thawing was not suppressed by proteinase inhibitors, and gp91-phox, a large subunit of cytochrome b558, was not degraded by freeze-thawing. Freeze-thawed specific granules did not affect the ability in plasma membranes, indicating the absence of an inactivating factor in specific granules. The increase in the amount of cytosol in the cell-free assay mixture did not compensate for the markedly decreased ability of freeze-thawed specific granules. Translocation of p47-phox, one of the cytosolic activation factors, to specific granules was not affected by freeze-thawing. We found that the ability of specific granules to activate NADPH oxidase was fragile, though it is unclear what is responsible for the instability, at present.

NADPH oxidase was activated by arachidonate in a cell-free system consisting of membrane and cytosol fractions prepared from guinea pig neutrophils. Vanadate apparently inhibited the NADPH oxidase activity in the cell-free system (IC 50 =2 μM) without phosphotyrosine accumulation. The pH dependency and stability of the inhibitory effect observed for vanadate solution indicated that decavanadate, an isopolyanion of vanadate, was responsible for the inhibition. Pervanadate (vanadyl hydroperoxide) also inhibited the oxidase activity but at a higher concentration (IC 50 =0.2 mM). Decavanadate lowered the V max but did not affect the K m value of NADPH oxidase for NADPH. Decavanadate inhibited the activation process of NADPH oxidase but not the oxidase activity itself. Decavanadate-pretreatment of membrane and cytosol fractions irreversibly decreased the abilities of both fractions to activate NADPH oxidase in the cell-free system. Translocation of p47-phox, one of the cytosolic activation factors of NADPH oxidase, from cytosol to membrane, was little affected by decavanadate. These results suggest that decavanadate inhibits the activation of NADPH oxidase in the cell-free system without affecting the phosphotyrosine phosphatase, and that decavanadate can bind to both the membrane and cytosolic activation factors when they are in a dormant state, but not to the active oxidase complex.

We studied differences between the NADPH oxidase activation pathways triggered by pervanadate, a protein tyrosine phosphatase inhibitor, and phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, in guinea pig neutrophils. Previously, pervanadate has been shown to activate NADPH oxidase via the tyrosine kinase-dependent pathway (Yamaguchiet al. Arch. Biochem. Biophys.323, 382–386, 1995). Both pervanadate and PMA induced superoxide anion (O−2) production, translocation of the 47-kDa protein component of the phagocyte oxidase (p47-phox) to the plasma membrane, and phosphorylation of p47-phox in the membrane. A selective protein kinase C inhibitor, GF 109203X, markedly inhibited PMA-induced O−2production, p47-phox translocation, and p47-phox phosphorylation, but did not inhibit pervanadate-induced O−2production and only slightly suppressed pervanadate-induced translocation and phosphorylation. These results demonstrate that pervanadate activates NADPH oxidase independently of protein kinase C. Phosphopeptide mapping of p47-phox revealed differences in the mechanism between pervanadate-induced and PMA-induced phosphorylation. Furthermore, some protein kinases which phosphorylate p47-phox-derived peptide are activated by pervanadate. These results suggest the existence of novel protein kinases responsible for the phosphorylation of p47-phox and the activation of these protein kinases by tyrosine kinase.

The effect of insulin on the translocation of GLUT4 (glucose transporter isoform 4) from the intracellular membranes to the plasma membranes was compared in the hearts of 4-8 week old SAMP8, a substrain of senescence-accelerated prone mouse and those of SAMR1, a substrain of senescence-accelerated resistant mouse. After 20 min of the intravenous injection of insulin, the blood glucose levels in SAMR1 and SAMP8 were decreased by 50 and 68%, respectively. Under this condition, the concentrations of GLUT4 protein in the plasma membranes in the hearts of SAMR1 and SAMP8 were increased 1.8- and 2.1-fold, respectively. Concomitantly, the concentrations of the GLUT4 protein in the intracellular membranes in the hearts of SAMR1 and SAMP8 were decreased by about 70 and 50%, respectively. These results suggest that the heart of 4-8 week old SAMP8 is more sensitive to insulin than that of age-matched SAMR1.

