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Biomedical subjects

K Negishi

Publications and source records attributed to K Negishi.

At least 127 records · Page 7Linked to original sources
PubMed1989

Spectrum of N4-aminocytidine mutagenesis.

N4-Aminocytidine, a nucleoside analog, is a potent mutagen towards phages, bacteria, Drosophila and mammalian cells in culture. In vitro, biochemical studies indicate that this reagent acts by being incorporated into DNA. To elucidate the mechanism of N4-aminocytidine mutagenesis, it is essential to identify the nature of DNA sequence alterations taking place during the mutagenesis. We have analyzed the nucleotide sequence changes in the lac promoter-lacZ alpha region of M13mp2 phage induced by treatment of phage-infected Escherichia coli with N4-aminocytidine. The sequence alterations of DNA samples from 89 mutants of the phage were determined. These mutants had single point mutations, except one mutant, in which a double point mutation was detected. Several hot spots were found: however, there are no apparent relations to particular DNA sequences regarding the locations of these spots. All the mutations are transitions; neither transversions nor deletions/insertions were found. A feature in these transitions is that the A/T to G/C and G/C to A/T changes occur at approximately equal rates. The overall picture of the mutagenesis is consistent with a scheme in which misincorporation and misreplication caused by the modified cytosine structure are the key steps in the DNA replication leading to transitions. Similar nucleotide alterations were found for the mutagenesis induced by an alkylated derivative, N'-methyl-N4-aminocytidine. N4-Aminocytidine also induced reversions of these mutants; both A/T to G/C and G/C to A/T transitions again took place.

Bacteriophages↗
PubMed1989

[Measurement of coronary flow velocity with Doppler catheter: evaluation of coronary flow reserve in successful angioplasty].

To assess the therapeutic effect of percutaneous transluminal coronary angioplasty (PTCA) on coronary flow reserve, coronary flow velocity (CFV) was measured with a Doppler catheter before and immediately after PTCA in 11 patients, who underwent elective PTCA for critical stenosis in proximal or mid portion of the left anterior descending artery (LAD). A Doppler catheter was positioned at the proximal portion of the LAD and the CFV was measured at rest and after intracoronary injection of 6 ml of contrast material (Iopamidol), 6 ml of saline or 3 mg of Isosorbide Dinitrate (ISDN). Peak to resting velocity ratio (PRVR) was calculated as an estimate of coronary flow reserve. Percent diameter stenosis (%S) was measured from cineangiogram. A translesional pressure gradient was obtained with an angioplasty catheter. These parameters measured in PTCA candidates were compared with those in 11 patients whose LAD had no critical stenosis. After PTCA, %S was decreased (94.2 +/- 1.4 vs 34.1 +/- 5.1%; mean +/- SEM). Pressure gradient was also decreased (59.5 +/- 4.9 vs. 25.1 +/- 3.3 mmHg). There was no difference between mean CFV at rest in patients before PTCA and that in patients without stenosis (4.52 +/- 0.63 vs. 5.46 +/- 0.61 cm/sec). By successful PTCA, CFV at rest was increased (7.39 +/- 1.32, p less than 0.05 vs. before PTCA). PRVRs in patients before PTCA were smaller than those in patients without stenosis (1.5 +/- 0.1, 1.4 +/- 0.1, 1.6 +/- 0.2 vs. 2.8 +/- 0.1, 2.5 +/- 0.2, 2.8 +/- 0.2, p less than 0.01; by contrast material, saline, ISDN, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1988

Dopamine cells and rod bipolar cells contain protein kinase C-like immunoreactivity in some vertebrate retinas.

The localization of cells immunoreactive to a monoclonal antibody against protein kinase C (PKC) and to polyclonal antibodies against tyrosine hydroxylase (TH) was investigated in the retina of fish (carp, goldfish, dace and catfish), frog, turtle, chick and some mammalians (guinea pig, rat, cat and rabbit) by means of fluorescence microscopy. PKC-like immunoreactivity was found in dopamine (DA) or TH-like immunoreactive (IR) cells in all the species examined and also in rod bipolar cells in the fish (except for catfish), and in presumed rod bipolar cells in the other animals (except for frog and turtle). In the catfish, frog and turtle retinas, no PKC-like IR bipolar cells were found. In the rat retina, some other amacrine cells in addition to TH-like IR amacrine cells were reactive to the anti-PKC antibody. It is of interest that PKC-like immunoreactivity is commonly found in DA cells and probably in rod bipolar cells in most animal species, although the functional significance is unknown at present.

