Biomedical subjects

H Kasai

Publications and source records attributed to H Kasai.

At least 145 records · Page 8Linked to original sources

PubMed1997

Kinetic diversity in the fusion of exocytotic vesicles.

The speed at which secretory vesicles fuse with the plasma membrane is a key parameter for neuronal and endocrine functions. We determined the precise time courses for fusion of small clear and large dense-core vesicles in PC12 and chromaffin cells by simultaneously measuring both plasma membrane areas and release of vesicular contents. We found that instantaneous increases in cytosolic Ca2+ concentration evoked vesicle fusion, but with time constants that varied over four orders of magnitude among different types of vesicles and cells. This indicates that the molecular machinery for the final Ca2+-dependent fusion steps of exocytosis is highly variable and is as critical as Ca2+ signalling processes in determining the speed and amount of secretion of neurotransmitters and hormones. Our results suggest a new possibility that the molecules responsible for the final fusion reaction that leads to vesicle fusion are key determinants for neuronal plasticity and hormonal disorders.

Acetylcholine↗

PubMed1997

Incorporation of the guanosine triphosphate analogs 8-oxo-dGTP and 8-NH2-dGTP by reverse transcriptases and mammalian DNA polymerases.

We have measured the efficiencies of utilization of 8-oxo-dGTP and 8-NH2-dGTP by human immunodeficiency virus type 1 and murine leukemia virus reverse transcriptases and compared them to those of DNA polymerases alpha and beta. Initially, we carried out primer extension reactions in the presence of dGTP or a dGTP analog and the remaining three dNTPs using synthetic DNA and RNA templates. These assays revealed that, in general, 8-NH2-dGTP is incorporated and extended more efficiently than 8-oxo-dGTP by all enzymes tested. Second, we determined rate constants for the incorporation of each analog opposite a template cytidine residue using steady state single nucleotide extension kinetics. Our results demonstrated the following. 1) Both reverse transcriptases incorporate the nucleotide analogs; discrimination against their incorporation is a function primarily of Km or Vmax depending on the analog and the enzyme. 2) Discrimination against the analogs is more stringent with the DNA template than with a homologous RNA template. 3) Polymerase alpha exhibits a mixed kinetic phenotype, with a large discrimination against 8-oxo-dGTP but a comparatively higher preference for 8-NH2-dGTP. 4) Polymerase beta incorporates both analogs efficiently; there is no discrimination with respect to Km and a significantly lower discrimination with respect to Vmax when compared with the other polymerases.

DNA Polymerase II↗

PubMed1997

Four novel KVLQT1 and four novel HERG mutations in familial long-QT syndrome.

BACKGROUND: Familial long-QT syndrome (LQTS) is characterized by prolonged ventricular repolarization. Clinical symptoms include recurrent syncopal attacks, and sudden death may occur due to ventricular tachyarrhythmias. Three genes responsible for this syndrome (KVLQT1, HERG, and SCN5A) have been identified so far. We investigated mutations of these genes in LQTS families. METHODS AND RESULTS: Thirty-two Japanese families with LQTS were brought together for screening for mutations. Genomic DNA from each proband was examined by the polymerase chain reaction-single-strand conformation polymorphism technique followed by direct DNA sequencing. In four of the families, comprising 16 patients, mutations were identified in KVLQT1; five other families (9 patients) segregated mutant alleles of HERG. All 25 of these patients carried the specific mutations present in their respective families, and none of 80 normal individuals carried these alleles. Mutations were confirmed by endonuclease digestion or hybridization of mutant allele-specific oligonucleotides. No mutation in SCN5A was found in any family. CONCLUSIONS: We identified nine different mutations among 32 families with LQTS. Eight of these were novel and account for 25% of all types of mutations reported to date. Such a variety of mutations makes it difficult to screen high-risk groups using simple methods such as endonuclease digestion or mutant allele-specific amplification.

Alleles↗

PubMed1997

Presence of human cellular protein(s) that specifically binds and cleaves 8-hydroxyguanine containing DNA.

