Biomedical subjects

K Takuma

Publications and source records attributed to K Takuma.

At least 19 recordsLinked to original sources

PubMed2001

Anti-apoptotic effect of cGMP in cultured astrocytes: inhibition by cGMP-dependent protein kinase of mitochondrial permeable transition pore.

Reperfusion of cultured astrocytes with normal medium after exposure to H(2)O(2)-containing medium causes apoptosis. We have recently shown that ibudilast, which has been used for bronchial asthma and cerebrovascular disorders, attenuated the H(2)O(2)-induced apoptosis of astrocytes via the cGMP signaling pathway. This study examines the mechanism underlying the protective effect of cGMP. The membrane-permeable cGMP analog dibutyryl-cGMP attenuated the H(2)O(2)-induced decrease in cell viability, DNA ladder formation, nuclear condensation, reduction of the mitochondrial membrane potential, cytochrome c release from mitochondria, and caspase-3 activation in cultured astrocytes. These effects of dibutyryl-cGMP were almost completely inhibited by the cGMP-dependent protein kinase (PKG) inhibitor KT5823. In isolated rat brain mitochondria, cGMP in the presence of cytosolic extract from astrocytes inhibited the mitochondrial permeability transition pore (PTP) as determined by monitoring Ca(2+)-induced mitochondrial swelling. This ability of the cytosolic extract was inactivated by heat treatment and was mimicked by exogenous PKG. The effect of cGMP on the mitochondrial swelling was blocked by KT5823. The PTP inhibitors cyclosporin A and bongkrekic acid prevented the H(2)O(2)-induced decrease in cell viability and caspase-3 activation. These findings demonstrate that cGMP inhibits the mitochondrial PTP via the activation of PKG, and the prevention of mitochondrial dysfunction contributes to its anti-apoptotic effect.

Animals↗

PubMed2001

Phosphorylation of histones triggers DNA fragmentation in thymocyte undergoing apoptosis induced by protein phosphatase inhibitors.

The treatment of thymocytes with protein phosphatase inhibitors such as calyculin A and okadaic acid resulted in apoptosis with a concomitant increase in phosphorylation of nuclear proteins. The phosphorylated protein in the thymocyte nuclei induced by protein phosphatase inhibitors was identified as histones by the use of two-dimensional polyacrylamide gel electrophoresis. These compounds accelerated the phosphorylation of histone H2A, H3, and H1. On the other hand, little phosphorylation of H2B and H4 by these compounds was observed. The effect of these compounds on the level of nuclear histones was also examined using high-performance capillary electrophoresis. No significant changes in the level of histones were seen in the nuclei of thymocytes treated with calyculin A and okadaic acid. Thus, the induction of thymocyte apoptosis is involved in the chemical modification of histones but not the change in their quantity. Moreover, the treatment of thymocytes with calyculin A increased the sensitivity toward endogenous DNase in the nuclei. These results suggest that phosphorylation of histones, especially H2A, H3, and H1, is an early step of triggering DNA fragmentation in thymocyte apoptosis.

Animals↗

PubMed2001

Ibudilast attenuates astrocyte apoptosis via cyclic GMP signalling pathway in an in vitro reperfusion model.

We examined the effect of 3-isobutyryl-2-isopropylpyrazolo[1,5-a]pyridine (ibudilast), which has been clinically used for bronchial asthma and cerebrovascular disorders, on cell viability induced in a model of reperfusion injury. Ibudilast at 10 - 100 microM significantly attenuated the H(2)O(2)-induced decrease in cell viability. Ibudilast inhibited the H(2)O(2)-induced cytochrome c release, caspase-3 activation, DNA ladder formation and nuclear condensation, suggesting its anti-apoptotic effect. Phosphodiesterase inhibitors such as theophylline, pentoxyfylline, vinpocetine, dipyridamole and zaprinast, which increased the guanosine-3',5'-cyclic monophosphate (cyclic GMP) level, and dibutyryl cyclic GMP attenuated the H(2)O(2)-induced injury in astrocytes. Ibudilast increased the cyclic GMP level in astrocytes. The cyclic GMP-dependent protein kinase inhibitor KT5823 blocked the protective effects of ibudilast and dipyridamole on the H(2)O(2)-induced decrease in cell viability, while the cyclic AMP-dependent protein kinase inhibitor KT5720, the cyclic AMP antagonist Rp-cyclic AMPS, the mitogen-activated protein/extracellular signal-regulated kinase inhibitor PD98059 and the leukotriene D(4) antagonist LY 171883 did not. KT5823 also blocked the effect of ibudilast on the H(2)O(2)-induced cytochrome c release and caspase-3-like protease activation. These findings suggest that ibudilast prevents the H(2)O(2)-induced delayed apoptosis of astrocytes via a cyclic GMP, but not cyclic AMP, signalling pathway.

