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Biomedical subjects

M Hirose

Publications and source records attributed to M Hirose.

At least 217 records · Page 12Linked to original sources
PubMed1998

Multiple processing of procathepsin L to cathepsin L in vivo.

Three amino-terminal-specific peptidic antibodies against cathepsin L were generated. These antibodies recognize in vitro processing products of procathepsin L in time-course-dependent fashion. Immunoblot analyses with these antibodies followed by immunoprecipitation with anti-cathepsin L antibody showed that the amino terminal amino acid sequences of intracellular cathepsin L are heterogeneous: the single chain form of cathepsin L starts with either EPLML, LKIPK or IPKSV, and the heavy chain of the two chain form with IPKSV. Percoll density gradient and fluorescence immunohistochemistry suggested that these three species of cathepsin L localize in the lysosomes where procathepsin L processing occurs.

3T3 Cells↗
PubMed1998

Immunohistochemical demonstration of carcinogen-DNA adducts in tissues of rats given 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP): detection in paraffin-embedded sections and tissue distribution.

A polyclonal antibody against 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-DNA adducts was raised for their immunohistochemical demonstration in paraffin-embedded sections. Specificity of this antibody was confirmed by competitive ELISA. Positive signals were immunohistochemically detected in acetone-fixed but not in formalin- or ethanol-fixed sections from F344 rats treated by gavage with a single dose of PhIP at 37.5-300 mg/kg and killed at 1, 2, and 7 days thereafter. Dose-dependent positive staining was observed in almost all organs of both sexes, including the colon, prostate, and mammary gland but largely independent of the tumor response. Repair activity, judged by disappearance of adducts with time, differed according to the organ or cell type. One exception was hepatocytes, the liver incidentally being a nontarget organ. The results suggest that the generated antibody is applicable for detection of cells targeted by PhIP in paraffin-embedded sections and also for the investigation of the mechanisms of PhIP-carcinogenesis.

Administration, Oral↗
PubMed1998

A novel hepatic stellate (Ito) cell-derived protein, epimorphin, plays a key role in the late stages of liver regeneration.

Limited data exist regarding morphogenesis and differentiation during liver regeneration. We examined the role of epimorphin on liver regeneration. After 70% partial hepatectomy, mouse liver was collected on days 1, 3, 7, and 14 for immunohistochemistry and the detection of epimorphin mRNA and connexin 32. Using primary cultured rat hepatocytes, morphogenesis and differentiation of cells were tested with or without epimorphin. Seven days after cell inoculation, the expression of connexin 32 and the cell-cell communication was tested as a marker of differentiation. Epimorphin was detected exclusively in hepatic stellate cells. Connexin 32 was detected only in hepatocytes. After partial hepatectomy, epimorphin mRNA was detected on day 3 and peaked at day 7, followed by protein expression. Connexin 32 expression showed a similar time course. Cultured hepatocytes formed multicellular spheroids in an active epimorphin-coated culture dish and showed positive dye coupling, whereas the cell-cell communication was lost without active epimorphin. Because epimorphin was expressed late in liver regeneration, it might play a role in morphogenesis and differentiation.

Animals↗
PubMed1998

Conformational state of ovalbumin at acidic pH as evaluated by a novel approach utilizing intrachain sulfhydryl-mixed disulfide exchange reactions.

