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M Yabuki

Publications and source records attributed to M Yabuki.

At least 37 records · Page 2Linked to original sources
PubMed1998

Activation of caspase-3-like protease by digitonin-treated lysosomes.

Apoptosis, a naturally occurring programmed cell death or cell 'suicide', has been paid much attention as one of the critical mechanisms for morphogenesis and tissue remodeling. Activation of cysteine aspartases (caspases) is one of the critical steps leading to apoptosis. Although a mitochondria-mediated pathway has been postulated to be one of the activation mechanism of caspase-3, another subcellular compartment might be involved in the activation of the enzyme. The present study shows that the supernatant fraction of digitonin-treated lysosomes strongly activates Ac-DEVD-CHO inhibitable caspase-3-like protease. Activation of caspase-3-like protease by digitonin-treated lysosomal fractions was specifically suppressed by leupeptin and E-64, inhibitors of cysteine protease. These results indicate that leakage of lysosomal cysteine protease(s) into the cytosolic compartment might be involved in the activation of caspase-3-like protease.

Animals↗
PubMed1998

Molecular regulation of phospholamban function and gene expression.

Ca-ATPase regulates intracellular Ca levels by pumping Ca into sarcoplasmic reticulum. Phospholamban (PLN) functions as an inhibitory cofactor for cardiac Ca-ATPase (SERCA2). To define the molecular mode of interaction between two proteins, interaction sites have been identified. Studies using photoactivated cross-linker and chimeric Ca-ATPase between SERCA2 and nonmuscle Ca-ATPase (SERCA3) indicated that potential binding residues are located just downstream of the active ATPase site (Asp351) of SERCA2. Site-directed mutagenesis study of this region showed that six residues, Lys-Asp-Asp-Lys-Pro-Val402, of SERCA2 are functionally important for the interaction. Further, mutagenesis study of PLN showed that the cytoplasmic region of PLN contains a potential binding site with SERCA2. The unique expression of PLN in cardiac cells has been analyzed by the transcriptional level of its gene using luciferase activity and Gel shift assays. CCAAT-box in the 5'-upstream region was found to be essential for its expression by associating with Y-box binding transcriptional factors.

Amino Acid Sequence↗
PubMed1998

Direct association of the gap junction protein connexin-43 with ZO-1 in cardiac myocytes.

The gap junction protein connexin-43 is normally located at the intercalated discs of cardiac myocytes, and it plays a critical role in the synchronization of their contraction. The mechanism by which connexin-43 is localized within cardiac myocytes is unknown. However, localization of connexin-43 likely involves an interaction with the cytoskeleton; immunofluorescence microscopy showed that in cardiac myocytes, connexin-43 specifically colocalizes with the cytoskeletal proteins ZO-1 and alpha-spectrin. In transfected HEK293 cells, immunoprecipitation experiments using coexpressed epitope-tagged connexin-43 and ZO-1 indicated that ZO-1 links connexin-43 with alpha-spectrin. The domains responsible for the protein-protein interaction between connexin-43 and ZO-1 were identified using affinity binding assays with deleted ZO-1 and connexin-43 fusion proteins. Immunoblot analysis of associated proteins showed that the C-terminal domain of connexin-43 binds to the N-terminal domain of ZO-1. The role of this linkage in gap junction formation was examined by a dominant-negative assay using the N-terminal domain of ZO-1. Overexpression of the N-terminal domain of ZO-1 in connexin-43-expressing cells resulted in redistribution of connexin-43 from cell-cell interfaces to cytoplasmic structures; this intracellular redistribution of connexin-43 coincided with a loss of electrical coupling. We therefore conclude that the linkage between connexin-43 and alpha-spectrin, via ZO-1, may serve to localize connexin-43 at the intercalated discs, thereby generating functional gap junctions in cardiac myocytes.

Amino Acid Sequence↗
PubMed1998

Culture-positive Lyme borreliosis.

