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Biomedical subjects

K Negishi

Publications and source records attributed to K Negishi.

At least 163 records · Page 9Linked to original sources
PubMed1986

Quantitative estimation of islet cell surface antibodies in sera of patients with diabetes mellitus, using BK virus-induced insulinoma, rat islet or fish islet cells and 125I-antihuman IgG antibody.

Using a radioligand assay with 125I-antihuman IgG antibody as well as an 125I-protein A binding assay, we quantitatively evaluated islet cell surface antibodies (ICSA) in the sera of diabetic patients already proven to contain antibodies by the immunofluorescence method (IF). It was possible to use not only rat islet but also BK virus-induced hamster insulinoma cells and yellow tail fish (Serolia quinqueradiata) islet cells as target cells in these radioassays. 7 out of 8 diabetic subjects, positive for ICSA by IF, showed levels of binding of 125I-antihuman IgG antibody greater than 2SD above the mean for normal controls. Additionally 3 diabetic subjects negative for ICSA by IF showed levels of binding of 125I-antihuman IgG antibody less than 2SD above the mean for normal controls, and 2 subjects negative for ICSA by IF had levels slightly higher than 2SD above the mean for normal controls. The quantitative ICSA radioligand assay reported here is both sensitive and specific in the detection of the IgG of ICSA. It is also more convenient to use than other methods, and may use any of several different kinds of target cells, each of which may be easily and reproducibly obtained. These radioimmunological methods for the detection of ICSA appear to be ideal for screening large numbers of patients for ICSA.

Adenoma, Islet Cell↗
PubMed1986

Evaluation of the mutagenicity of 1,N6-ethenoadenine- and 3,N4-ethenocytosine-nucleosides in Salmonella typhimurium.

To provide supporting evidence for the hypothetical involvement of etheno-derivative formation in DNA in chloroacetaldehyde-mediated mutagenesis, etheno nucleosides were examined for their direct-acting mutagenicity in Salmonella typhimurium strain TA100. The results, however, have shown that 1,N6-ethenoadenosine, 1,N6-ethenodeoxyadenosine, 3,N4-ethenocytidine and 3,N4-ethenodeoxycytidine lack mutagenicity in this test system. A lesson learned in this study is that 3,N4-ethenocytosine nucleosides prepared synthetically or obtained from commercial sources can give false positive mutagenicity due to mutagenic contaminants.

Adenine↗
PubMed1986

Synthesis of a mutagenic nucleoside, 2'-deoxy-2-(p-nitrophenyl)-adenosine.

The reaction of 2-amino-6-chloropurine riboside with i-amyl nitrite in benzene in the presence of Cu2O, followed by treatment with NH3/MeOH gave 2-phenyladenosine (1). The crude sample of 1 was found to be mutagenic to bacteria (Salmonella typhimurium TA 98 and TA 100, without metabolic activation). When this material was subjected to high pressure liquid chromatography, the mutagenic activity was found only in contaminating minor components, whose structures were assigned as 2-(m- and p-nitrophenyl)-adenosines (2m,p). In order to study structure-activity relationships, several nucleoside and base analogues were synthesized. Among them, 2'-deoxy-2-(p-nitrophenyl)-adenosine (8) was the most potent mutagen as tested either with TA 98 or TA 100.

Adenosine↗
PubMed1985

Direct-acting mutagenicity of N4-aminocytidine derivatives bearing alkyl groups at the hydrazino nitrogens.

To investigate the mechanism of N4-aminocytidine-induced mutagenesis, N'-alkyl-N4-aminocytidines and N4-alkyl-N4-aminocytidines were prepared and their mutagenicity on bacteria were assayed. N'-Methyl-N4-aminocytidine, N'-(2-hydroxyethyl)-N4-aminocytidine and N',N'-dimethyl-N4-aminocytidine showed direct-acting mutagenicity on S. typhimurium TA100 and E. coli WP2 uvrA, tester strains that are sensitive to base-pair substitutions. In contrast, N4-methyl-N4-aminocytidine, N4-(2-hydroxyethyl)-N4-aminocytidine and N4,N'-dimethyl-N4-aminocytidine were not mutagenic on these bacteria. Since N'-methyl-N4-aminocytidine does not form hydrazones, the possibility that N4-aminocytidine causes mutation due to its reactivity with carbonyl compounds has been excluded. Furthermore, the fact that only those alkyl N4-aminocytidines having a hydrogen on the nitrogen at position 4 are mutagenic is consistent with the previously proposed mechanism in which the tautomerization between the amino and the imino forms of N4-aminocytosine allowing an ambiguous base pairing is the cause of the mutagenesis.

