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Biomedical subjects

K Negishi

Publications and source records attributed to K Negishi.

At least 19 recordsLinked to original sources
PubMed1997

The mechanism of mutation induction by a hydrogen bond ambivalent, bicyclic N4-oxy-2'-deoxycytidine in Escherichia coli.

The triphosphate of the nucleoside deoxyribosyl dihydropyrimido[4,5-c][1,2]oxazin-7-one (dP) is known to be incorporated into DNA efficiently by Taq polymerase and is a useful tool for polymerase-mediated in vitro mutagenesis. It is shown here that dP is a potent mutagen in Escherichia coli and Salmonella typhimurium . In E.coli , this deoxycytidine analog induces both GC-->AT and AT-->GC transitions. No induced transversions are observed. It is highly mutagenic in wild-type E.coli, but this is much reduced in a strain lacking thymidine kinase. Mutagenesis induced by dP is efficiently inhibited by the addition of thymidine. Partially purified thymidine kinase from E.coli catalyzes phosphorylation of dP to its 5'-monophosphate. When E.coli was grown in the presence of dP, the nucleoside analog was incorporated into its DNA. The content of dP in DNA was dependent on the concentration of dP added to the medium. The incorporation characteristics of the 5'-triphosphate of dP (dPTP) were also studied using E.coli DNA polymerase I large fragment. The results confirm that this triphosphate can be incorporated opposite A and G in the template with similar efficiencies. This indicates that dP is metabolized as a thymidine analog and that the resulting triphosphate induces a high rate of mutagenesis through replicational errors.

Base Sequence↗
PubMed1997

Origins of horizontal cell spectral responses in the retina of marine teleosts (Centropomus and Mugil sp).

Intracellular recording and marking of horizontal cells (HCs) were carried out in isolated retinas of marine teleosts (Centropomus and Mugil sp.) to identify the cellular origins of different spectral types of S-potential. The spectral responses recorded under mesopic conditions were classified into four types: photopic L, biphasic C(R/G), biphasic C(G/R), and scotopic L(sL). Intracellular marking with Lucifer yellow (LY) or Procion yellow (PY) revealed that in the Centropomus retina an L-type response was recorded from H1 and H2 cells, C(R/G)-type from H3 cells, and sL-type from H4 cells. However, in 20% of microelectrode penetrations from the photoreceptor surface, the sequential order of response appearances was found to be L-, sL-, and C(R/G)-types. In this species, thick dendritic processes of H3 and H4 cells are distributed at the same level as the cell body. In the Mugil retina, on the other hand, L-type response was recorded from H1 cells, C(G/R)-type from H2 cells, C(R/G)-type from H3 cells, and sL-type from H4 cells, this sequential arrangement being very regular. In both species, the cone-connected H1 cells are small and possess an axon, while the two other cone HCs as well as rod-connected H4 cells are axonless. Earlier (Negishi et al., 1988) and present findings indicate that there is a wide variety of HC morphology and functional organization in the teleost retina.

Animals↗
PubMed1996

Mutational analysis of the CitA citrate transporter from Salmonella typhimurium: altered substrate specificity.

The CitA citrate transporter in Salmonella typhimurium is encoded by the citA gene and consists of 434 amino acid residues that probably include 12 membrane-spanning segments [Shimamoto. T., et al. (1991) J. Biochem. 110, 22-28]. CitA mutants with altered substrate specificities were isolated by in vitro mutagenesis using nitrous acid. The mutants could grow on isocitrate as a sole carbon source which normally cannot be transported well by the CitA transporter of S. typhimurium. The mutation sites in the citA gene of the nine mutants were determined to involve single residues at seven sites (one mutation per mutant). The original amino acid residues at these sites (Arg-19, Ala-38, Glu-51, Gly-132, Ala-169, Pro-262 and Leu-271) were identified to be responsible for the altered substrate specificity. All these amino acid residues were conserved in four other homologous citrate transporters from Escherichia coli, Citrobacter amalonaticus and Klebsiella pneumoniae and are suggested to be involved in substrate recognition by the CitA transporter.

Amino Acid Sequence↗
PubMed1996

Effects of glutamic acid and related agents on horizontal cells in a marine teleost retina.

