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Biomedical subjects

A Hara

Publications and source records attributed to A Hara.

At least 199 records · Page 11Linked to original sources
PubMed1997

Involvement of two basic residues (Lys-270 and Arg-276) of human liver 3 alpha-hydroxysteroid dehydrogenase in NADP(H) binding and activation by sulphobromophthalein: site-directed mutagenesis and kinetic analysis.

A human liver 3 alpha-hydroxysteroid dehydrogenase isoenzyme, a member of the aldo-keto reductase family, shows a marked preference for NADP(H) over NAD(H), and is activated by sulphobromophthalein, which increases the Km values for both NADP(H) and substrates. Here we report kinetic alterations in binding of the coenzymes and the activator to the enzyme caused by site-directed mutagenesis of Lys-270 and Arg-276, which are strictly conserved among the aldo-keto reductase family of enzymes. The mutated enzymes, K270M and R276M, showed increases in the Km for NADP+ of 22- and 290-fold respectively; the Km for alcohol substrate and the kca1 of the NADP(-)-linked reaction were also elevated, by 9- and 5-fold respectively. No kinetic constant of the NAD(+)-linked reaction was altered by more than 3-fold. Calculation of the free-energy changes showed that the 2'-phosphate group of NADP+ contributes 16.3 kJ/mol (3.9 kcal/mol) of binding energy to its interaction with the wildtype enzyme, and the mutagenesis to K270M and R276M destabilized the binding energy of NADP+ by 6.3 and 13.0 kJ/mol (1.5 and 3.1 kcal/mol) respectively. In addition, the mutations attenuated enzyme activation by sulphobromophthalein, which bound to the mutant enzymes as an inhibitor. The inhibition for the R276M mutant was competitive with respect to NADP+ and non-competitive with respect to the substrate, whereas that for the K270M mutant was mixed-type, showing activation at coenzyme concentrations greater than 20 x Km. These results suggest that the two basic residues in the 3 alpha-hydroxysteroid dehydrogenase isoenzyme play crucial roles in binding both the negatively charged 2'-phosphate group of NADP+ and the sulphonic groups of sulphobromophthalein.

3-Hydroxysteroid Dehydrogenases↗
PubMed1997

Switch of coenzyme specificity of mouse lung carbonyl reductase by substitution of threonine 38 with aspartic acid.

Mouse lung carbonyl reductase, a member of the short-chain dehydrogenase/reductase (SDR) family, exhibits coenzyme specificity for NADP(H) over NAD(H). Crystal structure of the enzyme-NADPH complex shows that Thr-38 interacts with the 2'-phosphate of NADPH and occupies the position spatially similar to an Asp residue of the NAD(H)-dependent SDRs that hydrogen-bonds to the hydroxyl groups of the adenine ribose of the coenzymes. Using site-directed mutagenesis, we constructed a mutant mouse lung carbonyl reductase in which Thr-38 was replaced by Asp (T38D), and we compared kinetic properties of the mutant and wild-type enzymes in both forward and reverse reactions. The mutation resulted in increases of more than 200-fold in the Km values for NADP(H) and decreases of more than 7-fold in those for NAD(H), but few changes in the Km values for substrates or in the kcat values of the reactions. NAD(H) provided maximal protection against thermal and urea denaturation of T38D, in contrast to the effective protection by NADP(H) for the wild-type enzyme. Thus, the single mutation converted the coenzyme specificity from NADP(H) to NAD(H). Calculation of free energy changes showed that the 2'-phosphate of NADP(H) contributes to its interaction with the wild-type enzyme. Changing Thr-38 to Asp destabilized the binding energies of NADP(H) by 3.9-4.5 kcal/mol and stabilized those of NAD(H) by 1.2-1.4 kcal/mol. These results indicate a significant role of Thr-38 in NADP(H) binding for the mouse lung enzyme and provide further evidence for the key role of Asp at this position in NAD(H) specificity of the SDR family proteins.