8-hydroxyguanine (oh8Gua) is a major form of oxygen free radical-induced DNA damage. The oh8Gua nucleotide can pair with cytosine (C) and adenine (A) nucleotides which can cause G:C to T:A transversions. It is known that multiple repair systems for the correction of the oh8Gua exist in both mammalian and bacterial cells. Using the technique of gel mobility shift assay, protein(s) bound to the oh8Gua:C base pair in short fragments of DNA was detected in cell-free extracts of a human small-cell lung cancer cell line. This DNA binding activity was specific, since it was poorly detected with an unmodified G:C base pair containing oligonucleotide duplex and was affected by neither the unmodified G:C base pair nor an oh8Gua:A base pair containing oligonucleotide duplex. The partially purified protein which selectively binds to the oh8Gua:C base pair was shown by gel filtration column chromatography to have an apparent molecular mass of 52 kDa. The column fraction which showed the highest binding activity to the oh8Gua:C base pair was found to possess an enzymatic activity that specifically cleaves the oh8Gua containing oligonucleotide strand at both the 5' and 3' sides of the oh8Gua residue. These results indicate the presence of a protein(s) that is involved in a DNA repair pathway for the correction of the oh8Gua residue in human cells.

DNA Repair↗

PubMed1997

The biochemical studies on phalloidin-induced cholestasis in rats.

We investigated sequential changes in bile flow, serum and biliary biochemical parameters in phalloidin-induced cholestasis in rats. Intrahepatic cholestasis was induced by administration with phalloidin (500 microg/kg) for 7 days, and then the animals were allowed to survive for 1, 2, 4, 7, 14 and 28 days after the last treatment. In phalloidin-treated rats, bile flow significantly decreased up to 4 days of recovery, compared with the control animals. In contrast, serum ALP activity, LAP activity, cholesterol concentration and phospholipid concentration exhibited a marked elevation throughout the recovery periods. For biliary parameters, bilirubin excretion rate was unchanged but, cholesterol excretion rate showed a marked decrease throughout the recovery periods. These results demonstrate that some parameters, particularly important indexes of cholestasis (serum ALP, cholesterol, bile flow and so on), continued significant changes at least 4 days after the last administration of phalloidin. These results demonstrate that successive treatment with phalloidin can cause damage in most of serum and biliary parameters at a chronic stage of cholestasis. Thus, our findings may provide useful information for diagnosis of drug-induced cholestasis and help to further elucidate the biochemical mechanisms of drug-induced cholestasis in humans.

Animals↗

PubMed1997

Micromolar and submicromolar Ca2+ spikes regulating distinct cellular functions in pancreatic acinar cells.

Agonists induce Ca2+ spikes, waves and oscillations initiating at a trigger zone in exocrine acinar cells via Ca2+ release from intracellular Ca2+ stores. Using a low affinity ratiometric Ca2+ indicator dye, benzothiazole coumarin (BTC), we found that high concentrations of agonists transiently increased Ca2+ concentrations to the micromolar range (>10 microM) in the trigger zone. Comparison with results obtained with a high affinity Ca2+ indicator dye, fura-2, indicated that fura-2 was in fact saturated with Ca2+ during the agonist-induced Ca2+ spikes in the trigger zone. We further revealed that the micromolar Ca2+ spikes were necessary for inducing exocytosis of zymogen granules investigated using capacitance measurements. In contrast, submicromolar Ca2+ spikes selectively gave rise to sequential activation of luminal and basal ion channels. These results suggest new functional diversity in Ca2+ spikes and a critical role for the micromolar Ca2+ spikes in exocytotic secretion from exocrine acinar cells. Our data also emphasize the value of investigating the Ca2+ signalling using low affinity Ca2+ indicators.

Acetylcholine↗

PubMed1997

Substitution and deletion mutations induced by 2-hydroxyadenine in Escherichia coli: effects of sequence contexts in leading and lagging strands.