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 causes of the early increase in oxidative stress observed in the cerebral cortex of 4–8 weeks old SAMP8.

d-Glucose metabolism in cerebral cells prepared from aged senescence-accelerated mouse (SAM), was investigated in consideration of a sex difference. The production of 14CO2 from 6-[14C]D-glucose was reduced in female senescence-accelerated-prone mouse (SAMP) 8, a prone substrain, in comparison with that in female senescence-accelerated-resistant mouse (SAMR) 2, a control substrain, whereas there was no difference in males. The 2-deoxy-D-glucose uptake into cerebral cells from female SAMP8 was also lower than that of control mice. But, the 3-O-methyl-D-glucose uptake in SAMP8 was higher than that of SAMR2, suggesting that the low hexokinase activity was involved in the decreased glucose metabolism in cerebrum of SAMP8 females irrespective of glucose transporter. This possibility was supported by the finding that the contents of glucose 6-phosphate produced from glucose added to cerebral cells from SAMP8 was lower than that in ICR mice.

Protein kinases are stimulated during the microbicidal responses of neutrophils. In particular, the activation of protein kinase C causes the phosphorylation of p47-phox, one of the cytosolic components of NADPH oxidase. Phosphorylated p47-phox was accumulated not only by PMA treatment, but also by calyculin A treatment. However, p47-phox phosphorylated by calyculin A treatment lost its ability to activate NADPH oxidase. Several protein kinases were activated by calyculin A treatment. Furthermore the phosphorylation sites of p47-phox by calyculin A treatment were different from those by PMA treatment. These results indicated that calyculin A-activated kinases phosphorylated p47-phox at the site different from that phosphorylate by protein kinase C, resulting in suppression of NADPH oxidase activation.

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.

Mono(ADP-ribose) synthetase activity in the nuclear fraction of immature rat testis was investigated in reference to the effect of testosterone. When the nuclear fraction was incubated with [ 32 P] NAD in the presence of nicotinamide, an inhibitor of poly (ADP-ribose) synthetase, some proteins with a molecular mass of 14.4∼21.5 kDa were ADP-ribosylated. In these proteins, H2B and H3 histones were found by sequencing 20 amino acids in the N-terminus. Their incorporations of ADP-ribose moiety were significantly decreased in immature rats at 4 hr after the subcutaneous injection of testosterone. Furthermore, the treatments with LH and FSH of the immature rats reduced mono(ADP-ribosyl)ations of H2B and H3 histones in a time-dependent manner. These results suggest that the testicular mono(ADP-ribose) synthetase activity is under the control of gonadotropin-testosterone system and is possibly related to differentiation of the testis.

The effect of plant extracts on D-glucose uptake by Ehrlich ascites tumour cells was examined. Among the 23 extracts of medicinal plants, five samples inhibited, and six samples activated, the uptake significantly. From one of the active plants, Lagerstroemia speciosa, two triterpenoids, colosolic acid and maslinic acid were isolated. Colosolic acid was shown to be a glucose transport activator. Since this compound was known to have hypoglycemic activity, our simple in vitro bioassay method can at least be used as a first screening for anti-diabetic activity.

Protein phosphatase inhibitors, okadaic acid and calyculin A, potentiated and elongated N -formyl-methionyl-leucyl-phenylalamine-induced superoxide anion (O − 2 ) production in guinea pig neutrophils. The activity of NADPH oxidase in the membrane fraction prepared from phorbol 12-myristate 13-acetate-stimulated neutrophils was inactivated by the addition of the cytosol from resting neutrophils, such inactivation of NADPH oxidase was also suppressed by the protein phosphatase inhibitors. We previously reported that phosphorylation of the 46-kDa protein by protein kinase C is one of the activation mechanisms of NADPH oxidase-dependent superoxide anion production. In the cytosol fraction, we found protein phosphatase activity that catalyzed dephosphorylation of 32 P-labeled phosphoproteins including the 46-kDa protein. Dephosphorylation of the 46-kDa protein was inhibited by the addition of okadaic acid and calyculin A. These results indicate that dephosphorylation of the 46-kDa protein by protein phosphatase is involved in the inactivation of NADPH oxidase. NADPH oxidase activity in guinea pig neutrophil may be regulated by the phosphorylation/dephosphorylation state of the 46-kDa protein by protein kinase C and protein phosphatase.

