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Biomedical subjects

M Tsuda

Publications and source records attributed to M Tsuda.

At least 595 records · Page 33Linked to original sources
PubMed1981

Qualitative and quantitative abnormalities of argininosuccinate synthetase in citrullinemia.

Enzymological and immunochemical analyses of the liver were preformed in seven Japanese patients with citrullinemia. Among the urea cycle enzymes in the liver, only the activity of argininosuccinate synthetase was specifically decreased to 2 to 50% of normal controls. Liver argininosuccinate synthetase of patients was indistinguishable from that of controls when tested immunochemically by Ouchterlony's double immunodiffusion technique with anti-rat argininosuccinate synthetase antiserum. Immunochemical analysis by means of the single radial immunodiffusion revealed that the decrease in the activity of liver argininosuccinate synthetase was explainable by a decrease in the amount of the enzyme protein in five patients, while the decrease in the activity in the other two patients was not accompanied by a decrease of enzyme protein. The Km values for the substrates of liver argininosuccinate synthetase of the former five were similar to those of the control, while the kinetic properties of the latter two were quite different in terms of higher Km values and negative cooperativity. From these results, we consider that citrullinemia may consist of more than one type including qualitative or quantitative abnormalities of argininosuccinate synthetase caused by some defects in certain genes or in the epigenetic processes in the liver.

Adolescent↗
PubMed1981

Tn1 insertion mutagenesis in Escherichia coli K-12 using a temperature-sensitive mutant of plasmid RP4.

A method for Tn1 insertion mutagenesis in Escherichia coli has been developed using pTH10, a mutant plasmid of RP4 temperature-sensitive for maintenance. The mutagenesis involves three steps. Firstly, from strains carrying pTH10 showing resistance to the antibiotics kanamycin, tetracycline, and ampicillin at 30 degrees C but not at 42 degrees C, clones are isolated resistant to kanamycin at 42 degrees C. Such temperature-independent, drug resistant clones probably carry pTH10 integrated into the host chromosome. Secondly, they are cultivated at 30 degrees C. At this temperature segregants carrying pTH10, which has been excised from the host chromosome, accumulate. Thirdly, to cure such segregants of autonomous pTH10, they are cultivated at 42 degrees C. By these procedures, clones free of pTH10, but carrying Tn1 insertions on the host chromosome, were obtained. About 3% of the clones carrying Tn1 insertions were auxotrophic. Distribution of auxotrophic mutations was not random, indicating the existence of preferential integration sites of Tn1 on the host chromosome. The frequency of precise excision of Tn1 was less than 10(-10). The pTH10 plasmid has a wide host range among Gram-negative bacteria and thus may serve as a excellent vector for insertion mutagenesis of Tn1 in many Gram-negative bacterial species.

DNA Transposable Elements↗
PubMed1981

Nitrite converts 2-amino-alpha-carboline, an indirect mutagen, into 2-hydroxy-alpha-carboline, a non-mutagen, and 2-hydroxy-3-nitroso-alpha-carboline, a direct mutagen.

2-Amino-alpha-carboline [26148-68-5] which was isolated from a pyrolysate of soybean globulin and which was mutagenic to Salmonella typhimurium in the presence of a rat-liver microsomal fraction (S9 mix), was converted into non-mutagenic 2-hydroxy-alpha-carboline by treatment with nitrite in acidic conditions. However, on prolonged treatment with nitrite and acid, 2-hydroxy-alpha-carboline was further converted into a new mutagen which did not require S9 mix for exhibition of the mutagenicity. This direct-acting mutagen was found to be 2-hydroxy-3-nitroso-alpha-carboline by mass and proton magnetic resonance spectroscopies.

Carbolines↗
PubMed1981

Faithful transcription initiation of fibroin gene in a homologous cell-free system reveals an enhancing effect of 5' flanking sequence far upstream.

For the faithful initiation of transcription of exogenously added fibroin gene, a crude cellular extract was prepared from the posterior silk glands where fibroin gene is specifically transcribed. The faithful initiation was demonstrated by the assay with use of truncated fibroin gene templates and confirmed by nuclease S1 mapping. The transcription initiation of fibroin gene was detected also in the extract from the middle silk glands where fibroin gene is not transcribed, indicating that an expected suppression machinery for the gene was left behind during the extraction procedures. The promoter sequence of fibroin gene required in these homologous extracts (the nucleotide position -29 to +6) is the same as that in a heterologous HeLa cell extract. This result guarantees a limited usefulness of the HeLa cell extract in determining promotor sequence or analyzing general transcription machinery. In the homologous extracts, but not in the heterologous extract, however, it has been shown for the first time in vitro that the 5' flanking sequence upstream from -74 enhances the transcription initiation of fibroin gene. In addition, a remarkable fibroin gene preference in transcription over mouse beta globin and adenovirus 2 major late genes was observed in the homologous extracts. The fibroin gene preference is partly caused by the sequence element upstream from -74 of fibroin DNA.

