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Biomedical subjects

M Miyano

Publications and source records attributed to M Miyano.

At least 37 records · Page 2Linked to original sources
PubMed1997

Restoration of Leydig cells after repeated administration of ethane dimethanesulfonate in adult rats.

Adult male rats were repeatedly treated with ethane dimethanesulfonate (EDS), an agent known to destroy Leydig cells selectively. Following a second injection, changes in serum testosterone levels and histological and morphometric changes of Leydig cells showed the time course to be similar to those after the first treatment. The number and volume of Leydig cells markedly decreased at day 2, began to increase from day 7, and recovered to the values of the control rats at day 30, concomitant with the changes of serum testosterone levels. Cells in the interstitial tissue labeled with bromodeoxyuridine markedly increased in number at day 2, gradually decreased thereafter, and returned to the values of the controls at day 14. During this period, cells undergoing mitosis were seen, their type unable to be determined, but were presumed to be regenerating Leydig cells. Even 30 days following four treatments with intervals of 30 days each, serum testosterone levels were the same as those in the controls. Also the numerical and volume densities of Leydig cells and the volume of an average Leydig cell were the same as those of the controls. Mitosis was observed in mature Leydig cells at this period, if any. It appears that new Leydig cells began to proliferate by division earlier than 14 days after EDS, allowing that there were several stages of proliferation, and that the source of reappearing Leydig cells may not be a limited number of precursor cells, implying the presence of stem cells for Leydig cells.

Animals↗
PubMed1997

[Late phase reaction in atopic dermatitis--immunological parameters and immunohistological analysis].

We studied the correlation between late phase reaction (LPR) and laboratory data in atopic dermatitis (AD) and evaluated the pathological findings in LPR. We studied the correlation between LPR and the severity of AD, total IgE, specific IgE, total IgG4, specific IgG4 and eosinophils. There were correlations between immediate reaction (IR) and some laboratory data, but not between LPR and laboratory data. Pathological findings at 15 min 24 hrs and 48 hrs after dermal injection showed edema at 15 min, followed by epidermal eczematous changes in almost all cases at 24 hrs, edema and degeneration of dermal endothelial cells and surrounding connective tissue, and leukocytoclasia in half of the cases and deposition of C3 and IgG in parts. We found CD4+ cells in all layers of dermis, a greater ratio of EG2+ cells in deeper layers of dermis as time passed, and decrease of CD23+ cells after 24 hrs throughout the dermis. These changes were found in cases of IR-LPR+, too. It appears that the mechanism of LPR is complex and that IR is not indispensable, for LPR and that infiltration of CD4+ and EG2+ cells throughout all layers of dermis are important, and these condition were related to subsequent reactions.

Adolescent↗
PubMed1996

Physiochemical characterization of ATP binding to human 5-lipoxygenase.

Human 5-lipoxygenase requires ATP as a stimulatory factor. At the two preferred concentrations of the free Ca2+, 0.02 microM with a resting cell and 20 microM with a stimulated cell, Scatchard analysis revealed that 5-lipoxygenase has one affinity ATP binding site with a Kd of 4.6 microM at the low Ca2+ concentration but has two affinity ATP binding sites with a higher Kd of 4.4 microM and a lower Kd of 14.5 microM at the high Ca2+ concentration. In contrast, in a Tween 20 reaction system, 5-lipoxygenase had similar activation coefficients for ATP at both Ca2+ concentrations; these were 12.7 microM at the low Ca2+ concentration and 12.0 microM at the high Ca2+ concentration. These results showed that 5-lipoxygenase has an ATP binding site and suggest that self-association of 5-lipoxygenase in 20 microM Ca2+ may affect ATP binding affinity as measured by Scatchard analysis.

Adenine Nucleotides↗
PubMed1995

A novel series of orally active antiplatelet agents.

