Biomedical subjects

Y Kumada

Publications and source records attributed to Y Kumada.

16 recordsLinked to original sources

PubMed1992

[A case of late-onset SLE complicated with EDTA-dependent pseudothrombocytopenia].

A 57-year-old man was admitted to our clinic with complaints of proximal myalgia in extremities. He was diagnosed as late-onset SLE based on the findings of pleuritis, pericarditis, arthritis and antibodies to DNA and cardiolipin. Aggregation of the platelets and the decreased counts of platelets were observed when EDTA was used as anticoagulant for the blood tests. However, the platelet aggregation was not noted with normal counts of platelets when Heparin-Theophylline was used as anticoagulant. From this observation, EDTA-dependent pseudothrombocytopenia was diagnosed and IgM class of EDTA-dependent anti-platelet antibody was detected by means of flow cytometry. Administration of prednisolone at 40mg/day reduced the symptoms and EDTA-dependent pseudothrombocytopenia, and EDTA-dependent anti-platelet antibody disappeared. His clinical course suggested that EDTA-dependent pseudothrombocytopenia was closely associated with the disease activity of SLE.

Autoantibodies↗

PubMed1992

[The effects of potassium concentration in reperfusion solution upon myocardial protection].

The effects of potassium in reperfusion solution (RS) and the influence of sodium on this effect were studied. Experimental time course was as followed: 20 min working perfusion, 3 min cardioplegic infusion with St. Thomas Cardioplegic Solution followed by global ischemia for 33 or 35 min at 37.5 degrees C, 15 min early Langendorff reperfusion with several different potassium concentration modified with Krebs Henseleit Bicarbonate Buffer (KHBB) containing 145 mM and 110 mM sodium and 5 min late reperfusion with KHBB, followed by 20 min working perfusion. Potassium in RS possessed bell shaped dose response nature with optimal concentration of 10 mM in the condition of 145 mM sodium but 6 m in the condition of 110 mM in terms of percent recovery of aortic flow. Although higher potassium reperfusion produced less Creatine Kinase leakage.

Animals↗

PubMed1992

[The effects of osmolarity in reperfusion solution upon myocardial protection].

The effects of several different osmolarity in reperfusion solution were studied. Experimental time course was as follows: 20 min working perfusion, 3 min cardioplegic infusion with St. Thomas Cardioplegic Solution (STS) followed by global ischemia for 33 min at 37.5 degrees C, 15 min early Langendorff reperfusion with different osmolarity by adding sucrose and 5 min late reperfusion with Krebs Henseleit bicarbonate buffer, followed by 20 min working perfusion. Percent recoveries of aortic flow showed that 290 mOsm/L in reperfusion solution possessed optimal protective properties with bell shaped dose response characteristics.

Animals↗

PubMed1991

The bialaphos biosynthetic genes of Streptomyces viridochromogenes: cloning, heterospecific expression, and comparison with the genes of Streptomyces hygroscopicus.

The bialaphos resistance gene, bar, was used as a selectable marker to isolate the bialaphos production genes (bap) from the Streptomyces viridochromogenes genome. The S. viridochromogenes bar gene was cloned on overlapping restriction fragments using pIJ680 and pIJ702 in the bialaphos-sensitive host, S. lividans. Although the restriction endonuclease cleavage map of these fragments was not similar to the bap cluster of S. hygroscopicus, the presence and location of bar and four other bap genes as well as a gene required for the transcriptional activation of the cluster (brpA) was demonstrated by heterologous cloning experiments using a series of previously characterized bialaphos-nonproducing S. hygroscopicus mutants. Since recombination-deficient mutants of streptomycetes have not been isolated, restored function provided by cloned homologous DNA results from both recombination (marker rescue) and complementation in trans. In contrast to our previously reported homologous cloning experiments where we were able to define the position of mutant alleles by recombination, in these heterologous cloning experiments we observed little if any recombination between plasmid-cloned genes and the chromosome. As a result, this approach allowed us to define the location and orientation of functional genes using a genetic complementation test. The organization of the clustered S. viridochromogenes bap genes was indistinguishable from the corresponding S. hygroscopicus mutant alleles. The fact that the S. viridochromogenes transcriptional regulatory gene, brpA, functioned in S. hygroscopicus implied that some transcriptional regulatory signals may also be interchangeable. In these two Streptomyces species, which have considerable nucleotide sequence divergence, the complex biochemical and genetic organization of the bialaphos biosynthetic pathway is conserved.

