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Biomedical subjects

A Hara

Publications and source records attributed to A Hara.

At least 145 records · Page 8Linked to original sources
PubMed1999

Beta-catenin (Ctnnb1) gene mutations in diethylnitrosamine (DEN)-induced liver tumors in male F344 rats.

Alterations in multiple phosphorylation sites on exon 3 of the beta-catenin gene have recently been implicated in hepatocarcinogenesis in humans as well as mice. To identify genetic alterations which could be involved in the chemical-induced hepatocarcinogenesis of rats, we analyzed the status of the sites in the beta-catenin gene (Ctnnb1) of liver neoplasms induced by diethylnitrosamine (DEN) in male F344 rats, using the polymerase chain reaction-single strand conformation polymorphism method. In the present investigation, we examined 35 hepatocellular neoplasms (28 adenomas and 7 carcinomas) for the expression of mutations in the region of the beta-catenin gene. Point mutation at codon 32, 35, 37 or 41, which has been reported in human and mouse liver cell carcinomas and/or other cancers, was recognized in eleven (31%) out of 35 lesions (8 adenomas and 3 carcinomas). Our results indicate that Ctnnb1 mutations may contribute to hepatocarcinogenesis in rats. Our finding that Ctnnb1 mutation was present in adenomas as well as carcinomas also suggests that the mutation is a relatively early event in DEN-induced hepatocarcinogenesis in rats.

Adenoma↗
PubMed1999

Effect of 5-(N,N-Dimethyl)-amiloride, a specific inhibitor of Na(+)/H(+) exchanger, on the palmitoyl-L-carnitine-induced mechanical and metabolic derangements in the isolated perfused rat heart.

The present study was carried out to determine the effect of 5-(N, N-dimethyl)-amiloride (DMA), a specific inhibitor of the Na(+)/H(+) exchanger, on the cardiac mechanical and metabolic derangements induced by palmitoyl-L-carnitine (PALCAR). Rat hearts were perfused aerobically at a constant flow according to the Langendorff technique, while being paced electrically. PALCAR (5 micromol/l) increased the left-ventricular end-diastolic pressure, decreased the left ventricular developed pressure (i.e. mechanical dysfunction), decreased the tissue level of ATP and increased the tissue level of AMP (i.e. metabolic change). DMA (10 or 20 micromol/l) attenuated the mechanical and metabolic alterations induced by PALCAR in a concentration-dependent way. Nevertheless, DMA (10 or 20 micromol/l) did not affect the mechanical function or energy metabolism in the normal (PALCAR-untreated) heart. These results suggest that inhibition of the Na(+)/H(+) exchanger with DMA is effective in attenuating the PALCAR-induced mechanical and metabolic derangements in the heart.

Amiloride↗
PubMed1999

Inhibition of Na+ channel or Na+/H+ exchanger attenuates the hydrogen peroxide-induced derangements in isolated perfused rat heart.

The effect of tetrodotoxin, a specific inhibitor of the Na+ channel, and 5-(N,N-dimethyl)-amiloride, a specific inhibitor of the Na+/H+ exchanger, on the mechanical and metabolic derangements induced by hydrogen peroxide (H2O2) was studied in the isolated perfused rat heart. The isolated rat heart was perfused aerobically at a constant flow rate and driven electrically. H2O2 (600 microM) decreased the left ventricular developed pressure and increased the left ventricular end-diastolic pressure (i.e. mechanical dysfunction), decreased the tissue levels of adenosine triphosphate and adenosine diphosphate (i.e. metabolic derangement), and increased the tissue level of malondialdehyde (i.e. lipid peroxidation). These mechanical and metabolic derangements induced by H2O2 were significantly attenuated by tetrodotoxin (3 microM) or 5-(N,N-dimethyl)-amiloride (15 microM). Neither tetrodotoxin nor 5-(N,N-dimethyl)-amiloride modified the tissue malondialdehyde level, which was increased by H2O2. In the normal (H2O2-untreated) heart, neither tetrodotoxin nor 5-(N,N-dimethyl)-amiloride affected the mechanical function and energy metabolism. These results suggested that inhibition of the Na+ channel or Na+/H+ exchanger was effective in attenuating the H2O2-induced mechanical dysfunction and metabolic derangements in the isolated perfused rat heart.

Amiloride↗
PubMed1999

Inhibition of human aldehyde reductase by drugs for testing the function of liver and kidney.

