A Method to Measure Hydrolytic Activity of Adenosinetriphosphatases (ATPases).

A method to measure hydrolytic activity of adenosinetriphosphatases (ATPases).

PLoS One. 2013; 8(3): e58615
Bartolommei G, Moncelli MR, Tadini-Buoninsegni F

The detection of small amounts (nanomoles) of inorganic phosphate has a great interest in biochemistry. In particular, phosphate detection is useful to evaluate the rate of hydrolysis of phosphatases, that are enzymes able to remove phosphate from their substrate by hydrolytic cleavage. The hydrolysis rate is correlated to enzyme activity, an extremely important functional parameter. Among phosphatases there are the cation transporting adenosinetriphosphatases (ATPases), that produce inorganic phosphate by cleavage of the ?-phosphate of ATP. These membrane transporters have many fundamental physiological roles and are emerging as potential drug targets. ATPase hydrolytic activity is measured to test enzyme functionality, but it also provides useful information on possible inhibitory effects of molecules that interfere with the hydrolytic process. We have optimized a molybdenum-based protocol that makes use of potassium antimony (III) oxide tartrate (originally employed for phosphate detection in environmental analysis) to allow its use with phosphatase enzymes. In particular, the method was successfully applied to native and recombinant ATPases to demonstrate its reliability, validity, sensitivity and versatility. Our method introduces significant improvements to well-established experimental assays, which are currently employed for ATPase activity measurements. Therefore, it may be valuable in biochemical and biomedical investigations of ATPase enzymes, in combination with more specific tests, as well as in high throughput drug screening. HubMed – drug


Transscleral sustained vasohibin-1 delivery by a novel device suppressed experimentally-induced choroidal neovascularization.

PLoS One. 2013; 8(3): e58580
Onami H, Nagai N, Kaji H, Nishizawa M, Sato Y, Osumi N, Nakazawa T, Abe T

We established a sustained vasohibin-1 (a 42-kDa protein), delivery device by a novel method using photopolymerization of a mixture of polyethylene glycol dimethacrylate, triethylene glycol dimethacrylate, and collagen microparticles. We evaluated its effects in a model of rat laser-induced choroidal neovascularization (CNV) using a transscleral approach. We used variable concentrations of vasohibin-1 in the devices, and used an enzyme-linked immunosorbent assay and Western blotting to measure the released vasohibin-1 (0.31 nM/day when using the 10 ?M vasohibin-1 delivery device [10VDD]). The released vasohibin-1 showed suppression activity comparable to native effects when evaluated using endothelial tube formation. We also used pelletized vasohibin-1 and fluorescein isothiocyanate-labeled 40 kDa dextran as controls. Strong fluorescein staining was observed on the sclera when the device was used for drug delivery, whereas pellet use produced strong staining in the conjunctiva and surrounding tissue, but not on the sclera. Vasohibin-1 was found in the sclera, choroid, retinal pigment epithelium (RPE), and neural retina after device implantation. Stronger immunoreactivity at the RPE and ganglion cell layers was observed than in other retinal regions. Significantly lower fluorescein angiography (FA) scores and smaller CNV areas in the flat mounts of RPE-choroid-sclera were observed for the 10VDD, VDD (1 ?M vasohibin-1 delivery device), and vasohibin-1 intravitreal direct injection (0.24 ?M) groups when compared to the pellet, non-vasohibin-1 delivery device, and intravitreal vehicle injection groups. Choroidal neovascularization can be treated with transscleral sustained protein delivery using our novel device. We offer a safer sustained protein release for treatment of retinal disease using the transscleral approach. HubMed – drug


Sedentary behaviour and physical activity in South asian women: time to review current recommendations?

PLoS One. 2013; 8(3): e58328
Waidyatilaka I, Lanerolle P, Wickremasinghe R, Atukorala S, Somasundaram N, de Silva A

Our aims were to describe activity and sedentary behaviours in urban Asian women, with dysglycaemia (diagnosed at recruitment), and without dysglycaemia and examine the relative contribution of these parameters to their glycaemic status.2800 urban women (30-45 years) were selected by random cluster sampling and screened for dysglycaemia for a final sample of 272 newly diagnosed, drug naive dysglycaemic and 345 normoglycaemic women. Physical activity and sedentary behaviours were assessed by the International Physical Activity Questionnaire (IPAQ). Demographic data, diet and anthropometry were recorded. Logistic regression analysis assessed contribution of all parameters to dysglycaemia and exposure attributable fractions were calculated.The mean energy expenditure on walking (2648.5±1023.7 MET-min/week) and on moderate and vigorous physical activity (4342.3±1768.1 MET-min/week) for normoglycemic women and dysglycaemic women (walking;1046.4±728.4 MET-min/week, moderate and vigorous physical activity; 1086.7±1184.4 MET-min/week) was above the recommended amount of physical activity per week. 94.3% of women spent >1000 MET-minutes/week on activity. Mean sitting and TV time for normoglycaemic and dysglycaemic women were 154.3±62.8, 38.4±31.9, 312.6±116.7 and 140.2±56.5 minutes per day respectively. Physical activity and sedentary behaviour contributed to dysglycaemia after adjustment for family history, diet, systolic blood pressure and Body Mass Index. Exposure attributable fractions for dysglycaemia were; lower physical activity: 78%, higher waist circumference: 94%, and TV viewing time: 85%.Urban South Asian women are at risk of dysglycaemia at lower levels of sedentary behaviour and greater physical activity than western populations, indicating the need for re-visiting current physical activity guidelines for South Asians. HubMed – drug


Diversity and taxonomy of endophytic xylariaceous fungi from medicinal plants of dendrobium (orchidaceae).

PLoS One. 2013; 8(3): e58268
Chen J, Zhang LC, Xing YM, Wang YQ, Xing XK, Zhang DW, Liang HQ, Guo SX

spp. are traditional Chinese medicinal plants, and the main effective ingredients (polysaccharides and alkaloids) have pharmacologic effects on gastritis infection, cancer, and anti-aging. Previously, we confirmed endophytic xylariaceous fungi as the dominant fungi in several species of tropical regions from China. In the present study, the diversity, taxonomy, and distribution of culturable endophytic xylariaceous fungi associated with seven medicinal species of (Orchidaceae) were investigated. Among the 961 endophytes newly isolated, 217 xylariaceous fungi (morphotaxa) were identified using morphological and molecular methods. The phylogenetic tree constructed using nuclear ribosomal internal transcribed spacer (ITS), large subunit of ribosomal DNA (LSU), and beta-tubulin sequences divided these anamorphic xylariaceous isolates into at least 18 operational taxonomic units (OTUs). The diversity of the endophytic xylariaceous fungi in these seven species was estimated using Shannon and evenness indices, with the results indicating that the dominant Xylariaceae taxa in each species were greatly different, though common xylariaceous fungi were found in several species. These findings implied that different host plants in the same habitats exhibit a preference and selectivity for their fungal partners. Using culture-dependent approaches, these xylariaceous isolates may be important sources for the future screening of new natural products and drug discovery. HubMed – drug