Anti-Bacterial Agents↗

PubMed1991

Conversion of bialaphos to other oligopeptides containing phosphinothricin by Streptomyces hygroscopicus.

Two oligopeptides containing phosphinothricin were accumulated in the culture of bialaphos (BA) producer Streptomyces hygroscopicus SF1293 when large amount of BA was added to the culture at idio phase. One of the oligopeptides was a new substance, BA dimer (phosphinothricyl-alanyl-alanyl-phosphinothricyl-alanyl-alan ine), and the other was a known substance called If (phosphinothricyl-alanyl-alanyl-phosphinothricin). Though BA none producing mutants which were blocked at the steps 1, 10 and 13, respectively in BA biosynthesis also converted BA to BA dimer and If, the mutants blocked at the step 11 (alanylation) could not curry out the conversion. Antibiotic activities of BA dimer and If were lower than 1/100 of BA.

Aminobutyrates↗

PubMed1990

Streptomyces hygroscopicus has two glutamine synthetase genes.

Streptomyces hygroscopicus, which produces the glutamine synthetase inhibitor phosphinothricin, possesses at least two genes (glnA and glnB) encoding distinct glutamine synthetase isoforms (GSI and GSII). The glnB gene was cloned from S. hygroscopicus DNA by complementation in an Escherichia coli glutamine auxotrophic mutant (glnA). glnB was subcloned in Streptomyces plasmids by insertion into pIJ486 (pMSG3) and pIJ702 (pMSG5). Both constructions conferred resistance to the tripeptide form of phosphinothricin (bialaphos) and were able to complement a glutamine auxotrophic marker in S. coelicolor. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of S. lividans(pMSG5) revealed a highly overexpressed 40-kilodalton protein. When GS was purified from this strain, it was indistinguishable in apparent molecular mass from the 40-kilodalton protein. The nucleic acid sequence of the cloned region contained an open reading frame which encoded a protein whose size, amino acid composition, and N-terminal sequence corresponded to those of the purified GS. glnB had a high G + C content and codon usage typical of streptomycete genes. A comparison of its predicted amino acid sequence with the protein data bases revealed that it encoded a GSII-type enzyme which had previously been found only in various eucaryotes (47 to 50% identity) and nodulating bacteria such as Bradyrhizobium spp. (42% identity). glnB had only 13 to 18% identity with eubacterial GSI enzymes. Southern blot hybridization experiments showed that sequences similar to glnB were present in all of the five other Streptomyces species tested, as well as Frankia species. These results do not support the previous suggestion that GSII-type enzymes found in members of the family Rhizobiaceae represent a unique example of interkingdom gene transfer associated with symbiosis in the nodule. Instead they imply that the presence of more than one gene encoding GS may be more common among soil microorganisms than previously appreciated.

Amino Acid Sequence↗

PubMed1989

Intestinal parasitic infections among schoolchildren in Chiang Mai, northern Thailand: an analysis of the present situation.

Stool examination from primary schoolchildren in Chiang Mai Province, north Thailand, was performed to determine the present state of parasitic infections in this area. Out of a total of 491 children, 239 proved positive (48.7%). The most common type of parasite was found to be soil-transmitted helminths such as hookworm (26.3%) or Strongyloides stercoralis (11.2%), while Ascaris lumbricoides was not so prevalent (1.2% being positive in one school out of three). These results are in contrast to earlier reports showing higher prevalence rates, leading the authors to hypothesize that improvements in sanitary conditions and eradication projects have been effective. Opisthorchiasis is another parasitic disease with a relatively high prevalence rate of 7.5%. This disease rate increases with age and it was found in two out of three schools (8.3-15.8%) and was the most common type of helminth infection. Ascariasis was not seen in these two schools, but strongyloidiasis was found to be the second most prevalent helminthiasis, having a higher infection rate than hookworm. Therefore, eradication efforts now need to be directed toward eliminating opisthorchiasis and strongyloidiasis in addition to continuing to eradicate ascariasis and hookworm infections. The most common protozoal infection with a high pathogenicity in this region was found to be giardiasis (7.7%).

