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K Negishi

Publications and source records attributed to K Negishi.

At least 73 records · Page 4Linked to original sources
PubMed1995

Mechanism of mutagenesis induced by cytosine analogs bearing N(4)-substitutions.

The mechanism of mutagenesis induced by dihydropyrimido [4,5-c][1,2]oxazin-7-one deoxyriboside, P-nucleoside, was studied. This analog is highly mutagenic toward Escherichia coli and Salmonella typhimurium. In E. coli, it induces GC-to-AT and AT-to-GC transitions specifically. No transversions are inducible. P-nucleoside was highly mutagenic to a wild-type E. coli, but little mutagenic in a strain lacking thymidine kinase. This indicates that P-nucleoside may be phosphorylated by thymidine kinase after its uptake into bacteria. The mutagenesis induced by P-nucleoside was efficiently inhibited by the addition of thymidine. This inhibition further confirmed the involvement of thymidine kinase in the first step of the metabolism of P-nucleoside in the bacterial cells. These findings indicate that P-nucleoside is a mutagen of a nucleoside-analog type, causing mutations by the erroneous incorporation and replication. The experiments to prove its ambiguous nature in DNA synthesis is now under way.

Adenine↗
PubMed1994

Reciprocal changes in left ventricular collagen alpha 1 chain gene expression between types I and IV in spontaneously diabetic rats.

The characteristic features of diabetic cardiomyopathy have been reported to be increased collagen formation associated with impairment of ventricular performance, based on experimental models of diabetes. The present study was therefore designed to clarify collagen gene expression in hearts obtained from female spontaneously diabetic BioBreeding Worcester Tokyo (BB/W@Tky) rats. Cardiac hypertrophy was observed as early as 14 weeks in diabetic BB/W@Tky rats, i.e. 4 weeks after the onset of diabetes. Left ventricular gene expression of collagen alpha 1 (I) was decreased to 10.6% of the control level. In 24-week-old diabetic rats, which had more marked cardiac hypertrophy, the level of alpha 1 Type I collagen mRNA was further decreased to 5.7% of the control level, whereas collagen alpha 1 (IV) mRNA demonstrated a 3-fold increase. As a result, a ratio of collagen alpha 1 (IV) to actin mRNA was positively correlated with plasma glucose concentration. These results suggest that hyperglycemia may alter the gene expression of extracellular matrix proteins, resulting in the morphological and functional changes seen in diabetic cardiomyopathy.

Actins↗
PubMed1994

Mutagenicity of 5-bromouracil and N6-hydroxyadenine studied by yeast oligonucleotide transformation assay.

The mutagenicity of 5-bromouracil (BrU) and N6-hydroxyadenine (HA) was tested by means of the yeast oligonucleotide transformation procedure. BrU-containing oligonucleotide was not mutagenic; although two mutants (per 200 micrograms oligonucleotide) were obtained, they were attributed to base insertion or base substitution at positions different from BrU. This result supports the view that BrU mutagenesis is dependent on intracellular nucleotide pool imbalance. In contrast, HA-containing oligonucleotide was highly mutagenic; 56 mutants (per 140 micrograms oligonucleotide) were obtained. Of 21 induced mutants examined, 20 had G and one had C at the HA position, a result indicating that HA-->G changes took place. To provide back-up evidence, we carried out a general reversion assay for base HA using a set of yeast tester strains, and the results showed that HA induces exclusively AT-to-GC and GC-to-AT transitions. We conclude that in S. cerevisiae HA is a classic base analog mutagen, causing AT-to-GC and GC-to-AT transitions by ambiguous base pairing. The present work has clearly demonstrated the usefulness of the oligonucleotide transformation procedure for elucidating mutagenicity of modified bases.

Adenine↗
PubMed1994

8-Hydroxyguanine is not produced by permanganate oxidation of DNA.

Single-stranded M13mp2 DNA was oxidized with 0.8 mM KMnO4 at pH 8.6 and 4 degrees C for 5 min. These are conditions used previously by Akman et al. (Arch. Biochem. Biophys., 1990, 282, 202) for the oxidation of a denatured DNA and the authors reported that 8-hydroxyguanine was formed in the DNA with this treatment. We decomposed the oxidized DNA with heating in formic acid and the resulting bases were analyzed by HPLC. No significant 8-hydroxyguanine formation was detected in this sample. We also investigated the consumption of KMnO4 by nucleosides. Under conditions in which thymidine consumed KMnO4 rapidly, very little consumption was found with deoxyguanosine (less than 1/100, in rate, compared to that by thymidine). These results show that the permanganate oxidation of DNA does not result in the formation of 8-hydroxyguanine.

