Exercise training rescues impaired H2O2-mediated vasodilation in porcine collateral-dependent coronary arterioles through enhanced K+ channel activation.

We previously reported that exercise training drives enhanced agonist-stimulated hydrogen peroxide (H2O2) levels and restores endothelium-dependent dilation via an increased reliance on H2O2 in arterioles isolated from ischemic porcine hearts. In this study, we tested the hypothesis that exercise training would correct impaired H2O2-mediated dilation in coronary arterioles isolated from ischemic myocardium through increases in protein kinase G (PKG) and protein kinase A (PKA) activation and subsequent colocalization with sarcolemmal K+ channels. Female adult Yucatan miniature swine were surgically instrumented with an ameroid constrictor around the proximal left circumflex coronary artery, gradually inducing a collateral-dependent vascular bed. Arterioles (∼125 µm) supplied by the left anterior descending artery served as nonoccluded control vessels. Pigs were separated into exercise (treadmill; 5 days/wk for 14 wk) and sedentary groups. Collateral-dependent arterioles isolated from sedentary pigs were significantly less sensitive to H2O2-induced dilation compared with nonoccluded arterioles, whereas exercise training reversed the impaired sensitivity. Large conductance calcium-activated potassium (BKCa) channels and 4AP-sensitive voltage-gated (Kv) channels contributed significantly to dilation in nonoccluded and collateral-dependent arterioles of exercise-trained but not sedentary pigs. Exercise training significantly increased H2O2-stimulated colocalization of BKCa channels and PKA, but not PKG, in smooth muscle cells of collateral-dependent arterioles compared with other treatment groups. Taken together, our studies suggest that with exercise training, nonoccluded and collateral-dependent coronary arterioles better use H2O2 as a vasodilator through increased coupling with BKCa and 4AP-sensitive Kv channels; changes that are mediated in part by enhanced colocalization of PKA with BKCa channels.NEW & NOTEWORTHY The current study reveals that coronary arterioles distal to stenosis display attenuated dilation responses to H2O2 that are restored with endurance exercise training. Enhanced H2O2 dilation after exercise is dependent on Kv and BKCa channels and at least in part on in colocalization of BKCa channel and PKA and independent of PKA dimerization. These findings expand our earlier studies which demonstrated that exercise training drives beneficial adaptive responses of reactive oxygen species in the microvasculature of the ischemic heart.

Exercise induces superoxide and NOX4 contribution in endothelium-dependent dilation in coronary arterioles from a swine model of chronic myocardial ischemia.

Exercise training is an effective, nonpharmacologic therapy and preventative measure for ischemic heart disease. While recent studies have examined reactive oxygen species (ROS) as mediators of exercise training-enhanced coronary blood flow, specific oxidants and their sources have yet to be fully elucidated. We investigated the hypothesis that NADPH oxidase (NOX)-derived superoxide anion would contribute to vasodilation effects in the coronary microcirculation of swine and that these effects would be impaired by chronic ischemia and rescued with exercise training. Adult Yucatan miniature swine were instrumented with an ameroid occluder around the proximal left circumflex coronary artery, resulting in a collateral-dependent myocardial region. Eight weeks post-operatively, swine were randomly assigned to either a sedentary or exercise training (treadmill run; 5 days/week for 14 weeks) protocol. Coronary arterioles were isolated from nonoccluded and collateral-dependent myocardial regions and pressure myography was performed. Exercise training resulted in enhanced endothelium-dependent dilation after occlusion. Scavenging of superoxide via the superoxide dismutase (SOD)-mimetic, tempol, attenuated dilation in both nonoccluded and collateral-dependent arterioles of exercise-trained, but not sedentary swine. NOX1/4 inhibition with GKT136901 attenuated dilation after exercise training but only in collateral-dependent arterioles. High performance liquid chromatography revealed that neither ischemia nor exercise training significantly altered basal or bradykinin-stimulated superoxide levels. Furthermore, superoxide production was not attributable to NOX isoforms nor mitochondria. Immunoblot analyses revealed significantly decreased NOX2 protein after exercise with no differences in NOX1, NOX4, p22phox, SOD proteins. Taken together, these data provide evidence that superoxide and NOX4 independently contribute to enhanced endothelium-dependent dilation following exercise training.

Association of personal characteristics and cooking skills with vegetable consumption frequency among university students.

Most university students are transitioning to adulthood, and tend to adopt unhealthy eating habits characterised by a low intake of fruits and vegetables. Few studies have specifically addressed the consumption of vegetables, which have a high content of minerals, fibre, and active compounds. The aim of this investigation was to assess vegetable consumption frequency among university students in a Brazilian capital and examine associations with individual characteristics and cooking skills. This crosssectional study used a online questionnaire to collect data. Cooking skills were evaluated in eight dimensions. Vegetable consumption was recorded in five frequency groups and then categorised into daily and non-daily consumption. Data were subjected to Pearson's chi-squared or analysis of variance followed by Bonferroni post hoc test. Associations between cooking skill dimensions and daily vegetable consumption were identified by crude and adjusted logistic regression analyses.Adjustment was performed for sex, age, paternal education, and overweight/obesity.Results are expressed as odds ratios (p < 0.05). Less than half of the respondents (N = 237; 45%) reported consuming vegetables daily. Higher level of paternal education, not being overweight or obese, and higher scores on seven cooking skill dimensions were positively associated with daily vegetable consumption. Given the scarcity of research on the topic, the findings of this study make a significant contribution to knowledge and may support public health strategies for promoting vegetable consumption among university students.

