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Biomedical subjects

T Imanaka

Publications and source records attributed to T Imanaka.

At least 271 records · Page 15Linked to original sources
PubMed1983

Molecular cloning of a thermostable neutral protease gene from Bacillus stearothermophilus in a vector plasmid and its expression in Bacillus stearothermophilus and Bacillus subtilis.

The structural gene for a thermostable protease from Bacillus stearothermophilus was cloned in plasmid pTB90. It is expressed in both B. stearothermophilus and Bacillus subtilis. B. stearothermophilus carrying the recombinant plasmid produced about 15-fold more protease (310 U/mg of cell dry weight) than did the wild-type strain of B. stearothermophilus. Some properties of the proteases that have been purified from the transformants of B. stearothermophilus and B. subtilis were examined. No significant difference was observed among the enzyme properties studied here despite the difference in host cells. We found that the protease, neutral in pH characteristics and with a molecular weight of 36,000, retained about 80% of its activity even after treatment of 65 degrees C for 30 min.

Bacillus subtilis↗
PubMed1983

Lipid composition of low-density lysosomal membrane fraction prepared from atheromatous aorta of cholesterol-fed rabbits.

The lipid composition of low-density lysosomal membranes prepared from atheromatous aorta was analyzed. Results showed that the molar ratio of free cholesterol to phospholipids was 2.4, the phospholipids contained 49% sphingomyelin and 4% phosphatidylethanolamine and the phosphatidylcholine contained 22% polyunsaturated fatty acids. These values for the lipid composition are very different from those for normal biological membranes. The involvement of alteration of lysosomal membranes in accumulation of cholesterol ester in atherosclerosis is discussed.

Animals↗
PubMed1982

Ultrastructural analysis of membrane-bound polysomes in human myeloma cells.

We prepared ultra-thin sections of human myeloma cells, in which the rER was cut tangentially, and studied the make-up and distribution of membrane-bound polysomes electromicroscopically. In IgG myeloma large and small polysomes were detected. The polysome distribution curve showed a high peak at 7 ribosomes and a lower peak at 17-18 ribosomes. IgA-, IgD- and IgE myeloma, as well as macroglobulinemia, showed peaks at 7 and 13 ribosomes. BJP myeloma manifested a sharp peak only at 7 ribosomes. Our results suggest that BJP myeloma has only small polysomes participating in L-chain synthesis, while the other myelomas exhibited large and small polysomes participating in H- and L-chain synthesis, respectively. The quantitative ratio of small and large polysomes was determined on the basis of an analytically corrected direct count.

Humans↗
PubMed1982

Phenotypic variations in re-lysogenization of Bacillus licheniformis with temperate phage.

A bacitracin-producing strain Bacillus licheniformis ATCC 10716 harbors two types of inducible phages (LP52 and DLP 10716). 156 strains re-lysogenized with phage LP52 were independently isolated from a cured strain UM12 of B. licheniformis. Those strains were divided into 12 groups based on colony morphology and pigment production. Some of the re-lysogenized strains grew faster than UM12 and others produced more bacitracin than the cured strain. For example, the production of bacitracin by one of the re-lysogenized strains, L89, was enhanced by about 70% in comparison with UM12. The phenotypic variations observed with re-lysogenized strains might be due to the re-insertion of the phage genome at different sites of the chromosome in addition to the pleiotropic effect assumed.

Bacillus↗
PubMed1982

New approach to tryptophan production by Escherichia coli: genetic manipulation of composite plasmids in vitro.

For the purpose of studying the production of L-tryptophan by Escherichia coli, the deletion mutants of the trp operon (trpAE1) were transformed with mutant plasmids carrying the trp operon whose anthranilate synthase and phosphoribosyl anthranilate transferase (anthranilate aggregate), respectively, had been desensitized to tryptophan inhibition. In addition to release of the anthranilate aggregate from the feedback inhibition required for plasmids such as pSC101 trp.I15, the properties of trp repression (trpR) and tryptophanase deficiency (tnaA) were both indispensable for host strains such as strain Tna (trpAE1 trpR tnaA). The gene dosage effects on tryptophan synthase activities and on production of tryptophan were assessed. A moderate plasmid copy number, approximately five per chromosome, was optimal for tryptophan production. Similarly, an appropriate release of the anthranilate aggregate from feedback inhibition was also a necessary step to ward off the metabolic anomaly. If the mutant plasmid pSC101 trp-I15 was further mutagenized (pSC101 trp.I15.14) and then transferred to Tna cells, an effective enhancement of tryptophan production was achieved. Although further improvement of the host-plasmid system is needed before commercial production of tryptophan can be realized by this means, a promising step toward this goal has been established.