DNA Damage↗
PubMed1994

Nucleoside and nucleobase analog mutagens.

Compounds with structures close to those of normal nucleosides or nucleobases may be incorporated into cells and then become constituents of their DNA. Proliferation of such cells could yield mutants. In this article, the current status of studies on such nucleoside and nucleobase analogs is described. Base mispairing mechanisms for these analogs are discussed in light of recent biochemical and biophysical findings.

Animals↗
PubMed1994

5-Fluorouracil reduces proliferating cell nuclear antigen immunoreactive cells in goldfish retina.

A pyrimidine analogue, 5-fluorouracil (5-FU), was injected intravitreally into one eye of the goldfish, either alone, or before or after injection of the same eye with the dopaminergic neurotoxin, 6-hydroxydopamine (6-OHDA). Effects of these agents were explored by measuring their actions on numbers of proliferating cell nuclear antigen-immunoreactive (PCNA-ir) cells, representing mitotically active rod precursor cells (RPCs) in normal conditions, in both treated and untreated retinas. At various intervals (5-45 days after the first injection of drug), the retinas (n = 6 for each treatment group) were isolated and processed as wholemounts by an indirect immunohistochemical method for PCNA. Some retinas were cryosectioned and processed for certain immunoreactive cells other than PCNA. Changes in the mean density of PCNA-ir cells, following intravitreal 5-FU (10 micrograms/2 microliter saline on 3 consecutive days) alone or in combination with intravitreal 6-OHDA (2.5 micrograms/2 microliter), were statistically compared for interval vs. day 0 (control from intact retinas) and for treated vs. contralateral (untreated or treated) retinas, in both outer and inner nuclear layers (ONL and INL, respectively). 5-FU at this dose drastically reduced the densities of endogenous and 6-OHDA-induced PCNA-ir cells in the ONL of treated retinas, but transiently increased them in the contralateral untreated retinas, probably reflecting an injury influence from the treated retina. The density of PCNA-ir cells at the retinal margin was also greatly reduced in treated retinas. Such changes peaked on days 10-15, and gradually disappeared over days 30-45.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Improvement of glucose tolerance by bezafibrate in non-obese patients with hyperlipidemia and impaired glucose tolerance.

Glucose intolerance or diabetes mellitus, hyperlipidemia, obesity and hypertension may have a close interrelation based on insulin resistance. We selected 28 impaired glucose tolerance (IGT) patients with hyperlipidemia. The IGT patients demonstrated hypertriglyceridemia associated with hyperinsulinemia, a typical manifestation of insulin resistance. Administration of bezafibrate at 400 mg/day for 4 weeks to the IGT patients with hypertriglyceridemia resulted in an improvement of the plasma glucose level and insulin response to 75 g oral glucose loading associated with a concomitant decrease in non-esterified fatty acids. The ratio of the level of serum C-peptide to that of insulin after a 75 g oral glucose tolerance test (OGTT) was augmented after 4 weeks of bezafibrate administration. However, reduction of the cholesterol level with pravastatin did not alter these parameters. These results suggest that treatment to reduce the level of serum triglycerides, but not that of cholesterol, may have a beneficial effect for improving insulin resistance even in the non-obese subjects with IGT and decreasing the risk of coronary heart disease.

Bezafibrate↗
PubMed1994

Effects of bromocriptine in Huntington chorea. Case report.

1. The effects of bromocriptine (BC) on choreiform movement were compared with those of bromperidol (BP) and fluphenazine (FLZ) in a patient with Huntington disease. The patient (male, 42 years old) was treated with BP (15 mg/day, 4 weeks), FLZ (3 mg/day, 4 weeks), low dose of BC (5 mg/day, 4 weeks) and relatively high dose of BC (10 mg/day, 8 weeks). The CSF content of homovanilic acid (HVA) was assayed at last day of the each drug trial. The efficacy of the drugs was evaluated by electromyography. 2. Although BP and FLZ did not succeed to ameliorate the choreiform movement, both low dose and high dose BC showed rapid improvement of the involuntary movement. The CSF HVA concentration was 35.0 ng/ml before beginning treatment. Whereas FLZ and high dose of BC substantially increased the levels of HVA after the dosage (49.3 and 53.1 ng/ml, respectively), moderate increase of HVA (41.5 ng/ml) was observed when the low dose of BC was administered. These observations suggest that increase of CSF HVA might be necessary for clinical improvement of choreiform movement but not correlate with the degree of improvement and dopamine agonists could be useful drug for the treatment of choreiform movement which is refractory to the administration of neuroleptics.

