Different applications of polymerases with and without proofreading activity in single-nucleotide polymorphism analysis

With the completion of the human genome project, single-nucleotide polymorphisms (SNPs) have become the focus of intense study in biomedical research. Polymerase-mediated primer extension has been employed in a variety of SNP assays. However, these SNP assays using polymerase without proofreading function are compromised by their low reliability. Using a newly developed short amplicon harboring restriction enzyme site, EcoR-I, we were able to compare the single-base discrimination abilities of polymerases with and without proofreading function in primer extension in a broad range of annealing temperatures. Thermodynamic analysis demonstrated a striking single-nucleotide discrimination ability of polymerases with proofreading function. Using unmodified 3'-end allele-specific primers, only template-dependent products were generated by polymerase with proofreading activity. This powerful single-base discrimination ability of exo(+) polymerases was further evaluated in primer extension using three types of 3' terminally modified allele-specific primers. As compared with the poor fidelity in primer extension of polymerases lacking 3' exonuclease activity, this study provides convincing evidence that the use of proofreading polymerases in combination with 3'-end modified allele-specific primers can be a powerful new strategy for the development of SNP assays.     

Universally conserved and yeast-specific U1 snRNA sequences are important but not essential for U1 snRNP function

To study the contribution of the large, 568-nucleotide yeast (Saccharomyces cerevisiae) U1 snRNA to pre-mRNA splicing, we generated mutations in two regions of the molecule and introduced each mutant gene back into yeast as the sole copy of the U1 snRNA gene. We mutagenized the "A loop," a subregion highly conserved in primary sequence in all U1 snRNA molecules analyzed to date. We also mutagenized a portion of the yeast core subdomain, a region conserved in primary and secondary structure among several yeast species but absent from the much smaller metazoan U1 molecule. Surprisingly, mutations in these two regions had little or no effect on growth rate, yet several of them affected an inefficiently spliced reporter gene construct. In addition, combinations of mutants in both regions gave rise to reduced growth rates. Using the latter assay, we confirmed some of the proposed secondary structure of the yeast core domain. The experiments indicate that both regions contribute to U1 snRNP activity but that mutations in a single region do not have a substantial effect on growth rate because U1 snRNP activity is not rate-limiting for growth.     

假鹰爪化学成分研究(Ⅱ)

目的：寻找番荔枝科假鹰爪属植物假鹰爪Ｄｅｓｍｏｓ ｃｈｉｎｅｎｓｉｓ抗肿瘤活性成分。方法：采用硅胶柱色谱分离化学成分。根据理化性质及波谱分析等方法鉴定结构。结果：从氯仿萃取物中分离鉴定了７个化合物,分别是ｕｎｏｎ－ａｌ（Ⅰ）,黄芩素－７－甲醚（Ⅱ）,去甲氧杜鹃花素（Ⅲ）,去甲氧杜鹃花素－７－甲醚（Ⅳ）,苯甲酸（Ⅴ）,β－谷甾醇（Ⅵ）和豆甾醇（Ⅶ）。结论：该种植中鉴定的黄酮类化合物均为β－环未取代,     

Physical activity and incident hypertension in black and white adults: the Atherosclerosis Risk in Communities Study

BACKGROUND: The epidemiologic observation that physical activity reduces the risk for hypertension has only been made for white men who self-reported hypertension. This study examined physical activity and clinically determined incident hypertension in black and white men and women of the Atherosclerosis Risk in Communities (ARIC) Study. METHODS: ARIC is a population-based prospective study with four U.S. clinic centers. The present analyses included 7,459 black and white adults 45-65 years of age. Hypertension (systolic/diastolic blood pressure >/= 140/90 mm Hg) was defined by blood pressure measured by a random-zero device or medication use. Physical activity was assessed with the Baecke questionnaire. RESULTS: After adjustment for age, baseline blood pressure, ARIC center, education, body mass index, waist-hip ratio, parental history of hypertension, cigarette smoking, alcohol consumption, and diet, white men in the highest quartile of leisure activity (primarily cycling and walking) had a 34% lower odds of developing hypertension over 6 years compared to the least active (OR = 0.66, 95% CI = 0.47-0.94; P for quartile trend = 0.01). Baseline activity was not associated with incident hypertension in white women or blacks. CONCLUSIONS: Leisure-time physical activity reduces the odds of hypertension in middle-aged white men. Additional studies in women and blacks are needed.     

Prevalence and anatomic characteristics of infarct-like lesions on MR images of middle-aged adults: the atherosclerosis risk in communities study.