A novel series of orally active fibrinogen receptor antagonists has been discovered through structural modification of our lead intravenous (iv) antiplatelet agent, 5-(4-amidinophenyl)pentanoyl-Asp-Phe 1 (SC-52012). The Asp-Phe amide bond was removed through truncation to a 3-substituted beta-amino acid aspartate mimetic which resulted in a tripeptide mimetic inhibitor of lower molecular weight (from 482 to the 330-390 g mol-1). The zwitterionic nature of the inhibitor was masked through the preparation of an ethyl ester prodrug. A lead compound from this series, 5-(4-amidinophenyl)pentanoyl-3-(3-pyridyl)propanoic acid 19a, was found to be a potent inhibitor of canine platelet aggregation in vitro (collagen, platelet rich plasma, PRP, IC50 = 270 nM). In further canine studies, oral administration of different ester pro-drugs of 19a at 10 mg kg-1 resulted in the following oral systemic activities: pivaloyloxymethyl ester derivative 19p (5.1 +/- 1.5% OSA), cyclohexyl ester derivative 19c (9.2 +/- 1.9% OSA), and ethyl ester derivative 19e (9.9 +/- 2.3% OSA).

Administration, Oral↗
PubMed1994

Human 5-lipoxygenase associates with phosphatidylcholine liposomes and modulates LTA4 synthetase activity.

A Ca2+ and a phosphatidylcholine (PC) as stimulatory factors to human 5-lipoxygenase (5-LO) were assessed to examine aspects of the regulatory mechanism of 5-LO. In the presence of Ca2+ (1 microM or less), PC liposomes distinctly stimulated the dual activities of 5-LO for the production of 5-HPETE from arachidonate and for its subsequent conversion to LTA4. At the same concentration of Ca2+, 5-LO was found to bind to PC liposomes. As with 5-LO activities, the binding was dependent on the range of Ca2+ concentration. The conversion ratios of 5-HPETE to LTA4 were dependent on PC liposome concentration and reached a maximum of 50% conversion. Among the four cell membrane lipids examined, PC liposomes demonstrated the highest conversion ratio of 5-HPETE to LTA4 by 5-LO. Most of the arachidonate added to the reaction mixture localized in PC liposomes. These results confirm that the intracellular increase of Ca2+ concentration causes 5-LO to associate with the cell membrane and perform an interfacial reaction. They also suggest that this binding of 5-LO to the cell membrane enhances the subsequent conversion from 5-HPETE to LTA4.

Adenosine Triphosphate↗
PubMed1994

X-ray structure of a pokeweed antiviral protein, coded by a new genomic clone, at 0.23 nm resolution. A model structure provides a suitable electrostatic field for substrate binding.

We have determined the crystal structure of alpha-pokeweed antiviral protein, a member of ribosome-inactivating proteins, at 0.23 nm resolution, by the molecular-replacement method. The crystals belong to the space group P2(1)2(1)2 with unit-cell dimensions a = 4.71, b = 11.63 and c = 4.96 nm, and contain one protein molecule/asymmetric unit based on a crystal volume/unit protein molecular mass of 2.1 x 10(-3) nm3/Da. The crystallographic residual value was reduced to 17.2% (0.6-0.23 nm resolution) with root-mean-square deviations in bond lengths of 1.9 pm and bond angles of 2.2 degrees. The C alpha-C alpha distance map shows that alpha-pokeweed antiviral protein is composed of three modules, the N-terminal (Ala1-Leu76), the central (Tyr77-Lys185) and the C-terminal (Tyr186-Thr266) modules. The substrate-binding site is formed as a cleft between the central and C-terminal modules and all the active residues exist on the central module. The electrostatic potential around the substrate-binding site shows that the central and C-terminal module sides of this cleft have a negatively and a positively charged region, respectively. This charge distribution in the protein seems to provide a suitable interaction with the substrate rRNA.

Amino Acid Sequence↗
PubMed1994

Improved crystals of the toxic protein MAP by protein engineering towards the host specificity.

Mirabilis anti-viral protein (MAP) is a ribosome-inactivating protein from Mirabilis jalapa L. Since MAP is effective over a broad spectrum of species, the protein is difficult to express in heterologous hosts such as Escherichia coli. Recently, we obtained a MAP mutant, Y72F which exhibits a lower (1/100) activity against E. coli ribosomes while retaining almost full activity against mammalian cells [Habuka, Miyano, Kataoka, Tsuge & Noma (1992). J. Biol. Chem. 267, 7758-7760]. For the crystallographic studies, the Y72F MAP expression vector with an OmpA leading sequence was constructed and expressed in E. coli. The Y72F MAP mutant was then isolated and purified from the cell culture medium. Crystals were grown using the crystallization conditions for the native MAP crystals [Miyano et al. (1992). J. Mol. Biol. 226, 281-283]: 50% ammonium sulfate containing 50 mM ammonium citrate and 2 mM adenine sulfate, pH 5.4. The crystals belong to space group P3(1)21 (or P3(2)21) with a = b = 104.1 and c = 134.3 A. The crystals are isomorphous with the wild-type crystals but diffract to higher resolution. Imaging-plate photographs of the Y72F mutant showed sharp intense spots without the streaking observed in the native crystals.

