BnP1, a novel P-I metalloproteinase from Bothrops neuwiedi venom: Biological effects benchmarking relatively to jararhagin, a P-IIISVMP

Snake venom metalloproteinases (SVMPs) have been extensively studied and their effects associated with the local bleeding observed in human accidents by viper snakes. Representatives of P-I and P-III classes of SVMPs similarly hydrolyze extracellular matrix proteins or coagulation factors while only P-III SVMPs induce significant hemorrhage in experimental models. In this work, the effects of P-I and P-III SVMPs on plasma proteins and cultures of muscle and endothelial cells were compared in order to enlighten the mechanisms involved in venom-induced hemorrhage. To reach this comparison, BnP1 was isolated from B. neuwiedi venom and used as a weakly hemorrhagic P-I SVMPs and jararhagin was used as a model of potently hemorrhagic P-III SVMP. BnP1 was isolated by size exclusion and anion-exchange chromatographies, showing apparent molecular mass of approximately 24kDa and sequence similarity with other members of SVMPs, which allowed its classification as a group P-I SVMP. The comparison of local effects induced by SVMPs showed that BnP1 was devoid of significant myotoxic and hemorrhagic activities and jararhagin presented only hemorrhagic activity. BnP1 and jararhagin were able to hydrolyze fibrinogen and fibrin, although the latter displayed higher activity in both systems. Using HUVEC primary cultures, we observed that BnP1 induced cell detachment and a decrease in the number of viable endothelial cells in levels comparable to those observed by treatment with jararhagin. Moreover, both BnP1 and jararhagin induced apoptosis in HUVECs while only a small increase in LDH supernatant levels was observed after treatment with jararhagin, suggesting that the major mechanism involved in endothelial cell death is apoptosis. Jararhagin and BnP1 induced little effects on C2C12 muscle cell cultures, characterized by a partial detachment 24h after treatment and a mild necrotic effect as evidenced by a small increase in the supernatants LDH levels. Taken together, our data show that P-I and P-III SVMPs presented comparable effects except for the hemorrhagic activity, suggesting that hydrolysis of coagulation factors or damage to endothelial cells are not sufficient for induction of local bleeding. (C) 2007 Elsevier Ltd. All rights reserved.

Autolytic Mycobacterium leprae Hsp65 fragments may act as biological markers for autoimmune diseases

Investigating the proteolytic activity of the recombinant Mycobacterium leprae Heat Shock Protein of 65 kDa (rHsp65), chaperonin 2 (cpn2), we observed that it displays high instability. The fragmentation process starts at the C-terminus followed by progressive degradation of the N-terminus, which leads to a stable fragment comprising the middle region of the molecule. Urea was able to prevent autolysis, probably due to its denaturing action, while EDTA increased degradation levels indicating the need for metal ions. Peptides originated from autolysis were purified and analyzed by mass spectrometry, generating a continuous map. Since the bacteria and mammalian Hsp60 are known to be targets of the immune response and have been implicated in autoimmune diseases and chronic inflammation, the in vivo effect of rHsp65 peptides was evaluated in the spontaneous Systemic Lupus Erythematosus (SLE) model developed by the (NZB/NZW)F(1) mouse hybrids, and their individual anti-rHsp65 IgG2a/IgG1 antibody titer ratio was determined. The results showed orientation toward a T(H)1 responsiveness, and the treatment with the rHsp65 peptides diminished the environmental variance of the survival time of treated animals. These results outline the fact that environmental factors may also act through the modified stability expression of Heat Shock Proteins intervening during autoimmune processes. (C) 2011 Elsevier Ltd. All rights reserved.

Phylogeographical, ecological and biological patterns shown by nuclear (ssrRNA and gGAPDH) and mitochondrial (Cyt b) genes of trypanosomes of the subgenus Schizotrypanum parasitic in Brazilian bats