To evaluate the mutation frequency and the mutation spectrum of 2-hydroxyadenine (2-OH-Ade), an oxidative DNA lesion, the modified base was site-specifically incorporated into a unique restriction enzyme site (SalI, GTCGA*C or AflII, CTTA*AG where A* represents 2-OH-Ade) in single- and double-stranded vectors. The 2-OH-Ade residues were introduced into (+)- and (-)-strands of the double-stranded vectors and into the (+)-strand of single-stranded vectors. When the vectors were transfected intoEscherichia coli, the modified base showed little to no cytotoxicity. The mutation frequencies of 2-OH-Ade in the SalI and AflII sites were approximately 0.8 and 0.07%, respectively, with double-stranded (+)-vectors. An increase in the mutation frequencies was not observed with single-stranded vectors. When incorporated into the (-)-strand, the mutation frequencies of 2-OH-Ade in the SalI and AflII sites were approximately 0.3 and 0.1%, respectively. The mutations observed most frequently were -1 deletions at both positions, in the case of the (+)-strand. On the other hand, we observed that 2-OH-Ade in the (-)-strand induced A-->G and A-->T substitutions. These results indicate that 2-OH-Ade residues in DNA induce substitution and deletion mutations without blocking replication inE.coli.

DNA↗

PubMed1997

Inhibition of 8-hydroxyguanine repair in testes after administration of cadmium chloride to GSH-depleted rats.

The main goal of this study is to investigate the mechanism of cadmium (Cd)-induced carcinogenesis by reactive oxygen species. Rats were divided into four groups and were treated with (i) saline (control), (ii) cadmium chloride (CdCl2), (iii) l-buthionine-[S, R]-sulfoximine (BSO, an inhibitor of GSH biosynthesis), and (iv) CdCl2 and BSO, respectively. They were euthanized at 0, 24, 48, and 72 hr after these treatments, and the lungs and testes were analyzed. After treatment with both CdCl2 and BSO, the testicular 8-OH-Gua level increased (48 hr), its repair activity decreased (48 and 72 hr), the GSH content was markedly suppressed (48 and 72 hr), the superoxide dismutase activities slightly (48 and 72 hr) decreased, and the lipid peroxidation level increased (24 and 72 hr) in the testes as compared to the control levels. These results suggest that under GSH-depleted conditions, CdCl2 inhibits 8-OH-Gua repair activity in the rat testis and 8-OH-Gua accumulates in the DNA, which may pertain to testicular carcinogenesis.

Animals↗

PubMed1997

A histopathological study of craniopharyngioma with special reference to its stroma and surrounding tissue.

The numerous pathology reports on craniopharyngioma describe the classical features of squamous epithelium and keratin. Relatively little attention has been paid to its stroma and the surrounding tissue. We focused our observation on these two areas in a retrospective study of craniopharyngiomas in 36 operative cases and 5 autopsy cases. The tumor interdigitated with the brain and pituitary gland. At the interface, the brain was markedly gliotic with Rosenthal fibers simulating pilocytic astrocytoma. The tumor stroma had reactive changes and commonly showed cyst formation and calcification.

Adolescent↗

PubMed1997

Determination of carcinogenic potential of mineral fibers by 8-hydroxydeoxyguanosine as a marker of oxidative DNA damage in mammalian cells.

8-Hydroxydeoxyguanosine (8-OH-dG) is a typical form of oxidative DNA damage, which causes mutations in vitro and in vivo. To develop a simple method of testing the carcinogenicity of fibrous materials, the formation of 8-OH-dG was determined in the DNA of J774 cells, an established reticulum cell sarcoma line, after treatment with various natural and man-made mineral fibers. The amount of 8-OH-dG was determined using high-pressure liquid chromatography (HPLC) equipped with an electrochemical detector (ECD). We tested three natural mineral fibers (crocidolite, amosite, and chrysotile) and three man-made mineral fibers (ceramic, glass, and potassium octatitanate). Among them, a significant increase in 8-OH-dG formation was observed in the crocidolite- and amosite-treated cells. We also measured the amount of tumor necrosis factor (TNF) produced by J774 cells incubated with the fibrous materials. Cellular TNF production increased after treatment with all the fibers tested, but it was not statistically significant except in the case of chrysotile. Therefore, these results indicate that the mechanism of TNF production is different from that of 8-OH-dG formation, and that the carcinogenicity of various fibrous materials can be better evaluated by measuring the 8-OH-dG level in J774 cellular DNA after treatment with these fibers.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1997

Cytoplasmic Ca2+ gradients evoked by acetylcholine and peptides in pancreatic acinar cells of the guinea-pig.

