Dereplication of Bromotyrosine-derived Metabolites by LC-PDA-MS and Analysis of the Chemical Profile of 14 Aplysina Sponge Specimens from the Brazilian Coastline

In order to investigate the chemical profile of 14 specimens of Aplysina spp. marine sponges, we have developed a method based on LC-PDA-MS for the detection of bromotyrosine-derived metabolites. The method enabled the dereplication of three distinct chemotypes of bromotyrosine-derived compounds based on UV absorptions, which were further refined by electrospray ionization-mass spectrometry analysis of the brominated quasi-molecular ion clusters. This procedure led to either a single compound assignment, or a maximum of two possible isobaric compounds. The dereplication study indicated that the chemical profile of the 14 specimens of Aplysina spp. analyzed presented practically the same dibromotyrosine-derived compounds. The results obtained suggested a possible biogenetic pathway for the formation of dibromotyrosine-derived compounds of wide occurrence in Verongida sponges.

Pyrodysinoic Acid Derivatives from the Marine Sponge Dysidea robusta

Three new nitrogen-containing terpenes related to pyrodysinoic acid (1) have been isolated from the sponge Dysidea robusta collected in Brazil. Isopyrodysinoic acid (2), 13-hydroxyisopyrodysinoic acid (3), and pyrodysinoic acid B (4) were obtained from the crude extract of D. robusta and identified by analysis of spectroscopic data. Pyrodysinoic acid B (4) is the first furodysin or furodysinin sesquiterpene derivative with a trans junction between the two six-membered rings of the 1,2,3,4,4a,7,8,8a-octahydro-1,1,6-trimethylnaphthalene moiety.

Crystallization and preliminary X-ray diffraction analysis of a eumenine mastoparan toxin: a new class of mast-cell degranulating peptide in the wasp venom

Mastoparans are tetradecapeptides found to be the major component of vespid venoms. A mastoparan toxin isolated from the venom of Anterhynchium flavomarginatum micado has been crystallized and X-ray diffraction data collected to 2.7 Angstrom resolution using a synchrotron-radiation source. Crystals were determined to belong to the space group P6(2)22 (P6(4)22). This is the first mastoparan to be crystallized and will provide further insights into the conformational significance of mastoparan toxins with respect to their potency and activity in G-protein regulation.

Preliminary cryocrystallography analysis of an eumenine mastoparan toxin isolated from the venom of the wasp Anterhynchium flavomarginatum micado

Mastoparans are tetradecapeptides found to be the major component of vespid venoms. These peptides present a wide spectrum of biological activities, such as mast cell degranulation, hemolytic activity and also reveals antimicrobial activity. A mastoparan toxin isolated from the venom of Anterhynchium flavomarginatum micado has been crystallized. At room temperature these crystals diffracted to 2.8 Angstrom resolution. However, upon cooling to cryogenic temperature around 85 K, the original resolution limit could be improved to 2.0 Angstrom. Crystals were determined to belong to the space group P3(1) (P3(2)). This is the first mastoparan to be crystallized and it will provide further insights in the conformational significance of mastoparan toxins, with respect to their potency and activity in G protein regulation. (C) 3001 Elsevier B.V. B.V. All rights reserved.

Xylella fastidiosa gene expression analysis by DNA microarrays

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

Retrospective analysis of post-exposure to human anti-rabies treatment in Botucatu, São Paulo State, Brazil

This analysis aimed to identify characteristics of accidents that would, probably, provoke rabies infection. A total of 14,409 survey questionnaires for surveillance of human rabies from the Brazilian Information System for Disease Notification (SINAN), from 2000 to 2005, were analyzed. Regarding demographics, it was observed that 7,377 (51.5%) of the victims were white, 4,458 (30.93%) were children and 8,008 (55.58%) were males. Urban cases were prevalent (88.10%) while dogs were the animals most frequently involved in accidents, in 11,700 cases (81.19%). Bites (84.35%) and scratches (19.15%) were the most prevalent exposure types, and occurred predominantly on victims' extremities (38.79%). The prophylactic measure taken in 6,179 cases comprised anti-rabies vaccine; of these victims, 421 (2.92%) showed systemic reactions while 693 (4.80%) reported no response. The importance of developing awareness in professionals that should correctly report post-exposure immunoprophylaxis cases is emphasized given the high number of individuals who receive this type of treatment annually.

Neutralization of snake venom phospholipase A(2) toxins by aqueous extract of Casearia sylvestris (Flacourtiaceae) in mouse neuromuscular preparation

