Biomedical subjects

Y Inui

Publications and source records attributed to Y Inui.

At least 37 records · Page 2Linked to original sources

PubMed1996

Inhibition of cell growth of human hepatoma cell line (Hep G2) by a farnesyl protein transferase inhibitor: a preferential suppression of ras farnesylation.

So far, treatment with anti-cancer agents has failed to achieve satisfactory results in hepatocellular carcinoma. In the process of hepatocarcinogenesis, ras has been shown to play a role. ras requires a farnesyl moiety for activation. It has been found that UCFI-C (manumycin), an antibiotic, inhibits farnesyl protein transferase, an enzyme that catalyzes farnesylation. Therefore, we investigated the effects of UCFI-C on cell growth, prenylation of cellular proteins including ras and Rapl, MAP kinase activity, activities of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and synthesis of cholesterol in a ras-activated human hepatoma cell line, Hep G2. Treatment with varying concentrations of UCF1-C(10-30 microM for 24 and 72 hr resulted in a time- and dose-dependent inhibition of cell numbers. 3H-Thymidine incorporation was also inhibited in a dose-dependent manner, with 50% inhibition after 44 hr being observed at a concentration of 17 microM. UCFI-C dose-dependently inhibited ras farnesylation and MAP kinase activity, but did not decrease Rap 1++ geranylgeranylation or prenylation of 21-to 26-kDa proteins. Neither the activities of 3-hydroxy-3-methylglutaryl-coenzyme A reductase nor cholesterol synthesis were inhibited. These results suggest that UCFI-C antagonizes the growth of Hep G2 via the suppression of ras farnesylation and could be a lead for the development of new anti-cancer agents blocking the function of oncogenic ras associated with human cancer, including hepatocellular carcinoma.

Alkyl and Aryl Transferases↗

PubMed1996

Distribution and subcellular localization of a growth inhibitory factor in hamster liver and its intracellular partner(s).

The subcellular, intralobular distributions and intracellular partner(s) of a factor which inhibits the proliferation of cell growth (Hashimoto C. et al. (1994) Biochim. Biophys. Acta 1221, 107-117) were determined in hamster livers, using a combination of immunological and biochemical techniques. The IgG fraction from an antiserum raised against the growth inhibitory factor with 37 kDa was shown to be highly specific for the antigen. The nuclear and cytosolic fractions demonstrated inhibitory effects on cell growth and Western blot analysis revealed that both fractions contained the immunoreactive 37 kDa protein with the anti-inhibitory factor IgG but microsomal and mitochondrial fractions did not. The nuclear and cytoplasmic localization of the inhibitory factor were further confirmed by immunochemical staining mediated through the immune IgG and an avidin-biotinylated horseradish peroxidase complex, the parenchymal liver cells were clearly stained, but endothelial and connective tissue cells were not. Although some staining was evident throughout the liver parenchyma, the hepatocytes with most intensively stained nuclei were located in the periportal region. In the liver from hamsters 6 days old or the regenerating hamster livers 3 days after partial hepatectomy, the staining intensity was low and the number of hepatocytes with the inhibitory factor positive nuclei was very few compared with the adult hamster livers. In primary cultures of the isolated hepatocytes from adult hamster the inhibitory factor disappeared from nuclei after incubation for 24-48 h. The extracts of hepatic nuclei from adult hamsters were immunoprecipitated with either the anti-growth inhibitory factor IgG or a monoclonal antibody to the RM protein. The growth inhibitory factor and the RB protein coprecipitated in each case, implying that the proteins were complexed with each other in the nuclei. The RB protein family is composed of two sets of species, an un- or underphosphorylated species and a hyperphosphorylated one. It was suggested that the factor bound preferentially to the un- or underphosphorylated member of the family.

Animals↗

PubMed1996

Efficacy of ursodeoxycholic acid therapy in chronic viral hepatitis C with high serum gamma-glutamyltranspeptidase levels.

