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Biomedical subjects

K Negishi

Publications and source records attributed to K Negishi.

At least 181 records · Page 10Linked to original sources
PubMed1985

DNA strand cleavage in vitro by 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]-indole, a direct-acting mutagen formed in the metabolism of carcinogenic 3-amino-1-methyl-5H-pyrido[4,3-b]indole.

3-Hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (N-OH-Trp-P-2) is a direct-acting mutagenic compound derived by metabolic activation from 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), a strongly mutagenic carcinogen. The action of N-OH-Trp-P-2 on DNA in vitro was investigated. N-OH-Trp-P-2 inactivated Bacillus subtilis transforming DNA and produced single-strand cuts in a supercoiled circular DNA (phi X174RFI) under neutral conditions. When mouse FM3A cells in culture were treated with a noncytotoxic dose of N-OH-Trp-P-2 and then the cellular DNA was examined by the alkaline elution technique, chain cleavages of the DNA were observed. Cysteamine inhibited the spontaneous degradation of N-OH-Trp-P-2 and enhanced the covalent binding of [3H]N-OH-Trp-P-2 to DNA. This finding offered an explanation for the previously observed enhancement of Trp-P-2 mutagenicity by cysteamine. In contrast cysteamine inhibited the N-OH-Trp-P-2-mediated inactivation of B. subtilis DNA as well as the strand cleavage in phi X174RFI DNA. The cleavage in phi X174RFI DNA was also inhibited by catalase. These observations indicate that the mutagenicity and DNA-cleaving activity of N-OH-Trp-P-2 are distinct from each other, that the inactivation of transforming DNA was caused mainly by strand cleavage, and that the DNA cleavage was probably caused by active oxygen radicals produced in the oxidative degradation of N-OH-Trp-P-2.

Animals↗
PubMed1984

Dye coupling between amacrine cells in carp retina.

Amacrine cells in isolated retinas of the carp (Cyprinus carpio) were intracellularly recorded and marked with a fluorescent dye, Lucifer yellow. On occasion, dye coupling was found to occur between amacrine cells when the dye was iontophoretically injected into an amacrine cell, generating one of the transient or sustained types of photoresponse. Dye-coupled cells in the vicinity of the marked cell were very similar to the marked cell in soma shape and dendritic stratification. Dye diffusion is assumed to take place at gap junctions between dendrities of amacrine cells which belong to a population of similar type cells in morphology and possibly in function.

Animals↗
PubMed1984

Regular orientation of horizontal cells in the river lamprey retina.

In isolated retinas of the river lamprey (Lampetra japonica), two types (fast and slow) of light-induced responses (S-potentials) were recorded from two distinct classes of axon-bearing horizontal cells. After the spectral responses of recording cells were examined, a fluorescent dye Lucifer yellow CH (LY) was ionophoretically injected into individual cells. Such a cellular marking was made for 15-25 points per retina successively in time and in space. Then the retinas were processed as flatmounts for fluorescence microscopic observation. Horizontal cells marked with LY were correlated with the recordings and mapped in the retinal field. Both classes of horizontal cells were found to be regularly arranged in space around the optic disc; the long axes of somata and axonal processes are oriented in parallel with the latitude line of the eyeball through the optic disc.

Animals↗
PubMed1984

Sensitive and insensitive states of cultured glioma cells to glutamate damage.

Cytotoxic effects of L-glutamate and related compounds were investigated on rat glioma C6 cells in vitro. Within 12-24 h, addition of glutamate to the culture medium, resulted in degeneration of the C6 cells. The ED50 for glutamate-induced damage was about 4 mM. Seventeen structural analogues of glutamate, including agonists and antagonists for glutamate receptors as well as glutamate-uptake inhibitor, were examined concerning their toxicity on C6 cells. Among them, L-aminoadipic acid, DL-aminopimelic acid, DL-homocysteic acid, L-cysteic acid, quisqualic acid, L-glutamic acid diethyl ester and 2-amino-4-phosphonobutyric acid elicited similar degeneration at comparable concentrations. The D-isomer of glutamate was not cytotoxic. Following differentiation of C6 cells with 1 mM dibutyryl cyclic AMP or 3 mM sodium butyrate, they were no longer susceptible to L-glutamate and L-aminoadipate. C6 cells treated with 10 microM hydrocortisone, which is known to induce glutamine synthetase activity, were also resistant to L-glutamate, but not to L-aminoadipate. The decomposition of cellular DNA in glutamate-treated cultures was confirmed by flow cytometer analysis. The results demonstrate that the sensitivity of C6 cells to glutamate-induced cytotoxicity was modified by cellular metabolic conditions. This indicates that cultured glioma C6 cells are a useful model system to investigate the molecular mechanism of glutamate gliotoxicity in vitro.