Alcohol Oxidoreductases↗
PubMed1997

Chemoprevention of 4-nitroquinoline 1-oxide-induced oral carcinogenesis in rats by flavonoids diosmin and hesperidin, each alone and in combination.

The modifying effects of the two flavonoids diosmin and hesperidin given during the initiation and postinitiation phases of oral carcinogenesis initiated with 4-nitroquinoline 1-oxide (4-NQO) were investigated in male F344 rats. The compounds were tested alone and in combination. At 6 weeks of age, animals were divided into experimental and control groups and fed diets containing 1000 ppm diosmin and 1000 ppm hesperidin and a diet containing both compounds (900 ppm diosmin and 100 ppm hesperidin). At 7 weeks of age, all animals except those treated with each test chemical alone and control groups were given 4-NQO (20 ppm) in the drinking water for 8 weeks to induce oral cancer. Starting 7 days before the 4-NQO exposure, groups of animals were fed the diets containing test chemicals for 10 weeks and then switched to the basal diet. Starting 1 week after the cessation of 4-NQO exposure, the groups given 4-NQO and a basal diet were switched to the diets containing diosmin, hesperidin, or diosmin combined with hesperidin and maintained on these diets for 22 weeks. The other groups consisted of rats given diosmin (1000 ppm), hesperidin (1000 ppm), and the combination regimen of these two compounds (900 ppm diosmin with 100 ppm hesperidin) alone, and untreated rats. All animals were necropsied at the termination of the study (week 32). The incidences of tongue lesions (neoplasms and preneoplasms), polyamine levels in the tongue tissue, and cell proliferation activity estimated by a 5-bromodeoxyuridine-labeling index and by morphometric analysis of silver-stained nucleolar organizer regions protein were compared among the groups. Feeding of both compounds singly or in combination during the initiation phase caused a significant reduction in the frequency of tongue carcinoma [diosmin, 68% reduction (P < 0.01); hesperidin, 75% reduction (P < 0.005); and the combination regimen, 69% (P < 0.05)]. When fed the test compounds singly or the combination regimen after 4-NQO exposure, the frequency of tongue cancer was also decreased [diosmin, 77% reduction (P < 0.005); hesperidin, 62% reduction (P < 0.05); and the combination regimen, 77% (P < 0.005)]. The incidences of oral preneoplasia (hyperplasia and dysplasia) in these groups were also decreased when compared with carcinogen controls (P < 0.05-P < 0.001). There were no pathological alterations in rats treated with test compounds or the combined regimen alone or those in an untreated control group. Dietary administration of these compounds significantly decreased the expression of cell proliferation biomarkers (5-bromodeoxyuridine-labeling index and silver-stained nucleolar organizer regions protein number) of the nonlesional tongue squamous epithelium (P < 0.05). Also, polyamine concentrations in the oral mucosa were lowered in rats given the carcinogen and test compounds, alone and in combination, compared with those of rats given 4-NQO alone (P < 0.05). These findings suggest that supplementation with the flavonoids diosmin and hesperidin, individually and in combination, is effective in inhibiting the development of oral neoplasms induced by 4-NQO, and such inhibition might be related to suppression of increased cell proliferation caused by 4-NQO in the oral mucosa.

4-Nitroquinoline-1-oxide↗
PubMed1997

Dominant hereditary conductive hearing loss due to an ossified stapedius tendon.

Familial conductive deafness is rare. This report confirms the existence of a lineage with congenital conductive hearing loss in 3 generations. The results of otologic evaluations, including pure-tone audiometry, tympanometry, acoustic reflex test, and liquid test, in 14 patients in this family were consistent with the findings of ossicular fixation in 10 patients. Tympanotomies performed in both ears of 4 patients indicated stapes ankylosis caused by ossification of the stapedius tendon. The patients gained normal hearing levels using a simple surgical procedure.

Adolescent↗
PubMed1997

Palmitoyl-L-carnitine modifies the myocardial levels of high-energy phosphates and free fatty acids.