We administered ursodeoxycholic acid (UDCA) orally, at a daily dose of 600 mg, for 4 months to 36 patients with chronic viral hepatitis C. Another 36 patients with chronic viral hepatitis C, treated with placebo for 4 months, served as controls. None of the patients were alcoholics and none suffering from autoimmune hepatitis. Of the 36 patients in the UDCA-treated group, 13 had high levels of serum gamma-glutamyltranspeptidase (GGT), i.e., exceeding 150 U/l (normal < 50 U/l). Histological examination of liver biopsy specimens obtained from 10 patients in this group before treatment suggested that damage of the interlobular bile ducts was prominent in patients with higher levels of serum GGT. After 1 month of UDCA treatment, significant decreases in the levels of serum GGT, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were observed (P < 0.05 for GGT and AST), and the decreases continued for the 4-month treatment period. The reduction of GGT levels was the most prominent change in the liver function indices; the percent change in the GGT level was -25.2 +/- 4.4 (mean percent change +/- SE) at 1 month and -38.0 +/- 5.0 at 4 months. A significant correlation was observed between the serum delta GGT level (GGT value before treatment minus value after 3 months of treatment) and the total score for morphological injury of the bile ducts (P < 0.05). These results suggested that UDCA has the potential to reverse hepatocellular damage in patients with chronic viral hepatitis C, in whom high GGT levels may be due, in part, to a damaged interlobular bile duct. UDCA may be useful for the treatment of chronic viral hepatitis C, especially in patients exhibiting a high level of GGT.

Administration, Oral↗

PubMed1996

A nonbenzodiazepine partial agonist, S-(+)-DN-2327, has minimal physical dependence-producing liability, but shows cross-dependence on barbital in rats.

1. Physical dependence and cross-physical dependence on barbital of the benzodiazepine receptor partial agonist S-(+)-DN-2327 and the benzodiazepine receptor full agonist diazepam were compared in male Fischer 344 rats. 2. In the physical dependence study, rats were treated with S-(+)-DN-2327 (30, 100, 300 and 1000 mg/kg/day) or diazepam (30, 100 and 300 mg/kg/day) for 4 weeks by the drug admixed with food method. After stopping the treatment, the body weight and food consumption in the diazepam 100 and 300 mg/kg groups tended to decrease or decreased to values lower than those in the control group, whereas these parameters in the S-(+)-DN-2327 30, 100 and 300 mg/kg groups were comparable to the control group values. 3. In the cross-dependence study, rats were treated with increasing doses of barbital by admixing the drug with food for 4 weeks, after which the diet admixed with barbital was replaced by basal diet alone or admixed with S-(+)-DN-2327 or diazepam (target doses: 100 and 300 mg/kg/day for each compound). During the substitution period, the decreases in body weight and food consumption in both S-(+)-DN-2327 and both diazepam groups were suppressed compared with those in the basal diet group. 4. These results suggest that S-(+)-DN-2327 possesses minimal physical dependence-producing liability, but shows cross-dependence on barbital, as do benzodiazepine receptor full agonists.

Animals↗

PubMed1996

Biotin deficiency decreases ornithine transcarbamylase activity and mRNA in rat liver.

Biotin deficiency is well known as a cause of hyperammonemia, but there has been no report on the effect of biotin deficiency on hepatic ureagenesis. In this study, we examined the changes in the activities and gene expression of urea cycle enzymes using rats fed raw egg white as a model of biotin deficiency. All rats were made biotin-deficient by feeding them an avidin-containing diet for 6 wk. The rats were divided into two groups at the beginning of this experiment: biotin-deficient rats (BD rats) and biotin-supplemented rats (BS rats) which were treated with biotin once a day at a dose of 1 mg per rat intraperitoneally. The plasma ammonia concentration of the BD rats (92.8 +/- 12 mumol/L) was significantly higher than that of BS rats (63.9 +/- 16 mumol/L, P < 0.05). The activities of ornithine transcarbamylase (OTC) was significantly lower in the liver of the BD (110.2 +/- 5.5) rats than in the BS rats (154 +/- 3.8 U/mg protein, P < 0.01). Activities of the other urea cycle enzymes were not significantly different in the two groups. OTC gene expression in the liver of BD rats was 40% lower than in BS rats (P < 0.05). These data suggest that biotin deficiency decreases OTC activity and the amount of OTC mRNA.

Amino Acids↗

PubMed1996

Expression of heparin-binding epidermal growth factor-like growth factor in neointimal cells induced by balloon injury in rat carotid arteries.