Adult↗
PubMed1994

The effect of age on plasma level of setiptiline maleate in depressed patients.

1. Setiptiline maleate (SPT) was administered orally to 45 subjects aged 22-86 years and steady state plasma levels were determined by mass fragment chromatography (GC-MF) to examine the effect of aging on those values. 2. There was a significant correlation between the plasma levels and daily dose. However, there was a wide interindividual variability. 3. Dose-corrected plasma level (DC-PL), or values corrected by dividing the plasma level by daily dose/body weight, was used as the systemic drug clearance parameter. 4. DC-PL was compared among 7 age groups of the subjects distributed in 10-year-intervals. DC-PL showed no difference among groups of subjects between the > 29 years bracket to the 70 years bracket, but showed significantly higher values in those in the > 80 bracket compared to all age groups and subjects in the < 79 bracket. 5. There was a significant correlation between the age of patients and DC-PL according to polynomial response curve analysis. Regression analysis yielded the equation y = -52.72 + 7.05 x -0.17 x2 + 0.01 x3 (n = 45, r = 0.49, p < 0.01).

Adult↗
PubMed1994

Double-staining of horizontal and amacrine cells by intracellular injection with lucifer yellow and biocytin in carp retina.

Horizontal and amacrine cells in the isolated carp retina were impaled with micropipette electrode, identified by their characteristic light responses, and injected iontophoretically with markers for morphological study. Both Lucifer Yellow CH and biocytin were injected simultaneously. Lucifer Yellow was seen by its own fluorescence while biocytin was visualized by binding with Texas Red-linked or horseradish peroxidase-conjugated avidin. For cone-connected horizontal cells, biocytin-coupled cells were found to be approximately five-times more numerous than Lucifer Yellow-coupled cells. Coupling for both tracers was consistently hampered by intravitreally applied dopamine. In untreated retinas, the injected Lucifer Yellow was restricted within one rod-connected horizontal cell, while biocytin revealed several coupled neighbors. Amacrine cells, labeled by the tracers, were morphologically grouped into eight types, based on our earlier classification. Among them, amacrine cells, belonging to three types (Fnd, Pmb or Pma), were confirmed to be Lucifer Yellow-coupled, and the number of biocytin-coupled cells was more numerous (about 2.5 times) than that of Lucifer Yellow-coupled cells. Most amacrine cells (i.e. Pwd, Fnb and Fna) showed biocytin-coupling with no Lucifer Yellow-coupling. A few classified (i.e. Pwb and Fwa) and unclassified cells did not show any coupling. Since the tracer coupling takes place via gap junctions, the majority of amacrine cells, belonging to certain homologous types, appear to be functionally coupled with each other in the inner plexiform layer. However, dopamine did not influence the range of tracer coupling between amacrine cells in the carp retina under the present experimental conditions.

Animals↗
PubMed1994

[Efficacy and safety of electroshock therapy without convulsions under general anesthesia in senile depression].

We examined the efficacy and safety of electroshock therapy (EST) under general anesthesia in 26 depressed patients (9 males, 17 females, age range 55-79). The scores of the Hamilton Psychiatric Rating Scale for depression were significantly decreased after EST and clinical symptoms such as depressive mood. psychomotor retardation, anxiety, agitation, suicidal tendencies, hypochondria and sleeplessness improved in all of the patients. Complications included amnesia (16/26), delirium (3/26) and transient arrhythmia (1/26) after EST, but neither lethal nor lasting complications were observed. In summary, EST was an effective and convenient method of treatment for senile depression.

Aged↗
PubMed1993

Fibroblast growth factor induces proliferating cell nuclear antigen-immunoreactive cells in goldfish retina.

