Amino acid sequence and crystal structure of BaP1, a metalloproteinase from Bothrops asper snake venom that exerts multiple tissue-damaging activities

BaP1 is a 22.7-kD P-I-type zinc-dependent metalloproteinase isolated from the venom of the snake Bothrops asper, a medically relevant species in Central America. This enzyme exerts multiple tissue-damaging activities, including hemorrhage, myonecrosis, dermonecrosis, blistering, and edema. BaP1 is a single chain of 202 amino acids that shows highest sequence identity with metalloproteinases isolated front the venoms of snakes of the subfamily Crotalinae. It has six Cys residues involved in three disulfide bridges (Cys 117-Cys 197, Cys 159-Cys 181, Cys 157-Cys 164). It has the consensus sequence H(142)E(143)XXH(146)XXGXXH(152), as well as the sequence C164I165M166, which characterize the metzincin superfamily of metalloproteinases. The active-site cleft separates a major subdomain (residues 1-152), comprising four a-helices and a five-stranded beta-sheet, from the minor subdomain, which is formed by a single a-helix and several loops. The catalytic zinc ion is coordinated by the N-epsilon2 nitrogen atoms of His 142, His 146, and His 152, in addition to a solvent water molecule, which in turn is bound to Glu 143. Several conserved residues contribute to the formation of the hydrophobic pocket, and Met 166 serves as a hydrophobic base for the active-site groups. Sequence and structural comparisons of hemorrhagic and nonhemorrhagic P-I metalloproteinases from snake venoms revealed differences in several regions. In particular, the loop comprising residues 153 to 176 has marked structural differences between metalloproteinases with very different hemorrhagic activities. Because this region lies in close proximity to the active-site microenvironment, it may influence the interaction of these enzymes with physiologically relevant substrates in the extracellular matrix.

A micelle nucleation model for the interaction of dodecyl sulphate with Lys49-phospholipases A(2)

Bothropstoxin-I (BthTx-I) is a Lys49-PLA(2) from the venom of Bothrops jararacussu that lacks detectable catalytic activity, yet causes rapid Ca2+-independent membrane damage. With the aim of understanding the interaction between BthTx-I and amphiphilic molecules, we have studied the interaction of sodium dodecyl sulphate (SDS) with the protein. Circular dichroism and attenuated total reflection Fourier-transform infrared spectra of BthTx-I reveal changes in the alpha-helical organization of the protein at an SDS/BthTx-I molar ratio of 20-25. At SDS/BthTx-I ratios of 40-45 the alpha-helices return to a native-like conformation, although fluorescence emission anisotropy measurements of 2-amino-N-hexadecyl-benzamide (AHBA) demonstrate that the total SDS is below the critical micelle concentration when this transition occurs. These results may be interpreted as the result of SDS accumulation by the BthTx-I homodimer and the formation of a pre-micelle SDS/BthTx-I complex, which may subsequently be released from the protein surface as a free micelle. Similar changes in the alpha-helical organization of BthTx-I were observed in the presence of dipalmitoylphosphatidylcholine liposomes, suggesting that protein structure transitions coupled to organization changes of bound amphiphiles may play a role in the Ca2+-independent membrane damage by Lys49-PLA(2)s. (c) 2006 Elsevier B.V. All rights reserved.

Nonlinear Lattice Within Supersymmetric Quantum Mechanics Formalism

In the last decades, the study of nonlinear one dimensional lattices has attracted much attention of the scientific community. One of these lattices is related to a simplified model for the DNA molecule, allowing to recover experimental results, such as the denaturation of DNA double helix. Inspired by this model we construct a Hamiltonian for a reflectionless potential through the Supersymmetric Quantum Mechanics formalism, SQM. Thermodynamical properties of such one dimensional lattice are evaluated aming possible biological applications.

