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Biomedical subjects

C Ishii

Publications and source records attributed to C Ishii.

At least 55 records · Page 3Linked to original sources
PubMed1995

Identification and expression of the Neurospora crassa mei-3 gene which encodes a protein homologous to Rad51 of Saccharomyces cerevisiae.

The mei-3 gene of Neurospora crassa encodes a homolog of the Escherichia coli RecA and Saccharomyces cerevisiae Rad51 proteins, which are required for recombination and repair of DNA double-strand breaks. To determine the molecular function of MEI3 protein, anti-MEI3 antibody was prepared and used in Western blot analysis. The antibody cross-reacted only with crude extracts prepared from perithecia, the fruiting bodies of Neurospora. The molecular weight of the MEI3 protein was estimated to be 38 kDa. Transformation experiments showed that a DNA fragment longer than previously reported was needed to complement the mei-3 mutation. On sequencing cDNA and genomic DNA, one open reading frame (ORF) was found, which consists of three exons interrupted by two small introns. This ORF encoded a MEI3 protein of 353 amino acids, and the inferred MW of 38 kDa is in good agreement with the results from Western blot analysis. Comparisons of MEI3 with other Rad51 homologs indicated that MEI3 protein contains the two conserved core domains (I and II) generally observed in Rad51 homologs in eukaryotes. Northern blot analysis showed that expression of mei-3 was raised remarkably after UV-irradiation or methyl methanesulfonate (MMS)-treatment. The transcript size was 1.6 kb and this was also larger than was reported previously.

Amino Acid Sequence↗
PubMed1995

A eukaryotic gene encoding an endonuclease that specifically repairs DNA damaged by ultraviolet light.

Many eukaryotic organisms, including humans, remove ultraviolet (UV) damage from their genomes by the nucleotide excision repair pathway, which requires more than 10 separate protein factors. However, no nucleotide excision repair pathway has been found in the filamentous fungus Neurospora crassa. We have isolated a new eukaryotic DNA repair gene from N.crassa by its ability to complement UV-sensitive Escherichia coli cells. The gene is altered in a N.crassa mus-18 mutant and responsible for the exclusive sensitivity to UV of the mutant. Introduction of the wild-type mus-18 gene complements not only the mus-18 DNA repair defect of N.crassa, but also confers UV-resistance on various DNA repair-deficient mutants of Saccharomyces cerevisiae and a human xeroderma pigmentosum cell line. The cDNA encodes a protein of 74 kDa with no sequence similarity to other known repair enzymes. Recombinant mus-18 protein was purified from E.coli and found to be an endonuclease for UV-irradiated DNA. Both cyclobutane pyrimidine dimers and (6-4)photoproducts are cleaved at the sites immediately 5' to the damaged dipyrimidines in a magnesium-dependent, ATP-independent reaction. This mechanism, requiring a single polypeptide designated UV-induced dimer endonuclease for incision, is a substitute for the role of nucleotide excision repair of UV damage in N.crassa.

Amino Acid Sequence↗
PubMed1995

Incidence of various gyrA mutants in 451 Staphylococcus aureus strains isolated in Japan and their susceptibilities to 10 fluoroquinolones.

Point mutations in the gyrA genes of 451 clinical strains of Staphylococcus aureus isolated in Japan were detected by a combination of nonradioisotopic single-strand conformation polymorphism analysis and restriction fragment length polymorphism analysis and by direct sequencing. Six types of gyrA mutations were observed in 149 of the 451 strains (33%), and ofloxacin MICs were greater than 6.25 micrograms/ml for 147 of the 149 strains (98.7%). These mutations were localized between codons 84 and 88, and they were associated with fluoroquinolone resistance. Two types of silent mutations were also found. Among these eight types of mutations, three types are novel, i.e., the serine at position 84 (Ser-84)-->Val (TCA-->GTA), Ser-84-->Leu (TCA-->TTA) plus Ile-86 (ATT-->ATC, silent), and Phe-110 (TTT-->TTC, silent). Among GyrA mutants, strains with a Ser-84-->Leu alteration and strains with a Glu-88-->Lys alteration were dominant. In contrast, few strains had Ser-84-->Ala and Glu-88-->Gly alterations. All fluoroquinolones tested showed greater than a fourfold decrease in their activities in terms of their MICs that inhibited 50% of strains tested for each GyrA mutant, in comparison with their MICs that inhibited 50% of strains tested for susceptible strains. Most of the currently available fluoroquinolones, such as norfloxacin, enoxacin, ofloxacin, ciprofloxacin, tosufloxacin, lomefloxacin, sparfloxacin, and fleroxacin, were ineffective against each mutant. Mutants containing a Ser-84-->Leu or Val alteration showed high-level resistance to fluoroquinolones, and one containing a Ser-84-->Ala alteration showed relatively low-level resistance. Double mutations were associated with a higher level of resistance than single mutations.

