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K Hotta

Publications and source records attributed to K Hotta.

At least 235 records · Page 13Linked to original sources
PubMed1988

Mechanism of increased kanamycin-resistance generated by protoplast regeneration of Streptomyces griseus. II. Mutational gene alteration and gene amplification.

Genetic mechanisms involved in the kanamycin (KM)-hyper-resistance of Streptomyces griseus SS-1198PR and NP1-1PR generated by protoplast regeneration were investigated. Southern hybridization of Sph I-digested genomic DNA with a KM-resistance gene (kan) probe revealed that the strain SS-1198PR and its KM-sensitive parent (SS-1198) had the same copy number of a 4.2-kb Sph I fragment hybridizing to the kan probe, while the kan gene of the strain NP1-1PR was located on a highly amplified DNA sequence (100-200 copies/chromosome) consisting of the 6.7 kb amplifiable unit with Sph I site at the junction site region. There was no difference in the restriction endonuclease cleavage map of the kan gene region of the Sph I fragments from the three strains. However, the level (50 micrograms/ml) of KM-resistance conferred by the cloned NP1-1PR kan gene was much lower than that (1,000 micrograms/ml) conferred by the cloned SS-1198PR kan gene in Streptomyces lividans TK21 although the strain TK21 harboring these kan genes produced an aminoglycoside acetyltransferase, AAC(3)-V, with the same substrate specificity. It seemed, therefore, likely that a mutational alteration of the kan gene and a DNA amplification involving the kan region played major roles for the KM-hyper-resistance depending on AAC(3)-V in the strains SS-1198PR and NP1-1PR, respectively.

Base Sequence↗
PubMed1988

Strain- and species-specific distribution of the streptomycin gene cluster and kan-related sequences in Streptomyces griseus.

The streptomycin (SM) gene cluster was investigated for its distribution in streptomycetes by Southern hybridization using nick-translated DNA probes, which were isolated from the SM-6-phosphotransferase (SPH) and amidinotransferase (ADT) regions of the SM gene cluster of Streptomyces griseus SS-1198. Bgl II-digested genomic DNAs from SM-producing strains of S. griseus yielded the same size fragment (7.0 kb) which hybridized to both the SPH and ADT probes as expected from the restriction endonuclease cleavage map of the SM gene cluster. By contrast, no genomic DNA fragments from heterologous Streptomyces strains hybridized to the probes. Thus, only SM-producing strains of S. griseus possess the highly homologous SM gene cluster. Similarly, distribution of DNA sequences homologous to the kanamycin (KM)-resistance determinant (kan) from a KM-resistant regenerant of S. griseus SS-1198 protoplasts was also examined. Using the kan gene fragment as the probe it was revealed that the kan-related sequences are present in all the strains of S. griseus tested, irrespective of the type of antibiotics they produce. However, no hybridization to the kan gene probe (KAN) was observed with DNA digests derived from other Streptomyces species.

Anti-Bacterial Agents↗
PubMed1988

Cloning of macromomycin apoprotein gene from Streptomyces macromomyceticus by use of 50-mer deoxynucleotide probes.

A mixed probe consisting of two synthetic deoxynucleotides (52 and 54 mers referred to as 50-mer) with arbitrarily chosen C or G for the third letters was prepared based on the amino acid sequences No. 31-48 and No. 72-90 of macromomycin (MCM) apoprotein and successfully used to clone the MCM apoprotein gene. Digestion with Sph I of total DNA of MCM-producing Streptomyces macromomyceticus M480-M1 yielded a 2.6-kb fragment that hybridized strongly to the probes. The hybridized probe was stable to washing with 3 x SSC at 75 degrees C. Radioactivity derived from the hybridized probe was comparable to that expected theoretically from hybridization between the probe and the true target sequence. The 2.6-kb fragment was cloned into Escherichia coli RR1 with pBR322 and subsequently subcloned into Streptomyces lividans TK21 with pIJ702. Nucleotide sequence analysis of the cloned fragment verified the existence of the sequence corresponding to the amino acid sequence of MCM apoprotein and about 90% homologies with the probes. Thus, the use of relatively long deoxynucleotide probes with arbitrarily chosen C or G for the third letters will be advantageous in cloning Streptomyces protein genes where more than 90% of the third letters have been known to be C or G. In addition, theoretical diagnosis of hybridization should be a great help to distinguish true positives from false ones.

Amino Acid Sequence↗
PubMed1988

Mechanism of increased kanamycin-resistance generated by protoplast regeneration of Streptomyces griseus. I. Cloning of a gene segment directing a high level of an aminoglycoside 3-N-acetyltransferase activity.