Long-chain acylcarnitines, such as palmitoyl-L-carnitine (PALCAR), are known to accumulate in the myocardium during ischemia. We examined whether exogenous PALCAR modifies the myocardial levels of high-energy phosphates (HEP) and free fatty acids (FFA) in the heart, and whether d-cis-diltiazem and l-cis-diltiazem, an optical isomer having less potent Ca2+ channel blocking action than d-cis-diltiazem, attenuate the PALCAR-induced myocardial changes. Rat hearts were perfused aerobically at a constant flow according to the Langendorff's technique, while being paced electrically. PALCAR (5 microM) decreased the tissue levels of adenosine triphosphate and creatine phosphate and increased the tissue level of adenosine monophosphate, and produced mechanical dysfunction. In addition, PALCAR (5 microM) increased markedly the tissue levels of FFA, especially those of arachidonic and palmitoleic acids, and the release of creatine kinase (CK) from the myocardium. These alterations in the myocardial levels of HEP and FFA induced by PALCAR were significantly attenuated by d-cis-diltiazem (15 microM) or l-cis-diltiazem (15 microM). Both drugs also attenuated the PALCAR-induced CK release. The present study demonstrates that PALCAR modifies the tissue levels of HEP and FFA in the heart and that both d-cis- and l-cis-diltiazem protect the myocardium against the PALCAR-induced changes through mechanisms other than Ca2+ channel blocking action.

Adenine Nucleotides↗
PubMed1997

Germinoma in cerebral hemisphere associated with Down syndrome.

A Down syndrome patient with germinoma developing in the cerebral hemisphere is reported. A review of the literature yielded only 14 cases of Down syndrome with brain tumors, including our case. This finding of brain tumors in patients with Down syndrome may reflect chance occurrence. However, it is of interest in this regard that in 6 of the 14 (43%) reported cases the lesions were intracranial germ cell tumors.

Adolescent↗
PubMed1997

Lysophosphatidylcholine induces Ca2+-independent cellular injury attenuated by d-propranolol in rat cardiomyocytes.

In isolated rat cardiomyocytes, exogenous lysophosphatidylcholine (LPC) (15 microM) increased the intracellular Ca2+ concentration (Ca2+]i) from 72 +/- 5 to 3042 +/- 431 nM accompanied by cell injury as indicated by the hypercontracture of the cells and the increase in creatine phosphokinase (CPK) release. In order to understand whether the cell injury induced by LPC was a consequence of the elevation of [Ca2+]i, the effect of LPC was examined in the Ca2+-free solution containing EGTA. Under the Ca2+ -free conditions, LPC did not increase [Ca2+]i, whereas it still inflicted injury on the cells in terms of cell-shape change and CPK release to the same degree as that under the Ca2+-present condition. Addition of ryanodine (10 microM) failed to prevent the changes in cell-shape and CPK release induced by LPC under both Ca2+-free and Ca2+-present conditions. Preincubation of the myocytes with d-propranolol (50 microM) inhibited the LPC-induced changes in cell-shape and CPK release under both Ca2+ -free and Ca2+ -present conditions (p < 0.05). Our study provides clear evidence that the cellular injury induced by LPC could be independent of the increase in [Ca2+]i, and the Ca2+-independent cellular injury induced by LPC could be attenuated by d-propranolol, although the mechanism remains unknown.

Adrenergic beta-Antagonists↗
PubMed1997

Comparison of susceptibility to apoptosis induced by rhTNF-alpha and cycloheximide between human circulating and exudated neutrophils.

To determine whether exudated neutrophils differ from circulating ones in their apoptosis, rhTNF-alpha plus cycloheximide-induced apoptosis in human salivary neutrophils was compared to that in human neutrophils in peripheral blood. Concomitant treatment of peripheral blood neutrophils with rhTNF-alpha and cycloheximide-induced apoptosis in blood neutrophils within 3 hr, as evaluated both by light microscopic changes characteristic to apoptosis and by DNA fragmentation, whereas the same treatment failed to induce any apoptosis in salivary neutrophils. These results indicate that the exudation of neutrophils from blood into tissue is associated with marked changes in their functions such as alteration in their sensitivity to apoptosis-inducing stimuli.

