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N Yoshida

Publications and source records attributed to N Yoshida.

At least 19 recordsLinked to original sources
PubMed1992

Characterization of recombinant human and rat pancreatic phospholipases A2 secreted from Saccharomyces cerevisiae: difference in proteolytic processing.

An expression plasmid for human pancreatic phospholipase A2 in Saccharomyces cerevisiae was constructed by insertion of cDNA encoding its preprophospholipase A2 into a yeast expression vector pAM82. The resulting product secreted in the yeast culture medium was mainly prophospholipase A2, which was the same as the natural proenzyme in all aspects examined, including the higher order structure. However, when the rat preprophospholipase A2 cDNA was manipulated in the same manner, the active phospholipase A2 of the intact mature form was secreted with the proenzyme being hardly detected in the medium. This unexpected favorable result would occur due to cleavage of rat phospholipase A2 pro-peptide by a trypsin-like proteinase in S. cerevisiae. Based on this finding, we constructed a plasmid carrying the sequence coding for the prepro-peptide of rat pancreatic phospholipase A2 behind the PHO5 promoter in the pAM82 vector, which leads to the secretion of heterologous proteins as their mature form. The use of this plasmid led to secretion of biologically active human pancreatic secretory trypsin inhibitor and a glutamic acid-specific endopeptidase from Staphylococcus aureus ATCC 12600, which are eukaryote and prokaryote proteins, respectively, in the culture medium of S. cerevisiae.

Amino Acid Sequence↗
PubMed1992

Heterozygous abnormal fibrinogen Osaka III with the replacement of gamma arginine-275 by histidine has an apparently higher molecular weight gamma-chain variant.

Congenitally abnormal fibrinogen Osaka III with the replacement of gamma Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of alpha- and gamma-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal gamma-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 gamma remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

Adult↗
PubMed1992

A nuclear gene encoding beta-amylase of sweet potato.

A nuclear AmyB gene from sweet potato encoding beta-amylase (beta Amy) that is abundant in tuberous roots and inducible in other organs by an exogenous supply of sucrose or polygalacturonic acid, was isolated and characterized. Genomic Southern blot hybridization, restriction maps of independently isolated phage lambda genomic clones, and the nucleotide sequence of AmyB compared with that of the cDNA, all suggested that beta Amy of sweet potato is encoded by a gene that is present in a single copy per haploid genome. In the sequence of AmyB, the sequence that is identical to that of the cDNA was split into seven exons by six introns, and the transcription of this gene starts from multiple sites 26 to 30 bp downstream from a potential TATA-box sequence, 5'-TATATAA. In the 5'-upstream region of AmyB, there are sequences homologous to those conserved in the 5'-upstream regions of genes encoding sporamin, which are regulated similarly to AmyB. The 5'-upstream region of AmyB also contains sequences to which several previously known plant nuclear factors bind.

Amino Acid Sequence↗
PubMed1992

5-HT3 receptor antagonist effects of DAT-582, (R) enantiomer of AS-5370.

The serotonin 5-HT3 receptor antagonist effects of DAT-582, the (R) enantiomer of AS-5370 ((+/-)-N-[1-methyl-4-(3-methyl-benzyl)hexahydro-1H-1,4-diazepin-6- yl]-1H- indazole-3-carboxamide dihydrochloride), and its antipode were compared with those of AS-5370 and existing 5-HT3 receptor antagonists. In anesthetized rats, DAT-582 antagonized 2-methyl-5-HT-induced bradycardia with an ED50 value of 0.25 microgram/kg i.v., whereas the (S) enantiomer was without effect even at 1000 micrograms/kg i.v. In antagonizing the bradycardia, DAT-582 was as potent as granisetron, slightly more potent than AS-5370, and 2, 5 and 18 times more potent than ondansetron, ICS 205-903 and renzapride, respectively, although it was less potent than zacopride. DAT-582 inhibited cisplatin (10 mg/kg i.v.)-induced emesis in ferrets with an ED50 value of 3.2 micrograms/kg i.v. twice. The antiemetic activity of DAT-582 was more potent than that of the existing 5-HT3 receptor antagonists examined, except zacopride. In contrast, the (S) enantiomer had little effect at 1000 micrograms/kg i.v. twice. In isolated guinea-pig ileum, DAT-582 inhibited 5-HT-induced contractions with an IC50 value of 91 nM, whereas the (S) enantiomer hardly inhibited them even at 1000 nM. These results suggest that DAT-582, the (R) enantiomer of AS-5370, potently and selectively blocks 5-HT3 receptors.

