Kazuhito Yamaguchi

Phosphoinositide 3-kinase accelerates autophagic cell death during glucose deprivation in the rat cardiomyocyte-derived cell line H9c2

We investigated cell death during glucose deprivation in rat cardiomyocyte-derived H9c2 cells. Electron microscopic analysis revealed accumulation of autophagic vacuoles during glucose deprivation. The addition of 3-methyladenine or LY294002, which are known to inhibit autophagosome formation, reduced cell death while Z-VAD-FMK, a caspase inhibitor, slightly affected cell death. Thus, cell death during glucose deprivation is not type I programmed cell death (apoptotic cell death) but type II programmed cell death (autophagic cell death). Moreover, we found that both insulin-like growth factor-I and the adenovirus-mediated overexpression of wild-type class I PI 3-kinase accelerated cell death as well as accumulation of autophagic vacuoles during glucose deprivation while dominant-negative PI 3-kinase reduced these phenomena. The results indicate that IGF-I/PI 3-kinase accelerates the accumulation of autophagic vacuoles and subsequent autophagic cell death during glucose deprivation, revealing the opposing role of IGF-I/PI 3-kinase in two distinct types of programmed cell death (apoptotic and autophagic cell death).

Cutting edge: the B cell chemokine CXC chemokine ligand 13/B lymphocyte chemoattractant is expressed in the high endothelial venules of lymph nodes and Peyer's patches and affects B cell trafficking across high endothelial venules.

While CCR7 ligands direct T cell trafficking into lymph nodes (LNs) and Peyer’s patches (PPs), chemokines that regulate B cell trafficking across high endothelial venules (HEVs) remain to be fully elucidated. Here we report that CXC chemokine ligand (CXCL)13 (B lymphocyte chemoattractant) is detected immunohistologically in the majority of HEVs in LNs and PPs of nonimmunized mice. Systemically administered anti-CXCL13 Ab bound to the surface of ∼50% of HEVs in LNs and PPs, but not to other types of blood vessels, indicating that CXCL13 is expressed in the HEV lumen. In CXCL13-null mice, B cells rarely adhered to PP HEVs, whereas T cells did efficiently. Superfusion of CXCL13-null PPs with CXCL13 restored the luminal presentation of CXCL13 and also B cell arrest in PP HEVs at least partially. Collectively, these results indicate that CXCL13 expressed in the HEV lumen plays a crucial role in B cell trafficking into secondary lymphoid tissues such as PPs.

Infection of the erythroid cell line, KU812Ep6 with human parvovirus B19 and its application to titration of B19 infectivity.

A human parvovirus B19 (B19) infectivity assay was developed using the erythroid cell line, KU812Ep6. KU812Ep6 was cloned for high efficiency infection with B19 in vitro, in the presence of erythropoietin by a limiting dilution method from the parent cell line, KU812. B19 was effectively propagated in KU812Ep6 and was detected for B19 antigens, VP1 and VP2. The titers of B19 positive sera measured with KU812Ep6 cells were in the range of 10(6) to 10(8) TCID50 ml. This KU812Ep6 infectivity assay had a 10(3)-10(4.5) higher sensitivity than the colony forming unit-erythroid (CFU-e) injury assay. It was calculated that one TCID50 needed 10(3) B19 genome copies, judging from the infectivity assay and semi-quantitative PCR. The KU812Ep6 infectivity assay was also used to determine infectivity of B19 in vitro, and to evaluate inactivation, as well as clearance of the virus. The inactivation of B19 by heating was carried out and infectivity declined from 10(4) TCID50 ml to < 10 TCID50 ml (lower limit of detection) at 60 degrees C for 3 h or at 70 degrees C for 30 min, but only decreased to 10(2.5) TCID50 ml at 50 degrees C for 8 h.