Cloning, Molecular↗
PubMed1982

Transformation of Bacillus stearothermophilus with plasmid DNA and characterization of shuttle vector plasmids between Bacillus stearothermophilus and Bacillus subtilis.

A thermophilic bacterium Bacillus stearothermophilus IFO 12550 (ATCC 12980) was transformed with each of the following plasmids, pUB110 (kanamycin resistance, Kmr), pTB19 (Kmr and tetracycline resistance [Tcr]), and its derivative pTB90 (Kmr Tcr), by the protoplast procedure in the presence of polyethylene glycol at 48 degrees C. The transformation frequencies per regenerant for pUB110, pTB19, and pTB90 were 5.9 x 10(-3), 5.5 x 10(-3), and 2.0 x 10(-1), respectively. Among these plasmids, pTB90 was newly derived, and the restriction endonuclease cleavage map was constructed. When tetracycline (5 micrograms/ml) was added into the culture medium, the copy number of pTB90 in B. stearothermophilus was about fourfold higher than that when kanamycin (5 micrograms/ml) was added instead of tetracycline. Bacillus subtilis could also be transformed with the plasmids extracted from B. stearothermophilus and vice versa. Accordingly, pUB110, pTB19, and pTB90 served as shuttle vectors between B. stearothermophilus and B. subtilis. The requirements for replication of pTB19 in B. subtilis and B. stearothermophilus appear to be different, because some deletion plasmids (pTB51, pTB52, and pTB53) derived from pTB19 could replicate only in B. subtilis, whereas another deletion plasmid pTB92 could replicate solely in B. stearothermophilus. Plasmids pTB19 and pTB90 could be maintained and expressed in B. stearothermophilus up to 65 degrees C, whereas the expression of pUB110 in the same strain was up to 55 degrees C.

Bacillus subtilis↗
PubMed1981

Purification and properties of rabbit liver acid-lipase (4-methylumbelliferyl oleate hydrolase).

An acid lipase was purified from rabbit liver lysosomes by, in sequence, osmotic treatment of the lysosomal fraction, Sephadex LH-20, DEAE-Sephadex A-50, Bio-Gel A-5m, hydroxyapatite and, finally, Sephadex G-200 column chromatography. The substrate was 4-methylumbelliferyl oleate. The enzyme was solubilized by Sephadex LH-20 column chromatography instead of detergents and organic solvents, to obtain an intrinsic macromolecule. 4-Methylumbelliferyl oleate hydrolase, osmotically released from lysosomal particles, had a very high molecular weight (greater than 800 000) which was reduced by gel filtration on a Sephadex LH-20 column; the final molecular weight of the purified enzyme was 58 000. The specific activity of 4-methylumbelliferyl oleate hydrolase increased at almost the same rate as acid cholesterol esterase and triacylglycerol lipase after Sephadex LH-20 column chromatography; the thermal stability of the activity of the three enzymes was almost identical. We also discuss the properties of the enzyme molecule and the interaction between the enzyme and the lysosomal membrane.

Animals↗
PubMed1981

TnA-directed deletion of the trp operon from RSF2124-trp in Escherichia coli.

Plasmid pMT-trp was constructed by digestion of RSF2124-trp with restriction endonuclease PstI and ligation with T4 ligase. In pMT-trp about 78% of the DNA of transposon TnA from RSF2124-trp was deleted, and hence the gene for ampicillin resistance was lost. All Trp- segregants from pMT-trp carriers in Escherichia coli W3110 and its derivatives were found to have lost the entire plasmid. On the other hand, deletion plasmids which had lost the trp operon were found among Trp- segregants from RSF2124-trp carriers, particularly from the mutant strain trpAE1 trpR tnaA. The experimental fact that deletion occurred exclusively in RSF2124-trp suggests that the presence of TnA in the plasmid (RSF2124-trp) was responsible for the deletion.

