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MicroRNAs have been shown to play a role in B‐cell differentiation and activation. Here, we found miR‐182 to be highly induced in activated B cells. However, mice lacking miR‐182 have normal B‐cell and T‐cell development. Interestingly, mutant mice exhibited a defective antibody response at early time‐points in the immunization regimen when challenged with a T‐cell‐dependent antigen. Germinal centres were formed but the generation of extrafollicular plasma cells was defective in the spleens of immunized miR‐182‐deficient mice. Mutant mice were also not able to respond to a T‐cell‐independent type 2 antigen, which typically elicited an extrafollicular B‐cell response. Taken together, the data indicated that miR‐182 plays a critical role in driving extrafollicular B‐cell antibody responses.

The environmental factors such as aging, smoking, and alcohol consumption have been reported to influence DNA methylation (DNAm). However, the versatility of DNAm measurement by DNAm array systems is low in clinical use. Thus, we developed the MethyLight assay as a simple method to measure DNAm. In the present study, we isolated peripheral blood DNA from 33 healthy volunteers and selected cg25809905, cg02228185, and cg17861230 as aging, cg23576855 as smoking, and cg02583484 as alcohol consumption biomarkers. The predicted age by methylation rates of cg25809905 and cg17861230 significantly correlated with chronological age. In immortalized B-cells, DNAm rates of two sites showed a younger status than the chronological age of donor. On the other hand, the predicted age of the patients with myocardial infarction (MI) was not accelerated. The methylation rate of cg23576855 was able to discriminate the groups based on the smoking status. The DNAm rate of cg02583484 was reduced in subjects with habitual alcohol consumption compared to that of subjects without habitual alcohol consumption. In conclusion, our MethyLight assay system reconfirms that aging, smoking, and alcohol consumption influenced DNAm in peripheral blood in the Japanese. This MethyLight system will facilitate DNAm measurement in epidemiological and clinical studies.

MicroRNAs (miRNAs) are small noncoding RNAs of 18–23 nucleotides that regulate gene expression. Recently, plasma miRNAs have been investigated as biomarkers for various physiological and pathological conditions. The present study details the conserved miRNA expression profiles of tubular tissues, and discusses whether they could be used to distinguish between proximal tubule injury, diagnose acute kidney injury (AKI), and the early-stage renal tubular dysfunction. miRNA expression was assessed with miRNA array and real-time reverse transcription polymerase chain reaction using the TaqMan system. The expression profiles of miR-200a/b/c, miR-145, miR-192, miR-194, miR-216a/b, miR-217, and miR-449a in human and rat tubular tissues such as the kidneys, lung, small intestine, and various exocrine glands were adequate for discriminating tubular tissues. In the kidney, miR-192 and miR-194 were highly expressed, whereas miR-145 and miR-449a were absent. miR-145 and miR-449a were relatively specifically expressed in small intestine and lung, respectively. Therefore, the combined levels of miR-200a/b/c, miR-192, and miR-194 in plasma were very useful in diagnosing AKI induced by contact freezing in mice. Moreover, urinary miR-200a levels were useful for the diagnosis of renal tubular dysfunction in Dahl salt-sensitive rat with high salt administration. Our results indicate that miRNA expression profiles are useful as biomarkers for identification of various kidney injuries.

The Dahl salt-sensitive (DS) rat is the most prevalently used animal model of salt-sensitive hypertension. The purpose of the present study was to test the hypothesis that mitochondrial DNA (mtDNA) polymorphisms influence blood pressure in DS rats. We produced two strains of female F1 rats, one from female DS and male Lewis rats (DL) and the other from Lewis female and DS male rats (LD). These two strains had the same autosomal genetic background, but their mitochondria had different origins. The DL and LD rats had DS and Lewis mitochondria, respectively. A high-salt diet was started at 4 weeks of age. Radiotelemetry devices were implanted into the lower abdominal aorta of these F1 rats at 9 weeks of age. Blood pressure was monitored for 24 h at 11, 12, 13, 14, and 19 weeks of age. No significant differences were observed in blood pressure levels between the strains. Although more than 100 polymorphisms were detected between DS and Lewis rats, it is unlikely that polymorphisms in mtDNA contribute to hypertension in DS rats. Moreover, we found no difference between DS and Lewis rats in the mtDNA copy number in the kidneys, the liver, and the ventricles of the heart before and after salt loading. Thus, it is unlikely that mitochondrial dysfunction due to high blood pressure exacerbated target organ damage. Intriguingly, the time course of body weight gain differed significantly between DL and LD F1 rats, suggesting the influence of mitochondrial polymorphisms on body composition.

