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Masayasu Inoue

Publications and source records attributed to Masayasu Inoue.

At least 19 recordsLinked to original sources
PubMed2006

Identification and characterization of endoplasmic reticulum-associated protein, ERp43.

Disposal of misfolded proteins from the lumen of the endoplasmic reticulum (ER) is one of the quality control mechanisms present in the protein secretory pathway. Through ER-associated degradation, misfolded substrates are targeted to the cytosol where they are degraded by proteasomes. Here we describe the identification of a human ER-associated 43-kD protein (ERp43) by sequencing of the subtraction suppression hybridization cDNA library from ER stress-treated cells. The ERp43 gene encodes a protein of 383 amino acid residues that contains a potential transmembrane domain. Analysis revealed that ERp43 is primarily located in the ER. Quantitative reverse transcriptase-polymerase chain reaction demonstrated that gene expression was relatively high in the neuronal tissues and in the kidney, with ERp43 protein highly expressed in the spinal cord and in the kidney. In cultured cells, overexpression of ERp43 accelerated cell growth and inhibited ER stress-induced cell death, while down-regulation of ERp43 expression decreased proliferation rate and enhanced this type of cell death. These findings indicate that ERp43 plays important roles in cell growth and ER stress-induced cell death.

Amino Acid Sequence↗
PubMed2006

Lack of mitochondrial DNA enhances growth of hepatocellular carcinoma in vitro and in vivo.

To elucidate the role of mitochondrial DNA (mtDNA) in determination of growth of hepatocellular carcinoma, we examined wild-type Hepa1-6 cells and their rho(0) cells with depleted mtDNA in vitro and in vivo. Cultured rho(0) cells grew more rapidly than did wild-type cells. Production of reactive oxygen species (ROS) was higher in wild-type cells than in rho(0) cells. Hypoxia inhibited the growth of wild-type cells more markedly than that of rho(0) cells. Resistance to mitochondrial respiratory inhibitor-induced cell death was stronger in rho(0) cells than in wild-type cells. rho(0) cells subcutaneously inoculated in the hind thigh of mice grew more rapidly and formed larger solid tumors. These findings indicate that lack of mtDNA increases growth of hepatocellular carcinoma by decreasing ROS production and increasing resistance to mitochondrial respiratory inhibition.

Journal Article↗
PubMed2006

Mannose-conjugated alendronate selectively depletes Kupffer cells and inhibits endotoxemic shock in the mice.

To elucidate the roles of Kupffer cells in the host-defense mechanisms and liver injury, we synthesized a mannose-conjugated alendronate (MANA) and examined its effects on Kupffer cells and lipopolysaccharide (LPS)-induced liver injury in the mice. Intravenous administration of a small amount of MANA (50mumol/kg) rapidly and selectively depleted Kupffer cells in the mice. The depletion of Kupffer cells by MANA resulted in a marked decrease of the production of both TNF-alpha and IL-1beta in the plasma during the liver injury induced by low (1mg/kg) and lethal (75mg/kg) doses of LPS. The effect was so remarkable that all animals treated with a lethal dose of LPS survived without any sign of endotoxemia. These findings indicate that Kupffer cells play critical roles in the development of endotoxemia, and that MANA will be useful in studies to elucidate pathophysiological roles of Kupffer cells in various liver diseases.

Journal Article↗
PubMed2006

L-carnitine suppresses the onset of neuromuscular degeneration and increases the life span of mice with familial amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal disease caused by progressive degeneration of motor neurons in the spinal cord and motor cortex. Although the etiology of ALS remains unknown, a mutation of the gene encoding Cu,Zn-superoxide dismutase (SOD1) has been reported in 20% of familial cases of ALS (FALS). Transgenic mice that overexpress a mutated human SOD1 exhibit a phenotype and pathology similar to those observed in patients with FALS. Mitochondrial abnormality has been reported in patients with ALS and in animal models of FALS. We recently reported that L-carnitine, an essential cofactor for the beta-oxidation of long-chain fatty acids, effectively inhibits various types of mitochondrial injury and apoptosis both in vitro and in vivo. The present study demonstrates that oral administration of L-carnitine prior to disease onset significantly delayed the onset of signs of disease (log-rank P=0.0008), delayed deterioration of motor activity, and extended life span (log-rank P=0.0001) in transgenic mice carrying a human SOD1 gene with a G93A mutation (Tg). More importantly, subcutaneous injection of L-carnitine increased the life span of Tg mice (46% increase in male, 60% increase in female) even when given after the appearance of signs of disease.

