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K Ikenaka

Publications and source records attributed to K Ikenaka.

123 records · Page 7Linked to original sources
PubMed1985

Primary characterization of the protein products of the Escherichia coli ompB locus: structure and regulation of synthesis of the OmpR and EnvZ proteins.

The ompB operon of Escherichia coli contains the structural genes for two proteins, OmpR and EnvZ, which control the osmoregulated biosynthesis of the porin proteins OmpF and OmpC. By inserting XbaI octamer linkers into the cloned ompB locus, four distinct frameshift mutants were isolated and subsequently characterized for their OmpR and EnvZ protein products and their outer membrane porin phenotype. In a minicell expression system, the wild-type products of the ompR and envZ genes were found to be approximately 28 and 50 kilodaltons in size, respectively, whereas the mutant proteins were either truncated or extended due to the frame shift. The identity of the envZ gene product was confirmed by immunoprecipitation. M13 dideoxy sequencing of the DNA around the wild-type ompR-envZ junction revealed an error in the sequence published for this operon; the complete corrected sequence is presented. A sequence, ATGA, was found that forms the termination codon for the OmpR reading frame and a possible initiation codon for the EnvZ protein; these sequences are consistent with the sizes of the proteins observed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The translational activity of this ATG codon was confirmed by fusing the lacZ gene in frame with the putative EnvZ coding sequence. The implications of these results are discussed with respect to the regulation of synthesis of the ompB gene products.

Amino Acid Sequence↗
PubMed1983

Effect of an exogenous energy source and amino acids on DNA synthesis in regenerating rat liver.

Rats on a protein-free diet synthesized less DNA after partial hepatectomy than rats on a normal diet. In regenerating livers of animals on the protein-free diet, induction of several enzymes involved in the DNA precursor synthetic pathway, and especially ribonucleotide reductase, were depressed. When young rats were maintained solely by parenteral nutrition after partial hepatectomy, exogenous amino acids were more important than the exogenous energy source for induction of enzymes involved in synthesis of DNA and its pyrimidine nucleotide precursors. In particular, induction of ribonucleotide reductase appeared to be controlled by exogenous amino acids. Tryptophan, methionine, phenylalanine, leucine, valine, isoleucine and threonine seemed to stimulate the induction of this enzyme most after partial hepatectomy.

Amino Acids↗
PubMed1983

[Studies on the mechanism of cytotoxicity of 5-fluorouracil--through impairment of metabolism].

Mechanism of cytotoxicity of 5-fluorouracil (5-FU) in relation to impairment of RNA metabolism was studied. 5-FU depressed the levels of ribonucleotide reductase and thymidine kinase, which are two key enzymes in DNA synthesis, in solid AH-130 tumor when given intraperitoneally at a dose of 60 mg/kg, and this inhibition seemed to be very toxic against cells. But this dose of 5-FU exceeded the amount that could be degraded by the liver, which was much higher than the clinical dose. We concluded that the impairment of RNA metabolism caused by 5-FU played a minor role in its anti-tumor actions.

Animals↗
PubMed1981

Metabolism of pyrimidine nucleotides in various tissues and tumor cells from rodents.

The metabolism of pyrimidine nucleotides in various tissues and tumor cells of rodents was investigated. Ribonucleoside diphosphate reductase, thymidine monophosphate kinase and DNA polymerase (alpha, beta) were specifically localized in tumor cells, i.e., the activities of these enzymes in tumor cells were at least three times higher than those in normal tissues, including rapidly growing tissues, such as bone marrow, thymus, and spleen. The activities of deoxycytidine monophosphate deaminase and all the nucleoside kinase were high not only in tumor cells, but also in rapidly growing normal tissues, so that these enzymes are unsuitable as targets for cancer chemotherapy. The tissue distribution of other enzymes, including orotate phosphoribosyltransferases, cytidine triphosphate synthetase, thymidine monophosphate synthase, nucleoside phosphorylases and cytidine deaminase had no relation with the cell growth rate. AH130 tumor cells and the thymus showed specific increases in the activities of enzymes involved in de novo DNA synthesis. In contrast, Yoshida sarcoma and bone marrow showed high activities of enzymes in the salvage pathway of DNA synthesis, which suggested that the two tumors have different patterns of nucleotide metabolism.

Animals↗
PubMed1981

Possible regulation of ribonucleoside diphosphate reductase.