Drugs for testing the function of liver and kidney (sulfobromophthalein, phenolsulfonphthalein, indigo carmine and indocyanine green) and other organic anions (rose bengal and haematin) were found to potently inhibit human liver aldehyde reductase that is involved in the detoxification of 3-deoxyglucosone and methylglyoxal, reactive intermediates, during the formation of advanced glycation end products. The inhibition patterns by the compounds were non-competitive with respect to both coenzyme (NADPH) and substrate (D-glucuronate). The kinetics of the inhibition by a mixture of the 2 inhibitors suggests that all the inhibitory compounds bind to overlapping sites on the enzyme. The binding of rose bengal, sulfobromophthalein and phenylsulfonphthalein to the free enzyme was detected by ultrafiltration assay. However, in the reverse reaction, the enzyme was inhibited competitively with respect to the alcohol substrate by rose bengal, haematin, phenolsulfonphthalein, sulfobromophthalein, indigo carmine and indocyanine green, which showed Ki values of 0.1, 1, 3, 4, 4 and 10 microM, respectively. The results suggest that these potent inhibitors bind weakly to the free enzyme and tightly to the enzyme-NADP binary complex.

Aldehyde Reductase↗
PubMed1999

Protective effect of quinaprilat, an active metabolite of quinapril, on Ca2+-overload induced by lysophosphatidylcholine in isolated rat cardiomyocytes.

We examined the effects of quinaprilat, an active metabolite of quinapril (an angiotensin converting enzyme (ACE) inhibitor) on the increase in intracellular concentration of Ca2+ ([Ca2+]i) (Ca2+-overload) induced by lysophosphatidylcholine (LPC) in isolated rat cardiomyocytes. LPC (15 microM) produced Ca2+-overload with a change in cell-shape from rod to round. Quinaprilat but not quinapril at 20 or 50 microM attenuated the LPC-induced increase in [Ca2+]i and the change in cell-shape in a concentration-dependent manner. Since quinaprilat has an inhibitory action on ACE and quinapril has practically no inhibitory action on ACE, it is likely that the inhibitory action of quinaprilat on ACE is necessary for the protective effect of the drug against LPC-induced changes. We therefore examined the effects of enalapril (another ACE inhibitor with the weak inhibitory action on ACE) and enalaprilat (an active metabolite of enalapril with an inhibitory action on ACE) on the LPC-induced changes. Both enalapril and enalaprilat attenuated the LPC-induced Ca2+-overload, suggesting that the inhibitory action on ACE may not mainly contribute to the protective effect of ACE inhibitors against LPC-induced Ca2+-overload. This suggestion was supported by the fact that neither ACE (0.2 U/ml) nor angiotensin II (0.1-100 microM) increased [Ca2+]i in isolated cardiomyocytes. Furthermore, application of bradykinin (0.01-10 microM) did not enhance the protective effect of quinaprilat against LPC-induced changes. LPC also increased release of creatine kinase (CK) from the myocyte markedly, and quinaprilat but not quinapril attenuated the LPC-induced CK release. Unexpectedly, both enalapril and enalaprilat did not attenuate the LPC-induced CK release. Neither quinapril nor quinaprilat changed the critical micelle concentration of LPC, suggesting that these drugs do not directly bind to LPC. We conclude that quinaprilat attenuates the LPC-induced increase in [Ca2+]i, and that the protective effect of quinaprilat on the LPC-induced change may not be related to a decrease in angiotensin II production or an increase in bradykinin production.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1999

The protective effect of cyclosporine A on anti-Fas antibody-induced hepatitis in mice.

The effect of cyclosporine A (CsA) on anti-Fas antibody-induced hepatitis was studied. The administration of anti-Fas antibody (250 microg/kg) to mice elevated plasma alanine aminotransferase (ALT) activity at 3 hr. This anti-Fas antibody-induced elevation of ALT was inhibited by treatment with CsA (10, 30 and 100 mg/kg) in a dose-dependent manner. Anti-Fas antibody administration elevated CPP32-like protease activity at 3 hr in mouse liver, and this elevation of CPP32-like activity was inhibited by treatment with CsA. The present results show that CsA treatment inhibits the anti-Fas antibody-induced apoptotic process of hepatitis, at least in part, by affecting a reaction upstream of CPP32-like protease activation.

