Analysis of Bothrops jararacussu venomous gland transcriptome focusing on structural and functional aspects: I - gene expression profile of highly expressed phospholipases A(2)

Snake venom glands are a rich source of bioactive molecules such as peptides, proteins and enzymes that show important pharmacological activity leading to in local and systemic effects as pain, edema, bleeding and muscle necrosis. Most studies on pharmacologically active peptides and proteins from snake venoms have been concerned with isolation and structure elucidation through methods of classical biochemistry. As an attempt to examine the transcripts expressed in the venom gland of Bothrops jararacussu and to unveil the toxicological and pharmacological potential of its products at the molecular level, we generated 549 expressed sequence tags (ESTs) from a directional cDNA library. Sequences obtained from single-pass sequencing of randomly selected cDNA clones could be identified by similarities searches on existing databases, resulting in 197 sequences with significant similarity to phospholipase A(2) (PLA(2)), of which 83.2% were Lys49-PLA(2) homologs (BOJU-1), 0.1% were basic Asp49-PLA(2)s (BOJU-II) and 0.6% were acidic Asp49-PLA(2)s (BOJU-III). Adjoining this very abundant class of proteins we found 88 transcripts codifying for putative sequences of metalloproteases, which after clustering and assembling resulted in three full-length sequences: BOJUMET-I, BOJUMET-II and BOJUMET-III; as well as 25 transcripts related to C-type lectin like protein including a full-length cDNA of a putative galactose binding C-type lectin and a cluster of eight serine-proteases transcripts including a full-length cDNA of a putative serine protease. Among the full-length sequenced clones we identified a nerve growth factor (Bj-NGF) with 92% identity with a human NGF (NGHUBM) and an acidic phospholipase A2 (BthA-I-PLA(2)) displaying 85-93% identity with other snake venom toxins. Genetic distance among PLA(2)s from Bothrops species were evaluated by phylogenetic analysis. Furthermore, analysis of full-length putative Lys49-PLA(2) through molecular modeling showed conserved structural domains, allowing the characterization of those proteins as group II PLA(2)s. The constructed cDNA library provides molecular clones harboring sequences that can be used to probe directly the genetic material from gland venom of other snake species. Expression of complete cDNAs or their modified derivatives will be useful for elucidation of the structure-function relationships of these toxins and peptides of biotechnological interest. (C) 2004 Elsevier SAS. All rights reserved.

The use of biodiversity as source of new chemical entities against defined molecular targets for treatment of malaria, tuberculosis, and T-cell mediated diseases: a review

The modern approach to the development of new chemical entities against complex diseases, especially the neglected endemic diseases such as tuberculosis and malaria, is based on the use of defined molecular targets. Among the advantages, this approach allows (i) the search and identification of lead compounds with defined molecular mechanisms against a defined target (e.g. enzymes from defined pathways), (ii) the analysis of a great number of compounds with a favorable cost/benefit ratio, (iii) the development even in the initial stages of compounds with selective toxicity (the fundamental principle of chemotherapy), (iv) the evaluation of plant extracts as well as of pure substances. The current use of such technology, unfortunately, is concentrated in developed countries, especially in the big pharma. This fact contributes in a significant way to hamper the development of innovative new compounds to treat neglected diseases. The large biodiversity within the territory of Brazil puts the country in a strategic position to develop the rational and sustained exploration of new metabolites of therapeutic value. The extension of the country covers a wide range of climates, soil types, and altitudes, providing a unique set of selective pressures for the adaptation of plant life in these scenarios. Chemical diversity is also driven by these forces, in an attempt to best fit the plant communities to the particular abiotic stresses, fauna, and microbes that co-exist with them. Certain areas of vegetation (Amazonian Forest, Atlantic Forest, Araucaria Forest, Cerrado-Brazilian Savanna, and Caatinga) are rich in species and types of environments to be used to search for natural compounds active against tuberculosis, malaria, and chronic-degenerative diseases. The present review describes some strategies to search for natural compounds, whose choice can be based on ethnobotanical and chemotaxonomical studies, and screen for their ability to bind to immobilized drug targets and to inhibit their activities. Molecular cloning, gene knockout, protein expression and purification, N-terminal sequencing, and mass spectrometry are the methods of choice to provide homogeneous drug targets for immobilization by optimized chemical reactions. Plant extract preparations, fractionation of promising plant extracts, propagation protocols and definition of in planta studies to maximize product yield of plant species producing active compounds have to be performed to provide a continuing supply of bioactive materials. Chemical characterization of natural compounds, determination of mode of action by kinetics and other spectroscopic methods (MS, X-ray, NMR), as well as in vitro and in vivo biological assays, chemical derivatization, and structure-activity relationships have to be carried out to provide a thorough knowledge on which to base the search for natural compounds or their derivatives with biological activity.

