Objectives To examine the expression and activity of the calcium dependent NADPH oxidase in human atherosclerotic coronary arteries. Background The Nox based NADPH oxidases are major sources of reactive oxygen species (ROS) in human vessels. Several Nox homologs have been identified but their relative contribution to vascular ROS production in coronary artery disease (CAD) is unclear. Nox5 is a unique homolog in that it is calcium dependent and thus could be activated by vasoconstrictor hormones. Its presence has not yet been studied in human vessels. Methods Coronary arteries from patients undergoing cardiac transplant with CAD or without CAD were studied. Nox5 was quantified and visualized using Western blotting, immunofluorescence and quantitative real-time PCR. Calcium dependent NADPH oxidase activity, corresponding greatly to Nox5 activity was measured by electron paramagnetic resonance. Results Both western blotting and quantitative real time PCR indicated a marked increase in Nox5 protein and mRNA in CAD vs non CAD vessels. Calcium dependent NADPH driven production of reactive oxygen species in vascular membranes, reflecting Nox5 activity was increased 7 fold in CAD and correlated significantly with Nox5 mRNA levels among subjects. Immunofluorescence demonstrated that Nox5 was expressed in the endothelium in the early lesions and in vascular smooth muscle cells in the advanced in coronary lesions. Conclusions These studies identify Nox5 as a novel, calcium dependent source of reactive oxygen species in atherosclerosis.

Objective-Impaired endothelial function, characterized by nitric oxide scavenging by increased superoxide production, is a hallmark of vascular disease states. However, molecular mechanisms regulating superoxide production in human blood vessels remain poorly defined. Methods and Results-We compared endothelial function, vascular superoxide production, and the expression of NAD(P)H oxidase subunits in arteries and veins from patients undergoing coronary bypass surgery (nϭ86). Superoxide release was similar in arteries and veins. Inhibitor studies revealed that the NAD(P)H oxidase system was a quantitatively and proportionately greater source of superoxide in veins, whereas xanthine oxidase also contributed significantly to superoxide production in arteries. Moreover, NAD(P)H oxidase molecular composition differed in veins and arteries; veins expressed more nox2 and p22phox, whereas the relative expression of nox4 was greater in arteries. However, there were strong correlations between p22phox and nox4 expression and between superoxide production, NAD(P)H oxidase activity, and endothelial function in arteries and veins from the same patient. Conclusions-In individuals with coronary artery disease, changes in vascular superoxide production, endothelial function, and NAD(P)H oxidase activity and expression are related in veins and arteries. These findings highlight the importance of systemic effects on the molecular regulation of the NAD(P)H oxidases in human vascular disease. Key Words: endothelium Ⅲ oxidant stress Ⅲ reactive oxygen species Ⅲ nitric oxide Ⅲ NAD(P)H oxidase O xidative stress plays an important role in the pathogenesis of atherosclerosis, hypertension, and other vascular diseases. In particular, overproduction of superoxide anion may be detrimental because of its rapid interaction with nitric oxide (NO), which leads to the loss of NO bioavailability and reduces its anti-atherogenic effects. 1 Superoxide also regulates redoxsensitive signaling pathways, acts as a direct vascular smooth muscle cell (VSMC) mitogen, and modulates vessel remodeling and plaque stability. 1 Recent studies indicate that patients with endothelial dysfunction in whom arterial superoxide production is increased are at highest risk for vascular morbidity and mortality. 2 The sources of vascular superoxide include NAD(P)H oxidases, xanthine oxidase, cyclooxygenases, nitric oxide synthases, or mitochondrial oxidases. 1,3,4 In particular, NAD(P)H oxidases have been identified as a major enzyme system involved in the generation of vascular oxidative stress. 5 Recent studies have revealed several molecular homologs of the NAD(P)H oxidase large subunit (termed nox-nonphagocytic oxidase). The molecular composition of vascular NAD(P)H oxidases appears to vary in different cell types and at different stages of atherosclerotic plaques. 3 However, the molecular regulation of the NAD(P)H oxidases within atherosclerotic plaques may be more relevant to plaque events, such as rupture, rather than reflecting systemic changes related to global ...

Background: PCR methods are the most commonly used DNA-based identity tool in the commercial food, beverage, and natural health product markets. These methods are routinely used to identify foodborne pathogens and allergens in food. Proper validation methods for some sectors have been established, while there are none in other markets, such as botanicals. Results: A survey of the literature indicates that some validation criteria are not addressed when developing PCR tests for botanicals. Objective: We provide recommendations for qualitative real-time PCR methods for validating identity tests for botanical ingredients. Methods: These include common criteria that underpin the development and validation of rigorous tests, including (1) the aim of the validation test, (2) the applicability of different matrix variants, (3) specificity in identifying the target species ingredient, (4) sensitivity in detecting the smallest amount of the target material, (5) repeatability of methods, (6) reproducibility in detecting the target species in both raw and processed materials, (7) practicability of the test in a commercial laboratory, and (8) comparison with alternative methods. In addition, we recommend additional criteria, according to which the practicability of the test method is evaluated by transferring the method to a second laboratory and by comparison with alternative methods. Conclusions and Highlights: We hope that these recommendations encourage further publication on the validation of PCR methods for many botanical ingredients. These properly validated PCR methods can be developed on small, real-time biotechnology that can be placed directly into the supply chain ledger in support of highly transparent data systems that support QC from the farm to the fork of the consumer.