2-Aminoadipic Acid↗
PubMed1984

Embryonic development of monoaminergic neurons in the chick retina.

By means of a histofluorescence technique, embryonic and postnatal development of monoaminergic neurons was followed in the chicken retina with or without intravitreal injection of monoamines 30-60 minutes before eye removal. Fluorescent cells were tentatively classified into five subsets with respect to the soma shape, localization, migration of somata during retinal development, uptake capacity (color and intensity in fluorescence), and sensitivity to neurotoxins. The five subsets of cells were endogenous dopaminergic (DA), catecholamine-accumulating (CA), indoleamine-accumulating (IA), CA-bipolarlike, and IA-bipolarlike cells. Greenish endogenous DA-cells first appeared at the 14-15th embryonic day. The cell body of DA-cells was initially fusiform and located slightly distal to the innermost level of the inner nuclear layer (INL). They became round or oval and migrated to the innermost level of the INL by day 20. Both large and small bottle-shaped CA-cells were visualized at an intermediate portion of INL by intravitreal injection of exogenously applied dopamine or noradrenaline (1-2 micrograms/eye) at day 10. Large bottle-shaped cells, like the DA cells, changed to round or oval and migrated to the innermost level of the INL by day 20. On the other hand, small bottle-shaped CA-cells retained their cell shape and location in the INL as retinal development progressed. Therefore, the large bottle-shaped CA-cells seen in an early developmental stage correspond to the DA-cells. IA-cells were visualized one or two cell rows outward in the INL first at day 13-14 by intravitreal injection of 5,6-dihydroxytryptamine or 5-hydroxytryptamine (1-5 micrograms).(ABSTRACT TRUNCATED AT 250 WORDS)

5,7-Dihydroxytryptamine↗
PubMed1984

Regional density of monoamine-accumulating amacrine cells in the rabbit retina.

The spatial distribution of noradrenaline (NA)- and indoleamine-accumulating (IA) amacrine cells was studied by fluorescence microscopy in flatmounts and tangential cryosections (15 microns thickness) of albino rabbit retinas. The two classes of cells were found to be distributed over the retinal field in a mixed and random fashion. The regional density (mean +/- S.D. cells/mm2) of monoamine accumulating cells was highest in the visual streak (NA cells, 88 +/- 5; IA cells, 1507 +/- 92), and lowest (41 +/- 7; 625 +/- 105, respectively) in the inferior periphery. The density ratio of NA:IA cells was 1:15 on average. Among cells located in the amacrine cell layer, NA and IA cells accounted for 0.3 and 3.9% of the total cell population, respectively. Monoamine-accumulating amacrine cells displaced into the ganglion cell layer were few; these displaced cells were only 2% and 0.6% of the cell number of normally situated cells in the amacrine cell layer for NA and IA cells, respectively.

5,6-Dihydroxytryptamine↗
PubMed1984

A new reaction useful for chemical cross-linking between nucleic acids and proteins.

Cytosine in nucleic acids can be converted into N4-aminocytosine by treatment with a mixture of hydrazine and bisulfite. The hydrazino group thus formed at position 4 of the pyrimidine ring can be linked to a sulhydryl group in proteins by the use of bromopyruvate as a linker. Successful use of this scheme of chemical cross-linking between nucleic acid and protein was demonstrated in the linking of poly(C) with glutathione, and of RNA with protein in the E. coli 30 S ribosomal subunit.

Cross-Linking Reagents↗
PubMed1984

Effects of dopamine on photopic L-type S-potentials in the catfish retina.

Photopic L-type responses were recorded from the soma and the axon terminal of horizontal cells of the catfish (Ictalurus punctatus) retina in eye-cup preparations. The responses were produced by a spot of light with 100-micron diameter (intensity, 10 microW/cm2), which was flashed or steadily illuminated and swept along a 6-mm length over the retinal surface at a speed of 0.95 mm/sec. In some experiments, a traveling random bar stimulus was used instead of the sweeping spot. While recording the responses, dopamine (DA) was applied in a jet form via a nebulizer over the retinal preparation or as superfusate to the eye-cup preparation. DA increased the response amplitude by about 50% and markedly narrowed the spatial profile of the responses from the soma but not from the axon terminal. These DA effects were observed in both normal retinas and those from which DA cells had been deprived by prior intraocular injection of 6-hydroxydopamine. Deprival of DA cells from the retina resulted in a slightly wider spatial profile of the soma and axon terminal responses than that in normal retinas. The results indicate that the spatial properties of photopic L-type responses are modulated by DA at the soma level of horizontal cells.

