Conformational stability of peanut agglutinin using small angle X-ray scattering

In this work, quaternary conformational studies of peanut agglutinin (PNA) have been carried out using small-angle X-ray scattering (SAXS). PNA was submitted to three different conditions: pH variation (2.5, 4.0, 7.4 and 9.0), guanidine hydrochloride presence (0.5-2M) at each pH value, and temperature ranging from 25 to 60°C. All experiments were performed in the absence and presence of T-antigen to evaluate its influence on the lectin stability. At room temperature and pH 4.0, 7.4 and 9.0, the SAXS curves are consistent with the PNA scattering in its crystallographic native homotetrameric structure, with monomers in a jelly roll fold, associated by non-covalent bonds resulting in an open structure. At pH 2.5, the results indicate that PNA tends to dissociate into smaller sub-units, as dimers and monomers, followed by a self-assembling into larger aggregates. Furthermore, the conformational stability under thermal denaturation follows the pH sequence 7.4>9.0>4.0>2.5. Such results are consistent with the conformational behavior found upon GndHCl influence. The presence of T-antigen does not affect the protein quaternary structure in all studied systems within the SAXS resolution.     

Effects of microplusin, a copper-chelating antimicrobial peptide, against Cryptococcus neoformans

Microplusin is an antimicrobial peptide isolated from the cattle tick Rhipicephalus (Boophilus) microplus. Its copper-chelating ability is putatively responsible for its bacteriostatic activity against Micrococcus luteus as microplusin inhibits respiration in this species, which is a copper-dependent process. Microplusin is also active against Cryptococcus neoformans (MIC(50) = 0.09 μM), the etiologic agent of cryptococcosis. Here, we show that microplusin is fungistatic to C. neoformans and this inhibitory effect is abrogated by copper supplementation. Notably, microplusin drastically altered the respiratory profile of C. neoformans. In addition, microplusin affects important virulence factors of this fungus. We observed that microplusin completely inhibited fungal melanization, and this effect correlates with the inhibition of the related enzyme laccase. Also, microplusin significantly inhibited the capsule size of C. neoformans. Our studies reveal, for the first time, a copper-chelating antimicrobial peptide that inhibits respiration and growth of C. neoformans and modifies two major virulence factors: melanization and formation of a polysaccharide capsule. These features suggest that microplusin, or other copper-chelation approaches, may be a promising therapeutic for cryptococcosis.     

Synthesis and antitumor activity of β-carboline 3-(substituted-carbohydrazide) derivatives

A series of β-carboline derivatives bearing a substituted-carbohydrazide moiety at C-3 were synthesized and evaluated for their antitumor activity against eight human cancer cell lines. The β-carboline N-(substituted-benzylidene)carbohydrazides showed, in general, a greater antitumor activity than their N-(alkylidene)carbohydrazide analogues. The N(9)-methylation of β-carboline N-(substituted-benzylidene) carbohydrazides resulted in a decrease of antitumor activity. Among compounds tested, the benzylidene-carbohydrazides 3, 4, 11, 13, 16, 21 and 22 were the most active, possessing IC(50) less than 10 μM for six of the eight tumor cell lines assayed. The derivative 4 displayed the most significant activity toward all tested cell lines, with a remarkable cytotoxicity against renal (786-0) cell lines (IC(50)=0.04 μM). Compound 4 was assayed for its in vivo antineoplastic activity in the Ehrlich solid carcinoma assay.     

Participation of the liver gluconeogenesis in the glibenclamide-induced hypoglycaemia in rats

We previously demonstrated an increased liver gluconeogenesis (LG) during insulin‐induced hypoglycaemia. Thus, an expected effect of sulphonylureas induced hypoglycaemia (SIH) could be the activation of LG. However, sulphonylureas infused directly in to the liver inhibits LG. Considering these opposite effects we investigated herein LG in rats submitted to SIH. For this purpose, 24 h fasted rats that received glibenclamide (10 mg kg^?1) were used (SIH group). Control group received oral saline. Glycaemia at 30, 60, 90, 120 and 150 min after oral administration of glibenclamide were evaluated. Since the lowest glycaemia was obtained 120 min after glibenclamide administration, this time was chosen to investigate LG in situ perfused livers. The gluconeogenesis from precursors that enters in this metabolic pathway before the mitochondrial step, i.e. L ‐alanine (5 mM), L ‐lactate (2 mM), pyruvate (5 mM) and L ‐glutamine were decreased (p < 0·05). However, the gluconeogenic activity using glycerol (2 mM), which enters in the gluconeogenesis after the mitochondrial step was maintained. Taken together, the results suggest that the inhibition of LG promoted by SIH overcome the activation of this metabolic pathway promoted by IIH and could be attributed, at least in part, to its effect on mitochondrial function. Copyright © 2011 John Wiley & Sons, Ltd.     

