Studies of the reduction and protonation behavior of tetraheme cytochromes using atomic detail

A comparative study of tetraheme cytochrome c3 molecules from several species was carried out using recently developed theoretical methods based on continuum electrostatics. The binding joint equilibrium of electrons and protons was simulated, revealing the complete thermodynamic aspects of electron-proton coupling in these molecules. The method yields excellent accuracy in terms of midpoint potentials, giving the correct reduction orders in all molecules examined, except for one heme site. The coupling between electrons and protons is shown to be present and significant at physiological pH in all cases. This phenomenon, known as the redox-Bohr effect, though of thermodynamic nature, is shown to have an intrinsic "dynamic" character at the molecular level (in the sense of the empty/occupied fluctuations at the microscopic level), with the binding states of redox and protonatable sites displaying both correlated averages and correlated fluctuations. The protonatable sites more directly involved in the redox-Bohr effect are identified using, among other properties, the statistical correlation between pairs of sites, which automatically reflects indirect effects mediated by other sites. Several sites are identified in this analysis. Propionate D of heme I seems to be the most interesting, generally showing a high correlation not only with its own heme, but also with heme II, corresponding to an indirect stabilization of the reduced forms of both hemes. Other interesting sites are the free histidines of two of the cytochromes and propionate D of heme IV, the latter being potentially associated with redox-induced structural changes. Among the set of cytochromes c3 analyzed in this study, significant differences are observed for several properties of the acidic cytochrome included in the set, from Desulfovibrio africanus, supporting the hypothesis of a different functional role.     

Virulence factors in food,clinical and reference Enterococci: A common trait in the genus?

The occurrence of several virulence traits (cytolysin, adhesins and hydrolytic enzymes) was investigated in a collection of 164 enterococci, including food and clinical isolates (from human and veterinary origin), as well as type and reference strains from 20 enterococcal species. Up to fifteen different cyl genotypes were found, as well as silent cyl genes. The occurrence of the cyl operon and haemolytic potential seems to be widespread in the genus. A significant association of this virulent trait with clinical isolates was found (p < 0.05). High levels of incidence were also observed for genes encoding surface adhesins (esp, efaA(fs), efaA(fm)), agg and gelE, irrespectively of species allocation and origin of strains. Although gelE behaves as silent in the majority of the strains, gelatinase activity predominates in clinical isolates, whereas lipase and DNase were mainly detected in food isolates pointing to their minor role as virulence determinants. No hyaluronidase activity was detected for all strains. Numerical hierarchic data analysis grouped the strains in three main clusters, two of them including a total of 50 strains with low number of virulence determinants (from 2 to 7) and the other with 114 strains with a high virulence potential (up to 12 determinants). No statistical association was found between virulence clusters and species allocation (p > 0.10), strongly suggesting that virulence determinants are a common trait in the genus Enterococcus. Clinical strains seem to be significantly associated with high virulence potential, whereas food, commensal and environmental strains harbour fewer virulence determinants (p < 0.01). A high level of relative diversity in virulence patterns was observed (Shannon's index varies from 0.95 to 1.0 among clusters), reinforcing the strain-specific nature of the association of virulence factors. Although a low risk seems to be associated with the use of enterococci in long-established artisanal cheeses, screening of virulence traits and their cross-synergies must be performed, particularly for commercial starters, probiotic strains and products to be used by high risk population groups.     

Hepatic stellate cells uptake of retinol associated with retinol-binding protein or with bovine serum albumin

Retinol is stored in liver, and the dynamic balance between its accumulation and mobilization is regulated by hepatic stellate cells (HSC). Representing less than 1% total liver protein, HSC can reach a very high intracellular retinoid (vitamin-A and its metabolites) concentration, which elicits their conversion from the myofibroblast to the fat-storing lipocyte phenotype. Circulating retinol is associated with plasma retinol-binding protein (RBP) or bovine serum albumin (BSA). Here we have used the in vitro model of GRX cells to compare incorporation and metabolism of BSA versus RBP associated [(3)H]retinol in HSC. We have found that lipocytes, but not myofibroblasts, expressed a high-affinity membrane receptor for RBP-retinol complex (KD = 4.93 nM), and both cell types expressed a low-affinity one (KD = 234 nM). The RBP-retinol complex, but not the BSA-delivered retinol, could be dislodged from membranes by treatments that specifically disturb protein-protein interactions (high RBP concentrations). Under both conditions, treatments that disturb the membrane lipid layer (detergent, cyclodextrin) released the membrane-bound retinol. RBP-delivered retinol was found in cytosol, microsomal fraction and, as retinyl esters, in lipid droplets, while albumin-delivered retinol was mainly associated with membranes. Disturbing the clathrin-mediated endocytosis did not interfere with retinol uptake. Retinol derived from the holo-RBP complex was differentially incorporated in lipocytes and preferentially reached esterification sites close to lipid droplets through a specific intracellular traffic route. This direct influx pathway facilitates the retinol uptake into HSC against the concentration gradients, and possibly protects cell membranes from undesirable and potentially noxious high retinol concentrations.     

