Development of bacterial cell-based system for intracellular antioxidant activity screening assay using green fluorescence protein (GFP) reporter

The novel bacterial cell-based assay was developed for evaluating the intracellular antioxidant activity. The genetically engineered Escherichia coli strains harboring the fusions of sodA::gfp and fumC::gfp were constructed and applied as reporters in response to cellular superoxide stress. Using this assay, twelve pure compounds and three Thai medicinal plants were investigated for intracellular antioxidant activity in comparison with conventional chemical-based assays; 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide dismutase (SOD) activity assays. Both strains demonstrated that quercetin and α- tocopherol exhibited the most potent and significant antioxidant activity with more than 60% reduction of intracellular superoxide. These compounds also showed high DPPH radical scavenging activity. Interestingly, gallic, caffeic and protocatechuic acids had the most significant DPPH radical scavenging and SOD-like activities but with moderate to weak intracellular antioxidant activity. Our hypothesis was that the lower intracellular antioxidant activity possibly occurs due to poor permeability of compounds into biological membrane based on their structures. Moreover, our results demonstrated that intracellular antioxidant activity of three plant extracts well correlated to results from DPPH assay. Our bacterial-based assay is simple, reproducible, very specific and applicable as an alternative screening tool for assessing the activity of compounds and plant extracts affecting cellular oxidative stress.Key words: Bacterial cell-based assay, antioxidant activity, oxidative stress, superoxide dismutase, fumarase, green fluorescence protein (GFP) reporter, plant extracts

Increase membrane vesiculation in essential hypertension

Background: A hypertensive condition is known to be an important risk factor for arterial disease and thrombotic events. The detailed understanding of the pathophysiology in thrombotic events is crucial for the development of both preventive measures and the treatment during the early stage. Microparticles are submicron cell membrane vesicle shed from the cell surface in response to cell injury or apoptosis, and they are an essential element in the process leading to the pathogenesis of thrombosis and hemostasis dysfunction in patients. Activation of blood cells can result in the formation of microparticles, which carry a negatively charged, phosphatidylserine (PS), with a diameter of <1.0 micron. The biological and clinical functions of microparticles have been highlighted in coronary artery disease and heart failure. Objective: To elucidate the functional role of microparticles in essential hypertension. Methods: We quantitated the total number of circulating PS+ microparticles and studied the role of PS+ microparticles to see whether they can shorten the plasma recalcification time in patients with essential hypertension. Results: The PS+ microparticles were detectable at a low level in healthy blood and significantly increased in the patients with essential hypertension. With regard to PS+ microparticles affecting prothrombotic state in hypertension, we enriched microparticles and determined their procoagulant activity with a plasma recalcification time. The clotting time was significantly reduced after the addition of enriched microparticles to plasma poor microparticles (PPMP). A significant negative correlation was detected between numbers of enriched-PS+ microparticles and plasma-clotting time (r=-0.43, P=0.01). Conclusion: Taken together, high levels of PS+ microparticles are present in the circulating blood of essential hypertension and may contribute to the generation and perpetuation of a thrombotic state

High-fat diet-induced plasma protein and liver changes in obese rats can be attenuated by melatonin supplementation

Obesity triggers changes in protein expression in various organs that might participate in the pathogenesis of obesity. Melatonin has been reported to prevent or attenuate such pathological protein changes in several chronic diseases. However, such melatonin effects on plasma proteins have not yet been studied in an obesity model. Using a proteomic approach, we investigated the effect of melatonin on plasma protein profiles after rats were fed a high-fat diet (HFD) to induce obesity. We hypothesized that melatonin would attenuate abnormal protein expression in obese rats. After 10weeks of the HFD, animals displayed increased body weight and fat accumulation as well as increased glucose levels, indicating an obesity-induced prediabetes mellitus-like state. Two-dimensional gel electrophoresis and liquid chromatography-mass spectrometry/mass spectrometry revealed 12 proteins whose expression was altered in response to the HFD and the melatonin treatment. The altered proteins are related to the development of liver pathology, such as cirrhosis (α1-antiproteinase), thrombosis (fibrinogen, plasminogen), and inflammation (mannose-binding protein A, complement C4, complement factor B), contributing to liver steatosis or hepatic cell death. Melatonin treatment most probably reduced the severity of the HFD-induced obesity by reducing the amplitude of HFD-induced plasma protein changes. In conclusion, we identified several potential biomarkers associated with the progression of obesity and its complications, such as liver damage. Furthermore, our findings reveal melatonin's beneficial effect of attenuating plasma protein changes and liver pathogenesis in obese rats.journal article2017 Jun2017 05 05importe