Biomedical subjects

H Kasai

Publications and source records attributed to H Kasai.

At least 55 records · Page 3Linked to original sources

PubMed2000

Two DNA polymerases of Escherichia coli display distinct misinsertion specificities for 2-hydroxy-dATP during DNA synthesis.

The insertion specificities of an oxidized dATP analogue, 2-hydroxydeoxyadenosine 5'-triphosphate (2-OH-dATP), were determined using the alpha (catalytic) subunit of Escherichia coli DNA polymerase III and the exonuclease-deficient Klenow fragment of DNA polymerase I. In contrast to our previous observation that mammalian DNA polymerase alpha incorporated the oxidized nucleotide opposite T and C, these two E. coli DNA polymerases incorporated 2-OH-dATP opposite T and G on the DNA template. Steady-state kinetic studies indicated that the alpha subunit incorporated 2-OH-dATP 10 times more frequently opposite T than opposite G. On the other hand, the incorporation of 2-OH-dATP opposite T by the exonuclease-deficient Klenow fragment was 2 orders of magnitude more efficient than that opposite G. These results indicate that the misinsertion specificity of 2-OH-dATP differs between replicative and repair-type DNA polymerases, and provide a biochemical basis for the mutations induced by 2-OH-dATP in E. coli.

Adenosine Triphosphate↗

PubMed2000

STAM2, a new member of the STAM family, binding to the Janus kinases.

We here cloned a cDNA encoding STAM2, a new member of the STAM family, which contains an SH3 domain and ITAM. STAM2 like STAM1 is associated with Jak2 and Jak3, and involved in the signaling for DNA synthesis and c-myc induction mediated by IL-2 and GM-CSF. Co-expression of the SH3 deletion mutants of STAM1 and STAM2 induces an additive effect on suppressing DNA synthesis upon stimulation with IL-2 and GM-CSF, suggesting that STAM1 and STAM2 exhibit compensatory effects on the signaling pathways downstream of Jak2 and Jak3 upon stimulation with GM-SCF and IL-2, respectively.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Methylglyoxal induces G:C to C:G and G:C to T:A transversions in the supF gene on a shuttle vector plasmid replicated in mammalian cells.

We previously reported that the majority of base-pair substitutions induced by an endogenous mutagen, methylglyoxal, were G:C-->T:A transversions and G:C-->A:T transitions in wild-type and nucleotide excision repair (NER)-deficient (uvrA or uvrC) Escherichia coli strains. To investigate the mutation spectrum of methylglyoxal in mammalian cells and to compare the spectrum with those detected in other experimental systems, we analyzed mutations in a bacterial suppressor tRNA (supF) gene in the shuttle vector plasmid pMY189. We treated pMY189 with methylglyoxal and immediately transfected it into simian COS-7 cells. The cytotoxicity and the mutation frequency (MF) increased according to the dose of methylglyoxal. In the mutants induced by methylglyoxal, multi-base deletions were predominant (50%), followed by base-pair substitutions (35%), in which 89% of the substitutions occurred at G:C sites. Among them, G:C-->C:G and G:C-->T:A transversions were predominant. The overall distribution of methylglyoxal-induced mutations detected in the supF gene was different from that for the spontaneous mutations. These results suggest that methylglyoxal may take part in causing G:C-->C:G and G:C-->T:A transversions in vivo.

Animals↗

PubMed2000

Classical conditioning of oxidative DNA damage in rats.

This study investigated whether the formation of 8-hydroxydeoxyguanosine (8-OH-dG), a known oxidative DNA modification relevant to carcinogenicity, can be classically conditioned to a novel taste in order to clarify the possible role of the central nervous system (CNS) or psychological stress on cancer initiation via a classical conditioning mechanism. Male Wistar rats underwent one or two conditioned taste aversion (CTA) experiments in which ferric nitrilotriacetate (Fe-NTA), which has renal toxicity and can induce renal cell carcinoma, served as a visceral unconditioned stimulus (US), and a saccharin solution (SAC) was used as a conditioned stimulus (CS). The 8-OH-dG levels in the group conditioned with the combination of SAC and Fe-NTA significantly increased as compared to those of the uncombined groups by two repeats of the conditioning procedure (P=0.013). The rats that showed a painful response at the Fe-NTA administration had significantly higher values of 8-OH-dG than those without pain (P=0. 003). These results not only provide the first evidence regarding classical conditioning of oxidative DNA damage using the CTA procedure, but also suggest the involvement of the CNS and psychological stress in the pathogenesis of cancer via oxidative DNA damage.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2000

2-Hydroxy-dATP is incorporated opposite G by Escherichia coli DNA polymerase III resulting in high mutagenicity.

