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Biomedical subjects

M Hirose

Publications and source records attributed to M Hirose.

At least 271 records · Page 15Linked to original sources
PubMed1997

A novel method for making nested deletions and its application for sequencing of a 300 kb region of human APP locus.

We developed a novel in vitro method for making nested deletions and applied it to a large-scale DNA sequencing. A DNA fragment to be sequenced (up to 15 kb long) was cloned with a new vector possessing two unique Sfi I sites, digested by Sfi I and ligated to generate a large head-to-tail concatemer. The large concatemer was randomly fragmented by sonication and then redigested by Sfi I to separate insert and vector DNAs. The fragments of various length were then cloned into the other vector(s) specifically designed for selective cloning of insert-derived DNA fragments to generate a library of nested deletions. This method allowed a single person to generate >20 nested deletion libraries sufficient to cover 100 kb in a few days. We applied the method for sequencing of P1 clones and successfully determined the complete sequence of approximately 300 kb of the human amyloid precursor protein (APP) locus on chromosome 21 with a redundancy of 3.8, reasonably low cost and very few gaps remaining to be closed. Development of some new instruments and software is also described which makes this method more applicable for large-scale sequencing.

Amyloid beta-Protein Precursor↗
PubMed1997

Effects of n-tritriacontane-16,18-dione, curcumin, chlorphyllin, dihydroguaiaretic acid, tannic acid and phytic acid on the initiation stage in a rat multi-organ carcinogenesis model.

The modifying effects of the naturally occurring antioxidants n-tritriacontane-16,18-dione (TTAD), curcumin, dihydroguaiaretic acid (DHGA), chlorophyllin, tannic acid and phytic acid on the initiation stage in a rat multi-organ carcinogenesis model were examined in male F344 rats. Animals were initiated with two i.p. injections of 2,2'-dihydroxy-di-n-propylnitrosamine (DHPN), followed by two i.g. administrations of N-ethyl-N-hydroxyethylnitrosamine (EHEN), and then three s.c. injections of 3,2'-methyl-4-aminobiphenyl (DMAB) during the first 3 weeks. Starting 1 day before the first carcinogen application, groups of rats received diet containing one of the antioxidants (0.2% TTAD, the others at 1% each) until 1 week after the last carcinogen exposure. Surviving animals were killed and complete autopsies were performed at the end of week 36. Histological examination revealed no inhibitory effects in terms of the multiplicities and/or incidences of neoplastic lesions in any of the organs examined, other than a significant increase in seminal vesicle atypical hyperplasia observed in rats treated with tannic acid. Thus, the antioxidants, with the exception of tannic acid, did not show any modifying effects on the initiation stage in the present multi-organ carcinogenesis model and at the present dose levels applied.

Aminobiphenyl Compounds↗
PubMed1997

Refolding process of ovalbumin from urea-denatured state. Evidence for the involvement of nonproductive side chain interactions in an early intermediate.

Ovalbumin contains one cystine disulfide (Cys73-Cys120) and four cysteine sulfhydryls (Cys11, Cys30, Cys367, and Cys382) in a single polypeptide chain of 385 amino acid residues. The refolding mechanism of ovalbumin was investigated under disulfide-bonded and disulfide-reduced conditions using the denatured protein state, DA, as the starting protein sample. For the preparation of DA, the disulfide-intact and disulfide-reduced forms of ovalbumin were denatured by protein incubation in 9 M urea at pH 2.2. When DA was placed in a refolding buffer, pH 8.2, an intermediate state IN was produced in either the disulfide-bonded or the disulfide-reduced condition; IN showed about 60% of the native CD ellipticity at 222 nm and the intrinsic tryptophan fluorescence with the native spectrum peak but with decreased intensity. The formation of IN as detected by far UV CD ellipticity was quite rapid and finished within a mixing dead time of 20 ms. When DA was diluted with an acidic buffer, pH 2.2, a partially folded equilibrium intermediate IA with the structural characteristics equivalent to those of IN was formed. After the formations of IN and IA, the regains in CD ellipticity and tryptophan fluorescence at pH 8.2 followed biphasic kinetics in the disulfide-bonded condition but monophasic kinetics in the disulfide-reduced condition. As unexpected findings, the native disulfide in DA and IA underwent nonproductive disulfide rearrangements in the disulfide-bonded condition at an early refolding stage and then was recovered during the subsequent refolding. The integrity of overall refolding was confirmed by the observation that the proteins refolded for 20 h in the disulfide-bonded and disulfide-reduced conditions showed, on differential scanning calorimetry analyses, almost exactly the same denaturation temperatures as their native protein counterparts. These results were consistent with a refolding process for ovalbumin which includes nonproductive side chain-side chain interactions in the early intermediate IN, which requires subsequent reorganization for the correct refolding.