BACKGROUND AND PURPOSE: MR imaging has revealed putative evidence of subclinical cerebrovascular disease (CVD) as reflected by white matter signal changes and infarct-like lesions (ILLs). Nonetheless, the prevalence of this condition in the general population has been defined only to a limited extent. We herein report the prevalence and anatomic characteristics of ILLs seen on cranial MR images obtained as part of a population-based study of cardiovascular disease in middle-aged adults. These results are contrasted to those of previous similar studies, particularly those of an elderly population in the Cardiovascular Health Study (CHS). METHODS: This Atherosclerosis Risk in Communities (ARIC) cohort consists of a probability sample of community-living persons who were 55 to 72 years old at the time of MR examination. MR imaging of 1890 participants was performed at two ARIC field centers, based on a common protocol. MR studies were evaluated by trained readers at the MR Reading Center using original digital data displayed on a high-resolution workstation. The measures of lesion size, anatomic location, and signal intensity were collected. The definition for an ILL was a non-mass, hyperintense region with an arterial vascular distribution on spin-density and T2-weighted images. RESULTS: Two hundred ninety participants had ILLs, for an overall prevalence of 15.3%. Eighty-two percent of participants with ILLs had lesions that were 3 mm or larger in maximal dimension, although 87% of these lesions were 20 mm or smaller in maximal dimension. The prevalence of ILLs increased with age, from 7.9% in the 55- to 59-year-old age group to 22.9% in the 65- to 72-year-old age group (P < .001). Lesion prevalence was greater in black (20.7%) than in white persons (10.2% [P < .0001]), but did not differ significantly between male and female participants. The basal ganglia and thalamic region was the most commonly affected anatomic site, accounting for 78.9% of the lesions. CONCLUSION: Considering that the prevalence of self-reported stroke or transient ischemic attack in ARIC participants is 1.5%, these results suggest that there is significantly more subclinical than clinical CVD in the general population. Furthermore, the prevalence of this subclinical disease increases with age, and is greater in black persons. ILLs are dominated by "lacunae" in the basal ganglia and thalamus. These results are, in general, similar to those of a comparable study of elderly participants in the CHS, except for a 60% lower prevalence of ILLs in this younger population.     

Ambulatory urodynamic monitoring of external urethral sphincter behavior in chronic prostatitis patients

Aim: To study the behavior of external urethral sphincter in chronic prostatitis (CP) patient under natural filling.Methods: Twenty-one CP patients and 17 normal volunteers were involved in the study. Both the patients andvolunteers underwent ambulatory urodynamic monitoring (AM) and conventional medium filling cystometry (CMG).Urodec 500 was used for AM and Menuet for CMG. AM findings from CP patients were compared with those fromnormal volunteers, and the results from AM were compared with those from CMG. Results: In AM, the restingand voiding external urethral sphincter (EUS) pressures and maximum urethral closure pressures (MUCP) weresignificantly higher in CP patients [ ( 121.5 ±10.3) and (85.6±3.5) cm water, respectively ] than in normalvolunteers [ (77.6±11.4) and (10.3±1.6) cm water, respectively)]. Conclusion: The behavioral changes ofEUS in CP patients included spasm and instability of EUS, which were demonstrated using AM under natural filling;the findings were also in accord with the res     

CD18-mediated neutrophil recruitment contributes to the pathogenesis of reperfused but not nonreperfused stroke

BACKGROUND AND PURPOSE: Neutrophil (PMN) recruitment mediated by increased expression of intercellular adhesion molecule-1 expression (ICAM-1, CD54) in the cerebral microvasculature contributes to the pathogenesis of tissue injury in stroke. However, studies using blocking antibodies against the common beta2-integrin subunit on the PMN, the counterligand for ICAM-1 (CD18), have demonstrated equivocal efficacy. The current study tested the hypothesis that mice deficient in CD18 would be protected in the setting of reperfused but not nonreperfused stroke. METHODS: Two groups of mice were studied, those whose PMNs could express CD18 (CD18 +/+) and those mice hypomorphic for the CD-18 gene (CD18 -/-). PMNs obtained from CD18 -/- or CD18 +/+ mice were fluorescently labeled and tested for binding to murine brain endothelial monolayers. Using a murine model of focal cerebral ischemia in which an occluding suture placed in the middle cerebral artery (MCA) is removed after 45 minutes (transient ischemia, reperfused stroke) or left in place (permanent ischemia, nonreperfused stroke), cerebral infarct volumes (% ipsilateral hemisphere by TTC staining), cerebral blood flow (CBF, % contralateral hemisphere by laser-Doppler flowmetry), and survival (%) were examined 24 hours after the initial ischemic event. Adoptive transfer studies used 111In-labeled PMNs (from either CD18 +/+ or CD18 -/- mice) to examine the relative accumulation of PMNs in the ischemic region. RESULTS: PMNs obtained from CD18 -/- mice exhibit reduced adhesivity (compared with CD18 +/+ PMNs) for both quiescent and cytokine-activated endothelial monolayers. CD18 -/- mice (n=14) subjected to transient focal cerebral ischemia demonstrated a 53% decrease in infarct volumes versus CD18 +/+ mice (n=26, P<0.05), improved penumbral CBF at 24 hours (1.8-fold, P=0.02), and a 3.7-fold decrease in mortality (P=0.02). However, when CD18 -/- mice (n=12) were subjected to permanent focal cerebral ischemia, no differences were noted in infarct volume, mortality, or CBF versus similarly treated CD18 +/+ mice (n=10). There was a greater accumulation of CD18 +/+ PMNs in the ischemic zone of CD18 +/+ animals than CD18 -/- animals subjected to reperfused stroke (82% increase, P=0.02), although there was no difference between groups when subjected to permanent MCA occlusion. CONCLUSIONS: Deficiency for the CD18 gene confers cerebral protection in a murine model of reperfused stroke, but this benefit does not extend to CD18-deficient animals subjected to permanent MCA occlusion. These data suggest that anti-PMN strategies should be targeted to reperfused stroke and may perhaps be used in conjunction with thrombolytic therapy that establishes reperfusion.