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.     

Effect of temperature and surfactant on the control release of microencapsulated dye in lecithin liposomes. I

The objective of our work has been the microencapsulation of dyes with lecithin from soybean, with the formation of liposomes, as a substitute for synthetic auxiliaries so as to improve the quality of the effluent. Current scenarios promote the disintegration and leakage of the liposomes, such as, changes in temperature, pH and the use of surfactants. Since dyeing process is a mix of all these parameters, we pretended to study each one separately. Rhodamine 6G fluorescence is known to be concentration quenched through the formation of non-fluorescent dimmers and, additionally, through the energy transfer from rhodamine monomer to these dimmers (Baptista ALF, Coutinho PJG, Real Oliveira MECD, Gomes JINR. Proceedings of 13th International Symposium of Surfactants, SIS 2000, Gainesville, USA, 2000). The temperature, the surfactant and pH induce a release of the encapsulated dye resulting in rhodamine dilution and consequently alterations in the dimerization/binding equilibrium. The experimental spectra indicate that rhodamine binds almost completely to liposomes. The decomposition of the rhodamine fluorescence spectra allowed us to determine the percentage of released dye during a simulated dyeing process, and allowed us to conclude that the dimerization process occurs mainly at the inner interfaces. The amount of dye released induced by temperature changes was greater in the presence of surfactant.     

Trehalose delays the reversible but not the irreversible thermal denaturation of cutinase

The effect of trehalose (0.5 M) on the thermal stability of cutinase in the alkaline pH range was studied. The thermal unfolding induced by increasing temperature was analyzed in the absence and in the presence of trehalose according to a two-state model (which assumes that only the folded and unfolded states of cutinase were present). Trehalose delays the reversible unfolding. The midpoint temperature of the unfolding transition (Tm) increases by 4.0 degrees C and 2. 6 degrees C at pH 9.2 and 10.5, respectively, in the presence of trehalose. At pH 9.2 the thermal unfolding occurs at higher temperatures (Tm is 52.6 degrees C compared to 42.0 degrees C at pH 10.5) and a refolding yield of around 80% was obtained upon cooling. This pH value was chosen to study the irreversible inactivation (long-term stability) of cutinase. Temperatures in the transition range from folded to unfolded state were selected and the rate constants of irreversible inactivation determined. Inactivation followed first-order kinetics and trehalose reduced the observed rate constants of inactivation, pointing to a stabilizing effect on the irreversible inactivation step of thermal denaturation. However, if the contribution of reversible unfolding on the irreversible inactivation of cutinase was taken into account, i.e., considering the fraction of cutinase molecules in the reversible unfolded conformation, the intrinsic rate constants can be calculated. Based on the intrinsic rate constants it was concluded that trehalose does not delay the irreversible inactivation. This conclusion was further supported by comparing the activation energy of the irreversible inactivation in the absence and in the presence of trehalose. The apparent activation energy in the absence and in the presence of trehalose were 67 and 99 Kcal/mol, respectively. The activation energy calculated from intrinsic rate constants was higher in the absence (30 Kcal/mol) than in the presence of trehalose (16 Kcal/mol), showing that kinetics of the irreversible inactivation step increased in the presence of trehalose. In fact, trehalose stabilized only the reversible step of thermal denaturation of cutinase.     

Sewage input effects on the macroinvertebrate community associated to Typha domingensis Pers in a coastal lagoon in southeastern Brazil.

This study was carried out at Imboassica Lagoon, located in an urban zone in the municipality of Macaé, Rio de Janeiro state, Brazil. This lagoon has been subject to anthropogenic impacts due to the increasing city population, such as the input of sewage. Areas of variable degree of anthropogenic influence in the lagoon were compared regarding the structure of the macroinvertebrate community associated to Typha domingensis leaves. For sampling, we used 35 x 20 cm net plastic bags, with 6.8 mm mesh containing T. domingensis leaves for colonization. Two different sampling stations were selected: station A, under direct input of sewage; and station B with lesser sewage influence. The bags were removed after 20, 40 and 75 days of colonization. For each sample the Shannon-Wiever Diversity, Pielou Evenness, Jaccard Similarity Indices, Correspondence Analysis and taxonomic richness were calculated. A total of 31,874 individuals were sampled, belonging to 34 taxa. The main taxonomical groups were: Oligochaeta (41%), Chironomidae (40%), Ancylidae (4.6%), Polymitarcyidae (4%) and Thiaridae (3%). At station A, the taxonomic richness, the Evenness and Diversity values were lower than in station B. On the other hand, the total density was three times higher in station A than in B. It was already possible to discriminate the community structure of each sampling station in the first sampling. Trichoptera and Ephemeroptera were the main exclusive groups of station B and are considered good water quality indicators due to their high sensibility to contamination. The major contribution to discriminate between the macroinvertebrate communities of the two sample stations came from Chironomidae, Oligochaeta and Ephemeroptera.     

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.     