Anti-Infective Agents↗
PubMed1995

Increased expression of a regenerating (reg) gene protein in neonatal rat pancreas treated with streptozotocin.

We examined the expression of reg protein in neonatal rat pancreas treated with streptozotocin (STZ) by means of the immunohistochemical technique and northern blotting. Seven days after STZ injection, the plasma glucose levels in STZ-treated neonatal rats were significantly higher than those in control rats. Scattered distribution of reg protein in pancreatic islet cells was clearly observed in STZ-treated rats, but not in control rats. On the other hand, reg proteins was positively stained in the exocrine cells in both groups of rats. Northern blot analyses revealed that the expression of insulin mRNA markedly decreased in STZ-treated rat pancreas, but a significant increase in reg mRNA expression was recognized in the STZ-treated rat pancreas compared with that of control rats. Rats treated with STZ during the neonatal period have been used as a model of non-insulin-dependent diabetes mellitus (NIDDM) and beta cell regeneration. Thus, the increased reg gene expression in neonatal STZ-treated rat pancreas was therefore described for the first time, and thus would be a useful model for studying the relationship between NIDDM and beta cell regeneration or reg gene protein.

Animals↗
PubMed1994

The role of insulin in coronary atherosclerosis.

In 197 patients with coronary artery disease (CAD) who underwent coronary angiography, 75 g oral glucose tolerance test (OGTT) was performed, measuring plasma glucose(PG) and plasma insulin (IRI) at 4 time points (0, 30, 60 and 120 min). Subjects were separated into two groups by their insulinogenic index (I.I. = delta IRI/delta PG from 0 up to 30 min), 99 cases with good insulin response (I.I. > or = 0.4) and 98 cases with poor insulin response (I.I. < 0.4). Only two subjects were diabetic in good insulin response, while 37 were diabetic in poor insulin response. The severity of coronary atherosclerosis was expressed as a coronary index (CI), calculated according to Balcon's method. Fasting PG and the sum of PG were significantly higher in the latter group, while the sum of IRI was significantly lower. CI was not significantly different, however. In the group with good insulin response, the severity of CAD was significantly correlated to fasting IRI (n = 99, r = -0.387, P < 0.02), but, there was no such relationship in the other group. We conclude that hyperinsulinemia might be a risk factor for ischemic heart disease, but in diabetics it is difficult to link the relationship between fasting IRI and CI.

Adult↗
PubMed1994

Mutagenesis and epistatic grouping of the Neurospora meiotic mutants, mei-2 and mei-3, which are sensitive to mutagens.

To understand the possible roles of the Neurospora meiotic genes mei-2 and mei-3 in DNA repair, the frequencies of spontaneous and UV-induced mutation at the ad-3 loci were investigated and double mutants between mei mutants and other DNA-repair mutants were analyzed for mutagen sensitivity. Spontaneous mutation frequency in mei-2 was similar to that of the wild type, while the frequencies were high in both of two mei-3 strains which contained different mei-3 alleles. In addition, the frequency of spontaneous mutation in mei-3 varied greatly from experiment to experiment, which clearly showed a mutator phenotype for mei-3 mutation. UV irradiation increased mutation frequencies in both mei-2 and mei-3. The mutagen sensitivity of the double mutant, mei-2 mei-3, was no greater than that of the single mutants when treated with UV and methyl methanesulfonate (MMS). Thus, both mei genes appear to be involved in the same repair group. When analyzed in combination with other mutations, both mei mutations showed an epistatic relationship to uvs-6 and a synergistic relationship to mus-18 and uvs-2. However, uvs-3 was epistatic to mei-2, but the relationship of uvs-3 to mei-3 could not be tested directly, because the double mutant was barely viable. These results indicate that mei-2 and mei-3 are involved in the same repair group as uvs-6, and uvs-3 is possibly involved in the same group. Alternatively, the mei genes, or uvs-3 itself, may be related to more than one DNA repair group.