Animals↗
PubMed1984

Immunological events in new onset diabetes.

Data from our laboratory are reviewed showing that both cell-mediated cytotoxicity, complement-dependent antibody-mediated cytotoxicity, antibody-dependent cellular cytotoxicity, and complement-augmented antibody-dependent cellular cytotoxicity may provide mechanisms which kill pancreatic islets during pathogenesis of insulin-dependent (type I) diabetes. Xenogenic test systems employing dispersed rat islet target cells were employed. Most of the findings were reversible during clinical remission of diabetes.

Animals↗
PubMed1984

Regulatory effect of dopamine on spatial properties of horizontal cells in carp retina.

Three types of light-induced response (L-, RG-, and YRB -type S-potentials) recorded from isolated retinas of the carp (Cyprinus carpio) were identified by their spectral response and later by morphological localization of the recording sites marked with an intracellular Lucifer Yellow (LY). Horizontal cells in a given layer, generating one of the above response types, are electrically coupled via gap junctions, so that the injected LY normally diffused to several neighboring cells. The spatial property of the three types of responses was examined by enlarging the diameter of a light spot (0.25 to 4.0 mm) and displacing the spot (0.5 mm diameter) along a straight 4-mm line which passed over the recording point at the middle. In normal retinas, the half-decay distance of response amplitude with spot displacement was shorter in the order of L-, RG-, and YRB -type responses, and correspondingly the dye diffusion area was narrower in the same order of cells. Dopamine (DA; 10 to 20 microM), applied to the vitreous fluid beneath the isolated retina, altered the spatial property of all types of responses by increasing the amplitude of responses to central spots and decreasing that of those to distant spots, and it restricted the intracellular LY to single injected cells. In contrast, in retinas from which DA interplexiform cells had been deprived by prior destruction with a neurotoxin, 6-hydroxydopamine, the amplitude of responses became smaller while the half-decay distance was longer by 1.1- to 1.6-fold, depending upon the cell type, and the dye diffusion area in all types of cells became wider by 2-fold as compared to those in normal retinas.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

N4-aminocytidine, a nucleoside analog that has an exceptionally high mutagenic activity.

The reaction of cytidine with hydrazine to give N4-aminocytidine was greatly promoted by addition of a less-than-stoichiometric amount of bisulfite, and the product was isolated in a good yield. N4-Aminocytidine was strongly mutagenic to bacteria (Salmonella typhimurium TA100 and TA1535, and E. coli WP2 uvrA) and to phage (phi X174 am3). The activity did not require the presence of mammalian microsomal fraction in the system. The mutagenic potency of N4-aminocytidine in these systems was two orders of magnitude greater than that of N4-amino-2'-deoxycytidine, and more than two orders of magnitude greater than that of N4-hydroxycytidine. The greater activity of the riboside than the deoxyriboside was ascribed to the lack of deoxycytidine kinase in these cells. This compound may be useful as a powerful mutagen to induce a transition mutation in microorganisms.

Bacteriophage phi X 174↗
PubMed1983

A GABA antagonist, bicuculline, exerts its uncoupling action on external horizontal cells through dopamine cells in carp retina.

External horizontal cells in isolated retinas of the carp (Cyprinus carpio) were intracellularly recorded and marked with the fluorescent dye Lucifer Yellow (LY). These cells are electrically coupled via gap junctions, so that the injected dye normally diffused to neighboring cells. A GABA antagonist, bicuculline (Bcc, 20 microM), applied to the vitreous fluid beneath the isolated retina, altered the spatial properties of light-induced responses, by increasing the amplitude of responses to central spots and decreasing that of those to distant spots. Bcc also restricted to injected dye to single recorded cells. These uncoupling actions of Bcc were similar to those of amphetamine (20 microM) or dopamine (10-20 microM) and were abolished by the presence of a dopaminergic blocker, haloperidol (20-40 microM). Both the actions of Bcc and amphetamine were not observed when applied to retinas deprived of dopaminergic cells by prior destruction with 6-hydroxydopamine. Therefore, Bcc exerts its uncoupling actions on external horizontal cells indirectly through the dopaminergic system in the carp retina.