Alkaloids↗

PubMed2001

[Delayed apoptosis and its regulation in astrocytes].

Astrocytes, the most abundant glial cell type in the brain, are considered to have physiological and pathological roles in neuronal activities. We found that reperfusion of cultured astrocytes after Ca2+ depletion causes Ca2+ overload followed by delayed cell death and the Na(+)-Ca2+ exchanger in the reverse mode is responsible for this Ca(2+)-mediated cell injury (Ca2+ paradox injury). The Ca2+ paradox injury of cultured astrocytes is considered to be an in vitro model of ischemia/reperfusion injury, since a similar paradoxical change in extracellular Ca2+ concentration is reported in ischemic brain tissue. This review summarizes the mechanisms underlying the Ca(2+)-mediated injury of astrocytes and the protective effects of drugs against Ca2+ reperfusion injury. This study shows that Ca2+ reperfusion injury of astrocytes is accompanied by apoptosis as evidenced by DNA fragmentation and nuclear condensation. Calpain, reactive oxygen species, calcineurin, caspase-3, and NF-kappa B are involved in Ca2+ reperfusion-induced delayed apoptosis of astrocytes. Several drugs including CV-2619, T-588 and ibudilast protect astrocytes against the delayed apoptosis. CV-2619 prevents astrocytes from the delayed apoptosis by production of nerve growth factor, resulting in an activation of mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) and phosphatidylinositol-3 (PI3) kinase signal pathways. The protective effect of T-588 is mainly mediated by an activation of MAP/ERK signal cascade. Moreover, ibudilast prevents the Ca2+ reperfusion-induced delayed apoptosis of astrocytes via cyclic GMP signaling pathway. Further studies in this system will contribute to the development of new drugs that attenuate ischemia/reperfusion injury via modulation of astrocytes.

Animals↗

PubMed2001

SEA0400, a novel and selective inhibitor of the Na+-Ca2+ exchanger, attenuates reperfusion injury in the in vitro and in vivo cerebral ischemic models.

The effect of the newly synthesized compound 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline (SEA0400) on the Na+-Ca2+ exchanger (NCX) was investigated and compared against that of 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea (KB-R7943). In addition, the effects of SEA0400 on reperfusion injury in vitro and in vivo were examined. SEA0400 was extremely more potent than KB-R7943 in inhibiting Na+-dependent Ca2+ uptake in cultured neurons, astrocytes, and microglia: IC50s of SEA0400 and KB-R7943 were 5 to 33 nM and 2 to 4 microM, respectively. SEA0400 at the concentration range that inhibited NCX exhibited negligible affinities for the Ca2+ channels, Na+ channels, K+ channels, norepinephrine transporter, and 14 receptors, and did not affect the activities of the Na+/H+ exchanger, Na+,K+-ATPase, Ca2+-ATPase, and five enzymes. SEA0400, unlike KB-R7943, did not inhibit the store-operated Ca2+ entry in cultured astrocytes. SEA0400 attenuated dose- dependently paradoxical Ca2+ challenge-induced production of reactive oxygen species, DNA ladder formation, and nuclear condensation in cultured astrocytes, whereas it did not affect thapsigargin-induced cell injury. Furthermore, administration of SEA0400 reduced infarct volumes after a transient middle cerebral artery occlusion in rat cerebral cortex and striatum. These results indicate that SEA0400 is the most potent and selective inhibitor of NCX, and suggest that the compound may exert protective effects on postischemic brain damage.