Cytoplasmic Ca2+ levels ([Ca2+]i) in guinea-pig pancreatic acinar cells stimulated with acetylcholine (ACh) as well as cholecystokinin octapeptide (CCK-8), its analogue CCK-JMV-180, bombesin and substance P (SP) have been compared by confocal Ca2+ imaging. We found that at intermediate and high concentrations all agonists evoked [Ca2+]i waves emanating from the secretory pole of the cell. The [Ca2+]i gradients evoked by ACh and SP were often unidirectional whereas CCK-8- and bombesin-elicited [Ca2+]i responses consisting of [Ca2+]i waves were normally followed by a secondary phase with reversed [Ca2+]i gradients. At low agonist concentrations the [Ca2+]i responses appeared diffuse or localized to the granular part of the cells. The lag times to the [Ca2+]i rises were shorter in response to ACh and SP than for other peptides. Thus, we found that agonists induced different spatio-temporal patterns of Ca2+ waves in pancreatic acinar cells of the guinea-pig. The reactions evoked by SP and bombesin resembled those evoked by ACh and CCK-8, respectively.

Acetylcholine↗

PubMed1997

Increased oxidative deoxyribonucleic acid damage in the spermatozoa of infertile male patients.

OBJECTIVE: To investigate whether a high level of oxidative DNA damage in spermatozoa occurs in infertile male patients and to examine the influence of antioxidant treatments on the levels of this damage. DESIGN: Controlled clinical study and uncontrolled pilot study. SETTING: Department of Obstetrics and Gynecology, Akita University School of Medicine. PATIENT(S): Nineteen infertile male and 17 control patients. INTERVENTION(S): The levels of oxidative DNA damage in spermatozoa of infertile male and control patients were compared. In addition, 14 infertile males were given antioxidants for 2 months. MAIN OUTCOME MEASURE(S): The levels of 8-hydroxy-2'-deoxyguanosine, a form of oxidative damage, in the spermatozoa were determined using high-performance liquid chromatography with electrochemical detection. RESULT(S): The levels of 8-hydroxy-2'-deoxyguanosine in sperm DNA were significantly higher in male infertile patients than in the control patients (1.5 +/- 0.2 versus 1.0 +/- 0.1 per 10(5) deoxyguanosine) and were correlated with sperm concentrations in ejaculates. Antioxidant treatment resulted in significant positive effects on sperm concentrations, with a significant reduction in sperm 8-hydroxy-2'-deoxyguanosine levels (from 1.5 +/- 0.2 to 1.1 +/- 0.1 per 10(5) deoxyguanosine). CONCLUSION(S): Our present data demonstrate an association between the level of oxidative DNA damage in spermatozoa and male infertility and point to the possible use of antioxidants to reduce this damage.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1997

Separation of multiple yellow fluorescent lipofuscin components in rat kidney and their characterization.

Yellow fluorescent lipofuscin deposited in rat kidney was extracted in an aqueous solution and characterized after separation. Centrifugal fractionation of the extract revealed that most of the yellow fluorescence was detected in the 105,000 x g-supernatant, and little in nuclei, cell debris, mitochondria, lysosomes, microsomes and plasma membrane. The yellow fluorescence in the supernatant was fractionated by gel filtration through Sephadex columns into 5 yellow fluorescent fractions A, B (B1, B2 and B3) and C showing the same fluorescence spectra with excitation maximum/emission maximum at 400/620 nm. The components in fraction A were converted into the smaller molecular-weight components in fraction B on treatment with 4 M urea or protease, suggesting that they were proteinaceous. The smallest molecular-weight fluorescent components in fraction C were adherent to solid cellulose materials. The fluorescent components in all the fractions were soluble in water and insoluble in chloroform-methanol, indicating that they were not lipidic materials. The fluorophores in these fractions were kept stable on borohydride treatment, but readily converted into non-fluorescent components on heavy-metal ion treatment. The characteristics of the yellow fluorescence in these fractions were quite different from those of bluish lipofuscin-like fluorophores that may be generated in tissues during lipid peroxidation.

