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.

Influence of the riparian zone phytophysiognomies on the longitudinal distribution of fishes: evidence from a Brazilian savanna stream

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

Resource use and coexistence of two syntopic hylid frogs (Anura, Hylidae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

A molecular mechanism for Lys(49)-phospholipase A(2) activity based on ligand-induced conformational change

Agkistrodon contortrix laticinctus myotoxin is a Lys(49)- phospholipase A(2) (EC 3.1.1.4) isolated from the venom of the serpent A contortrix laticinctus (broad-banded copperhead). We present here three monomeric crystal structures of the myotoxin, obtained under different crystallization conditions. The three forms present notable structural differences and reveal that the presence of a ligand in the active site (naturally presumed to be a fatty acid) induces the exposure of a hydrophobic surface (the hydrophobic knuckle) toward the C terminus. The knuckle in A contortrix laticinctus myotoxin involves the side chains of Phe(121) and Phe(124) and is a consequence of the formation of a canonical structure for the main chain within the region of residues 118-125. Comparison with other Lys(49)-phospholipase A(2) myotoxins shows that although the knuckle is a generic structural motif common to all members of the family, it is not readily recognizable by simple sequence analyses. An activation mechanism is proposed that relates fatty acid retention at the active site to conformational changes within the C-terminal region, a part of the molecule that has long been associated with Ca2+-independent membrane damaging activity and myotoxicity. This provides, for the first time, a direct structural connection between the phospholipase active site and the C-terminal myotoxic site, justifying the otherwise enigmatic conservation of the residues of the former in supposedly catalytically inactive molecules.

Crystallographic and pre-steady-state kinetics studies on binding of NADH to wild-type and isoniazid-resistant enoyl-ACP(CoA) reductase enzymes from Mycobacterium tuberculosis

An understanding of isoniazid (INH) drug resistance mechanism in Mycobacterium tuberculosis should provide significant insight for the development of newer anti-tubercular agents able to control INH-resistant tuberculosis (TB). The inhA-encoded 2-trans enoyl-acyl carrier protein reductase enzyme (InhA) has been shown through biochemical and genetic studies to be the primary target for INH. In agreement with these results, mutations in the inhA structural gene have been found in INH-resistant clinical isolates of M. tuberculosis, the causative agent of TB. In addition, the InhA mutants were shown to have higher dissociation constant values for NADH and lower values for the apparent first-order rate constant for INH inactivation as compared to wild-type InhA. Here, in trying to identify structural changes between wild-type and INH-resistant InhA enzymes, we have solved the crystal structures of wild-type and of S94A, I47T and I21V InhA proteins in complex with NADH to resolutions of, respectively, 2.3 angstrom, 2.2 angstrom, 2.0 angstrom, and 1.9 angstrom. The more prominent structural differences are located in, and appear to indirectly affect, the dinucleotide binding loop structure. Moreover, studies on pre-steady-state kinetics of NADH binding have been carried out. The results showed that the limiting rate constant values for NADH dissociation from the InhA-NADH binary complexes (k(off)) were eleven, five, and tenfold higher for, respectively, I21V, I47T and S94A INH-resistant mutants of InhA as compared to INH-sensitive wildtype InhA. Accordingly, these results are proposed to be able to account for the reduction in affinity for NADH for the INH-resistant InhA enzymes. (c) 2006 Elsevier Ltd. All rights reserved.

Structure of magnetic poly(oxyethylene)-siloxane nanohybrids doped with Fe-II and Fe-III

Hybrid organic - inorganic nanocomposites doped with Fe-II and Fe-III ions and exhibiting interesting magnetic properties have been obtained by the sol - gel process. The hybrid matrix of these ormosils ( organically modified silicates), classed as di-ureasils and termed U( 2000), is composed of poly( oxyethylene) chains of variable length grafted to siloxane groups by means of urea crosslinkages. Iron perchlorate and iron nitrate were incorporated in the diureasil matrices, leading to compositions within the range 80 greater than or equal to n greater than or equal to 10, n being the molar ratio of ether-type O atoms per cation. The structure of the doped diureasils was investigated by small-angle X-ray scattering (SAXS). For Fe-II-doped samples, SAXS results suggest the existence of a two-level hierarchical structure. The primary level is composed of spatially correlated siloxane clusters embedded in the polymeric matrix and the secondary, coarser level consists of domains where the siloxane clusters are segregated. The structure of Fe-III-doped hybrids is different, revealing the existence of iron oxide based nanoclusters, identified as ferrihydrite by wide-angle X-ray diffraction, dispersed in the hybrid matrix. The magnetic susceptibility of these materials was determined by zero-field-cooling and field-cooling procedures as functions of both temperature and field. The different magnetic features between Fe-II- and Fe-III-doped samples are consistent with the structural differences revealed by SAXS. While Fe-II-doped composites exhibit a paramagnetic Curie-type behaviour, hybrids containing Fe-III ions show thermal and field irreversibilities.

