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Biomedical subjects

M Kuwano

Publications and source records attributed to M Kuwano.

At least 307 records · Page 17Linked to original sources
PubMed1979

The cross-resistance of mouse blasticidin S-resistant cell lines to puromycin and sparsomycin, inhibitors of ribosome function.

The antibiotic blasticidin S inhibits peptide-chain elongation in extracts of bacteria and mammalian cells. After spontaneous or nitrosoguanidine mutagenesis, we have isolated 46 blasticidin S-resistant (Blar) cell lines independently from mouse mammary carcinoma cells (FM3a). Among those Blar clones, we studied two clones, a spontaneously induced one (S501) and a nitrosoguanidine mutagenized one (N742) in more detail. The resistant phenotype of these Blar cells is retained without change for at least four months in the absence of the antibiotic. These Blar cells are 10- to 20-fold more resistant to the cytotoxic action of the antibiotic than their parental cells in vivo. Polyuridylate dependent polyphenylalanine synthesis in vitro with S-30 extracts either from N742 or S501 is 10- to 50-fold more resistant to the inhibitory action of blasticidin S compared to the parental FM3a cells. Ribosomes from FM3a and N742 are fractionated into 40-S and 60-S subunits, and polyphenylalanine synthesis by mixing them in various combinations with S-100 fraction from mouse leukemia L5178Y cells indicating that the resistant phenotype of Blar cells is due to the alteration of 60 S ribosomal subunit. We also found that these two Blar cell lines (N742 and S501) show cross-resistance to gougerotin, puromycin and sparsomycin, but not to emetine or cycloheximide. The polyribosomal pattern of FM3a (Blas) and N742 was compared when the cells were incubated with 3 microgram/ml puromycin for 6 h. Puromycin treatment of Blas cells induced accumulation of monosomes and ribosomal subunits, while little if any transition of polyribosomes into monosome and ribosomal subunits appeared in its counterpart N742 treated with the same dose of puromycin.

Animals↗
PubMed1979

Genetic analysis of a mutation affecting ribosomal protein S1 in Escherichia coli.

Ribosomal protein S1 from a newly isolated Escherichia coli mutant has a molecular weight of about 54,000 which is smaller than the wild type S1 (M.W. 65,000). The isoelectric points of the smaller and the wild type S1 species are similar in the gel electrophoresis system of O'Farrell (1975). Genetic analyses by Hfr conjugation and P1 phage transduction indicate that the mutation affecting S1 (rpsA) is located close to the serC gene [20 min on the E. coli genetic map of Bachmann et al. (1976)], with a co-transduction frequency of 61%. The most probable gene order is serC-rpsA-cmlB.

Chromosome Mapping↗
PubMed1979

Synergistic effect of vitamin A and fusidic acid on Hela cells in vitro.

Examinations were made to see if various vitamin A compounds, including retinol, retinal, retinol palmitate, retinol acetate, and retinoic acid, enhance the action of fusidic acid, an antibiotic and inhibitor of protein synthesis, on human carcinoma HeLa cells in vitro. Among these vitamins, retinal and retinol were found to potentiate strongly the effect of fusidic acid on HeLa cells. In this system, however, retinol palmitate, retinoic acid, and retinol acetate in 10 approximately 20 microgram/ml concentration were not found to enhance the effect of fusidic acid significantly.

Cell Survival↗
PubMed1979

Control of permeation of bleomycin A2 by polyene antibiotics in cultured Chinese hamster cells.

Control of permeation of bleomycin A2, a well-known antitumor antibiotic, in combination with various polyene macrolide antibiotics was analyzed in cultured Chinese hamster cells in vitro. Three polyene antibiotics, filipin, pentamycin, and pimaricin, were found to enhance the action of bleomycin A2 remarkably, while amphotericin B or nystatin could not. Although DNA synthesis and colony-forming activity of polyene-sensitive Chinese hamster V79 cells were synergistically inhibited by the combination of filipin and bleomycin A2, in a polyene-resistant subline (AMBR-1) derived from V79, they were only slightly affected in the presence of both drugs. The cellular uptake of [14C]bleomycin A2 by V79 was enhanced 2- to 4-fold in the presence of increasing doses of filipin or pentamycin, but not in the presence of amphotericin B. The treatment of V79 cells with filipin for 20 to 30 min was enought to block DNA synthesis almost completely when combined with 20 microgram belomycin A2 per ml. The pretreatment of the hamster cells with 6 microgram filipin per ml for 60 min continued to enhance the inhibitory action by bleomycin A2 of DNA synthesis up to 5 hr after the removal of filipin from the cultured medium.