Ampicillin↗
PubMed1981

Isolation and characterization of antibiotic resistance plasmids from thermophilic bacilli and construction of deletion plasmids.

Ten plasmids were isolated as covalently closed circular deoxyribonucleic acid from antibiotic-resistant thermophilic bacteria. Of the 10 plasmids tested, 2 could transform Bacillus subtilis, yielding resistance to specific antibiotics. Plasmid pTB20 (2.8 X 10(6) daltons, approximately 24 copies per chromosome) specifies resistance to tetracycline (Tcr), whereas pTB19 (17.2 X 10(6) daltons, approximately 1 copy per chromosome) renders the host resistant to both kanamycin and tetracycline (KMrTcr). Three plasmids were not self-transmissible. The restriction endonuclease cleavage maps of the two plasmids, pTB19 and pTB20, were constructed. pTB19 and pTB20, both of which were originally isolated from thermophilic bacilli, were tested for stability in B. subtilis. Digestion of pTB19 followed by ligation yielded deletion plasmids pTB512 (Kmr), pTB52 (Tcr), and pTB53 (KmrTcr). Determinants of Kmr, Tcr, and DNA replication were associated with EcoRI fragments R1b (4.2 X 10(6) daltons), R3 (2.8 X 10(6) daltons), and R1a (4.2 X 10(6) daltons), respectively. Restriction endonuclease cleavage maps of pTB51, pTB52, and pTB53 were constructed. Tetracycline resistance of pTB20 was confirmed to be in the EcoRI fragment (1.85 X 10(6) daltons).

Bacillus subtilis↗
PubMed1981

Cloning of the genes for penicillinase, penP and penI, of Bacillus licheniformis in some vector plasmids and their expression in Escherichia coli, Bacillus subtilis, and Bacillus licheniformis.

By using plasmid pMB9, penicillinase genes (penP and penI) from both the wild-type and constitutive strains of Bacillus licheniformis 9945A were cloned in EScherichia coli. When a low-copy-number plasmid was used, both wild-type and constitutive penicillinase genes could be transferred into Bacillus subtilis. However, when a high-copy-number plasmid was used, only the genes of the wild type could be transferred. These recombinant plasmids in B. subtilis could all be transferred by the protoplast transformation procedure into B. licheniformis. Transformants of E. coli were resistant to ampicillin (20 micrograms/ml) in spite of the low penicillinase activities (7 U/mg of cells). However, transformants of B. subtilis and B. licheniformis were sensitive to ampicillin (20 micrograms/ml) even in high penicillinase activities (more than 10,000 U/mg of cells). The secretion of penicillinase was rarely observed in E. coli. In contrast, penicillinases secreted from transformants of B. subtilis and B. licheniformis were around 30 and 60% of the total activities, respectively. We took advantage of the plasmids to permit the construction of hetero- and mero-polyploid structures in host cells, and we discuss a regulatory mechanism of penicillinase synthesis in B. licheniformis.

Bacillus↗
PubMed1980

Phenotypic stability of trp operon recombinant plasmids in Escherichia coli.

The recombinant plasmids RSF2124-trp and pSC101-trp were examined for their phenotypic stability in Escherichia coli W3110 and its derivatives under various culture conditions. RSF2124-trp and pSC101-trp were stable in a trpAE1 strain. In an amber mutant of the tryptophan repressor gene, RSF2124-trp was fairly stable, whereas pSC101-trp was unstable. All Trp- segregants from the pSC101-trp carrier had lost the entire plasmid. In a mutant carrying the tnaA mutation, RSF2124-trp was unstable in rich media. Most Trp- segregants that appeared under these conditions were deleted in trp genes as well as in the cI gene on the recombinant plasmid. pSC101-trp in this tnaA mutant was also unstable. All Trp- segregants had lost the plasmid. Studies of enzyme activities revealed that the greater the activity of anthranilate synthase and tryptophan synthase in bacteria, the more segregants tended to appear in the stability test. RSF2124 and pSC101 without the trp gene were completely stable in the same bacteria. The apparent instability of bacteria carrying the recombinant plasmid could be explained by the lower growth rate compared with bacteria carrying only the vector plasmid, resulting in the enrichment of Trp- bacteria during culture.

Anthranilate Synthase↗