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Biomedical subjects

M Inoue

Publications and source records attributed to M Inoue.

At least 37 records · Page 2Linked to original sources
PubMed1992

Induction of uterine cervical neoplasias in mice by human papillomavirus type 16 E6/E7 genes.

We constructed a recombinant retrovirus containing the E6/E7 genes of human papillomavirus (HPV) 16 (ZE67) and examined the morphological changes in the cervicovaginal epithelium induced by inoculation of this virus into the vagina of mice. The ZNeo virus without HPV genes was used as a negative control. Moreover, cotreatment with phorbo-13-myristate-12-acetate or N-methyl-N'-nitro-N-nitrosoguanidine and these viruses was carried out. At the end of the observation period (9 months for CD-1 nu/nu and 15 months for C57BL/6J), of 31 CD-1 nude mice treated with ZE67, 7 and 19 had low-grade and high-grade dysplasia, respectively, while 5 of 22 mice treated with ZNeo had low-grade dysplasia (rank sum test, P less than 0.001). Similarly, 4 and 3 of 8 C57BL mice treated with ZE67 had low-grade and high-grade dysplasias, respectively, whereas 3 of the 8 control mice had low-grade dysplasias (P = 0.049). ZE67 plus phorbol-13-myristate-12-acetate and ZE67 plus N-methyl-N'-nitro-N-nitrosoguanidine cotreatments induced cervical cancers in 2 of 13 CD-1 nude mice and 6 of 15 C57BL mice, respectively. On the contrary, none of 6 CD-1 and 2 of 10 C57BL mice had cancer in the control groups (P = 0.0142 for phorbol-13-myristate-12-acetate treatment; P = 0.0173 for N-methyl-N'-nitro-N-nitrosoguanidine treatment). In addition, the existence and expression of HPV 16 E6/E7 genes were detected in the lesions induced by ZE67 but not in the lesions of the control mice by analysis by polymerase chain reaction and mRNA in situ hybridization. The present results suggest that HPV 16 E6/E7 genes induce dysplastic changes but require additional promoting or mutagenic stimulation for the development of cancer.

Animals↗
PubMed1992

Effect of the D-Phe2 residue on molecular conformation of an endogenous neuropeptide achatin-I. Comparison of X-ray crystal structures of achatin-I (H-Gly-D-Phe-Ala-Asp-OH) and achatin-II (H-Gly-Phe-Ala-Asp-OH).

The molecular conformation of achatin-II neutral form (H-Gly-Phe-Ala-Asp-OH), an endogenous peptide from the Achatina fulica ganglia, was elucidated by X-ray crystal analysis. The molecule takes an extended beta-pleated structure stabilized by 5 intermolecular hydrogen bonds with the antiparallely arranged molecules. This is in contrast with the turn conformation of a neuroactive achatin-I (H-Gly-D-Phe-Ala-Asp-OH) [(1992) FEBS Lett. 276,95-97]. The conformational comparison of both of the molecules makes clear the structural role which D-Phe residue of achatin-I plays in forming a definite active form.

Amino Acid Sequence↗
PubMed1992

alpha 1(VIII)-collagen gene transcripts encode a short-chain collagen polypeptide and are expressed by various epithelial, endothelial and mesenchymal cells in newborn mouse tissues.

Type-VIII collagen is a major constituent of Descemet's membrane and contains two genetically distinct alpha chains, alpha 1(VIII) and alpha 2(VIII). We have previously cloned the human alpha 1(VIII) and alpha 2(VIII) genes and determined that they are located on chromosomes 3 and 1, respectively. Comparison of the alpha 1(VIII) and alpha 2(VIII) genes with the alpha 1(X)-collagen gene showed that the structure of the three genes and the sequence of their collagen polypeptides were strikingly similar. Therefore, we have grouped the three genes in a common subclass of collagens which we have named the short-chain collagens because of the relatively small size of their triple-helical domains. In the present study, we have isolated and characterized a mouse gene fragment encoding the entire mouse alpha 1(VIII)-collagen chain and determined the complete primary structure of the polypeptide chain. The size of mouse alpha 1(VIII)-collagen mRNA, as estimated by Northern-blot analysis, was 4.2 kb, compared with 2.8 kb previously reported for the corresponding rabbit mRNA. By cloning and sequencing of four overlapping cDNA, we demonstrate that this larger size of mouse alpha 1(VIII) mRNA is due to a larger 3' untranslated region in the mouse transcript. Using the gene fragment as a probe, we performed Northern-blot hybridization analysis of RNA prepared from newborn mice and demonstrated that alpha 1(VIII) collagen mRNA is expressed at high levels in the calvarium, eye and skin. In situ hybridization revealed that alpha 1(VIII) RNA is present in skin keratinocytes, corneal epithelial and endothelial cells, lens epithelial cells, as well as mesenchymal cells surrounding cartilage and calvarial bone and in the meninges surrounding the brain.

