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Biomedical subjects

Y Tsujimoto

Publications and source records attributed to Y Tsujimoto.

At least 73 records · Page 4Linked to original sources
PubMed1998

Ceramide formation leads to caspase-3 activation during hypoxic PC12 cell death. Inhibitory effects of Bcl-2 on ceramide formation and caspase-3 activation.

PC12 cells undergo apoptosis as well as necrosis following exposure to hypoxia. Following a 6-h hypoxic treatment, a time-dependent increase in intracellular ceramide level was observed with a concurrent decrease in sphingomyelin. It was also shown that the hypoxia-induced ceramide accumulation resulted from activation of neutral magnesium-dependent sphingomyelinase. Comparative kinetic analyses of the neutral sphingomyelinase in the cells under normoxia and hypoxia showed that hypoxia increased Vmax but did not affect Km of the enzyme. In PC12 cells overexpressing Bcl-2 which show strong resistance to hypoxia, sphingomyelin hydrolysis was decreased and activation of neutral sphingomyelinase was reduced. Addition of exogenous C2-ceramide induced cell death and activated caspase-3 as markedly as the hypoxia treatment. On the other hand, in PC12 cells overexpressing Bcl-2, significant decreases in cell death and inhibition of caspase-3 activation were observed after exogenous addition of C2-ceramide. The inhibitors of caspase-3 prevented cell death by either hypoxia or C2-ceramide. These results suggest that ceramide generated by activation of neutral magnesium-dependent sphingomyelinase mediates hypoxic cell death and that Bcl-2 has inhibitory effects on ceramide formation and caspase activation.

Animals↗
PubMed1998

Bcl-2 prevents apoptotic mitochondrial dysfunction by regulating proton flux.

We and others have recently shown that loss of the mitochondrial membrane potential (Deltapsi) precedes apoptosis and chemical-hypoxia-induced necrosis and is prevented by Bcl-2. In this report, we examine the biochemical mechanism used by Bcl-2 to prevent Deltapsi loss, as determined with mitochondria isolated from a cell line overexpressing human Bcl-2 or from livers of Bcl-2 transgenic mice. Although Bcl-2 had no effect on the respiration rate of isolated mitochondria, it prevented both Deltapsi loss and the permeability transition (PT) induced by various reagents, including Ca2+, H2O2, and tert-butyl hydroperoxide. Even under conditions that did not allow PT, Bcl-2 maintained Deltapsi, suggesting that the functional target of Bcl-2 is regulation of Deltapsi but not PT. Bcl-2 also maintained Deltapsi in the presence of the protonophore SF6847, which induces proton influx, suggesting that Bcl-2 regulates ion transport to maintain Deltapsi. Although treatment with SF6847 in the absence of Ca2+ caused massive H+ influx in control mitochondria, the presence of Bcl-2 induced H+ efflux after transient H+ influx. In this case, Bcl-2 did not enhance K+ efflux. Furthermore, Bcl-2 enhanced H+ efflux but not K+ flux after treatment of mitochondria with Ca2+ or tert-butyl hydroperoxide. These results suggest that Bcl-2 maintains Deltapsi by enhancing H+ efflux in the presence of Deltapsi-loss-inducing stimuli.

Animals↗
PubMed1998

Prevention of hypoxic liver cell necrosis by in vivo human bcl-2 gene transfection.

Prevention of hypoxic cell death is a key to successful liver transplantation. We developed a new method for preventing liver hypoxic cell death by introducing an anti-cell death gene directly into rat livers. When the human bcl-2 gene (hbcl-2) was directly transfected into rat livers together with non-histone chromosomal protein high mobility group 1 (HMG1) by the hemagglutinating virus of Japan (Sendai virus; HVJ)-liposome method, human Bcl-2 protein (hBcl-2) was efficiently expressed. Electron microscopy and fluorescence microscopy revealed that hepatocytes expressing exogenous hBcl-2 were almost completely protected the hypoxic cell necrosis. The expression of the hBcl-2 also inhibited activation of caspase-3 (-like) proteases and liver dysfunction. Thus, we conclude that transfection of the hbcl-2 gene through HVJ-liposome method is useful to prevent liver cell necrosis induced by hypoxia. This finding could lead to new strategies to avoid the hypoxic cell death, the major problem in liver transplantation.