Enhanced KCl-mediated contractility and Ca2+ sensitization in porcine collateral-dependent coronary arteries persist after exercise training.

We have previously reported enhanced Ca2+ sensitivity of coronary arteries that is dependent upon collateral circulation for their blood supply. For the current study, we hypothesized that small collateral-dependent arteries would exhibit an enhanced KCl-mediated contractile response attributable to Ca2+ sensitization and increased Ca2+ channel current. Ameroid constrictors were surgically placed around the left circumflex (LCX) artery of female Yucatan miniature swine. Eight weeks postoperatively, pigs were randomized into sedentary or exercise-trained (treadmill run; 5 days/wk; 14 wk) groups. Small coronary arteries (150-300 µm luminal diameter) were isolated from myocardial regions distal to the collateral-dependent LCX and the nonoccluded left anterior descending arteries. Contractile tension and simultaneous measures of both tension and intracellular free Ca2+ levels (fura-2) were measured in response to increasing concentrations of KCl. In addition, whole cell Ca2+ currents were also obtained. Chronic occlusion enhanced contractile responses to KCl and increased Ca2+ sensitization in collateral-dependent compared with nonoccluded arteries of both sedentary and exercise-trained pigs. In contrast, smooth muscle cell Ca2+ channel current was not altered by occlusion or exercise training. Ca2+/calmodulin-dependent protein kinase II (CaMKII; inhibited by KN-93, 0.3-1 µM) contributed to the enhanced contractile response in collateral-dependent arteries of sedentary pigs, whereas both CaMKII and Rho-kinase (inhibited by hydroxyfasudil, 30 µM or Y27632, 10 µM) contributed to increased contraction in exercise-trained animals. Taken together, these data suggest that chronic occlusion leads to enhanced contractile responses to KCl in collateral-dependent coronary arteries via increased Ca2+ sensitization, a response that is further augmented with exercise training.NEW & NOTEWORTHY Small coronary arteries distal to chronic occlusion displayed enhanced contractile responses, which were further augmented after exercise training and attributable to enhanced calcium sensitization without alterations in calcium channel current. The calcium sensitization mediators Rho-kinase and CaMKII significantly contributed to enhanced contraction in collateral-dependent arteries of exercise-trained, but not sedentary, pigs. Exercise-enhanced contractile responses may increase resting arterial tone, creating an enhanced coronary flow reserve that is accessible during periods of increased metabolic demand.

Over-expression of the cucumber expansin gene (Cs-EXPA1) in transgenic maize seed for cellulose deconstruction.

Plant cell wall degradation into fermentable sugars by cellulases is one of the greatest barriers to biofuel production. Expansin protein loosens the plant cell wall by opening up the complex of cellulose microfibrils and polysaccharide matrix components thereby increasing its accessibility to cellulases. We over-expressed cucumber expansin in maize kernels to produce enough protein to assess its potential to serve as an industrial enzyme for applications particularly in biomass conversion. We used the globulin-1 embryo-preferred promoter to express the cucumber expansin gene in maize seed. Expansin protein was targeted to one of three sub-cellular locations: the cell wall, the vacuole, or the endoplasmic reticulum (ER). To assess the level of expansin accumulation in seeds of transgenic kernels, a high throughput expansin assay was developed. The highest expressing plants were chosen and enriched crude expansin extract from those plants was tested for synergistic effects with cellulase on several lignocellulosic substrates. Activity of recombinant cucumber expansin from transgenic kernels was confirmed on these pretreated substrates. The best transgenic lines (ER-targeted) can now be used for breeding to increase expansin expression for use in the biomass conversion industry. Results of these experiments show the success of expansin over-expression and accumulation in transgenic maize seed without negative impact on growth and development and confirm its synergistic effect with cellulase on deconstruction of complex cell wall substrates.

A Mobile Phone App for the Provision of Personalized Food-Based Information in an Eating-Out Situation: Development and Initial Evaluation.

