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Background: Thrombin is a serine protease known to be the final product of the coagulation cascade. However, thrombin plays other physiological roles in processes such as gastric contractions and vessel wound healing, and a state of coagulability is increased in patients with dilated cardiomyopathy (DCM). In this study, we investigate the role of thrombin in the pathogenesis of DCM. The purpose of this study is to clarify the role of thrombin in the pathogenesis of DCM and investigate the possibility of treatment against DCM by thrombin inhibition., Methods: We investigated the expression of thrombin in the left ventricles of five patients with DCM who underwent the Batista operation and four patients without heart disease. Furthermore, we investigated the involvement of thrombin in the development of DCM using knock-in mice with a deletion mutation of cardiac troponin T that causes human DCM (∆K210 knock-in mouse) (B6;129-Tnnt2 tm2Mmto ) and assessed the effects of a direct thrombin inhibitor, dabigatran on ∆K210 knock-in mice using echocardiographic examinations, the Kaplan-Meier method and Western blotting., Results: The immunohistochemical analysis showed a strong thrombin expression in the DCM patients compared to the patients without heart disease. In immunohistochemical analysis, a strong thrombin expression was observed in the heart tissues analysis in the ∆K210 knock-in mice. Dabigatran administration significantly improved fractional shortening according to the echocardiographic examination and the survival outcomes in ∆K210 knock-in mice., Conclusion: Tissue thrombin is involved in the pathogenesis of DCM and thrombin inhibition can be beneficial for the treatment of DCM., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

It is unknown how very short introns (<65 nt; termed 'ultra-short' introns) could be spliced in a massive spliceosome (>2.7 MDa) without steric hindrance. By screening an annotated human transcriptome database (H-InvDB), we identified three model ultra-short introns: the 56-nt intron in the HNRNPH1 (hnRNP H1) gene, the 49-nt intron in the NDOR1 (NADPH dependent diflavin oxidoreductase 1) gene, and the 43-nt intron in the ESRP2 (epithelial splicing regulatory protein 2) gene. We verified that these endogenous ultra-short introns are spliced, and also recapitulated this in cultured cells transfected with the corresponding mini-genes. The splicing of these ultra-short introns was repressed by a splicing inhibitor, spliceostatin A, suggesting that SF3b (a U2 snRNP component) is involved in their splicing processes. The 56-nt intron containing a pyrimidine-rich tract was spliced out in a lariat form, and this splicing was inhibited by the disruption of U1, U2, or U4 snRNA. In contrast, the 49- and 43-nt introns were purine-rich overall without any pyrimidine-rich tract, and these lariat RNAs were not detectable. Remarkably, shared G-rich intronic sequences in the 49- and 43-nt introns were required for their splicing, suggesting that these ultra-short introns may recruit a novel auxiliary splicing mechanism linked to G-rich intronic splicing enhancers., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Late gadolinium enhancement (LGE) of cardiac magnetic resonance imaging (MRI) represents myocardial fibrosis and may be related to the clinical outcome of various heart diseases. This study evaluated the relationship between LGE and cardiac events in hypertrophic cardiomyopathy (HCM) using a new scoring method., Methods and Results: This study retrospectively followed 46 HCM patients without heart failure symptoms for 3.8 ± 1.8 years. Gadolinium-enhanced cardiac MRI was performed in all patients. Cardiac events including newly developed heart failure or ventricular tachyarrhythmia were evaluated during the follow-up period. We evaluated the predictive factors to identify the patients with cardiac events. None of the risk factors reported to be related to poor outcome or the existence of LGE alone could predict cardiac events, which might be due to the small number of subjects investigated in this study. A new scoring method for LGE-positive areas (LGE score) was applied and higher LGE score can predict cardiac events in this study population., Conclusions: The proposed LGE score for cardiac MRI is considered to be a potentially valid method for assessing cardiac events in HCM patients., (Copyright © 2011 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.)

Background and Purpose: Metabolic syndrome and chronic kidney disease (CKD) have received attention as new risk factors for cardiovascular disease. This study evaluated differences in key risk factors between acute coronary syndrome (ACS) and stable angina pectoris (SAP) by using traditional coronary risk factors, metabolic syndrome, and CKD., Methods: Among 1890 consecutive patients admitted to our institution, we studied 140 patients with initially diagnosed ACS and 163 patients with initially diagnosed SAP and compared risk factors between the two groups. Next, the relationship between smoking status after the initial diagnosis of coronary artery disease (CAD) and the incidence of subsequent cardiac event was examined after discharge in 284 patients., Results: Adjusted multivariate analysis showed that only current smoking was an independent predictor of ACS (odds ratio, 2.20; 95% CI, 1.28-3.78; p=0.004) among all risk factors we examined. Treatment with a calcium-channel blocker had a preventive effect on ACS (odds ratio, 0.44; 95% CI, 0.26-0.75; p=0.003), but treatment with a beta-blocker did not. Patients who continued to smoke after CAD was diagnosed had a risk of cardiac events about 5 times that of smokers who quit (adjusted hazard ratio, 5.05; 95% CI, 1.33-19.20; p=0.02)., Conclusions: The risk factors were significantly different between initially diagnosed ACS and SAP. Smoking was a more important risk factor of initially diagnosed ACS. Smoking cessation might have a preventive effect on subsequent cardiac events. Also, we found that treatment with a calcium-channel blocker would help prevent ACS in Japanese patients., (Copyright 2010 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.)