Balloon catheter injury of rat carotid arteries induces migration and proliferation of smooth muscle cells (SMCs), with subsequent neointimal formation. Several growth factors, such as platelet-derived growth factor and basic fibroblast growth factor, have been shown to be involved in this process, but the mechanisms that modulate the growth and/or migratory properties of SMCs remain unclear. In this study, we investigated whether heparin-binding epidermal growth factor-like growth factor (HB-EGF), which is known to be a potent SMC stimulator from in vitro study, is associated with the proliferative response of SMCs to arterial injury. Northern blot analysis showed that the transcript levels of HB-EGF increased rapidly approximately 12-fold within 2 hours after injury and declined by 2 days but remained 3-fold at 14 days. In situ hybridization analysis demonstrated that the transcript of HB-EGF remained strongly expressed in the neointima, especially near the luminal surface, at 14 days after injury. Immunohistochemical staining showed that HB-EGF protein was positive in the endothelium and only faintly visible in medial SMCs in uninjured vessels. In contrast, 2 days after injury, positive HB-EGF immunostaining was detected in the medial SMCs along the luminal surface. At 7 days, the neointimal SMCs exhibited strong immunostaining for HB-EGF, and at 14 days, they exhibited a gradient of HB-EGF expression with strong immunoreactivity in the most luminal cells. SMCs labeled with 5-bromo-2'-deoxyuridine in their nuclei showed strong immunostaining for HB-EGF protein. Furthermore, the epidermal growth factor receptor to which HB-EGF can bind was also immunostained positively in neointimal SMCs. These data suggest that HB-EGF may play an important role of the proliferation and migration of SMCs in the process of neointimal accumulation induced by arterial injury, probably in an autocrine, paracrine, and/or juxtacrine manner.

Animals↗

PubMed1996

Characterization of microsomal cytochrome P450 enzymes involved in the oxidation of xenobiotic chemicals in human fetal liver and adult lungs.

Levels and catalytic activities of cytochrome P450 (P450) enzymes involved in the oxidation of drugs and carcinogens were determined in human adult lungs and fetal livers and compared with those in microsomes from adult livers. P450s immunoreactive with anti-human P4501A1 and anti-human P4503A antibodies were detected in fetal liver microsomes by immunoblotting analysis, and P450s related P4501A1, 2A6, 2C9, 2E1, and 3A4 were determined in adult lung microsomes; all of these P450 enzymes were detected in much higher amounts in adult liver microsomes except that P4501A2 was only the 1A subfamily of P450 found in adult livers. Drug oxidation activities with the substrates ethoxyresorufin, coumarin, 7-ethoxycoumarin, bufuralol, and testosterone were determined in these microsomes, and we found that none of the activities were higher in microsomes of adult lungs and fetal livers than in adult livers. Activation of procarcinogens to reactive metabolites that induce umu gene expression in Salmonella typhimurium TA1535/pSK1002 or NM2009 was also examined and it was found that activities with (+)- and (-)-enantiomers of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene were higher in fetal liver microsomes than adult lung or liver microsomes. The adult liver and lung activities for these two procarcinogens were similar on the basis of microsomal protein contents despite the fact that p450 contents are higher in liver than lung microsomes. alpha-Naphthoflavone, a known inhibitor of P4501A-related activities, did not affect these procarcinogen activation in fetal liver microsomes. Fetal liver microsomes catalyzed activation of aflatoxin B1 and sterigmatocystin, two procarcinogens known to be activated by P4503A4/7 in humans, although activation of carcinogenic arylamines that are good substrates for P4501A2 was much lower in microsomes of fetal livers and adult lungs than in adult livers. These results suggest that in human fetal livers at least two P450 enzymes, a form of P450 that is immunoreactive P4501A1 and P4503A7, are actually expressed and these enzymes are suggested as being involved in the activation of the (+)- and (-)-enantiomers of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene and the carcinogenic mycotoxins, respectively. The exact nature of the former enzyme in fetal livers is unknown. In adult human lungs, several P450 enzymes are expressed, although the precise roles of these enzymes in the oxidation of xenobiotics were not determined due to the low level of expression of these P450s.

