[Show abstract][Hide abstract] ABSTRACT: We describe the identification and characterization of a GTP-binding protein with a molecular weight of 45 kD (Gbp45). Gbp45 cDNA was found to overlap with a hypothetical human protein, PTD004, the sequence of which was previously deposited in the databases. The gene for PTD004 was recently found to be one of the ATPases, hOLA1 (human Obg-like ATPase 1). The Gbp45 gene encodes a protein of 396 amino acid residues. Immunocytochemical analysis and examination with GFP-tagged protein revealed that Gbp45 is primarily located in the cytosolic compartment. Immunoblot analysis showed that the Gbp45 protein is strongly expressed in the neuronal tissues and pancreas. T43N and T56N mutations resulted in a loss of Gbp45's ability to bind to GTP and a loss of GTPase activity. In cultured cells, the transfection of wild-type Gbp45 accelerated cell proliferation, though T43N and T56N mutations induced cell death. Down-regulating Gbp45 expression decreased the cell proliferation rate and increased the rate of cell death induced by the inhibition of mitochondrial electron transport. These findings indicate that Gbp45 plays important roles in cell proliferation and death related to mitochondrial function.
[Show abstract][Hide abstract] ABSTRACT: The mechanism of hepatocarcinogenesis remains unclear in patients in whom hepatitis C virus (HCV) disappears after interferon (IFN) therapy. We compared molecular alterations in hepatocellular carcinoma (HCC) between patients with a sustained virological response (SVR) to IFN and patients with HCV.
The study group comprised 44 patients with HCV and 13 patients with SVR. One patient in the SVR group had two tumour nodules, both of which were examined. Mitochondrial DNA (mtDNA) mutations in displacement-loop lesions were directly sequenced. Mutation of the TP53 gene was examined by direct sequencing. The methylation status of p16, p15, p14, RB and PTEN genes was evaluated by a methylation-specific polymerase chain reaction.
The average number of mtDNA mutations was 4.2 in 44 HCCs with HCV and 2.0 in 14 HCCs with SVR (P=0.0021). mtDNA mutation was less frequently detected in HCCs from patients with SVR than in patients with HCV. TP53 mutations were detected in 12 (27%) of 44 HCCs with HCV and 2 (14%) of 14 SVR-HCCs. Hypermethylation of the p16, p15, p14, RB and PTEN promoters was, respectively, detected in 34, 13, 8, 12 and 11 of 44 HCCs from patients with HCV and 14, 0, 0, 2 and 2 of 14 HCCs from patients with SVR (P=0.049, 0.021, 0.085, 0.322 and 0.402). Hypermethylation of p16 was one of the most important alterations in SVR-HCC.
Molecular alterations in hepatocarcinogenesis of patients with SVR-HCC were different from those of patients with continuous HCV infection.
No preview · Article · Jun 2008 · Liver international: official journal of the International Association for the Study of the Liver
[Show abstract][Hide abstract] ABSTRACT: Production of reactive oxygen species (ROS) and defense systems against them are balanced well in the living body. This balance is very important for the maintenance of physiological condition. The collapse of the balance such as in inflammation causes ROS toxicity and induces oxidative injury in various tissues. In this report, the relation between oxidative stress and tissue injury is discussed.
No preview · Article · Apr 2008 · Masui. The Japanese journal of anesthesiology
[Show abstract][Hide abstract] ABSTRACT: To elucidate the roles of enteric bacteria and immunological interactions among liver, spleen and intestine in the pathogenesis of liver injury during obstructive jaundice, we studied the effects of antibiotics and splenectomy on bile-duct-ligated C57BL mice. When animals were subjected to bile-duct-ligation (BDL), plasma levels of bilirubin, alanine aminotransferase and aspartate aminotransferase increased markedly. However, the increases in plasma transaminases were significantly lower in splenectomized or antibiotics-treated groups than in the control BDL group. Histological examination revealed that liver injury was also low in the two groups. BDL markedly increased plasma level of interferon-gamma (IFN-gamma) and the expression of inducible nitric oxide synthase (iNOS) in liver and spleen. These changes were suppressed either by splenectomy or administration of antibiotics. Kinetic analysis revealed that BDL-induced liver injury and the increase of interleukin-10 (IL-10) and INF-gamma were lower in iNOS(-/-) than in wild type animals. BDL markedly increased the expression of IgA in colonic mucosa. These observations suggest that enteric bacteria, nitric oxide and cytokines including IFN-gamma and IL-10 derived from spleen and intestines form a critical network that determines the extent of liver injury during obstructive jaundice.
