Larval stages of Paradasygyius depressus (Bell, 1835) (Crustacea : Decapoda : Brachyura : Majidae) and a phylogenetic analysis for 21 genera of Majidae

All larval stages and the first crab instar of Paradasygyius depressus (Bell) were obtained in laboratory culture. Larval development consists of two zoeal stages, followed by the megalopa. Each larval stage is described in detail. Beginning with the first zoea, the duration of each stage was 4--7 (4.5 +/- 0.7), 4-5 (4.5 +/- 0.5), and 7 days, the megalopa and first crab instar appearing 11 +/- 1 and 15 days after hatching, respectively. A phylogenetic analysis of 21 genera of Majidae is provided based on 34 zoeal and three megalopal characters. The phylogenetic analysis resulted in four equally parsimonious trees 173 steps long (CI = 0.66, RI = 0.71, and RC = 0.47) supporting the monophyly of Oregoniinae, Majinae, and Inachinae (with the exclusion of Macrocheira de Haan incertae sedis). Based on general agreement of sister-group hypotheses, we provide sets of larval characters that define Oregoniinae, Majinae, and Inachinae. Our phylogenetic hypothesis suggests that Oregoniinae is the most basal clade within the Majidae, and Majinae and the clade (Epialtus H. Milne Edwards + Inachinae [excluding Macrocheira incertae sedis]) are sister taxa. Within Inachinae, all trees suggest that Inachus Weber and Macropodia Leach are sister taxa nested as the most derived clade, followed by Achaeus Leach, Pyromaia Stimpson, Paradasygyius Garth, Anasimus A. Milne-Edwards, and the most basal Stenorhynchus Lamarck. The sister-group relationships of the clade (Pisa Leach (Taliepus A. Milne-Edwards + Libinia Leach)), Mithrax Latreille and Microphrys H. Milne Edwards remained unresolved.

Crystallization and preliminary X-ray crystallographic analysis of chorismate synthase from Mycobacterium tuberculosis

The enzymes of the shikimate pathway are potential targets for the development of new therapies because they are essential for bacteria but absent from mammals. The last step in this pathway is performed by chorismate synthase (CS), which catalyzes the conversion of 5-enolpyruvylshikimate-3-phosphate to chorismate. Optimization of crystallization trials allowed the crystallization of homogeneous recombinant CS from Mycobacterium tuberculosis (MtCS). The crystals of MtCS belong to space group P6(4)22 (or P6(2)22) and diffract to 2.8 Angstrom resolution, with unit-cell parameters a = b = 129.7, c = 156.8 Angstrom. There are two molecules in the asymmetric unit. Molecular-replacement trials were not sucessful. Heavy-atom derivative screening is in progress.

Structural and biological characterization of three novel mastoparan peptides from the venom of the neotropical social wasp Protopolybia exigua (Saussure)

The venom of the Neotropical social wasp Protopolybia exigua(Saussure) was fractionated by RP-HPLC resulting in the elution of 20 fractions. The homogeneity of the preparations were checked out by using ESI-MS analysis and the fractions 15, 17 and 19 (eluted at the most hydrophobic conditions) were enough pure to be sequenced by Edman degradation chemistry, resulting in the following sequences:Protopolybia MPI I-N-W-L-K-L-G-K-K-V-S-A-I-L-NH2 Protopolybia-MP II I-N-W-K-A-I-I-E-A-A-K-Q-A-L-NH2 Protopolybia-MP III I-N-W-L-K-L-G-K-A-V-I-D-A-L-NH2All the peptides were manually synthesized on-solid phase and functionally characterized. Protopolybia-MP I is a hemolytic mastoparan, probably acting on mast cells by assembling in plasma membrane, resulting in pore formation; meanwhile, the peptides Protopolybia-MP II and -MP III were characterized as a non-hemolytic mast cell degranulator toxins, which apparently act by virtue of their binding to G-protein receptor, activating the mast cell degranulation. (C) 2004 Elsevier Ltd. All rights reserved.

Structural and biological characterization of two novel peptides from the venom of the neotropical social wasp Agelaia pallipes pallipes

The venom of the neotropical social wasp Agelaia pallipes pallipes was fractionated by RP-HPLC resulting in the elution of seven fractions; the last two were re-fractionated under RP-HPLC by using isocratic elution conditions and the purity of the fractions were confirmed by using ESI-MS analysis. Both fractions are constituted of peptide components, which were sequenced by Edman degradation chemistry, resulting in the following sequences:Protonectin I-L-G-T-I-L-G-L-L-K-G-L-NH2Agelaia-MP I-N-W-L-K-L-G-K-A-I-I-D-A-L-NH2Both peptides are manually synthesized on solid-phase and functionally characterized by using Wistar rats cells. Protonectin is a non-hemolytic chemotactic peptide for polymorphonucleated leukocytes (PMNL), presenting some mast cell degranulating activity and potent antimicrobial action both against Gram-positive and Gram-negative bacteria. Agelaia-MP was characterized as a hemolytic mast cell degranulator toxin, presenting a poor antimicrobial action and no chemotaxis for PMNL. (C) 2004 Elsevier Ltd. All rights reserved.

