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J Aikawa

Publications and source records attributed to J Aikawa.

At least 37 records · Page 2Linked to original sources
PubMed1995

Synthesis of N-acetylglucosaminyl asparagine-substituted puromycin analogues.

As part of our project aimed to introduce specifically glycosylated amino acids into proteins, new glycosylated puromycin analogues were chemically synthesized. Introduction of a free N-acetylglucosaminyl asparaginyl side chain abolished the activity of puromycin completely, but when the sugar OH groups were rendered increasingly hydrophobic by acetylation or benzylation, up to 8% of the activity was recovered. The results of our preliminary inhibition tests suggest that the interaction of puromycin analogues and therefore also of glycosylated aminoacyl tRNA, with the ribosomal A site increase with hydrophobicity of the modifying protecting groups.

Acetylglucosamine↗
PubMed1994

A Mucor pusillus mutant defective in asparagine-linked glycosylation.

A Mucor pusillus mutant defective in asparagine-linked glycosylation was found in our stock cultures. This mutant, designated 1116, secreted aspartic proteinase (MPP) in a less-glycosylated form than that secreted by the wild-type strain. Analysis of enzyme susceptibility, lectin binding, and carbohydrate composition indicated that this mutant secreted three glycoforms of MPPs, one of which contained no carbohydrate; the other two had truncated asparagine-linked oligosaccharide chains such as Man0-1GlcNAc2. Further analysis using oligosaccharide processing inhibitors, such as castanospermine, 1-deoxynojirimycin and N-methyldeoxynojirimycin, suggested that MPPs in the mutant were glycosylated through a transfer of the truncated lipid-linked oligosaccharides, Man0-1GlcNAc2, to the MPP protein but not through an aberrant processing. In addition, genetic studies with forced primary heterokaryons indicated that the mutation in strain 1116 was recessive.

1-Deoxynojirimycin↗
PubMed1994

Endothelium-dependent relaxation by angiotensin-converting enzyme inhibitors in canine femoral arteries.

The effects of angiotensin-converting enzyme (ACE) inhibitors on vascular reactivity were investigated using isolated canine femoral arteries with and without endothelium. N-N-(S)-1-carboxy-3-phenylpropyl-L-alanyl-N-(indan-2-yl)glycine (M-1; an active metabolite of delapril, a nonsulfhydryl ACE inhibitor) and captopril (a sulfhydryl ACE inhibitor, 10(-8) to 10(-5) M) relaxed in a dose-dependent manner canine femoral arterial rings precontracted with prostaglandin F2 alpha in the presence of endothelium only. The endothelium-dependent relaxations by M-1 and captopril were completely blocked by methylene blue, an inhibitor of soluble guanylate cyclase; NG-monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide synthesis; and oxyhemoglobin, an inactivator of nitric oxide; they were partially blocked by aspirin, an inhibitor of cyclooxygenase and were enhanced by superoxide dismutase, a radical scavenger. The inhibitory effect of L-NMMA on the relaxations by M-1 and captopril were reversed by a high dose of L-arginine. Moreover, a bradykinin antagonist partially inhibited these relaxations. These results suggest that endothelium-dependent relaxations by M-1 and captopril in canine femoral arteries are mediated through the release of both prostanoids and endothelium-derived nitric oxide via endogenous bradykinin.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1993

Characterization of an aspartic proteinase of Mucor pusillus expressed in Aspergillus oryzae.

The aspartic proteinase (MPP) gene from the zygomycete fungus Mucor pusillus was introduced into an ascomycete fungus, Aspergillus oryzae, by protoplast transformation using the nitrate reductase (niaD) gene as the selective marker. Southern blot analysis indicated that the MPP gene was integrated into the resident niaD locus at a copy number of 1-2. MPP secreted by the recombinant A. oryzae was correctly processed but was more highly glycosylated than that produced in the original M. pusillus strain. Treatment with endo-beta-N-acetylglucosaminidase H and analysis of the carbohydrate composition of the secreted MPP revealed that the extra glycosylation of the MPP secreted by the recombinant A. oryzae was due to altered processing of mannose residues. The extra glycosylation of MPP affected its enzyme properties including its milk-clotting and proteolytic activities.

Aspartic Acid Endopeptidases↗
PubMed1993

Vascular relaxing mechanism of denopamine in isolated canine coronary, femoral, mesenteric, and renal arteries.