Full-text · Article · Jun 2007 · Journal of Clinical Biochemistry and Nutrition
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Dec 2006 · Hepatology Research
[Show abstract][Hide abstract] ABSTRACT: To test the possible involvement of reactive oxygen species (ROS) in the etiology of diabetic complications and therapeutic potential of antioxidant biofactors, we studied the effects of Curcuma aromatica (C. aromatica) on the pathologic events in streptozotocin-treated diabetic rats. Administration of streptozotocin (100 mg/kg, i.p.) increased plasma levels of glucose, triglyceride, cholesterol, urea nitrogen (BUN), creatinine, and lipid peroxidation products but decreased plasma albumin levels and suppressed the growth of animals. Histological examination revealed a marked injury in renal glomeruli and proximal tubules with concomitant occurrence of 8-hydroxy-2′-deoxyguanosine (8-OHdG) and mitochondrial 4-hydroxy-2-nonenal (4-HNE). Urinary excretion of 8-OHdG was also increased in streptozotocin-treated animals. Administration of streptozotocin decreased the mitochondrial localization of both Cu/Zn-superoxide dismutase (SOD) and cytochrome C in the kidney without affecting the localization of Mn-SOD. When animals were given 1.5%C. aromatica-containing diet for 1 wk before and 8 wk after administration of streptozotocin, all the events induced by streptozotocin except for hyperglycemia decreased markedly. Thus, C. aromatica may have therapeutic potential for the prevention of hyperglycemia-associated diabetic complications.
No preview · Article · Nov 2006 · Journal of Food Science
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Oct 2006 · Hepatology Research
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Feb 2006 · Free Radical Biology and Medicine
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Jan 2006 · AJP Cell Physiology
[Show abstract][Hide abstract] ABSTRACT: We recently reported that oxidative stress elicited by chronic inflammation increases the mutation of mitochondrial DNA (mtDNA) and possibly correlates with precancerous status. Since severe oxidative stress is elicited in the colorectal mucosa of individuals with ulcerative colitis (UC), the possible occurrence of an mtDNA mutation in the inflammatory colorectal mucosa and colitic cancer was investigated. Colorectal mucosal specimens were obtained from individuals with UC with and without colitic cancer and from control subjects. The frequency of mtDNA mutations was higher in colorectal mucosal specimens from patients with UC than that from control subjects. The levels of 8-hydroxy-2'-deoxyguanosine, a DNA adduct by reactive oxygen species, were significantly higher in UC than in control. Specimens from patients with colitic cancer contained a significantly higher number of mtDNA mutations. The present observations suggest that the injury followed by the regeneration of colorectal mucosal cells associated with chronic inflammation causes accumulation of mtDNA mutations. The increased instability of genes, including those on the mtDNA, is consistent with the high and multicentric incidence of colorectal cancer in individuals with UC. Thus, analysis of mtDNA could provide a new criterion for the therapeutic evaluation, and may be useful for the prediction of risk of carcinogenesis.
Preview · Article · Sep 2005 · British Journal of Cancer
[Show abstract][Hide abstract] ABSTRACT: We recently reported that the genetic instability resulting in the high rate of mitochondrial DNA (mtDNA) mutation in noncancerous liver tissue is consistent with the multicentric hepatocarcinogenesis detected clinically. Interferon (IFN) has been reported to reduce hepatocarcinogenesis in individuals with hepatitis virus infection. Liver biopsy specimens were obtained from 26 patients with chronic hepatitis C virus (HCV) infection before and after IFN therapy (total dose: 252 million units). The mean (+/-SD) age of the study population was 45 +/- 9 years and 13 (50%) were male [mode of acquisition: blood transfusion (27%), unknown (73%); viral load: 5.2 +/- 1.1 k copies/mL; duration of infection: 17 +/- 9 years (65%), unknown (35%); genotype: I (4%), II (80%), III (8%), IV (8%); alcohol intake: positive (31%), negative (69%)]. DNA samples were extracted from the specimens and subjected to direct sequencing. The mtDNA mutation frequency in the D-loop was increased in liver specimens from individuals with HCV infection compared with 21 controls (2.5 vs 0.6, P < 0.001). IFN therapy decreased the mtDNA mutation (mean difference = 0.7, P < 0.001) and the decreased number of mtDNA mutations was positively correlated with suppression of the total histological activity index score (mean difference = 1.3, P < 0.01). These results clearly indicate that the mutational rate of mtDNA is strongly associated with IFN therapy. Thus, analysis of mtDNA could provide a new criterion for the therapeutic evaluation of the effect of IFN, and may be useful for the prediction of risk of carcinogenesis.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Preview · Article · Feb 2005 · International Journal of Cancer
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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).
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.
Full-text · Article · Dec 2004 · Journal of Gastroenterology
[Show abstract][Hide abstract] ABSTRACT: 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.