Catechol-O-methyltransferase (COMT) is an enzyme that inactivates catecholamines. Several studies have suggested that this enzyme may play a role in blood pressure regulation. We previously reported that the expression levels of Comt mRNA in Dahl salt-sensitive (DS) rats were lower than those in Lewis (LEW) rats. However, the physiological significance of this phenomenon has not been investigated. The purpose of the present study was to evaluate the significance of lower expression of Comt in Dahl salt-sensitive hypertension. The Comt gene in DS rats has a palindromic insertion in 3'-untranslated region, which appears to be responsible for reduced mRNA stability. A genome-wide quantitative trait loci (QTL) analysis of blood pressure using 107 F2 rats indicated that a statistically significant QTL for pulse pressure was located at the Comt locus in chromosome 11. Microarray analysis confirmed that Comt was the only gene differentially expressed between DS and LEW rats in this chromosomal region. However, COMT inhibitors had no significant effects on blood pressure in either DS or LEW rats. Thus, Comt was excluded from the candidate genes contributing to salt-sensitive hypertension in DS rats. A true gene responsible for pulse pressure in this chromosome 11 region remains to be determined.

Nanomaterials have numerous potential benefits for society, but the effects of nanomaterials on human health are poorly understood. In this study, we aim to determine the genotoxic effects of chronic exposure to nanomaterials in various cell lines.Chinese hamster ovary (CHO) cells, human epidermoid-like carcinoma (Hela) cells and human embryonic kidney 293 (HEK293) cells were treated with the water-soluble fullerence C(60)(OH)(24) for 33-80 days. Cell proliferation, cytotoxic analysis and micronucleus tests were performed.When treated with C(60)(OH)(24) (0, 10, 100, or 1000 pg/ml) for 33 days, both the HEK293 and Hela cells showed increased cell proliferation, but cellular lactate dehydrogenase (LDH) activity was not affected. After long-term exposure (80 days) to C(60)(OH)(24) (0, 10, 100, or 1000 pg/ml), the CHO, Hela and HEK293 cells showed increased genotoxicity on the micronucleus test.This study suggests that nanomaterials, such as C(60)(OH)(24), have genotoxic effects.

Nanoscale materials are presently under development for diagnostic (nanomedicine) and electronic purposes. In contrast to the potential benefits of nanotechnology, the effects of nanomaterials on human health are poorly understood. Nanomaterials are known to translocate into the circulation and could thus directly affect vascular endothelial cells (ECs), causing vascular injury that might be responsible for the development of atherosclerosis. To explore the direct effects of nanomaterials on endothelial toxicity, human umbilical vein ECs were treated with 1–100 μg/ml hydroxyl fullerene [C60(OH)24; mean diameter, 7.1 ± 2.4 nm] for 24 h. C60(OH)24 induced cytotoxic morphological changes such as cytosolic vacuole formation and decreased cell density in a dose-dependent manner. Lactate dehydrogenase assay revealed that a maximal dose of C60(OH)24 (100 μg/ml) induced cytotoxic injury. Proliferation assay also showed that a maximal dose of C60(OH)24 inhibited EC growth. C60(OH)24 did not seem to induce apoptosis but caused the accumulation of polyubiquitinated proteins and facilitated autophagic cell death. Formation of autophagosomes was confirmed on the basis of Western blot analysis using a specific marker, light chain 3 antibody, and electron microscopy. Chronic treatment with low-dose C60(OH)24 (10 μg/ml for 8 days) inhibited cell attachment and delayed EC growth. In the present study, we have examined, for the first time, the toxicity of water-soluble fullerenes to ECs. Although fullerenes changed morphology in a dose-dependent manner, only maximal doses of fullerenes caused cytotoxic injury and/or death and inhibited cell growth. EC death seemed to be caused by activation of ubiquitin-autophagy cell death pathways. Although exposure to nanomaterials appears to represent a risk for cardiovascular disorders, further in vivo validations are necessary.