Adenine↗
PubMed2006

Production of reactive oxygen species in peripheral blood is increased in individuals with Helicobacter pylori infection and decreased after its eradication.

BACKGROUND: Helicobacter pylori infection has been reported to cause gastroduodenal ulcer, mucosa-associated lymphoid tissue lymphoma, and gastric cancer. Recent studies have suggested that H. pylori infection may also associate with other diseases, including hematologic and dermatologic disorders, and cardiovascular injury, by unknown mechanisms. METHODS: Production of reactive oxygen species (ROS) was determined in peripheral blood samples from 86 patients (34 H. pylori-negative and 52 H. pylori-positive subjects) using a highly sensitive chemiluminescence probe, L-012 (8-amino-5-chloro-7-phenylpyrido(3,4-d) pyridazine-1 and 4 (2H, 3H) dione). Eleven H. pylori-positive individuals were also analyzed their ROS production in peripheral blood after H. pylori eradication. RESULTS: ROS production was significantly higher in individuals with H. pylori infection than in those without infection. Enhanced production of ROS was decreased significantly after eradication of H. pylori. No correlation was found between the extent of ROS production and sex, age, smoking status, alcohol ingestion, use of medications, or serum level of C-reactive protein. CONCLUSION: These findings suggest that ROS production was enhanced in peripheral blood by H. pylori infection. Chemiluminescence analysis of blood samples using L-012 permits evaluation of systemic oxidative stress in patients with H. pylori infection.

Adult↗
PubMed2005

A possible cooperation of SOD1 and cytochrome c in mitochondria-dependent apoptosis.

A small amount of reactive oxygen species (ROS) is generated through aerobic respiration even under physiological conditions. Because ROS are known to have various deteriorating actions, the way cells could evade the effects of ROS in and around mitochondria would determine the fate of cells. We previously reported that Cu,Zn-superoxide dismutase (SOD1), a cytosolic enzyme, is also localized in mitochondria in various types of cells. Therefore, we undertook this study to elucidate the physiological significance of SOD1 localization in and around mitochondria. We analyzed the effects of various reagents that could modulate mitochondrial respiration, ROS metabolism, and subcellular localization of SOD1 and cytochrome c. Using rat liver mitochondria, we have shown that Ca2+, Fe2+, or long-chain fatty acids increased the mitochondrial generation of ROS and that the resulting ROS oxidized the critical thiol groups in adenine nucleotide translocase (ANT). The oxidation of ANT induced mitochondrial swelling followed by the release of SOD1 and cytochrome c. Although inhibitors of electron transport, such as rotenone, antimycin A, and KCN, also increased ROS generation, they failed to (i) oxidize the critical thiol groups in ANT, (ii) induce swelling, and (iii) release SOD1 and cytochrome c. These results suggest that the oxidation of ANT thiols and the opening of the membrane permeability transition pores induce the release of both SOD1 and cytochrome c. We demonstrated that the loss of SOD1 increases the susceptibility of mitochondria to oxidative stresses and that the simultaneous release of SOD1 enhances the vicious cycle of apoptotic reactions triggered by the released cytochrome c. Therefore, SOD1 must have important roles in protecting mitochondria from ROS-induced injury. Our data also suggest that SOD1 release parallels cytochrome c release under all conditions. We propose that intramembranously localized SOD1 is a third reagent (along with AIF) that will regulate apoptosis.

Animals↗
PubMed2005

Mitochondrial density determines the cellular sensitivity to cisplatin-induced cell death.