Alkylation↗
PubMed1985

Spatial orientation of horizontal cell axon terminals in the carp retina.

In flatmounts of the carp (Cyprinus carpio) retina, 646 horizontal cells were singly marked by intracellular Lucifer yellow CH (LY) in the presence of dopamine or amphetamine, agents which were useful for restricting LY to single injected cells. Most axon terminals of cone-connected horizontal cells have a tendency to orient either radially or tangentially in the retinal field with respect to the optic disc as a center. Although the fluorescent cellular (dendritic field) area greatly varied depending upon the cell type (L-, RG- and YRB-type), the lengths of the axonal processes (axon plus terminal) were all comparable (400-600 microns). A few cells (4.1% of cells with visible axons) possessed a bifurcate axon with two axon terminals. Axons were not observed on rod-connected horizontal cells. The cellular area and the axonal length of L- and RG-type cells appeared to be smaller in the central than in the intermediate region of the retinal field.

Animals↗
PubMed1985

Mutagenesis by N4-aminocytidine: induction of AT to GC transition and its molecular mechanism.

N4-Aminocytidine is a potent mutagen toward Escherichia coli and Salmonella typhimurium. It induced reversion of an amber mutant of phi X174 phage (am3) to the wild type. This reversion was shown to be exclusively due to the AT to GC transition. It is likely that N4-aminocytidine is metabolized within the bacterial cells into N4-aminodeoxycytidine 5'-triphosphate and this nucleotide is incorporated into DNA during the multiplication of the cells and the phages, thereby causing base-pair transitions. The molecular basis for this erroneous replication was obtained in studies of in vitro incorporation of N4-aminodeoxycytidine 5'-triphosphate into polynucleotides catalyzed by the E. coli DNA polymerase I large fragment. The results have shown that this cytosine analogue can be efficiently incorporated as a substitute of cytosine and that it can also be incorporated as a substitute of thymine. The ratio in the rate of the N4-aminocytosine nucleotide incorporation to that of natural nucleotide incorporation was 1/2 to cytosine and 1/30 to thymine. Furthermore, the N4-aminocytosine residues in the polynucleotide templates can be read by the enzyme as efficiently as cytosines, and guanines were incorporated opposite to them.

Bacteriophage phi X 174↗
PubMed1985

So-called interplexiform cells immunoreactive to tyrosine hydroxylase or somatostatin in rat retina.

The morphology of so-called interplexiform (IP) cells immunoreactive to tyrosine hydroxylase (TH) or somatostatin (SOM) in the rat retina was described in comparison with those in the carp retina. In frozen cross-sections of the rat retina, many processes of TH-like immunoreactive cells were found to extend toward the outer plexiform layer (OPL), forming a thin layer of network fibers. A few of them further extended into the photoreceptor cell layer; such fibers were never found in the carp retina. Some processes of SOM-like immunoreactive cells in the rat retina were found to travel across the inner nuclear layer and appeared to poorly develop a network at the OPL. In the carp retina, on the other hand, only one exceptional cross-section contained such an ascending process.

Animals↗
PubMed1985

Induction of mutation in vitro in phage phi X174 am3 by N4-aminodeoxycytidine triphosphate.