Hexokinase II prepared from Ehrlich-Lettre hyperdiploid tumor cells (ELD cells) was subjected to a limited digestion by trypsin. After 60 min digestion, hexokinase II (100 kDa) was completely cleaved to two fragments with the molecular weight of about 60 kDa and 40 kDa as manifested in SDS-PAGE. It was noteworthy that the enzyme activity was observed even at the time when the native enzyme molecule was no more detectable. These fragments were separated by SDS-PAGE irrespective of the presence of a reducing agent, but neither by native PAGE nor by cellulose acetate membrane electrophoresis under the nondenaturing conditions. Neither kinetic parameters such as Km values for ATP and glucose nor an ability of binding to mitochondria were changed significantly by the tryptic digestion. These results indicate that an essential conformation of hexokinase II can be restored by the self-association of two fragments produced as a result of the cleavage by trypsin at the middle of the molecule. Affinity labeling with 2′-3′-dialdehyde ATP followed by the trypsin digestion showed that ATP binding site resided in the 40 kDa fragment. Furthermore, the mode of the response in the incorporation of this ATP analog to hexose phosphate, moreover, was similar to that in the catalytic activity.

Conditions for Superoxide anion (O−2) production were examined in guinea pig polymorphonuclear leukocytes (PMNL). When PMNL were suspended in the hypotonic medium, O−2 production was significantly enhanced by concurrent treatment with low concentrations of 1-oleoyl-2-acetylglycerol (OAG), a cell-permeable protein kinase C activator. Such hypotonicity or OAG alone had little effect on the production. Other protein kinase C activators also markedly enhanced O−2 production in combination with hypotonicity, but not in the isotonic medium. Protein kinase C inhibitors, H-7 and staurosporine, dose-dependently inhibited the production. These observations indicate that protein kinase C participates in such synergistic O−2 production with hypotonicity. Phosphorylation of 46-kDa protein(s), which was commonly enhanced in parallel with an activation of NADPH oxidase in guinea pig PMNL, was increased by treatment with 10 μ m OAG, but the phosphorylation was little altered by hypotonic treatment. Intracellular calcium concentration, arachidonate release, and 1,2-diacylglycerol and phosphoinositide concentrations were slightly altered by hypotonic treatment. A change in phosphatidate (PA) production in PMNL was induced by hypotonic treatment either by itself or in combination with OAG treatment. These results suggest that the combination of cell membrane changes by hypotonic treatment accompanied by the increase in PA and 46-kDa protein phosphorylation by protein kinase C provides the conditions required for a marked increase in O−2 production. Hypotonicity may be a good tool for studying the mechanism of priming in the activation of NADPH oxidase.

An aqueous extract of mesocarps of the fruits of Balanites aegyptiaca exhibited a prominent antidiabetic activity by oral administration in streptozotocin induced diabetic mice. From one of the active fractions of this extract, two new steroidal saponins were isolated, and their structures were determined as 26-O-beta-D-glucopyranosyl-(25R)-furost-5-ene-3 beta,22,26-triol 3-O-[alpha-L-rhamnopyranosyl-(1----2)]-[beta-D-xylopyranosyl-(1---- 3)]-[alpha-L-rhamnopyranosyl-(1----4)]-beta-D-glucopyranoside and its 22-methyl ether. In addition, two known saponins, 26-O-beta-D-glucopyranosyl-(25R)-furost-5-ene-3 beta,22,26-triol 3-O-(2,4-di-O-alpha-L-rhamnopyranosyl)-beta-D-glucopyranoside and its methyl ether were isolated and identified. It was revealed that the individual saponins did not show antidiabetic activity, while the recombination of these saponins resulted in significant activity. From an ethanolic extract of the epicarps, two known flavonol glycosides, isorhamnetin-3-O-robinobioside and isorhamnetin-3-O-rutinoside were isolated and identified.