Aging↗

PubMed1997

8-hydroxyguanine (7,8-dihydro-8-oxoguanine) DNA glycosylase and AP lyase activities of hOGG1 protein and their substrate specificity.

Recently we cloned a structural human homolog (hOGG1) of the yeast OGG1 (yOGG1) gene that is involved in the excision repair of 8-hydroxyguanine (also known as 7,8-dihydro-8-oxoguanine; oh8Gua), hOGG1 protein shares 38% amino acid identity with yOGG1 protein. In this paper, we define the substrate specificity of oh8Gua DNA glycosylase and AP lyase activities of the hOGG1 protein. The oh8Gua released from oh8Gua containing DNA was measured by analysis with HPLC coupled with electrochemical detector (ECD) and cleavage sites in the DNA were identified by cleavage assay using gel electrophoresis. GST-hOGG1 protein possessed the oh8Gua DNA glycosylase/AP lyase activity and weak delta-elimination activity, oh8Gua opposite the C in duplex oligonucleotide was most efficiently released by GST-hOGG1 protein and oh8Gua opposite the T was also released, while oh8Gua opposite the G or A was very slowly done. The rank order of DNA cleavage efficiency was the same as that of oh8Gua glycosylase activity. Glycosylase/AP lyase activities and their substrate specificities of the GST-hOGG1 protein was similar to GST-yOGG1 protein but different from MutM protein. These results indicate that the dominant function of hOGG1 protein is a oh8Gua glycosylase reaction by specifically recognizing oh8Gua and pyrimidine opposite the oh8Gua and delta-elimination reaction in the same manner as yOGG1 protein. Thus, the hOGG1 gene is a functional human homolog of the yOGG1 gene on oh8Gua excision repair in spite of the low structural identity at amino acid level between hOGG1 and yOGG1 proteins.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1997

Analysis of a form of oxidative DNA damage, 8-hydroxy-2'-deoxyguanosine, as a marker of cellular oxidative stress during carcinogenesis.

8-hydroxy-2'-deoxyguanosine (8-OH-dG) was first reported in 1984 as a major form of oxidative DNA damage product by heated sugar, Fenton-type reagents and X-irradiation in vitro. 8-OH-dG has been detected in cellular DNA using an HPLC-ECD method in many laboratories. Analyses of 8-OH-dG in animal organ DNA after the administration of oxygen radical-forming chemicals will be useful for assessments of their carcinogenic risk. Its analysis in human leucocyte DNA and in urine is a new approach to the assessment of an individual's cancer risk due to oxidative stress. The increase of the 8-OH-dG level in the cellular DNA, detected by HPLC-ECD method, was supported by its immunochemical detection and its enhanced repair activity. The validity of the general use of 8-OH-dG as a marker of cellular oxidative stress is discussed.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1997

Cigarette smoking induces an increase in oxidative DNA damage, 8-hydroxydeoxyguanosine, in a central site of the human lung.

To evaluate the effect of cigarette smoking on oxidative stress in lung tissues, we compared the levels of a type of oxidative DNA damage, 8-hydroxydeoxyguanosine (8-OH-dG), in tissue obtained from the central sites of lungs from 14 current smokers, seven ex-smokers and nine non-smokers. The mean level of 8-OH-dG in the lung tissues from smokers was 1.43-fold higher than that of the non-smokers (the difference was statistically significant, P = 0.0262). A positive correlation between the 8-OH-dG levels in normal lung tissues and the Brinkman index was obtained from smokers and ex-smokers (r = 0.525; P = 0.0134). A positive correlation was also obtained for the 8-OH-dG levels in normal lung tissues and the number of cigarettes smoked per day (r = 0.436; P = 0.0132). These results suggest that oxidative DNA damage is induced in lung DNA by cigarette smoking.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1997

Relationship between Asbestos Exposures and 8-Hydroxydeoxyguanosine Levels in Leukocytic DNA of Workers at a Chinese Asbestos-material Plant.