Ovalbumin contains four cysteine sulfhydryls (Cys11, Cys30, Cys367, and Cys382) and one cystine disulfide (Cys73-Cys120). A highly reactive aromatic disulfide, 2,2'-dipyridyl disulfide, reacts specifically with Cys367 of ovalbumin at pH 2.2 generating a mixed disulfide protein derivative [Tatsumi, E., and Hirose, M. (1997) J. Biochem. 122, 300-308]. The mode of conformational fluctuation in ovalbumin was investigated at pH 2.2 using the mixed disulfide derivatives of the cystine-intact and cystine-reduced protein forms. In the presence of a high concentration of urea, both the mixed disulfide derivatives underwent rapid cysteine sulfhydryl/mixed disulfide exchanges, thereby releasing the quantitative amount of 2-thiopyridone. A peptide mapping analysis for disulfide-forming cysteines revealed that this release was mostly accounted for by the nucleophile attack on the Cys367-mixed disulfide by the nearest cysteine residue in the primary structure, Cys382. At the acidic pH, the exchange reaction was practically restricted to the cysteine sulfhydryl/mixed disulfide exchanges; no other exchange reaction, such as the cysteine sulfhydryl/cystine disulfide exchange reaction, was detected. In the absence of urea, the cystine-reduced form, but not the cystine-intact form, underwent significant sulfhydryl/mixed disulfide exchange reactions at a physiological temperature, as determined by the release of 2-thiopyridone. A kinetic analysis for the generation of disulfide-forming cysteines with Cys367 at 37 degreesC revealed that the rate for the intrachain exchange reaction was quite different for the five cysteine sulfhydryls. The effective concentrations of the five cysteine sulfhydryls relative to the Cys367-mixed disulfide were determined by using three related model reactions: the obtained values were 11.4, 4.6, 15.2, 5.9, and 8.9 microM for Cys11, Cys30, Cys73, Cys120, and Cys382, respectively. Implications of the effective concentrations for the conformational state of acidic ovalbumin are discussed.

Acids↗
PubMed1998

Strong promoting activity of phenylethyl isothiocyanate and benzyl isothiocyanate on urinary bladder carcinogenesis in F344 male rats.

Post-initiation effects of phenylethyl isothiocyanate (PEITC) and benzyl isothiocyanate (BITC) on hepatocarcinogenesis and urinary bladder carcinogenesis were examined in rats pretreated with diethylnitrosamine (DEN) and N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Groups of 21 rats received a single intraperitoneal injection of 200 mg/kg body weight of DEN. Starting 2 days thereafter, they were administered 0.05% BBN in the drinking water for 4 weeks. Three days after completion of the carcinogen treatment, they were placed on a diet containing PEITC or BITC at a dose of 0.1%, or a basal diet alone for 32 weeks and then killed for autopsy. Further groups of 6 rats each were similarly treated with PEITC, BITC or basal diet alone for 32 weeks without prior DEN and BBN exposure. In the liver, although the incidences of liver tumors were not significantly affected, the multiplicity of foci larger than 0.5 cm in diameter was slightly increased by PEITC. In the urinary bladder, the incidences of papillary or nodular (PN) hyperplasias and carcinomas were significantly elevated by PEITC or BITC after DEN and BBN initiation. In the groups without initiation, PN hyperplasia was found in all rats of both PEITC and BITC groups, along with papillomas and carcinomas in some animals. Tumors and PN hyperplasias in the groups treated with PEITC and BITC are characterized by downward growth. Our results thus showed PEITC and BITC to be strong promoters of urinary bladder carcinogenesis with some complete carcinogenic potential.

Animals↗
PubMed1998

p53 may mediate the mdr-1 expression via the WT1 gene in human vincristine-resistant leukemia/lymphoma cell lines.

To investigate the regulatory mechanism of the expression of the multidrug resistance gene (mdr-1) and the multidrug resistance-associated protein gene (mrp), we investigated if p53, WT1, RB, C-myc, N-myc, cyclin D1, p16INK4 (p16) are involved in the acquirement of multidrug resistance phenotype (MDR) in human vincristine (VCR)-resistant cells of leukemia/lymphoma cell lines. By using RT-PCR, we observed that MDR in VCR-resistant cell lines was mediated by either mdr-1 or mrp genes. In cells that acquired the overexpression of mdr-1, downregulation of p53 and upregulation of WT-1 were observed. In contrast, no constant change of genes was observed in cells that overexpressed mrp. Although the change in the expression level of cyclin D1 and p-16 accompanied the development of VCR resistance, the mRNA of RB, C-myc and N-myc showed no correlation with the degree of VCR resistance or the level of mdr-1 expression. These results may provide a plausible diagnostic marker for determination of drug sensitivity in cancer patients and suggest that p53 may mediate directly or indirectly the expression of mdr-1 via WT1 in VCR-resistant hematologic cell lines.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Molecular dissection of the Rho-associated protein kinase (p160ROCK)-regulated neurite remodeling in neuroblastoma N1E-115 cells.