Journal Article↗
PubMed1994

Interfacial kinetic reaction of human 5-lipoxygenase.

The kinetics of human 5-lipoxygenase were investigated in the presence of Tween 20 using a continuous spectrophotometric assay. Using the mixture at a constant molar ratio of arachidonate/Tween 20 at pH 8.0, the steady-state velocity on a varied arachidonate concentration did not follow simple Michaelis-Menten-type kinetics and double-reciprocal plot analysis gave hyperbolic curves. However, by introducing the concept of a local pH change, it was possible to analyze the kinetics as simple Michaelis-Menten type. The concept of a local pH change implies that when utilizing an acidic and amphiphilic substance as a substrate, such as arachidonate, the medium around the substrate is acidified with an increased concentration of substrate. This concept was explained rationally by two experiments. Consequently, the data were transformed according to a local pH change and analyzed according to a dual phospholipid model as has been proposed for phospholipase A2 [Hendrickson, H. S. and Dennis, E. A. (1984) Kinetic analysis of the dual phospholipid model for phosphalipase A2, J. Biol. Chem. 259, 5734-5739]. It is concluded that 5-lipoxygenase performs an interfacial reaction in the arachidonate/Tween 20 mixed micelles in the same manner as phospholipase A2. The values of Km were almost constant (about 0.07 molar fraction), even when arachidonate molar ratios were changed in the surface of the mixed micelles. The values for Ks (the association constant of the enzyme to the micelle interface) ranged over 0.21-0.48 microM. The Vmax was 25.76 mumol.min-1.mg-1. This concept of a local pH change could be used extensively with enzymes which utilize both amphiphilic and acidic substances as substrates.

Arachidonate 5-Lipoxygenase↗
PubMed1994

Crystallization and preliminary X-ray crystallographic studies of recombinant human leukotriene A4 hydrolase complexed with bestatin.

Recombinant human leukotriene A4 hydrolase complexed with bestatin, an inhibitor of metalloprotease, has been crystallized by the hanging drop vapor diffusion method using 0.1 M phosphate buffer (pH 6.5) and 50 to 54% saturated ammonium sulfate. The orthorhombic crystals belong to the space group I222 or I2(1)2(1)2(1) with unit cell dimensions of a = 273.6 A, b = 261.3 A and c = 52.9 A. They diffract beyond 2.5 A resolution and a native data set up to 3 A resolution has been collected on an imaging plate Weissenberg camera using synchrotron radiation.

Amino Acid Sequence↗
PubMed1994

[A study on the visibility of different floor levels--experiments from a stumbling point of view].

Experiments were conducted to examine the visibility of different floor levels from a stumbling point of view. The test subjects were the elderly and the young, and we compared the difference between the two. We investigated eye movement while they were scanning the different floor levels. The results were as follows: 1) The height of different floor levels and illuminance had an influence on the visibility of different floor levels. Especially, the elderly were easily influenced by them. 2) The eye movements in scanning the different floor levels did not relate to the visibility of them, but were related to the personality of the test subjects. Eye movement patterns of the elderly differed from that of the young. The elderly were scanning mainly the lower part. The eye fixation time of the elderly was longer than that of the young. From the results, it was clarified that there were the difference between the elderly and the young. We could show it by the degree of influence. For example, the influence by the change of environmental condition.

Adult↗
PubMed1994

[Study on the aging influence on the recognizability for colored targets--black and color].

Recently spaces for the aged have become more wide spread as the population of them has increased. Most of the spaces, however, are originally provided for the young and, accordingly, can not be considered preferable environments for the aged. From this point of view, the authors aimed at the visual environments for the aged and carried out experimental research on the influences that aging gives to recognizability for colored targets, for the purpose of providing a preferable visual environment for the aged. The results show that the recognizability for colored targets declines drastically from about fifty years old, as just as the recognizability for uncolored targets. Moreover, the results show that, although the recognizability of the young is considerably influenced by differences of the illuminance and color of the target surfaces, aging made the recognizability less influencable by these differences.