Amino Acid Sequence↗
PubMed1992

Multiple role of hydrophobicity of tryptophan-108 in chicken lysozyme: structural stability, saccharide binding ability, and abnormal pKa of glutamic acid-35.

Trp108 of chicken lysozyme is in van der Waals contact with Glu35, one of two catalytic carboxyl groups. The role of Trp108 in lysozyme function and stability was investigated by using mutant lysozymes secreted from yeast. By the replacement of Trp108 with less hydrophobic residues, Tyr (W108Y lysozyme) and Gln (W108Q lysozyme), the activity, saccharide binding ability, stability, and pKa of Glu35 were all decreased with a decrease in the hydrophobicity of residue 108. Namely, at pH 5.5 and 40 degrees C, the activities of W108Y and W108Q lysozymes against glycol chitin were 17.3 and 1.6% of that of wild-type lysozyme, and their dissociation constants for the binding of a trimer of N-acetyl-D-glucosamine were 7.4 and 309 times larger than that of wild-type lysozyme, respectively. For the reversible unfolding at pH 3.5 and 30 degrees C, W108Y and W108Q lysozymes were less stable than wild-type lysozyme by 1.4 and 3.6 kcal/mol, respectively. As for the pKa of Glu35, the values for W108Y and W108Q lysozymes were found to be lower than that for wild-type lysozyme by 0.2 and by 0.6 pKa unit, respectively. The pKa of Glu35 in lysozyme was also decreased from 6.1 to 5.4 by the presence of 1-3 M guanidine hydrochloride, or to 5.5 by the substitution of Asn for Asp52, another catalytic carboxyl group. Thus, both the hydrophobicity of Trp108 and the electrostatic interaction with Asp52 are equally responsible for the abnormally high pKa (6.1) of Glu35, compared with that (4.4) of a normal glutamic acid residue.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Activation of calpain I in thrombin-stimulated platelets is regulated by the initial elevation of the cytosolic Ca2+ concentration.

The source and concentration of Ca2+ required to activate calpain I were investigated in thrombin-stimulated platelets. The concentration of cytosolic free Ca2+ ([Ca2+]i) was measured in platelets containing fura-2-AM, and exhibited a biphasic response after stimulation with 0.05, 0.1 or 0.5 NIH units of thrombin/ml. An initial transient elevation, which was predominantly dependent upon Ca2+ released from the internal stores into the cytosol, peaked at 15 s after stimulation, and a secondary sustained elevation, which was due to Ca2+ influx, was observed following the initial elevation. Calpain I was present at about 540 ng/10(8) unstimulated platelets, as measured by immunoblotting using rabbit anti-(human calpain I) IgG. Calpain I was activated 10 s after thrombin stimulation, as determined by the appearance of the 78 kDa and 76 kDa forms on immunoblots. The activation ratio of calpain I was calculated as the amount of the 78 + 76 kDa forms as a percentage of the total (80 + 78 + 76 kDa), and was influenced by the extent of the initial transient [Ca2+]i elevation after stimulation. An initial increase in [Ca2+]i of 300 nM was required to achieve the maximal activation (60%) of calpain I, and half-maximal activation occurred at 160 nM- Ca2+]i. These results suggest that the activation of calpain I in platelets is regulated by the initial elevation in Ca2+]i after thrombin stimulation, and does not necessarily require a Ca2+ influx.

Adult↗
PubMed1992

Dynamic aspects of ovarian superoxide dismutase isozymes during the ovulatory process in the rat.

To investigate the role of superoxide dismutase (SOD) in the ovulatory process, SOD isozymes and their mRNAs were determined in the ovary of 22-day-old rats. After treatment with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG), ovarian activity of Mn-SOD decreased markedly while Cu/Zn-SOD remained unchanged. However, the ovarian level of mRNA for Mn-SOD markedly increased after hCG-treatment while that for Cu/Zn-SOD decreased only slightly. Ovulation was inhibited by intravenous injection of a long-acting SOD. These results suggested that superoxide radicals in the ovary might play a critical role in the mechanism for hCG-induced ovulation.

Animals↗
PubMed1992

Synthesis of a cytochrome c derivative with prolonged in vivo half-life and determination of ascorbyl radicals in the circulation of the rat.