Amphotericin B↗
PubMed1978

The synthesis and degradation of presumptive messenger RNA in cultured mouse leukemia cells during the inhibition of protein synthesis.

RNA synthesis in mouse leukemia L5178Y cells was inhibited depending upon the time of treatment by blasticidin S or by ricin, which inhibits specifically protein synthesis. When blasticidin S or ricin blocked protein synthesis by more than 90% of the control, marked accumulation of monosome was accompanied by the decrease of pulse-labeled RNA (20% of that in the control) in the polysomes and monosome fraction. The size distribution of pulse-labeled RNA among polysomal fractions including monosome obtained from the cells treated with either blasticidin S, ricin of L-asparaginase showed that the size of presumptive mRNA was shifted from 18 S to 9--10 S. TReatment of a blasticidin S-resistant (Bla-R) subline derived from L5178Y cells (Kuwano, M., Matsui, K., Takenaka, K., Akiyama, S. and Endo, H. (1977) Int. J. Cancer 20, 296--302) with L-asparaginase or ricin induced smaller size (9--10 S) RNA, but treatment of Bla-R cells with blasticidin S did not. Such shorter RNA fragments could not be observed even when cellular protein synthesis was inhibited by treatment for short time with blasticidin S (40--80% of the control activity). Smaller RNA fragments accumulated after drastic inhibition of protein synthesis were composed of 74% of polyadenylate sequence lacking poly(A)(-)RNA with peak of approx. 10 S and 26% of polyadenylate sequence containing poly(A)(+)RNA with a peak of 18 S, whereas cytoplasmic polysomal RNA of the control contained 46% poly(A)(+) with a peak of 18 S and 54% poly(A)(-)RNA with a 10--18 S peak. Cytoplasmic poly(A)(+)RNA degraded biphasically with half-lives of approx. 2 h and 8--10 h in exponentially growing mouse cells. However, in degradation of poly(A)(+)RNA molecules being formed in the cells pretreated with blasticidin S for 3 h, the rapid phase of decay with a half-life of approx. 2 h was interrupted by successively appearing poly(A)(+)RNA with a longer half-life of 8--10 h in cytoplasm. However, when the cells were pretreated with blasticidin S for 6 h, there appeared no poly(A)(+)RNA population with the rapid-decay in cytoplasm.

Animals↗
PubMed1978

Isolation and characterization of amphotericin B-resistant cell lines in Chinese hamster cells.

Amphotericin B is a polyene macrolide antibiotic which interacts specifically with sterols in mammalian cell membranes. Amphotericin B-resistant (AMBr) lines of stable phenotype have been isolated from cultured Chinese hamster (V79) cells. Three AMBr clones (AMBr-1, -2 and -3) isolated independently after treatment with nitrosoguanidine were resistant to greater than or equal to microgram/ml of the antibiotic, while DNA synthesis as well as the colony-forming ability of the parental V79 cells was blocked by greater than 80% of control in the presence of 20--50 microgram/ml amphotericin B. The AMBr cell line also exhibited increased resistance to other polyene macrolide antibiotics such as nystatin and pentamycin. Other agents, however, such as cytosine arabinoside or ricin, blocked DNA synthesis in AMBr cells to the same extent as in V79 cells. The amphotericin B resistance phenotype was stably retained even after AMBr cells were cultured in the absence of the drug for over 200 generations. The control of free cholesterol or its esters was significantly decreased in all three resistant clones. Furthermore, cholesterol synthesis from acetate as well as mevalonate was partly defective in AMBr cells, compared with that in V79 cells.

Amphotericin B↗
PubMed1978

Synergistic combination therapy of 5-fluorouracil, vitamin A and cobalt-60 radiation upon head and neck tumors.

We have applied a triple combination of 5-fluorouracil (5FU), vitamin A and cobalt-60 radiation (FAR therapy) to clinical treatment of head and neck tumors since 1972. The treatment of 33 patients with cancer of larynx and 15 patients with hypopharyngeal or cervical esophageal cancer with FAR therapy during three years from 1972 to 1974 resulted in highly effective synergism in vivo, and the improved points of this therapy were presented.