Selectivity in the mechanism of action of antimicrobial mastoparan peptide Polybia-MP1

Many potent antimicrobial peptides also present hemolytic activity, an undesired collateral effect for the therapeutic application. Unlike other mastoparan peptides, Polybia-MP1 (IDWKKLLDAAKQIL), obtained from the venom of the social wasp Polybia paulista, is highly selective of bacterial cells. The study of its mechanism of action demonstrated that it permeates vesicles at a greater rate of leakage on the anionic over the zwitterionic, impaired by the presence of cholesterol or cardiolipin; its lytic activity is characterized by a threshold peptide to lipid molar ratio that depends on the phospholipid composition of the vesicles. At these particular threshold concentrations, the apparent average pore number is distinctive between anionic and zwitterionic vesicles, suggesting that pores are similarly formed depending on the ionic character of the bilayer. To prospect the molecular reasons for the strengthened selectivity in Polybia-MP1 and its absence in Mastoparan-X, MD simulations were carried out. Both peptides presented amphipathic alpha-helical structures, as previously observed in Circular Dichroism spectra, with important differences in the extension and stability of the helix; their backbone solvation analysis also indicate a different profile, suggesting that the selectivity of Polybia-MP1 is a consequence of the distribution of the charged and polar residues along the peptide helix, and on how the solvent molecules orient themselves according to these electrostatic interactions. We suggest that the lack of hemolytic activity of Polybia-MP1 is due to the presence and position of Asp residues that enable the equilibrium of electrostatic interactions and favor the preference for the more hydrophilic environment.

Three exopolysaccharides of the beta-(1 -> 6)-D-glucan type and a beta-(1 -> 3;1 -> 6)-D-glucan produced by strains of Botryosphaeria rhodina isolated from rotting tropical fruit

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

Influence of N-Terminus Modifications on the Biological Activity, Membrane Interaction, and Secondary Structure of the Antimicrobial Peptide Hylin-a1

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

Mechanism of Action and Relationship Between Structure and Biological Activity of Ctx-Ha: A New Ceratotoxin-like Peptide from Hypsiboas albopunctatus

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Spectrophotometric Evaluation of the Behavior of Disperse Red 1 Dye in Aqueous Media and its Interaction with Calf Thymus ds-DNA

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

Morfologia dos ovários, tubas uterinas e útero em catetos (Tayassu tajacu, Linnaeus, 1758) e queixadas (Tayassu pecari, Link, 1795)

Estudaram-se a morfologia externa, a sintopia e os dados métricos dos ovários, tubas uterinas e útero em 14 fêmeas adultas de cateto (12 adultas, uma prenhe e uma jovem) e em sete fêmeas de queixada (três jovens e quatro adultas), e o material, após fixação em solução aquosa de formol a 10%, foi dissecado. O material foi obtido na Universidade Federal de Uberlândia (UFU) - Campus Palotina e na Universidade Estadual Paulista - Campus de Ilha Solteira. Os ovários apresentaram-se ovalados e de superfície irregular, quando havia corpo lúteo ou folículo. As tubas uterinas mostraram-se longas, finas e enoveladas e terminando numa extremidade ovárica com fímbrias; a comunicação com o útero ocorreu de maneira contínua. Uma bolsa ovárica parcial, com um largo orifício, contém o ovário. O útero, bicórneo, apresentou um corpo curto e uma cérvix longa, com projeções anulares para o canal cervical, conferindo-lhe luz espiralada. Os cornos uterinos mostraram-se curtos, voltados ventralmente e em forma de hélice.