Investigating the effect of different positioning of lysine residues along the peptide chain of mastoparans for their secondary structures and biological activities

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

How C-terminal carboxyamidation alters the biological activity of peptides from the venom of the eumenine solitary wasp

Inflammatory peptides display different types of post-transcriptional modifications, such as C-terminal amidation, that alter their biological activity. Here we describe the structural and molecular dynamics features of the mast cell degranulating peptide, eumenine mastoparan-AF (EMP-AF-NH2), found in the venom of the solitary wasp, and of its carboxyl-free C-terminal form (EMP-AF-COO-) characterized by a reduced activity. Circular dichroism indicates that both peptides switch from a random coil conformation in water to a helical structure in TFE and SDS micelles. NMR data, in 30% TFE, reveal that the two peptides fold into an alpha-helix spanning most of their length, while they differ in terms of molecular rigidity. To understand the origins of the conformational flexibility observed in the case of EMP-AF-COO-, a 5 ns MD simulation was carried out for each peptide, in an explicit water/TFE environment. The results show that the two peptides differ in an H-bond between Leu14 NH2 and the backbone carbonyl of Ile11. The loss of that H-bond in EMP-AF-COO- leads to a significant modification of its structural dynamics. In fact, as evidenced by essential dynamics analysis, while EMP-AF-NH2 exists mainly as a rigid structure, EMP-AF-COO- presents two helical stretches that fluctuate in some sort of independent fashion. We conclude that the diverse biological activity of the two peptides is not simply due to the reduction of the net positive charge, as generally suggested, but also to a structural perturbation of the amphipathic alpha-helix that affects their ability to perturb the cell membrane.

Proteomic analysis of kidney in rats chronically exposed to fluoride

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

Spatial and temporal population interactions between the parasitoids Cotesia flavipes and Tachinidae flies: considerations on the adverse effects of biological control practice

Biological control of Diatraea saccharalis is regarded as one of the best examples of successful classical biological control in Brazil. Since the introduction of the exotic parasitoid Cotesia flavipes, the decrease of D. saccharalis infestation in sugarcane fields has been attributed to the effectiveness of this agent. Recently, the native tachinid fly parasitoids (Lydella minense and Paratheresia claripalpis) have also been implicated in the success. Here, we investigated the spatial and temporal population interactions between C. flavipes and the tachinid flies, and provide a critical analysis of the biological control practice, focusing on the undesirable effects of introductions of exotic natural enemies. To investigate these questions, a large data set comprising information from two sugarcane mills located in the state of São Paulo, Brazil (Barra and Sao Joao Mills), was analysed. Analysis of the correlation between C. flavipes and tachinid fly population densities through time revealed that such populations were inversely correlated in the Sao Joao Mill and not correlated in the Barra Mill. Logistic regressions were computed to investigate the proportion of sites occupied by the parasitoid species at both mills as a function of time. An increasing trend in the proportion of sites occupied by C. flavipes was observed, with a concomitant decrease of the sites occupied by tachinid flies. This effect was more intense in the Sao Joao Mill. Thus, there is a convincing possibility that constant releases of C. flavipes decreased the tachinid fly populations, resulting in an undesirable effect of biological control practice.

Spectral analysis of the electromyograph of the erector spinae muscle before and after a dynamic manual load-lifting test

The aim of the present study was to assess the spectral behavior of the erector spinae muscle during isometric contractions performed before and after a dynamic manual load-lifting test carried out by the trunk in order to determine the capacity of muscle to perform this task. Nine healthy female students participated in the experiment. Their average age, height, and body mass (± SD) were 20 ± 1 years, 1.6 ± 0.03 m, and 53 ± 4 kg, respectively. The development of muscle fatigue was assessed by spectral analysis (median frequency) and root mean square with time. The test consisted of repeated bending movements from the trunk, starting from a 45º angle of flexion, with the application of approximately 15, 25 and 50% of maximum individual load, to the stand up position. The protocol used proved to be more reliable with loads exceeding 50% of the maximum for the identification of muscle fatigue by electromyography as a function of time. Most of the volunteers showed an increase in root mean square versus time on both the right (N = 7) and the left (N = 6) side, indicating a tendency to become fatigued. With respect to the changes in median frequency of the electromyographic signal, the loads used in this study had no significant effect on either the right or the left side of the erector spinae muscle at this frequency, suggesting that a higher amount and percentage of loads would produce more substantial results in the study of isotonic contractions.