Since cytochrome c and acetylated cytochrome c disappear from the circulation with a half-life of 4 min, these proteins cannot be used for in vivo detection of superoxide radicals and related metabolites. To determine superoxide and other radicals in vivo, a cytochrome c derivative (SMAC) was synthesized by linking 1 mol of poly(styrene-co-maleic acid) butyl ester (SM) to cytochrome c, followed by acetylation of its lysyl amino groups. SMAC retained 8 and 80% of cytochrome c activity to react with ascorbyl and superoxide radicals, respectively. However, SMAC did not serve as a substrate for cytochrome c reductase and cytochrome c oxidase. When injected intravenously to the rat, SMAC circulated bound to albumin with a half-life of 130 min. SMAC was rapidly reduced in the circulation of intact animals. Treatment of animals with paraquat markedly enhanced the reduction of the circulating SMAC. We have synthesized an SM-conjugated superoxide dismutase (SOD) derivative (SM-SOD) that circulates bound to albumin with a half-life of 6 h. Kinetic analysis revealed that SM-SOD effectively inhibited the superoxide-dependent reduction of SMAC either in the presence or absence of 0.5 mM albumin. However, the reduction of the circulating SMAC was not inhibited by SM-SOD both in normal and paraquat-treated animals. Plasma samples from both animal groups also reduced cytochrome c and SMAC by an SOD-insensitive mechanism. However, after treatment with ascorbate oxidase, both plasma samples lost their activity to reduce cytochrome c and SMAC. These and other results suggest that ascorbyl radical might principally be responsible for the reduction of circulating SMAC and that plasma levels of ascorbyl radical might increase in paraquat-treated animals.

Acetylation↗
PubMed1992

Structure of acidic phospholipase A2 for the venom of Agkistrodon halys blomhoffii at 2.8 A resolution.

The crystal structure of acidic phospholipase A2 from the venom of Agkistrodon halys blomhoffii has been determined by molecular replacement methods based on the known structure of Crotalus atrox PLA2, a same group II enzyme. The overall structures, except the calcium-binding regions, are very similar to each other. A calcium ion is pentagonally ligated to two carboxylate oxygen atoms of Asp-49 and each carbonyl oxygen atoms of Tyr-28, Gly-30 and Ala-31. A reason why the former enzyme functions as monomeric form, while the latter one does as dimer, could be presumed by the structural comparison of these calcium-binding regions. Although Gly-32 is usually participated as a ligand in the coordination with calcium ion in group I PLA2, it is characteristically replaced to Ala-31 in the present structure, and thus the coordination geometry of calcium ion is rather different from the usually observed one.

Amino Acid Sequence↗
PubMed1992

Mutual assistance of hydrogen-bond pairing and aromatic stacking interactions for molecular recognition: spectroscopic study on the interaction of guanine base with cytosine and tryptophan molecules.

Fluorescence and 1H-NMR spectroscopic experiments on the interaction of guanine base with cytosine base and Trp indole ring using the model compounds have shown the first clear-cut evidence that both of the hydrogen-bond pairing and aromatic stacking interactions, which are recognized as important forces for the strict molecular recognition, do not function independently, but highly help each other for the recognition of guest molecule.

Cytosine↗
PubMed1992

A link between extracellular reactive oxygen and endotoxin-induced release of tumour necrosis factor alpha in vivo.

Pretreatment with the reactive oxygen species scavengers superoxide dismutase (SOD) and catalase or with the xanthine oxidase inhibitor allopurinol protected mice against hepatitis induced by the combined administration of lipopolysaccharide (endotoxin) and D-galactosamine. In the sera of protected animals no tumor necrosis factor (TNF alpha) was detectable in contrast to abundant amounts in the sera of injured control animals. A similar protection by the suppression of systemic TNF alpha was observed following the pretreatment of mice with polystyrene-coupled SOD prior to endotoxic challenge. Both pretreatments were ineffective when hepatitis was evoked by administration of the mediator TNF alpha instead of endotoxin. These findings indicate that the formation of extracellular reactive oxygen species is a condition needed to induce the release of TNF alpha and thus to mediate endotoxin-induced toxicity.

Alanine Transaminase↗
PubMed1992

Molecular conformation of ascidiacyclamide, a cytotoxic cyclic peptide from Ascidian: X-ray analyses of its free form and solvate crystals.