We investigated the mechanism of vascular relaxation produced by denopamine (deno), an oral positive inotropic agent that has selective beta 1-adrenergic action. Deno concentration-dependently (0.1 microM-30 microM) relaxed ring segments of canine femoral, mesenteric, and renal arteries which were partially precontracted with 1 micron phenylephrine or norepinephrine, but did not relax those precontracted with 5 microM prostaglandin F2 alpha or 40 mM K+. The relaxation was not significantly inhibited by pretreatment with 10 microM propranolol or metoprolol. Deno produced a parallel rightward shift in concentration-response curves to phenylephrine in femoral and renal arteries. The Schild plot yielded linear regressions of slopes of 1.301 +/- 0.106 and 0.823 +/- 0.122, respectively, which were not significantly different from unity. The pA2 values of Deno against phenylephrine in femoral and renal arteries were 5.41 +/- 0.03 and 5.76 +/- 0.06, respectively. On the other hand, Deno concentration-dependently (10 nM-10 microM) relaxed ring segments of canine coronary arteries which were partially precontracted with 5 microM prostaglandin F2 alpha. The relaxation was significantly inhibited by pretreatment with 10 microM metoprolol. In conclusion, vascular smooth muscle relaxation by Deno was mediated through beta 1-adrenergic action in canine coronary arteries and through the blocking effect of alpha-adrenoceptors in canine femoral, mesenteric, and renal arteries.

Adrenergic beta-Agonists↗
PubMed1992

Granulocyte-colony stimulating factor stimulates immunoreactive endothelin-1 release from cultured bovine endothelial cells.

We examined the effect of human recombinant granulocyte-colony stimulating factor (G-CSF) on the release of immunoreactive endothelin-1 (ET-1) from cultured bovine vascular endothelial cells. G-CSF dose dependently (10(-8)-10(-6) M) increased the release of immunoreactive ET-1 as a function of time under a serum-free condition. Coaddition of G-CSF and thrombin induced an additive effect on immunoreactive ET-1 release. Neither Ca2+ channel antagonist nor cyclooxygenase inhibitor affected immunoreactive ET-1 release stimulated by G-CSF. These results suggest that G-CSF, in addition to its effect on granulocyte progenitors, has a direct effect on vascular endothelium to induce the release of immunoreactive ET-1.

Animals↗
PubMed1992

Endothelin-1- and endothelin-3-induced vasorelaxation via common generation of endothelium-derived nitric oxide.

The vasorelaxant effects by endothelin-1 (ET-1) and endothelin-3 (ET-3), and their mechanisms of action were studied in isolated porcine pulmonary arterial strips. ET-1 and ET-3 dose-dependently (10(-9) - 10(-8) M) relaxed vascular strips precontracted with norepinephrine only in the presence of endothelium. The maximal vasorelaxant effect by ET-1 was about 70% of that by ET-3. The ET-1- and ET-3- induced vasorelaxation was blocked by NG-nitro-L-arginine, an inhibitor of nitric oxide synthesis, and methylene blue, an inhibitor of soluble guanylate cyclase. The present data suggest that vascular smooth muscle relaxation induced by ET-1 and ET-3 is mainly ascribed to synthesis and release of nitric oxide from L-arginine in endothelium.

Animals↗
PubMed1992

Prenatal diagnosis in high risk pregnancies for Zellweger syndrome.

Zellweger syndrome is a lethal disorder. At present, no effective therapies are known for the patients of Zellweger syndrome. Recently a typical case of Zellweger syndrome in Japan was observed. In spite of intensive care, the patient died at the age of 3 months. Following this, the parents requested prenatal diagnosis for their following two pregnancies. We investigated levels of very long chain fatty acids (VLCFA), levels of bile acids in amniotic fluid and immunoblotting of peroxisomal beta-oxidation enzymes in cultured amniocytes. We report that immunoblotting using cultured amniocytes is an effective method for prenatal diagnosis of Zellweger syndrome. Furthermore, if we use immunoblotting for prenatal diagnosis, we can discriminate pseudoZellweger syndrome from pseudoneonatal adrenoleucodystrophy. Following prenatal diagnosis, two healthy babies were delivered. After birth, no abnormal levels of VLCFA in either serum or red blood cell membranes were confirmed. In this paper, we report that we can diagnose a healthy fetus in a high risk pregnancy for Zellweger syndrome.

Amniocentesis↗
PubMed1992

Protein engineering of the milk-clotting aspartic proteinases.