Alanine Transaminase↗
PubMed1999

Suppressive effects of chlorogenic acid on N-methyl-N-nitrosourea-induced glandular stomach carcinogenesis in male F344 rats.

The modifying effects of chlorogenic acid (CA) on N-methyl-N-nitrosourea (MNU)-induced glandular stomach carcinogenesis were investigated in five groups of male F344 rats. Rats in Groups 1 through 3 were given MNU in drinking water at a concentration of 400 ppm for 12 weeks. Animals of Group 1 were then kept on the basal diet alone, and those of Group 2 or 3 were fed a diet containing 500 or 250 ppm CA for a subsequent 22 weeks. Group 4 was exposed to CA alone through the experimental period (36 weeks), and Group 5 was given the basal diet continuously and treated as a control. At the end of the experiment, the incidence of glandular stomach carcinoma of Group 3 was significantly smaller than that of Group 1 (p < 0.03). The incidence of adenomatous hyperplasia of Group 2 was also significantly lower than that of Group 1 (p < 0.02). In addition, the proliferating cell nuclear antigen (PCNA) labeling index of the epithelial cells from the non-neoplastic mucosa in rats of Group 2 or 3 was significantly smaller than that of Group 1 (p < 0.0001). These results suggest that CA has a chemopreventive effect on MNU-induced rat glandular stomach carcinogenesis by exposure during the post-initiation phase, and CA may be a promising agent for prevention of human stomach cancer.

Animals↗
PubMed1999

Characterization of a novel variant (S145C/L311V) of 3alpha-hydroxysteroid/dihydrodiol dehydrogenase in human liver.

Human liver 3alpha-hydroxysteroid/dihydrodiol dehydrogenase (DD) is involved in the metabolism of steroid hormones and polycyclic aromatic hydrocarbons, and is also responsible for the reduction of ketone-containing drugs. To account for the interindividual difference in the activity, we isolated and characterized clones for the human liver enzymes. The sequence of the cDNA clone coding for the variant differed from that coding for the wild-type DD by two nucleotides (substitutions of C with G at positions 434 and 931) which caused two amino acid replacements, Ser145 to Cys (S145C) and Leu311 to Val (L311V). The heterologous expression of the variant mRNA was confirmed in four of 31 liver samples from Japanese by an allele-specific polymerase chain reaction. The effects of the mutations on the catalytic properties were examined with the recombinant enzymes expressed in Escherichia coli. The introduction of S145C/L311V double mutations resulted in three- to five-fold decreased activities for xenobiotic and steroidal substrates, whereas no significant change was observed by an introduction of the S145C mutation alone. The results substantiate the existence of polymorphic forms for human liver DD, and also suggest the importance of the residue at position 311 for substrate binding to the enzyme.

Base Sequence↗
PubMed1998

Roles of the C-terminal domains of human dihydrodiol dehydrogenase isoforms in the binding of substrates and modulators: probing with chimaeric enzymes.

Human liver dihydrodiol dehydrogenase (DD; EC 1.3.1.20) exists in isoforms (DD1, DD2 and DD4) composed of 323 amino acids. DD1 and DD2 share 98% amino acid sequence identity, but show lower identities (approx. 83%) with DD4, in which a marked difference is seen in the C-terminal ten amino acids. DD4 exhibits unique catalytic properties, such as the ability to oxidize both (R)- and (S)-alicyclic alcohols equally, high dehydrogenase activity for bile acids, potent inhibition by steroidal anti-inflammatory drugs and activation by sulphobromophthalein and clofibric acid derivatives. In this study, we have prepared chimaeric enzymes, in which we exchanged the C-terminal 39 residues between the two enzymes. Compared with DD1, CDD1-4 (DD1 with the C-terminal sequence of DD4) had increased kcat/Km values for 3alpha-hydroxy-5beta-androstanes and bile acids of 3-9-fold and decreased values for the other substrates by 5-100-fold. It also became highly sensitive to DD4 inhibitors such as phenolphthalein and hexoestrol. Another chimaeric enzyme, CDD4-1 (DD4 with the C-terminal sequence of DD1), showed the same (S)-stereospecificity for the alicyclic alcohols as DD1, had decreased kcat/Km values for bile acids with 7beta- or 12alpha-hydroxy groups by more than 120-fold and was resistant to inhibition by betamethasone. In addition, the activation effects of sulphobromophthalein and bezafibrate decreased or disappeared for CDD4-1. The recombinant DD4 with the His314-->Pro (the corresponding residue of DD1) mutation showed intermediate changes in the properties between those of wild-type DD4 and CDD4-1. The results indicate that the binding of substrates, inhibitors and activators to the enzymes is controlled by residues in their C-terminal domains; multiple residues co-ordinately act as determinants for substrate specificity and inhibitor sensitivity.