A critical role for the small GTPase Rho and one of its targets, p160ROCK (a Rho-associated coiled coil-forming protein kinase), in neurite remodeling was examined in neuroblastoma N1E-115 cells. Using wild-type and a dominant-negative form of p160ROCK and a p160ROCK-specific inhibitor, Y-27632, we show here that p160ROCK activation is necessary and sufficient for the agonist-induced neurite retraction and cell rounding. The neurite retraction was accompanied by elevated phosphorylation of myosin light chain and the disassembly of the intermediate filaments and microtubules. Y-27632 blocked both neurite retraction and the elevation of myosin light chain phosphorylation in a similar concentration-dependent manner. On the other hand, suppression of p160ROCK activity by expression of a dominant-negative form of p160ROCK induced neurites in the presence of serum by inducing the reassembly of the intermediate filaments and microtubules. The neurite outgrowth by the p160ROCK inhibition was blocked by coexpression of dominant-negative forms of Cdc42 and Rac, indicating that p160ROCK constitutively and negatively regulates neurite formation at least in part by inhibiting activation of Cdc42 and Rac. The assembly of microtubules and intermediate filaments to form extended processes by inhibitors of the Rho-ROCK pathway was also observed in Swiss 3T3 cells. These results indicate that Rho/ROCK-dependent tonic inhibition of cell process extension is exerted via activation of the actomysin-based contractility, in conjunction with a suppression of assembly of intermediate filaments and microtubules in many cell types including, but not exclusive to, neuronal cells.

3T3 Cells↗
PubMed1998

Stage and organ dependent effects of 1-O-hexyl-2,3,5-trimethylhydroquinone, ascorbic acid derivatives, n-heptadecane-8-10-dione and phenylethyl isothiocyanate in a rat multiorgan carcinogenesis model.

The effects of 1-O-hexyl-2,3,5-trimethylhydroquinone (HTHQ), phenylethylisothiocyanate (PEITC), 3-O-ethylascorbic acid, 3-O-dodecylcarbomethylascorbic acid and n-heptadecane-8, 10-dione were analyzed in a rat multiorgan carcinogenesis model. Groups of 15 animals were given a single intraperitoneal (i.p.) injection of diethylnitrosamine and 4 i.p. injections of N-methylnitrosourea as well as N-butyl-N-(4-hydroxybutyl) nitrosamine in the drinking water during the first 2 weeks. Then 4 subcutaneous (s.c.) injections of dimethylhydrazine and 2,2'-dihydroxy-di-n-propylnitrosamine were given in the drinking water over the next 2 weeks for initiation. Test compounds were administered during the initiation or post-initiation periods. The dietary dose was 1% except for n-heptadecane-8, 10-dione and PEITC (0.1%). Complete autopsy was performed at the end of experimental week 28. All 5 compounds reduced the number of lung hyperplasia, particularly PEITC when given during the initiation period. In addition, HTHQ lowered the incidence of esophageal hyperplasia in the initiation period, and of small and large intestinal adenomas in the post-initiation period. However, it also enhanced the development of hyperplasia and papilloma in the forestomach and tongue. PEITC lowered the induction of esophageal hyperplasia, kidney atypical tubules and liver glutathione S-transferase placental form (GST-P)-positive foci when given during the initiation period but enhanced the development of liver GST-P positive foci and urinary bladder tumors in the post-initiation period. Moreover, it induced hyperplasia of the urinary bladder. Our results indicate minor adverse effects for HTHQ in the forestomach and tongue, and demonstrate that PEITC, which inhibits carcinogenesis at the initiation stage in several organs, also exhibits promotion potential in liver and urinary bladder in the post-initiation stage under the present experimental conditions.

Animals↗
PubMed1998

Regional differences in cardiac effects of pituitary adenylate cyclase-activating polypeptide-27 in the isolated dog heart.