Mecanismos celulares e moleculares que controlam o desenvolvimento e o crescimento muscular

O músculo estriado esquelético é formado pela associação de fibras musculares com a matriz extracelular. Esse tecido possui alta plasticidade e o conhecimento das características morfológicas, da miogênese, e da dinâmica do crescimento é importante para o entendimento da morfofisiologia bem como para a seleção de animais visando a melhoria na produção de carne. A maioria dos músculos estriados originam-se de células precursoras do mesoderma a partir dos somitos do embrião e o controle da diferenciação ocorre pela ação de fatores indutores ou inibidores. Um grupo de fatores transcricionais, pertencentes à família MyoD tem um papel central na diferenciação muscular. Coletivamente chamados de Fatores de Regulação Miogênica (MRFs), são conhecidos quatro tipos: MyoD, myf-5, miogenina e MRF4. Esses fatores ligam-se à seqüências de DNA conhecidas como Ebox (CANNTG) na região promotora de vários genes músculo-específicos, levando à expressão dos mesmos. As células embrionárias com potencial para diferenciação em células musculares (células precursoras miogênicas) expressam MyoD e Myf-5 e são denominadas de mioblastos. Essas células proliferam, saem do ciclo celular, expressam miogenina e MRF4, que regulam a fusão e a diferenciação da fibra muscular. Uma população de mioblastos que se diferencia mais tardiamente, as células miossatélites, são responsáveis pelo crescimento muscular no período pós natal, que pode ocorrer por hiperplasia e hipertrofia das fibras. As células satélites quiescentes não expressam os MRFs, porém, sob a ação de estímulos como fatores de crescimento ou citocinas, ocorre a ativação desse tipo celular que prolifera e expressa os MRFs de maneira similar ao que ocorre com as células precursoras miogênicas durante a miogênese. Os mecanismos de crescimento muscular são regulados pela expressão temporal dos (MRFs), que controlam a expressão dos genes relacionados com o crescimento muscular.

Purification, sequencing and structural characterization of the phospholipase A(1) from the venom of the social wasp Polybia paulista (Hymenoptera, Vespidae)

The biochemical and functional characterization of wasp venom toxins is an important prerequisite for the development of new tools both for the therapy of the toxic reactions due to envenomation caused by multiple stinging accidents and also for the diagnosis and therapy of allergic reactions caused by this type of venom. PLA(1) was purified from the venom of the neotropical social wasp Polybia paulista by using molecular exclusion and cation exchange chromatographies; its amino acid sequence was determined by using automated Edman degradation and compared to the sequences of other vespid venom PLA(1)'s. The enzyme exists as a 33,961.40 da protein, which was identified as a lipase of the GX class, liprotein lipase superfamily, pancreatic lipases (ab20.3) homologous family and RP2 sub-group of phospholipase. P. paulista PLA(1) is 53-82% identical to the phospholipases from wasp species from Northern Hemisphere. The use restrained-based modeling permitted to describe the 3-D structure of the enzyme, revealing that its molecule presents 23% alpha-helix, 28% beta-sheet and 49% coil. The protein structure has the alpha/beta fold common to many lipases; the core consists of a tightly packed beta-sheet constituted of six-stranded parallel and one anti-parallel beta-strand, surrounded by four alpha-helices. P. paulista PLA(1) exhibits direct hemolytic action against washed red blood cells with activity similar to the Cobra cardiotoxin from Naja naja atra. In addition to this, PLA(1) was immunoreactive to specific IgE from the sera of P. paulista-sensitive patients. (c) 2007 Elsevier Ltd. All rights reserved.

Commensal worm traces and possible juvenile thalassinidean burrows associated with Ophiomorpha nodosa, Pleistocene, southern Brazil

The Pleistocene Chui Formation at Osorio (Rio Grande do Sul, Brazil) consists of coastal marine and eolian sands, the former containing abundant and well-preserved Ophiomorpha nodosa burrow systems. Detailed ichnological study has revealed interesting features associated with them. Small-sized Ophiomorpha, here assigned to a new ichnospecies, O. puerilis, are interpreted as possible burrows of juvenile thalassinidean crustaceans probably belonging to the same species as the producers of larger O. nodosa. Additionally, helicoidal burrows with thick, concentrically laminated linings are associated with the walls of O. nodosa. They are assigned to the new ichnospecies Cylindrichnus helix, and they are interpreted as dwellings of commensal annelid worms. The association of these three icbnospecies constitutes a fossil example of the role of thalassinideans as ecosystem engineers able to modify their environment and to create new space and resources usable by other organisms. (c) 2005 Elsevier B.V. All rights reserved.

