Biomedical subjects
Publications and source records attributed to K Isono.
The DNA sequence of the gene rpsA as well as of its neighboring regions has been determined using the dideoxyribonucleotide method. It was found that there is an "open-reading-frame" of 350 bp which precedes the gene rpsA. Furthermore, an extensive internal repeats of nucleotide sequence have been found in this gene.
Extensive modifications have been incorporated into many of the computer programs written by Staden (1-4) to make them easier to cope with DNA and amino acid sequence data. These programs can be easily used by persons with minimal knowledge of computers.
An amber mutation has been induced in the gene rpsA (which codes for ribosomal protein S1) of Escherichia coli K-12 strain in the presence of an amber suppressor (supD) and mutations sueA, sueB and sueC that additively enhance the efficiency of suppression. That the amber mutation has occurred in the gene rpsA was confirmed by complementation with a plasmid which carried the wild-type allele of rpsA. The mutation is lethal in the absence of an amber suppressor, indicating that ribosomal protein S1 is indispensable to E. coli.
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The primary structure of proteins S1, the largest protein component of the Escherichia coli ribosome, has been elucidated by determining the amino acid sequence of the protein (from E. coli MRE600) and the nucleotide sequence of the S1 gene (rpsA, of a K-12 strain). The two methods gave results in perfect agreement except of two positions where possible strain specific differences were found. Protein S1 (MRE600) is composed of 557 amino acid residues (no modified amino acids were detected) and has Mr 61,159. The DNA sequence for protein S1 (K-12) suggests 556 amino acid residues. A computer survey of the sequence revealed three regions in S1 with a high degree of internal homology. The ribosome binding domain of S1 (NH2 terminus) does not show any preponderance of basic amino acids. The two cysteine and the majority of tryptophan residues of S1 as well as two od the three homologous regions were located in its middle region which contains the nucleic acid binding domain. The pattern of degenerate codon usage in the S1 gene is nonrandom and similar to that reported for other ribosomal protein genes.
For the purpose of verifying the effectiveness of peplomycin, one of the derivatives of bleomycin, against carcinoma of the esophagus and the safety of it, the analysis of the data for total 113 cases collected from 25 institutions in Japan was made. The results are as follows. It was effective in 19 out of 74 evaluable cases of carcinoma of the esophagus (25.7%). In case of treatment with peplomycin alone, it was effective in 6 out of 39 cases (15.4%). In case of the combination treatment with peplomycin and some other therapy, it was effective in 13 out of 35 cases (37.1%). As for the side effects, the incidence of fever was the highest in both the cases of peplomycin alone and the combination treatment such as 39.6% and 37.0%, respectively. Anorexia, nausea, vomiting, respiratory symptoms and tiredness were found in relatively many cases. In the clinical laboratory tests, the vital capacity after the treatment tended to be lower than that before the treatment, but there was little change in the hematological tests, pulmonary function test and renal function test.
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The present study was aimed at investigating the efficacy of a new oral anticancer agent, HCFU, that is, (1-hexylcarbamoyl-5-fluorouracil), against 14 cases of unresectable gastric cancers which were confirmed by the laparotomy. The daily dose was 600mg and given continuously as long as the patients could take the drug orally. The efficacy was examined by X-ray photographs according to the criteria for the judgement of direct effect of the chemotherapy for solid malignant tumor. For the diffuse invasive type of gastric cancer, the changes in the X-ray finding such as stiffness and poor elasticity of gastric wall and malignant relief were also taken into consideration for the judgement. Among 9 patients, in whom the efficacy was measurable, two cases showed "partial response (PR)", two cases were diagnosed as "no change (NC)" and other 5 cases were diagnosed as "progressive disease (PD)". Among 2 partial responders, 1 case showed 85.3% decrease of the tumor size on the X-ray film and the other, who had diffuse invasive gastric cancer, showed much improvement on the X-ray film described above. The survival was around 5 months in "progressive disease" group. On the other hand, the patients with the good response to HCFU such as PR patients and NC patients could obtain a relatively longer survival period.
(2RS,3S,4S)-[2-14C,4-3H]Isoleucine and (2S,3S,4R)-[U-14C,4-3H]isoleucine have been prepared by stereospecific syntheses. The addition of these substrates to Streptomyces cacaoi led to the isolation of polyoxins from which 3-ethylidene-L-azetidine-2-carboxylic acid was isolated by hydrolysis. The pro-R hydrogen at C-4 of isoleucine was lost and the pro-S hydrogen was retained in the biosynthesis of 3-ethylidene-L-azetidine-2-carboxylic acid. These results indicate that the enzymatic dehydrogenation of isoleucine to form 3-ethylidene-L-azetidine-2-carboxylic acid involves the antiperiplanar elimination of the hydrogen at C-3 and the pro-R hydrogen at C-4.
Among mutants of E. coli selected for temperature-sensitive growth, four were found to possess alterations in ribosomal proteins L7/L12. Of these, three apparently lack protein L7, the acetylated form of protein L12. Genetic analyses have revealed that the mutation responsible for this alteration maps at a locus around 34 min of the current E. coli genetic map, which is clearly different from the location for the structural gene for protein L7/L12 which is situated at 89 min. Hence, the gene affected in these mutants was termed rimL. Tryptic and thermolysin fingerprints of the protein L12 purified from the rimL mutants showed a profile indistinguishable from that of wild-type protein. It was found that the assayed in vitro, in the high-speed supernatant prepared from mutant cells. These results indicated that the three mutants contain mutations in the gene rimL codes for an acetylating enzyme specific for ribosomal protein L12.
The specialized transducing bacteriophage lambda dpyrE DNA was used as a source of DNA to clone two ribosomal protein genes rpmB (L28) and rpmG (L33) on the cloning vehicle pACYC184. Using one of these plasmids, the nucleotide sequence of these two genes and their flanking regions were determined. The amino acid sequences of both proteins deduced from the nucleotide sequences match with the amino acid sequences previously determined, with one exception. The nucleotide sequences suggest that these two ribosomal protein genes are cotranstribed. There was no expression of the second gene of the operon, rpmG, in the absence of the 5' sequences adjacent to the first gene, rpmB. Observation of the structure of mRNA also strongly supports the idea that rpmB and rpmG are in a single transcription unit whose order is: rpmBp-rpmB-rpmG-rpmGt.
An improved method for the two-dimensional electrophoretic analysis of ribosomal proteins on acrylamide gel slabs has been developed by combining the procedures for the first dimension of Mets and Bogorad (1974) and for the second dimension of Kaltschmidt and Wittmann (1970) and by introducing several modifications. Ribosomal proteins of various Escherichia coli mutants have been analyzed by the new method. Advantages are that (1) it requires only small amounts of protein (100-200 micrograms 70S ribosomal proteins), (2) reproducibility is very high, and (3) it makes it easier to identify mutational alterations in proteins S10, L4, L10, and L21 which hardly migrate out of the sample gel with our previous electrophoresis procedure. Furthermore, the new method can be nicely adapted to analysis of the ribosomal proteins from other organisms, such as Bacilli or yeast.