Phenolic and anthocyanin fractions from wild blueberries (V. angustifolium) differentially modulate endothelial cell migration partially through RHOA and RAC1

The present study investigates the effect of anthocyanin (ACN), phenolic acid (PA) fractions, and their combination (ACNs:PAs) from wild blueberry powder (Vaccinum angustifolium) on the speed of endothelial cell migration, gene expression, and protein levels of RAC1 and RHOA associated with acute exposure to different concentrations of ACNs and PAs. Time-lapse videos were analyzed and endothelial cell speed was calculated. Treatment with ACNs at 60 μg/mL inhibited endothelial cell migration rate ( P ≤ 0.05) while treatment with PAs at 0.002 μg/mL ( P ≤ 0.0001), 60 μg/mL ( P ≤ 0.0001), and 120 μg/mL ( P ≤ 0.01) significantly increased endothelial cell migration rate compared with control. Moreover, exposure of HUVECs to ACNs:PAs at 8:8 μg/mL ( P ≤ 0.05) and 60:60 μg/mL increased ( P ≤ 0.001) endothelial cell migration. Gene expression of RAC1 and RHOA significantly increased 2 hours after exposure with all treatments. No effect of the above fractions was observed on the protein levels of RAC1 and RHOA. Findings suggest that endothelial cell migration is differentially modulated based on the type of blueberry extract (ACN or PA fraction) and is concentration-dependent. Future studies should determine the mechanism of the differential action of the above fractions on endothelial cell migration.     

Effects of acute hypobaric hypoxia on the appearance of ingested deuterium from a deuterium oxide-labelled carbohydrate beverage in body fluids of humans during prolonged cycling exercise

To determine whether or not acute hypobaric hypoxia alters the rate of water absorption from a carbohydrate beverage ingested during exercise, six men cycled for 80 min on three randomly assigned different occasions. In one trial, exercise was performed in hypoxia (barometric pressure, P(B) = 594 hPa, altitude 4,400 m) at an exercise intensity selected to elicit 75% of the individual's maximal oxygen uptake (VO2max) previously determined in such conditions. In the two other experiments, the subjects cycled in normoxia (P(B) = 992 hPa) at the same absolute and the same relative intensities as in hypoxia, which corresponded to 55% and 75%, respectively, of their VO2max determined in normoxia. The subjects consumed 400 ml of a 12.5% glucose beverage just prior to exercise, and 250 ml of the same drink at 20, 40 and 60 min from the beginning of exercise. The first drink contained 20 ml of deuterium oxide to serve as a tracer for the entry of water into body fluids. The heart rate (HR) during exercise was higher in hypoxia than in normoxia at the same absolute exercise intensity, whereas it was similar to HR measured in normoxia at the same relative exercise intensity. Both in normoxia and hypoxia, plasma noradrenaline concentrations were related to the relative exercise intensity up to 40 min of exercise. Beyond that duration, when exercise was performed at the highest absolute power in normoxia, the noradrenaline response was higher than in hypoxia at the same relative exercise intensity. No significant differences were observed among experimental conditions, either in temporal profiles of plasma D accumulation or in elimination of water ingested in sweat. Conversely, elimination in urine of the water ingested appeared to be related to the severity of exercise, either high absolute power or the same relative power combined with hypoxia. We concluded that water absorption into blood after drinking a 12.5% glucose beverage is not altered during cycling exercise in acute hypobaric hypoxia. It is suggested that the elimination of water ingested in sweat and urine may be dependent on local circulatory adjustments during exercise.     

Particle-based multidimensional multispecies biofilm model

In this paper we describe a spatially multidimensional (two-dimensional [2-D] and three-dimensional [3-D]) particle-based approach for modeling the dynamics of multispecies biofilms growing on multiple substrates. The model is based on diffusion-reaction mass balances for chemical species coupled with microbial growth and spreading of biomass represented by hard spherical particles. Effectively, this is a scaled-up version of a previously proposed individual-based biofilm model. Predictions of this new particle-based model were quantitatively compared with those obtained with an established one-dimensional (1-D) multispecies model for equivalent problems. A nitrifying biofilm containing aerobic ammonium and nitrite oxidizers, anaerobic ammonium oxidizers, and inert biomass was chosen as an example. The 2-D and 3-D models generally gave the same results. If only the average flux of nutrients needs to be known, 2-D and 1-D models are very similar. However, the behavior of intermediates, which are produced and consumed in different locations within the biofilm, is better described in 2-D and 3-D models because of the multidirectional concentration gradients. The predictions of 2-D or 3-D models are also different from those of 1-D models for slowly growing or minority species in the biofilm. This aspect is related to the mechanism of biomass spreading or advection implemented in the models and should receive more attention in future experimental studies.     