Animals↗
PubMed1981

Flash photolysis and low temperature photochemistry of bovine rhodopsin with a fixed 11-ene.

Nonbleachable rhodopsins containing retinal moieties with fixed 11-ene structures have been prepared. When the nonbleachable rhodopsin analogue corresponding to the natural pigment was flash-photolysed at 20.8 degrees C, no absorption changes occurred at the monitoring wavelengths of 380, 480, and 580 nm for the time range of 2 microseconds--10 s. This observation is in contrast to that of natural rhodopsin which showed the formation of metarhodopsin I and its decay to meta II. Irradiation of the artificial rhodopsin, 77 K, with light of 460 and 540 nm, also gave no spectral changes; in the case of natural rhodopsin, however, the irradiation leads to formation of the red-shifted intermediate bathorhodopsin. The absence of photochemistry in the artificial pigment shows that an 11-cis to trans photoisomerization of the retinal moiety is a crucial step in inducing the chain of events in te photolysis of rhodopsin.

Animals↗
PubMed1981

Promoter sequence of fibroin gene assigned by in vitro transcription system.

We have shown that the silk fibroin gene from Bombyx mori is faithfully transcribed in an in vitro transcription system of the HeLa cell extract prepared by the method of Manley et al. [Manley, J. L., Fire, A., Cano, A., Sharp, P. A. & Gefter, M. L. (1980) Proc. Natl. Acad. Sci. USA 77, 3855-3859]. Using this system and a series of deletion mutants of fibroin gene, we have assigned the promoter sequence of fibroin gene. The 5' boundary of the promoter is around nucleotide position -29, indicating that most of the T-A-T-A-A-A-A sequence (-30 to -24) is essential for the promoter function, where the transcription initiation point of fibroin gene is assigned as nucleotide position +1 [Tsuda, M., Ohshima, Y. & Suzuki, Y. (1979) Proc. Natl. Acad. Sci. USA 76, 4872-4876]. The 3' boundary is around nucleotide position +6. However, to support the efficient, faithful transcription, some additional (more than 26 but less than 41) nucleotides of nonspecific origin are required at the 5' side of -29. Functions ascribed to the promoter region are discussed.

Animals↗
PubMed1981

Analysis of flagellar genes in Pseudomonas aeruginosa by use of Rfla plasmids and conjugations.

Over 300 flagellar mutants were isolated in Pseudomonas aeruginosa PAO. R-prime plasmids carrying segments of bacterial chromosome which can complement the mutant phenotypes were isolated by means of plasmid R68.45. Among the R-prime plasmids, pMT6 complemented 167 out of 307 mutants examined, and pMT19 complemented the remaining 140 mutants. We found no mutant which was complemented by both of these plasmids. Hence, the flagellar genes were divided into two clusters by these two plasmids, namely, region I on pMT19 and region II on pMT6. By FP5- and R68.45-mediated conjugation, these two regions were located on the P. aeruginosa PAO chromosome with an order of puuF--region I--region II--oru-325.

Chromosome Mapping↗
PubMed1981

Comparison of argininosuccinate synthetase from young and old rat livers.

Properties of argininosuccinate synthetases (ASS), including immunological properties and heat stability, from young and old rats were quite similar. However the degradation rates of ASS from young and old rats were found to be different as shown in the experiment using the double labeling technique. Three forms of this enzyme from young and old rat livers were separated by DEAE-Sephadex A-50 column chromatography, and they were called ASS 1, 2 and 3 in order of elution. The degradation rate of ASS 1 from young and old rat livers was very similar, but the rate of degradation of ASS 2 form old rat livers was greater than that from young rat livers.

Aging↗
PubMed1981

Hypercholesterolemia in ExHC rats and lipid-lowering drug: a screening method for new hypocholesterolemic agents.

The ExHC rat strain which was selected and bred from the Sprague-Dawley strain, develops severe hypercholesterolemia and is liable to aortic lipid deposition when the rats are fed an atherogenic diet. After only 4 days on the atherogenic diet, hypercholesterolemia was induced in these ExHC rats. Following a study of gel-filtration and SDS-disc electrophoresis, significant increases of broad-LDL (beta-VLDL) and of apo E and apo B were noted. On the other hand, plasma HDL and its major apolipoprotein, apo A-I, showed decreases. All these changes might be responsible for the future induction of aortic lipid deposition. A screening method for new hypocholesterolemic agents which takes advantage of these ExHC rat characteristics has been set up; it involves the evaluation of cholesterol lowering and HDL increasing effects by the determination of plasma cholesterol and by radioimmunoassay of apo A-I, respectively. Although clofibrate (100 mg/kg body weight) reduced plasma cholesterol levels significantly, there was no apo A-I increasing effect.