Four kinds of oxidatively damaged DNA precursors, 8-hydroxydeoxyguanosine 5'-triphosphate (8-OH-dGTP), 2-hydroxydeoxyadenosine 5'-triphosphate (2-OH-dATP), 5-hydroxydeoxycytidine 5'-triphosphate (5-OH-dCTP) and 5-formyldeoxyuridine 5'-triphosphate (5-CHO-dUTP), were employed in in vitro gap-filling reactions of the supF gene conducted by the Escherichia coli DNA polymerase III holoenzyme, and these treated DNAs were transfected into various E.coli strains. When the manipulated DNAs were transfected into the repair-proficient strain, supF mutants were obtained much more frequently by the purine nucleotides than by the pyrimidine nucleotides (2-OH-dATP > 8-OH-dGTP >> 5-OH-dCTP approximately 5-CHO-dUTP). This result is in contrast to our previous observation that these four oxidatively damaged nucleotides induce chromosomal gene mutations with similar frequencies when incorporated directly into E.coli. 2-OH-dATP elicited G-->T transversions, indicating the formation of G*2-OH-dATP pairs. These results demonstrate that 2-OH-dATP was highly mutagenic in this assay system containing the in vitro DNA synthesis by the E.coli replicative DNA polymerase, in addition to in the in vivo assay system reported previously. Slight increases in the mutant frequencies were observed when alkA (for 8-OH-dGTP and 2-OH-dATP) and mutY (for 2-OH-dATP) strains were used as hosts. This is the first report that clearly shows the formation of G*2-OH-dATP pairs.

Adenosine Triphosphate↗

PubMed2000

Hyperpolarization is not responsible for the acetylcholine-induced negative chronotropic action in the presence of isoproterenol.

It has been reported that acetylcholine hyperpolarizes the maximum diastolic potential of canine Purkinje fibers through a pathway involving a G protein and induces a decrease in their automaticity. It is unclear, however, whether the negative chronotropic action of acetylcholine in the presence of beta-adrenergic stimulation is due to the hyperpolarization of the maximum diastolic potential or a decrease in the slope of phase 4 depolarization. We used standard microelectrode techniques to study the negative chronotropic mechanism of acetylcholine in the presence of isoproterenol in adult canine Purkinje fibers. Fibers were incubated for 24 hours in Tyrode's solution alone (n = 10) or plus pertussis toxin (n = 10), and then superfused with acetylcholine (10(-9) to 10(-4) M) in the presence of isoproterenol (10(-7) M). Acetylcholine in the presence of isoproterenol significantly decreased automaticity without hyperpolarization of the maximum diastolic potential, and decreased the slope of phase 4 depolarization. The effects of acetylcholine on automaticity and the slope of phase 4 depolarization were attenuated by pertussis toxin. The present findings indicate that the negative chronotropic action of acetylcholine in the presence of isoproterenol is due to the decrease in the slope of phase 4 depolarization through a pathway involving a pertussis toxin-sensitive G protein and that it is not the result of hyperpolarization of the maximum diastolic potential.

Acetylcholine↗

PubMed2000

Corrigendum.

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Journal Article↗

PubMed2000

Action of chlorogenic acid in vegetables and fruits as an inhibitor of 8-hydroxydeoxyguanosine formation in vitro and in a rat carcinogenesis model.

Various plant extracts, such as carrot, burdock (gobou), apricot and prune, showed inhibitory effects in an in vitro assay of lipid peroxide-induced 8-hydroxydeoxyguanosine (8-OH-dG) formation. The major inhibitor purified from various plants extracts was identified as chlorogenic acid (CA), on the basis of UV- and mass-spectra and comparison with a standard sample. To examine whether CA also inhibits 8-OH-dG formation in animal organs, an oxygen radical-forming carcinogen, 4-nitroquinoline-1-oxide, was administered to rats, with or without CA. The 8-OH-dG level in the DNA of the rat tongue, the target organ, was significantly reduced in the CA-treated group.