Crosses, Genetic↗
PubMed1994

Uptake of remnant like particles (RLP) in diabetic patients from mouse peritoneal macrophages.

To investigate whether the remnant like particles (RLP), separated from serum by an immunoaffinity gel mixture of anti-apo B-100 and apo A-I monoclonal antibodies, are relevant to the initiation or progression of atherosclerosis, the incorporation of RLP into mouse macrophages was studied using histochemical and biochemical techniques. Remnant lipoproteins such as RLP are reported to contain a large quantity of chyloniron and very low density lipoprotein (VLDL) remnants, especially in diabetic patients. The RLP separated from the sera of 32 diabetic patients were found to be predominantly taken up into macrophages harvested from mouse abdominal cavities by the staining method applying oil red O. Furthermore, using 14C-oleate to prove the uptake of lipoproteins by macrophages, the uptake of RLP-VLDL, a VLDL fraction of RLP by ultracentrifugation, was the next highest to that of the oxidized LDL, which suggests that RLP-VLDL is also aggressively taken up by macrophages. The degree of uptake of RLP-VLDL by macrophages was positively correlated with HbA1c of these diabetic patients (r = 0.556, p < 0.01), irrespective of the ways of the treatment of diabetes. In conclusion, RLP can contribute to the foaming of macrophages, which in turn may explain the acceleration of atherosclerosis in diabetic patients.

Aged↗
PubMed1993

Genistein augments cyclic adenosine 3'5'-monophosphate(cAMP) accumulation and insulin release in MIN6 cells.

Effects of genistein on insulin release were studied using MIN6 cells, a glucose-sensitive insulinoma cell line. At the non-stimulatory concentrations of glucose, genistein did not affect insulin release, however, at the stimulatory concentrations of glucose, genistein significantly increased insulin release in a dose-dependent manner up to 20 micrograms/ml. The content of cAMP in MIN6 cells was also elevated significantly by genistein and the dose-response relationship between the genistein and cAMP accumulation was consistent with the relationship between the genistein and insulin release. These effects were inhibited by calcium antagonists or by the omission of extracellular calcium. Isobutylmethylxanthine (IBMX;0.1mM) increased both cAMP accumulation and insulin release in MIN6 cells and there were no additive effects by the addition of genistein. The accumulation of cAMP might have, at least in part, resulted from phosphodiesterase inhibition by genistein. These results suggest that genistein augments glucose-induced insulin release by the contribution of cAMP accumulation and calcium modulation which depends on extracellular calcium.

1-Methyl-3-isobutylxanthine↗
PubMed1993

Appearance of a regenerating (reg) gene protein in pancreatic islets of remission BB/Wor//Tky rats.

Remission of diabetes, i.e. significant amelioration from absolute insulin-dependency, has been sometimes observed in diabetic BB/Wor//Tky rats which were treated with insulin. In remission BB/Wor//Tky rats, plasma glucose levels improved to near normal level and insulin content was also preserved as much as that between diabetic and non-diabetic rats. In this process, we hypothesized that autoimmune insulitis was suppressed and remaining islet B-cells was restored from severe destruction by recovering in number and/or function. While, recently, a novel regenerating (reg) gene, identified in the regenerating pancreatic islets of surgical models, is reported to be related to the replication of pancreatic B-cells in vitro. Based on these findings, we histologically investigated whether the reg protein could be actually expressed or not in the islets from remission BB/Wor//Tky rats. As expected, reg protein was observed in the islets from remission BB/Wor//Tky rats mainly in accordance with pancreatic B-cells. Thus, the present findings suggested that the regeneration of pancreatic B-cells represented by the expression of reg protein might be, at least in part, relevant to remission induced by insulin therapy in spontaneously occurring Type 1 diabetes in BB/Wor//Tky rats.