Animals↗
PubMed1997

Zatebradine inhibits tachycardia induced by bronchodilators without affecting respiratory resistance in dogs.

Bronchodilators used for bronchial asthma reduce respiratory resistance but also increase heart rate to some extent. It is often difficult to use such bronchodilators with elderly patients and patients with heart disease. The object of our study was to investigate whether a specific bradycardic agent, zatebradine, inhibited the heart rate increased by bronchodilators without affecting respiratory resistance. We evaluated the effects of zatebradine on the increases in heart rate and inhibition of the respiratory resistance in response to the bronchodilators, isoproterenol, procaterol (a beta 2-adrenoceptor agonist), 6-(3-dimethylaminopropionyl)-forskolin, NKH 477 (an adenylyl cyclase activator) and aminophylline in the anesthetized and artificially ventilated dog. When zatebradine in doses of 0.05-1.5 mg/kg i.v. decreased heart rate without affecting arterial blood pressure, it dose dependently attenuated the increase in heart rate in response to isoproterenol, procaterol, NKH 477 and aminophylline but did not affect the inhibition by these substances of the increase in respiratory resistance induced by histamine. Propranolol (0.01-0.3 mg/kg i.v.) dose dependently inhibited not only the increase in heart rate but also the inhibition of the respiratory resistance induced by isoproterenol and procaterol. The present results indicate that zatebradine selectively inhibits the increase in heart rate in response to cyclic AMP-dependent bronchodilators without affecting their bronchodilator effects in anesthetized dogs and suggest that zatebradine may be a useful drug for prevention of the tachycardia induced by bronchodilators used for patients with bronchial asthma.

Airway Resistance↗
PubMed1997

Effects of green tea catechins on the progression or late promotion stage of mammary gland carcinogenesis in female Sprague-Dawley rats pretreated with 7,12-dimethylbenz(a)anthracene.

Effects of the green tea catechins (GTCs) on the late promotion or progression stage of mammary gland carcinogenesis were examined in female Sprague-Dawley (SD) rats pretreated with 7,12-dimethylbenz(a)anthracene (DMBA). A total of 84 7-week-old rats received a 50 mg/kg body weight intra-gastric dose of DMBA, and starting 13 weeks thereafter, when the tumor incidence had reached 50%, three groups of 28 animals each were placed on diet containing 0.5% Polyphenon E (58.4% content (-)-epigallocatechin gallate (EGCG)) (groups 1a and 1b), 0.5% EGCG-80 (81% content of EGCG) (groups 2a and 2b) or basal diet alone (groups 3a and 3b) for 23 weeks. The experiment was terminated at week 36. The growth (i.e. change in mean diameter) of mammary tumors present at week 13 (groups 1a, 2a and 3a) was not influenced by the treatment with EGCGs, with no significant intergroup differences in the lesion incidences, multiplicity or size being observed. Values for these parameters did show a tendency for decrease in group 2b (Polyphenon E) as compared to group 3b (control) during the study, but they were not significantly reduced at the sacrifice time point. These results indicate that GTCs are not effective at inhibiting progression of rat mammary carcinogenesis, but Polyphenon E may exert a weak inhibitory effect on the early promotion stage.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1997

The prostate: a target for carcinogenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) derived from cooked foods.

Prostate tissues obtained from rats given a food-derived carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), at a dose of 400 ppm in the diet for 52 weeks were histopathologically evaluated and found to contain prostate carcinomas limited to the ventral lobe in 18 of 27 cases. Atypical hyperplasias were also detected in the ventral and anterior prostate as well as the seminal vesicles. 32P-Postlabeling analysis of DNA demonstrated that PhIP-DNA adducts are produced in all lobes of the prostate of rats receiving PhIP. The findings indicate that PhIP is carcinogenic to rat prostate in addition to the previously demonstrated targeting of the colon and mammary glands, providing evidence of a possible role of PhIP in human prostate carcinogenesis and highlighting the potential importance of PhIP for man.