Adolescent↗

PubMed1988

The bialaphos resistance gene (bar) plays a role in both self-defense and bialaphos biosynthesis in Streptomyces hygroscopicus.

We inactivated the bialaphos (BA) resistance gene (bar) of a BA producer, Streptomyces hygroscopicus, by the gene replacement technique. The resulting BA-sensitive mutant (Bar-) was able to produce little BA but considerable amount of an intermediate demethylphosphinothricin (DMPT). The Bar- mutant was still able to convert the N-acetyl derivative (AcDMPT) of DMPT to BA. Introduction of normal bar containing plasmid restored both BA resistance and BA biosynthesis to levels as high as the parental BA producer. By contrast, introducing a multi copy glutamine synthetase gene (glnA) into the Bar- mutant restored BA resistance but not BA production. Thus, the bar gene plays a crucial role in both self-defense and a step of BA biosynthesis in the BA-producing S. hygroscopicus.

Anti-Bacterial Agents↗

PubMed1988

Replacement of Streptomyces hygroscopicus genomic segments with in vitro altered DNA sequences.

We have developed a method for gene replacement in Streptomyces hygroscopicus which permits introduction of an in vitro derived mutation carried on a plasmid into the chromosome. We constructed the plasmid pMSB212 which can replicate in S. hygroscopicus and contains the step5 gene of the bialaphos biosynthetic pathway which was inactivated by a frame-shift mutation caused by filling in the cohesive ends of the EcoR I site in the structural gene. pMSB212 was introduced into a bialaphos producer strain and by protoplast regeneration of the primary thiostrepton-resistant transformants, non-producing mutants, were obtained. Biochemical and genetical analyses indicated that these mutants were specifically blocked by introduction of the frame-shift mutation in the step5 gene on the chromosome. This method will enable us to obtain isogenic mutants of known genes and to identify new genes encoded on a cloned fragment.

Chromosomes, Bacterial↗

PubMed1988

The bialaphos biosynthetic genes of Streptomyces hygroscopicus: cloning and analysis of the genes involved in the alanylation step.

We have isolated and studied the genes involved in the alanylation step in the biosynthesis of a herbicide, bialaphos which is produced by Streptomyces hygroscopicus. Three bialaphos-nonproducing mutants, NP60, NP61 and NP62, isolated from S. hygroscopicus by treatment with N-methyl-N'-nitro-N-nitrosoguanidine were defective for the alanylation step and were not restored to productivity by any locus of the gene cluster previously cloned. Three plasmids were isolated using NP60, NP61 and NP62 as recipients. The genes which restored productivity to NP61 and NP62 hybridized to the contiguous region of the bialaphos biosynthetic gene cluster. The gene cluster involved in the bialaphos production was about 35 kb long. The gene which restored productivity to NP60 did not hybridize to the bialaphos biosynthetic gene cluster. VM3 and VM4, putative alanylation blocked mutants, were derived from a bialaphos producer by gene replacement of an unidentified region of the biosynthetic gene cluster with an in vitro altered DNA sequence. The genes which restored productivity to VM3 and VM4 were located between the genes which code for phosphinomethylmalic acid synthase and demethylphosphinothricin acetyltransferase in the cluster. These results suggest that multiple genes are involved in the alanylation step.

Alanine↗

PubMed1986

Cloning of a streptomycin-production gene directing synthesis of N-methyl-L-glucosamine.

A Streptomyces bikiniensis DNA fragment complementing a deficiency in streptomycin (Sm) production was cloned on the plasmid vector pIJ385. Host strain S. bikiniensis SD 1 used as the recipient in cloning displayed deficiency in biosynthesis of N-methyl-L-glucosamine, one of the moieties of Sm. The cloned fragment on the multicopy plasmid pIJ385 conferred sevenfold increase in Sm production in comparison with the wild-type parental strain. By subcloning, the region complementing the Sm deficiency of SD 1 was narrowed to a 3.0-kb fragment.

Cloning, Molecular↗

PubMed1979

Studies on auromomycin.