Recent observations indicate that several neuropeptides may be involved in the regulation of cardiac function, but the effects of these peptides on the atrium are not always the same as those on the ventricle. To compare the effect of pituitary adenylate cyclase-activating polypeptide (PACAP)-27 on the atrium with that on the ventricle, we investigated the effects of PACAP-27 on the sinus rate and atrial and ventricular contractility in isolated, blood-perfused dog heart preparations. PACAP-27 (0.01-0.3 nmol) caused transient positive followed by negative chronotropic and inotropic responses in a dose-dependent manner in the isolated right atrium, whereas it caused only a dose-dependent positive inotropic response in the left ventricle. After atropine treatment, PACAP-27 caused only positive cardiac responses in isolated atria. The order of the increase in response to PACAP-27 was atrial contractile force > sinus rate > or = ventricular contractile force. Tetrodotoxin blocked the negative chronotropic and inotropic responses to PACAP-27 in isolated atria. Propranolol did not affect the positive response. PACAP-(6-27), a type I PACAP receptor antagonist, attenuated the positive responses similarly in the atropine-treated right atrium and the left ventricle. Thus, we demonstrated that (1) PACAP-27 caused negative cardiac effects in the atrium and sinoatrial node by activation of intracardiac parasympathetic nerves, but had no negative effect on the ventricle; (2) PACAP-27 had positive effects in the atrium, sinoatrial node and ventricle mediated by type I PACAP receptors, but PACAP-27 was more effective in the atrium and sinoatrial node than in the ventricle of the dog heart.

Animals↗
PubMed1998

Glial cell line-derived neurotrophic factor/neurturin-induced differentiation and its enhancement by retinoic acid in primary human neuroblastomas expressing c-Ret, GFR alpha-1, and GFR alpha-2.

Neuroblastomas often undergo spontaneous differentiation and/or regression in vivo, which is at least partly regulated by the signals through neurotrophins and their receptors. Recently, glial cell line-derived neurotrophic factor (GDNF) and a second family member, neurturin (NTN), have been found to mediate their signals by binding to a heterotetrameric complex of c-Ret tyrosine kinase receptors and glycosylphosphatidylinositol-linked proteins, GFR alpha-1 (GDNFR-alpha) or GFR alpha-2 (TrnR2/GDNFR-beta/NTNR-alpha/RETL2). Here, we studied the effect of GDNF and NTN on human neuroblastomas in the short-term primary culture system, as well as the expression of c-Ret, GFR alpha-1, GFR alpha-2, GDNF, and NTN. GDNF (1-100 ng/ml) induced morphological differentiation in 34 of 38 primary neuroblastomas and an accompanying increase in c-Fos induction. These effects were markedly enhanced by treatment with 5 microM all-trans-retinoic acid. Although GDNF alone induced a rather weak differentiation independent of the disease stages, the enhancement of neurite outgrowth induced by treatment with both GDNF and all-trans-retinoic acid was significantly correlated with younger age (less than 1 year; P = 0.0039), non-stage 4 diseases (P = 0.0023), a single copy of N-myc (P = 0.027), and high levels of TRK-A expression (P = 0.0062). To examine the expression levels of GFR alpha-1, we cloned a short form of the human GFR alpha-1 gene with a 15-bp deletion by screening a human adult substantia nigra cDNA library. Many primary neuroblastomas expressed c-Ret, GFR alpha-1, and GFR alpha-2 as well as their ligands, GDNF and NTN, suggesting the presence of a paracrine or autocrine signaling system within the tumor tissue. The effect of NTN on primary culture cells of neuroblastoma was similar to that of GDNF. These imply that the GDNF(NTN)/c-Ret/GFR alpha-1(GFR alpha-2) signaling may have an important role in regulating the growth, differentiation, and cell death of neuroblastomas.

Antineoplastic Agents↗
PubMed1998

Patent ductus arteriosus and neonatal death in prostaglandin receptor EP4-deficient mice.