MicroRNAs constitute a growing class of non-coding RNAs that are thought to regulate gene expression via translational repression. MicroRNAs are initially transcribed as several hundred-nucleotide pri-miRNAs and are then processed to approximately 60-nucleotide hairpin pre-miRNAs. We hypothesized that polymorphisms in both pre-miRNA and mature microRNA modify various biological processes by influencing the processing and/or target selection of microRNAs. In the present study, we sequenced 173 human pre-miRNA genome regions in 96 subjects and found 10 polymorphisms in the 10 pre-miRNA hairpin regions. Although most of these polymorphisms seem to have no effect on microRNA processing, we identified a C to A polymorphism in the mature miR-30c-2 sequence. This polymorphism may alter target selection and thus exert profound biological effects. To the best of our knowledge, this is the first report of polymorphisms in pre-miRNAs.

Association studies were performed to confirm the effect of polymorphisms in apolipoprotein A1 ( ApoA1) on the high-density lipoprotein cholesterol (HDL-C) level and the incidence of myocardial infarction (MI). A sequence analysis identified nine polymorphisms in ApoA1. After considering linkage disequilibrium, four polymorphisms in ApoA1 and four polymorphisms in the 5'-flanking regions and 3'-flanking regions from the JSNP database were determined in 1,880 subjects recruited from the Suita study, which represents the general population in Japan. Of the eight polymorphisms tested, the ApoA1 T84C polymorphism had the greatest effect on the levels of HDL-C ( P=0.0005, P(c)=0.0040 corrected by the Bonferroni method) and triglyceride ( P0.0001, P(c)=0.0008). The ApoA1 MspI polymorphism was not associated with HDL-C or triglyceride levels. We confirmed that the ApoA1 T84C polymorphism was associated with the HDL-C level but not the triglyceride level in patients with MI ( n=637). Moreover, this polymorphism was associated with the incidence of MI in male subjects ( P=0.0326). A logistic analysis indicated that the frequency of MI in the CC genotype was lower than that in the CT+TT genotype ( P=0.0145, OR=0.4955, 95% CI: 0.2746-0.8525). The ApoA1 T84C polymorphism is an important marker for the HDL-C level and may be a new risk marker for MI in Japanese.

We performed association studies between 118 single-nucleotide polymorphisms (SNPs) of 22 candidate genes (or gene family) and hypertension in a Japanese population.The study population consisted of 1880 subjects representing the general population in Japan, recruited from the Suita study. The candidate genes were selected based on their functions, including insulin resistance (APM1, CD36, HSD11B1), oxidative stress (CYBA, GPX1, GSTMs), steroid hormone (ESR1, ESR2, HSD11B2), renal functions (PTGS2, KLK1, NPHS1, NPHS2, SGK, SLC12A1, PTGES), and others related to cardiovascular physiology (GJA4, NOS1, NTRK3, P2RX4, SPP1, ALDH2).Multiple logistic analyses, with age and body mass index as covariates, indicated that 13 SNPs (eight genes), six SNPs (four genes) and 11 SNPs (four genes) were associated with hypertension (P0.05) in the total, male, and female populations, respectively. PTGS2 seems to be a promising candidate gene for hypertension in men. GSTM3 and SLC12A1 seem to be promising candidate genes for hypertension in women. Especially, a polymorphism in SLC12A1 was significantly associated with hypertension in women even after correction by the Bonferroni method (corrected P = 0.0236). Multiple logistic analyses, with age and body mass index as covariates, indicated that the prevalence of hypertension in females was significantly higher in subjects with the CC genotype than in those with the TT + TC genotypes (P0.0001, odds ratio = 1.967, 95% confidence interval = 1.430-2.712).Although the present results should be replicated in other study populations for confirmation, the present results suggest that SLC12A1 may contribute to hypertension in Japanese women.