Labaditin,a cyclic peptide with rich biotechnological potential: preliminary toxicological studies and structural changes in water and lipid membrane environment

Cyclic peptides isolated from the plants of the Euphorbiaceae family have been largely studied due to their rigid conformation, which is considered significant for biologic activity. The peptide Labaditin (L₀) and its open chain analogs (L₁) were synthesized by the solid-phase peptide synthesis technique (Fmoc/tBu), and purified to elucidate its interaction with membrane models. A shift in λ_max emission and Stern–Volmer constants values indicate that both tryptophans migrate to a more apolar environment, with L₁ decreasing less than L₀. A circular dichroism (CD) study revealed that L₀ was kept unstructured in aqueous media as much as in the presence of dipalmitoilphosphatidylcholine liposomes. The thermodynamic studies by differential calorimetry (DSC) show a ΔH increase (50 and 18 kcal/mol, for L₀ and L₁, respectively) with peptide concentrations, which is indicative of lipids associating with peptides, resulting in the inability of the lipids to participate in the main transition. Therefore, all CD, DSC, and fluorescence data suggest a greater L₀ membrane insertion. A probable mechanism for Labaditin interaction is based initially on the hydrophobic interaction of the peptide with the lipid membrane, conformational change, peptide adsorption on the lipid surface, and internalization process. Peptide’s antibacterial effect was also evaluated and revealed that only L₀ showed reduction in viability in Gram-positive bacteria while no effects to the Gram-negative.     

Cave lithology determining the structure of the invertebrate communities in the Brazilian Atlantic Rain Forest

In Brazil, only limestone caves and a few caves in sandstone, iron ore and granite rocks had their invertebrate communities evaluated. Being such, the present study aimed to promote a comparative analysis of the structure of the invertebrate communities in caves associated to carbonatic, magmatic, siliciclastic and ferruginous rocks of the Brazilian Atlantic forest. Significant differences in the relative richness, abundance and diversity were observed between lithologies. The average relative richness was higher in the ferruginous caves (0.53 spp). The total number of troglomorphic species was significantly different among caves and the highest average richness occurred at ferruginous caves (5.79 spp/cave). Siliciclastic, carbonatic and magmatic caves presented a higher quantitative similarity of the fauna. Ferruginous caves revealed communities with a fauna composition different from the other lithologies. The total richness of invertebrates correlated significantly and positively with the linear development in the siliciclastic caves (R_s = 0.67, P < 0.05), carbonatic (R_s = 0.71, P < 0.05) and ferruginous (R_s = 0.74, P < 0.05). The rock type in which the cave is inserted can determine differences in the richness of invertebrate troglophyles and troglobites. Therefore, on creating value attributes, the size of the caves should always come related to their lithology by the fact that same sized caves associated to different lithologies, possess communities with quite diverse structures.     

Characteristics of the Human Host Have Little Influence on Which Local Schistosoma mansoni Populations Are Acquired

Background

Brazil remains the country in the Americas with the highest prevalence of schistosomiasis. A combination of control efforts and development, however, has sharply reduced its intensity and distribution. The acquisition of specific schistosome populations may be dependent on host characteristics such as sex, age, geography, work, habits and culture. How these and other host characteristics align with parasite subpopulations may guide approaches to improve control.

Methodology

A cohort of more than 90% of the residents in two rural communities in Brazil participated in an epidemiologic survey of demographic, socio-economic and behavioral characteristics. The variables sex, age, intensity of infection, socio-economic index, % lifetime spent on site, previous infection, and trips outside the district were used to group parasites infecting individuals. Schistosoma mansoni infection status was determined by examination of stools submitted on 3 different days. The aggregate of eggs collected from the whole stool was used to determine degree of population differentiation from allele frequencies for 15 microsatellites.

Conclusions/Significance

Infection prevalence was 41% for these communities, and the epidemiologic characteristics were similar to many of the endemic areas of Brazil and the world. Parasite population structuring was observed between the two communities (Jost''s D 0.046, CI95% 0.042–0.051), although separated by only 8 km and connected by a highway. No structuring was observed when infected individuals were stratified by host''s biologic, demographic or epidemiologic characteristics. Those most heavily infected best reflected the communities'' overall parasite diversity. The lack of differentiation within villages suggests that individuals are likely to get infected at the same sites or that the same parasite multilocus genotypes can be found at most sites. The geographic structuring between villages and the lack of structuring by age of the host further supports the impression of a population little affected by migration or drift.     