Two inhibitors of ribonucleoside diphosphate reductase (RR) (EC 1.17.4.1) in vitro were isolated from normal rat liver: they were a nondialyzable, heat-labile, high-molecular-weight ribonucleoside diphosphate reductase inhibitor (HRRI, and a dialyzable, heat-stable, low-molecular-weight ribonucleoside diphosphate reductase inhibitor (LRRI). The activities of both inhibitors varied inversely with the cell growth rate. HRRI from the cytosol fraction of rat liver was partially purified by ammonium sulfate fractionation (0 - 50%), and gel filtration on a Sepharose 6B column. It was eluted in the void volume from this column, together with ATP-hydrolyzing activity. The HRRI fraction also contained CDP kinase and CDPase activities, suggesting that HRRI is a complex of several enzymes that reduce the concentrations of the substrate of RR, CDP, and of the allosteric activator, ATP. LRRI was extracted from the cytosol of rat liver with ethanol (80% final concentration) and purified further by washing with organic solvent, and be chromatographies of Amberlite IR-45 and Dowex 50. Finally, it was identified as glucose, which was phosphorylated to glucose 6-phosphate by hexokinase present tin the RR enzyme solution ( 0 - 35% ammonium sulfate fraction of AH-130 cytosol), thus causing ATP depletion. Thus, neither inhibitor reacted directly with the RR enzyme, but both may regulate the enzyme activity in vivo by reducing the intracellular levels of substrates or cofactors.

Animals↗
PubMed1980

Effect of coadministration of thymine or thymidine on the antitumor activity of 1-(2-tetrahydrofuryl)-5-fluorouracil and 5-fluorouracil.

The antitumor activity of 1-(2-tetrahydrofuryl)-5-fluorouracil (FT-207) on sarcoma was enhanced by oral coadministration of uracil, thymine, or thymidine. The activity was enhanced equally by thymine and by uracil than by thymidine, but thymine caused loss in body weight. The antitumor activity of 5-fluorouracil (5-FU) was also enhanced by thymine or uracil, but both caused loss in body weight. Degradation of 5-FU in vitro was inhibited more by thymine than by uracil. Phosphorylation of 5-FU, however, was not inhibited by uracil, thymine, or thymidine, even at 100 times the concentration of 5-FU. These results suggest that the mechanism of enhancement of the antitumor activity of FT-207 by thymine or thymidine was similar to that by uracil, and that uracil had more effect than thymine or thymidine in enhancing antitumor effect of these drugs to FT-207 without toxicity.

Administration, Oral↗
PubMed1979

Effect of coadministration of uracil or cytosine on the anti-tumor activity of clinical doses of 1-(2-tetrahydrofuryl)-5-fluorouracil and level of 5-fluorouracil in rodents.

Concentration of 5-fluorouracil (5-FU) in the tumor, blood, and various organs of AH130-bearing rats after administration of clinical doses of 1-(2-tetrahydrofuryl)-5-fluorouracil (FT-207) and uracil was examined. The concentration of 5-FU in blood was less than 0.02 microgram/ml with all combinations of FT-207 and uracil except high molar ratios of uracil to FT-207 (ratio, 5 and 10), whereas high concentrations of up to a maximum of 0.200 microgram/g on administration of uracil plus 5 or 7.5 mg/kg of FT-207 (ratio, 4), was found in the tumor. On oral administration of FT-207 plus uracil in various combinations, the highest T/B (ratio of concentration of 5-FU in the tumor to that in blood) value was obtained at a ratio of uracil to FT-207 of 4. With this combination, 5-FU concentration in the tumor, muscle, and spleen was higher than that after administration of FT-207 alone (5 mg/kg). These results suggest that at the clinical doses the optimum molar ratio of uracil to FT-207 is 4. Coadministration of cytosine enhanced the antitumor activity of FT-207 on sarcoma-180 in mice. However, cytosine enhanced the antitumor activity of FT-207 less than uracil and its coadministration resulted in a lower concentration of 5-FU in the tumor than coadministration of uracil.

Administration, Oral↗
PubMed1979

Effect of uracil on metabolism of 5-fluorouracil in vitro.

The effect of uracil on the metabolism of 5-fluorouracil (5-FU) in vitro was studied. 5-FU was mainly phosphorylated in intact Yoshida sarcoma cells, whereas it was mainly degraded in liver slices. Uracil inhibited degradation of 5-FU much more than its phosphorylation; incubation of 2,500 microM of uracil with 2.5 microM of 5-FU (molar ratio, 1,000:1) inhibited the degradation of 5-FU by 70%, but did not affect its phosphorylation. With homogenates of Yoshida sarcoma or liver uracil inhibited degradation of 5-FU greatly, phosphorylation of 5-FU by alpha-D-ribose 1-phosphate (RiblP) and ATP to some extent, and phosphorylation by 5-phospho-alpha-D-ribosyl diphosphate (PPRibP) very little. The activities of the enzymes involved in the metabolism of 5-FU in various tissues were also determined. Degradation of 5-FU was much faster in liver than in other tissues and was very slow in tumor tissue. Phosphorylation of 5-FU with RiblP and ATP was rapid in Yoshida sarcoma and bone marrow. Phosphoribosyltransferase activity was high in Yoshida sarcoma and thymus, but low in bone marrow.

Animals↗
PubMed1979

Metabolism of 5-fluorouracil in sensitive and resistant tumor cells.