Animals↗
PubMed1988

Ab initio molecular orbital study of the mispairing ability of a nucleotide base analogue, N4-aminocytosine.

The intrinsic properties of N4-aminocytosine, a base analogue of cytosine, are analyzed by an ab initio molecular orbital method. Relative stabilities of four possible isomeric structures of N4-aminocytosine are shown. The more stable isomer has the smaller dipole moment, so the relative stabilities of the isomers in solutions are subject to solvent polarity. The mutagenicity of this base analogue must arise because it can behave like either cytosine or thymine. It can form a guanine-cytosine-like base pair more easily than cytosine, and an adenine-thymine-like base pair less easily than thymine.

Chemical Phenomena↗
PubMed1988

Regional difference in the dendritic morphology of dopamine cells in carp retina.

The dendritic morphology of dopamine (DA) cells in the inner plexiform layer of the retina of carp (body length, ca. 33 cm) was investigated by identifying their fluorescent cell bodies in isolated, aldehyde-fixed flat-mounts and injecting them iontophoretically with Lucifer yellow CH under microscopic control. Attention was paid to clarifying regional differences in their dendritic morphology. In the marginal zone within 0.25 mm from the retinal edge, the density of DA cells was extremely high (120 cells/mm2), their dendrites tended to extend in parallel with the retinal circumference, and the dendritic field size was small (2.5 X 10(-2) mm2). As the injection point was shifted centrally by steps, the dendrites of DA cells tended to extend toward the optic disc and subsequently toward the margin, finally forming a round or oval dendritic field for each cell. Concomitantly with such changes in the dendritic field, the cell density sharply decreased to about 30 cells/mm2, and the dendritic field size increased to 10 X 10(-2) mm2 in a zone 2-3 mm interior to the margin. However, the dendritic coverage factor was consistently about 3.0 over the entire retinal field. Such morphological changes observed sequentially from the retinal margin to the intermediate region represent a developmental course of DA cells in the carp retina.

Animals↗
PubMed1988

Identification of horizontal cells generating different spectral responses in the retina of a teleost fish (Eugerres plumieri).

Six different types of spectral responses were recorded from horizontal cells under mesopic conditions in perfused retina, isolated from the dark-adapted mojarra (Eugerres plumieri). They were tentatively termed photopic Lr-, Lg1-, Lg2-, Lb-, and C-type, and scotopic L-type. The Lr-, Lg-, and Lb-type responses showed a maximum peak at 605, 550, and 516 nm respectively, while the C-type was composed of hyperpolarizing potentials in response to shorter wavelengths and depolarizing potentials in response to longer wavelengths (so-called R/G-type). The scotopic L-type has a peak at 516 nm in the spectral response and a slow decay phase in the waveform response. Following a brief period of diffuse illumination, it was found that the Lg1-type response is altered to the Lr-type, while both Lg2- and Lb-type responses change to the C-type. Intracellular marking with Lucifer or Procion yellow identified the cellular origins of different response types: external (He) and medial horizontal (Hm) cells for the Lr-type, internal horizontal (Hi) cells for the C-type, and rod-horizontal (Hr) cells for the scotopic L-type. Only He cells were found to possess an axon, while dye coupling was seen between axonless Hm, Hi, or Hr cells but not between He cells. The morphology of these fluorescent dye-marked cells was the same as that of the respective cells observed in Golgi-stained materials.

Animals↗
PubMed1988

Superoxide dismutase-mediated reversible conversion of 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole, the N-hydroxy derivative of Trp-P-2, into its nitroso derivative.