Conformational features of cepacian: the exopolysaccharide produced by clinical strains of Burkholderia cepacia

Conformational energy calculations and molecular dynamics investigations, both in water and in dimethyl sulfoxide, were carried out on the exopolysaccharide cepacian produced by the majority of the clinical strains of Burkholderia cepacia, an opportunistic pathogen causing serious lung infection in patients affected by cystic fibrosis, the investigation was aimed at defining the structural and conformational features, which might be relevant for clarification of the structure-function relationships of the polymer. The molecular dynamics calculations were carried out by Ramachandran-type energy plots of the disaccharides that constitute the polymer repeating unit. The dynamics of an oligomer composed of three repeating units were investigated in water and in Me2SO, a non-aggregating solvent. Analysis of the time persistence of hydrogen bonds showed the presence of a large number of favourable interactions in water, which were less evident in Me2SO. The calculations on the cepacian chain indicated that polymer conformational features in water were affected by the lateral chains, but were also largely dictated by the presence of solvent. Moreover, the large number of intra-chain hydrogen bonds in water disappeared in Me2SO solution, increasing the average dimension of the polymer chains. (c) 2005 Elsevier Ltd. All rights reserved.

At the interface: Crystal structures of phospholipases A(2)

The protein content of many snake venoms often includes one or more phospholipases A(2) (PLA(2)). In recent years a growing number of venoms from snakes of Agkistrodon, Bothrops and Trimeresurus species have been shown to contain a catalytically inactive PLA(2)-homologue in which the highly conserved aspartic acid at position 49 (Asp49) is substituted by lysine (Lys49). Although demonstrating little or no catalytic activity, these Lys49-PLA(2)s disrupt membranes by a Ca2+-independent mechanism of action. In addition, this family of PLA(2)s demonstrates myotoxic and cytolytic pharmacological activities, however the structural bases underlying these functional properties are poorly understood. Through the application of X-ray crystallography in combination with biophysical and bioinformatics techniques, we are studying structure/function relationships of Lys49-PLA(2)s. We here present results of a systematic X-ray crystallographic and amino acid sequence analysis study of Lys49-PLA(2)s and propose a model to explain the Ca2+ independent membrane damaging activity. (C) 1998 Elsevier B.V. Ltd. All rights reserved.

Molecular determinants of binding of a wasp toxin (PMTXs) and its analogs in the Na+ channels proteins

The structural specificity of alpha-PMTX, a novel peptide toxin derived from wasp venom has been studied on the neuromuscular synapse in the walking leg of the lobster. alpha-PMTX is known to induce repetitive action potentials in the presynaptic axon due to sodium channel inactivation. We synthesized 29 analogs of alpha-PMTX by substituting one or two amino acids and compared threshold concentrations of these mutant toxins for inducing repetitive action potentials. In 13 amino acid residues of alpha-PMTX, Arg-1, Lys-3 and Lys-12 regulate the toxic activity because substitution of these basic amino acid residues with other amino acid residues greatly changed the potency. Determining the structure-activity relationships of PMTXs will help clarifying the molecular mechanism of sodium channel inactivation. (C) 2000 Elsevier B.V. Ireland Ltd. All rights reserved.