Animals↗
PubMed1997

A rapid, useful and quantitative method to measure telomerase activity by hybridization protection assay connected with a telomeric repeat amplification protocol.

Telomerase, a ribonucleoprotein enzyme, is expected to be a new marker for cancer diagnosis. TRAP (the telomeric-repeat amplification protocol) developed by Kim et al. is a sensitive method to detect telomerase activity. Telomerase activity is detected by TRAP in most malignant cells in vivo and in vitro, but it is not found, or found only in very low amounts, in normal somatic cells and tissues. TRAP and its modified protocols are, however, not always suitable for measuring the activity of a large number of clinical samples to diagnose cancer, because they generally require a time-consuming detection step such as gel electrophoresis with radioactive materials. To improve the procedure for mass diagnosis, we applied a hybridization protection assay (HPA) to replace the detection step. HPA, which employs an acridinium-ester-labelled probe, is radioactivity-free, easy to handle without electrophoresis, quick, and applicable to a quantitative format. In this work we have established and demonstrated the advantages of TRAP/HPA. The telomerase activity of various primary and established cells, differentiating cancer cells, and normal and tumour colorectal and liver tissues was quantitatively analysed by TRAP/HPA. The results indicate that HPA combined with TRAP is a rapid and simple method, easy to handle and quantify, for the clinical diagnosis of cancer.

Cell Line↗
PubMed1997

Multiple ulcers of the ileum due to Cytomegalovirus infection in a patient who showed no evidence of an immunocompromised state.

68-year-old woman presented with abdominal pain and vomiting. After initial conservative therapy, laparotomy showed multiple ulcers of the ileum, one of which had perforated and adhered to the uterus. The affected segment of the ileum was resected. Numerous cytomegalic cells, corresponding to endothelia and macrophages, with intranuclear inclusion bodies, were found in microscopic sections of the ulcerated lesions. These findings were consistent with cytomegalic vasculitis and enteritis. Cytomegalovirus infections of the alimentary tract have been reported mainly in severely immunocompromised patients or those with predisposing disorders such as ulcerative colitis; their prognosis is usually poor. In our patient, there was no obvious immunocompromised state or other gastrointestinal disorders. The postoperative course has been uneventful for 2 years after surgery. The prognosis of Cytomegalovirus-associated lesions in the alimentary tract may be quite good in the immunocompetent patient.

Aged↗
PubMed1997

Effects of epidermal growth factor and insulin on migration and proliferation of primary cultured rabbit gastric epithelial cells.

We assessed the influence of epidermal growth factor (EGF) and insulin on gastric epithelial restoration in vitro. Rabbit gastric epithelial cells were cultured and formed a complete monolayer cell sheet in 2 days. We created a wound (1.8 +/- 0.05 mm2) by denuding an area of cells, and EGF (0.1-30 ng/ml) and/or insulin (1 nM-1 microM) was added. The restoration process, which included cell migration and proliferation, was monitored by measuring the cell-free area every 12 h for 2 days. Proliferating cells were detected by sequential staining with bromodeoxyuridine (BrdU). Control cells showed complete repair in 36-48 h and restoration was accelerated dose-dependently by EGF or insulin. EGF plus insulin further accelerated restoration, which was then completed in 12-24 h. EGF and/or insulin increased the number of BrdU- positive cells. The results indicated that EGF and insulin additively accelerated gastric epithelial wound repair by stimulating both the migration and the proliferation of gastric epithelial cells (particularly the former).

Animals↗
PubMed1997

Hypertrophic cardiomyopathy in a common wombat (Vombatus ursinus).

A case of hypertrophic cardiomyopathy (HCM) in a common wombat (Vombatus ursinus) is described. At necropsy, the heart showed symmetrical ventricular hypertrophy, a narrow left ventricular cavity, and dilatation of the left atrium. Microscopical findings in the ventricular myocardium included a strikingly disordered arrangement of cardiac muscle cells, in which adjacent cells were aligned perpendicularly and obliquely to each other, interstitial myocardial fibrosis, and arteriosclerosis of small intramural coronary arteries (fibromuscular hyperplasia). The changes, which were widespread in both ventricles, resembled those of HCM in man and other animals.

Animals↗
PubMed1997

Effects of metrifonate on impairment of learning and dysfunction of cholinergic neuronal system in basal forebrain-lesioned rats.