To investigate the effects of polymorphisms in the ATP-binding cassette transporter A1 ( ABCA1) gene on the high-density lipoprotein cholesterol (HDL-C) level and the incidence of myocardial infarction (MI), we performed association studies. Sequence analysis identified 14 polymorphisms in the promoter region of ABCA1. After considering linkage disequilibrium, three polymorphisms in the promoter region and 11 polymorphisms from the JSNP database were determined in 1,880 subjects recruited from the Suita Study, representing the general population in Japan. We evaluated the association between the ABCA1 genotype and HDL-C level adjusted not only for standard factors, but also for genetic factors including ApoA1 and ApoE genotypes. Of the 14 polymorphisms tested, the G(-273)C ( P=0.0074), C(-297)T ( P=0.0195), and IMS-JST071749 ( P=0.0093) polymorphisms were significantly associated with the HDL-C level in the Suita population. We could reconfirm that the G(-273)C genotype was influential in another set of subjects ( P=0.0310, n=743). However, the distribution of the ABCA1 G(-273)C genotype in subjects with MI ( n=598) was not different from that in the control population ( n=801). These results indicate that ABCA1 G(-273)C has a significant effect on the HDL-C level in the general Japanese population, but not on the incidence of MI.

To identify candidate genes responsible for hypertension in Dahl salt-sensitive rats (Dahl-S), an oligonucleotide microarray analysis was performed to find differentially expressed genes in kidneys of Dahl-S and Lewis rats. We obtained 101 F2 male rats from Dahl-S and Lewis rats and performed precise measurements of blood pressure (BP) and heart rate by telemetric monitoring at 14 weeks of age after 9 weeks of salt-loading. The correlation analysis between genotypes of differentially expressed genes and BP in F2 rats indicated that pyridoxine 5'-phosphate oxidase (Pnpo) and catecholamine-O-methyltransferease (Comt) showed a highly significant association with BP. However, in the case of Comt, the Dahl-S genotype correlated with low BP. Short/branched chain acyl-CoA dehydrogenase and Sah also showed a significant association with systolic blood pressure. The present study provided evidence that Pnpo is a candidate gene responsible for hypertension in Dahl-S rats.

OBJECTIVE To examine the relationship between the gene for haeme oxygenase (HO)-1 (HMOX-1) and human essential hypertension, because both the acute and systemic induction of HMOX-1 have been suggested to attenuate vascular tone and blood pressure. METHODS We screened for sequence variations in HMOX-1 and conducted an association study, using these polymorphisms, in a large cohort (1998 individuals) representing the general Japanese population. RESULTS We sequenced HMOX-1 and found a T(-413)A polymorphism in the promoter region. The frequency of hypertensive individuals and the use of antihypertensive drugs were significantly greater in the AA genotype than in other genotypes among women: 45.5, 34.2, and 35.0% (P = 0.0099) and 23.4, 17.5, and 15.0% (P = 0.038), respectively, for the AA, AT, and TT genotypes, respectively. However, this association was not observed in men. Multiple logistic analyses indicated that the T(-413)A (AA/TA+TT) polymorphism, age, and body mass index affected the occurrence of hypertension in women. The odds ratio of the AA genotype for hypertension in women was 1.59 (P = 0.0058; 95% confidence interval 1.14 to 2.20). A luciferase reporter assay indicated that the A allele-promoter had eight-fold greater activity than the T allele promoter (P < 0.01). CONCLUSIONS The AA genotype of HMOX-1 is associated with an increased incidence of hypertension in women. Oestrogen attenuates vasoconstriction by increasing the expression of inducible nitric oxide synthase. As carbon monoxide, which is one of the products of HO-1, can attenuate nitric oxide-induced vasodilatation, a high expression of HO-1 may cause hypertension, especially in women.

Spontaneously hypertensive rats (SHR) are a well-known animal model for hypertension. We have previously identified eleven differentially expressed genes in the kidneys between SHR/Hos and Wistar-Kyoto rats (WKY/Hos) using an oligonucleotide microarray and analyzed the correlation between these genes and hypertension. In the present study, we analyzed the differentially expressed genes in the kidneys between SHR/NCrj and WKY/NCrj obtained from an other source to clarify the common and/or specific gene expression between the different sources. Furthermore, expression changes in the representative genes were characterized by Northern blot analysis using samples prepared from a third source, the Izm strain. The comparison revealed quite different changes in the differentially expressed genes among them. Sequence analysis of one of the differentially expressed genes, cytosolic epoxide hydrolase, revealed that two haplotypes could in part explain the expression level. Our study showed the complex nature of the genetic heterogeneity between SHR and WKY from different sources.