Animals↗
PubMed1992

Purification, characterization and gene cloning of a novel glutamic acid-specific endopeptidase from Staphylococcus aureus ATCC 12600.

Twenty strains of Staphylococcus aureus from ATCC type cultures and strains found in clinical studies were cultivated, and their endopeptidase activity specific for glutamic acid was surveyed using benzyloxycarbonyl-Phe-Leu-Glu-p-nitroanilide (Z-Phe-Leu-Glu-pNA) as a substrate. The activity was found in two of the strains, ATCC 12600 and ATCC 25923. A glutamic acid-specific proteinase, which we propose to call SPase, was purified from the culture filtrate of S. aureus strain ATCC 12600 by a series of column chromatographies on DEAE-Sepharose twice and on Sephacryl S-200. A single band was observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified SPase. The molecular weight of the proteinase was estimated to be 34000 by SDS-PAGE. When synthetic peptides and oxidized insulin B-chain were used as substrates, SPase showed the same substrate specificity as V8 proteinase, EC 3.4.21.9, which specifically cleaves peptide bonds on the C-terminal side of glutamic acid and aspartic acid. Examination with p-nitroanilides of glutamic acid and aspartic acid as substrates, however, revealed that both proteinases are highly specific for a glutamyl bond in comparison with an aspartyl bond. To elucidate the complete primary structure of SPase, its gene was cloned from genomic DNA of S. aureus ATCC 12600, and the nucleotide sequence was determined. Taking the amino acid sequence of SPase from the NH2-terminus to the 27th residue into consideration, the clones encode a mature peptide of 289 amino acids, which follows a prepropeptide of 68 residues. SPase was confirmed to be a novel endopeptidase specific for glutamic acid, being different from V8 proteinase which consists of 268 amino acids.

Amino Acid Sequence↗
PubMed1992

Structural studies on the glycosylphosphatidylinositol membrane anchor of Trypanosoma cruzi 1G7-antigen. The structure of the glycan core.

The 1G7-antigen is expressed by the infective metacyclic trypomastigote stage of the protozoan parasite Trypanosoma cruzi. The 1G7-antigen is a 90-kDa glycoprotein, present at about 40,000 copies/cell, which is anchored in the plasma membrane via a glycosylphosphatidylinositol (GPI) membrane anchor. The glycan of the GPI anchor has been isolated from immunopurified 1G7-antigen and its structure determined using a combination of methylation linkage analysis and exoglycosidase sequencing. The structure of the glycan is Man alpha 1-2Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcNH2. The glucosamine residue is in glycosidic linkage to a phosphatidylinositol moiety. The penultimate nonreducing alpha-Man residue is substituted with phosphate, which is most likely part of an ethanolamine phosphate bridge linking the GPI anchor to the 1G7-antigen polypeptide. The glycan sequence was obtained from 1.1 nmol of glycoprotein isolated from a detergent lysate of whole cells. The procedures reported here represent a high sensitivity protocol for determining GPI glycan structures from small quantities of biological material. The structure of the 1G7-antigen GPI anchor is consistent with the conserved core structure of all GPI anchors analyzed to date and is similar to that of the T. cruzi lipopeptidophosphoglycan. The biosynthesis of GPI anchors and lipopeptidophosphoglycan in T. cruzi is discussed in the light of this structural homology.

