George Koike

Expression and cloning of complementary DNA for a human enzyme that repairs O6-methylguanine in DNA.

A cell line with an increased resistance to alkylating agents and an extremely high level of O6-methylguanine-DNA methyltransferase activity was isolated after transfection of methyltransferase-deficient Mer- cells with a cDNA library, prepared from methyltransferase-proficient human Mer+ (Raji) cells. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis revealed that a protein, with a molecular weight of approximately 25,000, accepted 3H label from DNA that had been treated with [3H]methylnitrosourea. Since the cDNA for methyltransferase was integrated into the chromosomal DNA, it was recovered by using the polymerase chain reaction. When the cDNA placed in an expression vector p500 was introduced into Mer- cells, the cells acquired an increased resistance to alkylating agents and exhibited a high level of O6-methylguanine-DNA methyltransferase activity. From the transformants the cDNA could be recovered as a part of the autonomously replicating plasmid. The nucleotide sequence of the cDNA was determined, and an open reading frame comprising 207 amino acid residues was found. The molecular weight of methyltransferase, calculated from the predicted amino acid sequence, was 21,700. The predicted amino acid sequence of the human methyltransferase exhibits an intensive homology with those of the bacterial counterparts, Ada and Ogt proteins of Escherichia coli and Dat protein of Bacillus subtilis, especially around possible methyl acceptor sites.

Angiotensin in the Nucleus Tractus Solitarii Contributes to Neurogenic Hypertension Caused by Chronic Nitric Oxide Synthase Inhibition

Activation of the sympathetic nervous system and renin-angiotensin system has been suggested to contribute to the hypertension caused by chronic nitric oxide synthase inhibition. The aim of the present study was to determine whether angiotensin within the nucleus tractus solitarii (NTS) plays a role in activation of the sympathetic nervous system in this model. Rats were treated with N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 mg. kg(-1). d(-1) in drinking water) for 2 weeks. Experiments were performed on anesthetized rats with denervated arterial and cardiopulmonary baroreceptors. Arterial pressure, heart rate, and renal sympathetic nerve activity (RSNA) were measured. Microinjection of an angiotensin II type 1 (AT(1)) receptor antagonist (CV11974) or an angiotensin II type 2 (AT(2)) receptor antagonist (PD123319) into the depressor region within the NTS (identified by prior injection of L-glutamate) was performed. Microinjection of CV11974, but not of PD123319, produced greater decreases in arterial pressure, heart rate, and RSNA in L-NAME-treated rats than in control rats. The administration of hexamethonium resulted in a larger fall in arterial pressure in L-NAME-treated rats than in control rats. The ACE mRNA level in the brain stem was greater in L-NAME-treated rats than in control rats. These results suggest that increased sympathetic nerve activity plays a role in hypertension caused by chronic nitric oxide synthase inhibition and that activation of the renin-angiotensin system in the NTS is involved at least in part in this increased sympathetic nerve activity via AT(1) receptors.

Relationship Between Obesity and Physical Fitness and Periodontitis

BACKGROUND Obesity and exercise are important elements associated with lifestyle-related diseases, and studies suggested that these factors may also be related to periodontitis. This study investigates the relationship between obesity and physical fitness and periodontitis. METHODS One thousand, one hundred and sixty Japanese subjects, aged 20 to 77 years, who participated in health examinations at Fukuoka Health Promotion Center were analyzed. Periodontal conditions were evaluated using the Community Periodontal Index (CPI), and subjects with > or =3 sextants of CPI code 3 or 4 were defined as having severe periodontitis. We used the body mass index (BMI) and percentage of body fat as indicators of obesity and estimated the maximal oxygen consumption (VO(2max)) during exercise as an indicator of physical fitness. We divided these variables into quintiles. We examined the single effect and interactions of the obesity index and VO(2max) on severe periodontitis. RESULTS The lowest quintile in BMI and the highest quintile in VO(2max) were inversely associated with severe periodontitis, singly, in multivariate logistic regression analyses. Subjects with the combined lowest quintile in BMI and the highest quintile in VO(2max) had a significantly lower risk of severe periodontitis compared to subjects with other combined quintiles in BMI and in VO(2max) (odds ratio: 0.17; 95% confidence interval: 0.05 to 0.55). CONCLUSION This study suggests that obesity and physical fitness may have some interactive effect on periodontal health status.

Expression of the ada gene of Escherichia coli in response to alkylating agents. Identification of transcriptional regulatory elements.

Ada protein plays a central role in the regulatory synthesis of DNA repair enzymes, following exposure of Escherichia coli to alkylating agents. Methyl groups of alkylated DNA are transferred to Ada protein by its own methyltransferase activity and the methylated Ada protein then acts as a positive regulator to overproduce the ada and related gene products. To elucidate regulatory mechanisms for the expression of the ada gene by its own product, we analyzed the ada promoter region by random and site-directed mutagenesis. A series of deletion analyses revealed that a sequence up to 53 nucleotides upstream from the transcription initiation site is required for the controlled expression of the ada gene. Libraries of base substitution mutants were constructed by synthesizing oligonucleotides corresponding to the ada promoter region in the presence of a small amount of all possible sets of nucleotides. Internal deletion and insertion mutants were also constructed with the use of synthetic oligonucleotides. Using these mutants, the -10 and the -35 boxes of the promoter as well as the ada regulatory sequence were identified, the latter being an eight-nucleotide sequence, AAAGCGCA. A six-nucleotide stretch between the regulatory sequence and the -35 box, also affected levels of expression of the gene. When the promoter DNAs derived from wild type or base substitution mutants that showed normal expression in vivo were used as templates for transcription in vitro, the ada-specific RNA was formed in the presence of a methylated form of Ada protein. With the DNAs derived from mutants of defective type as templates, no or relatively small amounts of the RNA were synthesized. Some base substitution mutants showed a constitutive expression of the gene in vivo, but this observation did not reconcile with findings in experiments in vitro.

Disparity of MCP-1 mRNA and protein expressions between the carotid artery and the aorta in WHHL rabbits: one aspect involved in the regional difference in atherosclerosis.

Objective—This study was designed to examine why in WHHL rabbits, muscular arteries, such as the carotid artery, are relatively resistant to atherosclerosis compared with the aorta, with a special reference to monocyte chemoattractant protein (MCP)-1. Methods and Results—MCP-1 mRNA expression was quantitated by Northern blot analysis, and its protein expression was quantitated by immunostaining and ELISA at the age of 1, 3, 6, and 12 months (n=5 to 6 each). In the aorta, atherosclerotic lesions were progressively developed with aging, and MCP-1 was highly expressed in endothelial cells and infiltrating macrophages. By contrast, in the carotid artery, atherosclerotic lesions and MCP-1 immunoreactivity were not evident throughout the experimental period. Unexpectedly, however, the extent of MCP-1 mRNA expression was comparable between the aorta and the carotid artery throughout the experimental period. Endothelial cells in primary culture from the aorta and the carotid artery expressed the same extent of MCP-1 mRNA on stimulation by oxidized LDL. There was no abnormality in primary structure of MCP-1 cDNA in WHHL. Conclusions—These results suggest that in WHHL, the atherosclerosis process, including MCP-1 protein expression, may be reduced in the carotid artery (and possibly in other muscular arteries), accounting in part for the regional resistance to atherosclerosis.

Quality of Life and Psychological Factors in Patients with Implantable Cardioverter Defibrillator

INTRODUCTION: As indication for implantable cardioverter defibrillators (ICDs) has expanded, prophylactic implantations have increased. It has been well understood that some ICD recipients have psychological problems. Some of those problems are recognized as maladjustment syndromes.