Aqueous extract of Casearia sylvestris (Flacourtiaceae) has been shown to inhibit enzymatic and biological properties of some Bothrops and Crotalus venoms and their purified phospholipase A(2) (PLA(2)) toxins. In this work we evaluated the influence of C sylvestris aqueous extract upon neuromuscular blocking and muscle damaging activities of some PLA(2)S (crotoxin from C. durissus terrificus, bothropstoxin-I from B.jararacussu, piratoxin-I from B. pirajai and myotoxin-II from B. moojeni) in mouse phrenic-diaphragm preparations. Crotoxin (0.5 mu M) and all other PLA2 toxins (1.0 mu M) induced irreversible and time-dependent blockade of twitches. Except for crotoxin, all PLA2 toxins induced significant muscle damage indices, assessed by microscopic analysis. Preincubation of bothropstoxin-I, piratoxin-I or myotoxin-II with C. sylvestris extract (1:5 (w/w), 30 min, 37 degrees C significantly prevented the neuromuscular blockade of preparations exposed to the mixtures for 90 min; the extent of protection ranged from 93% to 97%. The vegetal extract also neutralized the muscle damage (protection of 80-95%). Higher concentration of the C. sylvestris extract (1: 10, w/w) was necessary to neutralize by 90% the neuromuscular blockade induced by crotoxin. These findings expanded the spectrum of C. sylvestris antivenom activities, evidencing that it may be a good source of potentially useful PLA2 inhibitors. (c) 2007 Elsevier B.V.. All rights reserved.

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.

Cryptic hammerhead shark lineage occurrence in the western South Atlantic revealed by DNA analysis

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

Chromosome banding and 18S rDNA in situ hybridization analysis of seven species of the family Achiridae (Teleostei : Pleuronectiformes)

Achiridae is an important family of the order Pleuronectiformes widely distributed in North, Central, and South America with freshwater and marine species. In the present study cytogenetic analyses comprising conventional and molecular techniques were carried out in seven species of this family. The following diploid numbers (2n) and fundamental numbers (FN) were obtained: Achirus declivis 2n = 34, FN = 52; Achirus lineatus 2n = 40, FN = 66; Catathyridium jenynsi 2n = 40 and FN = 50; Gymnachirus nudus 2n = 36 and FN = 50; Hypoclinemus mentalis 2n = 38 and FN = 54; Trinectes paulistanus 2n = 42 and FN = 52; and Trinectes sp. 2n = 38 and FN = 54. All species presented a single nucleolar organizer region (NOR) bearing chromosome pair and C-band positive segments mainly distributed at the pericentromeric position. The wide variation observed in chromosome number and FN suggests the occurrence of larger chromosome rearrangements in the family Achiridae if compared with other families of the same order.

DNA barcodes identify marine fishes of São Paulo State, Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Populations analysis of the Brazilian Sharpnose Shark Rhizoprionodon lalandii (Chondrichthyes: Carcharhinidae) on the São Paulo coast, Southern Brazil: inferences from mt DNA sequences

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

Structural description of humic substances from subtropical coastal environments using elemental analysis, FT-IR and C-13-solid state NMR data

Elemental composition and spectroscopic properties (FT-IR and CP/MAS C-13-NMR) of sedimentary humic substances (HS) from aquatic subtropical environments (a lake, an estuary and two marine sites) are investigated. Humic acids (HA) are relatively richer in nitrogen and in aliphatic chains than fulvic acids (FA) from the same sediments. Conversely, FA are richer in carboxylic groups and in ring polysaccharides than HA. Nitrogen is mostly present as amide groups and for lake and marine HS the FT-IR peaks around 1640 cm(-1) and 1540 cm(-1) identify polypeptides. Estuarine HS exhibit mixed continental-marine influences, these being highly influenced by site location. Overall, the data suggest that aquatic and mixed HS are more aliphatic than has been proposed in current models and also that amide linkages form an important part of their structural configuration.

The chelation of metal ions by the acylpolyamine toxins from the web-spider Nephilengys cruentata: effects in the intoxication/detoxification of preys

Orb-web-spiders present a series of different strategies for prey capture, involving the use of different types of silk for web building, the use of adhesive traps in the webs, the secretion of toxic compounds to the spider's preys in the adhesive coating of the capture web and the biosynthesis of a wide range of structurally related acylpolyamine toxins in their venoms. The polyamine toxins usually block neuromuscular junctions and/or the central nervous system (CNS) of Arthropods, targeting specially the ionotropic glutamate receptors; this way these toxins are used are as chemical weapons to kill / paralyze the spider's prey. Polyamine toxins contain many azamethylene groups involved with the chelation of metal ions, which in turn can interact with the glutamate receptors, affecting the toxicity of these toxins. It was demonstrated that the chelation of Ni+2, Fe+2, Pb+2, Ca+2 and Mg+2 ions by the desalted crude venom of Nephilengys cruentata and by the synthetic toxin JSTX-3, did not cause any significant change in the toxicity of the acylpolyamine toxins to the model-prey insect (honeybees). However, it was also reported that the chelation of Zn+2 ions by the acylpolyamines potentiated the lethal / paralytic action of these toxins to the honeybees, while the chelation of Cu+2 ions caused the inverse effect. Atomic absorption spectrometry and Plasma-ICP analysis both of N.cruentata venom and honeybee's hemolymph revealed that the spider's venom concentrates Zn+2 ions, while the honeybee's hemolymph concentrates Cu+2 ions. These results are suggesting that the natural accumulation of Zn+2 ions in N. cruentata venom favors the prey catching and/or its maintenance in the web, while the natural accumulation of Cu+2 ions in prey's hemolymph minimizes the efficiency of the acylpolyamine toxins as killing/paralyzing tool.