Calf Chymosin and a fungal protease from Mucor pusillus (Mucor rennin) are members of the aspartic proteinases used as milk-coagulants in cheese industry. A system for production of recombinant chymosin as inclusion bodies in Escherichia coli cells and its refolding into the active form was established. Another expression system for production of Mucor rennin in Saccharomyces cerevisiae was also established. Mucor rennin was efficiently excreted from the yeast host as a heavily glycosylated form. Glycosylation affected both the secretion and the enzyme properties. Site-directed mutagenesis of the Tyr residue at position 75 in chymosin and Mucor rennin revealed its crucial role in catalytic function of the aspartic proteinases. The results also suggested possibility to improve practical properties of the milk-clotting enzymes by site-directed mutagenesis.

Amino Acid Sequence↗
PubMed1991

Low-density particles (W-particles) containing catalase in Zellweger syndrome and normal fibroblasts.

By both histological and biochemical criteria, peroxisomes in patients with Zellweger syndrome appear to be absent or severely deficient. By using 15-30% (wt/vol) Nycodenz/sucrose gradients to study the subcellular localization of extraperoxisomal catalase activity, a commonly used marker for mature peroxisomes, we detected a single peak of activity in Zellweger syndrome fibroblasts at an equilibrium density of 1.13 g/cm3, lower than the expected 1.17 g/cm3 of mature peroxisomes. Upon recentrifugation in either the original gradient or one with a higher salt concentration, essentially all catalase activity was recovered in fractions of the original densities. The activity of the catalase peak was further analyzed by a digitonin titration and filtration assay in combination with Triton X-100 treatment. The catalase activity passed through 0.1-microns and 0.22-microns but was retained on 0.025-microns membrane filters (mean pore size). After treatment with Triton X-100 nearly all catalase activity passed through the filters. The results from fractionations data, digitonin latency measurement, and the detergent effect on the filtration behavior suggest that catalase is not free in the cytosol of Zellweger syndrome fibroblasts as commonly thought but in particles (W-particles). Similar low-density catalase-containing particles, distinct from peroxisomes, are also found in normal fibroblasts. We found that L-alpha-hydroxyacid oxidase, another peroxisomal matrix enzyme, is also present in W-particles derived from normal and Zellweger syndrome fibroblasts. We speculate that the low-density catalase-containing W-particle may represent an immature or incomplete form of peroxisome distinct from previously described "peroxisomal ghosts" in Zellweger syndrome fibroblasts.

Biomarkers↗
PubMed1991

Import of human bifunctional enzyme into peroxisomes of human hepatoma cells in vitro.

A polypeptide containing the carboxyl-terminal fragment of human peroxisomal enoyl-CoA hydratase:3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme was synthesized in vitro from its cDNA clone. This expression polypeptide was transported into purified rat liver peroxisomes. When the expression polypeptide was incubated with postnuclear supernatant fractions of human hepatoma cells and analyzed by Nycodenz gradient SDS-PAGE and fluorography, it was imported specifically into peroxisomes as indicated by its resistance to proteinase K degradation. A deletion of the last nine amino acid residues at the carboxyl-terminus of this polypeptide prevents its peroxisomal import. A tripeptide sequence, SKL, located at the carboxyl-terminus of human bifunctional enzyme appears to be the targeting signal for the peroxisomal importation of bifunctional enzyme in human cells.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1991

Acetylcholine-induced endothelium-dependent vascular smooth muscle relaxation in nitroglycerin-tolerant isolated rat aorta.

Nitroglycerin (NTG) tolerance is recognized clinically, and its pharmacological mechanism has been thought to be due to a decrease in the accumulation of cyclic GMP (cGMP) which is a second messenger of NTG. Endothelium-derived relaxing factor (EDRF) also relaxes vascular smooth muscle through the activation of soluble guanylate cyclase and the production of cGMP. The purpose of this study was to investigate acetylcholine (ACh)-induced endothelium-dependent relaxation and cGMP response in NTG-tolerant isolated rat aorta. Ring strips prepared from the thoracic aorta of male Wistar rats were mounted in tissue baths and contracted with 10(-6) M norepinephrine. NTG and ACh relaxation responses were compared before and after 1 h treatment with 5 x 10(-4) M NTG. The chronological changes in tissue cGMP levels by 10(-6) M NTG and ACh were compared between a control group (untreated) and NTG-tolerant group (treated with 5 x 10(-4) M NTG for 1 h). The NTG dose-response curve shifted markedly to the right, but the ACh dose-response curve shifted to the left after the induction of NTG tolerance. In the control group, both NTG and ACh elevated the tissue cGMP levels, but in the NTG-tolerant group only ACh elevated cGMP significantly. However, in the NTG-tolerant group, the cGMP increase induced by ACh was smaller than that in the control group. These results suggest that NTG tolerance does not decrease, but rather augments ACh-induced endothelium-dependent vascular smooth muscle relaxation in isolated rat aorta.

Acetylcholine↗