The objective of the study was to evaluate the level of 8-hydroxydeoxyguanosine (8-OHdG) in DNA of peripheral-blood leukocytes as a biological marker of asbestos exposure and/or its fibrotic effects in an occupational population exposed to asbestos. The setting was a large-scale asbestos plant in China producing brake linings, asbestos rubber, and textile using chrysotile. From a base population of active and retired workers with various levels of cumulative exposure to asbestos and grades of asbestosis, 39 study subjects were randomly selected to reflect incremental grades of asbestosis based on Chinese diagnostic standards. They consisted of 19 "normal" (control) and ten "suspected" and ten "definite" asbestosis-grade subjects, group-matched for age and sex. Leukocytic DNA was extracted from 5-mL samples of peripheral blood and 8-OHdG level measured by high-pressure liquid chromatography. A cumulative asbestos exposure index (CEI) was calculated for each subject as the summed product of duration and level of asbestos exposure per job, incorporating a job-exposure matrix. Geometric mean 8-OHdG levels showed a positive gradient in relation to increasing grades of asbestosis (control: 1.78, suspected: 2.21, definite: 2.58), with a significant difference between the control and definite-asbestosis subgroups (p < 0.05). The 8-OHdG level of the two subgroups combined as one "asbestosis" group was significantly higher than that of the control group (control: 1.78, asbestosis: 2.39, p = 0.01). Further, 8-OHdG levels were moderately correlated with CEIs for all subjects (r = 0.35, p < 0.05) and with grades of asbestosis for all (r = 0.47, p < 0.01) and for male subjects (r = 0.43, p < 0.05). In multiple regression analyses, grade of asbestosis explained 27% of the total variation in 8-OHdG and was a better predictor than CEI or duration of exposure. Thus, the 8-OHdG level in leukocytic DNA is related to grade of asbestosis and to individual cumulative exposure and may serve as a biologic marker reflecting the status of oxidative DNA damage by asbestos.

Journal Article↗

PubMed1997

Comparison of oxidation products from DNA components by gamma-irradiation and Fenton-type reactions.

The four 2'-deoxyribonucleosides were gamma-irradiated or were aerobically treated with Fenton-type-reagents, Fe(II)-EDTA or a renal carcinogen Fe(II)-nitrilotriacetic acid (NTA) under the neutral conditions. The reaction mixtures were immediately analyzed by reverse-phase HPLC. Major products detected were 2-hydroxydeoxyadenosine (2-OH-dA), 8,5'-cyclodeoxyadenosine (cyclo-dA), 8-hydroxydeoxyadenosine (8-OH-dA). 5-formyldeoxyuridine (5-CHO-dU), 5-hydroxydeoxycytidine (5-OH-dC), 8-hydroxydeoxyguanosine (8-OH-dG), 8,5'-cyclodeoxyguanosine (cyclo-dG), and glyoxal and its adduct with dG. Ratio of these oxidized products were dramatically changed depending upon the agents used. For example, 2-OH-dA was a modified nucleoside produced most efficiently by Fe(II)-EDTA, while 5-CHO-dU and 5-OH-dC were the major products by the Fe(II)-NTA treatment and gamma-irradiation, respectively. Glyoxal itself was estimated to be produced most frequently (13 folds of 8-OH-dG) when treated with Fe(II)-EDTA, but its formation was not detected by the treatment with Fe(II)-NTA or by gamma-irradiation. 8-OH-dA was not produced by Fe-EDTA or Fe-NTA but was produced by gamma-irradiation. In contrast, 2-OH-dA was not produced by gamma-irradiation. These results suggest that triphosphates of 2-OH-dA, cyclo-dA, 8-OH-dA, cyclo-dG, 5-CHO-dU, 5-OH-dC, and glyoxal-dG as well as 8-OH-dG may be produced in cells with different ratio by various types of oxidative stress and involved in mutagenesis and carcinogenesis.

Chromatography, High Pressure Liquid↗