Novel allosteric conformation of human HB revealed by the hydration and anion effects on O-2 binding

The effect of anions on the stability of different functional conformations of Hb is examined through the determination of the dependence of O-2 affinity on water activity (a(w)). The control of a(w) is effected by varying the sucrose osmolal concentration in the bathing solution according to the osmotic stress method. Thus, the hydration change following Hb oxygenation is determined as a function of Cl- and of DPG concentration. We find that only similar to 25 additional water molecules bind to human Hb during the deoxy-to-oxy conformation transition in the absence of anions, in contrast with similar to 72 that bind in the presence of more than 50 mM Cl- or more than 15 mu M DPG. We demonstrate that the increase in the hydration change linked with oxygenation is coupled with anion binding to the deoxy-Hb. Hence, we propose that the deoxy-Hb coexists in two allosteric conformations which depend on whether anion is bound or not: the tense T-state, with low oxygen affinity and anion bound, or a new allosteric P-state, with intermediate oxygen affinity and free of bound anions. The intrinsic oxygen affinity of this unforeseen P-state and the differential binding of Cl-, DPG, and H2O between states P and T and P and R are characteristics which are consistent with those expected for a putative intermediate allosteric state of Hb. These findings represent a new opportunity to explore the structure-function relationships of hemoglobin regulation.

SEQUENCE OF A CDNA-ENCODING BOTHROPSTOXIN-I, A MYOTOXIN FROM THE VENOM OF BOTHROPS-JARARACUSSU

With the aim of further understanding the structure/function relationships in the membrane-damaging activity of the Lys(49) phospholipase A(2) (Lys(49)-PLA(2)) sub-family, we used PCR (polymerase chain reaction) on total venom gland cDNAs from Bothrops jararacussu with degenerate oligodeoxyribonucleotides encoding the N- and C-termini of myotoxin II, a Lys(49)-PLA(2) from Bothrops asper. A 350-bp cDNA coding for bothropstoxin I (BtxtxI) was amplified. Sequencing of the amplified fragment shows that BtxtxI has a Lys(49), and comparison with the known structure of myotoxin II showed that the amino acids involved in the formation of a novel dimeric structure in this protein were also conserved.

Functional and structural analysis of two fibrinogen-activating enzymes isolated from the venoms of Crotalus durissus terrificus and Crotalus durissus collilineatus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Developing descriptors to predict mechanical properties of nanotubes

Descriptors and quantitative structure property relationships (QSPR) were investigated for mechanical property prediction of carbon nanotubes (CNTs). 78 molecular dynamics (MD) simulations were carried out, and 20 descriptors were calculated to build quantitative structure property relationships (QSPRs) for Young's modulus and Poisson's ratio in two separate analyses: vacancy only and vacancy plus methyl functionalization. In the first analysis, C N2/CT (number of non-sp2 hybridized carbons per the total carbons) and chiral angle were identified as critical descriptors for both Young's modulus and Poisson's ratio. Further analysis and literature findings indicate the effect of chiral angle is negligible at larger CNT radii for both properties. Raman spectroscopy can be used to measure CN2/C T, providing a direct link between experimental and computational results. Poisson's ratio approaches two different limiting values as CNT radii increases: 0.23-0.25 for chiral and armchair CNTs and 0.10 for zigzag CNTs (surface defects <3%). In the second analysis, the critical descriptors were CN2/CT, chiral angle, and MN/CT (number of methyl groups per total carbons). These results imply new types of defects can be represented as a new descriptor in QSPR models. Finally, results are qualified and quantified against experimental data. © 2013 American Chemical Society.