Antibodies↗

PubMed1995

cDNA cloning of thyroid hormone receptor beta for the Japanese flounder.

cDNA encoding the beta type of thyroid hormone receptor (THR) was cloned from a lambda gt10 library prepared from the whole bodies of metamorphosing flounder (Paralichthys olivaceus). The deduced amino acid sequence of the flounder THR beta (fTHR beta 1) showed higher homologies to the known THR beta s of other vertebrate animals than to THR alpha s, including flounder THR alpha s. Unlike any other THR, fTHR beta 1 possessed an insertion sequence composed of nine amino acids in the region prior to the hormone-binding domain. PCR analyses suggested the presence of transcripts for another THR beta (fTHR beta 2) that had a longer insertion sequence than fTHR beta 1. The analyses further suggested that the cDNA sequences of the two flounder THR beta s most likely shared a constant sequence except for a 60-base additional sequence found only in fTHR beta 2. The flounder genome possessed a single gene for both fTHR beta 1 and beta 2, suggesting that the two THR beta variants are produced from the same gene through an alternative splicing system.

Amino Acid Sequence↗

PubMed1995

Procarcinogen activation by cytochrome P450 3A4 and 3A5 expressed in Escherichia coli and by human liver microsomes.

Recent studies indicate that cytochrome P450 (P450) 3A4 plays important roles in the activation of procarcinogens such as aflatoxin B1 and sterigmatocystin, as well as in the oxidation of a number of structurally diverse chemicals and endogenous compounds. Since P450 3A5 has been reported to be present at significant levels in liver microsomes in approximately 25% of human adults, we examined and compared the role of P450 3A4 and 3A5 in procarcinogen activation in humans. Immunoblot experiments with liver microsomes from 60 human samples suggested that 4/30 Japanese and 4/30 Caucasians contained considerable levels of P450 3A5, although P450 3A4 could be determined at relatively high levels in all of the human samples examined. Good correlation was observed between P450 3A4, but not P450 3A5, levels versus activation of aflatoxin B1 and stergmatocystin in these human samples. Comparisons of the activation of procarcinogens in reconstituted monooxygenase systems containing modified P450 3A4 and 3A5 enzymes expressed in Escherichia coli were carried out in Salmonella typhimurium TA1535/pSK1002 or NM2009 tester strain for genotoxicity assay, and it was found that P450 3A4 had similar activities to or higher rates than P450 3A5 for the 24 procarcinogens tested.

Adult↗

PubMed1995

Increased mitogenic response to heparin-binding epidermal growth factor-like growth factor in vascular smooth muscle cells of diabetic rats.

We investigated the mitogenic effects of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in vascular smooth muscle cells (SMCs) obtained from rats with streptozotocin (STZ)-induced diabetes and evaluated the role of heparan sulfate proteoglycan (HSPG) in inducing these effects. HB-EGF significantly increased DNA synthesis in the SMCs of diabetic rats (STZ-SMCs) compared with control rats (control SMCs). However, the mitogenic effects of EGF, which shares EGF receptors with HB-EGF, and basic fibroblast growth factor, another heparin-binding growth factor, were similar in STZ-SMCs and control SMCs. The mitogenic response to HB-EGF in SMCs of insulin-treated diabetic rats was similar to the response in control SMCs. HB-EGF-induced autophosphorylation of EGF receptors was increased in STZ-SMCs compared with control SMCs, although the number of EGF receptors in STZ-SMCs was 40% of that in controls. This increased mitogenic response to HB-EGF in STZ-SMCs was completely inhibited by treatment with heparitinase, chlorate, and a synthetic peptide corresponding to the heparin-binding domain of HB-EGF. Compared with heparan sulfate isolated from control SMCs, heparan sulfate isolated from STZ-SMCs was of smaller molecular size and caused a greater mitogenic effect of HB-EGF. These findings suggest that the mitogenic response to HB-EGF is increased in SMCs of diabetic rats. Changes in cell-associated heparan sulfate in STZ-SMCs may be related to the increased mitogenic response to HB-EGF.

Animals↗

PubMed1995

Localization of heparin-binding EGF-like growth factor in the smooth muscle cells and macrophages of human atherosclerotic plaques.