A 65-year-old man with advanced renal cell carcinoma was admitted due to continuing chest pain at rest. Two weeks before his admission, sorafenib had been started. He was diagnosed with non-ST-elevation myocardial infarction by laboratory data and electrocardiogram. Enhanced heart magnetic resonance imaging also showed subendocardial infarction. However, there was no stenosis in coronary arteries on angiography. Coronary artery spasm was induced by a provocative test. Cessation of sorafenib and administration of Ca-channel blocker and nitrates ameliorated his symptoms, but relapse occurred after resumption of sorafenib. Addition of oral nicorandil reduced his symptoms and maintained stable angina status. We report the first case of sorafenib-induced coronary artery spasm. Sorafenib is a multikinase inhibitor that targets signaling pathways necessary for cellular proliferation and survival. On the other hand, the Rho/ROCK pathway has an important role in the pathogenesis of coronary artery spasm. Our report may show an adverse effect on the Rho/ROCK pathway by sorafenib use.

Ruthenium (II) complex of mesoporphyrin-IX was incorporated into apomyoglobin to prepare artificial ruthenium myoglobin (RuMb) containing the ruthenium porphyrin in place of protoheme. The electrochemical and spectral characteristics (i.e., UV and CD spectra) of RuMb were investigated in comparison with wild type myoglobin. The effect of the metal center on the redox properties of myoglobin is directly observed by electrochemical analysis, all of which may be compared with similar measurements of the wild type myoglobin. Unlike other metal reconstituted myoglobins, i.e., cobalt myoglobin and manganese myoglobin, fast and reversible electron transfer properties were observed for RuMb, which is comparable with wild type myoglobin. The formal potential of 170 (+/-10) mV vs. Ag|AgCl (sat. KCl) of RuMb was directly determined for the first time by cyclic voltammetry, where the k(0)' value was estimated to be about 3(+/-0.2)x10(-4) cm s(-1) at pH 6.8. Mediatorless and reversible spectroelectrochemical behaviors were also observed using an optically transparent thin-layer electrode cell (OTTLE). The present results suggest that the major redox properties of the protein result from both the metal porphyrin center and globin environment. The novel redox properties predict that the engineered RuMb has analogous biofunctionalities to the wild type myoglobin in contrast to other metal reconstituted myoglobins.

Purine-rich exonic splicing enhancers (ESEs) stimulate splicing of the adjacent introns with suboptimal splice sites. To elucidate the mechanism regarding ESEs, factors specifically associated with ESEs in HeLa cell nuclear extracts were previously investigated, and shown to include SR (serine/arginine-rich) proteins. However, factors associated with ESEs in vivo have not yet been explored. Here we show that a GAA repeat RNA sequence, a typical ESE, is associated in Xenopus oocyte nuclei with at least one SR protein, SF2/ASF, as was expected. Moreover, components of SF3a/b complexes, U2 snRNA, and U2AF(65) were also found to be associated with the ESE in the nucleus. Since SF3a/b complexes are the constituents of the 17S U2 snRNP, these results suggest that the 17S U2 snRNP is associated with the ESE in the nucleus, probably through bridging interactions of U2AF and SR proteins. The identified factors may represent a functional splicing enhancer complex in vivo.

Background: Drugs that inhibit the renin-angiotensin-aldosterone system benefit patients at risk for or with existing cardiovascular disease. However, evidence for this effect in Asian populations is scarce. We aimed to investigate whether addition of an angiotensin receptor blocker, valsartan, to conventional cardiovascular treatment was effective in Japanese patients with cardiovascular disease., Methods: We initiated a multicentre, prospective, randomised controlled trial of 3081 Japanese patients, aged 20-79 years, (mean 65 [SD 10] years) who were undergoing conventional treatment for hypertension, coronary heart disease, heart failure, or a combination of these disorders. In addition to conventional treatment, patients were assigned either to valsartan (40-160 mg per day) or to other treatment without angiotensin receptor blockers. Our primary endpoint was a composite of cardiovascular morbidity and mortality. Analysis was by intention to treat. The study was registered at clintrials.gov with the identifier NCT00133328., Findings: After a median follow-up of 3.1 years (range 1-3.9) the primary endpoint was recorded in fewer individuals given valsartan than in controls (92 vs 149; absolute risk 21.3 vs 34.5 per 1000 patient years; hazard ratio 0.61, 95% CI 0.47-0.79, p=0.0002). This difference was mainly attributable to fewer incidences of stroke and transient ischaemic attack (29 vs 48; 0.60, 0.38-0.95, p=0.028), angina pectoris (19 vs 53; 0.35, 0.20-0.58, p<0.0001), and heart failure (19 vs 36; 0.53, 0.31-0.94, p=0.029) in those given valsartan than in the control group. Mortality or tolerability did not differ between groups., Interpretation: The addition of valsartan to conventional treatment prevented more cardiovascular events than supplementary conventional treatment. These benefits cannot be entirely explained by a difference in blood pressure control.