Aerobic oxidation of 3-hydroxyamino-1-methyl-5H-pyrido-[4,3-b]indole [Trp-P-2(NHOH)] in neutral aqueous solution was greatly accelerated by copper-zinc superoxide dismutase (SOD). The major product in this SOD-mediated reaction was identified as 3-nitroso-1-methyl-5H-pyrido[4,3-b]indole [Trp-P-2(NO)]. This conversion was accompanied by a decrease of the mutagenicity of the mixture, as monitored by the direct-acting mutagenicity on Salmonella typhimurium TA98; a rapid change to approximately 1/3 of the original mutagenicity was followed by no further decrease of the activity. In contrast, in the spontaneous aerobic oxidation of Trp-P-2-(NHOH), the mutagenicity slowly and continuously decreased, until it was finally lost almost completely. Similar acceleration by SOD of aerobic oxidation was found for 2-hydroxyamino-6-methyldipyrido[1,2-a:3',2'-d]imidazole [Glu-P-1(NHOH)]. Again, mutagenicity of approximately 1/4 that of the original was retained in the SOD-mediated decomposition, while a complete loss of the mutagenicity was observed in the spontaneous decomposition. When Trp-P-2(NO) was treated with the superoxide-generating system, xanthine oxidase plus xanthine, Trp-P-2(NHOH) was formed. Therefore, the role of SOD in the conversion of Trp-P-2(NHOH) into Trp-P-2(NO) is the removal of superoxide anions generated by reduction of aerobic oxygen, thereby inhibiting the reverse reactions, i.e. the reduction of Trp-P-2(NO) and that of the putative intermediate nitroxide radical. In support of this proposed mechanism, phenylhydroxylamine underwent a SOD-accelerated conversion to nitrosobenzene, and nitrosobenzene was reduced to phenylhydroxylamine by the action of the xanthine oxidase-xanthine system. Hence, this reversible interchange between an arylhydroxylamine and its nitroso compound, coupled with the oxygen-superoxide cycle, may be a general phenomenon. A consequence of this finding is that the xenobiotic N-hydroxylamines may be converted by the action of SOD in the biological settings into nitroso compounds, which are chemically more stable, serving as a reservoir for mutagenicity.

Carbolines↗
PubMed1988

The genotoxicity of N4-aminocytidine in the Drosophila wing spot test.

The nucleoside analogue N4-aminocytidine is known to induce mutations in bacteria, and was shown to induce somatic mutations in Drosophila melanogaster after larval administration. The assay system employed was a wing-hair mutation spot test developed by Würgler and co-workers. The potency of N4-aminocytidine to induce somatic mutation is comparable to those of several food-pyrolysate mutagens previously reported. The occurrence of twin spots, i.e. two types of recessive mutant-hair clones in adjacent positions, suggests that N4-aminocytidine induces somatic recombination in Drosophila. Another feature of the mutagenicity of N4-aminocytidine is that both the acute and the chronic larval feedings gave rise to mutant hair formation of similar patterns with respect to the spot-size distributions: small single spots were formed predominantly and the larger the spot-size, the lower their frequency.

Animals↗
PubMed1988

Generation of intracellular active oxygens in mouse FM3A cells by 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole, the activated Trp-P-2.

Mouse FM3A cells in culture were treated with a reactive metabolite of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2-(NHOH]. When the treated cells, which were judged as viable on the basis of trypan-blue exclusion, were subjected to nitroblue tetrazolium staining, formazan was formed inside the cells, a fact suggesting the intracellular presence of superoxide. No formazan formation was detected on treatment of the cells with Trp-P-2. Single-strand breaks in the cellular DNA took place during this treatment with Trp-P-2(NHOH). Since Trp-P-2(NHOH) in solution generates superoxide anion accompanying its oxidative degradation, we conclude that the Trp-P-2(NHOH) treatment produces intracellular active oxygens that can damage DNA.

Animals↗
PubMed1988

GABAergic inhibition on dopamine cells of the fish retina: a [3H]dopamine release study with isolated fractions.

Inner retinal cells including dopamine (DA) cells were isolated and fractionated from the carp (Cyprinus carpio) retina by an enzyme cell dissociation and metrizamide gradient centrifugation method. When gamma-aminobutyric acid (GABA) antagonists (bicuculline and picrotoxin) were added into the perfusate over such a cell fraction, they stimulated the release of [3H]DA which had been preloaded in the cell fraction. The action of GABA antagonists was dose and Ca2+ dependent. Their minimal effective concentration was very low (0.5 microM). A similar action was elicited by high K+. In the presence of excess GABA, this stimulatory action of GABA antagonists and high K+ on [3H]DA release was completely abolished. To interpret the action of GABA antagonists on DA cells, isolated cell fractions were preincubated with GABAse. After such a treatment, the stimulatory effects of GABA antagonists and high K+ on [3H]DA release were differentiated from each other; the former disappeared whereas the latter remained unchanged. The data strongly suggest that GABA inhibits the DA release from retinal DA cells and thus the GABA antagonists affect [3H]DA release from cell fractions not by a direct membrane action but by a disinhibition mechanism via GABA receptors on the DA cell bodies.