4-Nitroquinoline-1-oxide↗

PubMed2000

Mutations induced by glyoxal and methylglyoxal in mammalian cells.

To investigate the mutation spectra of glyoxal and methylglyoxal in mammalian cells, we analyzed mutations in a bacterial suppressor tRNA (supF) gene in the shuttle vector plasmid pMY189. The cytotoxicity and the mutation frequency increased according to the doses of glyoxal and methylglyoxal. The majority of glyoxal-induced mutations (65%) were base-pair substitutions, in which G:C-->C:G transversions were predominant. In the mutants induced by methylglyoxal, multi-base deletions were predominant (50%), followed by base-pair substitutions (35%), in which G:C-->C:G and G:C-->T:A transversions were predominant.

Animals↗

PubMed2000

Mutagenicity of 5-formyluracil in mammalian cells.

5-Formyluracil, a major oxidized form of thymine, was incorporated into a predetermined site of one of the leading and lagging template strands of a double-stranded vector, and the DNA replication efficiency and the mutation frequency of 5-formyluracil in simian COS-7 cells were investigated. 5-Formyluracil did not block DNA replication and was weakly mutagenic in simian cells. 5-Formyluracil primarily elicited base substitutions at the modified positions.

Animals↗

PubMed2000

Intrageneric relationships among Micromonospora species deduced from gyrB-based phylogeny and DNA relatedness.

The phylogenetic structure of genus Micromonospora within actinomycetes was examined by analysing the gyrB sequences of 15 validly described species and four subspecies. All but one of the Micromonospora strains formed a tight cluster, as had previously been demonstrated by a 16S rDNA-based phylogenetic analysis. However, the intrageneric relationships deduced from the gyrB-based phylogeny were different from those based on their 16S rDNA sequences. To examine which phylogeny would be more relevant for classifying genus Micromonospora, DNA-DNA hybridization experiments were performed. The gyrB-based classification agrees with the results of the DNA-DNA hybridization studies, indicating that this classification method is useful for analysing the phylogenetic relationships of high G+C Gram-positive bacteria at the level of the genomic species. Genus Micromonospora was reclassified into the following 14 species: Micromonospora echinospora, Micromonospora pallida, Micromonospora nigra, Micromonospora purpureochromogenes, Micromonospora aurantiaca, Micromonospora carbonacea, Micromonospora chalcea, Micromonospora chersina, Micromonospora coerulea, Micromonospora gallica, Micromonospora halophytica, Micromonospora inositola, Micromonospora olivasterospora and Micromonospora rosaria.

DNA Gyrase↗

PubMed2000

Increase in 8-hydroxyguanine and its repair activity in the esophagi of rats given long-term ethanol and nutrition-deficient diet.

Epidemiological studies have shown that an increased risk of esophageal cancer is associated with the chronic consumption of alcoholic beverages, although alcohol itself is not a carcinogen in animal models. Reactive oxygen species produced by the metabolism of ethanol or by chronic inflammation may play an important role in the carcinogenic process. In this study, we analyzed one type of oxidative DNA damage, 8-hydroxyguanine (8-OH-Gua), and its repair activity in the esophagus as indicators of cellular oxidative stress in rats given long-term ethanol and an autoclaved diet (nutrition-deficient diet). Three-week-old male Sprague-Dawley rats were fed an ethanol beverage whose concentration was increased from 12 to 70% over 20 weeks. When the concentration reached 50%, the diet of one group was changed from the regular diet to an autoclaved diet. At the feeding periods of 20, 25, 30, and 35 weeks, the rats were sacrificed and the 8-OH-Gua levels and repair activities within the esophagi were measured. After 30 weeks of ethanol- and autoclaved diet-feeding, significant increases of 8-OH-Gua and its repair activity were observed in the esophagi, but not in those of the ethanol- and normal diet-fed rats. This result indicates that the combined effects of long-term ethanol consumption and nutritional deficiency may be involved in inducing oxidative stress in the rat esophagus.

Animals↗

PubMed2000

Analyses of oxidative DNA damage and its repair activity in the livers of 3'-methyl-4-dimethylaminoazobenzene-treated rodents.