The genetic diversity and phylogeographical patterns of Trypanosoma species that infect Brazilian bats were evaluated by examining 1043 bats from 63 species of seven families captured in Amazonia, the Pantanal, Cerrado and the Atlantic Forest biomes of Brazil. The prevalence of trypanosonne-infected bats, as estimated by haemoculture, was 12.9%, resulting in 77 Cultures of isolates, most morphologically identified as Trypanosoma cf. cruzi, classified by barcoding using partial sequences from ssrRNA gene into the subgenus Schizotrypanum and identified as T. cruzi (15), T cruzi marinkellei (37) or T. cf. dionisii (25). Phylogenetic analyses using nuclear ssrRNA, glycosomal glyceraldehyde 3-phosphate dehydrogenase (gGAPDH) and mitochondrial cytochrome b (Cyt b) gene sequences generated three clades, which clustered together forming the subgenus Schizotrypanum. In addition to vector association, bat trypanosomes were related by the evolutionary history, ecology and phylogeography of the bats. Tryponosoma cf. dionisii trypanosomes (32.4%) infected 12 species from four bat families captured in all biomes, from North to South Brazil, and clustered with T. dionisii from Europe despite being separated by some genetic distance. Trypanosoma cruzi marinkellei (49.3%) was restricted to phyllostomid bats from Amazonia to the Pantanal (North to Central). Trypanosoma cruzi (18.2%) was found mainly in vespertilionid and phyllostomid bats from the Pantanal/Cerrado and the Atlantic Forest (Central to Southeast), with a few isolates from Amazonia. (C) 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Effect of 3,4-ethylenedioxy-extension of thiophene core on the DNA/RNA binding properties and biological activity of bisbenzimidazole amidines

Novel bisbenzimidazoles (4-6), characterized by 3,4-ethylenedioxy-extension of thiophene core, revealed pronounced affinity and strong thermal stabilization effect toward ds-DNA. They interact within ds-DNA grooves as dimmers or even oligomers and agglomerate along ds-RNA. Compounds 4-6 have shown moderate to strong antiproliferative effect toward panel of eight carcinoma cell lines. Compound 5 displayed the best inhibitory potential and in equitoxic concentration (IC(50) = 1 x 10 (6) M) induced accumulation of cells in G2/M phase after 48 h of incubation. Fluorescence microscopy showed that 5 entered into live HeLa cells within 30 min, but did not accumulate in nuclei even after 2.5 h. Compound 5 inhibited the growth of Trypanosome cruzi epimastigotes (IC(50) = 4.3 x 10 (6) M). (C) 2009 Elsevier Ltd. All rights reserved.

Metallopeptidase inhibitors arrest vital biological processes in the fungal pathogen Scedosporium apiospermum

P>Scedosporium apiospermum is an emerging agent of opportunistic mycoses in humans. Previously, we showed that mycelia of S. apiospermum secreted metallopeptidases which were directly linked to the destruction of key host proteins. In this study, we analysed the effect of metallopeptidase inhibitors on S. apiospermum development. As germination of inhaled conidia is a crucial event in the infectious process of S. apiospermum, we studied the morphological transformation induced by the incubation of conidia in Sabouraud-dextrose medium at 37 degrees C. After 6 h, some conidia presented a small projection resembling a germ-tube. A significant increase, around sixfold, in the germ-tube length was found after 12 h, and hyphae were exclusively observed after 24 h. Three distinct metallopeptidase inhibitors were able to arrest the transformation of conidia into hyphae in different ways; for instance, 1,10-phenanthroline (PHEN) completely blocked this process at 10 mu mol l-1, while ethylenediamine tetraacetic acid (EDTA) and ethylene glycol-bis (beta-aminoethyl ether; EGTA) only partially inhibited the differentiation at up to 10 mmol l-1. EGTA did not promote any significant reduction in the conidial growth, while PHEN and EDTA, both at 10 mmol l-1, inhibited the proliferation around 100% and 65%, respectively. The secretion of polypeptides into the extracellular environment and the metallopeptidase activity secreted by mycelia were completely inhibited by PHEN. These findings suggest that metallo-type enzymes could be potential targets for future therapeutic interventions against S. apiospermum.

Investigation of a new GRASP-based clustering algorithm applied to biological data

A large amount of biological data has been produced in the last years. Important knowledge can be extracted from these data by the use of data analysis techniques. Clustering plays an important role in data analysis, by organizing similar objects from a dataset into meaningful groups. Several clustering algorithms have been proposed in the literature. However, each algorithm has its bias, being more adequate for particular datasets. This paper presents a mathematical formulation to support the creation of consistent clusters for biological data. Moreover. it shows a clustering algorithm to solve this formulation that uses GRASP (Greedy Randomized Adaptive Search Procedure). We compared the proposed algorithm with three known other algorithms. The proposed algorithm presented the best clustering results confirmed statistically. (C) 2009 Elsevier Ltd. All rights reserved.