When phi X174 am3-phage-infected E. coli is treated with N4-aminocytidine, reversion of the phage to the wild type is efficiently induced. The mechanism of this reversion is considered to consist of metabolic conversion of N4-aminocytidine into its deoxynucleoside 5'-triphosphate followed by incorporation of the nucleotide into the replicating phage DNA, thereby causing AT-to-GC transition at the am3 locus. The second half of this mechanism has now been experimentally proved, using an in vitro mutagenesis system. Thus, by nick-translation, N4-aminodeoxycytidine 5'-triphosphate was incorporated into the replicative form of phi X174 am3 DNA, and the DNA was used to transfect CA++-treated E. coli HF4714 (sup+). The reversion frequency of the phage produced was up to one-order of magnitude greater than that of the control in which the nick-translation had been done without the addition of N4-aminodeoxycytidine triphosphate. This nucleotide analog may be useful as a reagent for in vitro site-directed mutagenesis.

Bacteriophage phi X 174↗
PubMed1985

Opposite effects of ammonia and carbon dioxide on dye coupling between horizontal cells in the carp retina.

Effects of ammonia (NH3) and carbon dioxide (CO2) on the membrane potential of horizontal cells and on dye coupling between the cells in isolated retinas of the carp (Cyprinus carpio) were investigated. Ammonia (less than 300 ppm NH3 in air) initially depolarized and subsequently hyperpolarized, while CO2 (10% in air) hyperpolarized the membrane potential of horizontal cells, accompanied by a diminution of both center and surround responses to spot and annular light stimuli. During the course of amplitude diminution, the center response consistently became smaller with NH3 and larger with CO2 than the surround response. In the presence of intravitreally applied DA (50 microM) or amphetamine (100 microM), a fluorescent dye Lucifer Yellow CH (LY) was found to be restricted to single injected horizontal cells. The presence of intravitreal haloperidol (100 microM) for 20-25 min or an exposure of the retina to NH3 for 5-10 min diffused the restricted LY from single injected cells to numerous neighboring cells. On the other hand, CO2 was found to restrict the injected dye to single cells, an effect similar to that of DA and opposite to that of NH3 and haloperidol. The results suggest that NH3 appears to act as a coupler while CO2 acts as an uncoupler on gap junctions between horizontal cells in the carp retina, presumably by changing the intracellular pH. In addition, a brief exposure of cells, marked with LY in the presence of DA, to the exciting light 426 nm was found to prevent the NH3-induced dye diffusion from single cells to their neighbors; the reason is unknown.

Ammonia↗
PubMed1985

L-Glutamate depolarizes ON-OFF transient type of amacrine cells in the carp retina: an ionophoretic study.

Amacrine cells generating the ON-OFF transient type of photoresponse, which is most frequently encountered, were identified by intracellular recording and staining with a fluorescent dye Lucifer Yellow in the carp (Cyprinus carpio) retina. L-Glutamate applied ionophoretically slowly depolarized the amacrine cells by about 10 mV and reduced their photoresponses during the application. Such changes gradually recovered following the cessation of L-glutamate injection. The results indicate that this amino acid directly activates the ON-OFF type amacrine cells.

Action Potentials↗
PubMed1985

A bifurcate axon of horizontal cells in the carp retina.

In flatmounts of the carp retina, among 368 cone-connected horizontal cells, which were singly marked by intracellular Lucifer yellow under various experimental conditions, 15 cells (4.1%) were found to possess a bifurcate axon and two terminals. The lengths of single and bifurcate axonal processes (an axon plus its terminal) are all comparable, ranging from 400 to 600 micron.

Animals↗
PubMed1985

Growth rate of a peripheral annulus defined by neurotoxic destruction in the goldfish retina.

Fifteen goldfish of similar size (6.8 cm in body length on the average) were reared in an aquarium (about 22 degrees C) for 5 months after neurotoxic destruction of retinal dopamine (DA) cells with intravitreal injection of 6-hydroxydopamine, and then further for 8 months after destruction of indoleamine-accumulating cells with 5,7-dihydroxytryptamine. By means of a histofluorescence technique for retinal wholemounts, the growth rate and the number of DA cells were estimated for a peripheral annulus which had been well defined by the successive neurotoxic destructions of monoamine-accumulating cells. As the fish grew from 8.4 to 11.6 cm in body length on the average over the 8-month period, the defined peripheral annulus of the retina was found to be enlarged radially by approximately 1.5 X and circumferentially by 1.3 X while the number of DA cells in the annulus remained unchanged.