Crystallization and preliminary X-ray analysis of bucain, a novel toxin from the Malayan krait Bungarus candidus

Bucain is a three-finger toxin, structurally homologous to snake-venom muscarinic toxins, from the venom of the Malayan krait Bungarus candidus. These proteins have molecular masses of approximately 6000-8000 da and encompass the potent curaremimetic neurotoxins which confer lethality to Elapidae and Hydrophidae venoms. Bucain was crystallized in two crystal forms by the hanging-drop vapour-diffusion technique in 0.1 M sodium citrate pH 5.6, 15% PEG 4000 and 0.15 M ammonium acetate. Form I crystals belong to the monoclinic system space group C2, with unit-cell parameters a = 93.73, b = 49.02, c = 74.09 Angstrom, beta = 111.32degrees, and diffract to a nominal resolution of 1.61 Angstrom. Form II crystals also belong to the space group C2, with unit-cell parameters a = 165.04, b = 49.44, c = 127.60 Angstrom, beta = 125.55degrees, and diffract to a nominal resolution of 2.78 Angstrom. The self-rotation function indicates the presence of four and eight molecules in the crystallographic asymmetric unit of the form I and form II crystals, respectively. Attempts to solve these structures by molecular-replacement methods have not been successful and a heavy-atom derivative search has been initiated.

Initial structural analysis of an alpha(4)beta(4) C-type lectin from the venom of Crotalus durissus terrificus

Convulxin, an alphabeta C-type lectin, is a potent platelet activator isolated from the venom of the South American rattlesnake Crotalus durissus terrificus. It is a 26.5 kDa alphabeta heterodimer consisting of two homologous disulfide-linked chains. The crystals belong to space group I4, with unit-cell parameters a = b = 131.61, c = 121.85 Angstrom, and diffraction data were collected to 2.7 Angstrom. The structure was solved by molecular replacement and the asymmetric unit contains two alphabeta heterodimers, each of which forms a disulfide-linked cyclic alpha(4)beta(4) tetramer in the unit cell. These alpha(4)beta(4) tetramers are stacked to form a large solvent channel.

Crystallization and preliminary X-ray diffraction analysis of the catalytic subunit of ADP-glucose pyrophosphorylase from potato tuber

ADP-glucose pyrophosphorylase is the key regulatory enzyme in the biosynthesis of starch in plants and glycogen in bacteria. The enzyme from potato tuber is comprised of a regulatory subunit and a catalytic subunit and is present as a heterotetramer (alpha(2)beta(2)) the catalytic subunit from potato tuber (50 kDa) was crystallized in four different forms, two of which are suitable for structural studies. A tetragonal crystal form obtained in the presence of the substrate analog Cr-ATP diffracted to 2.2 Angstrom and belongs to space group P4(1) (or its enantiomorph), with unit-cell parameters a = b = 110.57, c = 190.14 Angstrom. A second crystal form obtained diffracted to 2.8 Angstrom and belongs to space group PZ, with unit-eel parameters a = 80.06, b = 138.84, c = 92.20 Angstrom, beta = 112.40 degrees. As this protein displays no significant homology to any currently known protein structure, a search for heavy-atom derivatives has been initiated.

Crystallization and preliminary crystallographic analysis of SMase I, a sphingomyelinase from Loxosceles laeta spider venom

SMase I, a 32 kDa sphingomyelinase found in Loxosceles laeta venom, is responsible for the major pathological effects of spider envenomation. This toxin has been cloned and functionally expressed as a fusion protein containing a 6 x His tag at its N-terminus to yield a 33 kDa protein [Fernandes-Pedrosa et al. (2002), Biochem. Biophys. Res. Commun. 298, 638 - 645]. The recombinant protein possesses all the biological properties ascribed to the whole L. laeta venom, including dermonecrotic and complement-dependent haemolytic activities. Dynamic light-scattering experiments conducted at 291 K demonstrate that the sample possesses a monomodal distribution, with a hydrodynamic radius of 3.57 nm. L. laeta SMase I was crystallized by the hanging-drop vapour-diffusion technique using the sparse-matrix method. Single crystals were obtained using a buffer solution consisting of 0.08 M HEPES and 0.9 M trisodium citrate, which was titrated to pH 7.5 using 0.25 M sodium hydroxide. Complete three-dimensional diffraction data were collected to 1.8 Angstrom at the Laboratorio Nacional de Luz Sincrotron (LNLS, Campinas, Brazil). The crystals belong to the hexagonal system ( space group P6(1) or P6(5)), with unit-cell parameters a = b = 140.6, c = 113.6 Angstrom. A search for heavy-atom derivatives has been initiated and elucidation of the crystal structure is currently in progress.

SMase II, a new sphingomyelinase D from Loxosceles laeta venom gland: Molecular cloning, expression, function and structural analysis

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

Crystallization and preliminary X-ray diffraction analysis of a class II phospholipase D from Loxosceles intermedia venom

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