We report a case of Lyme borreliosis. Culture of skin biopsy was positive for Borrelia garinii, despite repeated prior treatment with antibiotics. The patient had travelled in Europe 17 months before the onset of symptoms, but the clinical details indicate that the organism could have been acquired in Australia. The results of conventional serological and histopathological tests were negative, despite an illness duration of at least two years.

Adult↗
PubMed1998

Intercellular calcium signaling via gap junction in connexin-43-transfected cells.

In excitable cells, intracellular Ca2+ is released via the ryanodine receptor from the intracellular Ca2+ storing structure, the sarcoplasmic reticulum. To determine whether this released Ca2+ propagates through gap junctions to neighboring cells and thereby constitutes a long range signaling network, we developed a cell system in which cells expressing both connexin-43 and ryanodine receptor are surrounded by cells expressing only connexin-43. When the ryanodine receptor in cells was activated by caffeine, propagation of Ca2+ from these caffeine-responsive cells to neighboring cells was observed with a Ca2+ imaging system using fura-2/AM. Inhibitors of gap junctional communication rapidly and reversibly abolished this propagation of Ca2+. Together with the electrophysiological analysis of transfected cells, the observed intercellular Ca2+ wave was revealed to be due to the reconstituted gap junction of transfected cells. We next evaluated the functional roles of cysteine residues in the extracellular loops of connexin-43 in gap junctional communication. Mutations of Cys54, Cys187, Cys192, and Cys198 to Ser showed the failure of Ca2+ propagation to neighboring cells in accordance with the electrical uncoupling between transfected cells, whereas mutations of Cys61 and Cys68 to Ser showed the same pattern as the wild type. [14C]Iodoacetamide labeling of free thiols of cysteine residues in mutant connexin-43s showed that two pairs of intramolecular disulfide bonds are formed between Cys54 and Cys192 and between Cys187 and Cys198. These results suggest that intercellular Ca2+ signaling takes place in cultured cells expressing connexin-43, leading to their own synchronization and that the extracellular disulfide bonds of connexin-43 are crucial for this process.

Calcium↗
PubMed1998

Met-Gly-Cys motif from G-protein alpha subunit cannot direct palmitoylation when fused to heterologous protein.

To determine whether the N-terminal Met-Gly-Cys motif from G-protein alpha subunit can direct palmitoylation of protein, we have generated heterologous fusion proteins containing the first 10 amino acids of Gi1 alpha and Gs alpha using tumor necrosis factor as a model protein and determined their ability to incorporate palmitate using in vitro and in vivo expression systems. DNA sequences coding for the N-terminal 10 amino acids of Gi1 alpha and Gs alpha were fused to the 5'-end of the cDNA coding for the mature domain of tumor necrosis factor (TNF) to give Gi1 alpha-TNF and Gs alpha-TNF cDNA. In vitro translation of the mRNA coding for the Gi1 alpha-TNF cDNA gave rise to an N-myristoylated fusion TNF with a molecular mass of 18 kDa as determined by the incorporation of [3H]myristic acid and by immunoprecipitation with anti-TNF antibody. In contrast, no incorporation of fatty acid was detected for Gs alpha-TNF. Baculovirus expression of the Gi1 alpha-TNF cDNA in Sf-9 cells gave rise to an N-myristoylated but not palmitoylated fusion TNF. This myristoylation was inhibited by replacement of Gly-2 with Ala but not Cys-3 with Ala, indicating the acylation reaction is entirely dependent on the N-myristoylation signal (Met-Gly-X-X-X-Ser) and Cys-3 is not involved. As is the case with in vitro translation, no incorporation of fatty acid was detected for Gs alpha-TNF. These results indicated that unlike the myristoylation signal Met-Gly-X-X-X-Ser/Thr/Cys, the Met-Gly-Cys motif found in G-protein alpha subunits itself is not sufficient to direct palmitoylation even if Gly-2 is myristoylated after removal of initiating Met. Thus, another structural determinant is implicated in this modification.

Amino Acid Sequence↗
PubMed1997

Catalytic and structural importance of Gly-454, Tyr-455, and Leu-456 in the carboxy-terminal region of Escherichia coli F1-ATPase alpha subunit.