Animals↗
PubMed1981

Clinical significance of a serum DNA binding protein (64-DP) in evaluating malignant diseases.

A DNA-binding protein with a molecular weight of 64,000 (64-DP) has been found in elevated concentrations in the serum of patients with malignant diseases, and a quantitative assay system (column-SDS disc electrophoresis method) for it has been developed. The average serum 64-DP level in 49 normal controls was 48 +/- 26 microgram/ml, whereas in 96 patients with untreated malignant disease it was 167 +/- 67 microgram/ml. Sera from patients with non-neoplastic diseases showed normal or only slightly elevated 64-DP concentrations. No organ or tissue specificity seems to be required for malignant growths to result in elevated serum 64-DP concentration. Of particular interest was the finding that all 11 patients with early gastric carcinoma and two patients with early esophageal carcinoma, all having histologically proven pT1 lesions, showed elevated levels of serum 64-DP. Following successful surgical resection and/or chemotherapy, 64-DP tends to fall toward the normal value. Although confirmation must await the results of larger clinical trials, our preliminary results strongly suggest that 64-DP evaluated by the column-SDS disc electrophoresis method may prove to be a useful tumor marker in patients with a wide variety of malignancies.

Adenocarcinoma↗
PubMed1981

Inhibitors of sterol biosynthesis. Carbon-13 nuclear magnetic resonance studies of 9 alpha-fluoro-5 alpha-cholest-8(14)-en-3 beta-ol-15-one and related compounds.

The natural abundance 13C nuclear magnetic resonance spectra of a number of 9 alpha-fluoro and 9 alpha-hydroxy-delta 8(14)-15-ketosterols and their derivatives have been studied. Peak assignments for individual carbons of 9 alpha-fluoro-5 alpha-cholest-8(14)-en-3 beta-ol-15-one, 9 alpha-hydroxy-5 alpha-cholest-8(14)-en-3 beta-ol-15-one, 5 alpha-cholest-8(14)-ene-3,15-dione, 9 alpha-fluoro-5 alpha-cholest-8(14)-ene-3,15-dione, 9 alpha-hydroxy-5 alpha-cholest-8(14)-ene-3,15-dione, 3 beta-benzoyloxy-5 alpha-cholest-8(14)-en-3 beta-ol-15-one, 3 beta-benzoyloxy-9 alpha-fluoro-5 alpha-cholest-8(14)-en-15-one, 3 beta-benzoyloxy-5 alpha-cholest-8(14)-en-9 alpha-ol-15-one, 3 beta-acetoxy-9 alpha-fluoro-5 alpha-cholest-8(14)-en-15-one, and 3 beta-acetoxy-5 alpha-cholest-8(14)-en-9 alpha-ol-15-one have been made. Also presented herein are: 1) considerations of the substituent effects of the 9 alpha-hydroxy and 9 alpha-fluoro groups on carbon shieldings, 2) demonstration that the state of oxidation of C-3 in the various delta 8(14)-15-ketosteroids affects the olefinic carbon shieldings due to an apparent long range through space effect of the electric field on the olefinic carbon shieldings, 3) the results of analyses of 13C-19F spin-spin couplings, and 4) the results of considerations of 13C nuclear magnetic resonance studies of the concerned compounds with respect to the conformation of ring B in the various 9 alpha-substituted sterols.

Cholestenes↗
PubMed1980

Light isomerizes the chromophore of bacteriorhodopsin.

The primary photochemical event in the two light-transducing pigments whose chromophore is retinal, rhodopsin or bacteriorhodopsin, is a source of controversy. It was originally proposed that the primary photoevent in the bleaching of rhodopsin is the photoisomerization of the chromophore from 11-cis to all-trans retinal. Photochemical considerations suggested that a photoisomerization is the primary event in both rhodopsin and bacteriorhodopsin. However, this description of bacteriorhodopsin's photochemistry has been questioned. To elucidate this problem, we determined the isomeric conformation of retinal for two of the photolytic intermediates of bacteriorhodopsin, using a method that enables us to extract chromophores from the photocycle intermediates L and M at low temperatures (-74 degrees C), and have determined the isomeric conformation of the extracted retinals by HPLC. Here we provide direct evidence that isomerization of the chromophore has taken place in two of the early photocycle intermediates (L and M) of bacteriorhodopsin.

Bacteriorhodopsins↗