We studied the relationship between the mitochondrial density in the cells and the cellular sensitivity to the toxicity of cis-diaminedichloroplatinum II (cisplatin), a potent anticancer agent. Biochemical analyses revealed that the density of mitochondria in the intestinal epithelium changed markedly along its entire length. The density was the highest at the duodenum, medium at the jejunum, and the lowest at the ileum. The sensitivity of epithelial cells to cisplatin toxicity was the highest at the duodenum, medium at the jejunum, and the lowest at the ileum as judged from the occurrence of apoptosis. Similar correlation between the cisplatin sensitivity and mitochondrial density was also observed with in vitro experiments, in which intestinal epithelial cells (IEC-6) and their rho0 cells with reduced number of mitochondria were used. The rho0 cells had a strong resistance to cisplatin compared with the control cells. Cisplatin markedly increased mitochondrial generation of reactive oxygen species in IEC-6 but not in rho0 cells. We analyzed the sensitivity of eight cell lines with different density of mitochondria to cisplatin and found the same positive correlation. These observations clearly show that cellular density of mitochondria is the key factor for the determination of the anticancer activity and side effects of cisplatin.

Animals↗
PubMed2005

L-carnitine inhibits hypoglycemia-induced brain damage in the rat.

Hypoglycemia sometimes occurs in patients with diabetes mellitus who receive excessive doses of insulin. Severe hypoglycemia has been known to induce mitochondrial swelling followed by neuronal death in the brain. Since L-carnitine effectively preserves mitochondrial function in various cells both in vitro and in vivo, we investigated its effects on the neuronal damage induced by hypoglycemic insult in male Wistar rats. Animals were given L-carnitine-containing water (0.1%) for 1 week and then received insulin (20 U/kg, i.p.) to induce hypoglycemia. Although L-carnitine did not affect the mortality of animals that developed hypoglycemic shock, it improved the cognitive function of the survived animals as assessed by the Morris water-maze test. L-carnitine effectively inhibited the increase in oxidized glutathione and mitochondrial dysfunction in the hippocampus and prevented neuronal injury. L-carnitine also inhibited the decrease in mitochondrial membrane potential and the generation of reactive oxygen species in hippocampal neuronal cells cultured in glucose-deprived medium. These results suggest that L-carnitine prevents hypoglycemia-induced neuronal damage in the hippocampus, presumably by preserving mitochondrial functions. Thus, L-carnitine may have therapeutic potential in patients with hypoglycemia induced by insulin overdose.

Aldehydes↗
PubMed2005

Enhanced oxidative stress in neutrophils from hyperlipidemic guinea pig.

Inhibitors of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase are antilipidemic agents (statins) widely used for the prevention of cardiovascular diseases. Recent studies have suggested that the overall benefits of statin therapy cannot be accounted for solely by its antilipidemic effect. To obtain further insight into the mechanism of action of statins, we studied the effect of pitavastatin on the generation of reactive oxygen species (ROS) by peritoneal polymorphonuclear leukocytes (PMN) obtained from control and hyperlipidemic guinea pigs. Flow cytometric analysis revealed that the amount of ROS generated by PMN from the hyperlipidemic animals that had been administered a laurate-containing diet (LD) for 4 weeks was larger than that from the normal diet (ND) group (837% increase, ND; 82.17 arbitrary units, LD; 688.10 arbitrary units, P < 0.01, n = 6). Administration of pitavastatin to the LD group significantly decreased plasma levels of total cholesterol (TC) and low-density lipoprotein (LDL) with a reduction in ROS generation by PMN (19% decrease, LD control; 688.10 arbitrary units, LD + pitavastatin; 556.87 arbitrary units, P < 0.01, n = 6). Western blotting analysis revealed that the expression of protein kinase C alpha (PKC alpha) and betaI was higher in PMN from the LD group than in PMN from the ND group (PKC alpha; 74% increase, PKC betaI; 339% increase, P < 0.05, n = 4, respectively). Furthermore, expression of NADPH oxidase gp91phox in PMN from the LD group was higher than that in PMN from the ND group (18% increase, P < 0.05, n = 4). By administration of pitavastatin to the LD group, the expression of PKC alpha, betaI and gp91phox was suppressed compared with the control LD group (PKC alpha; 41% decrease, PKC beta; 28% decrease, gp91phox; 56% decrease, P < 0.05, n = 4, respectively). These results indicate that PMN from hyperlipidemic animals is associated with an accelerated respiratory burst of ROS by increasing the expression of PKC alpha, betaI and gp91phox, and pitavastatin inhibits this by suppressing the expression of those proteins.