Aniline Compounds↗

PubMed2000

CV-2619 protects cultured astrocytes against reperfusion injury via nerve growth factor production.

In this study, we examined the effect of the neuroprotective agent 2, 3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone (CV-2619) on reperfusion injury in cultured rat astrocytes after exposure to hydrogen peroxide (H(2)O(2))-containing medium. CV-2619 (10 nM to 10 microM) significantly attenuated the reperfusion-induced decrease in cell viability. The compound showed an anti-apoptotic effect in this astrocyte injury model. Antioxidants such as ascorbic acid, alpha-tocopherol and reduced glutathione also inhibited H(2)O(2) exposure-induced cytotoxicity. CV-2619 did not affect the levels of reactive oxygen species, but it increased nerve growth factor (NGF) production. The effect of CV-2619 on H(2)O(2) exposure-induced cytotoxicity was blocked by cycloheximide and anti-NGF antibody. The protective effect of CV-2619 was antagonized by the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase inhibitor 2'-amino-3'-methoxyflavone and the phosphatidylinositol-3 kinase inhibitor wortmannin. These findings suggest that the effect of CV-2619 is mediated at least partly by NGF production in astrocytes and that ERK and phosphatidylinositol-3 kinases play a role in the downstream mechanism.

Animals↗

PubMed2000

T-588 inhibits astrocyte apoptosis via mitogen-activated protein kinase signal pathway.

The effect of (1R)-1-benzo[b]thiophen-5-yl-2-[2-(diethylamino)ethoxy]ethan -1-ol hydrochloride (T-588), a cognition enhancer, on reperfusion injury was studied in cultured rat astrocytes. T-588 at 1-10 microM partially protected astrocytes against reperfusion injury after exposure to Ca(2+)-free medium or hydrogen peroxide. Nerve growth factor (NGF) had a similar protective effect. Addition of both T-588 and NGF resulted in complete protection against Ca(2+) reperfusion injury. T-588 did not stimulate NGF production in astrocytes. The effect of T-588 on Ca(2+) reperfusion injury including apoptosis was inhibited by the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase inhibitor 2'-amino-3'-methoxyflavone (PD98059), but not by the phosphoinositide 3-kinase inhibitor wortmannin. The effect of NGF was inhibited by PD98059 and wortmannin. T-588 stimulated rapidly the phosphorylation of ERK, but did not affect that of Akt in astrocytes. These findings suggest that the ERK MAP kinase pathway has a role in the protective effects of T-588 and NGF.

Androstadienes↗

PubMed1999

Apoptosis in Ca2 + reperfusion injury of cultured astrocytes: roles of reactive oxygen species and NF-kappaB activation.

We previously reported that incubation of cultured astrocytes in Ca2 + -containing medium after exposure to Ca2 + -free medium caused Ca2 + influx followed by delayed cell death. Here, we studied the mechanisms underlying the Ca2 + -mediated injury of cultured astrocytes. Our results show that Ca2 + reperfusion injury of astrocytes appears to be mediated by apoptosis, as demonstrated by DNA fragmentation and prevention of death by caspase-3 inhibitors. Paradoxical Ca2 + challenge stimulated rapidly reactive oxygen species (ROS) production. Ca2 + reperfusion injury of astrocytes was influenced by several reagents which modified ROS production. When astrocytes were exposed to hydrogen peroxide (H2O2) for 30 min and then incubated without H2O2 for 1-5 days, cell toxicity including apoptosis was observed. Ca2 + reperfusion injury induced by Ca2 + depletion or H2O2 exposure was blocked by the iron chelator 1, 10-phenanthroline, the NF-kappaB inhibitor pyrrolidinedithiocarbamate and the calcineurin inhibitor FK506. Incubation in normal medium after H2O2 exposure rapidly increased the level of nuclear NF-kappaB p65 subunit, and the effect was blocked by 1,10-phenanthroline, pyrrolidinedithiocarbamate and FK506. These findings indicate that Ca2 + reperfusion-induced apoptosis is mediated at least partly by ROS production and ROS cause NF-kappaB activation in cultured astrocytes.