Excitatory amino acids (EAAs) such as glutamic and aspartic acids, considered as the most likely neurotransmitters at the photoreceptor-horizontal cell synapse of teleost retinas, as well as agonists such as kainic acid and several of their antagonists, were applied to isolated and superfused retinas of the teleost Eugerres plumieri. Intracellular recordings from horizontal cells reveal that EAA receptors are of the kainate-quisqualate type. There is competitive inhibition between the agonist and antagonist agents used, and under their combined effect, the synapse under study remains operational, in a functional state, able to modulate the horizontal cell membrane potential upon retinal illumination.

Animals↗
PubMed1996

HAM1, the gene controlling 6-N-hydroxylaminopurine sensitivity and mutagenesis in the yeast Saccharomyces cerevisiae.

The ham1 mutant of yeast Saccharomyces cerevisiae is sensitive to the mutagenic and lethal effects of the base analog, 6-N-hydroxylaminopurine (HAP). We have isolated a clone from a centromere-plasmid-based genomic library complementing HAP sensitivity of the ham1 strain. After subcloning, a 3.4 kb functional fragment was sequenced. It contained three open reading frames (ORFs) corresponding to proteins 353, 197 and 184 amino acids long. LEU2+ disruptions of the promoter and N-terminal part of the gene coding 197 amino acids long protein led to moderate and strong sensitivity to HAP, respectively, and were allelic to the original ham1-1 mutation. Thus this ORF represents the HAM1 gene. The deduced amino acid sequence of HAM1 protein was not similar to any protein sequence of the SwissProt database. The HAM1 gene was localized on the right arm of chromosome X between cdc8 and cdc11. Spontaneous mutagenesis was not affected by the ham1::LEU2 disruption mutation.

Adenine↗
PubMed1996

Elevated erythrocyte sodium-lithium counter-transport in hypertensive patients with non-insulin-dependent diabetes mellitus.

Increased erythrocyte (RBC) sodium-lithium (Na-Li) counter transport (CT) has been reported to be a genetic marker for essential hypertension (EHT). In addition, increased RBC Na-Li CT has been demonstrated in insulin-dependent diabetic (IDDM) patients with nephropathy, indicating that a predisposition to hypertension may cause renal damage and impaired renal function. Therefore, the present study was designed to determine RBC Na-Li CT in subjects with essential hypertension (EHT) and non-insulin-dependent diabetics (NIDDM) with or without hypertension (NIDDMHT or NIDDMNT), using the method of Canessa et al. with a slight modification by flame photometry and expressed as nmol Li/5 x 10(6) RBC/h. Na-Li CT in patients with EHT (0.159 +/- 0.051 (S.D.), n = 26) or NIDDMHT (0.168 +/0 0.083, n = 42) was higher than that in NIDDMNT patients (0.127 +/- 0.059, n = 27, P < 0.05). Among the NIDDMHT patients, those with clinical nephropathy had the same levels of Na-Li CT as those without nephropathy. When the NIDDM patients were divided into two groups with or without insulin treatment, the Na-Li CT in hypertensives was higher than that in normotensives, irrespective of whether or not they were on insulin therapy. Addition of insulin to RBCs in vitro did not augment the Na-Li CT activity. These results suggest that an increase of Na-Li CT may not be due to the stimulatory effect of endogenous or exogenous insulin, and reflect a genetic predisposition for hypertension, and hence diabetic nephropathy, not only in IDDM but also NIDDM patients.

Antiporters↗
PubMed1996

[Density distribution of dopaminergic neurons in the retina of a marine teleost, Thamnoconus (Navodon) modestus].

By means of tyrosine hydroxylase immnocytochemical detection, regional densities of dopaminergic (DAergic) neurons were measured in 6 whole-mounted retinas of a marine teleost, Thamnoconus (Navodon) modestus. Isodensity lines of DAergic cells in the right retina were drawn to make a contour map. Depending upon the range of cell densities, the retinal field was divided into 3 regions: zone A, containing more than 300 per mm square; zone B, from 200 to 299; and zone C, below 200. Zone A was located in the small temporal (A1) and nasal (A2) areas as well as in the peripheral margin (A3). Zone B formed a horizontal streak-like band between regions A1 and A2. Low density areas (C) were located dorsally or ventrally to zone B. Where the densities were higher, soma sizes appeared to be smaller. DAergic neurons in the peripheral zone A3 were bipolar and the dendrites ran parallel to the retinal margin, while DAergic neurons in the other zones were multipolar and the dendrites ran in various directions. The presence of the small temporal (A1) and nasal areas (A2) with high densities of DAergic neurons, as well as the streak-like area (B) with a relatively high density, may correspond to similar areas in the isodensity contour map of ganglion cells.