Animals↗
PubMed1998

Expression of the glyoxalase I gene of Saccharomyces cerevisiae is regulated by high osmolarity glycerol mitogen-activated protein kinase pathway in osmotic stress response.

Methylglyoxal is a cytotoxic metabolite derived from dihydroxyacetone phosphate, an intermediate of glycolysis. Detoxification of methylglyoxal is performed by glyoxalase I. Expression of the structural gene of glyoxalase I (GLO1) of Saccharomyces cerevisiae under several stress conditions was investigated using the GLO1-lacZ fusion gene, and expression of the GLO1 gene was found to be specifically induced by osmotic stress. The Hog1p is one of the mitogen-activated protein kinases (MAPKs) in S. cerevisiae, and both Msn2p and Msn4p are the transcriptional regulators that are thought to be under the control of Hog1p-MAPK. Expression of the GLO1 gene under osmotic stress was completely repressed in hog1Delta disruptant and was repressed approximately 80 and 50% in msn2Delta and msn4Delta disruptants, respectively. A double mutant of the MSN2 and MSN4 gene was unable to induce expression of the GLO1 gene under highly osmotic conditions. Glucose consumption increased approximately 30% during the adaptive period in osmotic stress in the wild type strain. On the contrary, it was reduced by 15% in the hog1Delta mutant. When the yeast cell is exposed to highly osmotic conditions, glycerol is synthesized as a compatible solute. Glycerol is synthesized from glucose, and a rate-limiting enzyme in glycerol biosynthesis is glycerol-3-phosphate dehydrogenase (GPD1 gene product), which catalyzes reduction of dihydroxyacetone phosphate to glycerol 3-phosphate. Expression of the GPD1 gene is also under the control of Hog1p-MAPK. Methylglyoxal is also synthesized from dihydroxyacetone phosphate; therefore, induction of the GLO1 gene expression by osmotic stress was thought to scavenge methylglyoxal, which increased during glycerol production for adaptation to osmotic stress.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1998

High performance liquid chromatographic determination of mercapturic acids in urine of rats administered with m- or p-xylene.

The determination of urinary mercapturic acid, N-acetyl-S-(m-xylyl)-L-cysteine or N-acetyl-S-(p-xylyl)-L-cysteine, was undertaken by HPLC after i.p. administration of m- or p-xylene to rats in order to confirm the structure of thioether which was known to be excreted in small amounts related with the metabolism of m- or p-xylene. The excretion rates of these mercapturic acids were much lower than those previously estimated from thioether determinations. The present study clearly demonstrates the mercapturic acid pathway operates only to limited degree as a metabolic route of m- or p-xylene.

Acetylcysteine↗
PubMed1998

Effect of nonsteroidal anti-inflammatory drugs on lipid peroxidation by hydroxyl radical.

1. Effects of acetaminophen and sodium salicylate on hydoxyl radical elimination were studied using an electron-spin-resonance spin-trapping method. The effects of these agents on lipid peroxidation of the human erythrocyte membrane were also investigated by the thiobarbituric acid method. 2. Acetaminophen and sodium salicylate depressed hydroxyl radical generated by Cu2+/H2O2. 3. Acetaminophen inhibited Cu2+/H2O2-dependent lipid peroxidation; however, sodium salicylate enhanced Cu2+/H2O2-dependent lipid peroxidation.

Acetaminophen↗
PubMed1998

Type I interferons are essential mediators of apoptotic death in virally infected cells.