Thymidine kinase, dTMP kinase, and DNA polymerase activities were determined in cell lines of AH hepatoma, L1210 leukemia, and Yoshida sarcoma that were sensitive and resistant to 5-fluorouracil (5-FU). It was found that the levels of these enzymes in tumor cells were not consistently related to the property of sensitivity to 5-FU. A marked difference was observed between sensitive and resistant cell lines of L1210 leukemia and Yoshida sarcoma in the uptake of labeled 5-FU into the acid-soluble, nucleotide, and RNA fractions, the rate of incorporation of 5-FU into these fractions being 3 to 5 times greater in sensitive tumor cells than in resistant tumor cells. The radioactivities in the acid-soluble fractions of AH44 (sensitive) and AH109A (resistant) were similar after incubation of these cells with labeled 5-FU in vitro. However, a smaller volume of ascites and lower cell number were observed in AH44 (sensitive)-bearing rats than in AH109A (resistant)-bearing rats. These in vivo results indicate that the 5-FU injected intraperitoneally was diluted by ascites more in AH109A (resistant)-bearing rats than in AH44 (sensitive)-bearing rats.

Animals↗
PubMedDate not supplied

Brain malformations caused by retroviral vector-mediated gene transfer of hippocampal cholinergic neurostimulating peptide precursor protein into the CNS via embryonic mice ventricles.

Hippocampal cholinergic neurostimulating peptide precursor protein (HCNP-pp) is a unique multifunctional protein, being not only the precursor of HCNP, which promotes the phenotype development of septo-hippocampal cholinergic neurons, but also the binding protein of phosphatidylethanolamine, ATP, Raf-1 kinase (known as "Raf-1 kinase inhibitory factor" in peripheral organs), and serine protease. We obtained a high-titer retroviral vector harboring HCNP-pp cDNA by the use of a modified packaging cell line and centrifugation, and by injecting it into embryonic mouse ventricles, we investigated the function of its gene product within the central nervous system (CNS). We found that efficient transduction into hippocampal pyramidal neurons can be achieved by injecting the vector into embryonic brain ventricles on embryonic day 14 (E14). Three days after receiving the intraventricular injection of the high-titer HCNP-pp retrovirus vector on E14, the tissues around the ventricles showed an overexpression of HCNP-pp. This was accompanied by a reduced amount of activated MEK and Erk (as analyzed by histochemical and Western blot methods), suggesting that HCNP-pp also regulates the MAP-kinase cascade within the CNS. Surprisingly, mouse brains that received the HCNP-pp retroviral vector showed massive malformation of the hippocampus and cerebellum when examined 30 days after birth. This shows that strictly regulated HCNP-pp gene expression is necessary for the normal development of the mouse brain, and that the moderate overexpression achieved by retroviral vector-mediated gene transfer is sufficient to cause severe abnormality of entire brain structures.

Animals↗
PubMedDate not supplied

Evaluation of prodrugs ability to induce effective ablation of cells transduced with viral thymidine kinase gene.

Transduction of the herpes simplex virus thymidine kinase (HSV-tk) gene into tumor cells followed by treatment with prodrugs is one of the most promising approaches for gene therapy in cancer. The choice of prodrugs is important in order to obtain maximum anticancer effects with minimum adverse reactions. We retrovirally transduced the HSV-tk gene into murine and rat hepatocellular carcinoma (HCC) cells, and investigated their sensitivity to ganciclovir and acyclovir. Retrovirally-mediated HSV-tk transduction did not affect cell proliferation, but led to both ganciclovir- and acyclovir-dependent cytotoxicity in the HCC cells. Ganciclovir exhibited much stronger cytotoxicity on HSV-tk transduced cells than acyclovir. Importantly, HSV-tk transduced cells were completely abrogated at a ganciclovir concentration which was lower than the minimum plasma level achieved in the clinical usage of ganciclovir. Furthermore, HSV-tk transduced cells induced stronger killing of neighboring untransduced cells in the presence of ganciclovir than acyclovir. Ganciclovir may be preferable to acyclovir in the HSV-tk transduction system.

Acyclovir↗
PubMedDate not supplied

Bystander effect caused by cytosine deaminase gene and 5-fluorocytosine in vitro is substantially mediated by generated 5-fluorouracil.

Because it appears impossible to transfer toxic genes to all the cells of a cancer, the bystander effect is critical to induce effective antitumor effects. In the present study, possible in vitro mechanisms of the bystander effect by the cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) were investigated. CD-transduced cancer cells exhibited much higher sensitivity to 5-FC compared to parental cells. CD-transduced cells caused killing of neighboring parental cells in the presence of 5-FC, irrespective of direct cell-to-cell contact. Media conditioned by CD-transduced cells and 5-FC contained considerable amounts of 5-fluorouracil (5-FU) and exhibited profound cytotoxicity on parental cells. Furthermore, this killing ability of conditioned media correlated well with 5-FU levels converted from 5-FC by CD-transduced cells. CD was shown not to be secreted into media from cells. These results indicate that diffusible 5-FU plays the substantially causative role in the in vitro bystander effect caused by the CD/5-FC system.

Animals↗