Animals↗
PubMed1996

Metformin decreases blood pressure and obesity in OLETF rats via improvement of insulin resistance.

To determine whether improvement of insulin resistance decreases blood pressure as well as obesity, metformin (100 mg/kg/d) or vehicle was administered for 20 weeks to 12-week-old male Otsuka Long-Evans Tokushima Fatty (OLETF) rats (n = 10 each), a newly developed animal model of non-insulin-dependent diabetes mellitus (NIDDM) with mild obesity, hyperinsulinemia, and hypertriglyceridemia. Oral administration of metformin ameliorated glucose intolerance and attenuated the insulin response to glucose loading (2 g/kg, i.p.), as evidenced by a decrease in the area under the curve for glucose and insulin at 24 weeks by 19% and 37%, respectively. At 21 weeks, systolic blood pressure was significantly lower in the metformin group than in controls (130 +/- 1.9 vs. 143 +/- 2.7 mmHg, p < 0.01), despite no difference in body weight. Subsequently, blood pressure tended to be slightly but insignificantly lower in the metformin group, and body weight was significantly lower in the metformin group (532 +/- 9.8 vs. 587 +/- 10.3 g at 31 weeks, p < 0.01). Metformin treatment also lowered the level of serum triglycerides (9.4 +/- 0.6 vs. 13.2 +/- 0.5 mmol/l, p < 0.01) and the plasma norepinephrine concentration (4,222 +/- 373 vs. 7,548 +/- 1,058 pg/ml, p < 0.01). These results suggest that metformin-induced improvement of insulin resistance in obese rats with NIDDM may lower blood pressure, as well as decrease sympathetic activity and reduce body weight.

Aging↗
PubMed1995

Differentiation of photoreceptors, glia, and neurons in the retina of the cichlid fish Aequidens pulcher; an immunocytochemical study.

Light-microscopic immunocytochemistry was carried out to investigate the developmental dynamics of several neurochemical markers in the retina of blue acara (Aequidens pulcher). As a rule, double-label experiments were performed in order to determine the absolute and relative timing of the appearance of these markers. The diameter of eye-ball (from 0.6 to 1.2 mm) and the body length (from 4.6 to 9.4 mm) enlarged in parallel during the observation period of 2 to 9 days after spawning (day 2-9); hatching took place usually on day 2. Immunoreactive proliferating cell nuclear antigen (ir-PCNA) was present in all neuroblasts (the embryonic homogeneous cell stage; day 1.0-2.0), but was lost progressively in a center-to-periphery and apparent proximal-to-distal sequence as the cells and layers differentiated. In late larvae and juveniles, ir-PCNA was confined to a ring of dividing neuroblasts at the retinal margin and to a population of scattered rod precursors in the outer nuclear layer. Immunoreactive structures of representative antigens progressively appeared after ir-PCNA had decayed. Around hatching, at the synaptic separation stage (day 2.0-2.5), luteinizing hormone-releasing hormone-ir centrifugal fibers, visinin-ir cones, glial fibrillary acidic protein-ir structures and gamma-aminobutyric acid-ir cell bodies appeared, which were followed by the emergence of rhodopsin-ir rods and tyrosine hydroxylase-ir interplexiform cells (on day 2.5-3.0) and serotonin-, neuropeptide Y- and substance P-ir amacrine cells (on day 3.0-4.0). The results indicate that photoreceptor cells, and especially rods start to differentiate at an earlier stage of retinogenesis than has previously been proposed. In addition, an extraretinal tissue in the brain identified as the prospective pineal organ was found to be visinin- and rhodopsin-immunoreactive on day 1.5-2.0 before these photoreceptor-specific antigens became positive in the retina.

