Background: and Purpose To investigate the association between derivatives of reactive oxygen metabolites (d-ROMs), biological antioxidant potential (BAP) and the risk of stroke by subtype, and ischemic heart disease (IHD). Methods: We employed a case cohort design consisting of cardiovascular disease cases (n=1,521; stroke, n=1,271; IHD, n=265) and a random sub-cohort (n=4,761) in a large Japanese population-based study. d-ROMs and BAP were measured in plasma samples collected between 1995 and 1999. Hazard ratios (HRs) were estimated using weighted Cox proportional hazards methods according to d-ROMs and BAP quartiles, adjusted for age, sex, area, and potential confounding factors. Results: Analysis revealed a positive association between d-ROMs and the risk of total stroke, ischemic stroke, IHD, and the composite outcome of ischemic stroke and IHD. The multivariable HRs and 95% confidence intervals (CIs) for the highest versus lowest quartiles for d-ROMs were 1.34 (95% CI: 1.11–1.63) for total stroke (P for trend<0.001), 1.47 (1.16–1.86) for ischemic stroke (P for trend<0.001), 1.47 (1.02–2.11) for IHD (P for trend=0.072), 1.47 (0.87–2.49) for subarachnoid hemorrhage (P for trend=0.101), and 1.01 (0.72–1.41) for intraparenchymal hemorrhage (P for trend=0.618). In contrast, no significant association was detected between BAP levels and the risk of cardiovascular disease. Conclusions Analysis revealed that d-ROMs levels were positively associated with the risk of ischemic stroke and heart disease but not with hemorrhagic stroke.

OBJECTIVESmall conductance calcium activated potassium channels type 2 (SK2) play a crucial role in atrial repolarization. It is difficult to acquire the full-length of its coded gene KCNN2 by RT-PCR with one step. We aim to get the full-length of KCNN2 gene and construct the plasmid by Overlapping PCR, and further more discuss the application of Overlapping PCR.

METHODSTotal RNA was extracted from human right atrial tissue and cDNA was acquired with reverse transcription. Overlapping PCR was conducted with three pairs of primers which were designed according to the sequence of KCNN2 (AY258141) gene. The expression plasmid of pIRES-hrGFP-SK2 was constructed by directed cloning with restriction enzyme site and identified by enzyme cutting and sequencing.

RESULTSThree parts of PCR amplification were consistent with predicted size. The sequence of the plasmid was consistent with the gene-bank data except two sites, however, which were the same as gene in different tissues.

CONCLUSIONThe expression plasmid pIRES-hrGFP-SK2 was constructed successfully. Overlapping PCR is a good choice for amplifying these genes with long size or low expression.

Base Sequence ; Gene Expression ; Humans ; Myocytes, Cardiac ; Plasmids ; genetics ; Polymerase Chain Reaction ; methods ; Small-Conductance Calcium-Activated Potassium Channels ; genetics