BACKGROUND: The interferons (IFNs) have been extensively studied in the context of host defence against viral infection. In the established model of IFN action, virally infected cells secrete type I IFNs (IFN-alpha/beta) which induce an antiviral state in uninfected cells. However, it is not clear how IFNs function on the infected cells. It has been reported that cells infected by some viruses die by apoptosis. RESULTS: In the present study, we found that three types of viruses commonly induce apoptosis in primary cell cultures. Importantly, we observed that virus-induced apoptosis was inhibited by anti-IFN-alpha/beta antibodies, and in cells lacking either the type I IFN receptor 1 (IFNAR1) or its downstream mediator, Stat1 (Signal transducer and activator of transcription 1). IFN-alpha treatment by itself did not induce apoptosis unless it was combined with transfection by double-stranded RNA (dsRNA), which is normally generated during the course of viral infection. CONCLUSION: These results indicate a novel antiviral function of the type I IFNs, i.e. the selective induction of apoptosis in virally infected cells. In effect, these IFNs have a bifunctional role in limiting the spread of virus; eliciting an antiviral state in uninfected cells while promoting apoptosis in infected cells. Our results may help explain why IFNs are sometimes useful in the treatment of viral diseases and will provide further insight into the mechanisms of virus-induced pathogenesis.

Animals↗
PubMed1998

Role of Bcl-2 family proteins in apoptosis: apoptosomes or mitochondria?

Apoptosis is an essential physiological process for the selective elimination of cells, which is involved in a variety of biological events. The Bcl-2 family is the best characterized protein family involved in the regulation of apoptotic cell death, consisting of anti-apoptotic and pro-apoptotic members. The anti-apoptotic members of this family, such as Bcl-2 and Bcl-XL, prevent apoptosis either by sequestering proforms of death-driving cysteine proteases called caspases (a complex called the apoptosome) or by preventing the release of mitochondrial apoptogenic factors such as cytochrome c and AIF (apoptosis-inducing factor) into the cytoplasm. After entering the cytoplasm, cytochrome c and AIF directly activate caspases that cleave a set of cellular proteins to cause apoptotic changes. In contrast, pro-apoptotic members of this family, such as Bax and Bak, trigger the release of caspases from death antagonists via heterodimerization and also by inducing the release of mitochondrial apoptogenic factors into the cytoplasm via acting on mitochondrial permeability transition pore, thereby leading to caspase activation. Thus, the Bcl-2 family of proteins acts as a critical life-death decision point within the common pathway of apoptosis.

Animals↗
PubMed1998

Death-signalling cascade in mouse cerebellar granule neurons.

Molecular mechanisms of neuronal cell death are still largely unknown. In the present study, the signal transduction pathway of cell death in cerebellar granule neurons was examined by employing various death-preventative agents. When death was induced by the depletion of serum and a depolarizing level of potassium, transient increase in active c-Jun, mitochondrial membrane potential (deltapsi) loss, activation of caspase-3 (-like) proteases, and nuclear condensation and fragmentation were observed. The protein synthesis inhibitor cycloheximide blocked all these phenomena, whereas RNA synthesis inhibitor actinomycin-D, survival factor such as insulin-like growth factor-1, brain-derived neurotrophic factor, high K+ (25 mM) and overproduced antiapoptotic protein Bcl-2, prevented deltapsi, loss, caspase activation, and nuclear change, but not an increase in active c-Jun. The caspase inhibitor z-Asp-CH2-DCB (carbobenzoxy-L-aspartyl-alpha-[(2,6-dichlorobenzoyl) oxy]methane) only inhibited activation of caspases and nuclear change. These results suggest that the death signal in cerebellar granule neurons is sequentially transduced in the order of c-Jun activation, de novo RNA synthesis, mitochondrial deltapsi loss, activation of caspase-3 (-like) proteases and nuclear change.

Animals↗
PubMed1998

Amelioration of hippocampal neuronal damage after global ischemia by neuronal overexpression of BCL-2 in transgenic mice.