BACKGROUND: Increasing pressure from governments, public health bodies, and consumers is driving a need for increased food-based information provision in eating-out situations. Meals eaten outside the home are known to be less healthy than meals eaten at home, and consumers can complain of poor information on the health impact and allergen content of meals eaten out. OBJECTIVE: This paper aimed to describe the development and early assessment of a mobile phone app that allows the provision of accurate personalized food-based information while considering individual characteristics (allergies, diet type, and preferences) to enable informed consumer choice when eating out. METHODS: An app was designed and developed to address these requirements using an agile approach. The developed app was then evaluated at 8 public engagement events using the System Usability Scale (SUS) questionnaire and qualitative feedback. RESULTS: Consideration of the literature and consultation with consumers revealed a need for information provision for consumers in the eating-out situation, including the ability to limit the information provided to that which was personally relevant or interesting. The app was designed to provide information to consumers on the dishes available in a workplace canteen and to allow consumers the freedom to personalize the app and choose the information that they received. Evaluation using the SUS questionnaire revealed positive responses to the app from a range of potential users, and qualitative comments demonstrated broad interest in its use. CONCLUSIONS: This paper details the successful development and early assessment of a novel mobile phone app designed to provide food-based information in an eating-out situation in a personalized manner.

Subcellular targeting is a key condition for high-level accumulation of cellulase protein in transgenic maize seed.

Ethanol from lignocellulosic biomass is being pursued as an alternative to petroleum-based transportation fuels. To succeed in this endeavour, efficient digestion of cellulose into monomeric sugar streams is a key step. Current production systems for cellulase enzymes, i.e. fungi and bacteria, cannot meet the cost and huge volume requirements of this commodity-based industry. Transgenic maize (Zea mays L.) seed containing cellulase protein in embryo tissue, with protein localized to the endoplasmic reticulum, cell wall or vacuole, allows the recovery of commercial amounts of enzyme. E1 cellulase, an endo-beta-1,4-glucanase from Acidothermus cellulolyticus, was recovered at levels greater than 16% total soluble protein (TSP) in single seed. More significantly, cellobiohydrolase I (CBH I), an exocellulase from Trichoderma reesei, also accumulated to levels greater than 16% TSP in single seed, nearly 1000-fold higher than the expression in any other plant reported in the literature. The catalytic domain was the dominant form of E1 that was detected in the endoplasmic reticulum and vacuole, whereas CBH I holoenzyme was present in the cell wall. With one exception, individual transgenic events contained single inserts. Recovery of high levels of enzyme in T2 ears demonstrated that expression is likely to be stable over multiple generations. The enzymes were active in cleaving soluble substrate.

A sperm-associated WD repeat protein orthologous to Chlamydomonas PF20 associates with Spag6, the mammalian orthologue of Chlamydomonas PF16.

cDNAs were cloned for the murine and human orthologues of Chlamydomonas PF20, a component of the alga axoneme central apparatus that is required for flagellar motility. The mammalian genes encode transcripts of 1.4 and 2.5 kb that are highly expressed in testis. The two transcripts appear to arise from alternative transcription start sites. The murine Pf20 gene was mapped to chromosome 1, syntenic with the location of the human gene on chromosome 2. An antibody generated against an N-terminal sequence of mouse Pf20 recognized a 71-kDa protein in sperm and testis extracts. Immunocytochemistry localized Pf20 to the tails of permeabilized sperm; electron microscope immunocytochemistry showed that Pf20 was located in the axoneme central apparatus. A murine Pf20-green fluorescent protein fusion protein expressed in Chinese hamster ovary cells accumulated in the cytoplasm. When coexpressed with Spag6, the mammalian orthologue of Chlamydomonas PF16, Pf20 was colocalized with Spag6 on polymerized microtubules. Yeast two-hybrid assays demonstrated interaction of the Pf20 WD repeats with Spag6. Pf20 was markedly reduced in sperm collected from mice lacking Spag6, which are infertile due to a motility defect. Our observations provide the first evidence for an association between mammalian orthologues of two Chlamydomonas proteins known to be critical for axoneme structure and function.

Analysis of the maize polyubiquitin-1 promoter heat shock elements and generation of promoter variants with modified expression characteristics.

The maize polyubiquitin-1 (Ubi-1) promoter is one of a few select promoters used to express foreign genes in monocots, such that recombinant proteins can be produced at commercially viable levels. Modifying the activity, specificity and responsiveness of such promoters provides a means to achieve desired levels and patterns of expression of genes encoding target products. Ubi-1 is constitutively expressed but is further induced by heat shock. The promoter contains two overlapping sequences with similarity to defined heat shock elements and we show that these sequences are also present upstream of the Ubi-1 homologue isolated from teosinte. Both the maize and teosinte promoters can mediate a heat shock response in transgenic maize. We have dissected the overlapping maize Ubi-1 promoter heat shock elements and demonstrate that the 3' element is required to mediate a heat shock response. The Ubi-1 promoter is particularly active in tissues consisting of rapidly dividing cells, and within the seed it is strongly biased towards driving expression in the embryo. However, replacement of the heat shock elements with a trimer of a basic domain/leucine zipper factor binding site of a pea lectin promoter shifts the balance in seed expression towards the endosperm. The Ubi-1 variants described here differ in their overall activity in the seed, but they all show potential for driving high levels of heterologous gene expression in maize.