Jahn-Teller effect on the structure of the Sm-doped PbTiO3: A theoretical approach

Samarium doped PbTiO3 (PT:Sm) and pure PbTiO3 (PT) powders were obtained by polymeric precursor method. These powders were characterized by X-ray diffraction (XRD) and theoretical calculations using the CRYSTAL98 program. The effect of the samarium atom is taken into account only indirectly. The experimental models were compared with the cubic (ideal) and tetragonal theoretical models. The structure deformations existent in the experimental compounds were analyzed from the tiny structural differences that lead to perturbations in the crystal orbital splittings. This paper proposes an efficient alternative methodology for defining structural distortions. (c) 2007 Elsevier B.V. All rights reserved.

Molecular model of cyclin-dependent kinase 5 complexed with roscovitine

Here is described a structural model for the binary complex CDK5-roscovitine. Roscovitine has been shown to potently inhibit cyclin-dependent kinases 1, 2 and 5 (CDK1, 2, and 5), and the structure of CDK2 complexed with roscovitine has been reported; however, no structural data, are available for complexes of CDK5 with inhibitors. The structural model indicates that roscovitine strongly binds to the ATP-binding pocket of CDK5 and structural comparison of the CDK2-roscovitine complex correlates the structural differences with differences in inhibition of these CDKs by this inhibitor. This structure opens the possibility of testing new inhibitor families, in addition to new substituents for the already known lead structures of adenine derivatives. (C) 2002 Elsevier B.V. (USA). All rights reserved.

New X-ray powder diffraction data and Rietveld refinement for Gd2O3 monodispersed fine spherical particles

Nominally pure Gd2O3 C-form structure from basic carbonate fine spherical particles and its differences concerning the XRD data among literature patterns using Rietveld method is reported. Gd2O3: Eu3+ from basic carbonate and Gd2O3 from oxalate were also investigated. All samples, except the one from oxalate precursor, are narrow sized, 100-200 nm. Only non-doped Gd2O3 from basic carbonate presents XRD data with smaller d(hkl) values than the literature ones. From Rietveld refinement, non-doped Gd2O3 from basic carbonate has the smallest crystallite size and from oxalate shows the greatest one. Also, the unit cell parameters indicate a plan contraction of the Gd2O3 from basic carbonate. The presence of Eu3+ increases crystallite size when basic carbonate precursor is used to prepare Gd2O3 and avoids plan contraction. The structural differences observed among Gd2O3 samples obtained are related to the type of precursor and to the presence or not of doping ion. (C) 2003 Elsevier B.V. (USA). All rights reserved.

Comparative genomics of two Leptospira interrogans serovars reveals novel insights into physiology and pathogenesis

Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in accidental hosts, including humans. Complete genome sequencing of Leptospira interrogans serovar Copenhageni and comparative analysis with the available Leptospira interrogans serovar Lai genome reveal that despite overall genetic similarity there are significant structural differences, including a large chromosomal inversion and extensive variation in the number and distribution of insertion sequence elements. Genome sequence analysis elucidates many of the novel aspects of leptospiral physiology relating to energy metabolism, oxygen tolerance, two-component signal transduction systems, and mechanisms of pathogenesis. A broad array of transcriptional regulation proteins and two new families of afimbrial adhesins which contribute to host tissue colonization in the early steps of infection were identified. Differences in genes involved in the biosynthesis of lipopolysaccharide 0 side chains between the Copenhageni and Lai serovars were identified, offering an important starting point for the elucidation of the organism's complex polysaccharide surface antigens. Differences in adhesins and in lipopolysaccharide might be associated with the adaptation of serovars Copenhageni and Lai to different animal hosts. Hundreds of genes encoding surface-exposed lipoproteins and transmembrane outer membrane proteins were identified as candidates for development of vaccines for the prevention of leptospirosis.

Resonant x-ray diffraction as a tool to calculate mixed valence ratios: Application to Prussian Blue materials

An approach is proposed here to calculate mixed valence ratios in molecular compounds. Synchrotron x-ray powder diffraction experiments were conducted to determine the Fe(+3)/Fe(+2) ratio in Prussian Blue compounds, which were elected as an example of the use of this approach. As a result, a resonant x-ray diffraction measurement provided direct evidence that the vacant [Fe(CN)(6)] group was randomly absent from similar to 31% of the structure, which was indicated by structural differences caused by variations in the anomalous dispersion term. These findings are very important for a deeper understanding of the changes occurring in properties during in situ compositional variations. (c) 2008 American Institute of Physics.