BACKGROUND AND PURPOSE: Reports suggesting the involvement of apoptosis in ischemic neuronal damage have been accumulating, and protection against apoptotic death by BCL-2 has been shown in many types of cells. Overexpression of BCL-2 has been shown to reduce infarct size after focal ischemia. The purpose of the present study was to assess whether BCL-2 exerted its effect on selective neuronal vulnerability after transient global ischemia. METHODS: Transgenic mice overexpressing BCL-2 in neurons and their littermates were subjected to transient forebrain ischemia for 12 minutes, and the hippocampus was examined 7 days later with conventional histology, immunohistochemistry, and in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling of fragmented DNA. RESULTS: Although both types of mice showed a similar degree of ischemic insult, transgenic mice showed a lesser degree of neuronal death together with DNA fragmentation in the hippocampus than their littermates. CONCLUSIONS: Overexpression of BCL-2 in neurons mitigates selective neuronal vulnerability in the hippocampus of transgenic mice after transient global ischemia.

Animals↗
PubMed1998

Isolation, mapping, and functional analysis of a novel human cDNA (BNIP3L) encoding a protein homologous to human NIP3.

We have isolated a novel cDNA that encodes a product showing significant sequence homology (56% identity) to human NIP3, a protein thought to interact with adenovirus E1B19kD and human BCL2 proteins. This cDNA contains an open reading frame of 657 nucleotides encoding a 219 amino acid polypeptide. The gene, designated BNIP3L, was expressed in all 16 normal human tissues examined; we mapped it to chromosome band 8p21 by fluorescence in situ hybridization. Introduction of the BNIP3L gene into six different cancer-cell lines caused significant growth suppression in each of them, while no such effect occurred when the antisense cDNA or the vector DNA was transfected, indicating that BNIP3L may function as a tumor suppressor.

Amino Acid Sequence↗
PubMed1997

Synergistic anti-apoptotic activity between Bcl-2 and SMN implicated in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a motor neuron disease characterized by degeneration of the anterior horn cells of the spinal cord. It is a common fatal autosomal recessive disorder and linkage studies have identified two candidate genes, SMN and NAIP, both on chromosome 5q13. Although NAIP protein is known to have an anti-apoptotic function, the function of SMN has been unclear and it shows no significant sequence similarity to any other protein. The SMN gene is deleted or interrupted on both chromosomes in nearly all SMA patients. Here we show that SMN interacts with Bcl-2, another anti-apoptotic protein, and that co-expression of SMN with Bcl-2 confers a synergistic preventive effect against Bax-induced or Fas-mediated apoptosis, although SMN itself has only a weak anti-apoptotic activity. SMN(Y272C), which carries a missense mutation and was found in an SMA patient who exceptionally retained SMN on one allele, exerts no synergism with Bcl-2. Furthermore, the product of a truncated transcript lacking exon 7, which was derived from an SMN gene carrying an intragenic mutation or from the SMN copy gene cBCD541 retained in all SMA patients, had no synergistic activity but instead had a dominant-negative effect on full-length SMN. Our results indicate that an absent or decreased anti-apoptotic activity of SMN in concert with Bcl-2 underlies the pathogenesis of SMA.

Animals↗
PubMed1997

Evidence against a functional site for Bcl-2 downstream of caspase cascade in preventing apoptosis.

Apoptotic cell death is driven by ICE family proteases (caspases) and negatively regulated by Bcl-2 family proteins. Although it has been shown that Bcl-2 exerts anti-apoptotic activity by blocking a step(s) leading to the activation of caspases, a role for Bcl-2 and Bcl-xL downstream of the caspase cascade has remained unclear. Here, we show that purified active caspase-3 (CPP32/Yama/apopain) and caspase-1 (ICE) induces apoptosis when microinjected into the cytoplasm of cells, confirming our recent observations, and that the apoptosis is not at all prevented by Bcl-2 and Bcl-xL, which are overexpressed more than sufficiently to prevent Fas-mediated and overexpressed procaspase-1-mediated apoptosis. Thus, Bcl-2 and Bcl-xL do not act downstream of the caspase cascade.

Apoptosis↗
PubMed1997

Caspase-dependent apoptosis of COS-7 cells induced by Bax overexpression: differential effects of Bcl-2 and Bcl-xL on Bax-induced caspase activation and apoptosis.