In order to investigate the conformational variation of ascidiacyclamide, a cytotoxic cyclic peptide from marine tunicate Ascidian, single crystals were prepared from ethanol and aqueous ethanol solutions as its free form (crystal I) and H2O/0.5 C2H5OH solvate (crystal II), respectively, and were determined by the x-ray diffraction method. Crystal I showed a pseudo C2-symmetric saddle-shaped rectangular conformation. Similar conformations were also observed in crystal II, where there were two crystallographically independent C2-symmetric molecules (named Mol-A and -B) per asymmetric unit. Mol-A and -B included H2O and H2O/C2H5OH solvents within their ring structures, respectively. These water and ethanol molecules were located on the crystallographic dyad axes, and were stabilized by the van der Waals contacts (including hydrogen bonds) with the polar-ring N atoms and nonpolar D-Val side-chain atoms. The conformational characteristics of ascidiacyclamide and its fluctuation/variation were discussed based on the present and previously reported x-ray results.

Animals↗
PubMed1992

Contribution of no-reflow phenomenon to hepatic injury after ischemia-reperfusion: evidence for a role for superoxide anion.

Controversy exists as to the role of oxygen-derived free radicals in tissue injury and the no-reflow phenomenon in reperfusion injury after ischemia. In this study using an experimental rat model, left hepatic lobar ischemia followed by reperfusion resulted in an increase of serum glutamic pyruvic transaminase at 30 min with concomitant histological evidence of hepatocellular necrosis at 24 hr. In the in vivo liver microcirculation, reperfusion after ischemia resulted in an initial transient return of blood flow, but stasis of blood flow later developed in the liver sinusoids. Thus a no-reflow phenomenon in the microcirculation was demonstrated. Intravenous administration of a long-acting form of superoxide dismutase (half-life 6 hr, dose 4 or 8 mg/kg) significantly decreased the hepatocellular necrosis and reduced the microcirculatory stasis in the liver sinusoids. These studies established the important contribution of the no-reflow phenomenon in ischemia-reperfusion injury to the liver and the participation of superoxide anions in mediating the no-reflow phenomenon.

Animals↗
PubMed1992

Defective biliary excretion of epinephrine metabolites in mutant (TR-) rats: relation to the pathogenesis of black liver in the Dubin-Johnson syndrome and Corriedale sheep with an analogous excretory defect.

Dubin-Johnson patients, mutant Corriedale sheep and TR- and EHBR mutant rats have recessively inherited defective bile canalicular secretion of many nonbile acid organic anions. The human and ovine mutants have black livers and lysosomal pigment accumulation. The livers in TR- and EHBR mutant rats are not black, and sparse lysosomal pigment accumulation is seen. Previously, we postulated that the unidentified pigment in the Dubin-Johnson syndrome results from the accumulation of tyrosine, phenylalanine and tryptophan metabolites, such as metanephrine, which are normally secreted in bile as organic anions. We tested this hypothesis in TR- rats. 3H-epinephrine was injected intravenously; control rats secreted 2.80% +/- 0.52% of the injected dose in bile as compared with 0.19% +/- 0.07% in TR- rats. From 82% to 90% of biliary radioactivity was due to polar conjugates in control rats and mutant rats. TR- rats retained more of the injected dose in the liver, particularly in lysosomes, and secreted more in urine than did control rats. After feeding control and TR- rats for 4 mo with a rat chow diet supplemented with 4% tyrosine, tryptophan and phenylalanine, the liver did not become grossly black; however, histological and electron microscopic study revealed dense lysosomal pigment accumulation in TR- rats. Intraportal injection of metanephrine resulted in the appearance of black liver in TR- rats that persisted for at least 2 hr and was not associated with pigment accumulation by light or electron microscopic examination.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Altered expression of sialyl-Tn, Lewis antigens and carcinoembryonic antigen between primary and metastatic lesions of uterine cervical cancers.

Immunohistochemical examination was performed of serial sections of 24 normal human adult cervical tissues and 53 human cervical carcinomas including 36 cases with lymph node metastasis. For this investigation, monoclonal antibodies directed to Lewis-X, Lewis-Y, sialyl-dimeric Lewis-X (SDLX), sialyl-Tn (STn) and carcinoembryonic antigen (CEA) were used. STn and CEA antigens were expressed very weakly in the normal cervical epithelium but strongly in the cancer cells, indicating the antigens to be oncogenic antigens of cervical squamous cell carcinoma. No significant difference in immunoreactivity was observed between primary and metastatic lesions of carcinoma or between primary lesions with and without metastasis. However, the expression patterns of STn and Lewis-Y antigens were quite different between primary lesions and metastatic lesions. In primary lesions the cancer cell nests tended to be stained centrally, but in metastatic lesions the cancer cell nests tended to be stained peripherally. This finding may reflect an important role of these carbohydrate chains in the process of metastasis of cervical squamous cell carcinoma to regional lymph nodes.