Animals↗
PubMed1992

Characterization of an abnormal fibrinogen Osaka V with the replacement of gamma-arginine 375 by glycine. The lack of high affinity calcium binding to D-domains and the lack of protective effect of calcium on fibrinolysis.

Prolonged thrombin time was completely corrected by the addition of millimolar concentrations of calcium in a new abnormal fibrinogen, Osaka V. Analysis of lysyl endopeptidase digests of A alpha-, B beta-, or gamma-chains by high performance liquid chromatography, and the following amino acid sequence analysis of relevant peptides revealed that about 50% of the gamma-chain has a replacement of gamma-arginine 375 by glycine. When fibrinogen was digested with plasmin in the presence of millimolar concentration of calcium, the amount of fragment D1 was about 50% of the normal control, and the rest was further cleaved to fragment D2, D3, or D62 with an apparent Mr of 62,000. Plasmic digestion of cross-linked fibrin in the presence of calcium resulted in the appearance of an abnormal fragment with an apparent Mr of 123,000 as well as fragments D2, D3, and D62, concomitant with the decrease of D dimer. The gamma-remnant of the abnormal fragment proved to be a cross-linked complex of the normal D1 gamma-remnant and residues 374-406/411 of the abnormal gamma-chain. The number of high affinity Ca(2+)-binding sites for the normal fibrinogen and fibrinogen Osaka V obtained by equilibrium dialysis was 2.88 (about 3) and 1.85, respectively, and that for the abnormal molecules was calculated as 0.9 (about 1) from their relative amounts in the samples, suggesting the lack of two Ca(2+)-binding sites in the D-domains. These data suggest that the normal structure of the COOH-terminal portion of the gamma-chain including residue 375 is required for the full expression of high affinity calcium binding to D-domains, the ability to be protected by calcium against plasmic digestion, and fibrin polymerization. During these studies, we found that the NH2-terminal amino acid of the gamma-remnant in fragments D or D dimer which were obtained after prolonged digestion with plasmin is gamma-Met89.

Adult↗
PubMed1992

Production of recombinant human glucagon in the form of a fusion protein in Escherichia coli; recovery of glucagon by sequence-specific digestion.

Recombinant human glucagon was successfully produced with a high level of expression in Escherichia coli as a fusion protein with human interferon gamma. The synthetic gene was designed to release glucagon, which does not contain glutamic acid residues, from fusion protein with the Staphylococcus aureus strain V8 protease that specifically cleaves the peptide bond on the carboxyl side of the glutamic acid residue. The resulting glucagon was purified to homogeneity by a combination of C18 reverse-phase HPLC and ion-exchange HPLC. The yield of intact glucagon obtained from 11 of culture was approximately 12 mg. The structure of recombinant human glucagon was confirmed by HPLC and amino acid composition/sequence analyses.

Amino Acid Sequence↗
PubMed1992

Recombinant human glucagon: large-scale purification and biochemical characterization.

Recombinant glucagon was expressed in Escherichia coli as a fusion protein including the glucagon sequence therein as previously reported [Ishizaki et al. (1992). Appl. Microbiol. Biotechnol. 36, 483-486]. We developed a large-scale method for the isolation and purification of recombinant glucagon. After cell disruption, the resultant pellets were solubilized with 2 M guanidine-HCl, to which Staphylococcus aureus V8 protease had been added, and were digested into intermediates composed of 53- and 60-residue peptides containing the glucagon moiety. After the digestion came to an end, the solution was desalted, and the remaining V8 protease was allowed to resume digestion of the intermediates into glucagon, followed by partial purification by S-Sepharose and Sephacryl S-100 chromatographies. The glucagon obtained was found to be not less than 99.5% pure by analytical HPLC. One liter of culture produced about 180 mg of pure glucagon. The amino acid composition and the sequence agreed well with the theoretical values. Radioreceptor assay gave an affinity constant similar to that of pancreatic glucagon, and similar activities in cAMP production and glycogenolysis were also observed. Thus, the recombinant glucagon was confirmed to be biochemically identical with pancreatic glucagon.