Bcl-2 family proteins and ICE/CED-3 family proteases (caspases) are regarded as the basic regulators of apoptotic cell death. They are evolutionarily conserved and implicated in a variety of apoptosis. However, the precise mechanism by which these two families interact to regulate cell death is not yet known. In this study, we found that the overexpression of the Bcl-2 family member Bax induced apoptotic cell death in COS-7 cells through the activation of CPP32 (caspase-3)-like proteases that cleaved the DEVD tetrapeptide. This apoptotic cell death was suppressed by the viral proteins CrmA and p35, as well as by the chemically synthesized caspase inhibitors Z-Asp-CH2-DCB and zVAD-fmk. We also found that the Bax-induced apoptosis of COS-7 cells was suppressed by Bcl-xL and Bcl-2, though both Bcl-xL and Bcl-2 similarly prevented etoposide-induced apoptosis in COS-7 cells. In addition, Bcl-xL inhibited the activation of caspase-3-like proteases accompanying Bax-induced COS-7 cell death but Bcl-2 did not. These results indicate that the caspase activation is essential for Bax-induced apoptosis, and that the ability of Bcl-2 and Bcl-xL to prevent the Bax-induced caspase activation and apoptosis in COS-7 cells could be differentially regulated. Our results also suggest that Bcl-2 family proteins function upstream of caspase activation and control apoptosis through the regulation of caspase activity.

Animals↗
PubMed1997

Inhibition of apoptosis by the actin-regulatory protein gelsolin.

Gelsolin is an actin-regulatory protein that modulates actin assembly and disassembly, and is believed to regulate cell motility in vivo through modulation of the actin network. In addition to its actin-regulatory function, gelsolin has also been proposed to affect cell growth. Our present experiments have tested the possible involvement of gelsolin in the regulation of apoptosis, which is significantly affected by growth. When overexpressed in Jurkat cells, gelsolin strongly inhibited apoptosis induced by anti-Fas antibody, C2-ceramide or dexamethasone, without changing the F-actin morphology or the levels of Fas or Bcl-2 family proteins. Upon the induction of apoptosis, an increase in CPP32(-like) protease activity was observed in the control vector transfectants, while it was strongly suppressed in the gelsolin transfectants. Pro-CPP32 protein, an inactive form of CPP32 protease, remained uncleaved by anti-Fas treatment in the gelsolin transfectants, indicating that gelsolin blocks upstream of this protease. The tetrapeptide inhibitor of CPP32(-like) proteases strongly inhibited Fas-mediated apoptosis, but only partially suppressed both C2-ceramide- and dexamethasone-induced apoptosis. These data suggest that the critical target responsible for the execution of apoptosis may exist upstream of CPP32(-like) proteases in Jurkat cells and that gelsolin acts on this target to inhibit the apoptotic cell death program.

Actins↗
PubMed1997

Involvement of caspase-4(-like) protease in Fas-mediated apoptotic pathway.

Proteases of the caspase family, especially caspase-1 (ICE)(-like), caspase-3 (CPP32/Yama/apopain)(-like) and caspase-8 (MACH/FLICE/Mch5) proteases, are implicated in Fas (APO-1/CD95)-mediated apoptosis. Here, we show that the caspase-4 (TX/ICH-2/ICE(rel)II)(-like) protease, another member of the caspase family, is also involved in Fas-mediated apoptosis, based upon the observations: (i) caspase-4 is processed in response to an agonistic anti-Fas antibody treatment, (ii) overexpression of a mutant caspase-4 with active site mutations in both p20 and p10 subunits delays Fas-mediated apoptosis, (iii) microinjected anti-caspase-4 antibodies inhibit Fas-mediated apoptosis. Together with our observations that the mutant caspase-4 inhibits the Fas-mediated activation of caspase-3(-like) proteases and purified caspase-4 cleaves pro-caspase-3 to generate a subunit of active form, these results suggest that Fas-mediated apoptosis is driven by a caspase cascade in which the caspase-4(-like) protease transmits a death signal from caspase-8 to caspase-3(-like) proteases probably through directly cleaving pro-caspase-3(-like) proteases.

Antibodies↗