Objectives: Stress thallium-201 (201Tl) myocardial scintigraphy can demonstrate perfusion abnormalities, especially in the septum in patients with complete left bundle branch block (CLBBB) even with angiographically normal coronary arteries. Differences in the images between exercise and pharmacological stress 201Tl myocardial scintigraphy were evaluated in patients with CLBBB and normal coronary arteries., Methods: Forty-five patients with CLBBB underwent exercise stress using treadmill or pharmacological (adenosine triphosphate) stress 201Tl myocardial scintigraphy from October 1997 to February 2003. Patients with myocardial diseases were excluded, such as cardiomyopathy and coronary artery diseases detected by echocardiography and/or cardiac catheterization. The myocardial segment was classified according to the American Heart Association style for coronary artery disease., Results: Peak blood pressure levels and heart rates were significantly higher in the exercise stress group than in the pharmacological stress group (p < 0.001). The rate of defects in stress images was significantly higher in the exercise stress group (72.4%; 21/29 cases) than in the pharmacological stress group (18.8%; 3/16 cases) (p < 0.01). The rate of redistribution of observed defects in delayed images was 76.2% (16/21 cases) in the exercise stress group, and 0% (0/3 cases)in the pharmacological stress group (p < 0.01). The myocardial segments showing defects were different between the exercise stress group and the pharmacological stress group., Conclusions: Patients with CLBBB showed different frequencies of defects by stress 201Tl myocardial scintigraphy according to the stress method. Moreover, defects also occured in areas other than the septum. Blood pressure and heart rate were involved in the mechanisms of defects in left bundle branch block.

The rapid and reversible electron transfer reaction of cytochrome b562 was observed at an In2O3 electrode. The estimated heterogeneous electron transfer rate constant (k0') was k0' > or = 5.0 x 10(-3) cm s(-1) at pH 6.5. When the methionine-7 (Met-7) residue, which coordinates to the heme iron as an axial ligand, of the wild-type cytochrome b562 was replaced by an Ala or Gly residue, a water molecule bound to the heme iron and the electron transfer rate constants decreased to 1.3 x 10(-3) and 1.8 x 10(-3) cm s(-1), respectively. This decrease in the electron transfer rate would be due to the larger reorganization energy for the structural change at the redox site. The midpoint potential of cytochrome b562 was shifted negatively by approximately 135 mV by replacing Met-7 with Ala or Gly. Similar dissociation kinetics of cyanide for the mutated molecules as compared to native myoglobin was obtained.

Chromosomal translocation t(6;14)(p21.1;q32.3) has been reported as a rare but recurrent event not only in myeloma and plasma cell leukemia but also in diffuse large B-cell non-Hodgkin lymphoma (B-NHL) (diffuse large B-cell lymphoma [DLBCL]) and splenic lymphoma with villous lymphocytes (SLVL); however, the nature of the target gene(s) has not been determined. This study identified t(6;14)(p21.1;q32.3) in 3 cases of transformed extranodal marginal zone B-NHL, in 1 case of SLVL, and in 1 case of a low-grade B-cell lymphoproliferative disorder. In a sixth case, a CD5(+) DLBCL, the translocation was identified by molecular cloning in the absence of cytogenetically detectable change. Two chromosomal translocation breakpoints were cloned by using long-distance inverse polymerase chain reaction methods. Comparison with the genomic sequence for chromosome 6p21.1 showed breakpoints approximately 59 and 73.5 kilobases 5' of the cyclin D3 (CCND3) gene with no other identifiable transcribed sequences in the intervening region. Although Southern blotting with derived genomic 6p21.1 probes failed to detect other rearrangements, fluorescent in situ hybridization assays, using BAC (bacterial artificial chromosome) clones spanning and flanking the CCND3 locus, along with probes for IGH confirmed localization of 6p21.1 breakpoints within the same region, as well as fusion of the CCND3 and IGH loci. Furthermore, in all cases, high-level expression of CCND3 was demonstrated at RNA and/or protein levels by Northern and Western blotting and by immunohistochemistry. These data implicate CCND3 as a dominant oncogene in the pathogenesis and transformation in several histologic subtypes of mature B-cell malignancies with t(6;14)(p21.1;q32.3) and suggest that CCND3 overexpression seen in about 10% of DLBCL cases may have a genetic basis.

A simple flow injection analysis (FIA) system for residual chlorine in tap water has been developed by using a Pb(II) ion-selective electrode (ISE) detector. The method is based on a specific response of the Pb(II)-ISE to residual chlorine. The FIA system consists of a millivolt meter, a peristaltic pump, a Pb(II)-ISE detector and a recorder. A linear working curve between peak height and concentration of residual chlorine was obtained from 0.1 to 1 mg l(-1) for the developed FIA system. The relative standard deviation for repeated injections of a 0.2 mg l(-1) residual chlorine sample was 2%. The regression line and its correlation factor between the conventional o-tolidine colorimetric method and the present method were Y=0.75X+0.17 and 0.967, respectively, for this determination.