Monoclonal antibody alpha110 recognizes Leu-456 in the alpha subunit of the Escherichia coli F1-ATPase. Binding of this antibody to the alpha subunit or mutation of this residue to Pro caused enhancement of the ATPase activity, suggesting that this residue is involved in the catalytic mechanism of this molecule (H. Kanazawa et al. (1995) Arch. Biochem. Biophys. 317, 348-356). Leu-456 together with Gly-454 and Tyr-455 are the only residues in the carboxy-terminal 75 amino acids conserved among various species, suggesting that these three residues play important roles in catalysis by the ATPase. Here, we introduced site-directed mutations into these residues. Not only L456P but also G454L, Y455K, Y455L, and L456N mutations caused enhancement of the ATPase activity. Surprisingly, Y455V, L456H, and L456S caused assembly defects of F1 subunits on the membrane. Reconstitution of the alpha betagamma complex from the wild-type beta and gamma subunits with the mutant alpha subunit (L4gamma6P) exhibited enhanced ATPase activity. Addition of delta or epsilon fused to glutathione S-transferase which are functionally similar to the delta and epsilon subunits, respectively, to the reconstituted F1-ATPase did not cause significant enhancement of its activity. Decreased interaction between alpha and beta subunits with the L456P mutation was detected by the yeast two-hybrid system. According to the deduced three-dimensional structure of the bovine a subunit, Leu-456, Gly-454, and Tyr-455 are included in a small alpha helix. These results suggest that this alpha helix affects interaction of the alpha subunit with the beta subunit but not with delta or epsilon, which may be important for the catalytic mechanism and F1 assembly.

Amino Acid Sequence↗
PubMed1997

A phylogenetic analysis of Saccharomyces species by the sequence of 18S-28S rRNA spacer regions.

Sequences of two internally transcribed spacer regions between 18S and 28S rRNA genes were determined to assess the phylogenetic relationship in the strains belonging to the genus Saccharomyces. The sequences of S. bayanus and S. pastorianus were quite similar, but not identical. Two phylogenetic trees constructed by the neighbor-joining method showed that all the species examined were distinguished from one another. The Saccharomyces sensu stricto species: S. cerevisiae, S. bayanus, S. paradoxus and S. pastorianus, were closely related and far from the Saccharomyces sensu lato species including S. barnetti, S. castellii, S. dairensis, S. exiguus, S. servazzii, S. spencerorum and S. unisporus, and an outlying species, S. kluyveri.

DNA, Fungal↗
PubMed1997

Do reactive oxygen species underlie the mechanism of apoptosis in the tadpole tail?

Although "programmed cell death" has been postulated to underlie the mechanism for the disappearance of the tadpole tail, critical factors that trigger this phenomenon remain to be elucidated. To investigate the mechanism for the disappearance of the tail, changes in morphology, DNA status and the enzymes which metabolize reactive oxygen species were determined in the tail of Rana rugosa tadpoles. Histological examination revealed that apoptotic cell death was apparent in the tail myocytes during metamorphosis. Electrophoretic analysis of the tail DNA revealed a marked fragmentation. During the apoptotic changes, the activity of Cu/Zn-type superoxide dismutase (SOD) in the tail markedly increased with a concomitant decrease in its catalase activity. The apoptotic process was markedly enhanced by adding hydrogen peroxide (H2O2) and aminotriazole, a catalase inhibitor, to the medium. These observations suggested that apoptotic cell death in the tadpole tail might be triggered, at least in part, by a mechanism depending on active oxygen species, such as H2O2.

Amitrole↗
PubMed1997

Reexamination of yeast strains classified as Torulaspora delbrueckii (Lindner).