In order to examine the involvement of insulin in the activity of Na+/glucose cotransporter in rat small intestine, we compared Na+-dependent uptake of d -glucose by brush-border membrane vesicles prepared from control, streptozotocin-induced diabetic, insulin-treated diabetic and starved diabetic rats. In four groups, the uptake of d -glucose showed a transient overshoot in the presence of Na+ gradient between medium and vesicles (medium > vesicles). The overshoot magnitude was increased (1.8-fold of controls) in diabetic brush border membrane vesicles and recovered to the control level by the treatment of diabetic rats with insulin. In contrast, increased uptake of d -glucose in diabetic rats was not recovered by the starvation of diabetic rats although the blood glucose level was the same as that of controls. Furthermore, we attempted to examine phlorizin binding activities among four groups. Scatchard analysis indicated that phlorizin binding to diabetic brush border membrane vesicles was increased (1.6-fold of controls) without a change of the affinity for phlorizin as compared with controls. Increased binding of phlorizin to diabetic brush border membrane vesicles was also recovered to the control level by the treatment of diabetic rats with insulin, but not by starvation. These results suggested that the increased activity of Na+/glucose cotransporter in diabetic rats was due to the increase of the number of cotransporter and that intestinal cotransporter was physiologically controlled by insulin, but not by blood glucose levels.

tsJT16 is a cell cycle temperature-sensitive (ts) mutant from a Fischer rat cell line. When it is growth-stimulated from G0 phase it enters S phase at the permissive temperature (34 degrees C) but not at the nonpermissive temperature (40 degrees C). It induces a nuclear labile protein, p70, when it is stimulated from G0 phase at 34 degrees C, but not at 40 degrees C. In growing cell cycle it progresses through the S, G2 and M phases at both temperatures but fails to pass through G1 phase at 40 degrees C. Here we described that p70 was synthesized neither in the randomly growing cycle nor in the G1 phase synchronously progressing from M phase. The cells synchronized at early G1 phase by culturing in serum-free medium for 7.5 h from G1/S boundary induced c-fos and c-myc following serum addition, but under the same condition p70 was not synthesized. These results indicate that the synthesis of p70 is not required for progression of the G1 phase of the growing cycle and can be used as an exclusive marker of G0-S transition.

When the homogenate prepared from immature rat testes was incubated with [32P]NAD, several proteins (90, 39 and 20 kDa) were ADP-ribosylated in the absence of bacterial toxins. This observation suggested the existence of an endogenous ADP-ribosyltransferase and substrates. The data that the digested product by phosphodiesterase of ADP-ribosylated 20 kDa protein was 5'-AMP suggested that 20 kDa protein was mono(ADP-ribosyl)ated. In addition, the mono(ADP-ribosyl)ation of 20 kDa protein was enhanced by guanine nucleotides such as GTP, GDP and GTP[gamma S], and decreased by the concentrations of 10 mM Mg2+. In contrast, the incorporation of ADP-ribose moiety from NAD to both 90 and 39 kDa proteins was not changed by guanine nucleotides. On the other hand, mono(ADP-ribosyl)ation of 20 kDa protein was not observed in the homogenate prepared from other tissues of the same rats. Furthermore, we found that mono(ADP-ribosyl)ation of 20 kDa protein was decreased with the maturation of the rats and that an endogenous mono(ADP-ribosyl)transferase and 20 kDa protein were located in the nuclei.