Heparin-binding EGF-like growth factor (HB-EGF) is a potent chemoattractant and mitogen for smooth muscle cells (SMC) in culture. To elucidate whether HB-EGF is implicated in the pathogenesis of human atherosclerosis, we examined immunohistochemical localization of HB-EGF in human aortic walls and atherosclerotic plaques. The medial SMC of the aorta in babies and children synthesized HB-EGF protein, while the number of SMC producing HB-EGF was dramatically decreased in young and middle-aged adults. In atherosclerotic plaques, however, marked production of HB-EGF protein was detected in SMC and macrophages of the plaques. Furthermore, EGF receptors, to which HB-EGF is known to bind, were detected in plaque SMC. These data suggest that HB-EGF may be implicated in the migration and proliferation of SMC that occurs in the normal development of arterial walls, and in the formation of atherosclerotic plaques.

Adult↗

PubMed1994

Isolation and characterization of a growth inhibitory factor from hamster liver.

Among lysates from various organs and tissues of adult hamsters only lysates from liver demonstrated an inhibitory effect on the cell growth of SV40-transformed hamster fibroblasts in culture. Lysates from the liver of fetal hamsters and those from 7-day-old hamsters did not demonstrate any inhibitory effect on the cell growth. Lysates from the remnant liver 3 days after partial hepatectomy did not show any inhibitory effect on the cell growth but lysates from the remnant liver 14 days after the operation came to show an appreciable inhibitory effect on the cell growth. An inhibitor of the cell growth was purified from adult hamster liver by ammonium sulfate precipitation, DEAE-, hydroxyl apatite-, phenyl Sepharose- and Sephadex G75 column chromatography. The cell growth inhibitor thus prepared was shown to be pure by an ion-exchange chromatography, SDS-PAGE and analytical isoelectric focusing. The inhibitor was found to have a molecular mass of 37 kDa and an isoelectric point of approx. 7.5 and to cause reversible arrest of the transformed fibroblasts predominantly in the G0/G1 phase of the cell cycle at the concentration of approx. 0.9 microgram per ml.

Animals↗

PubMed1994

Cloning of thyroid hormone receptor genes expressed in metamorphosing flounder.

Two distinct cDNAs encoding thyroid hormone receptors (THRs) were cloned from a lambda gt10 library prepared from the whole bodies of metamorphosing flounder larvae (Paralichthys olivaceus). Deduced amino acid sequences of the two isolated cDNAs shared 96% and 92% homologies in their DNA- and hormone-binding domains, respectively. These were highly conserved when compared to THRs for other vertebrates: 88-96% in the DNA-binding domain and 84-94% in the hormone-binding domain. Other receptors in the nuclear receptor family showed lower homologies than those of THRs. Both THRs for the flounder had higher homologies with the alpha-type THRs of other vertebrates than with the beta-type. Thus, the two THRs for flounder were designated as fTHR alpha A and fTHR alpha B.

Amino Acid Sequence↗

PubMed1994

The antimetamorphic effect of prolactin in the Japanese flounder.

Prolactin has an antimetamorphic action in amphibian tadpoles. In the Japanese flounder, Paralichthys olivaceus, ovine prolactin (oPRL, 100 ng/ml) antagonized the stimulatory effect of triiodothyronine (T3, 1 ng/ml) on the resorption of the dorsal fin rays of prometamorphic larvae in vitro. Ovine growth hormone (oGH) at the same dose was without effect. Injections with oPRL (50 ng/fish, six times) into prometamorphic larvae also delayed the resorption of the dorsal fin rays without affecting the rates of eye migration and settling, while oGH was again without effect. The changes in the expression of both PRL and GH mRNAs in the pituitary during metamorphosis were monitored by in situ hybridization using cDNA probes. Both PRL and GH genes were increasingly expressed during successive metamorphic stages. These results are discussed in light of the possible interactions of PRL and GH with thyroid hormones in the control of development in the flounder.

Animals↗

PubMed1994

Effect of thyroid hormone on developmental transition of myosin light chains during flounder metamorphosis.