Animals↗
PubMed1995

Effect of troglitazone (CS-045) and bezafibrate on glucose tolerance, liver glycogen synthase activity, and beta-oxidation in fructose-fed rats.

To clarify the relationship between lipid and glucose metabolism abnormalities in fructose-fed rats, we examined whether an improvement of insulin sensitivity by troglitazone (CS-045) or a decrease in plasma lipids by bezafibrate affects the relationship between serum levels of lipid and glucose. In addition, we also examined changes in liver glycogen metabolism and beta-oxidation in fructose-fed rats. Troglitazone ameliorated fasting hyperlipidemia, hyperglycemia, and hyperinsulinemia. In addition, it augmented glycogen synthase activity by 53%, and decreased the mitochondrial palmitic acid beta-oxidation rate and ketone body production rate by 27% and 55%, respectively. However, hyperglycemia and liver glycogen synthase activity were not improved by bezafibrate treatment despite a marked reduction of serum triglyceride (TG) levels resulting from a 1.76-fold increase in mitochondrial oxidation and a 2.04-fold increase in hepatic ketone body production. These results suggest that abnormalities in glucose and lipid metabolism in fructose-fed rats, which are ameliorated by troglitazone, may be closely linked to reduced glycogen synthase activity in the liver.

Animals↗
PubMed1995

Spectra of superoxide-induced mutations in the lacI gene of a wild-type and a mutM strain of Escherichia coli K-12.

We have analyzed the spectra of superoxide-induced mutations in the chromosomal lacI gene of a wild-type and a mutM strain of Escherichia coli K-12. The mutM strain is known to be deficient in removing 8-hydroxyguanine from DNA. An intracellular superoxide-generating agent, menadione, was used to cause the mutation. Analysis of the mutated DNA showed marked differences between the mutants from the wild type and those from the mutM strain. In the mutants from the wild type, all possible base-pair substitutions were present and their proportions were similar to each other, whereas in those from the mutM bacteria there was a 90% bias in favor of transversion. Furthermore, in the mutM strain GC-to-CG transversion rather than GC-to-TA was predominantly induced. 64% of the GC-to-CG transversions in the mutM strain occurred at the site of (CT/GC)GGC (mutated base underlined). The favorable mutation site, CTGGC, was the same as that of the UV- and sunlight-induced mutations previously reported: the mutations observed there were also G-to-C transversions. We speculate from these results that the superoxide in the cells may lead to production of a modified guanine that can pair with guanine and is subject to removal by the MutM protein.

Base Sequence↗
PubMed1995

Patterns of rod proliferation in deep-sea fish retinae.

In a sample of 37 species of deep-sea fish species from the sea floor of the Porcupine Seabight and the Gobal spur (North Atlantic) we investigated the overall structure of the retina with special respect for the organization of rods, their length and their arrangement in multiple banks. Using an immunocytochemical marker for cell proliferation (PCNA) we studied the mechanisms of rod proliferation, and, by means of serial section reconstruction followed their integration into the existing population of rods. Furthermore, in three different species we have observed growth related changes in retinal thickness, rod density and proliferation activity. We found evidence for two different principles for the organization of rods in these deep-sea fish retinae. In the first group of species represented by Nematonurus armatus and Coryphaenoides guentheri we found rods to be rather short (20-30 microns) and arranged in three and more banks. In these species rod proliferation occurred in a single band of cells immediately vitread of the external limiting membrane, thus showing a high degree of spatial and temporal order. In these species, young rods are inserted just sclerad of the external limiting membrane and the older outer segments pushed away from the incoming light towards the back of the eye. This may be linked to a progressive loss of function of the older rods and might represent an alternative mechanism to the disk shedding in other vertebrates. In the second population (e.g. Conocara macroptera, Alepocephalus agassizii) we observed considerably longer rod outer segments (60-80 microns) forming no more than two layers. These retinae had rod precursors arranged in disseminated clusters throughout the outer nuclear layer indicating the lack of clear spatio-temporal order in mitotic activity along with a more statistical pattern of integration of the newly formed outer segments. In our sample of species both populations were of about equal size suggesting that the two mechanisms are equally effective and may have arisen independently.