Apoptosis↗
PubMed1997

Development of an ELISA for chum salmon (Oncorhynchus keta) growth hormone.

A specific and sensitive enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of low levels of serum growth hormone (GH) in chum salmon (Oncorhynchus keta). The antiserum to GH (a-rsGH) was obtained from a rabbit immunized with recombinant chum salmon GH. The noncompetitive ELISA was performed by a sandwich method using a-rsGH rabbit IgG as the first antibody, its biotinylated Fab' fragment as the second antibody, and the avidin-biotin reaction for signal amplification. This assay could be run in 3 days and routinely detected GH at concentrations as low as 0.5 ng/ml. The development of an ELISA for GH made possible quantification of serum GH levels. In this assay system, parallel dilution curves were obtained using purified chum salmon GH and GH's from several species of the genus Oncorhynchus.

Animals↗
PubMed1997

Transition of serum vitellogenin cycle in Sakhalin taimen (Hucho perryi).

A specific and sensitive enzyme-linked immunosorbent assay (ELISA) and a single radial immunodiffusion (SRID) were developed for measurement of serum vitellogenin (Vg) levels in Sakhalin taimen (Hucho perryi). Regarding specificity for serum Vg, an antiserum raised against lipovitellin of taimen (a-Lv) was adequate for both assays. ELISA and SRID could detect Vg in serum at concentrations as low as 10 ng/ml and 25 micrograms/ml, respectively. In estrogen administration experiments, the level of serum Vg began clearly increasing within 12 to 24 hr after injection of immature females with estradiol-17 beta (E2). The appearance and levels of Vg in males treated with E2 were delayed and smaller, respectively, than for females. Vg levels varied throughout natural vitellogenesis from 0-4 micrograms/ml (3 years old) to approximately 30 mg/ml (5-6 years old). We observed an early transitory peak of serum Vg levels (primary reaction) at the time of early vitellogenesis and chronic high Vg levels (for 6-7 months) in winter period before ovulation. Changes of serum E2 levels were correlated with Vg levels. However, E2 levels decreased a month earlier than Vg levels near ovulation. It appears that the duration of vitellogenesis in taimen is considerably longer than that in other salmonids, lasting more than 2 years.

Animals↗
PubMed1997

Clinical trial of external beam-radiotherapy combined with daily administration of low-dose cisplatin for supratentorial glioblastoma multiforme--a pilot study.

This trial tested the assumed efficacy and safety of external beam-radiotherapy combined with daily administration of low dose cisplatin (CDDP) (ERCLC therapy) for patients with glioblastoma multiforme (GBM). Thirty adult patients with supratentorial GBM received daily postoperative treatment with low dose intravenous CDDP (4-6 mg/m2) administered 30 minutes before external irradiation. In 10 patients, intraoperative radiotherapy (IORT) following surgery was given prior to ERCLC therapy. Tumor response on MRI, interval to tumor progression, survival, and toxicities were analyzed. None of the patients showed a tumor response to ERCIC therapy. Overall, the median time to tumor progression was 6 months with a 1-year tumor progression-free rate of 26.7% and a 2-year rate of 0%. The median survival time was 15 months with a 1-year survival rate of 69.9% and a 2-year rate of 31.5%. The survival rate of patients with IORT was better than that of those without IORT, however, there was no significant difference. Anorexia associated with nausea occurred in 70% and general fatigue in 10.0%. Leukopenia and thrombocytopenia occurred in 26.7% and 33.3%, respectively. However, none of the patients had to be withdrawn from therapy due to these toxicities. Other toxicities were not observed. This clinical study showed that daily administration of low dose CDDP did not enhance tumor response to irradiation for GBM on MRI. Regarding toxicity, however, ERCLC therapy was well tolerated. Although this trial did not provide sufficient data to determine whether ERCLC therapy was effective for GBM due to the small number of patients, additional clinical trials of this therapy may be warranted because that the survival rate in this study was equal to the better results recently reported for newly diagnosed GBM.