The physiological role of the prostaglandin E2 receptor EP4 subtype was investigated by generation of EP4-deficient-mice by gene targeting. Loss of the EP4 receptor was not lethal in utero, but most EP4 (-/-) neonates became pale and lethargic approximately 24 h after birth and died within 72 h. Less than 5% of the EP4 (-/-) mice survived and grew normally more than a year. Histological examination revealed that the ductus arteriosus in dead neonates remained open, while it was partially closed in the survivors. In situ hybridization study showed that EP4 mRNA was strongly expressed in the ductus. These results suggest that neonatal death is at least partly due to patent ductus arteriosus and that the EP4 receptor plays a role in regulation of the patency of this vessel. They also indicate that normal function of the EP4 receptor is essential in neonatal adaptation of the circulatory system.

Animals↗
PubMed1998

Effects of catechol, sodium chloride and ethanol either alone or in combination on gastric carcinogenesis in rats pretreated with N-methyl-N'-nitro-N-nitrosoguanidine.

Combined effects of catechol, sodium chloride (NaCl) and ethanol on the post-initiation stage of gastric carcinogenesis were examined in rats pretreated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). F344 male rats were given a single intragastric dose of 150 mg/kg b.w. MNNG at 6 weeks of age. Starting 1 week thereafter, groups of 15 rats were administered 0.8% catechol, 5% NaCl and 10% ethanol either individually or in combination, or basal diet alone for 51 weeks. Further groups of animals were similarly treated with these chemicals without the MNNG pretreatment. All rats were killed at the end of week 52 for histopathological examination. In the forestomach, treatment with catechol alone after MNNG initiation caused a 100% incidence of papillomas (versus 67% in the controls) as well as carcinomas (versus 0% in the controls). On the other hand, the treatment with ethanol alone significantly lowered the incidence of papillomas (13 versus 67% in the controls). The combined treatment with catechol, NaCl and ethanol significantly lowered the incidence of squamous cell carcinomas (57%) as compared to the catechol alone group value (100%). In the glandular stomach, catechol enhanced the development of adenocarcinomas (73 versus 0% in the controls), but this was decreased to 29% by the combined treatment with ethanol and NaCl. NaCl without MNNG pretreatment slightly enhanced epithelial cell proliferation in the forestomach. These results indicate that combined treatment with NaCl and ethanol exerts protective effects against catechol-induced forestomach and glandular stomach carcinogenesis, this apparently being largely due to the ethanol.

Animals↗
PubMed1998

Effects of teprenone on gastric epithelial restoration in a rabbit cultured cell model.

The mechanism of action of gastrocytoprotective agents is not fully understood. We assessed the effects of an anti-ulcer agent, teprenone, and bile acid on epithelial restoration. Teprenone with or without deoxycholic acid was added to a complete confluent cultured gastric epithelial cell sheet after wounding. Restoration was monitored for 48 h, and the wound size was assessed. Migration velocity was measured, and proliferation was detected by sequential staining with bromodeoxyuridine. The labeling index was calculated. Restoration was completed within 48h in controls, whereas deoxycholic acid retarded repair. The migration velocity was suppressed by deoxycholic acid treatment. The number of proliferative cells peaked at 36 h (labeling index, 1.7%) in controls. In the deoxycholic acid group, the maximal labeling index was 0.5% at 48 h. Teprenone abolished the bile acid-induced retardation. Teprenone showed cytoprotective effects against deoxycholic acid-induced inhibition of epithelial cell migration and proliferation.

Animals↗
PubMed1998

Differential Ca2+ signaling in neonatal and adult rat hepatocyte doublets.