Inovação do método MIA-QSAR: aplicação para doenças tropicais negligenciadas

Multivariate image analysis applied to the quantitative structure-activity relationships (MIA-QSAR) is a 2D QSAR technique that has been presenting promising outcomes for the development of new drug candidates, due to its simplicity, rapidity and low cost. In this way, the present study aims at introducing, consolidating and improving the new dimensions named aug-MIA-QSAR and aug-MIA-QSARcolor, as well as applying them to the study of neglected diseases, in order to obtain new drug targets using chemico-biological interpretation of the MIA molecular descriptors. Four compound data sets with experimental bioactivities against Chagas disease, malaria, dengue and schistosomiasis were evaluated using three approaches: MIA-QSARt, aug-MIA-QSAR and aug-MIA-QSARcolor. In general, representations of atoms as spheres with different colors and sizes proportional to the corresponding van der Waals radii (aug-MIA approaches) improved the predictive ability and interpretability in all data sets. The use of colors proportional to the Pauling´s electronegativity showed that MIA descriptors are capable of identifying periodic properties relevant for the studied activity. Finally, solid colors instead of spotlighted atoms allowed a correct identification of atoms by means of pixel values in the studies for malaria, dengue and schistosomiasis, which were, subsequently, useful for the chemical interpretation related to the bioactivity. It can be inferred that semicarbazones and thiosemicarbazones derivative with a tri-substituted ring in R1 group and a trifluoro methyl group in the R 3 position instead of a chlorine antitripanossoma resulted in higher activity. The antimalarial activity of quinolon-4(1H)imines can be improved if: 1) R1 and R2 are electron donor groups, 2) R3 has long aminoalkyl chains, and 3) R4 possesses substituents with big atomic volume. In the study for dengue, it was found that tetrapeptides with unbranched small size amino acids in the A1 and A4 positions can increase the substrate affinity (Km) to the NS3 protein, and when in A1 and A2 positions, the substrate cleavage rate (kcat). On the other hand, acidic amino acids in the A2 and A4 positions were found to be related with low substrate affinity to the NS3 protein and when present in A1, with low substrate cleavage rate. Finally, the presence of metoxy substituents in R1 (or R2) and R5 in the neolignan backbone can favor their antischistosomal activity.

Análise funcional e estrutural comparativa da fastuosaina com papaína e bromelinas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Infidelidades: representações femininas e masculinas

O amor desperta nas pessoas lembranças. sentimentos e emoções prazerosas. associadas a histórias romanescas que. dificilmente. referem os conflitos que permeiam as relações amorosas. Cotidianamente, o amor é vivido e narrado não apenas a partir do belo mas. sobretudo. em função dos conflitos que produzem desarmonia O amor vivido em conjugalidade é regido pela monogamia. cultivada a partir do desejado ideal de fidelidade enquanto opção. Entretanto. muitas pessoas estabelecem relações afetivo e/ou sexual fora do vínculo conjugal, despertado por insatisfação afetiva e/ou sexual: pela falta de carinho, amor e/ou atenção ou. quem sabe por impulsos. desejos. prazer. paixão, vingança e/ou competição. comportamentos socialmente não desejáveis. Tentou-se desvendar como as relações de infidelidade são vivenciadas cotidianamente por oito mulheres e cinco homens, em Belém Pará , que se dispuseram a compartilhar histórias e segredos com a pesquisadora sobre o assunto. Como há uma circularidade de idéias, os informantes permitem. a pesquisadora. a partir de suas narrativas, encontrar diversas visões e inúmeras justificativas dadas à prática da infidelidade afetiva e/ou sexual: assim como nuanças diferenciadas que informam as representações femininas e masculinas sobre infidelidade, possibilitando ler os limites que estruturam as relações de conjugalidade. pois a infidelidade pensada como elemento desestruturador, é. em geral, escamoteada e serve de estratégia ao jogo amoroso garantindo. algumas vezes, quando não descoberta, a manutenção dos laços estabelecidos entre os casais.