Adult↗
PubMed1997

Expression of Bax and bcl-2 proteins, regulators of programmed cell death, in human brain tumors.

Expression of Bcl-2, a programmed cell death (PCD)-suppressing molecule, and Bax, the Bcl-2 related PCD-accelerating protein was investigated in varied human brain tumors. Thirty-six cases of human brain tumors comprising 4 astrocytomas, 3 anaplastic astrocytomas, 4 glioblastomas multiforme, 5 medulloblastomas, 1 ependymoma, 2 choroid plexus papilloma, 1 ganglioglioma, 1 central neurocytoma, 4 meningotheliomatous meningiomas, 3 transitional meningiomas, 4 fibroblastic meningiomas, 3 acoustic neurinomas and 1 craniopharyngioma were analyzed for the localization of Bax and Bcl-2 proteins. No relationship between the degree of the histological malignancy and the presence of Bax or Bcl-2 proteins was found in varied human brain tumors. However, it is suggested that reduced expression of Bax protein is necessary for the malignant transformation and progression of the brain tumors, since no histologically malignant brain tumors with positive Bax protein were present. Our findings indicate that the expression pattern of Bax and Bcl-2 may reflect histogenetic difference of each type of brain tumors.

Adolescent↗
PubMed1997

Expression of Bax and Bcl-2 protein in the gerbil hippocampus following transient forebrain ischemia and its modification by phencyclidine.

To determine the effect of phencyclidine (a noncompetitive NMDA receptor antagonist) on expression of Bax and Bcl-2 (apoptosis-regulating proteins) in gerbil hippocampus after transient forebrain ischemia, brain sections were immunohistochemically evaluated 48, 72, 96 h and 7 days following ischemia. In ischemic control animals, the expression of Bax in CA1 neurons was increased with time and peaked at 72 h, then disappeared at 96 h. In the phencyclidine (5 mg kg-1, intraperitoneally)-treated animals, the intensity of Bax expression at 72 h was weaker than that of ischemic control animals. Furthermore, at 96 h, Bax expression was still observed in CA1 neurons. No expression of Bcl-2 in the CA1 neurons was detected in either control or phencyclidine-treated animals. From these results, it is possible that NMDA receptor antagonists exert their preventive effect against delayed neuronal death through inhibition of Bax protein expression, although the precise relationship between the function of Bax protein and delayed neuronal death is still unclear.

Animals↗
PubMed1997

Citrus auraptene inhibits chemically induced colonic aberrant crypt foci in male F344 rats.

The modifying effect of dietary administration of auraptene isolated from the peel of citrus fruit (Citrus natsudaidai Hayata) on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) was investigated in rats. Male F344 rats were given s.c. injections of AOM (15 mg/kg body wt) once a week for 3 weeks to induce ACF. They also received diets containing 100 or 500 p.p.m. auraptene for 5 weeks, starting 1 week before the first dose of AOM. At termination of the study (week 5) dietary administration of auraptene caused a significant reduction in the frequency of ACF in a dose-dependent manner (P < 0.05). Feeding of auraptene suppressed expression of cell proliferation biomarkers (5-bromo-2'-deoxyuridine labeling-index, ornithine decarboxylase activity, polyamine content and number of silver stained nucleolar organizer region protein particles) in the colonic mucosa and the occurrence of micronuclei caused by AOM. Also, auraptene increased the activities of phase II enzymes (glutathione S-transferase and quinone reductase) in the liver and colon. These findings might suggest that inhibition of AOM-induced ACF may be associated, in part, with increased activity of phase II enzymes in the liver and colon and suppression of cell proliferation in the colonic mucosa.

Animals↗
PubMed1997

Modulation of N-methyl-N-amylnitrosamine-induced rat oesophageal tumourigenesis by dietary feeding of diosmin and hesperidin, both alone and in combination.