Amino Acid Sequence↗
PubMed1998

Removal of hydroxyl radical facilitates Ca2+-dependent [3H]GABA release by peroxynitrite.

We investigated mechanisms for enhancement of peroxynitrite (OONO-; 5 microM)-evoked [3H] gamma-aminobutyric acid (GABA) release. Hydroxyl radical scavengers such as N,N'-dimethylthiourea (DMTU), mannitol, and uric acid, significantly increased OONO--evoked [3H]GABA release, whereas urea showed no effects on the release. Removal of Ca2+ from incubation buffer abolished the enhancement of the release by DMTU, although DMTU showed no effects on the basal release with and without Ca2+ in extracellular space. These results indicate that hydroxyl radical scavengers facilitate OONO--evoked [3H]GABA release dependent on Ca2+.

Animals↗
PubMed1998

Evidence for the presence of multiple forms of Sph kinase in human platelets.

The intracellular distribution of sphingosine (Sph) kinase activity was examined in human platelets. A large proportion (72%) of the total activity was found to be associated with the membrane fraction, and the membrane-associated fraction had higher specific activity compared with the cytosolic enzyme. Most of the membrane-associated activity could be extracted with 1 M NaCl. The cytosolic activity was unstable upon heat treatment, with 80% of the activity being lost during incubation at 45 degreesC for 1 h, whereas the NaCl-extractable fraction was stable under the same conditions. When subjected to Mono Q column chromatography, the cytosolic fraction produced two activity peaks and the NaCl-extractable fraction gave a single peak. These three Sph kinase activities showed different responses to stimulation by beta-octylglucoside and inhibition by N, N-dimethylsphingosine and l-threo-dihydrosphingosine, suggesting the presence of multiple enzyme forms in human platelets.

Blood Platelets↗
PubMed1998

Purification of the precursors to vitelline envelope proteins from serum of Sakhalin taimen, Hucho perryi.

High and low molecular weight vitelline envelope-related proteins (hVERP and lVERP) were purified from serum of vitellogenic female Sakhalin taimen (Hucho perryi) by a combination of ion-exchange, hydroxylapatite and gel filtration chromatography. The molecular weight of hVERP was estimated to be 83 kDa by gel filtration, and 48 kDa and 54 kDa in SDS-PAGE under non-reduced and reduced conditions, respectively. The molecular weight of lVERP was 56 kDa by gel filtration, and 42 kDa and 46 kDa on SDS-PAGE (non-reduced and reduced, respectively). Amino acid composition of hVERP was characterized by high content of proline (15.9%) and glutamic acid (13.8%). The lVERP had high contents of glutamic acid (10.8%) and aspartic acid (10.5%). Specific antibodies against hVERP and against lVERP were prepared by immunizing rabbits. The antiserum to hVERP stained bands corresponding to 98 kDa and 48 kDa of vitelline envelope (VE) in SDS-PAGE without reduction, whereas the antiserum to lVERP immunostained 98 kDa and 42 kDa bands. Both specific antibodies recognized the vitelline envelope of vitellogenic oocytes immunocytochemically. Thus, hVERP and lVERP are precursors to vitelline envelope proteins in this species.

Amino Acids↗
PubMed1998

Transport of fragmented DNA in apical dendrites of gerbil CA1 pyramidal neurons following transient forebrain ischemia.

Transport of fragmented DNA in apical dendrites of the CA1 pyramidal neurons of gerbil hippocampus is observed in the apoptotic process following transient forebrain ischemia. The time-course of specific DNA fragmentation was examined after the ischemic insult by in situ nick-end-labeling method and fluorescence detection technique by DAPI. Although the role of the fragmented DNA movement is unclear, the transport mechanism of fragmented DNA is still active in the late phase of apoptotic process.

Animals↗
PubMed1998

Sequence of the cDNA of a human dihydrodiol dehydrogenase isoform (AKR1C2) and tissue distribution of its mRNA.