Animals↗
PubMed2005

L-carnitine inhibits hepatocarcinogenesis via protection of mitochondria.

Hepatocellular carcinoma is usually preceded by chronic inflammation. However, the molecular mechanism in hepatocarcinogenesis is not well known. Recently, we reported that mitochondrial dysfunction plays an important role in hepatocarcinogenesis via the production of free radicals. Furthermore, we proved that L-carnitine effectively protects mitochondrial function in vivo. Therefore, we investigated whether long-term administration of L-carnitine could prevent hepatitis and subsequent hepatocellular carcinoma in Long-Evans Cinnamon rats that are often analyzed as a model of hepatocarcinogenesis. The results indicated that oxidative stress elicited from abnormally accumulated copper increased the amount of free fatty acids, thereby inducing mitochondrial dysfunction, resulting in cell death and enhanced secondary generation of reactive oxygen species, which were significantly inhibited by carnitine treatment. Finally, the occurrence of placental glutathione S-transferase-positive foci as a marker for preneoplastic lesions and hepatocarcinogenesis were significantly inhibited by L-carnitine. These facts suggest that mitochondrial injury plays an essential role in the development of hepatocarcinogenesis and that the clinical use of carnitine has excellent therapeutic potential in individuals with chronic hepatitis.

Animals↗
PubMed2005

Male gametic cell-specific histone gH2A gene of Lilium longiflorum: genomic structure and promoter activity in the generative cell.

A genomic clone containing the gH2A gene, a histone variant specifically expressed in male gametic cells within the pollen of Lilium longiflorum, was isolated. Sequence analysis revealed that the coding region of the gene is interrupted by one intron, as is the case with the somatic type of plant histone H2A genes, suggesting derivation from the same ancestral gene containing one intron. In addition, a 2.8-kbp fragment of the 5' upstream region of gH2A contained TATA and CAAT boxes, but neither a plant histone-specific regulatory DNA element nor vegetative cell-specific cis-elements were found. A histochemical study of stable transformants demonstrated that the 5' upstream region of the gene can drive gene expression specifically in the generative cell of pollen; no activity was detectable in the vegetative cell or in other reproductive and vegetative tissues of transgenic Nicotiana tabacum. These results strongly suggest that the generative cell can direct specific gene expression, that this expression may be regulated by a putative male gametic factor, and that the gH2A promoter may therefore serve as a useful male gametic cell fate marker in angiosperms.

Amino Acid Sequence↗
PubMed2004

Effect of endocrine disruptor para-nonylphenol on the cell growth and oxygen radical generation in Escherichia coli mutant cells deficient in catalase and superoxide dismutase.