Animals↗

PubMed1999

[Cell injury and its protection in astrocytes].

Incubation of cultured astrocytes in Ca(2+)-containing medium after exposure to Ca(2+)-free medium causes Ca2+ influx followed by delayed cell death. Here, we summarize the mechanisms underlying the Ca(2+)-mediated injury of cultured astrocytes and the protective effects of drugs against Ca(2+)-reperfusion injury. Our results show that Ca(2+)-reperfusion injury of astrocytes appears to be mediated by apoptosis as evidenced by DNA fragmentation and nuclear condensation. Calpain, reactive oxygen species (ROS) production, calcineurin, caspase-3, and NF-kappa B activation are involved in Ca(2+)-reperfusion injury. Several drugs including T-588 and idebenone protect astrocytes against Ca(2+)-reperfusion injury. The protective effect of idebenone is mediated by nerve growth factor production, whereas that of T-588 is mediated mainly by the mitogen-activated protein/extracellular signal-regulated kinase signal cascade.

Animals↗

PubMed1998

Involvement of calcineurin in Ca2+ paradox-like injury of cultured rat astrocytes.

The Ca2+/calmodulin-dependent phosphatase calcineurin may have physiological and pathological roles in neurons, but little is known about the roles of the enzyme in glial cells. We have previously reported that reperfusion of cultured astrocytes in Ca2+-containing medium after exposure to Ca2+-free medium caused Ca2+ influx followed by delayed cell death. In this study, we examined if calcineurin is involved in this Ca2+-mediated astrocytic injury. FK506, an inhibitor of calcineurin, protected cultured rat astrocytes against paradoxical Ca2+ challenge-induced injury in a dose-dependent manner (10(-10)-10(-8) M). Cyclosporin A at 1 microM mimicked the effect of FK506. Rapamycin (1 microM) did not affect astrocyte injury, but it blocked the protective effect of FK506. Deltamethrin (20 nM), another calcineurin inhibitor, had a similar protective effect, whereas okadaic acid did not. FK506 affected neither paradoxical Ca2+ challenge-induced increase in cytosolic Ca2+ level nor Na+-Ca2+ exchange activity in the cells, suggesting that the calcineurin is involved in processes downstream of increased cytosolic Ca2+ level. Immunochemical studies showed that both calcineurin A (probably the A beta2 isoform) and B subunits were expressed in the cells. It is concluded that calcineurin is present in cultured astrocytes and it has a pathological role in the cells.

Animals↗

PubMed1998

Usefulness and limitations of ultrasonography in the initial evaluation of blunt abdominal trauma.

BACKGROUND: In the assessment of blunt abdominal trauma, the reliability of ultrasonography (US) in identifying individual organ injuries remains uncertain, in spite of its usefulness in detecting hemoperitoneum. This study was designed to evaluate the overall diagnostic value of US, including identification of individual organ injuries. METHODS: The accuracy of US in the detection of intra-abdominal injuries and the identification of individual organ injuries was evaluated in 1,239 patients seen during a 15-year period. Accuracy was based on detection of intraperitoneal fluid, free air, or irregular parenchymal lesions. RESULTS: For the detection of injuries, US was 94.6% sensitive, 95.1% specific, and 94.9% accurate. Individual organ injuries were identified with sensitivities of 92.4, 90.0, 92.2, 71.4, and 34.7% for the liver, spleen, kidneys, pancreas, and intestine, respectively. CONCLUSION: US is reliable for the detection of injuries and the identification of solid-organ injuries despite its poor sensitivity for intestinal injuries.

Abdominal Injuries↗

PubMed1998

[Apoptosis of astroglial cells].