Synthesis of dihydronaphthofurandiones and dihydrofuroquinolinediones with trypanocidal activity and analysis of their stereoelectronic properties

The synthesis of dihydronaphthofurandione and dihydrofuroquinolinedione derivatives 4-11 was performed through Diels-Alder reactions of dihydrobenzofurandione 1 with several carbodienes and acrolein N,N-dimethylhydrazone. Then, the use of 5-bromobenzofurandione 2 toward 1,3-pentadiene and the 1-azadiene afforded quinones 6 and 11 with a total regioselectivity. All the prepared quinones were tested for trypanocidal activity in vitro against Trypanosoma epimastigotes, Tulahuen strain. Among the tested compounds, the furoquinolinediones 10 and 11 have shown potent trypanocidal activities but, only the 1,5-regioisomer (11) was found active as a redox cycling agent. Calculation of their stereoelectronic properties by the density-functional theory method provided a new insight for the trypanocidal activity of these heterocyclic quinones.     

Determination of membrane protein stability via thermodynamic coupling of folding to thiol-disulfide interchange

Although progress has been made in understanding the thermodynamic stability of water-soluble proteins, our understanding of the folding of membrane proteins is at a relatively primitive level. A major obstacle to understanding the folding of membrane proteins is the discovery of systems in which the folding is in thermodynamic equilibrium, and the development of methods to quantitatively assess this equilibrium in micelles and bilayers. Here, we describe the application of disulfide cross-linking to quantitatively measure the thermodynamics of membrane protein association in detergent micelles. The method involves initiating disulfide cross-linking of a protein under reversible redox conditions in a thiol-disulfide buffer and quantitative assessment of the extent of cross-linking at equilibrium. The 19-46 alpha-helical transmembrane segment of the M2 protein from the influenza A virus was used as a model membrane protein system for this study. Previously it has been shown that transmembrane peptides from this protein specifically self-assemble into tetramers that retain the ability to bind to the drug amantadine. We used thiol-disulfide exchange to quantitatively measure the tetramerization equilibrium of this transmembrane protein in dodecylphosphocholine (DPC) detergent micelles. The association constants obtained agree remarkably well with those derived from analytical ultracentrifugation studies. The experimental method established herein should provide a broadly applicable tool for thermodynamic studies of folding, oligomerization and protein-protein interactions of membrane proteins.     

Synthesis and in vitro trypanocide activity of several polycyclic drimane-quinone derivatives

The Diels-Alder reaction between two polygodial-derived dienes and simple quinones to yield substituted naphtho- and anthraquinones, is described. The in vitro trypanocide activity for the series was determined. Two of the new compounds showed an activity ten and two times higher, respectively, than nifurtimox and benznidazole, the medicines of choice for the treatment of the acute Chagas' disease.     

Metabolism of the unnatural anticancer lipid safingol,L-threo-dihydrosphingosine,in cultured cells

We studied the metabolism of radioactively labeled safingol (l-threo-dihydrosphingosine) in primary cultured neurons, B104 neuroblastoma cells, and Swiss 3T3 fibroblasts, and compared it to that of its natural stereoisomer d-erythro-dihydrosphingosine. Both sphingoid bases are used as biosynthetic precursors for complex sphingolipids, albeit to different rates. Whereas a considerable amount of the natural sphingoid base is also directed to the catabolic pathway (20-66%, cell type dependent), only a minor amount of the nonnatural safingol is subjected to catabolic cleavage, most of it being N-acylated to the respective stereochemical variant of dihydroceramide. Interestingly, N-acylation of safingol to l-threo-dihydroceramide is less sensitive to fumonisin B1 than the formation of the natural d-erythro-dihydroceramide. In addition, safingol-derived l-threo-dihydroceramide, unlike its physiologic counterpart, is not desaturated. Most of it either accumulates in the cells (up to 50%) or is used as a biosynthetic precursor of the respective dihydrosphingomyelin (up to 45%). About 5% is, however, glucosylated and channeled into the glycosphingolipid biosynthetic pathway. Our results demonstrate that, despite its nonnatural stereochemistry, safingol is recognized and metabolized preferentially by enzymes of the sphingolipid biosynthetic pathway. Furthermore, our data suggest that the cytotoxic potential of safingol is reduced rather than enhanced via its metabolic conversion.