Akt activation by insulin treatment attenuates cachexia in Walker-256 tumor-bearing rats

Cancer-bearing often exhibits hypoinsulinemia, insulin (INS) resistance and glutamine depletion associated with cachexia. However, INS and glutamine effects on cachexia metabolic abnormalities, particularly on tumor-affected proteins related to INS resistance, are poorly known. The main purpose of this study was to investigate the effects of INS and glutamine dipeptide (GDP) treatments on phospho-protein kinase B (p-Akt), and phospho-hormone sensitive lipase (p-HSL) in Walker-256 tumor-bearing rats. INS (NPH, 40 UI/kg, subcutaneous), GDP (1.5 g/kg, oral), INS+GDP or vehicle (control rats) were administered for 13 days, once a day, starting at the day of inoculation of tumor cells. The experiments were performed 4 hours after the last treatment to evaluate acute effects of INS and GDP, besides the chronic effects. INS and/or INS+GDP treatments, which markedly increased the insulinemia, increased the p-Akt: total Akt ratio and prevented the increased p-HSL^Ser552: total HSL ratio in the retroperitoneal fat of tumor-bearing rats, without changing the INS resistance and increased expression of factor tumor necrosis-α (TNF-α) in this tissue. INS and INS+GDP also increased the p-Akt: total Akt ratio, whereas GDP and INS+GDP increased the GLUT4 glucose transporter gene expression, in the gastrocnemius muscle of the tumor-bearing rats. Accordingly, treatments with INS and INS+GDP markedly reduced glycemia, increased retroperitoneal fat and attenuated the body mass loss of tumor-bearing rats. In conclusion, hyperinsulinemia induced by high-dose INS treatments increased Akt phosphorylation and prevented increased p-HSL^Ser552: total HSL ratio, overlapping INS resistance. These effects are consistent with increased fat mass gain and weight loss (cachexia) attenuation of tumor-bearing rats, evidencing that Akt activation is a potential strategy to prevent loss of fat mass in cancer cachexia.     

Noncompetitive tight-binding inhibition of Anticarsia gemmatalis trypsins by Adenanthera pavonina protease inhibitor affects larvae survival

The economic loss in soybean crops caused by the Lepidoptera insects has encouraged the search for new strategies to control this pest, which are currently based on synthetic insecticides. This paper evaluated the ability of ApTI (Adenanthera pavonina trypsin inhibitor) to inhibit trypsin-like proteins from Anticarsia gemmatalis by docking, molecular dynamics, and enzymatic and survival assay. The docking and molecular dynamic simulation between trypsin and ApTI were performed using the program CLUSPRO and NAMD, respectively. The inhibitory constant K_i and the inhibition type were determined through chromogenic assays. The survival assay of neonatal larvae under treatment with artificial diet supplemented with ApTI was also performed. The ApTI binding site was predicted to block substrate access to trypsin due to four interactions with the enzyme, producing a complex with a surface area of 1,183.7 Ų. The kinetic analysis revealed a noncompetitive tight-binding mechanism. The survival curves obtained using Kaplan–Meier estimators indicated that the highest larvae mortality was 60%, using 1.2 mg of ApTI per 100 ml of artificial diet. The in vitro, in vivo, and in silico studies demonstrated that ApTI is a strong noncompetitive inhibitor of trypsin with biotechnological potential for the control of A. gemmatalis insect.     

Complex Coacervates Formed between Whey Protein Isolate and Carboxymethylcellulose for Encapsulation of β-Carotene from Sacha Inchi Oil: Stability,In Vitro Digestion and Release Kinetics

The present work aimed to study the influence of the pH and protein ratio on the formation of complex coacervates of carboxymethylcellulose (CMC) and whey protein isolated nanoparticles (WPIN). These biopolymers and transglutaminase, as a cross-linking agent, were used to encapsulate sacha inchi oil (SIO) containing β-carotene (β-C). The stability of β-C from SIO microcapsules (β-SIO microcapsules) was evaluated under in vitro digestion using an INFOGEST 2.0 in vitro digestion protocol. The release of β-C in a simulated food model was studied, and mathematical models were used to determine the mechanism. A ratio of 1:6 (CMC/WPIN) at pH 3.5 was used for the formation of the complex. Chemical and morphological analyses suggested that SIO was microencapsulated and that a high encapsulation efficiency was obtained. The β-C from β-SIO microcapsules was preserved in vegetable oil (food model), and Fickian diffusion occurred. The β-C from β-SIO microcapsules was preserved under oral and gastric conditions, and higher release occurred during intestinal digestion when samples were subjected to in vitro digestion simulation. After in vitro digestion, the β-C from β-SIO microcapsules presented higher stability (83.37%) and acceptable bioaccessibility (31.16%). There are few studies in the literature of encapsulated SIO using the CMC/WPIN complex or studies of the release of β-carotene from SIO during in vitro digestion and in food simulants. The knowledge obtained in this study will facilitate the use and applications of β-C-loaded microcapsule delivery systems.