Cobalt Radioisotopes↗
PubMed1978

Differential control of synergistic effect with polyene macrolide antibiotics upon Chinese hamster cells in vitro.

An amphotericin B-resistant cell (AMBR-1), which was isolated from aneuploid Chinese hamster cells (V79), was found to show much higher resistance than the parent V79 cells to other polyene antibiotics, such as pentamycin and filipin. To obtain the 50 to 60% inhibition of the control protein synthesis activity by a synergistic combination of fusidic acid and amphotericin B, 50 microgram fusidic acid per ml were combined with 10 microgram amphotericin B in V79 cells, whereas in AMBR cells 50 microgram fusidic acid per ml were combined with 100 microgram polyene antibiotic per ml. Bleomycin (10 microgram/ml), which alone did not affect cellular DNA synthesis, inhibited DNA synthesis of V79 cells by more than 90% of the control activity when combined with only 1 microgram pentamycin per ml, whereas a similar extent of inhibition in AMBR cells was observed by combination with more than 5 microgram pentamycin per ml.

Amphotericin B↗
PubMed1977

Gene affecting longevity of messenger RNA: a mutant of Escherichia coli with altered mRNA stability.

We have screened 897 temperature sensitive growth mutants of E. coli for mutant strains showing longer mRNA half-life. The fate of pulse-labelled RNA was examined at 42 degrees C after cessation of RNA synthesis and with prior exposure to nonpermissive temperature (42 degrees C). Eight stains showed altered turnover of RNA (presumably mRNA), and further analysis on mutant strain JE15144 indicated that the stability of pulse-labeled RNA as well as of tryptophan (trp) mRNA increased four to seven fold over its parental strain at 42 degrees C. At 4 min or 10 min after addition of rifampicin, some 70 to 80% of polyribosome in the growing cells could still be conserved in JE15144 cultured at the nonpermissive temperature while little, if any, polyribosomes remained in its parental strain (PA3092) under the same condition. Two generation times were required for complete stoppage of growth of this mutant strain after shifting to 42 degrees C, and protein synthesis continued at a significant, but slightly reduced, rate at 42 degrees C. However, functional decay of mRNA in the mutant strain, with respect to the capacity for producing peptides, appeared to be similar to the parent strain, with half-lives of 3.5 min in PA3092 and 4.7 min in JE15144.

Bacterial Proteins↗
PubMed1977

A mouse leukemia cell mutant resistant to blasticidin S.

After nitrosoguanidine mutagenesis we isolated from mouse leukemia L5178Y cells a mutant cell (Bla-R) resistant to blasticidin S, an inhibitor of protein synthesis. Neither growth nor leucine incorporation into hot-acid insoluble fraction of Bla-R cell was inhibited by 5 to 20 microgram/ml blasticidin S, which almost completely blocked protein synthesis as well as growth of the parental L5178Y cells. However, other inhibitors such as fusidic acid, cycloheximide, ricin D or L-asparaginase blocked protein synthesis in Bla-R cells to the same extent as in L5178Y cells. Protein synthesis in vitro using S-30 extracts from the parental cell line L5178Y was almost completely blocked in the presence of the antibiotic, while no inhibition by blasticidin S occurred when S-30 extracts -rom Bla-R mutant cells were used. Protein synthesis assays were made by using the S100 fraction from rat liver together with ribosomes from either L5178Y cells or Bla-R cells. Blasticidin S inhibited protein synthesis when ribosomes were derived from L5178Y cells, but not from Bla-R mutant.

Animals↗
PubMed1977

Genetic analysis of mutations affecting ribonuclease II in Escherichia coli.

Exonuclease activity in an Escherichia coli K12 mutant S296 is less than 1% of that in the wild type strain (Nikolaev et al., 1976). Another mutant N464 has thermolabile ribonuclease II (Castles and Singer, 1968; Kuwano et al., 1969). Genetic analysis of these mutants by Hfr conjugation and P1 transduction indicates that the structural gene (rnb) for ribonuclease II is located near the pyrF gene (28 min on the E. coli genetic map of Bachmann, Low and Taylor (1976)), and the most probable gene order is tyrT-trp-pyrF-rnb.