Astrocytes, the most abundant glial cell type in the brain, are considered to have physiological and pathological roles in neuronal activities. We found that reperfusion of cultured astrocytes after Ca2+ depletion causes delayed cell death and that the Na(+)-Ca2+ exchanger in the reverse mode is responsible for this Ca(2+)-mediated cell injury (Ca2+ paradox injury). The Ca2+ paradox injury of cultured astrocytes is considered to be an in vitro model of ischemia/reperfusion injury, since a similar paradoxical change in extracellular Ca2+ concentration is reported in ischemic brain tissue. Furthermore, we demonstrated that heat shock proteins, glutathione and calcineurin inhibitors protected astrocytes against Ca2+ paradox-induced cell toxicity. We also observed DNA fragmentation, a typical apoptotic ladder, 2-3 days after hydrogen peroxide exposure. In addition, laser microscopic observation showed that reperfusion after the exposure to hydrogen peroxide caused nuclear condensation of astrocytes. Hydrogen peroxide-induced cell injury and DNA fragmentation were attenuated by the NF-kappa B inhibitor ammonium pyrrolidinedithiocarbamate, 1,10-phenanthroline and a caspase 3 inhibitor. These findings suggest that astrocytes are one of the targets for ROS and the oxidative stress-induced delayed death of astrocytes is at least due to apoptosis.

Animals↗

PubMed1998

[Sepsis in extensive burned patients].

The prognosis of extensively burned patients is dependent upon the presence of sepsis. The more extensive the burns, the more likely patients are to become septic. Although recently the frequency of burn wound sepsis has been decreased due to the early excision of necrotic tissue, that of sepsis resulting from respiratory tract infection has increased. Staphylococcus aureus (methicillin-resistant S. aureus) and Pseudomonas aeruginosav are the agents most likely to cause infections. Sepsis syndrome also results from bacterial translocation (BT), in which gut bacteria and/or endotoxins are thought to enter the portal bloodstream and/or lymphosystem. The pathophysiological mechanism of sepsis is the increased release of inflammatory mediators and resulting imbalances between these substances and their antagonists. In cases of severe sepsis, the sequelae of the imbalance between inflammatory mediators and their antagonists can lead to endothelial injury, DIC, and finally MODS. Strategies against the occurrence of sepsis include hospital-wide infection control measures, blockage of infection routes, and administration of antibiotics. The early initiation of nutritional management, preferably by the enteral route, to enhance immune system function and minimize the occurrence of BT is recommended. Several drugs to control inflammatory mediator release are currently under development and are expected to be used clinically in future.

Burns↗

PubMed1997

Relationship between pupil size and acetylcholinesterase activity in patients exposed to sarin vapor.

OBJECTIVE: To elucidate the effect of sarin vapor on pupil size and erythrocyte acetylcholinesterase activity (AchE). DESIGN: Retrospective observational survey. SETTING: Emergency department of an urban teaching hospital. PATIENTS: 80 patients who were exposed to sarin in a terrorist attack in Tokyo subways. MEASUREMENTS AND RESULTS: Pupil size and AchE activity on the day of exposure were measured. Among the 80 patients, the pupils were miotic (< 3 mm) in 50 patients (62.5%), while AchE activity was below the normal range (< 1.2 U) in 34 patients (42.5%). AchE was significantly lower in the miotic group than in the group with normal pupils (1.0 +/- 0.5 U vs 1.5 +/- 0.3 U, p < 0.01). In the miotic group, AchE activity was lower than normal in 32 patients (64.0%) but was decreased in only 2 patients in the normal pupil group (6.7%) (p < 0.01). CONCLUSIONS: Miosis is a more sensitive index of exposure to sarin vapor than erythrocyte AchE. Systemic poisoning is apparently less likely to develop if the patient's pupil size is normal on arrival at the hospital.

Acetylcholinesterase↗

PubMed1997

Isoform-specific up-regulation by ouabain of Na+,K+-ATPase in cultured rat astrocytes.

There are two alpha-subunit isoforms (alpha1 and alpha2) and two beta-subunit isoforms (beta1 and beta2) of Na+,K+-ATPase in astrocytes, but the functional heterodimer composition is not known. Ouabain (0.5-1.0 mM) increased the levels of alpha1 and beta1 mRNAs, whereas it decreased those of alpha2 and beta2 mRNAs in cultured rat astrocytes. The increases in alpha1 and beta1 mRNAs were observed at 6-48 h after addition of the inhibitor. Immunochemical analyses showed that ouabain increased alpha1 and beta1, but not alpha2 and beta2, proteins, and that the isoforms in control and ouabain-treated cultures were of glial origin. Low extracellular K+ and monensin (20 microM) mimicked the effect of ouabain on alpha1 mRNA. The ouabain-induced increase in alpha1 mRNA was blocked by the protein synthesis inhibitor cycloheximide (10 microM), the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (30 microM), and the calcineurin inhibitor FK506 (1 nM). These findings indicate that chronic inhibition of Na+,K+-ATPase up-regulates the alpha1 and beta1, but not alpha2 and beta2, isoforms in astrocytes, suggesting a functional coupling of alpha1beta1 complex. They also suggest that intracellular Na+, Ca2+, and calcineurin may be involved in ouabain-induced up-regulation of the enzyme in astrocytes.