Animals↗
PubMed1995

Induction of DNA recombination by activated 3-amino-1-methyl-5H-pyrido[4,3-b]indole.

To investigate the genotoxic properties of a food-derived carcinogen, 3-amino-1-methyl-5H-pyrido-[4,3-b]indole (Trp-P-2), we have tested whether Trp-P-2 and its metabolically transformed products can induce DNA recombinations. Trp-P-2 is a strong mutagen and its activated form, the N-hydroxylated derivative, Trp-P-2(NHOH), is known to form DNA adducts and cause DNA chain cleavage. Using a system in which phage lambda undergoes recombination inside host Escherichia coli, we have found that Trp-P-2(NHOH), but not Trp-P-2 itself, can induce recombination. A nitroso derivative of Trp-P-2, Trp-P-2(NO), which can be reduced intracellularly to form Trp-P-2(NHOH), also induced recombination. Active oxygens are implicated in this recombinogenic action, since Trp-P-2(NHOH) is known to undergo spontaneous oxidative degradation, generating active oxygen radicals which can cause DNA chain cleavages. 4-Hydroxyaminoquinoline N-oxide and phenyl-hydroxylamine also showed recombinogenic actions in this assay system; hence, it is suspected that aromatic amine-type carcinogens have this property in common.

Bacteriophage lambda↗
PubMed1995

Growth factors influence contractility and alpha-smooth muscle actin expression in bovine lens epithelial cells.

PURPOSE: To determine whether basic fibroblast growth factor (bFGF) and transforming growth factor-beta 2 (TGF-beta 2) influence the contractile activity and the expression of alpha-smooth muscle actin (alpha-SMA) in bovine lens epithelial cells (LECs). To examine whether modulation of contractile activity by these growth factors depends on changes of alpha-SMA expression. METHODS: Bovine LECs were cultured in collagen gel in MED 5 medium (F-12 nutrient mixture supplemented with 5% fetal bovine serum) with or without bFGF (1 to 100 ng/ml) or TGF-beta 2 (0.01 to 10 ng/ml). To evaluate collagen gel contraction, the longest and shortest diameters of the gels were measured daily for 7 days, and the area was determined. Detection of alpha-SMA in the gels was performed immunohistochemically using a mouse monoclonal antibody against alpha-SMA. The percentage of alpha-SMA-positive cells to the total number of cells was determined. RESULTS: Control gels cultured with MED 5 medium alone contracted to 15.8% +/- 3.4% of their original area after 7 days. TGF-beta 2 significantly increased this contraction in a dose-dependent manner, whereas bFGF significantly decreased it. Approximately 30% of cells in the control gels were alpha-SMA positive. TGF-beta 2 significantly increased the alpha-SMA positivity dose dependently, whereas bFGF significantly decreased it. The percent positivity for alpha-SMA and the gel area showed a significant negative correlation. CONCLUSIONS: TGF-beta 2 increased collagen gel contraction and alpha-SMA expression in bovine LECs, whereas bFGF decreased these parameters. Because collagen gel contraction was correlated with alpha-SMA expression, the modulation of LEC contractile activity by growth factors may be related to an effect on alpha-SMA.

Actins↗
PubMed1995

[Intermittent mini-dose vancomycin intravenous administration and closed continuous irrigation technique applied to mediastinitis caused by MRSA following mitral valve replacement].

Mediastinitis caused by MRSA (Methicillin-Resistant Staphylococcus aureus) remains an intractable infection producing high mortality even in these days of advanced chemotherapy. The authors report a case of mediastinitis due to MRSA complicated with acute renal failure following mitral valve replacement. The patient's mediastinum had been thoroughly cleaned with physiological saline solution with 0.2% povidone iodine, and underwent a chemotherapy regimen of mini-dose vancomycin. The patient made favorable progress and recovered completely. Our patient's progress confirmed that when chemotherapy using vancomycin is administered in a patient whose condition is complicated with acute renal failure, closely monitoring the vancomycin serum concentration is essential. Intermittent mini-dose intravenous administration is sufficient to maintain an effective vancomycin serum concentration. In our case, vancomycin serum concentration measured before and at completion of dialysis revealed no appreciable decline.

Acute Kidney Injury↗
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↗