Adolescent↗
PubMed1993

Potent in vitro and in vivo inhibitors of platelet aggregation based upon the Arg-Gly-Asp-Phe sequence of fibrinogen. A proposal on the nature of the binding interaction between the Arg-guanidine of RGDX mimetics and the platelet GP IIb-IIIa receptor.

Peptide mimetics of the RGDF sequence in which Arg-Gly has been replaced with 5-(4-amidinophenyl)pentanoyl mimetic has led to a 1000-fold increase in inhibitory potency over the natural RGDF ligand. The guanidine residue of the arginine may be involved in a reinforced ionic interaction with a carboxylate of the receptor which could explain the dramatic increase in potency upon replacement with benzamidine. This hypothesis is supported by the observation of low inhibitory potency of the corresponding benzylamine (18) and no activity with the corresponding imidazoline derivative (19); plus, ab initio calculations on the respective complexes suggest that the benzamidine-carboxylate is more favorable than the guanidine-carboxylate interaction. The ED50 for the inhibition of ex vivo collagen induced platelet aggregation in the dog for SC-52012 (1) was 0.32 microgram/kg/min by iv infusion with a pharmacodynamic half-life for recovery of approximately 40 min.

Amino Acid Sequence↗
PubMed1993

Expression of a pokeweed antiviral protein in Escherichia coli and its characterization.

Two expression vectors were constructed to produce a putative mature alpha-pokeweed antiviral protein (alpha-PAP) in Escherichia coli with its NH2- and COOH-terminal extrapeptides excised. One was for its intracellular expression with a methionine at its NH2-terminal. The other was for its secretion using an ompA signal peptide. The former product was purified from the total soluble proteins of the transformant with a yield of 1.74 mg/liter and the latter had a yield of 5.55 mg/liter. Both products exhibited RNA N-glycosidase activity on wheat ribosomes and inhibitory activity to protein synthesis in a rabbit reticulocyte system.

Amino Acid Sequence↗
PubMed1993

Nucleotide sequence of a genomic gene encoding tritin, a ribosome-inactivating protein from Triticum aestivum.

A genomic gene of tritin, a ribosome-inactivating protein (RIP) from Triticum aestivum, was cloned using a barley RIP gene as a probe. The 5'-non-coding region has potential TATA boxes and three sequences homologous to the binding sequence of the transcriptional activator protein Opaque-2 which activates maize RIP gene expression. The cloned DNA encoded tritin consists of 275 amino acids with no secretion signal sequence. The coding region of tritin was expressed in Escherichia coli using lac promoter and yielded a protein similar to the native one, as determined by SDS-polyacrylamide gel electrophoresis and immunological analysis.

Amino Acid Sequence↗
PubMed1993

Amino acids and peptides. XVII. Synthesis of peptides related to N-terminal portion of fibrin alpha-chain and their inhibitory effect on fibrinogen/thrombin clotting.

Various peptides related to N-terminal portion of fibrin alpha-chain were synthesized by the solution method and the solid-phase method, and their inhibitory effect on fibrinogen/thrombin clotting was examined. Extension of peptide chain from N-terminal tripeptide decreased the inhibitory effect. The most potent effect was shown by N-terminal decapeptide analog, H-Gly-Pro-Arg-Pro-Pro-Glu-Arg-His-Gln-Ser-NH2.

Amino Acid Sequence↗
PubMed1993

[Studies on development of evaluation method for wood-based floors. Part 1. Evaluating the attenuational capacity of human locomotor system due to the acceleration at several parts of the human body].

Experiments were conducted to compare the impact on several parts of the body caused by the striking of the heel during human gait. In these experiments, five kinds of wood flooring that have differing levels of light weight-impact sound insulating efficiency were used. The obtained results were as follows: 1) The generated acceleration of calcaneus varied with the difference of flooring. According to the regression analysis, the open polygonal relationship between bound back coefficient of floor and acceleration of calcaneus was determined. 2) The impact force given to the calcaneus propagated from the heel to the forehead after most of the force being absorbed at the ankle and the knee. We found direct correlation between the subjective feeling of floor pliabilty and the force to the leg. Therefore, it was suggested that the measurement of the force to the ankle and the knee was important in evaluating the efficiency of floors.

Acceleration↗