Amino Acid Sequence↗
PubMed1992

Effects of a platelet-activating factor antagonist, CV-6209, on gastric mucosal lesions induced by ischemia-reperfusion.

Recent research was shown that oxygen-derived free radicals are involved in the pathogenesis of various diseases, including ischemia-reperfusion injury. We have also reported that oxygen-derived free radicals and lipid peroxidation may play an important role in gastric mucosal injury induced by ischemia-reperfusion. The hypoxanthine-xanthine oxidase system and neutrophils are considered important sources of oxygen-derived free radicals in this process. In recent years, it also has been shown that serum platelet-activating factor (PAF) levels increased during ischemia-reperfusion, and that induction of superoxide generation by neutrophils is one of the important biological effects of PAF. In the present study, we examined the effect of CV-6209, a specific PAF receptor antagonist, on gastric mucosal injury induced by ischemia-reperfusion, to shed some light on the possible involvement of PAF in such lesions. CV-6209 significantly attenuated the gastric mucosal injury induced by ischemia-reperfusion, and inhibited both an increase of thiobarbituric acid reactive substances and a decrease of alpha-tocopherol in gastric mucosa after ischemia-reperfusion. However, CV-6209 had no effect on gastric mucosal blood flow during ischemia-reperfusion. These results suggest that endogenous PAF may play an important role in gastric mucosal injury induced by ischemia-reperfusion, and that CV-6209 exerts its beneficial effect mainly by inhibiting neutrophil superoxide production induced by PAF.

Animals↗
PubMed1992

Ischemic but not reperfusion arrhythmias depend upon serum potassium concentration.

The effects of variations in serum concentrations of potassium on the occurrence and severity of ischemia- and reperfusion-induced arrhythmias have been studied in conscious rats. Serum potassium concentrations were modified by maintaining rats on diets which varied in potassium concentration, by treatment with hydrochlorothiazide, amiloride, spironolactone or infusions of potassium chloride. An inverse linear relationship was demonstrated between ischemia-induced arrhythmias and log(e) serum potassium concentration such that a 50% reduction in arrhythmias occur with a 3.8-fold increase in serum potassium concentration. On the other hand, the incidence of reperfusion-induced arrhythmias after 7 min of regional ischemia prior to reperfusion in previously untreated rats were not influenced by elevation of serum potassium concentrations prior to and after reperfusion. Effects on ischemia-induced arrhythmias could not be explained by changes in blood pressure or heart rate. It is speculated that modification of potassium concentration in non-ischemic or border zone ventricular tissue may directly modify arrhythmogenesis due to ischemia but not that due to reperfusion.

Animals↗
PubMed1992

Platelet activating factor in culture media as an indicator of human embryonic development after in-vitro fertilization.

Although human chorionic gonadotrophin can detect trophoblast after implantation of the conceptus, there is a need to detect the conceptus before implantation. We have investigated whether human embryo-derived platelet activating factor is formed during embryonic development after in-vitro fertilization. A total of 99 ova from 12 patients were cultured and the 54 media were analysed. Platelet activating factor was also measured by radioimmunoassay after extraction. Fertilization increased the amount of platelet activating factor 4-fold over non-fertilized ova to a level of 4 ng/ml. This increase was also dependent on the degree of embryonic development with a maximum level of platelet activating factor of 7 ng/ml at the 2-cell stage. The follicular inducing agent used to treat the patient also had an effect on platelet activating factor; buserelin treatment gave embryos with a higher level than did clomiphene citrate treatment. These results indicate that platelet activating factor may have a role in embryonic development before implantation and may serve as a useful marker for fertilization and the developmental stage of the embryo.

Adult↗
PubMed1992

Treatment of Langerhans cell histiocytosis in children with etoposide.