Animals↗
PubMed1993

[Effects of music on relieving pain associated with a compulsory posture].

To assess the role of music on pain management, we examined effects of music to relieve a pain associated with a compulsory posture. Classical musics, which are recognized to make persons feel well, were chosen in this study. Five healthy adult females kept a supine position for 2 hours without music. Complains, and variations of heart beat and respiration were observed in each individual during the 2 hour experiment. After 5 days or more, these subjects had the same experience with music. Frequency and intensity of complains were significantly diminished by music. Heart rate was not changed by music. Respiration rate was increased in 3 subjects with music. Frequency of irregular respiration was significantly decreased by music. There was a positive correlation between frequency of irregular respiration and number of complaints in subjects kept without music. The present study demonstrated that music is effective to relieve a pain associated with a compulsory posture. Our results suggest that music plays a significant role on pain management in palliative therapy.

Adult↗
PubMed1993

Effect of age on the development or progression of albuminuria in non-insulin-dependent diabetes mellitus (NIDDM) without hypertension.

To study the relationship between aging and development of diabetic nephropathy, we studied the time until the development of microalbuminuria in old onset (> 50 years old, n = 21) and young onset (< 40 years old, n = 26), normotensive NIDDM patients. Microalbuminuria which is associated with the early stage of diabetic nephropathy was defined as urinary albumin index (UAI; mg/g.creatinine) of more than 10mg/g.creatinine, using timed overnight urine. In these two groups, there were no significant differences in duration of diabetes, observation periods, glycemic control, systolic diastolic blood pressure, body mass index and creatinine clearance at the time of the last observation. Mean UAI +/- standard deviation of the two groups were 37.5 +/- 78.2 mg/g.cr and 93.0 +/- 127.2 mg/g.cr in the young onset group and the old onset group, and prevalences of microalbuminuria were 38% and 76% in the young onset and old onset group, respectively. Thus, UAI and prevalence of microalbuminuria in the old onset group are significantly higher than those of the young onset group (P < 0.05). These results suggest that aging, in itself, is one of the significant risk factors for the development of diabetic nephropathy in NIDDM patients.

Age of Onset↗
PubMed1991

A novel phenotype of an excision-repair mutant in Neurospora crassa: mutagen sensitivity of the mus-18 mutant is specific to UV.

A UV-sensitive mutant has been isolated from UV-mutagenized conidia of Neurospora crassa. The mutation responsible for the lesion was mapped in linkage group VL, proximal to the nucleolus organizer region. We designated the mutant mus-18. The sensitivity of the mus-18 mutant to UV-irradiation was not particularly high, being less than twice that of the wild-type strain. However, the frequency of mutations at the ad-3 loci induced by UV was extremely high even at low doses, under conditions where survival rates of mus-18 cells were almost identical to those of wild-type cells. Photo-reactivation of UV damage was normal in the mus-18 mutant. Sensitivity to other mutagens, such as gamma rays, 4-nitroquinoline-1-oxide, N-methyl-N'-nitro-N-nitrosoguanidine, mitomycin C and methyl methanesulfonate, was similar to that of the wild type. Fertility of the mus-18 mutant was normal in homozygous crosses. These results suggest that mus-18 is an excision-repair mutant. Measurement of endonuclease-sensitive sites (ESS) after liquid-holding recovery from UV damage revealed that ESS remained unrepaired for longer than 18 h in the mus-18 mutant, while most were eliminated within 6 h in wild-type cells and in other UV-sensitive mutants. This result suggests that mus-18 is defective in the incision step of dimer excision. The mus-18 mutant provides the first example of an excision-defective mutation in eukaryotes, which is specific to UV damage.

4-Nitroquinoline-1-oxide↗
PubMed1991

Somatic mutation frequencies in the stamen hairs of Tradescantia grown in soil samples from the Bikini Island.

