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Biomedical subjects

Y Eguchi

Publications and source records attributed to Y Eguchi.

At least 55 records · Page 3Linked 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

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

Maternal adrenocortical hormones maintain the early development of pancreatic B cells in the fetal rat.

To investigate the effect of maternal adrenocortical hormones on the development of fetal pancreatic islet cells, pregnant rats were adrenalectomised on d 6 of gestation. On d 12-16 the growth patterns of fetal insulin-producing B cells, glucagon-producing A cells, and somatostatin-producing D cells were observed histometrically. Maternal adrenalectomy resulted in growth retardation of fetal B cells on d 12-15. Maternal corticosterone therapy prevented this retardation. Maternal adrenalectomy, however, did not affect the developmental patterns of A and D cells. By Western blotting and immunohistochemistry, glucocorticoid receptors were demonstrated to be present in the islet cells from d 12 to d 15. These results suggest that maternal adrenocortical hormones, glucocorticoids in particular, maintain the early development of fetal pancreatic B cells through their specific intracellular glucocorticoid receptor.

Adrenal Cortex Hormones↗
PubMed1998

Lower risks of adverse outcome in twins conceived by artificial reproductive techniques compared with spontaneously conceived twins.

The outcomes of twins conceived by 136 women after medical assistance (MA) such as ovulation induction with or without assisted reproductive techniques, and twins conceived spontaneously (SP) by 72 women were compared. All 208 women were monitored from < 20 weeks gestation; they all delivered at > or = 24 weeks gestation. The chorionicity of the placenta was diagnosed antenatally and confirmed after delivery. There were 10 perinatal deaths; the physical and neurological status of the remaining 406 infants was assessed at 1 year of corrected age. There were no differences in gestational age at birth, the birth weights of the larger and smaller twins, the birth weight discordance, or the incidence of life-threatening major malformations between groups. Adverse infant outcomes, such as death, cerebral palsy and mental retardation occurred in nine (3.3%) of 272 MA twins compared with 12 (8.3%) of 144 SP twins (P < 0.05). The placenta was monochorionic in only three (2.2%) of 136 MA twin pregnancies compared with 41 (57%) of 72 SP twin pregnancies (P < 0.001). Of the 21 infants with adverse outcomes, nine had monochorionic placentas. Thus, the risk of an adverse outcome was 2.8-fold higher (95% confidence interval (CI) 1.2-6.4) in monochorionic twins than in dichorionic twins (10 versus 3.7%; P < 0.05). There was no difference in the incidence of adverse infant outcomes between SP (4.8%) and MA (3.4%) twins with dichorionic placentas. These findings suggest that ovulation induction in itself was not associated with an adverse outcome of twin pregnancies. The lower frequency of monochorionic placentas in MA twins may have been responsible for the lower risk of an adverse outcome in MA twins.

Birth Weight↗
PubMed1998

Effects of follicle-stimulating hormone on intermediate filaments and cell division of Sertoli cells of fetal rat testis in culture.

The present study was aimed to examine the effects of follicle-stimulating hormone (FSH) on cell division of Sertoli cells from rat fetal testes and on the kinetics of 2 kinds of intermediate filaments, cytokeratin and vimentin, which comprise the cytoskeleton of Sertoli cells. Testes from rat fetuses of different ages (from day 15 to day 17 of gestation ) were cultured for 48 hr, with or without added FSH. In 15-day testes, FSH influenced neither cell division of Sertoli cells nor kinetics of intermediate filaments. In 16-day testes, FSH promoted cell division of Sertoli cells and kinetic differentiation of intermediate filaments distributed toward the lumen of the seminiferous tubules. These findings suggest that 16-day testes in culture can respond to FSH in a fashion that cell division of Sertoli cells is promoted and that intermediate filaments increase in number and change in intracellular distribution. It is concluded that FSH influences both proliferation and morphological differentiation of Sertoli cells.

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

Expression of lipocalin-type prostaglandin D synthase (beta-trace) in human heart and its accumulation in the coronary circulation of angina patients.

Lipocalin-type prostaglandin D synthase (L-PGDS) is localized in the central nervous system and male genital organs of various mammals and is secreted as beta-trace into the closed compartment of these tissues separated from the systemic circulation. In this study, we found that the mRNA for the human enzyme was expressed most intensely in the heart among various tissues examined. In human autopsy specimens, the enzyme was localized immunocytochemically in myocardial cells, atrial endocardial cells, and a synthetic phenotype of smooth muscle cells in the arteriosclerotic intima, and accumulated in the atherosclerotic plaque of coronary arteries with severe stenosis. In patients with stable angina (75-99% stenosis), the plasma level of L-PGDS was significantly (P < 0.05) higher in the great cardiac vein (0.694 +/- 0.054 microg/ml, n = 7) than in the coronary artery (0.545 +/- 0.034 microg/ml), as determined by a sandwich enzyme immunoassay. However, the veno-arterial difference in the plasma L-PGDS concentration was not observed in normal subjects without stenosis. After a percutaneous transluminal coronary angioplasty was performed to compress the stenotic atherosclerotic plaques, the L-PGDS concentration in the cardiac vein decreased significantly (P < 0.05) to 0.610 +/- 0.051 microg/ml at 20 min and reached the arterial level within 1 h. These findings suggest that L-PGDS is present in both endocardium and myocardium of normal subjects and the stenotic site of patients with stable angina and is secreted into the coronary circulation.