A new antitumor antibiotic, named auromomycin, was isolated from the culture broth of Streptomyces macromomyceticus, a macromomycin-producing strain. The antibiotic was recovered from the culture filtrate by salting out with ammonium sulfate and further purified by successive application of ion-exchange chromatography on Amberlite IRA-93 (Cl form) and DEAE-Sephadex (OH form), Gel filtration on Sephadex G-50 and hydrophobic chromatography on Octyl-Sepharose CL-4B. The antibiotic is an acidic polypeptide with a molecular weitht of 12,500 and an isoelectric point of pH 5.4 and consists of 16 different amino acids. It has characteristic absorption maxima at 273 nm and 357 nm in the ultraviolet spectrum and two minima at 280 nm and 350 nm in the optical rotatory dispersion spectrum. Auromomycin exhibits antibacterial activity not only against Gram-positive bacteria, but also Gram-negative bacteria. Antitumor activities of auromomycin were revealed against EHRLICH ascites carcinoma, ascites sarcoma 180, L1210 leukemia and LEWIS lung carcinoma. Auromomycin was found to be converted into macromomycin by adsorption chromatography on Amberlite XAD.

Amino Acids↗

PubMed1979

Studies on inhibition of adenosine deaminase by isocoformycin in vitro and in vivo.

Isocoformycin is a structural isomer of coformycin which has been demonstrated to be a potent inhibitor of adenosine deaminase. Isocoformycin showed a weaker inhibition of this enzyme than coformycin; the binding of coformycin to enzyme was irreversible, but isocoformycin inhibition was competitive with substrate. The Ki value of isocoformycin was 4.5 approximately 10 X 10(-8) M. Following intraperitoneal injection of isocoformycin in mice, the adenosine deaminase activity of homogenates of several organs was determined and the following ED50 values (50% inhibition doses) were observed: 29 mg/kg for thymus, 13 mg/kg for spleen, 80 mg/kg for liver and 20 mg/kg for kidney. The inhibition of adenosine deaminase in rabbit blood in vitro was also tested in comparison with coformycin.

Adenosine Deaminase↗

PubMed1978

Biochemical activities of the derivatives of dehydrodicaffeic acid dilactone.

Activities of derivatives of dehydrodicaffeic acid dilactone (DDCAD) to inhibit catechol-O-methyltransferase (COMT), cyclic AMP phosphodiesterase (PDE) and DOPA decarboxylase (DDC) were examined. Among those tested, 2,6-bis-(5',6'-dibromo-4'-hydroxy-3'-methoxyphenyl)-3,7-dioxabicyclo-[3,3,0]-octane 4,8-dione was found to be the strongest inhibitor of both COMT and PDE. There were no derivatives which showed a stronger inhibition against DDC than the original compound, DDCAD.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1976

Dehydrodicaffeic acid dilactone, an inhibitor of catechol-O-methyl transferase.

In the screening of catechol-O-methyltransferase inhibitors, three compounds were isolated from the culture filtrate of a mushroom, Inonotus sp. One was 3,4-dihydroxycinnamic acid (caffeic acid) which had been reported as an inhibitor of this enzyme. The others were the dextrorotatory 2,6-bis-(3',4'-dihydroxyphenyl)-3,7-dioxabicyclo-[3,3,0]-octane 4,8-dione (dehydrodicaffeic acid dilactone) andits antipode. These new compounds inhibited both dopamine beta-hydroxylase and dopa decarboxylase and showed hypotensive activity in the SH rat.

Animals↗

PubMed1975

New isoflavones, inhibiting catechol-O-methyltransferase, produced by Streptomyces.

In the screening of catechol-O-methyltransferase inhibitors in streptomyces culture filtrates, three new isoflavones were isolated. Their structures were shown to be 3',5,7-trihydroxy-4',6-dimethoxyisoflavone (I), 3',5,7-trihydroxy-4',8-dimethoxyisoflavone (II), 3',8-dihydroxy-4',6,7-trimethoxyisoflavone (III). I and II inhibited both catechol-O-methyltransferase and dopa decarboxylase, and showed hypotensive action. III was a specific inhibitor of catechol-O-methyltransferase, and showed no hypotensive action.

Animals↗