The modifying effects of flavonoids diosmin and hesperidin during the initiation and post-initiation phases of oesophageal carcinogenesis initiated with N-methyl-N-amylnitrosamine (MNAN) were investigated in male Wistar rats. At 7 weeks of age, all animals except those treated each test chemical alone and control groups were given weekly intraperitoneal injections of MNAN (12.5 mg/kg body weight/injection) for 12 weeks to induce oesophageal neoplasms. For examining the modifying effects of 'initiation' treatment of test compounds, groups of animals were fed the diets containing 1000 ppm diosmin and 1000 ppm hesperidin, and the diet containing both compounds (900 ppm diosmin and 100 ppm hesperidin) for 13 weeks, starting 7 days before the MNAN dosing and then switched to the basal diet. For examining the modifying effects of 'post-initiation' treatment of these compounds, the groups given MNAN and a basal diet were switched to the experimental diets containing diosmin, hesperidin or diosmin combined with hesperidin at 1 week after the stop of MNAN injection, and maintained on these diets for 7 weeks. The other groups consisted of rats given test compounds alone or untreated rats. All animals were necropsied at the termination of the study (week 20) to determine the incidences of oesophageal neoplasms and preneoplasms, blood polyamine levels, and cell proliferation activity estimated by 5-bromodeoxyuridine (BrdU)-labelling index and by morphometric analysis of silver-stained nucleolar organizer regions' protein (AgNORs). A number of oesophageal neoplasms developed in rats treated with MNAN alone (75% and 100% incidences of carcinoma and papilloma, respectively). 'Initiation' feeding of diosmin significantly decreased the incidence of squamous cell carcinoma (P < 0.05). Also, 'initiation feeding' of both compounds singly or in combination caused a significant reduction in the multiplicities of oesophageal carcinoma and papilloma (diosmin, 78 and 58% reduction; hesperidin, 70 and 50% reduction; and the combination regimen, 70 and 30% reduction, P < 0.005). 'Post-initiation' feeding slightly decreased the multiplicities of these oesophageal neoplasms. Also, these dietary regimens reduced the multiplicities of preneoplastic lesions (hyperplasia and severe dysplasia; P < 0.05). There were no pathological alterations in rats treated with both compounds alone or the combined regimen alone or those in an untreated control group. Similarly, feeding of these compounds significantly decreased the expression of cell proliferation biomarkers (BrdU-labelling index and AgNORs number) of the non-lesional oesophageal epithelium (P < 0.05). Blood polyamine concentrations were also lowered in rats given the carcinogen and test compounds, both alone and in combination, when compared with those of rats given MNAN alone (P < 0.05). These findings suggest that diosmin and hesperidin supplementation, individually or in combination, is effective in inhibiting the development of oesophageal cancer induced by MNAN when given during the initiation phase, and such inhibition might be related to suppression of increased cell proliferation caused by MNAN in the oesophageal mucosa.

Administration, Oral↗
PubMed1997

A xanthine oxidase inhibitor 1'-acetoxychavicol acetate inhibits azoxymethane-induced colonic aberrant crypt foci in rats.

The modifying effect of dietary administration of a xanthine oxidase inhibitor 1'-acetoxychavicol acetate (ACA) present in an edible plant Languas galanga in Thailand on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) was investigated in rats. Male F344 rats were given s.c. injections of AOM (15 mg/kg body wt) once a week for 3 weeks to induce colonic ACF. They were fed the diets containing 100 or 200 ppm ACA for 5 weeks, starting 1 week before the first dosing of AOM. At the termination of the study (week 5), AOM induced 118 +/- 28 ACF/colon. Dietary administration of ACA caused significant reduction in the frequency of ACF (41% inhibition by 100 ppm ACA feeding and 37% inhibition by 200 ppm ACA feeding, P<0.01). Such inhibition might be associated with suppression of the proliferation biomarkers' expression such as ornithine decarboxylase activity in the colonic mucosa, number of silver-stained nucleolar organizer regions' protein in the colonic mucosal cell nuclei and blood polyamine content. These results indicate that ACA could inhibit the development of AOM-induced ACF through its suppression of cell proliferation in the colonic mucosa and ACA might be a possible chemopreventive agent against colon tumourigenesis.

Animals↗