Indocyanine green nanoparticles useful for photomedicine

Objective: the aim of this study was to evaluate the potential application of biodegradable nanoparticles (NPs) containing indocyanine green (ICG) in photodynamic therapy (PDT). Methods: Important parameters, such as particle size and external morphology, were established by dynamic light scattering (DLS) and scanning electron microscopy (SEM). Also, drug encapsulation efficiency and in vitro release behavior were evaluated by spectroscopic methods. Results: the particles are spherical in shape, they exhibit an 817-nm diameter, and they have a low tendency to aggregate. The loading efficiency was 65%. ICG photophysical parameters showed a bathocromic shift in ICG-loaded nanoparticles (ICG-NP). Analysis of the cell P388-D1 in the presence of the ICG-NP by SEM showed that the majority of the nanoparticles were uptaken by phagocytic cells after 2 h of incubation. After laser irradiation photodamage was observed in P388-D1 cells where ICG-NPs had been uptaken by phagocytic cells. Conclusion: Polymeric NPs work as an efficient drug delivery system for PDT drugs, and this approach can be used in the administration of amphiphilic photosensitizers in the treatment of neoplasic cells.

Aggregation behaviour of hydrophobically modified poly(allylammonium) chloride

The behaviour of hydrophobically modified poly(allylammonium) chloride having octyl, decyl, dodecyl and hexadecyl side chains has been studied in aqueous solution using fluorescence emission techniques. Micropolarity studies using the I-1/I-3 ratio of the vibronic bands of pyrene show that the formation of hydrophobic microdomains depends on both the length of the side chain and the polymer concentration. The I-1/I-3 ratio of the polymers with low hydrophobe content (less than 5% mel) changes substantially when reaching a certain concentration. These changes are assigned to aggregation originating from interchain interactions. This behaviour is also confirmed by the behaviour of the monomer/excimer emission intensities of pyrene- dodecanoic acid used as a probe. For polymers having dodecyl side chains and hydrophobe contents higher than 10%, aggregates are formed independently of the polymer concentration. Anisotropy measurements show that microdomains resulting from the inter- and/or intramolecular interactions are similar to those observed for cationic surfactants. Viscosity measurements show that the coil dimensions are substantially decreased for the polymers having high hydrophobe contents, indicating intramolecular associations.

Analysis of the ethidium bromide bound to DNA by photoacoustic and FTIR spectroscopy

Under physiological conditions B-form DNA is an exceedingly stable structure. However, experimental evidences obtained through nuclear magnetic resonance and fluorescence anisotropy suggest that the structure of the double helix fluctuates substantially. We describe photoacoustic phase modulation frequency measurements of ethidium bromide (Eb) with calf thymus, DNA. As in fluorescence phase modulation measurements, we used an intercalating dye as a probe; however, we monitored the triplet excited state lifetime at different ionic strengths. The triplet lifetime of Eb varied from about 0.30 ms, with no DNA present, to 20 ms, (at a DNA:Eb molar ratio of 5). With salt titration, this value falls, to about 2.0 ms. This result suggests, a strong coupling between the phenantridinium ring of the ethidium and the base pairs because of the stacking movement of the DNA molecule under salt effect. This, effect may be understood considering DNA as a polyelectrolyte. The counterions, in the solution shield the phosphate groups, reducing the electrostatic repulsion force between them, hence compacting the DNA molecule. The results from Fourier transform infrared demonstrated two important bands: 3187 cm(-1) corresponding to the symmetric stretching of the NH group of the bases, and 1225 cm(-1) corresponding to the asymmetric stretching of phosphate groups shifted toward higher wavenumbers, suggesting a proximity between the intercalant and base pairs and a modification of the DNA backbone state, both induced by salt accretion.