We measured the levels of 8-hydroxyguanine (8-OH-Gua) and its repair activity in the livers of the Donryu rat, the carcinogen-resistant DRH rat, and the ddy mouse, which were fed a 0.06% 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB)-containing diet. In a short-term rat experiment (maximum 2 months), 3'-MeDAB did not increase the 8-OH-Gua levels in the livers of the two rat strains, although it significantly increased the repair activity in only the Donryu rat liver at 1 and 2 months. After long-term 3'-MeDAB administration to the ddy mouse (8 months), the levels of 8-OH-Gua and its repair activity were increased in the liver by 3.6-fold and 1.6-fold, respectively. These experiments suggest that 3'-MeDAB increases 8-OH-Gua generation in rodent liver DNA and the 8-OH-Gua repair assay is a reliable marker of cellular oxidative stress induced by carcinogens.

Animals↗

PubMed2000

Differentiation of phylogenetically related slowly growing mycobacteria by their gyrB sequences.

The conventional methods for identifying mycobacterial species are based on their phenotypic characterization. Since some problematic species are slow growers, their taxonomy takes several weeks or months to identify. The ribosomal DNA (rDNA) sequence-based identification strategy has been adopted to solve this problem. More recently, the gyrB sequences have been shown to be useful phylogenetic markers for the identification of species. We determined the gyrB sequences of 43 slowly growing strains belonging to 15 species in the genus Mycobacterium. The frequencies of base substitutions in the gyrB sequences were comparable to those in the 16S-23S rDNA internal transcribed spacer (ITS) sequences. The ITS sequences of four species belonging to the M. tuberculosis complex (M. tuberculosis, M. bovis, M. africanum, and M. microti) were 100% identical, while four synonymous substitutions were found in the gyrB sequences of these strains. Based on the differences found in the gyrB sequences, we developed PCR and PCR-restriction fragment length polymorphism methods to discriminate these species.

Animals↗

PubMed2000

Lipoprotein(a), left atrial appendage function and thromboembolic risk in patients with chronic nonvalvular atrial fibrillation.

Lipoprotein(a) (Lp(a)) has a prothrombotic effect by modulating the fibrinolytic system. The purpose of the present study was to determine whether serum Lp(a) levels are associated with an increased risk of thromboembolism in chronic nonvalvular atrial fibrillation (NVAF). Clinical, laboratory and transesophageal echocardiographic data were collected in 172 consecutive, non-anticoagulated patients with chronic NVAF. Thirty-four patients (thromboembolic group) had a recent (<1 month) embolic event and/or a left atrial thrombus on transesophageal echocardiography. The thromboembolic group had a higher frequency of spontaneous echo contrast (94 vs. 58%, p<0.0001), increased concentrations of Lp(a) (median: 31.5 vs. 15.5 mg/dl, p<0.0001) and fibrinogen (median: 352 vs. 314 mg/dl, p = 0.0015), larger left atrial dimensions (median: 5.1 vs. 4.8cm, p = 0.0078), and reduced left atrial appendage (LAA) flow velocities (median: 9.5 vs. 21.2 cm/s, p<0.0001) than the nonthromboembolic group. Multivariate analysis identified 3 independent predictors of thromboembolism: Lp(a) level > or =30 mg/dl (odds ratio (OR) 9.5, 95% confidence interval (CI) 4.4-20.4, p<0.0001), LAA flow velocity of <20 cm/s (OR 8.7, 95% CI 3.3-23.0, p = 0.0003) and a fibrinogen concentration of <377mg/dl (OR 3.2, 95% CI 1.5-6.9, p = 0.0201). The Lp(a) elevations and reduced LAA flow velocities are independently associated with thromboembolism in chronic NVAF.

Aged↗

PubMed2000

2-hydroxyadenine in DNA is a very poor substrate of the Escherichia coli MutY protein.

To test the possibility that the Escherichia coli MutY or MutM protein acts as a 2-hydroxyadenine (2-OH-Ade) glycosylase, we treated double-stranded oligodeoxyribonucleotides containing 2-OH-Ade with the E. coli MutY or MutM protein in vitro. We found that a strand with 2-OH-Ade was a very poor substrate of MutY, irrespective of the base in the complementary strand. Moreover, a strand containing adenine or guanine opposite 2-OH-Ade was also rarely cleaved by MutY. The cleavage of oligonucleotides with 2-OH-Ade by MutM was not observed. These results indicate that neither MutY nor MutM plays an important role in the removal of 2-OH-Ade from DNA.