BACKGROUND/AIMS: Intracellular Ca2+ ([Ca2+]i) is important in various cellular functions, including cellular proliferation and differentiation. To elucidate the relationship between [Ca2+]i oscillations and physiological hepatocyte proliferation, phenylephrine-evoked [Ca2+]i responses were sequentially investigated using short-term cultured hepatocyte doublets obtained from 1-, 3-, 6- and 8-week-old rats. METHODS/RESULTS: DNA synthesis in hepatocytes, determined by BrdU incorporation, was approximately 20% in 1-week-old rats, and decreased to <1% as the rats aged. Correspondingly, [Ca2+]i responses evoked by 10 micromol/l phenylephrine in hepatocyte doublets shifted from transient to sinusoidal-type [Ca2+]i oscillations and then to a sustained increase in [Ca2+]i, followed by a gradual return to baseline. The incidence of [Ca2+]i oscillations was 100+/-0.0%, 83.3+/-16.7%, 38.7+/-0.6% and 5.5+/-5.0% in 1-, 3-, 6- and 8-week-old rats, respectively. Removal of extracellular Ca2+ did not abolish [Ca2+]i oscillations, indicating that [Ca2+]i oscillations were caused primarily by Ca2+ mobilization from internal sites of the cells. The [Ca2+]i level in each of the adjacent cells was synchronous in sustained increase in [Ca2+]i, but asynchronous in [Ca2+]i oscillations. In proliferating doublets obtained from 1-week-old rats, the frequency of oscillations increased in a dose-dependent manner for phenylephrine concentrations of 1 to 100 micromol/l. CONCLUSIONS: Phenylephrine-evoked [Ca2+]i oscillations were directly related to hepatocyte proliferation and were mediated by frequency modulation. These results suggest that phenylephrine-evoked [Ca2+]i oscillations may contribute to cell-cycle progression of hepatocytes in physiological liver growth.

Animals↗
PubMed1998

In vivo karyotypic evolution with altered biological characteristics in a case of neuroblastoma.

Molecular and biological analyses of a neuroblastoma case in which the original tumor contained a nodular region are described. No significant difference was observed between the nodular and the surrounding tumor tissue with respect to histopathologic examination, N-myc amplification, and trkA expression. However, flow cytometric analysis demonstrated that the nodular region consisted of a hypertetraploid clone, whereas the surrounding tissue mostly contained a hyperdiploid clone. Chromosome analysis showed that each clone had a similar chromosome acquisition pattern, suggesting that the hypertetraploid cells of the nodular region arose from the hyperdiploid cells of the surrounding tissue. Moreover, primary culture findings of the tumor cells showed that the responses to nerve growth factor or retinoic acid were different between the two. Collectively, this case suggests the possibility that neuroblastoma acquires novel biological characteristics through karyotypic evolution in vivo.

Abdominal Neoplasms↗
PubMed1998

Variation in susceptibility to the induction of forestomach tumours by butylated hydroxyanisole among rats of different strains.

The forestomach carcinogenicity of butylated hydroxyanisole (BHA) was compared in males of the F344, SHR, Lewis and Sprague Dawley rat strains. Groups of 30 6-wk-old animals were given a pellet diet containing 2% BHA for 104 wk. Forestomach squamous cell papillomas and hyperplasias developed in all rats given BHA, independent of the strain, but the incidences of squamous cell carcinomas (SCCs) differed considerably: F344, 26.7% (8/30) SHR, 76.7% (23/30) SD, 36.7% (1130) Lewis, 6.7% (2/30). Cytotoxic effects, reflected by inflammation, were also most severe in the SHR strain, correlating well with the development of SCCs. The present results indicate that major strain differences exist regarding BHA rat forestomach carcinogenesis and that sensitivity to cytotoxicity might be an important parameter.

Animals↗
PubMed1998

Mutation of the p53 gene in human astrocytic tumours correlates with increased resistance to DNA-damaging agents but not to anti-microtubule anti-cancer agents.

Astrocytic tumours often become resistant to a variety of chemotherapeutic agents in advanced stages and frequently possess mutations in the p53 tumour-suppressor gene. Previous studies using established cell lines to investigate the relation between mutated p53 genes and altered resistance to anti-cancer agents brought inconsistent results. In this report, we examined the status of the p53 gene in 56 astrocytic tumour specimens by single-strand conformation polymorphism and their in vitro chemosensitivity to 30 different kinds of anti-cancer agents. The chemosensitivity was determined by drug-induced cell death using flow cytometry. We found that the mutated p53 gene correlated with increased resistance to DNA-damaging agents but the sensitivity to anti-microtubule agents was independent of the mutation, suggesting a clinical significance of the status of p53 gene in astrocytic tumours and a rational application of anti-microtubule agents to the patients with p53-mutated astrocytic tumours.

Antineoplastic Agents↗