Several studies have indicated the possibility of using cholinesterase (ChE) inhibitors as therapeutic drugs for Alzheimer's disease. Metrifonate (MTF) is an organophosphorus compound that has been used in the treatment of schistosomiasis. In this study, we investigated the effects of MTF on the impairment of learning and memory, decreased ChE activity and extracellular acetylcholine (ACh) levels in basal forebrain (BF)-lesioned rats. The oral administration of MTF improved the BF-lesion-induced impairment of performance on passive avoidance task. Further, MTF reduced ChE activity in the cerebral cortex. In vivo brain microdialysis studies showed that MTF significantly increased the release of ACh, but decreased that of choline (Ch) in the cerebral cortex of BF-lesioned rats. These results indicated that MTF ameliorates the impairment of performance on passive avoidance task in BF-lesioned rats by increasing the extracellular ACh levels by inhibiting ChE. This suggested that MTF may be useful as a therapeutic drug for Alzheimer's disease.

Acetylcholine↗
PubMed1997

Characterization of a 180 kDa molecule apparently reactive with recombinant L-selectin.

In the present study we identified a 180 kDa molecule (p180) in rat lymph nodes (LN) apparently reactive with silkworm derived recombinant L-selectin (LEC-IgG) in a Ca(2+)-dependent manner. Analysis of amino acid sequence revealed that p180 has a strong homology to the macrophage mannose receptor (MMR), which was corroborated by the observation that p180 reacted with polyclonal anti-alveolar MMR antibody and mannosyl-BSA-agarose. In agreement with this notion, the binding of p180 to the silkworm LEC-IgG was inhibited by alpha-methyl-D-mannoside. However, in sharp contrast to its reactivity against the silkworm LEC-IgG, p180 failed to bind LEC-IgG produced by COS-7 cells, suggesting that p180 reacted with the silkworm LEC-IgG through the recognition of oligomannose-type oligosaccharides expressed on the silkworm products and that the lectin activity of L-selectin was not involved in the interaction. These results, together with the immunohistochemical studies showing that p180 was absent from the majority of high endothelial venules (HEV) but present in medullary macrophages, led us to conclude that p180 obtained from LN lysates by the use of the silkworm LEC-IgG is not a physiological ligand for L-selectin, warning against the use of recombinant proteins expressed in the baculovirus/ silkworm expression system for the detection of carbohydrate ligands.

Amino Acid Sequence↗
PubMed1997

Extradural buprenorphine suppresses breast feeding after caesarean section.

Satisfactory pain relief with postoperative extradural bupivacaine increases the amount of breast feeding after Caesarean section. To investigate the effect of extradural buprenorphine, we have evaluated the amount of breast feeding and the gain in infant weight for 11 days after Caesarean section in patients who received continuous extradural bupivacaine with or without buprenorphine. Extradural buprenorphine significantly decreased both measures although there was no significant difference in pain intensity. We suggest that extradural buprenorphine suppressed breast feeding after Caesarean section.

Analgesia, Epidural↗
PubMed1997

Refolding of urea-denatured ovalbumin that comprises non-native disulfide isomers.

Ovalbumin, which contains one cystine disulfide (Cys73-Cys120) and four cysteine sulfhydryls (Cys11, Cys30, Cys367, and Cys382) in the native state, undergoes intrachain sulfhydryl-disulfide exchanges at high concentrations of urea, generating many non-native disulfide isomers [E. Tatsumi, N. Takahashi, and M. Hirose (1994) J. Biol. Chem. 269, 28062-28067]. The refolding of ovalbumin from the urea-denatured state was investigated. When the denatured protein was diluted 20-fold with a refolding buffer (pH 8.2), an initial burst intermediate I(N) was produced within the 20 ms instrumental dead time; I(N) showed about 60% of the native CD ellipticity at 222 nm. The intrinsic tryptophan fluorescence of I(N) showed the same peak (338 nm), but with decreased intensity (57%), as compared to the native protein. After the rapid formation of I(N), most of the ovalbumin molecules correctly refolded into the native state with slow biphasic kinetics, as evaluated by far-UV CD, tryptophan fluorescence, and trypsin-resistance analyses. Furthermore, a peptide-mapping analysis revealed that sulfhydryl/disulfide exchange reactions occurred during the refolding, thereby increasing the formation of the native disulfide. The integrity of overall refolding was confirmed by a differential scanning calorimetry analysis. These data were consistent with the view that most, if not all, of the mispaired disulfide isomers in the urea-denatured ovalbumin can correctly refold into the native state via intrachain disulfide rearrangements.