The genetic and biochemical basis of a 200-fold increase in kanamycin (KM)-resistance shown in Streptomyces griseus SS-1198PR generated by protoplast regeneration was investigated. A 15-kb Bcl I-DNA segment responsible for the KM-resistance was cloned into pIJ61 with Streptomyces lividans TK21 as host. The KM-resistance segment was then subcloned into pIJ702 as a 1.8-kb BamH I-Bgl II fragment with a BamH I site essential for the KM-resistance. Both S. lividans TK21 containing the cloned segments and S. griseus SS-1198PR showed multiple resistance to KM, dibekacin and gentamicin C complex. Cell free extracts from these strains inactivated the antibiotics in the presence of acetyl CoA in agreement with their resistance pattern. The structure of the inactivated KM-A was determined as 3-N-acetyl-KM-A indicating acetylation by an aminoglycoside acetyltransferase, AAC(3). The substrate range of the enzyme was unique and was designated AAC(3)-V. No genetic linkage was found between the cloned 15 kb Bcl I segment and the separately cloned streptomycin resistance gene (str) segment (3.8 kb Sph I fragment). The str genes cloned from both the parent (SS-1198) and the strain SS-1198PR were identical in their size, restriction site and function. In addition, these strains showed no significant difference in the total DNA digestion pattern. These results indicate that protoplast regeneration may cause a critical change in a specific region of DNA resulting in a high activity of an AAC(3) with a novel substrate range.

Acetylation↗
PubMed1987

Similar inhibitory effects of dantrolene sodium on twitch tension and on silver ion-induced contracture in skeletal muscle.

To determine the mechanism by which Ag+ induces a transient contracture in skeletal muscle, the effect of dantrolene sodium on the Ag+ contracture was examined and the findings compared with those for the twitch, tetanus and caffeine contracture. The inhibition of twitch by dantrolene was equivalent to that of the Ag+ contracture at concentrations of 1, 2 or 5 microM of dantrolene. The tetanus tension was slightly inhibited by dantrolene, but not the caffeine contracture. These observations suggest that the Ag+ contracture may be governed by the same mechanism as that involved in the development of twitch tension.

Animals↗
PubMed1987

Subfractionation of the dansylated derivatives of glucosyl galactosyl hydroxylysine by liquid chromatography and its application to a specific alpha-1,2-glucosidase assay.

The dansyl derivative of glucosyl galactosyl hydroxylysine (GGH) was separated into two components, as GP-I (monodansyl GGH) and GP-II (didansyl GGH) by paper chromatography. GP-I was further fractionated into four peaks (a, b, c and d) by reversed-phase liquid chromatography. These peaks corresponded to the dansyl derivatives at the alpha-amino (a and b) and epsilon-amino (c and d) groups of their hydroxylysine residues. There is the possibility that the fractions for b and d are diastereoisomers of a and c, respectively, since the monodansyl derivative from human urine consists of a and c. GP-II was fractionated into two peaks, e and f, which may possibly be diastereoisomers of each other. Treatment of the a, b, c and d fractions with crude chicken liver enzyme resulted in the preferential cleavage of a and b and the production of monodansyl galactosyl hydroxylysine. Components c and d were also cleaved slowly, resulting in the production of monodansyl hydroxylysine by the successive action of beta-galactosidase on dansyl galactosyl hydroxylysine. The detected alpha-glucosidase activity was strongly inhibited by free mannosamine. The method developed using the monodansyl GGH fraction a (or b) and high-performance liquid chromatography facilitated the detection of alpha-1,2-glucosidase, which acts specifically toward GGH even in a crude enzyme preparation.

Animals↗
PubMed1987

Comparative study of specific alpha-1,2-glucosidase activity toward glucosyl galactosyl hydroxylysine in various animal species.

1. Assay of the activity of alpha-1,2-glucosidase was completed within 10 min using reversed-phase high performance liquid chromatography and purified dansylated glucosyl galactosyl hydroxylysine as the substrate. 2. A comparative study was made on the enzyme activity of liver homogenate from eight animal species, mouse, frog, chicken, rabbit, pig, rat, human, bovine and that of a spinach leaf homogenate. alpha-1,2-glucosidase activity in human and bovine liver was very low, and that of alpha-1,2-glucosidase could not be detected in the spinach homogenate as expected. 3. 1-deoxynojirimycin, a well known potent inhibitor of alpha-1,2-glucosidases which act on the N-glycosidic type carbohydrate chain, also inhibited alpha-1,2-glucosidase acting specificity on glucosyl galactosyl hydroxylysine derived from the collagen molecule.

Animals↗
PubMed1987

Effects of extracellular calcium and calcium channel blocker on silver-induced contractures in frog skeletal muscle fibers.

The mechanism by which Ag+ induces muscle contracture was elucidated by investigating the effect of external Ca2+ concentration and Ca2+ channel blocker on the maximum tension amplitude in single fibers from frog toe skeletal muscle. Five microM Ag+ induced two different types of contracture in the presence of external Ca2+ more than 0.1 mM, i.e., a phasic and a subsequent tonic contracture. The phasic contracture appeared only in fibers with intact T-tubules immersed in a solution with or without Ca2+ after a lag time of 5.7 +/- 0.9 s (N = 5). The maximum amplitude was 58% of the tetanus tension observed in the same fiber immediately before Ag+ exposure. Diltiazem at high-concentration (100 microM) inhibited the Ag+-induced phasic contracture only to a small extent (17%). The contracture was not affected by 1 microM TTX or 1 mM DAP at all. These results indicate that Na+, K+, and Ca2+ channels on the T-tubular membrane would not be attributed to the phasic tension development induced by Ag+. On the contrary, a tonic contracture did not require intact T-tubules. The amplitude and the rate of rise of the contracture depended on external Ca2+ concentrations and were inhibited by a high concentration of diltiazem. Neither 1 microM TTX nor 1 mM DAP affected them. Therefore, the tonic contracture seems to be triggered by Ca2+ which entered the muscle fiber through the surface but not T-tubular membranes.