tsJT60 is a nonlethal temperature-sensitive (ts) mutant of a Fischer rat cell line (3Y1) classified as a G0 mutant; i.e., the ts defect is not expressed within the cell growth cycle but is expressed only between the G0 and S phase. tsJT60 clones transformed with oncogenes such as adenovirus E1A, polyoma large T, polyoma middle T, v-Ki-ras, and LTR activated c-myc, or with a chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine, grew well at 34 degrees C. However, most of these clones grew slowly at 40 degrees C, producing many floating dead cells, and some clones were killed at 40 degrees C. When they were cultured under conditions inadequate for growth of untransformed cells, such as high cell density or serum restriction, they were killed at 40 degrees C. These and previous results from SV40- and adenovirus-transformed tsJT60 clones favour the idea that transformed tsJT60 cells occasionally enter the G0 phase and are metabolically imbalanced at 40 degrees C during self-stimulation from the G0 to S phase. We propose that a drug which exclusively block, G0-G1 transition would be cytocidal to transformed cells but cytostatic to normal cells.

A diacylglycerol (DG) kinase inhibitor, R 59 022, potentiated superoxide anion (O2-) production in guinea pig polymorphonuclear leukocytes (PMNL) induced by N-formyl-methionyl-leucyl-phenylalanine (FMLP). R 59 022 also potentiated O2- production induced by 1-oleoyl-2-acetylglycerol, a permeable DG. However, the production induced by phorbol 12-myristate 13-acetate (PMA), a direct activator for protein kinase C, was not potentiated by R 59 022. R 59 022 by itself had no significant effects on unstimulated O2- production. The potentiation of FMLP-induced O2- production by R 59 022 was correlated closely with increased formation of DG and decreased formation of phosphatidic acid, a product of DG kinase. R 59 022 had no effect on the breakdown of phosphoinositides. Phosphorylation of 46-kDa protein(s) by protein kinase C was also examined in relation to O2- production in PMNL. In coincidence with the increase in O2- production, the phosphorylation was potentiated by R 59 022 in the response to FMLP, but not in the response to PMA. In addition, staurosporine, a protein kinase C inhibitor, inhibited increases in both O2- production and phosphorylation of the 46-kDa protein(s) after PMA stimulation. Similar inhibitory effects of staurosporine were also observed upon stimulation with FMLP, irrespective of the presence of R 59 022. These results indicate that retention of DG as a result of the inhibition of further metabolism induces marked stimulation of O2- production via protein kinase C activation in PMNL. These results also provide further evidence for the close relationship between 46-kDa protein phosphorylation by protein kinase C and stimulation of O2- production in PMNL.

NADPH oxidase activity in a membrane fraction prepared from phorbol 12-myristate 13-acetate (PMA)-stimulated guinea pig polymorphonuclear leukocytes (PMNL) was inhibited by positively charged myristylamine. The inhibitory effect of myristylamine was significantly suppressed by simultaneous addition of a negatively charged fatty acid, such as myristic acid. However, the suppression by myristylamine was not sufficiently restored when myristic acid was added later. On the other hand, pretreatment of PMA-stimulated PMNL with glutaraldehyde, a protein crosslinking reagent, stabilized NADPH oxidase activity against inhibition by myristylamine, but not against that by p-chloromercuribenzenesulfonic acid. In a cell-free system of reconstituted plasma membrane and cytosolic fractions prepared from unstimulated PMNL, arachidonic acid-stimulated NADPH oxidase activity was also inhibited by myristylamine. During the activation of NADPH oxidase by PMA in intact PMNL and by arachidonic acid in the cell-free system, cytosolic activation factor(s) translocated to plasma membranes. The bound cytosolic activation factor(s) was released from the membranes by myristylamine, accompanied by a loss of NADPH oxidase activity. It is plausible from these results that the inhibitory effect of alkylamine on NADPH oxidase is due to induction of the decoupling and/or dissociation of the cytosolic activation component(s) from the activated NADPH oxidase complex by increments of positive charges in the membranes, and that the glutaraldehyde treatment prevents the dissociation of component(s).