Somatic pink mutation frequencies in the stamen hairs of Tradescantia BNL 02 clone grown for 76 days in two soil samples taken from the Bikini Island (where a hydrogen bomb explosion test had been conducted in 1954) were investigated. A significantly high mutation frequency (2.58 +/- 0.17 pink mutant events per 10(3) hairs or 1.34 +/- 0.09 pink mutant events per 10(4) hair-cell divisions) was observed for the plant grown in one of the two Bikini soil samples, as compared to the control plants (1.70 +/- 0.14 or 0.88 +/- 0.07, respectively) grown in the field soil of Saitama University. The soil sample which caused the significant increase in mutation frequency contained 6,880 +/- 330 mBq/g 137Cs, 62.5 +/- 4.4 mBq/g 60Co, and some other nuclides; a 150 microR/hr exposure rate being measured on the surface of the soil sample. The effective cumulative external exposures measured for the inflorescences of the plant grown in this soil sample averaged at most 60.8 mR, being too small to explain the significant elevation in mutation frequency observed. On the other hand, internal exposure due to uptake of radioactive nuclides was estimated to be 125 mrad (1.25 mGy) as an accumulated effective dose, mainly based on a gamma-spectrometrical analysis. However, it seemed highly likely that this value of internal exposure was a considerable underestimate, and the internal exposure was considered to be more significant than the external exposure.

Micronesia↗
PubMed1989

Effects of heat shock on the induction of mutations by chemical mutagens in Neurospora crassa.

Preheating of Neurospora conidia increased their susceptibility to mutation induction by chemical mutagens. Optimal conditions of heat shock for enhanced mutagenesis were determined in 2.5 X 10(7) conidia/ml 0.067 M KH2PO4-Na2HPO4 (pH 7.0) buffer to be treatment at 43 degrees C for 60 min. When protein synthesis during heat stock was eliminated by cycloheximide or by use of the temperature-sensitive mutation psi-1, induction of thermotolerance was inhibited while induction of the enhanced state of mutability was not. Therefore, inducible protein synthesis is not involved in this process. To discover whether DNA-repair systems are altered by heat shock and, as a result, whether reversion frequencies increase, DNA-repair mutants (upr-1, uvs-2, uvs-3, uvs-6, mus-7, mus-16) were heated and their reversion frequencies at the ad-8 locus were measured. All the DNA-repair mutants showed higher reversion frequencies with MNNG treatment after heat shock than in non-heated control. It therefore seems that DNA repair is not involved in the enhancement of chemical mutagenesis by heat shock. Heat shock does not increase frequencies of reversion induced by ultraviolet light, and heat shock after treatment with chemical mutagens does not affect reversion frequencies. These results suggest that heat shock may change the structure and function of cellular membranes and thereby increase the influx of mutagens into cells.

DNA Repair↗
PubMed1989

Epistasis, photoreactivation and mutagen sensitivity of DNA repair mutants upr-1 and mus-26 in Neurospora crassa.

Double mutants were constructed combining mus-26, formerly designated uvs-(SA3B), with other UV-sensitive mutants. Tests of sensitivity of these double mutants to UV and to chemical mutagens revealed that mus-26 and upr-1 belong to the same epistatic group. The UV dose-response curve of mus-26 showed a characteristic plateau in the range of 100-200 J/m2. The same characteristic was also shown in the dose-response curves of upr-1 and the double mutant, upr-1 mus-26. Photoreactivation of UV damage in mus-26, upr-1 and upr-1 mus-26 was defective but not null. Assays were made of the reversion rate of ad-8 in strains that also carried UV-sensitive mutations. The reversion frequencies of the strains with upr-1 and upr-1 mus-26 were very low for the UV dose range below 300 J/m2, similarly to mus-26. Previously reported homozygous sterility of mus-26 was not caused by the mus-26 locus itself, and fertile strains were obtained among progeny. The results of this study suggest that mus-26 and upr-1 have similar properties in DNA repair.

4-Nitroquinoline-1-oxide↗