Chromosome Mapping↗
PubMed1977

The effect of aurintricarboxylic acid on RNA polymerase from rat liver.

The effect of the dye aurintricarboxylic acid (ATA) on RNA polymerases [EC 2.7.7.6] solubilized from rat liver was studied. Complete inhibition of RNA synthesis in vitro was observed when 3-5 microng/ml of ATA was added to the reaction mixture at time 0, while 40-70 microng/ml of a rifampicin derivative, AF/013, was required to produce the same extent of inhibition. RNA formation, however, continued at a rate of one-half that of the control when ATA was given after the onset of RNA synthesis in a dose capable of completely blocking RNA formation if administered at time 0. ATA was found to interact with RNA polymerizing enzyme itself and competed specifically with the binding of RNA polymerase to template DNA. Preincubation of the enzyme with DNA at 37 degrees before adding dye made the DNA-enzyme complex partly resistant to the drug. RNA-synthesizing activity resistant to ATA increased when nucleoside triphosphates, especially GTP, were added to the preincubation mixture in the presence of Mn2+. However, ATA only slightly affected RNA synthesis in nuclei isolated from rat liver.

Animals↗
PubMed1976

Effect of stringent and relaxed control on transcription of the tryptophan operon from the ptrp promoter and the PL promoter in trp phage.

Tryptophan (trp) mRNA synthesis from the authentic trp promoter (Ptrp) is apparently arrested upon translation blockage, while trp mRNA synthesis as a result of read-through from the Pl promoter of the N gene in trp phage is not so affected. When translation is blocked at a nonpermissive temperature in the temperature-sensitive mutants of Escherichia coli rel carrying altered ribosomal elongation factors G (strain CP78G) and Ts (strain HAK88), CP78G and HAK88 show relaxed and stringent phenotypes respectively in control of RNA synthesis. Under conditions causing translation blockage in both mutants, Pl-promoted synthesis of trp mRNA is not depressed while Ptrp-promoted synthesis of trp mRNA is blocked. The insensitivity of Pl-promoted transcription of the translocated trp operon to stringent control is also confirmed by using a strain 10b6s rel carrying a temperature-sensitive valyl-tRNA synthetase. In contrast to the above observation, transcription of the trp operon from either the Pl and Ptrp promoters in a 10b6r rel infected with trp is inhibited greatly at the nonpermissive temperature. This effect occurs even if the level of trp transcription observed at 30 degrees is already low, thus suggesting that translational machinery is intrinsically abnormal in the rel strain.

Coliphages↗
PubMed1976

Initial trp operon sequence in Escherichia coli is transcribed without coupling to translation.

The transcription of the "leader" region (Bronson et al., 1973) of the trp operon in Escherichia coli was studied in normal mutants which delete most of the operator-distal region of the operon [a deletion strain (trp OAEG) retaining only about one third of the "leader" region and two deletion strains (trpOAE14 and trpOAE2) retaining the whole "leader" region and an initial portion of the trpE], as well as in a strain with an intact trp operon, but with a temperature-sensitive lesion in ribosomal protein factor EFTs (strain HAK88). In these deletion mutants, mRNA molecules corresponding to the "leader" region were detected as most of the trp-specific mRNA. Less inhibition of transcription, of the promoter-proximal portion of the trp "leader" region than that of more distal genes of the operon, was found in chloramphenicol-treated cells of strain trpOAE14. It was also observed that transcription of the initial one third portion of the "leader" region was not repressed by tryptophan in strains trpOAE6 and trp OAE14. A similar effect of a translation block on transcription of the distal part of the "leader" region was observed with strain HAK88 at the nonpermissive temperature. In sedimentation analysis of polyribosomes containing the trp mRNA molecules from the deletion mutants, trp mRNA from strain trpOAE14 was found in monosomes and small polyribosomes, whereas the majority of the trp mRNA from strain trpOAE6 was found joined to a single ribosome or ribosomal subunit. These results suggest that ribosomes bind in vivo to a site(s) located in the middle of the "leader" mRNA sequence, and that the initial transcription of the trp operon does not require any connection to functional translational machinery, while continuation of RNA synthesis beyond a first ribosome binding site seems indispensably coupled to ribosome function.

DNA, Bacterial↗