Lutzomyia sand flies (Diptera: Psychodidae) from middle and lower Putumayo Department, Colombia, with new records to the country

A total of 4,840 phlebotomine sand flies from 54 localities in Putumayo department (=state), in the Colombian Amazon region, were collected in Shannon traps, CDC light traps, resting places and from human baits. At least 42 Lutzomyia species were registered for the first time to the department. Psychodopygus and Nyssomyia were the subgenera with the greatest number of taxa, the most common species being L. (N.) yuilli and L. (N.) pajoti. They were sympatric in a wide zone of Putumayo, indicating that they should be treated as full species (new status). Among the anthropophilic sand flies, L. gomezi and L. yuilli were found in intradomiciliar, peridomestic, urban or forest habitats. L. richardwardi, L. claustrei, L. nocticola and L. micropyga are reported for the first time in the Colombian Amazon basin. L. pajoti, L. sipani and L. yucumensis are new records for Colombia.     

Nitrochalcones: Potential in vivo insulin secretagogues

In this study, the in vivo and in vitro anti-hyperglycemic activity of chalcone derivatives of 3,4-methylenedioxy, with a substituent electron-acceptor nitro group in the A or B ring, was investigated. As expected, the second generation sulfonylurea glipizide stimulated insulin secretion and reduced glycemia over the study period. Also, it was demonstrated for the first time that chalcones are able to increase insulin secretion and this event was coincident with serum glucose-lowering in the oral glucose tolerance test. Additionally, the chalcones studied had a similar effect on insulin secretion and serum glucose-lowering as glipizide. The effect of chalcones in terms of inducing insulin secretion was greater than that of glipizide after 30 min. Moreover, chalcones were not able to stimulate glucose uptake in soleus muscle, either in the presence of insulin or in the absence of this hormone. In addition, the oral treatment with chalcones did not alter glycemia in diabetic rats. These reports indicate that the effect of chalcones on serum glucose-lowering in hyperglycemic-normal rats is mainly a consequence of insulin secretion, highlighting these chalcones as novel compounds with strong anti-hyperglycemic properties.     

Determination of Parameters for the Supercritical Extraction of Antioxidant Compounds from Green Propolis Using Carbon Dioxide and Ethanol as Co-Solvent

The aim of this study was to determine the best processing conditions to extract Brazilian green propolis using a supercritical extraction technology. For this purpose, the influence of different parameters was evaluated such as S/F (solvent mass in relation to solute mass), percentage of co-solvent (1 and 2% ethanol), temperature (40 and 50°C) and pressure (250, 350 and 400 bar) using supercritical carbon dioxide. The Global Yield Isotherms (GYIs) were obtained through the evaluation of the yield, and the chemical composition of the extracts was also obtained in relation to the total phenolic compounds, flavonoids, antioxidant activity and 3,5-diprenyl-4-hydroxicinnamic acid (Artepillin C) and acid 4-hydroxycinnamic (p-coumaric acid). The best results were identified at 50°C, 350 bar, 1% ethanol (co-solvent) and S/F of 110. These conditions, a content of 8.93±0.01 and 0.40±0.05 g/100 g of Artepillin C and p-coumaric acid, respectively, were identified indicating the efficiency of the extraction process. Despite of low yield of the process, the extracts obtained had high contents of relevant compounds, proving the viability of the process to obtain green propolis extracts with important biological applications due to the extracts composition.     

Effects of Beta-Alanine Supplementation on Brain Homocarnosine/Carnosine Signal and Cognitive Function: An Exploratory Study

Objectives

Two independent studies were conducted to examine the effects of 28 d of beta-alanine supplementation at 6.4 g d^-1 on brain homocarnosine/carnosine signal in omnivores and vegetarians (Study 1) and on cognitive function before and after exercise in trained cyclists (Study 2).

Methods

In Study 1, seven healthy vegetarians (3 women and 4 men) and seven age- and sex-matched omnivores undertook a brain 1H-MRS exam at baseline and after beta-alanine supplementation. In study 2, nineteen trained male cyclists completed four 20-Km cycling time trials (two pre supplementation and two post supplementation), with a battery of cognitive function tests (Stroop test, Sternberg paradigm, Rapid Visual Information Processing task) being performed before and after exercise on each occasion.

Results

In Study 1, there were no within-group effects of beta-alanine supplementation on brain homocarnosine/carnosine signal in either vegetarians (p = 0.99) or omnivores (p = 0.27); nor was there any effect when data from both groups were pooled (p = 0.19). Similarly, there was no group by time interaction for brain homocarnosine/carnosine signal (p = 0.27). In study 2, exercise improved cognitive function across all tests (P<0.05), although there was no effect (P>0.05) of beta-alanine supplementation on response times or accuracy for the Stroop test, Sternberg paradigm or RVIP task at rest or after exercise.

Conclusion

28 d of beta-alanine supplementation at 6.4g d^-1 appeared not to influence brain homocarnosine/carnosine signal in either omnivores or vegetarians; nor did it influence cognitive function before or after exercise in trained cyclists.