Effects of Ebselen and its analogs (PZ-25, NAT06-123, NAT02-761, NAT02-801, NAT06-099, and NAT06-513) on superoxide anion (O2-) production induced by tetradecanoyl phorbol acetate (TPA) were examined in intact guinea pig polymorphonuclear leukocytes (PMNL). Four compounds having a structure of 1,2 benzoisoselenazol-3-(2H) one (Ebselen, NAT06-123, and NAT02-761) and its sulfur-substituted analog (PZ-25), had a potent inhibitory effect on O2- production as compared with others. Ebselen and NAT06-123 also markedly inhibited nicotinamide adenine dinuclestide phosphate (NADPH) oxidase activity, which is responsible for O2- production in intact cells, and in a particulate fraction prepared from TPA-stimulated PMNL, whereas PZ-25 inhibited this enzyme weakly and NAT02-761 did not. On the other hand, Ebselen and PZ-25 had the same degree of potent inhibitory effect on protein kinase C which was involved in the regulation of NADPH oxidase activation. Thus, it is plausible that inhibition of O2- production in intact PMNL by these compounds were due not only to direct inhibition of NADPH oxidase but also to inhibition of protein kinase C.

The effect of alkaline phosphatase (3.1.3.1) on desensitization of beta-adrenoceptor-responsive adenylate cyclase and the role of phosphorylation in desensitization were examined. Treatment of rat reticulocytes with isoproterenol, dibutyryl cAMP and tetradecanoyl phorbol acetate (TPA) caused the desensitization of beta-adrenoceptor-coupled adenylate cyclase. When the membranes from dibutyryl cAMP- and TPA-desensitized cells were incubated with alkaline phosphatase for 60 min at 30 degrees C, pH 8.0, the desensitization of isoproterenol-stimulated adenylate cyclase was markedly attenuated in both preparations. When the membranes from isoproterenol-desensitized cells were treated with alkaline phosphatase under the same conditions, the attenuation of the desensitization of alkaline phosphatase was less than in the case of treatment with dibutyryl cAMP or TPA. In other words, isoproterenol-induced desensitization was more resistant to alkaline phosphatase treatment. Isoproterenol- and dibutyryl cAMP-induced desensitization of NaF-stimulated adenylate cyclase were also attenuated by alkaline phosphatase treatment. Although the stability of the Gs-catalytic unit complex of adenylate cyclase was reduced by isoproterenol treatment, the reduction of stability was also decreased by alkaline phosphatase treatment.

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 V max of 4.8 nmol/mg protein per 30 s in diabetic BBMV was significantly decreased as compared with that of controls ( V max = 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.

The relationship between phagocytosis and bactericidal action of polymorphonuclear leukocytes was examined by comparing the functions of cytochalasin D-treated leukocytes with those of the control. Measurement of phagocytotic and bacterial DNA-degrading activities using Escherichia coli prelabeled with [3H]thymidine revealed that phagocytosis and bacterial DNA degradation were inhibited by treatment with cytochalasin D to about 50 and 10% of the control, respectively. Nevertheless, the bactericidal activity of the cytochalasin D-treated leukocytes was almost the same as that of the control leukocytes; almost all the bacteria were phagocytized by the latter leukocytes. Under the same experimental conditions, the production and release of superoxide anions and hydrogen peroxide, which are both known to be involved in the bactericidal action of the leukocytes, were markedly increased by cytochalasin D. Release of several lysosomal hydrolases was also increased markedly by cytochalasin D treatment, except for myeloperoxidase. However, lactate dehydrogenase, a typical cytosolic marker, was not released by the same treatment. Thus, it is unlikely that the increase in the release of the above-mentioned bactericidal factors was due to decomposition of the leukocytes. These results indicate that the site of bactericidal action of cytochalasin D-treated leukocytes is not necessarily intracellular but may be around the external surface of the cells.