Human liver contains three isoforms (DD1, DD2 and DD4) of dihydrodiol dehydrogenase with 20alpha- or 3alpha-hydroxysteroid dehydrogenase activity; the dehydrogenases belong to the aldo-oxo reductase (AKR) superfamily. cDNA species encoding DD1 and DD4 have been identified. However, four cDNA species with more than 99% sequence identity have been cloned and are compatible with a partial amino acid sequence of DD2. In this study we have isolated a cDNA clone encoding DD2, which was confirmed by comparison of the properties of the recombinant and hepatic enzymes. This cDNA showed differences of one, two, four and five nucleotides from the previously reported four cDNA species for a dehydrogenase of human colon carcinoma HT29 cells, human prostatic 3alpha-hydroxysteroid dehydrogenase, a human liver 3alpha-hydroxysteroid dehydrogenase-like protein and chlordecone reductase-like protein respectively. Expression of mRNA species for the five similar cDNA species in 20 liver samples and 10 other different tissue samples was examined by reverse transcriptase-mediated PCR with specific primers followed by diagnostic restriction with endonucleases. All the tissues expressed only one mRNA species corresponding to the newly identified cDNA for DD2: mRNA transcripts corresponding to the other cDNA species were not detected. We suggest that the new cDNA is derived from the principal gene for DD2, which has been named AKR1C2 by a new nomenclature for the AKR superfamily. It is possible that some of the other cDNA species previously reported are rare allelic variants of this gene.

Base Sequence↗
PubMed1998

Citrus auraptene exerts dose-dependent chemopreventive activity in rat large bowel tumorigenesis: the inhibition correlates with suppression of cell proliferation and lipid peroxidation and with induction of phase II drug-metabolizing enzymes.

In our previous short-term experiment, Citrus auraptene inhibited the development of azoxymethane (AOM)-induced aberrant crypt foci, which are precursor lesions for colorectal carcinoma. In the present study, the possible inhibitory effect of dietary administration of auraptene was investigated using an animal colon carcinogenesis model with a colon carcinogen AOM. Male F344 rats were given s.c. injections of AOM (15 mg/kg body weight) once a week for 3 weeks to induce colon neoplasms. They also received diets containing 100 or 500 ppm auraptene for 4 weeks in groups of "initiation" feeding, starting 1 week before the first dosing of AOM. The diets containing auraptene were also given to rats for 38 weeks in groups of "postinitiation" feeding. At the termination of the study (38 weeks), dietary administration of auraptene caused dose-dependent inhibition in AOM-induced large bowel carcinogenesis. Auraptene feeding during the initiation phase reduced the incidence of colon adenocarcinoma by 49% at 100 ppm (P = 0.099) and 65% at 500 ppm (P = 0.0075). Auraptene administration during the postinitiation phase inhibited the incidence of colon adenocarcinoma by 58% at 100 ppm (P = 0.021) and 65% at 500 ppm (P = 0.0075). Also, the multiplicity of colon carcinoma was significantly reduced by initiation feeding at a dose level of 500 ppm (P < 0.01) and postinitiation feeding at a level of 100 and 500 ppm (P < 0.05 and P < 0.01, respectively). Feeding of auraptene suppressed the expression of cell proliferation biomarkers (ornithine decarboxylase activity and polyamine content) in the colonic mucosa and reduced the production of aldehydic lipid peroxidation [malondialdehyde and 4-hydroxy-2(E)-nonenal]. In addition, auraptene increased the activities of Phase II drug-metabolizing enzymes (glutathione S-transferase and quinone reductase) in the liver and colon. These findings suggest that the inhibitory effects of auraptene on AOM-induced colon tumorigenesis at the initiation level might be associated, in part, with increased activity of Phase II enzymes, and those at the postinitiation stage might be related to suppression of cell proliferation and lipid peroxidation in the colonic mucosa.

Aldehydes↗
PubMed1998

Relationship between magnitude of hypothermia during ischemia and preventive effect against post-ischemic DNA fragmentation in the gerbil hippocampus.

Protective effect of hypothermia against DNA fragmentation in hippocampal CA1 field after transient forebrain ischemia in gerbils was evaluated by changing the magnitude of hypothermia. Inhibition of DNA fragmentation was proportional to the magnitude of hypothermia. The result indicates that, in terms of susceptibility to ischemia, hippocampal CA1 neurons are sensitive to a relatively small decrement of temperature, with temperatures </=35 degreesC being critical for the prevention of apoptotic process following transient forebrain ischemia.

Animals↗