Angina Pectoris↗
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

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

Isolation and sequencing of two alpha-globin genes alpha(A) and alpha(D) in pigeon and evidence for embryo-specific expression of the alpha(D)-globin gene.

By screening a pigeon genomic DNA library, we isolated a recombinant phage clone containing the alpha(A)-globin gene. The DNA sequence of the approximately 6kbp-long insert fragment of the phage clone was determined. The sequence suggested the existence of pigeon alpha(D)-globin gene located 3.1 kbp upstream from the alpha(A)-globin gene. The expression of the alpha(D)-globin in late embryo was also shown by the N-terminal amino-acid sequence of the intact globin chain. These results show that two adult alpha-globin genes, alpha(A) and alpha(D), exist in the pigeon genome, and the alpha(D)-globin is expressed at the late embryo stage. The stage-specific expression suggests the existence of regulatory elements and factors interacting to inhibit transcription at the adult stage.

Amino Acid Sequence↗
PubMed1997

Intracellular ATP levels determine cell death fate by apoptosis or necrosis.

Although apoptosis and necrosis are morphologically distinct manifestations of cell death, apoptosis and some necroses share common features in the death signaling pathway involving functional steps of death-driving interleukin 1beta-converting enzyme family proteases and anti-cell death protein Bcl-2. One evident physiological difference in cells undergoing apoptosis versus necrosis is in intracellular levels of ATP. In this study, we specifically addressed the question of whether apoptosis depends on intracellular ATP levels, since longer incubation under ATP-depleting conditions results in necrotic cell death. Incubation of cells in glucose-free medium with an inhibitor of mitochondrial F0F1-ATPases reduces intracellular ATP levels and completely blocks Fas/Apo-1-stimulated apoptosis. ATP supplied through glycolysis or oxidative phosphorylation restores the apoptotic cell death pathway. ATP depletion also leads to a block in Fas-induced activation of CPP32/Yama(-like) proteases, and when ATP is depleted after the activation of the proteases, subsequent apoptosis is significantly blocked. Thus, ATP-dependent steps exist both upstream and downstream of CPP32/Yama(-like) protease activation in apoptotic signal transduction. Treatment with the calcium ionophore induces apoptosis under ATP-supplying conditions but induces necrotic cell death under ATP-depleting conditions, indicating that ATP levels are a determinant of manifestation of cell death.

Adenosine Triphosphate↗
PubMed1997

Gene transfection of mouse primordial germ cells in vitro and analysis of their survival and growth control.

We evaluated electroporation, liposome-mediated transfection, and the calcium phosphate (CaPO4) coprecipitation method for gene transfection of mouse primordial germ cells (PGCs) in culture as a prelude to the investigation of molecular mechanisms of the germ cell development. We found that electroporation severely damaged PGCs, and the efficiency of liposome-mediated transfection was very low. In contrast, using the CaPO4 coprecipitation method, 18% of PGCs transfected with plasmid pSV-LT expressed simian virus 40 large tumor antigen (SV 40 T-Ag) transiently. However, we did not detect any effects on the proliferation and survival of PGCs obtained from the embryonic gonads at 11.5 days postcoitum (d.p.c.) during 2 days of culture after the transfection. PGCs isolated from the 11.5-d.p.c. gonads change from spread- to round-shape and exhibit growth arrest during a few days of culture, and these rounded PGCs quickly disappear from the culture. We found that the transfection and expression of Bcl-XL or adenovirus type 2 E1B 19,000-molecular-weight protein (E1B 19K) significantly promoted the survival of PGCs and retarded the disappearance of rounded PGCs from the culture system. These results suggest that the Bcl-XL or E1B 19K can prevent the apoptosis of PGCs and inhibit the cell death of the rounded PGCs in culture.

Adenovirus E1B Proteins↗
PubMed1997

Cellular localization of urokinase-type plasminogen activator, its inhibitors, and their mRNAs in breast cancer tissues.

The plasminogen activation (PA) system may participate in cancer invasion and metastasis. A series of breast cancer tissue specimens was analysed using in situ hybridization and immunohistochemistry. Urokinase-type plasminogen activator (u-PA) mRNA was detected in cancer cells and fibroblasts adjacent to them and its expression was found to be more intense in invasive than in intraductal regions. In invasive but not in intraductal regions, especially those with abundant stroma, plasminogen activator inhibitor-1 (PAI-1) mRNA was observed in cancer cells, fibroblasts, macrophages, and endothelial cells, and PAI-2 mRNA was present in cancer cells, and fibroblasts, macrophages, and lymphocytes around them. These PAI-1- and PAI-2-positive cancer cells were localized at the periphery of the invasive front. Immunohistochemistry yielded basically similar results. A retrospective study of surgically resected breast cancers from 73 patients revealed significant clinical differences associated with u-PA and PAI-2 expression in cancer cells, associated with a poor and a good prognosis, respectively. These findings indicate that breast cancer cells and fibroblasts express u-PA initially and then its inhibitors, and that this process is related to invasion. Expression of u-PA and PAI-2 in cancer cells themselves may serve to up-regulate and limit PA-mediated invasion and metastasis, respectively.

Adult↗