New rod photoreceptors are added to mature teleost retinas throughout life by regulated proliferation of rod precursor cells (RPCs). In this study, candidate regulators of RPC proliferation, acidic and basic fibroblast growth factors (aFGF and bFGF; 0.1 microgram/eye), interleukin-6 (IL-6; 0.1 microgram) and phytohaemagglutinin (HA15; 1.0 microgram), were injected intravitreally into one eye of goldfish (body length 5-6 cm), and mitotic RPCs in both retinas were detected and counted 3-50 days later by immunohistochemistry for proliferating cell nuclear antigen (PCNA). Retinal integrity after treatment was assessed by immunohistochemistry for tyrosine hydroxylase (TH) and other retinal antigens. All the agents applied altered the density of PCNA-immunoreactive (ir) cells in the outer and inner nuclear layers (ONL and INL) in both retinas as soon as 2-3 days after unilateral injection. Initially (2-20 days after injection), particularly in the treated retina, PCNA-ir cells appeared in clusters accompanied by various numbers of scattered individual cells, but subsequently the clusters of PCNA-ir cells disappeared while the density of singly distributed cells increased until 30 days after injection. At the doses given, these effects were most striking with aFGF and bFGF and less with IL-6 and HA15. In radial cryosections, other cellular elements immunoreactive to markers such as TH, serotonin, neuropeptide Y, substance P, glutamine synthetase, glial fibrillary acidic protein and protein kinase C, were found normal in terms of morphology. In addition, a monoclonal antibody (NN-2) was found to label some non-neuronal structures (macrophages, microglia and blood vessels) inside and outside the retina intoxicated with 6-hydroxydopamine, a few NN-2-ir cells being PCNA-positive. However, clustered PCNA-ir and marginal neuroblast cells were NN-2-negative. These results indicate that FGFs may play an important role in stimulating the proliferation of RPCs, for example, in the regeneration of fish retinas following neurotoxic destruction.

Animals↗
PubMed1993

[Coronary flow dynamics in the right coronary artery providing collateral circulation: comparison before and after successful angioplasty with a Doppler catheter].

Coronary blood flow dynamics were investigated in 9 patients with isolated coronary artery disease of the left anterior descending artery (LAD) with good collateral flow (grade 2 or 3 of Rentrop's classification) from the right coronary artery (RCA) and 20 patients with normal coronary arteries as controls. The coronary flow velocity (Vs: systolic peak, Vd: diastolic peak, Vm: mean) was measured with a Doppler catheter and the diameter (D) by the edge detection method in the proximal portion of the RCA. Vs/Vd was calculated. The areas under the velocity curve during systole (integral of s) and diastole (integral of d) were measured to obtain the ratio (integral of s/integral of d). Peak to resting velocity ratio (PRVR) was obtained as an index of coronary flow reserve, using intracoronary injections of 6 ml contrast medium or 8-12 mg papaverine. These parameters before percutaneous transluminal coronary angioplasty (PTCA), after PTCA, and in control subjects were compared. The collaterals disappeared angiographically immediately after successful PTCA. D (before PTCA: 3.2 +/- 0.1 mm, after PTCA: 3.3 +/- 0.1 mm, controls: 3.2 +/- 0.1 mm) and Vs did not vary between before and after PTCA and in control subjects (NS). The values of Vd, Vm decreased and Vs/Vd and integral of s/integral of d increased after PTCA to the values in the controls. PRVR obtained with contrast medium and papaverine also increased.

Aged↗
PubMed1992

Blockage of polymerase-catalyzed DNA chain elongation by chemically modified cytosine residues in templates and the release of blockage for readthrough.

The Klenow fragment-mediated in vitro DNA elongation was inhibited by the presence of a class of modified cytosines in the template DNA, i.e., the N4-amino(and -methoxy)-5,6-dihydrocytosine-6-sulfonate residues. We have studied the mechanism of the blockage, using as templates bisulfite-hydrazine (and -methoxyamine)- modified single strand phage-M13mp2 DNA and synthetic oligonucleotides. Both N4-amino-5,6-dihydrocytosine-6-sulfonate and N4-methoxy-5,6-dihydrocytosine-6-sulfonate residues blocked the elongation at one nucleotide before these sites. In this blockage, the idling of polymerase at the lesion site due to its 3'-5' exonuclease action appears not to play a major role, because Sequenase that lacks the 3'-5' exonuclease activity still could not readthrough these sites. It seems possible that conformational distortion of the template near these sites is responsible for the blockage, because on conversion of this 5,6-dihydropyrimidine-6-sulfonate structure into a planar pyrimidine, a complete restoration of polymerase-readthrough resulted. In the presence of RecA and SSB proteins, the Klenow fragment was able to partially readthrough these sites. Since there was no decrease in the 3'-5' exonuclease activity during this readthrough, it seems that the binding of these proteins relaxes the distortion in the modified template to allow the polymerase to readthrough the lesion site. These sites on phage DNA can be lethal but also are capable of inducing C-to-T transitions. This observation suggests that these sites can be read by E. coli DNA polymerases in vivo with accompanying errors.