Antibodies, Monoclonal↗
PubMed1992

Effect of flosequinan on exercise capacity and cardiac function in patients with chronic mild heart failure: a double-blind placebo-controlled study.

Although beneficial effects of a new vasodilating agent, flosequinan, have been demonstrated in patients with severe heart failure, its efficacy has not been studied in patients with a less severe form of chronic heart failure. In this study, the effects of 4 weeks' administration of flosequinan, 50 mg daily, and placebo on exercise capacity, cardiac function, and symptoms of heart failure were investigated in 24 patients with chronic mild heart failure (New York Heart Association functional class, mainly class II) in a double-blind clinical trial. When the parameter changes during the treatment period of the flosequinan and placebo groups were compared, no significant difference was found in any of the measurements except for left ventricular fractional shortening determined from M-mode echocardiograms; it was increased by 2.9 +/- 1.3% in the flosequinan group whereas it was decreased by 1.3 +/- 0.9% in the placebo group (P less than 0.05 vs flosequinan treatment). However, when compared to baseline values, flosequinan significantly increased exercise time in the symptom-limited maximal exercise test (704 +/- 103 to 763 +/- 107 s, P less than 0.05) and the oxygen uptake at the anaerobic threshold (13.8 +/- 1.3 to 16.7 +/- 1.4 ml/min kg, P less than 0.05), and improved symptoms assessed with a new heart failure severity classification (a median value of 2.0-1.5, P less than 0.05). These improvements were not observed in the placebo group. Serious adverse effects were not observed in either group. These results suggest that flosequinan is useful for the treatment of chronic mild heart failure as well as severe heart failure.

Adult↗
PubMed1992

Effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on cytotoxicity of PSK-induced peritoneal polymorphonuclear leukocytes (PMNs).

We investigated whether recombinant human granulocyte colony-stimulating factor (rhG-CSF) enhanced the cytotoxicity of PSK-induced polymorphonuclear leukocytes (PMNs) in the peritoneal cavity. Male C3H/He mice, 8- to 10-week-old, received single subcutaneous (s.c.) or intraperitoneal (i.p.) injection of 2.5 micrograms/animal of rhG-CSF at different time points before or after an i.p. administration of PSK. In other experiments, mice were s.c. or i.p. treated with the same dosage of rhG-CSF every day for 7 or 14 consecutive days and i.p. injected with 2.5 mg/animal of PSK on the last day. Peritoneal PMNs were harvested 6 hrs after the administration of PSK and purified to more than 95% by Ficoll-Paque for in vitro cytotoxic assay. In vitro cytotoxic assays with 51Cr labeled MM46 mammary carcinoma cells were added with 5-20 micrograms/ml of Nocardia rubra cell wall skeleton (N-CWS) at the beginning of the assay to augment the cytotoxic activity of PMNs. In vitro addition of rhG-CSF to the assay did not enhance the cytotoxicity of PSK-induced PMNs. However, the cytotoxicity was significantly increased when rhG-CSF was s.c. administered 12 hrs before a PSK injection or 2 or 5 hrs after that. On the other hand, the cytotoxicity was rather weak when mice s.c. or i.p. received consecutive injections of rhG-CSF. This cytotoxicity may be mediated by H2O2, since H2O2 production of PMNs during the cytotoxic assay appears to correlate with the levels of cytotoxicity under suppressed H2O2 generation by catalase or enhanced generation by rhG-CSF. These results suggest that rhG-CSF augments the cytotoxicity of PSK-induced PMNs when administered in vivo timely.

Animals↗
PubMed1992

In vitro augmentation of cytotoxicity by N-CWS in peritoneal polymorphonuclear leukocytes (PMNs) induced by PSK, OK-432 or N-CWS.

Peritoneal polymorphonuclear leukocytes (PMNs) were collected from the peritoneal cavity of C3H/He mice 6 hrs after intraperitoneal (i.p.) injection of 2.5 mg/head of PSK, 1 KE (100 micrograms)/head of OK-432 or 200 micrograms/head of Nocardia rubra cell wall skeleton (N-CWS). Without in vitro stimulation, these PMNs did not show cytotoxicity to syngeneic MM46 mammary carcinoma cells in 51Cr release assay. Cytotoxicity of these PMNs was augmented by the addition of 25 micrograms/ml of N-CWS but not of PSK or OK-432 to cultures for the assay at the beginning of the culture. H2O2 production of PSK-induced PMNs was increased by the in vitro addition of 25 micrograms/ml of N-CWS but not of PSK. These results suggest that PSK as well as OK-432 and N-CWS can induce PMNs capable of responding further to N-CWS as the second stimulant.

Adjuvants, Immunologic↗