Translocation of protein kinase C-α, δ and ϵ isoforms in ischemic rat heart

To explore the spatial and temporal localization of PKC isoforms during ischemia, we quantified PKC isoforms in the subcellular fractions in perfused rat heart by immunoblotting using specific antibodies against PKC isoforms. PKCs-alpha and epsilon translocated from the 100000 x g supernatant (S, cytosolic) fraction to the 1000 x g pellet (PI, nucleus-myofibril) and the 1000-100000 x g pellet (P2, membrane) fractions during 5-40 min of ischemia. PKC-delta redistributed from the P2 to the S fraction. A 50-kDa fragment of PKC-alpha appeared during ischemia possibly through calpain action. Immunohistochemical observations showed the different localizations of PKC-alpha, delta, and epsilon in the myocytes. The PKC assay displayed high basal levels of Ca(2+)-independent PKC, the activation of Ca(2+)-dependent PKC in the P1 and P2 fractions, and the activation of Ca(2+)-independent PKC in the P1 fraction after 20 min of ischemia. These observations show that ischemia induces different patterns of translocation of the three PKC isoforms, suggesting differences in their roles.

Interleukin‐1 beta production in the rabbit brain during endotoxin‐induced fever.

Interleukin‐1 beta (IL‐1 beta) production in the brain and the spleen was investigated in rabbits made febrile by intravenous (I.V.) injection of endotoxin, or human recombinant IL‐1 beta (hIL‐1 beta). The endotoxin used in the present study was the lipopolysaccharide (LPS) of Salmonella typhosa endotoxin. Monophasic fever was induced by I.V. injection of a low dose of LPS (0.02 micrograms kg‐1) and biphasic fever by I.V. injection of a large dose of LPS (4 micrograms kg‐1), a sublethal dose of LPS (40 micrograms kg‐1) or hIL‐1 beta (2 micrograms kg‐1). In situ hybridization and immunohistochemical studies revealed that, although no IL‐1 beta production was observed in the brain at 1 and 3 h after injection of a low dose of LPS (0.02 micrograms kg‐1) or of hIL‐1 beta (2 micrograms kg‐1), IL‐1 beta production was demonstrated in organum vasculosum laminae terminalis (OVLT) and some cells around the blood vessels in the parenchyma 1 h after 4 micrograms kg‐1 LPS. IL‐1 beta production was detected throughout the brain after 40 micrograms kg‐1 LPS. Pretreatment with indomethacin, an inhibitor of prostaglandin synthesis, did not affect IL‐1 beta production in the brain induced by 4 micrograms kg‐1 LPS. The cell type which produces IL‐1 beta in the OVLT following LPS injection was confirmed to be a macrophage by electron microscopy. The cells producing IL‐1 beta in the parenchyma were determined to be microglial cells. In the spleen, each dose of LPS induced a significant increase in IL‐1 beta production in polymorphonuclear cells and macrophages in the red pulp 1 h after injection. However, 2 micrograms kg‐1 hIL‐1 beta did not induce IL‐1 beta production in the spleen. The present results show clearly that systemic administration of LPS induces IL‐1 beta production in the OVLT which may be responsible for induction of the second phase of biphasic fever. The production of IL‐1 beta in the OVLT was not attributable to the action of peripherally synthesized IL‐1 beta or prostaglandins.

A novel method for removal of human immunodeficiency virus: Filtration with porous polymeric membranes

Abstract. We propose a new method to rid solutions of a virus by using a novel regenerated multilayered structured cellulose membrane (BMM). When the filtrate of human immunodeficiency virus (HIV) preparation was obtained through BMM it showed no infectivity. Electron microscopic observation revealed that HIV was completely caught by the multilayers of the BMM. Conveniently, BMM was seldomly found to adsorb protein molecules and also to have a high filtration rate. These characteristics may have a use in the removal of other variously sized pathogenic agents from plasma.

Phosphoinositide 3-kinase accelerates necrotic cell death during hypoxia.

Using H9c2 cells derived from rat cardiomyocytes, we investigated the mechanism of cell death during hypoxia in the presence of serum and glucose. Hypoxic cell death is by necrosis and is accompanied by metabolic acidosis. Moreover, hypoxic cell death is inhibited by Hepes buffer as well as by 2-deoxyglucose, an inhibitor of glycolysis, indicating that metabolic acidosis should play an essential role in hypoxic injury. The involvement of phosphoinositide 3-kinase (PI 3-kinase), which is known to activate glucose metabolism, was examined using its inhibitor, LY290042, or adenovirus-mediated gene transfer. Hypoxic cell death was inhibited by LY294002 in a dose-dependent manner. Overexpression of dominant negative PI 3-kinase was found to reduce cell death, whereas wild-type PI 3-kinase enhanced it. Dominant negative PI 3-kinase also reduced glucose consumption and acidosis, but this was stimulated by wild-type PI 3-kinase. The data indicate that PI 3-kinase stimulates cell death by enhancing metabolic acidosis. LY294002 significantly reduced glucose uptake, showing that PI 3-kinase regulates glycolysis at the step of glucose transport. These findings indicate the pivotal role of glucose metabolism in hypoxic cell death, and reveal a novel death-promoting effect of PI 3-kinase during hypoxia, despite this enzyme being considered to be a survival-promoting factor.