Bacteriophages↗
PubMed1992

An immunohistochemical study of regenerating newt retinas.

Light-microscopical examination was carried out to investigate the emergence and development of several classes of immunoreactive cells in regenerating retinas of the adult newt (Triturus pyrrhogaster) after total retinal ablation. Immunoreactive proliferating cell nuclear antigen (ir-PCNA, a marker for replicating cells) was present in nuclei of all neuroblasts in the early mono-layered to several-layered stages (15-20 days after retinal ablation; days 15-20), but was lost progressively in an intermediate-to-central/peripheral order as cells and layers increased (days 20-25). Cells, which had lost ir-PCNA, began to separate to form the outer nuclear, inner nuclear and ganglion cell layers around days 25-30 (the cell separation stage). Finally, the location of ir-PCNA was restricted to a band of neuroblast cells at the retinal margin (days 30-35) as seen in intact adult retinas. Visinin-immunoreactive (ir) cells, mainly destined to be cones, appeared first singly or as clusters at the most distal layer in the intermediate region of retinas multi-layered with PCNA-ir neuroblasts, which was followed by appearance of opsin-ir rod outer segments and tyrosine hydroxylase-ir amacrine cells around the cell separation stage. Shortly later, cells respectively immunoreactive to glutamic acid decarboxylase, neuropeptide Y, serotonin, glucagon, glutamine synthetase, glial fibrillary acidic protein, substance P and protein kinase C were found to emerge also in an intermediate-to-central/peripheral sequence. Some of the glucagon-ir cells appeared to be of an interplexiform type.

Animals↗
PubMed1992

Dendritic morphology of interstitial amacrine cells with monostratified dendrites in different-sized carp retinas.

The dendritic morphology of a class of interstitial (IS) amacrine cells in retinas of different-sized carp (body length, 9.1-32.3 cm) was investigated by identifying their fluorescent nuclei pre-loaded with 4,6-diamidino-2-phenylindole (DAPI), followed by iontophoretic injection of Lucifer yellow (LY) in isolated and formaldehyde-fixed flat-mounts under microscopic control. The LY-injected fusiform or pyriform cell bodies were found to locate at the middle of the inner plexiform layer (IPL) or immediately beneath the amacrine cell layer, and their dendrites monostratified in sublamina b of the IPL. The pyriform cells had a short stem from which extended 4-5 stout dendrites, while the fusiform cells extended similar dendrites from the soma. The dendrites of both types of cell were decorated with spines and a few long axon-like processes. The pyriform cells were found more frequently in smaller retinas than in larger retinas, suggesting that the former may migrate proximally during retinal growth. The dendritic field sizes of these IS amacrine cells were wider as the fish became larger, while the dendritic morphology, analyzed by the Sholl's branching model, was very similar in smaller and larger retinas. The results indicate that the IS amacrine cells do not add dendrites, but that their dendritic trees simply expand during retinal growth.

Aging↗
PubMed1992

Analysis of phage M13mp2 mutants produced from transfection of phage DNA having N4-aminocytosines at defined sequence positions.

N4-Aminocytidine is mutagenic in various organisms. In the cell, this cytidine analog is metabolized into N4-aminodeoxycytidine 5'-triphosphate, which will then be incorporated into DNA and mutation will result during the replication of the DNA. To prove that the N4-aminocytosine residue in DNA is indeed the site of mutagenesis, we prepared a series of phage M13mp2 DNA samples that bear N4-aminocytosine residues at a few defined positions in the lacZ alpha region, by carrying out in vitro limited extension of primed phage DNA. We then transfected the DNAs to Escherichia coli and examined the progeny phages for the forward mutations. The M13mp2 DNAs bearing N4-aminocytosines produced mutant phages at high frequencies. Furthermore, DNA sequencing of the resulting mutants demonstrated that both AT-to-GC and GC-to-AT mutations took place at those positions where N4-aminocytosine residues were originally present.

Bacteriophages↗