para-Nonylphenol (NP) had previously been found to have strong suppressive effects of growth of bacterial and yeast cells, and these effects were associated with NP-induced generation of radical oxygen species (ROS). In the present study, we determined that wild-type strains of Escherichia coli (CSH 7, SY-11, and IFO-3545) were resistant to NP compared with other sensitive microorganisms reported previously. To elucidate the relationship between NP-induced ROS generation and cell growth inhibition in more detail, we analyzed the effect of NP on cell growth and survival of wild-type and mutant E. coli strains deficient in ROS-scavenging enzymes such as catalase and superoxide dismutase (SOD). The SOD-deficient strain QC 774 (sod A- and sod B-) was much more sensitive to NP than wild-type (CSH 7) and catalase-deficient (UM 1 kat E- and kat G-) strains. As a comparative experiment, when hydrogen peroxide was applied to the same growth and survival assays, UM 1 cells were more sensitive to hydrogen peroxide than QC 774 and CSH 7. A chemiluminescence (CHL) experiment using MCLA (2-methyl-6-Lf-methylphenyl]-3,7-dihydroimidazc [1,2-alpha] pyrazin-3-one) reflecting predominantly superoxide generation showed that NP caused marked CHL generation in QC 774 cells, but not in CSH 7 and UM 1 cells. However, the CHL experiment using L-012 reflecting predominantly hydroxyl radical and hypochlorite did not exhibit significant CHL generation in QC 774 cells at the same concentrations of NP. Furthermore, supplementation with SOD prevented NP-induced ROS generation and cell survival inhibition of QC 774 cells, but the catalase and metal-chelating agent deferoxamine did not have significant effects. These results suggest that one of the primary actions of NP in cells is the generation of superoxide which may be responsible for NP-induced cell growth inhibition.

Catalase↗
PubMed2004

Inducible NO synthase inhibits the growth of free tumor cells, but enhances the growth of solid tumors.

To elucidate the role of nitric oxide (NO) in tumor cell growth in vivo, dynamic aspects of the growth of Ehrlich ascites tumor cells (EATCs) were studied in wild-type (WT) mice and in an inducible strain of NO synthase (iNOS)-deficient (iNOS(-/-)) mice. Kinetic analysis showed that the rate of free tumor cell growth in the peritoneal cavity was significantly higher in the iNOS(-/-) mice than in the WT mice. In contrast, EATCs inoculated subcutaneously rapidly grew and formed a solid tumor in WT mice, but failed to grow in iNOS(-/-) mice. These results clearly indicate that NO generated by iNOS predominantly inhibits the growth of tumor cells in their free form, but enhances the growth of solid tumors.

Animals↗
PubMed2004

Oxygen tension regulates reactive oxygen generation and mutation of Helicobacter pylori.

Although both bacillary and coccoid forms of Helicobacter pylori reside in human stomach, the pathophysiological significance of the two forms remains obscure. The present work describes the effect of oxygen tension on the transformation and reactive oxygen species (ROS) metabolism of this pathogen. Most H. pylori cultured under an optimum O2 concentration (7%) were the bacillary form, whereas about 80% of cells cultured under aerobic or anaerobic conditions were the coccoid form. The colony-forming unit of H. pylori decreased significantly under both aerobic and anaerobic culture conditions. The bacillary form of H. pylori generated predominantly superoxide radical, whereas the coccoid form generated preferentially hydroxyl radical. Specific activities of cellular respiration, urease, and superoxide dismatase decreased markedly after transformation of the bacillary form to the coccoid form, with concomitant generation of protein carbonyls and 8-hydroxyguanine. The frequency of mutation of cells increased significantly during culture under nonoptimum O2 conditions. These results indicate that ROS generated by H. pylori catalyze the oxidative modification of cellular DNA, thereby enhancing the transformation from the bacillary to the coccoid form. The enhanced generation of mutagenic hydroxyl radicals in the coccoid form might accelerate mutation and increase the genetic diversity of H. pylori.

Aerobiosis↗
PubMed2004

Correlation between clinical characteristics and mitochondrial D-loop DNA mutations in hepatocellular carcinoma.