Twenty-eight yeast strains presumed to represent Torulaspora delbrueckii were analyzed by randomly amplified polymorphic DNA-PCR analysis. Four strains (HUT 7161, IFO 1138, IFO 1145, and IFO 1956) that were considerably different from the type strain were further investigated. Morphological and physiological characteristics revealed that strains HUT 7161 and IFO 1145 belong to the genus Debaryomyces rather than the genus Torulaspora, and the former strain may represent Debaryomyces hansenii. Strains IFO 1138 and IFO 1956 were classified as either Saccharomyces castellii or Saccharomyces dairensis by identification keys involving physiological tests. On the basis of analysis of the sequences of two rRNA internal spacer regions, strains IFO 1138 and IFO 1956 were closely related to S. castellii and strains HUT 7161 and IFO 1145 were outside members of the genera Torulaspora, Zygosaccharomyces, and Saccharomyces.

Ascomycota↗
PubMed1997

Valinomycin induces apoptosis of ascites hepatoma cells (AH-130) in relation to mitochondrial membrane potential.

Valinomycin is a potassium ionophore, and is well known to cause the collapse of the mitochondrial membrane potential. It has been reported that loss of mitochondrial membrane potential is observed in the early stages of apoptosis induced by various agents. Thus, the effects of valinomycin on tumor cells were examined. Valinomycin induced uncoupling of respiration and depolarization of isolated mitochondria. Depolarization of intact mitochondria in AH-130 rat ascites hepatoma cells was also induced by valinomycin. Valinomycin induced apoptosis revealing the typical apoptotic characteristics such as fragmentation and ladder formation of DNA, shrinkage of cells, and formation of pycnotic nucleus. There was a correlation between the depolarization of mitochondria and DNA fragmentation. After depolarization of mitochondria, the activity of caspase-3-like protease but not caspase-1-like protease increased markedly. In contrast, this apoptosis did not involve the release of reactive oxygen species from mitochondria, increase in intracellular calcium concentration, or protein synthesis. In addition, anti-apoptotic members of the Bcl-2 family (Bcl-xL and Bcl-2) were not correlated with apoptosis. These results indicate that valinomycin might induce apoptosis through degradation of the mitochondrial membrane potential. Taken together, these observations suggest that there may be a mechanism that transmits the signal from mitochondrial depolarization to subsequent apoptosis execution steps.

Animals↗
PubMed1997

Molecular mechanisms of apoptosis in HL-60 cells induced by a nitric oxide-releasing compound.

Nitric oxide (NO) generated from 1-hydroxy-2-oxo-3, 3-bis(2-aminoethyl)-1-triazene (NOC 18), an NO-releasing compound, induced monocytic differentiation of human promyelocytic leukemia HL-60 cells as assessed by expression of nonspecific esterases and morphologic maturation. Simultaneously, DNA fragmentation and morphological alterations typical of apoptosis were also induced. To investigate the mechanisms of apoptosis during differentiation of HL-60 cells induced by NO, the endogenous levels of Bcl-2 and Bax were assessed by immunoblotting. Treatment of cells with NOC 18 slightly reduced the level of Bcl-2 followed by Bax. These changes might be involved in the induction of apoptosis. The involvement of the activation of the interleukin-1 beta converting enzyme (ICE) family of proteases (caspases), such as ICE and CPP32, in the pathways was also investigated. CPP32, but not ICE, was strongly activated in response to NOC 18 stimulation, thereby implicating CPP32-like activity in the induction of apoptosis. Moreover, the possible involvement of tyrosine phosphorylation in apoptosis was investigated. Pretreatment of cells with herbimycin A, an inhibitor of tyrosine kinases, suppressed DNA fragmentation and CPP32-like activity, whereas pretreatment with vanadate, an inhibitor of tyrosine phosphatases, enhanced both parameters, suggesting that tyrosine phosphorylation might be involved in the pathways of apoptosis in HL-60 cells induced by NO.

Apoptosis↗
PubMed1997

Oxygen-dependent fragmentation of cellular DNA by nitric oxide.