4-Aminopyridine↗
PubMed1987

A local anesthetic, tetracaine, similarly inhibits Ag+ and K+ contractures in frog skeletal muscle.

To evaluate usefulness of Ag+ contracture as a tool for elucidating the mechanism underlying the excitation-contraction coupling, the effects of tetracaine on Ag+ contracture were compared with those on K+ and caffeine contractures in frog skeletal muscle. Tetracaine less than 100 microM shortened the duration of 120 mM K+ contracture, without affecting tension amplitude. At higher concentrations of tetracaine, K+ contracture was inhibited dose-dependently and the duration shortened. Treatment of the fibers with 20-500 microM tetracaine for 3 min did not block the contracture induced by 25 mM caffeine. Effects of tetracaine on Ag+ contracture were similar to those on K+ contracture. In the presence of 200 microM tetracaine, 41% inhibition was observed in 120 mM K+ contracture, while 43% in 100 microM Ag+ contracture. Also, 200 microM tetracaine completely inhibited the contractures induced by 40 mM K+ or 5 microM Ag+. These findings suggest that the Ag+ may induce contractures via its action on the T/SR junction, not a direct action on the SR. Therefore, understanding the mechanism involved in the development of Ag+ contracture would be helpful to elucidate the mechanism of excitation-contraction coupling.

Action Potentials↗
PubMed1986

Anomer specificity of glucose-6-phosphatase and glucokinase.

The anomeric form of glucose produced by glucose-6-phosphatase was studied using an apparatus that specifically measures beta-D-glucose. The time course of beta-D-glucose formation from glucose-6-P by glucose-6-phosphatase is essentially linear. In the presence of mutarotase, this rate is reduced to 70% of that obtained in the absence of mutarotase. When detergent treated microsomes were used, the rate of beta-D-glucose formation is unaffected by mutarotase. These results suggest that only beta-anomer of glucose is produced by microsomal glucose-6-phosphatase and this specificity is determined by translocase for glucose-6-P or glucose. It was also demonstrated that alpha-D-glucose is the substrate for glucokinase.

Animals↗
PubMed1986

Characterization of a highly glycosylated biosynthetic intermediate of ovalbumin.

Ovalbumin extracted from oviduct slices incubated with [35S]methionine or [2-3]mannose contained two biosynthetic intermediates, OE and OF (Y. Kato, H. Iwase, and K. Hotta, 1984, J. Biochem. (Tokyo) 95, 455-463). In the present study, these intermediates are further characterized. The 3H dpm/35S dpm ratio of OF labeled with both [35S]methionine and [3H]mannose was twice that or greater than that of OE. The tritium-labeled OF migrated more slowly than OE on sodium dodecyl sulfate-gel electrophoresis and had a molecular weight exceeding that of OE by about 1500. These findings, along with the fact that [3H]OF had sugar chains similar to those of [3H]OE, suggest that OF may possibly have two sugar chains in one molecule. For confirmation of this, the glycosylation sites of OF were examined. Peptic and tryptic glycopeptides were prepared from [2-3H]mannose-labeled OF and the other ovalbumin subfractions--OA, OC, OD, and OE--and then analyzed by high-performance liquid chromatography. The peptic glycopeptides prepared from [3H]OF contained glycopeptides in addition to those derived from the other subfractions although tryptic glycopeptides obtained from [3H]OF were similar to those from the other subfractions. This shows that the above hypothesis is valid.

Acetylglucosaminidase↗
PubMed1986

Comparative study on mucus glycoproteins in rat stomach and duodenum.

The density of mucus glycoprotein compared to that of the corpus, antrum and duodenum was; 1.52, 1.49 and 1.57 g/ml respectively. Carbohydrate composition of gastrointestinal mucus glycoprotein consisted of N-acetylgalactosamine, N-acetylglucosamine, galactose, fucose and sialic acid. Ratios of carbohydrate composition among corpus, antral and duodenal mucus glycoproteins differed. The average length of an oligosaccharide was found to be about 12-13, 14 and 10 sugars in the corpus, antrum and duodenum, respectively. In the corpus, the amino acid content was found to have the following quantitative order: Thr greater than Ser greater than Glx = Pro; in the antrum: Thr greater than Ser greater than Glx; and in the duodenum: Thr greater than Ser greater than Pro. Corpus, antral and duodenal mucus glycoproteins have the blood-group A antigen; antral mucus glycoprotein in particular exhibited strong blood-group A activity.

Animals↗