Estudo teórico do mecanismo redox de derivados quinolínicos na atividade antimalárica

A malária é um sério problema de saúde pública mundial, acarretando perdas socioeconômicas e contribuindo para o subdesenvolvimento dos países afetados. Neste contexto, faz-se necessário estudar a relação entre as propriedades eletrônicas e a capacidade antioxidante de derivados quinolínicos na atividade antimalárica, o que servirá de subsídio para propor protótipos eficazes na terapêutica da doença. Nesta dissertação, foram utilizadas técnicas de modelagem molecular, no estudo da relação estrutura e atividade antioxidante correlacionada com a atividade antimalárica, no processo de seleção de grupamentos e parâmetros eletrônicos e conformacionais que permitam aperfeiçoar a atividade farmacológica e reduzir a toxicidade dos derivados. A análise dos valores de HOMO e PI indicou que o tautômero imino-quinolina é, provavelmente, melhor antioxidante que o tautômero amino-quinolina. Também se observou que o equilíbrio dos tautômeros é mais deslocalizado para a estrutura amino-quinolina na fase gasosa, e em água e clorofórmio no método PCM, apresentando valores de barreiras de energia da faixa de 10,78 Kcal/mol, 21,65 Kcal/mol e 22,04 Kcal/mol, respectivamente. Assim pôdese observar que nos derivados análogos de quinolina, os grupos elétrons-doadores mostraram destaque na redução do potencial de ionização, como os grupos amina na posição 8 substituído por um grupo alquilamina. Nos derivados da associação de 4- e 8-amino-quinolina notou-se que a presença de um segundo nitrogênio no grupo quinolina diminui seu potencial antioxidante, com exceção da posição 5, representando o grupo de maior destaque na redução do potencial de ionização e conseqüente provável elevada atividade antioxidante.

Maximizing the utility of bio-based diisocyanate and chain extenders in crystalline segmented thermoplastic polyester urethanes: effect of polymerization protocol

High molecular weight semi crystalline thermoplastic poly(ester urethanes), TPEUs, were prepared from a vegetable oil-based diisocyanate, aliphatic diol chain extenders and poly(ethylene adipate) macro diol using one-shot, pre-polymer and multi-stage polyaddition methods. The optimized polymerization reaction achieved ultra-high molecular weight TPEUs (>2 million as determined by GPC) in a short time, indicating a very high HPMDI diol reactivity. TPEUs with very well controlled hard segment (HS) and soft segment (SS) blocks were prepared and characterized with DSC, TGA, tensile analysis, and WAXD in order to reveal structure property relationships. A confinement effect that imparts elastomeric properties to otherwise thermoplastic TPEUs was revealed. The confinement extent was found to vary predictably with structure indicating that one can custom engineer tougher polyurethane elastomers by "tuning" soft segment crystallinity with suitable HS block structure. Generally, the HPMDI-based TPEUs exhibited thermal stability and mechanical properties comparable to entirely petroleum-based TPEUs. (C) 2014 Elsevier Ltd. All rights reserved.

Toxicity and loss of mitochondrial membrane potential induced by Alkyl Gallates in Trypanosoma cruzi

American trypanosomiasis or Chagas disease is a debilitating disease representing an important social problem that affects, approximately, 10 million people in the world. The main aggravating factor of this situation is the lack of an effective drug to treat the different stages of this disease. In this context, the search for trypanocidal substances isolated from plants, synthetic or semi synthetic molecules, is an important strategy. Here, the trypanocidal potential of gallates was assayed in epimastigotes forms of T. cruzi and also, the interference of these substances on the mitochondrial membrane potential of the parasites was assessed, allowing the study of the mechanism of action of the gallates in the T. cruzi organisms. Regarding the preliminary structure-activity relationships, the side chain length of gallates plays crucial role for activity. Nonyl, decyl, undecyl, and dodecyl gallates showed potent antitrypanosomal effect (IC50 from 1.46 to 2.90 μM) in contrast with benznidazole (IC50 = 34.0 μM). Heptyl gallate showed a strong synergistic activity with benznidazole, reducing by 105-fold the IC50 of benznidazole. Loss of mitochondrial membrane potential induced by these esters was revealed. Tetradecyl gallate induced a loss of 53% of the mitochondrial membrane potential, at IC50 value.