Although active oxygen species and related metabolites, such as nitric oxide (NO), have been postulated to play important roles in the apoptosis of various cells, a precise mechanism leading to cell death remains to be elucidated. Recently we found that the lifetime of NO depends greatly on the concentration of environmental oxygen and that NO reversibly inhibits mitochondrial respiration and ATP synthesis; the inhibitory effect is stronger at physiologically low oxygen tension than under atmospheric conditions (Arch. Biochem. Biophys. 323, 27-32, 1995). The present work describes the effects of the NO-generating agent, 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene (NOC 18) and oxygen tension on the respiration, ATP synthesis and apoptosis of HL-60 cells. When respiration was inhibited by NOC 18, cellular ATP levels decreased significantly and DNA fragmentation was elicited. Both events were enhanced by decreasing oxygen tension and suppressed by adding NO-trapping agents, such as 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) and oxyhemoglobin. The fragmentation of cellular DNA was inhibited in a dose dependent manner by herbimycin A, a tyrosine kinase inhibitor. Fragmentation of the DNA of HL-60 cells was also induced either by peroxynitrite, superoxide or hydroxyl radical by some mechanism which was diminished by lowering the oxygen tension. These results indicated that the decrease in cellular ATP and activation of tyrosine kinase might play important roles in NO-induced apoptosis particularly under physiologically low oxygen tensions.

Adenosine Triphosphate↗
PubMed1996

A research to develop a predicting system of mammalian subacute toxicity. II. Single dose detailed toxicity studies.

"Single dose detailed toxicity study" in rats was conducted with 6 chemical substances to establish a predicting system of subacute toxicity of non-congeneric industrial chemicals. In the detailed examinations, new and previously reported biological parameters were developed and utilized for the establishment and confirmation of the correlations for both qualitative and quantitative prediction of target organs and effects in repeated dose toxicity studies of non-congeners; the new parameters were concerned with liver, kidneys and blood and the established relating equations for prediction had a correlation coefficient of 0.83 (liver), 0.49 (kidneys) and 0.94 (blood), while the old parameters effectively confirmed the previous correlations. The serial changes in the study gave a biological explanation to the established relationships between acute and subacute toxicities in terms of extrapolation.

Animals↗
PubMed1996

A research to develop a predicting system of mammalian subacute toxicity. I. Prediction of subacute toxicity using the biological parameters of acute toxicities.

Predicting equations of subacute toxicity were developed by analyzing rat acute and subacute toxicity data of 56 chemicals of various structures. Minimum or 10% effect level in acute or subacute toxicity was estimated as a "biological parameter". Good regression equations were established between the geometrical means ("combined parameters") of any two of the parameters of acute and subacute toxicities and introduction of log P to the equations improved the correlations with a statistically significant multiple regression coefficient. The lowest predicted effect level of the subacute toxicity, which is selected from the data calculated by the above several correlations, can predict the upper limit of the no observed effect level. In recent years, for research and development of new chemical substances, it becomes one of the important factors that these have a lower environmental load in nature. Eventually, it becomes essential to evaluate not only their acute effects on human or environments, but also their chronic influences when they are to be exposed for a long period of time, and the cost for such verification is becoming a breaking factor for research and development. Thus, the development of a new technique which estimates the environmental load of a chemical substance including toxic effects on human with lower cost is now being attempted. For example, the development of in vitro new alternative methods using cultured cells, the utilization of a data base or software which relates mammalian and environmental toxicities and so on are internationally carried forwards. As for the latter, quantitative structure-activity relationship (QSAR) techniques have been applied practically to decide appropriate toxicity tests needed for regulatory purposes by EPA/TSCA, ITC/TSCA and FDA/FDAA in the USA. In addition, it was concluded recently in a joint meeting of EU and US-EPA that the approach by QSAR techniques was useful to specify the new chemical substances which are to be required toxicological examinations. In these QSAR techniques, however, while there are some considerations about common mechanisms of toxicity among chemicals with a similar structure (Congeneric chemicals, Congeners) for establishing of correlation formulas, for chemicals with various structures (Non-congeneric chemicals, Non-congeners) there often lack such common considerations. In addition, biological or physiological factors which are basic toxic indices are often ignored. In a previous study, we researched acute and subacute oral toxicities of industrial common chemicals in rats and reported the followings; 1) their subacute toxicological spectrum in target organs/tissues and morphologic changes was very limited and specific, 2) the important targets were liver, kidneys, blood (spleen) and stomach and these are considered the sites of dominant exposure due to the kinetics of chemical substances, 3) the morphological changes were hepatocellular hypertrophy, deposition of substance in renal tubules, extramedullary hematopoiesis in spleen and mucous lesion in stomach, and these are implying adaptation such as induction of drug-metabolizing enzymes, overload to renal function, anemia from erythrocyte destruction, and direct reaction, respectively, 4) there seems to occur a series of direct and adaptive reactions to exclude the "foreign compounds" which do not show any specific biological activities, 5) it is also considered that there is a possibility to establish a correlation between toxicological findings or target organs/tissues of both acute and subacute toxicities by their continuity. Therefore, in the present study, a predicting equation of subacute (28-day repeated dose) toxicity is attempted to develop from acute (single dose) toxicity data by considering both common mechanisms and biological factors for non-congeneric industrial chemical substances.