Biological effects of anionic meso-tetrakis (para-sulfonatophenyl) porphyrins modulated by the metal center. Studies in rat liver mitochondria

In this paper, we present a study about the influence of the porphyrin metal center and mesa ligands on the biological effects of meso-tetrakis porphyrins. Different from the cationic meso-tetrakis 4-N-methyl pyridinium (Mn(III)TMPyP), the anionic Mn(III) meso-tetrakis (para-sulfonatophenyl) porphyrin (Mn(III)TPPS4) exhibited no protector effect against Fe(citrate)-induced lipid oxidation. Mn(III)TPPS4 did not protect mitochondria against endogenous hydrogen peroxide and only delayed the swelling caused by tert-BuOOH and Ca(2+). Fe(III)TPPS4 exacerbated the effect of the tert-BuOOH, and both porphyrins did not significantly affect Fe(II)citrate-induced swelling. Consistently, Fe(III)TPPS4 predominantly promotes the homolytic cleavage of peroxides and exhibits catalytic efficiency ten-fold higher than Mn(III)TPPS4. For Mn(III)TPPS4, the microenvironment of rat liver mitochondria favors the heterolytic cleavage of peroxides and increases the catalytic efficiency of the manganese porphyrin due to the availability of axial ligands for the metal center and reducing agents such as glutathione (GSH) and proteins necessary for Compound II (oxomanganese IV) recycling to the initial Mn(III) form. The use of thiol reducing agents for the recycling of Mn(III)TPPS4 leads to GSH depletion and protein oxidation and consequent damages in the organelle. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Synthesis, characterization, crystal structure, and biological studies of vanadium complexes with glycolic acid

Synthesis, characterization, crystal structure, and biological studies of two complexes with glycolic acid are described. The solid complexes were formulated as K2[VO(C2H2O3)(C2H3O3)2] H2O (1) and K2[{VO2(C2H2O3)}2] (2) and characterized by X-ray studies, Fourier transform infrared spectroscopy (FTIR), Electron paramagnetic resonance (EPR), and magnetic susceptibility. Conversion of 1 to 2 was studied in aqueous solution by UV-Vis spectroscopy and in the solid state by diffuse reflectance spectroscopy. Complex 2 contains dinuclear [{VO2(C2H2O3)}2]2- anions in which glycolate(2-) is a five-membered chelating ring formed by carboxylate and -hydroxy groups. The geometry around the vanadium in 2 was interpreted as intermediate between a trigonal bipyramid and a square pyramid. Vanadium(IV) is pentacoordinate in 1 as a distorted square pyramid. Complex 1 contains a vanadyl group (V=O) surrounded by two oxygens from deprotonated carboxylate and hydroxy groups forming a five-membered ring. Two oxygens from different glycolates(1-) are bonded to the (V=O) also. Biological analysis for potential cytotoxic effects of 1 was performed using Human Cervix Adenocarcinoma (HeLa) cells, a human cervix adenocarcinoma-derived cell line. After incubation for 48 h, 1 causes 90 and 95% of HeLa cells death at 20 and 200 mol L-1, respectively.

Natural products biological screening and ligand-based virtual screening for the discovery of new antileishmanial agents

In the course of our research program to discover novel antileishmanial agents, a biological screening of natural products against Leishmania major promastigotes allowed the identification of a furoquinoline alkaloid (1) and a furanocoumarin (2) as new hits. Subsequently, an integrated ligand-based virtual screening approach was employed to search for new antileishmanial compounds using these naturally occurring molecules as templates. Fourteen out of 40 compounds selected from a database of about 800,000 compounds (extracted from ZINC, a free database for virtual screening) were experimentally confirmed to possess significant in vitro antileishmanial properties. The application of ligand-based virtual screening as a complementary approach to experimental natural product screening was a useful strategy to facilitate the identification of new promising lead candidates.

Density-Profile Processes Describing Biological Signaling Networks: Almost Sure Convergence to Deterministic Trajectories

We introduce jump processes in R(k), called density-profile processes, to model biological signaling networks. Our modeling setup describes the macroscopic evolution of a finite-size spin-flip model with k types of spins with arbitrary number of internal states interacting through a non-reversible stochastic dynamics. We are mostly interested on the multi-dimensional empirical-magnetization vector in the thermodynamic limit, and prove that, within arbitrary finite time-intervals, its path converges almost surely to a deterministic trajectory determined by a first-order (non-linear) differential equation with explicit bounds on the distance between the stochastic and deterministic trajectories. As parameters of the spin-flip dynamics change, the associated dynamical system may go through bifurcations, associated to phase transitions in the statistical mechanical setting. We present a simple example of spin-flip stochastic model, associated to a synthetic biology model known as repressilator, which leads to a dynamical system with Hopf and pitchfork bifurcations. Depending on the parameter values, the magnetization random path can either converge to a unique stable fixed point, converge to one of a pair of stable fixed points, or asymptotically evolve close to a deterministic orbit in Rk. We also discuss a simple signaling pathway related to cancer research, called p53 module.