Leptin activation of mTOR pathway in intestinal epithelial cell triggers lipid droplet formation,cytokine production and increased cell proliferation

Accumulating evidence suggests that obesity and enhanced inflammatory reactions are predisposing conditions for developing colon cancer. Obesity is associated with high levels of circulating leptin. Leptin is an adipocytokine that is secreted by adipose tissue and modulates immune response and inflammation. Lipid droplets (LD) are organelles involved in lipid metabolism and production of inflammatory mediators, and increased numbers of LD were observed in human colon cancer. Leptin induces the formation of LD in macrophages in a PI3K/mTOR pathway-dependent manner. Moreover, the mTOR is a serine/threonine kinase that plays a key role in cellular growth and is frequently altered in tumors. We therefore investigated the role of leptin in the modulation of mTOR pathway and regulation of lipid metabolism and inflammatory phenotype in intestinal epithelial cells (IEC-6 cells). We show that leptin promotes a dose- and time-dependent enhancement of LD formation. The biogenesis of LD was accompanied by enhanced CXCL1/CINC-1, CCL2/MCP-1 and TGF-β production and increased COX-2 expression in these cells. We demonstrated that leptin-induced increased phosphorylation of STAT3 and AKT and a dose and time-dependent mTORC activation with enhanced phosphorilation of the downstream protein P70S6K protein. Pre-treatment with rapamycin significantly inhibited leptin effects in LD formation, COX-2 and TGF-β production in IEC-6 cells. Moreover, leptin was able to stimulate the proliferation of epithelial cells on a mTOR-dependent manner. We conclude that leptin regulates lipid metabolism, cytokine production and proliferation of intestinal cells through a mechanism largely dependent on activation of the mTOR pathway, thus suggesting that leptin-induced mTOR activation may contribute to the obesity-related enhanced susceptibility to colon carcinoma.     

Do Brain Networks Evolve by Maximizing Their Information Flow Capacity?

We propose a working hypothesis supported by numerical simulations that brain networks evolve based on the principle of the maximization of their internal information flow capacity. We find that synchronous behavior and capacity of information flow of the evolved networks reproduce well the same behaviors observed in the brain dynamical networks of Caenorhabditis elegans and humans, networks of Hindmarsh-Rose neurons with graphs given by these brain networks. We make a strong case to verify our hypothesis by showing that the neural networks with the closest graph distance to the brain networks of Caenorhabditis elegans and humans are the Hindmarsh-Rose neural networks evolved with coupling strengths that maximize information flow capacity. Surprisingly, we find that global neural synchronization levels decrease during brain evolution, reflecting on an underlying global no Hebbian-like evolution process, which is driven by no Hebbian-like learning behaviors for some of the clusters during evolution, and Hebbian-like learning rules for clusters where neurons increase their synchronization.     

Characterization of genomic single nucleotide polymorphism via colorimetric detection using a single gold nanoprobe

Identification of specific nucleic acid sequences mediated by gold nanoparticles derivatized thiol-modified oligonucleotides (Au–nanoprobes) has been proven to be a useful tool in molecular diagnostics. Here, we demonstrate that, on optimization, detection may be simplified via the use of a single Au–nanoprobe to detect a single nucleotide polymorphism (SNP) in homo- or heterozygote condition. We validated this non-cross-linking approach through the analysis of 20 clinical samples using a single specific Au–nanoprobe for an SNP in the FTO (fat mass and obesity-associated) gene against direct DNA sequencing. Sensitivity, specificity, and limit of detection (LOD) were determined, and statistical differences were calculated by one-way analysis of variance (ANOVA) and a post hoc Tukey’s test to ascertain whether there were any differences between Au–nanoprobe genotyped groups. For the first time, we show that the use of a single Au–nanoprobe can detect SNP for each genetic status (wild type, heterozygous, or mutant) with high degrees of sensitivity (87.50%) and specificity (91.67%).     

Physical Damage on Giant Vesicles Membrane as a Result of Methylene Blue Photoirradiation

In this study we pursue a closer analysis of the photodamage promoted on giant unilamellar vesicles membranes made of dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), by irradiating methylene blue present in the giant unilamellar vesicles solution. By means of optical microscopy and electro-deformation experiments, the physical damage on the vesicle membrane was followed and the phospholipids oxidation was evaluated in terms of changes in the membrane surface area and permeability. As expected, oxidation modifies structural characteristics of the phospholipids that lead to remarkable membrane alterations. By comparing DOPC- with POPC-made membranes, we observed that the rate of pore formation and vesicle degradation as a function of methylene blue concentration follows a diffusion law in the case of DOPC and a linear variation in the case of POPC. We attributed this scenario to the nucleation process of oxidized species following a diffusion-limited growth regime for DOPC and in the case of POPC a homogeneous nucleation process. On the basis of these premises, we constructed models based on reaction-diffusion equations that fit well with the experimental data. This information shows that the outcome of the photosensitization reactions is critically dependent on the type of lipid present in the membrane.     

In vitro and in vivo fertility of ram semen cryopreserved in different extenders

Seminal traits of frozen-thawed (FT) ram semen and in vitro and field fertility in native Portuguese breeds were evaluated in 4 experiments. In exp. 1 and 2 the cryopreservation capacity of 2 extenders, E1 (15% egg yolk-EY) and E2 (4.5% EY and trehalose) was compared through morphological evaluation and in vitro fertilizability of FT ram semen. Exp. 3 aimed to determine the usefulness of in vitro homologous/heterologous fertilization tests as tools for predicting ram fertility. Exp. 4 was conducted to verify if the identified differences between the 2 extenders could be confirmed by field fertility. E1 showed a better cryoprotective action expressed by higher in vitro and field fertility results. In conclusion, EY is difficult to be replaced in ram semen extenders. Heterologous fertilization seems to be a useful tool for predicting fertility of FT ram semen.