4-Aminobutyrate Transaminase↗
PubMed1988

Mutagenic nucleoside analog N4-aminocytidine: metabolism, incorporation into DNA, and mutagenesis in Escherichia coli.

N4-Aminocytidine, a nucleoside analog, is strongly mutagenic to various organisms including Escherichia coli. Using E. coli WP2 (trp), we measured the incorporation of [5-3H]N4-aminocytidine into DNA and at the same time measured the frequency of reversion of the wild type, thereby attempting to correlate the incorporation with mutation induction. First, we observed that N4-aminocytidine uptake by the E. coli cells was as efficient as cytidine uptake. High-pressure liquid chromatographic analysis of nucleoside mixtures obtained by enzymatic digestion of isolated cellular DNA showed that the DNA contained [3H]N4-aminodeoxycytidine, corresponding to 0.01 to 0.07% of the total nucleoside; the content was dependent on the dose of N4-aminocytidine. There was a linear relationship between the N4-aminocytosine content in DNA and the mutation frequency observed. These results constitute strong evidence for the view that the N4-aminocytidine-induced mutation in E. coli is caused by the incorporation of this agent into DNA as N4-aminodeoxycytidine. We also found that the major portion of radioactivity in DNA of cells that had been treated with [5-3H]N4-aminocytidine was in the deoxycytidine fraction. We propose a metabolic pathway for N4-aminocytidine in cells of E. coli. This pathway involves the formation of both N4-aminodeoxycytidine 5'-triphosphate and deoxycytidine 5'-triphosphate; the deoxycytidine 5'-triphosphate formation is initiated by conversion of N4-aminocytidine into uridine. In support of this proposed scheme, a cytidine deaminase preparation obtained from E. coli catalyzed the decomposition of N4-aminocytidine into uridine and hydrazine.

Cytidine↗
PubMed1988

Induction of mutation in mouse FM3A cells by N4-aminocytidine-mediated replicational errors.

To explore the potential use of a nucleoside analog, N4-aminocytidine, in studies of cellular biology, the mechanism of mutation induced by this compound in mouse FM3A cells in culture was studied. On treatment of cells in suspension with N4-aminocytidine, the mutation to ouabain resistance was induced. The major DNA-replicating enzyme in mammalian cells, DNA polymerase alpha, was used to investigate whether the possible cellular metabolite of N4-aminocytidine, N4-aminodeoxycytidine 5'-triphosphate (dCamTP), can be incorporated into the DNA during replication. Using [3H]dCamTP in an in vitro DNA-synthesizing system, we were able to show that this nucleotide analog can be incorporated into newly formed DNA and that it can serve as a substitute for either dCTP or dTTP. dCamTP in the absence of dCTP maintained the activated calf thymus DNA-directed polymerization of deoxynucleoside triphosphates as efficiently as in its presence. Even in the presence of dCTP, dCamTP was incorporated into the polynucleotide. When dCamTP was used as a single substrate in the poly(dA)-oligo(dT)-directed polymerase reaction, it was incorporated into the polynucleotide fraction. The extent of incorporation was 4% of that of dTTP incorporation when dTTP was used as a single substrate. Even in the presence of dTTP, dCamTP incorporation was observed. A copolymer containing N4-aminocytosine residues was shown to incorporate guanine residues opposite the N4-aminocytosines. However, we were unable to observe adenine incorporation opposite N4-aminocytosine in templates. These cell-free experiments show that an AT-to-GC transition can take place in the presence of dCamTP during DNA synthesis, strongly suggesting that the mutation induced in the FM3A cells by N4-aminocytidine is due to replicational errors.

Animals↗
PubMed1988

In vitro mutagenesis by incorporation of N4-aminodeoxycytidine 5'-triphosphate.

We have investigated the possibility of introducing a new way to carry out in vitro mutagenesis. N4-Aminodeoxycytidine 5'-triphosphate was used in the Klenow enzyme-catalyzed chain elongation of a primer oligonucleotide hybridized to a lacZ alpha region of M13mp2 viral single strand DNA, and the possibility of inducing an efficient, randomly distributed point mutations into this particular genomic region was explored. On transfection of the resulting DNA into E. coli, mutant phages emerged at frequencies up to 1%. Analysis of the DNA sequences of the mutants has shown that single transitions, either A to G or G to A, were induced in a random fashion, thus providing data to show the possibility of using this method for production of mutant proteins having various single amino-acid changes in a defined domain.

Coliphages↗