BACKGROUND: Mitochondrial DNA (mtDNA) mutations are found in many kinds of human cancer. The aim of this study was to evaluate the relationship between mtDNA mutations in the liver and human hepatocarcinogenesis. METHODS: Direct sequencing of mtDNA was done in 54 hepatocellular carcinomas (HCCs) and 47 surrounding liver tissue samples, obtained from 54 patients with HCC, and in 5 liver samples without inflammation, obtained from 5 patients with metastatic liver tumors. We also examined p53 mutations in the 54 HCCs to examine the correlation between nuclear DNA mutations and mtDNA mutations. RESULTS: Mutations of mtDNA in the D-loop region were found in both HCC and noncancerous liver tissue. In normal liver without chronic inflammation, no mtDNA mutation was detected. In every case, the number of mtDNA mutations in HCC correlated with that in noncancerous liver tissue. Twelve of 52 mutation sites in the D-loop region of mtDNA were specific for HCC. The mean number of mtDNA mutations was 1.7 in well-differentiated HCC, as compared with 4.5 in moderately differentiated HCC and 4.6 in poorly differentiated HCC. The frequency of mtDNA mutations was thus higher in less differentiated HCC. We detected p53 mutations in 15 (28%) of 54 HCCs. The mean number of mtDNA mutations was 5.3 in HCC with p53 mutations and 3.8 in HCC with wild-type p53 (P = 0.024). CONCLUSIONS: A higher frequency of mtDNA mutations was found in less differentiated HCCs, and it is also possible that mtDNA mutations are related to nuclear DNA mutations in HCC. The accumulation of mtDNA mutations is a useful predictor of hepatocarcinogenesis.

Carcinoma, Hepatocellular↗
PubMed2004

Oxidative stress underlies the mechanism for Ca(2+)-induced permeability transition of mitochondria.

Recent studies demonstrated that the generation of intracellular reactive oxygen species (ROS) was enhanced prior to the onset of mitochondrial membrane permeability transition (MPT), a critical step for the induction of DNA fragmentation and apoptosis. Although Ca2+ induces typical MPT that involves depolarization and swelling of mitochondria and finally releases cytochrome c into cytosol, the mechanism by which ROS induce MPT remains unclear. In the presence of inorganic phosphate, Ca2+ increased the oxygen consumption and ROS production by isolated mitochondria as determined by a chemiluminescence (CHL) method using L-012. Ca2+ increased the generation of H2O2 by some mechanism that was inhibited by cyclosporin A but not by superoxide dismutase (SOD) and trifluoperazine. Ca2+ decreased the content of free thiols in adenine nucleotide translocase (ANT) in mitochondrial membranes with concomitant increase in ROS generation. The presence of cyclosporin A, trifluoperazine, or SOD inhibited the Ca(2+)-induced increase of L-012 CHL and decrease in the free thiols of ANT. These results indicate that Ca2+ increases the generation of ROS which oxidize the free thiol groups in mitochondrial ANT, thereby inducing MPT to release cytochrome c.

Animals↗
PubMed2004

Edaravone inhibits acute renal injury and cyst formation in cisplatin-treated rat kidney.

BACKGROUND: Although cis-diamminedichloroplatinum (II) (cisplatin) is an effective anticancer agent, its clinical use is highly limited predominantly due to its adverse effects on renal functions. The present work examined the therapeutic potential of edaravone, a free radical scavenger, for inhibiting cisplatin-induced renal injury. METHODS: Edaravone, 3-methyl-1-phenyl-pyrazolin-5-one, was administrated intravenously at a dose of 30 mg/kg of body weight to male Wistar rats (200-220 g). After 30 min, cisplatin was injected intraperitoneally at a dose of 5 mg/kg of body weight. At the indicated times after the treatment, functions and histological changes of the kidney were analyzed. To test the therapeutic potential of edaravone in chemotherapy, its effect on the anticancer action of cisplatin was examined in ascites cancer-bearing rats. RESULTS: We found that cisplatin rapidly impaired the respiratory function and DNA of mitochondria in renal proximal tubules, thereby inducing apoptosis of tubular epithelial cells within a few days and chronic renal dysfunction associated with multiple cysts one-year after the administration. Administration of edaravone inhibited the cisplatin-induced acute injury of mitochondria and their DNA and renal epithelial cell apoptosis as well as the occurrence of chronic renal dysfunction and multiple cyst formation. The anticancer effect of cisplatin remained unaffected by intravenous administrating of edaravone. CONCLUSIONS: These results indicate that edaravone may have therapeutic potential for inhibiting the acute and chronic injury of the kidney induced by cisplatin.

Animals↗