Ten children with Langerhans cell histiocytosis (LCH) were treated with etoposide. For five patients, this was the initial diagnosis. The other five had failed to respond to previous therapies. Etoposide (100 mg/m2) was given intravenously twice a week for 4 weeks, followed by maintenance therapy every 2 to 4 weeks for 2 years. All 10 patients responded to etoposide, and 6 of them (60%) have been in complete remission for 3 to 36 months without any side effects. One patient relapsed with diabetes insipidus, one with a soft tissue mass, and two others developed multiple bone lesions. Chemotherapy with etoposide appears to be effective and safe for the treatment of children with systemic LCH.

Adolescent↗
PubMed1992

The role of an anti-myeloperoxidase antibody in the diagnosis and classification of acute leukaemia: a comparison with light and electron microscopy cytochemistry.

The enzyme myeloperoxidase (MPO) is the hallmark of the myeloid lineage. We have analysed the presence of MPO in blasts from 180 cases of acute leukaemia (103 acute myeloid leukaemia (AML) and 77 acute lymphoid leukaemia (ALL) by means of monoclonal antibodies anti-MPO and immunocytochemistry (alkaline phosphatase anti-alkaline phosphatase method). The aim of the study was to investigate the specificity and sensitivity of this marker compared with MPO cytochemistry by light (LM) and electron microscopy (EM), and with the expression of myeloid antigens. Anti-MPO was positive (greater than 3% blasts) in all but one of the 90 AML positive by LM cytochemistry. Of 13 AML cases negative by MPO cytochemistry, six showed 3-10% blasts reactive with anti-MPO and were also positive with antibodies to CD13 and/or CD33. The presence of MPO was confirmed in four of these by EM. The overall positivity of anti-MPO in AML was 92%. Anti-MPO was negative in all but two ALL (6% and 8% positive blasts). The blasts in these two cases were also CD13, CD33 and MPO positive by EM; both were thus reclassified as biphenotypic. Another two ALL reinterpreted as biphenotypic were negative by MPO cytochemistry and anti-MPO but were MPO positive by EM and with CD13 and/or CD33. We conclude that anti-MPO is a sensitive and specific early marker of myeloid blasts and should be incorporated in the routine immunophenotyping of acute leukaemia.

Acute Disease↗
PubMed1992

Effect of mechanical pressure on the blood flow in human palatal mucosa measured by temperature controlled thermoelectrical method.

Mucosal blood flow in the human palate was measured by a temperature controlled thermoelectrical method based on the thermal conductivity of mucosal tissue using a blood flow monitor and a non-invasive surface probe. The effect of mechanical pressure on the palatal mucosal tissue was studied. Mechanical pressure (2.5, 5.0, 7.5, 10.0 or 15.0 g mm-2) to the mucosal surface was exerted circumferentially around the surface probe. In five out of seven subjects, the blood flow showed a tendency to be almost constant under the mechanical pressures which are 10.0 g mm-2 or higher. The effect of the duration of the pressure (10.0 g mm-2) exertion on the blood flow was also investigated and it was revealed that the blood flow tended to be constant after 60s in every subject. In the present study, it was also demonstrated that the temperature controlled thermoelectrical method is appropriate for studying the blood flow dynamics in oral mucosa.

Adult↗
PubMed1992

Polymorphism of the 35- and 50-kilodalton surface glycoconjugates of Trypanosoma cruzi metacyclic trypomastigotes.

In this study, we examined the immunochemical properties of the 35- and 50-kDa (35/50-kDa) surface glycoconjugates expressed on the surface of metacyclic trypomastigotes of Trypanosoma cruzi using three different monoclonal antibodies directed to this component. The 35/50-kDa surface antigen was expressed by metacyclic trypomastigotes of 11 different strains of T. cruzi and displayed a significant degree of molecular polymorphism. Results of immunoblotting and complement-mediated lysis were consistent with such diversity. Different monoclonal antibodies reacted with distinct epitopes on the 35/50-kDa antigen, which is resistant to proteases and behaves as an amphiphilic component.

Animals↗