Animals↗

PubMed1997

Na(+)-Ca2+ exchanger: physiology and pharmacology.

The Na(+)-Ca2+ exchanger in the plasma membrane is a bidirectional electrogenic ion transporter that couples the translocation of Na+ in one direction with that of Ca2+ in the opposite direction. This system is involved in regulation of intracellular Ca2+ concentration via the forward mode (Ca2+ extrusion) or the reverse mode (Ca2+ influx). There are two types of the plasma membrane Na(+)-Ca2+ exchanger in an animal, and they are called the cardiac type and rod outer segment type. In addition, there is an electroneutral Na(+)-Ca2+ exchanger present in mitochondria. Recent studies by the molecular biology technique show that there are at least 8 isoforms of the cardiac type (NCX1), and there are two other exchangers in the brain (NCX2 and NCX3). Due to new techniques of molecular biology and electrophysiology, much evidence is accumulating with respect to the structure, mechanism, regulation, and physiological and pathological roles of the Na(+)-Ca2+ exchanger. This review summarizes recent progress in this research field that is of pharmacological interest.

Animals↗

PubMed1997

[Bacteria isolated from surgical infections and their susceptibilities to antimicrobial agents. Special references to bacteria isolated between July 1995 and June 1996].

Isolated bacteria from infections in general surgery during the period from July 1994 to June 1995 were investigated in a multicenter study in Japan, and the following results were obtained. One hundred and sixty-four strains were isolated from primary infections, and 202 strains were isolated from postoperative infections. From primary infections, anaerobic Gram-positive bacteria were predominant, while from post operative infections, aerobic Gram-positive bacteria were predominant. Among aerobic Gram-positive bacteria, the isolation rate of Enterococcus faecalis was the highest, followed by that of Staphylococcus aureus from postoperative infections. Among anaerobic Gram-positive bacteria, the isolation rate of Peptostreptococcus spp. was the highest from both types of infections. Among anaerobic Gram-negative, Escherichia coli was the most predominantly isolated from primary infections, followed by Klebsiella pneumoniae and Pseudomonas aeruginosa in this order, and from postoperative infections, P. aeruginosa was the most predominantly isolated, followed by Enterobacter spp. and Klebsiella spp. Among anaerobic Gram-negative bacteria, the isolation rate of Bacteroides fragilis group was the highest from both types of infections. We noticed that MICs of cefazolin against three out of 23 strains of E. coli were higher than 100 micrograms/ml. Among anaerobic bacteria, there were many resistant strains against penicillins and cephems with MICs higher than 100 micrograms/ml, and the same trend was observed among other Bacteroides spp. and Prevotella spp.

Bacterial Infections↗

PubMed1997

[Isolation rate of Enterococcus spp. from surgical infections and their susceptibilities].

Enterococcus spp. isolated from surgical infections during the period from July 1982 to June 1995 were investigated in a multicenter study involving 19 hospitals in Japan, and the following results were obtained. 1. Though the isolation rate of Enterococcus faecalis and other Enterococcus spp. were not high from primary infections, and from postoperative infections the isolation rate of other Enterococcus spp. was also low, the isolation rate of E. faecalis was highest from postoperative infections after 1993. 2. Vancomycin (VCM) showed strongest activity against E. faecalis, and followed by those of ampicillin (ABPC), imipenem. levofloxacin (LVFX) and meropenem in this order. Against other Enterococcus spp., VCM showed strongest activity, and followed by those of ABPC and LVFX. There were no resistant strains against VCM.

Ampicillin↗