Bifurcation analysis of a model for biological control

In this paper we study the Lyapunov stability and Hopf bifurcation in a biological system which models the biological control of parasites of orange plantations. (c) 2007 Elsevier Ltd. All rights reserved.

Comparative electrochemical study of 5832-9 stainless steel at different media of biological interest

The electrochemical behavior of ISO 5832-9 stainless steel at 37 degrees C in 0.9% NaCl, Ringer Lactate and minimum essential medium (MEM) has been studied, using linear voltammetry, and surface analysis by SEM and EDS. Mechanical and toxicity tests were made. ISO 5832-9 is passivated at corrosion potential (E) and it does not present pitting corrosion on the media studied from to 50 in V above the transpassivation potential (Ei). SEM and EDS analysis have shown that the sample previously immersed in MEM presents a diffirent behavior at 50 in V above El: the manganese oxide inclusions are absent in the surface. E. values and passivation current density values j(pass) changed according to the following. E(corr, RL) < E(corr,NaCl) < E(corr, MEM) and J (MEM) << j(RL) congruent to j(NaCl) The stainless steel was characterized as non toxic in the cytotoxicity assay

Immobilization of Catalysts of Biological Interest on Porous Oxidized Silicon Surfaces

The present paper deals with the immobilization of redox mediators and proteins onto protected porous silicon surfaces to obtain their direct electrochemical reactions and to retain their bioactivities. This paper shows that MP-11 and viologens are able to establish chemical bonds with 3-aminopropyltriethoxylsilane-modified porous silicon surface. The functionalization of the surfaces have been fully characterized by energy dispersive X-ray analysis (EDX) and X-ray photoelectron spectroscopy (XPS) to examine the immobilization of these mediators onto the solid surface. Amperometric and open circuit potential measurements have shown the direct electron transfer between glucose oxidase and the electrode in the presence of the viologen mediator covalently linked to the 3-aminopropyltriethoxylsilane (APTES)-modified porous silicon surfaces.

Spectrophotometric determination of zinc in pharmaceutical and biological samples with di-2-pyridyl ketone benzoylhydrazone: A simple, fast, accurate and sensitive method

A simple, fast, accurate, and sensitive spectrophotometric method was developed to determine zinc(II). This method is based on the reaction of Zn(II) with di-2-pyridyl ketone benzoylhydrazone (DPKBH), at pH=5.5 and 50% (v/v) ethanol. Beers law was obeyed in the range 0.020-1.82 mu g mL(-1) with a molar apsorptivity of 3.64 x 10(4) L mol(-1) cm(-1), and a detection limit (3) of 2.29 mu g L-1. The action of some interfering ions was verified and the developed method applied to pharmaceutical and biological samples. The results were then compared with those obtained by using a flame atomic absorption technique.

Synthesis, characterization and biological analysis of the complex [VO(Hdhp)(2)] (H(2)dhp=2,3-dihydroxypyridine)

A new vanadium (IV) complex with the monoanion of 2,3-dihydroxypyridine (H(2)dhp), or 3-hydroxy-2(1H)-pyridone, was synthesized, characterized by physicochemical techniques and tested biologically. The EPR data for the [VO(Hdhp)(2)] complex in DMF are: g(x) = 1.9768, g(y) = 1.9768 and g(z) = 1.9390; A values (10(-4) cm(-1)): A(x), 59.4; A(y//), 59.4; A(z), 171.0. The vV=O band in the IR spectrum of the complex is at 986 cm(-1). The complex is paramagnetic, with mu(eff) = 1.65 BM (d(1), spin-only) at 25 degrees C. The irreversible oxidation process [V(V)/V(IV)] of the [VO(Hdhp)(2)] complex, as revealed in a cyclic voltammogram, occurs at 876 mV. The calculated molecular structure of [VO(Hdhp)(2)] shows the vanadium(IV) center in a distorted square pyramidal environment, with the oxo ligand in the apical position and the oxygen donor atoms of the Hdhp ligands in the basal positions. The ability of [VO(Hdhp)(2)] to mimic insulin, and its toxicity to hepato-biliary functions, were investigated in streptozotocin-induced diabetic rats and it was concluded that the length of treatment and the amount of [VO(Hdhp)(2)] administered were effective in reducing experimental diabetes.