A newly identified 3'-untranslated region (UTR) polymorphism of the gene for skeletal muscle-specific glycogen-targeting subunit of protein phosphatase 1 (PPP1R3) was associated with insulin resistance and type 2 diabetes in Pima Indians (Xia J, Scherers W, Cohen PTW, Majer M, Xi T, Norman RA, Knowler WC, Bogardus C, Prochazka M: A common variant in PP1R3 associated with insulin resistance and type 2 diabetes. Diabetes 47:1519-1524, 1998). Thus, we investigated the frequency of polymorphism of the adenine- and thymine-rich element (ARE-1 and its variant ARE-2) in 426 Japanese type 2 diabetic and 380 nondiabetic subjects using a polymerase chain reaction (PCR)-restriction enzyme fragment length polymorphism (RFLP) method. The allele frequency of the ARE-2 variant in diabetic subjects was higher than that in nondiabetic subjects (0.34 vs. 0.29; P < 0.05), even though its frequency in Japanese subjects was lower (P < 0.001) than the reported value in Pima Indians (0.56). An aspartate polymorphism at codon 905 was 100% coupled to the ARE-2 allele, and its allele frequency was higher also in diabetic subjects. Although a serine substitution at codon 883 was partially linked with the ARE-2 allele, there was no difference between diabetic and nondiabetic subjects. These results indicate that the frequency of polymorphism of the PPP1R3 gene (ARE-2 and Asp905) is different between two ethnic groups and is increased in Japanese people with type 2 diabetes, suggesting that these variants may be a possible marker for searching for diabetogenic genes.

Complementary DNAs for angiotensin II type 1 receptor isoforms AT1A and AT1B were cloned by expression cloning from bovine adrenal and rat vascular smooth muscles. Human AT1 receptor was also cloned. Seven transmembrane structures emerged. The AT1 type receptor interacted with more than one type of G-proteins. The ligand binding site of AT1 involving Arg167, Lys199, and Asp263 has been identified by site directed mutagenesis. The regulation of the receptors occur at many stages. The isoform, AT2, was also expression cloned from rat pheochromocytoma cells. Although its ligand binding is not affected by stable GTP analogs, it is a seven transmembrane domain receptor. It mediates the modulations of phosphotyrosine phosphatase by angiotensin II and AT2 specific CGP42112A. The modulation was abolished by pertussis toxin. Thus, AT2 belongs to a new class of angiotensin receptors with unique signalling and regulatory mechanisms. AT1 mediates cellular growth. Interestingly, AT2 expression is inversely related to the mitogenic activity of cells.

Molecular cloning of a complementary DNA to rat cyclophilin-like protein mRNA. Using the technique of differential plaque filter hybridization, a rat cDNA was isolated whose corresponding gene expression in the kidney was positively modulated up to threefold by sodium depletion. This mRNA was more abundantly expressed in the kidneys of 17-week-old spontaneously hypertensive rats than those of age-matched Wistar-Kyoto rats. The putative protein encoded by this cDNA is a homologue of cyclophilin, a cytosolic binding protein for cyclosporin A. This cyclophilin-like protein mRNA was expressed in all the tissues examined, including the adrenal, atrium, brain, kidney, liver, lung, spleen, and ventricle. Sodium depletion in rats increased the expression level of this mRNA not only in the kidney but also in the liver. The administration of cyclosporin A in rats increased the expression level of this mRNA in the kidneys and livers. By virtue of its possible involvement in sodium homeostosis and its homology to cyclophilin, this molecule might have significant implications in the mechanism of cyclosporine-induced renal insufficiency and hypertension.

Cultured human vascular smooth muscle cells (VSMC) produced an immunologically specific renin-like enzyme. The production of the renin-like enzyme was increased by 1 mM N6-O2'-dibutyryladenosine 3',5' cyclic monophosphoric acid sodium salt (db-cAMP). When co-cultured with human vascular endothelial cells (EC) and human skin fibroblast (FB), the basal production of the renin-like enzyme was increased. And the extent of the increase by db-cAMP was amplified by co-culture with EC, but not by the co-culture with FB.