DNA Glycosylases↗

PubMed2000

Antitachycardia burst pacing for pleomorphic reentrant ventricular tachycardias associated with non-coronary artery diseases: a morphology specific programming for ventricular tachycardias.

To study the role of antitachycardia burst pacing in patients with reentrant pleomorphic ventricular tachycardia (VT) associated with non-coronary artery diseases, the efficacy of antitachycardia pacing and appropriate antitachycardia pacing cycle length were evaluated in each pleomorphic VT morphology of seven patients. Seven patients were included in this study. Clinically documented pleomorphic VTs were reproduced in an electrophysiologic study. For each VT, rapid ventricular pacing was attempted from the apex of the right ventricle at a cycle length which was 20 ms shorter than that of VT and repeated after a decrement of the cycle length in steps of 10 ms until the VT was terminated or accelerated. All 16 VTs could be entrained by the rapid pacing, and 13 of the 16 VTs (81%) were terminated, whereas pacing-induced acceleration was observed in the other 3 VTs of the 3 patients. VT cycle length (VTCL), block cycle length (BCL) which was defined as the longest VT interrupting paced cycle length, %BCL/VTCL and entrainment zone which was defined as VTCL minus BCL, varied in each VT morphology of each patient. In two patients, antitachycardia pacing was effective in all VT morphologies and the maximum difference of the %BCL/VTCL among the pleomorphic VTs was less than 10%. Thus, antitachycardia pacing seemed to be beneficial for these patients. In the other 5 patients, a difference of more than 10% in %BCL/VTCL was observed among the pleomorphic VT morphologies and/or at least one VT morphology showed pacing-induced acceleration. Compared to the 13 terminated VTs, three accelerated VTs had a wide entrainment zone [160 +/- 44 vs 90 +/- 48 ms, p < 0.04] and small %BCL/VTCL [61 +/- 6 vs 77 +/- 11%,p<0.03]. In pleomorphic VTs associated with non-coronary artery diseases, responses to rapid pacing was not uniform; VT might be terminable or accelerated even in the same patient. We need to pay close attention when programming antitachycardia pacing in patients with pleomorphic VT.

Accelerated Idioventricular Rhythm↗

PubMed2000

Overexpression of the hOGG1 gene and high 8-hydroxy-2'-deoxyguanosine (8-OHdG) lyase activity in human colorectal carcinoma: regulation mechanism of the 8-OHdG level in DNA.

8-Hydroxy-2'-deoxyguanosine (8-OHdG) is one of the most abundant oxidatively modified lesions in DNA. Our previous study (Kondo et al, Free Radic. Biol. Med., 27: 401-410, 1999) revealed that human colorectal carcinoma cells are oxidatively stressed based on 8-OHdG determination. To elucidate 8-OHdG metabolism and its clinical significance in colorectal carcinoma, we studied the 8-OHdG repair system in DNA by measuring specific lyase activity and hOGG1 expression using quantitative-competitive reverse transcription-PCR. In addition, we searched for the presence of mutations and single nucleotide polymorphisms of the hOGG1 gene by single-strand conformational polymorphism and sequencing analyses. It was found that 8-OHdG-specific lyase activity and hOGG1 expression were significantly up-regulated in carcinoma, and a proportional association between 8-OHdG levels and either 8-OHdG lyase activity (r = 0.641, P < 0.05) or hOGG1 expression (r = 0.702, P < 0.05) was present. Whereas no difference was detected in the 8-OHdG level between early- and advanced-stage cancer, lyase activity (1.2-fold) and hOG1 expression (1.6-fold) were significantly increased in advanced-stage cancer. No mutation was found in the 25 tumors examined. Three kinds of single nucleotide polymor. phism were observed, including that of codon 326 (Ser/Cys) in exon 7. However, there was no correlation between any of the three polymorphic patterns and either 8-OHdG level or lyase activity. These results suggest that increased 8-OHdG levels in colorectal carcinoma are attributed to increased formation and are maintained by induced 8-OHdG repair activity at appropriate high levels. Our results may offer a unique approach in the development of preventive and therapeutic interventions as well as new insights into the pathogenesis of colorectal carcinoma.

8-Hydroxy-2'-Deoxyguanosine↗