5,7-Dihydroxytryptamine↗
PubMed1985

Retinal growth in carp of the same age: density and number of dopamine neurons.

Research has shown that an increase in the size of retina with fish growth is accompanied by a reduction in the density of monoamine neurons along with an increase in their cell number per retina. The age of fish, however, has not been considered in relation to the above. In the present study, juvenile carp (6.4 +/- 0.7 cm in body length), hatched 2 months earlier, were reared for 10 months under 2 different sets of conditions; one group was kept in a small aquarium at room temperature (RT, 9-26 degrees C), and the other was kept in a large aquarium in which the water temperature was constant (CT, 22 degrees C). The left eye of fish transferred into the RT aquarium was intravitreally injected with 5,7-dihydroxytryptamine (5,7-DHT; 1.0 microgram) to destroy indoleamine-accumulating (IA) neurons. The fish grew faster in the CT (16 +/- 1.6 cm) than in the RT aquarium (9.3 +/- 1.7 cm), resulting in a wide variation among fish of the same age. In fluorescent retinal flatmounts, the density of dopamine (DA) neurons was found to be lower, but their cell number per retina greater in the CT than in the RT group; these differences were statistically significant (P less than 0.001). The data suggest that the above parameters depend on the size of carp but not on their age. In the fish treated with 5,7-DHT and reared in the RT aquarium for 10 months, the growth zone of the retina was determined by circumferential addition of IA cells.(ABSTRACT TRUNCATED AT 250 WORDS)

5,7-Dihydroxytryptamine↗
PubMed1985

Dopamine inhibits calcium-independent gamma-[3H]aminobutyric acid release induced by kainate and high K+ in the fish retina.

Kainic acid (KA) at micromolar concentrations stimulated the release of gamma-[3H]aminobutyric acid [( 3H]GABA) from a particulate fraction of the carp (Cyprinus carpio) retina. The KA action was dose-dependent but Ca2+-independent. A similar response was elicited by another glutamate receptor agonist, quisqualic acid, and high K+, but not by an aspartate agonist, N-methyl-D-aspartic acid. The stimulatory action of KA on the [3H]GABA release was selectively blocked by the KA blockers gamma-D-glutamylglycine and cis-2,3-piperidine dicarboxylic acid. Dopamine (DA), which is contained in DA interplexiform cells in the carp retina, inhibited the [3H]GABA release induced by KA and high K+ in a dose-dependent manner. 5-Hydroxytryptamine and two well-known GABA antagonists, bicuculline (Bic) and picrotoxin (Pic), also mimicked the DA effect on the GABA release at a comparable concentration. This inhibitory effect of DA as well as Bic and Pic on the [3H]GABA release evoked by KA was clearly antagonized by a DA blocker, haloperidol. The action of these agents (KA, DA, GABA antagonist) belonging to three different receptor categories on the GABAergic neurons (possibly external horizontal cells; H1 cells) is discussed in relation to other electrophysiological studies on the lateral spread of S-potentials between H1 cells.

Animals↗
PubMed1985

Ambiguous incorporation of N4-aminodeoxycytidine 5'-triphosphate to DNA synthesized in vitro.

Molecular mechanism of the mutation induced by N4-aminocytidine was studied. The specificity of in vitro incorporation of N4-aminodeoxycytidine 5'-triphophate catalyzed by E. coli DNA polymerase large fragment was analyzed. The results have shown that this cytosine analog can be efficiently incorporated as a substitute of cytosine, and that it can also be incorporated with a low efficiency as a substitute of thymine. We have also shown that the N4-aminocytosine incorporated opposite adenine can be excised as its monophosphate at a high frequency. The N4-aminocytosine residues in the polynucleotide templates can be read by the enzyme as efficiently as cytosines, and guanines were incorporated opposite them.

DNA, Bacterial↗