Animals↗
PubMed1996

Research to develop a predicting system of mammalian subacute toxicity, (3). Construction of a predictive toxicokinetics model.

A new predictive toxicokinetics model was developed to estimate subacute toxicity (target organs, severity, etc.) of non-congeneric industrial chemicals, where the chemical structures and physico-chemical properties are only available. Thus, a physiological pharmacokinetics model, which consists of blood, liver, kidney (these were experimentally found as major toxicological targets), muscle and fat compartments, was established to simulate the chemical concentrations in organs/tissues with pharmacokinetic parameters by means of Runge-Kutta-Gill algorithm. The pharmacokinetic parameters, i.e. absorption rate, absorption ratio, hepatic extraction ratio of metabolism and renal clearance were calculated by using separately established Quantitative Structure-Pharmacokinetics Relationship equations. The developed predictive model was then applied to simulations of 43 non-congeneric industrial chemicals. The chemical concentrations in organs/tissues after single oral administration were simulated, and their maximum concentrations (Cmax's) and area under the concentration-time curves (AUC's) were calculated. Fast Inverse Laplace Transform was newly applied for the purpose of simulation of 28-day repeated dose toxicity. Simulated concentrations of 28 days repeated dose were, however, found to be the same as those of simple repetitions of a single administration per day because of the short half-lives of non-congeneric industrial chemicals. A comparison of subacute toxicity data with Cmax's and AUC's in a single dose scenario suggested that the organs/tissues with relatively high concentrations of tested chemical substances were the most sensitive targets within a chemical. Chemical concentrations in liver, for instance, were correlated with the severity of hepatotoxicity among the chemicals. It was also suggested that to improve and widen the present approach, data of metabolite and reactivity of non-congeneric industrial chemicals to organs/tissues, receptors, etc. should be incorporated into the model.

Adipose Tissue↗
PubMed1996

Oxygen-dependent-regulation of Ehrlich ascites tumor cell respiration by nitric oxide.

Effects of nitric oxide (NO) on oxygen uptake of Ehrlich ascites tumor cells (EATC) were examined in a study of the biological actions of NO on respiration and energy metabolism at the cellular level. Endogenous respiration of EATC was inhibited reversibly by NO in a dose dependent manner. Oxyhemoglobin, an NO trapping agent, restored the respiration promptly. The inhibitory action of NO also depended on oxygen-concentration, and the duration of suppression was prolonged remarkably at low oxygen tension. Similar inhibition was also observed in the presence of glucose. In this case, both lactate production and glucose consumption were promoted by NOC 18, an NO generating agent, and the activation was enhanced by lowering the oxygen-concentration. Furthermore, the membrane potential of EATC was depolarized transiently by adding NO, and the degree of depolarization was decreased in the presence of glucose. These results suggest that at physiologically low oxygen tension in ascites fluid, NO acts not only as a cytotoxic respiratory inhibitor but also as a regulatory factor in the energy metabolism of EATC.

Animals↗