Características anátomo-estruturais da pelé de ovinos (ovis áries L.) lanados e deslanados, relacionadas com o aspecto físico-mecânico do couro

The objective of the study was establishing the anatomical-structural differences between the skin of non-wool sheep of the Morada Nova breed and wool-on sheep of the Polwarth or Ideal breeds and their relations to the physical-mechanical characteristics of their leather after tanning. Ten animals of both breeds were used, with five animals of approximately one and four years of age of each breed. The animals were slaughtered, and immediately after skinning, samples were taken viewing the microtomy of the skin in the dorsal, lateral, ventral, hind and palette regions, parallel and perpendicular to the head-tail axle, and perpendicular to the grain of the skin. The skins were depilated, chromium-tanned and re-tanned. From the leather, three samples were taken from the two directions considered from the regions studied for physical-mechanical analysis, for tension and tear resistance and distension on the lastometer. The average of the results of the physical-mechanical analysis were compared by Tukey test at the levels of 1 and 5% probability. The breed, the age, the region and the position exerted a positive effect on the values of the resistance of the leather to tension for the Morada Nova four year old sheep, reaching a minimum of 200 kg/cm . The region and the position exerted a positive effect on the values of tear-resistence of the leather for both breeds studied, reaching a minimum of 40 kg/cm. The resistance of the leather on distension was superior to 8 mm, for all the animals studied, not influenced by breed, age, region or position. The skin of the Ideal sheep presented a thermostatic skin layer greater than the reticular layer with great folicular and glandular density, contrary to the skin of the Morada Nova sheep where the thermostatic and reticular layers are of approximately the same thickness, with less folicular and glandular density.

The behavior and natural history of Chartergellus, a little-known genus of neotropical social wasps (Vespidae Polistinae Epiponini)

This study reviews published data on the behavior and natural history of Chartergellus and presents the first observations on social interactions in this genus of tropical swarm-founding wasps. Observations of Chartergellus golfitensis in Costa Rica and C. punctatior in Colombia showed that queens perform a post-oviposition egg-guarding vigil, and a bending display like that characteristic of epiponine social wasps that lack consistent morphological differences between workers and queens and have caste determination in the adult stage. Young, old, and queen (egg-laying) females of C. golfitensis showed small differences that indicate color changes with age, and structural differences that could be due to seasonal or colony-cycle changes in developmental conditions, but do not rule out the possibility of pre-adult caste determination, a phenomenon that needs to be carefully distinguished from pre-adult caste bias. Sexual dimorphism and the behavior of males at the nest in C. golfitensis is described, as well as the aggressive and avoidance behavior of females toward males. Nest structure in both species is as described previously for Chartergellus species, but some anomalies and their possible evolutionary significance are discussed. Cell initiation by an egg-laying queen, a behavior never seen by workers, and by a young female with slightly developed ovaries, may be vestiges of ancestral solitary reproductive traits where developed ovaries are associated with cell construction. © 2010 Dipartimento di Biologia Evoluzionistica dell'Università, Firenze, Italia.

β-lactam antibiotics epitope mapping with STD NMR spectroscopy: A study of drug-human serum albumin interaction

Molecular recognition events are key issues in many biological processes. STD NMR (saturation transfer difference nuclear magnetic resonance spectroscopy) is one of the techniques used to understand such biological interactions. Herein, we have investigated the interactions of four β-lactam antibiotics belonging to two classes (cephalosporins and penicillins) with human serum albumin (HSA) by 1H STD NMR revealing that the interaction between the aromatic moiety and HSA is responsible for the binding efficiency. Thus, the structural differences from the five to six-membered thio ring in penicillins and cephalosporins do not seem to influence antibiotic-albumin interactions. © 2012 Sociedade Brasileira de Química.

Silver colloidal nanoparticles: Effect on matrix composition and structure of Candida albicans and Candida glabrata biofilms

Aim: The aim of this study was to assess the effect of different silver nanoparticles (SN) concentrations on the matrix composition and structure of Candida albicans and Candida glabrata biofilms. Methods and Results: Candida biofilms were developed in 6-well microtiter plates during 48 h. After, these biofilms were exposed to 13·5 or 54 μg SN ml-1 for 24 h. Then, extracellular matrices were extracted from biofilms and analysed chemically in terms of proteins, carbohydrates and DNA. To investigate the biofilm structure, scanning electron microscopy (SEM) and epifluorescence microscopy were used. SN interfered with the matrix composition of Candida biofilms tested in terms of protein, carbohydrate and DNA, except for the protein content of C. albicans biofilm. By SEM, Candida biofilms treated with SN revealed structural differences, when compared with the control groups. Further, SN showed a trend of agglomeration within the biofilms. Epifluorescence microscopy images suggest that SN induced damage on cell walls of the Candida isolates tested. Conclusions: In general, irrespective of concentration, SN affected the matrix composition and structure of Candida biofilms and these findings may be related to the mechanisms of biocide action of SN. Significance and Impact of the Study: This study reveals new insights about the behaviour of SN when in contact with Candida biofilms. SN may contribute to the development of therapies to prevent or control Candida infections. © 2012 The Society for Applied Microbiology.