A human diploid fibroblast strain, TIG-1, ceased to proliferate at about 60–62 population doubling level. The percentage of nucleic incorporating [3H]thymidine during 24-h culture in fresh medium containing 10% fetal bovine serum was less than 2% in the senescent cells used in this study. Infection of these cells with human cytomegalovirus (HCMV), strain AD-169, increased the percentage of [3H]thymidine-labeled cells by about ten-fold. Equilibrium density gradient centrifugation analysis of purified DNA from infected cells showed that cellular DNA synthesis was stimulated preceded by the viral DNA synthesis. Ultraviolet irradiation of HCMV reduced the ability to induce DNA synthesis. Equilibrium density gradient centrifugation analysis of DNA which was labeled with 5-bromodeoxy-uridine indicated semiconservative replication rather than repair synthesis. These results suggested that in a considerable fraction of human senescent cells host DNA replication could be reinitiated by infection with HCMV, but not by the addition of fetal bovine serum.

As a possible mechanism for the absence of mitochondria-bindable hexokinase in the liver, the presence of a protease similar in action to chymotrypsin, which specifically eliminates the binding ability of the bindable hexokinase without changing its catalytic properties, was investigated in rat liver. The lysosomal fraction prepared from the liver converted the bindable hexokinase prepared from rat brain to the nonbindable form with little change in catalytic activity. The activity of such a “processing protease” was much lower in rat brain, where the bindable form is predominant. The processing activity cosedimented with lysosomal marker enzyme activities in the subcellular fractionation of livers from normal and Triton WR-1339-injected rats. A fair portion of the activity was detected in the lysosomes without disruption. The activity was maximal at pH 6.0–7.0, inactivated almost completely by tosylphenylalanine chloromethyl ketone, tosyllysine chloromethyl ketone, leupeptin, antipain, and chymostatin, and dependent on dithiothreitol and mercaptoethanol. These results suggest that a protease, properties of which are fairly similar to those of cathepsin M, may be involved in the post-translational processing of original bindable hexokinase to the nonbindable form in rat liver.

Treatment of guinea pig polymorphonuclear leukocytes (PMNL) with arachidonate at concentrations of less than 20 μ m induced slight stimulation of Superoxide anion (O − 2 ) production with little enhancement of the phosphorylation of the 46K protein(s). The stimulation of the phosphorylation of those protein(s) has been observed in parallel with an activation of NADPH oxidase in our previous studies (N. Okamura et al. (1984) Arch. Biochem. Biophys. 228 , 270–277; T. Ohtsuka et al. (1986) Biochim. Biophys. Acta 888 , 332–337; T. Ohtsuka et al. (1987) J. Biochem . 101 , 897–903). On the other hand, the phosphorylation of the same protein(s) was increased by the treatment of PMNL with 10 μ m 1-oleoyl-2-acetylglycerol (OAG), a permeable diacylglycerol, with little change in O − 2 production. Treatment of PMNL with a combination of such low concentrations of arachidonate and OAG, induced marked increase in O − 2 production in accordance with the increase in the phosphorylation of 46K protein(s) which was probably due to OAG action. Thus, it is likely that this protein phosphorylation is a prerequisite or regulatory to the stimulation of the O − 2 production by arachidonate in PMNL.

New method for nitroblue-tetrazolium (NBT) test was developed by the following modifications. (1) After 10 N KOH treatment, bathochromic shift in the maximal absorption took place from 515 nm to 710 nm and the optical density of the new peak was 5 times higher than that of non-treated sample. In this assay, dimethylformamide extraction was substituted for pyridine. (2) Because of the smallness of the incubation mixture (100 μl), the background of the test was greatly reduced. (3) Increase in the concentration of latex particles elevated the NBT reducing activity. In consideration with the background absorption, 2.9-5.7×108 particles/assay was set as the optimal condition. The particles themselves, however, did not interrupt the reading of optical density in this system. In this modified method, 3 ml of blood was enough to estimate the NBT reducing activity in the duplicate tests (6 samples). This assay system is, therefore, applicable for weak children, from whom the collection of the large volume of the blood is not desirable.