Calorimetry, Differential Scanning↗
PubMed1997

Highly ordered molten globule-like state of ovalbumin at acidic pH: native-like fragmentation by protease and selective modification of Cys367 with dithiodipyridine.

Structural characteristics of ovalbumin at acidic pH were investigated by a variety of analytical approaches. At pH 2.2, the protein appeared to assume a partially denatured, molten globule-like conformation as evaluated by the binding of a hydrophobic probe, anilino-1-naphthalene-8-sulfonate. The protein was, however, resistant to proteolysis with pepsin under conditions in which the urea-denatured form was extensively hydrolyzed. Furthermore, under more drastic proteolytic conditions, the acid ovalbumin was specifically proteolyzed at the N-terminal site of Ala351, which is located in close proximity to the canonical serpin cleavage site Ala352-Ser353 that is known to be the cleavage site at neutral pH with subtilisin and elastase in native ovalbumin. Among the four cysteine residues (Cys11, Cys30, Cys367, and Cys382), which are all known to be buried in the native ovalbumin molecule, only Cys367 was specifically modified with 2,2'-dithiodipyridine, generating a mixed-disulfide protein derivative. Upon incubation of the derivative with a high concentration of L-cysteine, the thiopyridine mixed disulfide did not undergo any bimolecular exchange reaction with the thiol in the absence of an added denaturant, indicating that the mixed disulfide group is inaccessible. The far-UV CD spectra indicated that the native secondary structure is retained in either the modified or non-modified protein; but as evaluated by the near-UV CD spectra, the asymmetric nature of aromatic side chains in the non-modified ovalbumin and of the mixed-disulfide group in the modified protein was almost lost at pH 2.2. These results are consistent with a highly ordered molten globule-like state for OVA at pH 2.2, in which side chains, but not the backbone chain, significantly fluctuate.

2,2'-Dipyridyl↗
PubMed1997

Cellular proliferation and differentiation in rat atrophic gastric mucosa induced by N'-methyl-N'-nitro-N-nitrosoguanidine.

It has been demonstrated that mucosal cell proliferation in atrophic gastritis is significantly accelerated, although the dynamics of the cell proliferation and differentiation have not been well characterized. We sequentially analyzed the markers of proliferation and differentiation of rat atrophic gastritis induced by MNNG. Immunohistochemical staining by anti-BrdU, anti-PCNA, and anti-PDGF-BB antibodies demonstrated that cell proliferation in atrophic gastritis was accelerated not only in the parenchymal tissue but also in the mesenchymal tissue. Expression of a gap junction protein (connexin 32), which is a marker for differentiation of epithelial cells, was reduced during the progression of atrophy. Some apoptotic cells were observed in the lower to middle third of the atrophic mucosa, whereas apoptotic cells were rarely seen in normal mucosa, which suggests that apoptosis in these parts of the mucosa may be related to the occurrence of mucosal atrophy.

Animals↗
PubMed1997

Effects of PACAP-38 on the SA nodal pacemaker activity in autonomically decentralized hearts of anesthetized dogs.

Pituitary adenylate cyclase-activating polypeptide (PACAP) receptors exist, but the physiologic role of PACAP is unclear in the heart in situ. We investigated effects of PACAP-38 on sinus rate and on the negative chronotropic response to acetylcholine (ACh) or stimulation of the intracardiac parasympathetic nerve fibers to the sinoatrial nodal region in the automatically decentralized heart of the open chest, anesthetized dog. PACAP-38 (0.1-1 nmol) injected directly into the sinus node artery caused transient positive followed by negative chronotropic responses. Both pretreatment with atropine and tetrodotoxin inhibited the negative chronotropic responses to PACAP-38. However, hexamethonium did not block the negative responses to PACAP-38. After treatment with PACAP-38 (0.1-1 nmol), ACh induced atrial fibrillation significantly (p < 0.01). On the other hand, the negative chronotropic responses to intracardiac parasympathetic stimulation were not changed. These results suggest that (a) PACAP-38 induces negative chronotropic responses and liberates ACh from intracardiac postganglionic parasympathetic nerves, and that (b) PACAP-38 reduces ACh-induced atrial fibrillation threshold in the dog heart in situ.

Acetylcholine↗