Amphetamine↗
PubMed1983

Dopamine modulates S-potential amplitude and dye-coupling between external horizontal cells in carp retina.

Horizontal cells in the fish retina are electrically coupled and possess gap junctions so that intracellularly injected dye normally diffuses freely to neighbouring cells. Applied dopamine (DA) alters the spatial properties of the horizontal cell responses to light, increasing the amplitude of photopic L-type S-potentials but decreasing their lateral spread. These effects have been attributed to the action of DA on horizontal cell membrane resistance, particularly at the gap junctions, and our present study on the carp retina agrees with this in showing that DA also restricts intracellular Lucifer yellow (LY) to single injected horizontal cells, an effect, like those of DA on the S-potentials, which is antagonized by the dopamine blocker haloperidol. In addition, we present evidence that dopaminergic interplexiform cells in fish normally function to regulate the spatial properties of responses in horizontal cells, possibly acting on their junctional resistance via a DA-receptor-mediated mechanism. Previous destruction of the interplexiform cells with 6-hydroxydopamine (6-OHDA) resulted in much reduced L-type S-potentials to centred lights but wider lateral spread of these responses, while the dye injected spread extensively to neighbouring cells. After 6-OHDA treatment, however, applied DA retained its normal activity, restoring large-amplitude, narrow receptive-field S-potentials and restricting LY to the injected cells, effects which were both closely mimicked by dibutyryl cyclic AMP.

Action Potentials↗
PubMed1983

Regional difference in density of monoamine-accumulating cells of carp and catfish retinas.

By means of a histofluorescence technique, a comparative study was conducted on the regional density of dopaminergic (DA) and indoleamine-accumulating (IA) cells in carp (Cyprinus carpio) and catfish (Ictalurus punctatus) retinas. In order to enhance detection of fluorescent cells, noradrenaline (NA; 5.0 micrograms) or a mixture of NA (2.5 micrograms) and 5,6-dihydroxytryptamine (5,6-DHT; 2.5 micrograms) was intravitreally injected into the eyes 2-3 hr before enucleation. DA and IA cells were counted systematically in space on flat-mounted preparations. Both classes of cells were found to be distributed similarly in the two species of fish; the cell density is highest in the circumferential margin of the retina, and is slightly higher in a region dorsal to the optic disc than in the surrounding area. Differences in the distribution pattern of the cells between carp and catfish retinas were as follows: (a) the DA cell density is higher over the whole retinal field in carp (the mean density +/- SD = 34 +/- 16 cells/mm2) than in catfish (13 +/- 7 cells/mm2); (b) the region where the density is slightly higher than in the surrounding area is restricted to a small area immediately dorsolateral to the optic disc in carp, while it is relatively broadly placed dorsal to the optic discs, forming a horizontal band in catfish; (c) the density ratio of DA cells to IA cells is 1:1 in carp but 1:2 in catfish; and (d) catfish DA cells seem to be more irregular than carp DA cells in shape, size, dendritic arborization, uptake preference for monoamines intravitreally injected, and also in depth location seen in radial cryosections.

Animals↗
PubMed1983

5-hydroxytryptamine: its facilitative action on [3H]dopamine release from the retina.

5-Hydroxytryptamine (5-HT) at 5 X 10(-4) M caused a two-fold increase in [3H]dopamine (DA) release from retinal particulate fractions of the carp (Cyprinus carpio). The 5-HT action was dose- and Ca2+-dependent. The three 5-HT agonists examined (5-methoxytryptamine, 5-methoxy-N,N-dimethyltryptamine and tryptamine) were more effective than 5-HT on [3H]DA release. 5,6-Dihydroxytryptamine was the strongest competitor among drugs tested for [3H ]DA uptake, but did not evoke any significant release of [3H]DA. Noradrenaline (or DA) at 5 X 10(-4) M produced a large increase in [3H]DA release from the particulate fractions, but its action was Ca2+-independent. The 5-HT-induced DA release could be seen in the frog retina but not in the rat retina, which does not contain indoleamine-accumulating cells. The results obtained strongly suggest that 5-HT stimulates [3H ]DA release through a receptor mechanism on DAergic terminals and modifies the DA-cell's function in the retina.

Animals↗