Organum vasculosum laminae terminalis (OVLT) is a brain site to produce interleukin-1β during fever

The present study was carried out to determine whether interleukin-1 (IL-1) production occurs in the rabbit organum vasculosum laminae terminalis (OVLT) during fever induced by endotoxin. The intravenous (i.v.) injection of endotoxin (4 micrograms/kg) caused significant fever in rabbits. Through the use of in situ hybridization and immunohistochemical techniques, the synthesis of IL-1 was observed in the OVLT during the fever. The present results support the hypothesis that IL-1 is produced in the brain during fever.

Cocaine: evidence for NMDA-, β-carboline- and dopaminergic-mediated seizures in mice

Abstract The present study was undertaken to examine the role of the benzodiazepine/GABA and N-methyl- d -aspartate (NMDA) systems in the convulsive effect of cocaine in mice. When cocaine (3.5 mg/ml) solution was infused into the tail vein at a rate of 0.3 ml/min, mice showed clonic and tonic convulsions. These seizures were not affected by low doses of bicuculline or picrotoxin, a GABAA receptor antagonist and a Cl ion channel blocker, respectively. Aminooxyacetic acid (AOAA), a GABA deaminase inhibitor, and phenobarbital, a Cl ion channel activator, and baclofen, a GABAB receptor agonist, also had no effect on these convulsions. Benzodiazepine inverse agonist β-DMCM, at a dose which by itself had no convulsive effect lowered the convulsive threshold of cocaine. This lowered convulsive threshold was reversed by flumazenil, a benzodiazepine inverse antagonist, and diazepam, a benzodiazepine full agonist, which by themselves did not inhibit cocaine seizure. It is likely that cocaine seizure involves a benzodiazepine (β-carboline) recognition site other than the benzodiazepine/GABAA receptor–Cl ionophore complex system. CPP and MK-801, competitive and noncompetitive NMDA receptor antagonists, respectively, inhibited cocaine seizures. The inhibitory effects of CPP on cocaine convulsion were reversed by a low dose of NMDA, which by itself did not induce seizure. A dopamine D1 receptor agonist SKF38393 enhanced both clonic and tonic convulsions, while a dopamine D2 receptor agonist bromocriptine inhibited these convulsions. These stimulatory and inhibitory effects were reversed by the D1 and D2 receptor antagonists, SCH23390 and haloperidol, respectively. These results suggest that the cocaine-induced convulsion may involve an activation of the NMDA–Ca ionophore complex system, which is mediated by the dopaminergic system, and a β-carboline recognition site other than the benzodiazepine/GABAA receptor–Cl ionophore complex system.

Protein kinase C-ε protects PC12 cells against methamphetamine-induced death: possible involvement of suppression of glutamate receptor

The involvement of PKC isoform in the methamphetamine (MA)-induced death of neuron-like PC12 cell was studied. The death and the enhanced terminal dUTP nick end labeling (TUNEL) staining were inhibited by a caspase inhibitor, z-Val-Ala-Asp- (OMe)-CH(2)F (z-VAD-fmk). However, the cell death shows neither morphological nor biochemical features of apoptosis or necrosis. The cell death was suppressed by a protein kinase C (PKC) activator, 12,13-phorbol myristate acetate, but was enhanced by PKC specific inhibitor calphostin C or bisindolylmaleimide, not by PKC inhibitor relatively specific for PKC-alpha (safingol) or PKC-delta (rottlerin). Western blotting demonstrated the expression of PKC-alpha, gamma, delta, epsilon and zeta, of which PKC-epsilon translocated from the soluble to the particulate fraction after MA-treatment. Antisense to PKC-epsilon enhanced MA-induced death. A glutamate receptor antagonist MK801 abrogated the cell death, which is reversed by PKC inhibition. These data suggest that PKC-epsilon promotes PC12 cell survival through glutamate receptor suppression.