Thyroidal control of the transition of myosin isoforms during flounder metamorphosis was examined by the administration of either only thiourea (TU), a potent inhibitor of thyroid hormone synthesis, or thyroxine (T4) together with TU into premetamorphic larvae. Immersion of premetamorphic larvae in 400 microM of TU inhibited the appearance of the adult-type DTNB (5,5'-dithio-bis-nitrobenzoic acid) light chain, LC2, whereas administration of T4 with TU induced a precocious appearance of LC2 and decreased the relative amount of the larval-type DTNB light chain, LC2*. TU was administered into juveniles just after completion of metamorphosis. The treatment did not affect the composition of the myosin light chains. These results suggest that thyroid hormone irreversibly turns on the switch for transition of the DTNB light chains from larval to adult type during metamorphosis of the flounder.

Animals↗

PubMed1994

Effects of glucagon on urinary excretion of urea and on plasma ammonia level in argininosuccinate synthetase deficiency.

Glucagon, a potent inducer of urea cycle enzymes, was administered subcutaneously, at a dose of 0.5 mg once a day, for 7 days to two citrullinemic patients. During this period, plasma NH3 levels in case 1 decreased significantly (P < 0.05 compared to levels before administration) and daily urinary excretion of urea N increased significantly (P < 0.05). For 1 week after the cessation of administration, the daily urinary excretion of urea N was significantly higher than the level before administration (P < 0.05), the plasma citrulline level during glucagon administration was lower than that before administration. In case 2, glucagon administration also decreased the plasma NH3 level (although the decrease was not statistically significant), and significantly increased daily urinary excretion of urea N (P < 0.05 compared to levels before administration). For 1 week after the cessation of glucagon administration the plasma citrulline level was significantly lower than that before administration (P < 0.05). These results indicate that glucagon significantly increases the urinary excretion of urea in the late onset form of argininosuccinate synthetase deficiency and that it may also decrease plasma NH3 levels in some patients with the deficiency.

Adult↗

PubMed1994

Impaired ketogenesis in patients with adult-type citrullinemia.

BACKGROUND/AIMS: To clarify the mechanism causing fatty liver in adult-type citrullinemia, the effect of fasting on blood levels of free fatty acids, triglycerides, and ketone bodies was investigated in two cases. METHODS: Blood and urine samples were collected from two patients and healthy volunteers 12, 15, 17, 21.5, and 24 hours after their last meal. RESULTS: During 24-hour fasting free fatty acid concentrations increased in both cases to the concentrations found in the healthy volunteers. The levels of blood ketone bodies (beta-hydroxybutyrate and acetoacetate) were markedly suppressed throughout the fasting test without any increase in urinary excretion of ketone bodies or organic acids in both cases when plasma citrulline concentrations were more than 10-fold higher than in controls. Serum triglyceride concentrations in case 1 paradoxically increased from 185 mg/dL to 294 mg/dL during 24-hour fasting when the citrulline concentration was extremely high. When hemodialysis was performed and plasma citrulline consequently decreased to near the normal level in case 1, levels of both serum triglycerides and blood ketone bodies responded normally to 24-hour fasting. CONCLUSIONS: These data suggest that ketogenesis was impaired in adult-type citrullinemia.

Adult↗

PubMed1994

Expression of heparin-binding epidermal growth factor in human hepatocellular carcinoma.

BACKGROUND/AIMS: Growth factors are involved in the development and progression of cancer. The purpose of this study was to evaluate the possible role of heparin-binding epidermal growth factor-like growth factor (HB-EGF), which is a member of the EGF family, in the neoplastic transformation of hepatocytes. METHODS: Gene expression and protein production of HB-EGF were investigated in samples of human hepatocellular carcinoma (HCC) from 17 patients using Northern hybridization and immunohistochemical methods. RESULTS: The amount of HB-EGF messenger RNA was increased in the patients' HCC specimens compared with the surrounding liver tissues. In noncancerous hepatic tissues, HB-EGF was faintly positive in hepatocytes. Immunoreactive HB-EGF-producing cells were identified in HCC cells of all 17 patients with HCC, indicating that HB-EGF was produced in HCC cells themselves. However, none of the specimens from 10 patients with metastatic adenocarcinoma in the liver was positive for HB-EGF. The EGF receptor, which binds to HB-EGF, was also expressed on HCC cells. CONCLUSIONS: It is hypothesized that the enhanced expression of immunoreactive HB-EGF on the cell suggests a possible role of HB-EGF in the development or progression of human HCC in an autocrine and/or a juxtacrine manners.

Adult↗