Purification and characterization of the biological effects of phospholipase A(2) from sea anemone Bunodosoma caissarum

Sea anemones contain a variety of biologically active substances. Bunodosoma caissarum is a sea anemone from the Cnidaria phylum, found only in Brazilian coastal waters. The aim of the present work was to study the biological effects of PLA(2) isolated from the sea anemone B. caissarum on the isolated perfused kidney, the arteriolar mesenteric bed and on insulin secretion. Specimens of B. caissarum were collected from the Sao Vicente Channel on the southern coast of the State of São Paulo, Brazil. Reverse phase HPLC analysis of the crude extract of B. caissarum detected three PLA(2) proteins (named BcPLA(2)1, BCPLA(2)2 and BcPLA(2)3) found to be active in B. caissarum extracts. MALDI-TOF mass spectrometry of BcPLA(2)1 showed one main peak at 14.7 kDa. The N-terminal amino acid sequence of BcPLA(2)1 showed high amino acid sequence identity with PLA(2) group III protein isolated from the Mexican lizard (PA23 HELSU, HELSU, PA22 HELSU) and with the honey bee Apis mellifera (PLA(2) and 1POC_A). In addition, BcPLA(2)1 also showed significant overall homology to bee PLA(2). The enzymatic activity induced by native BCPLA(2)1 (20 mu g/well) was reduced by chemical treatment with p-bromophenacyl bromide (p-BPB) and with morin. BcPLA(2)1 strongly induced insulin secretion in presence of high glucose concentration. In isolated kidney, the PLA(2) from B. caissarum increased the perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate, and sodium, potassium and chloride levels of excretion. BcPLA(2)1, however, did not increase the perfusion pressure on the mesenteric vascular bed. In conclusion, PLA(2), a group III phospholipase isolated from the sea anemone B. caissarum, exerted effects on renal function and induced insulin secretion in conditions of high glucose concentration. (C) 2009 Elsevier Ltd. All rights reserved.

Enzymatic and structural characterization of new PLA2 isoform isolated from white venom of Crotalus durissus ruruima

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

Proteomic analysis of dog tears for potential cancer markers

The first reference map of the proteome of pooled normal dog tears was created using 2-dimensional polyacrylamide gel electrophoresis and the identity of a number of the major species determined using matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF) and peptide mass fingerprint matching on protein sequence databases. In order to understand the changes in protein expression in the tear film of dogs with cancer, tears from such animals were similarly examined. A number of differences were found between the tears of healthy dogs and the dogs with cancer. Differences were found in levels of actin and albumin and in an unidentified protein which may be analogous to human lacryglobulin. These findings suggest that it may be possible to develop tear film analysis to provide a simple non-invasive test for the diagnosis and/or management of canine cancers. (C) 2007 Elsevier Ltd. All rights reserved.

Jelleines: a family of antimicrobial peptides from the Royal Jelly of honeybees (Apis mellifera)

Four antimicrobial peptides were purified from Royal Jelly of honeybees, by using reverse phase-HPLC and sequenced by using Q-Tof-MS/MS: PFKLSLHL-NH2 (Jelleine-I), TPFKLSLHL-NH2 (Jelleine-II), EPFKLSLHL-NH2 (Jelleine-III), and TPFKLSLH-NH2 (Jelleine-IV). The peptides were synthesized on-solid phase, purified and submitted to different biological assays: antimicrobial activity, mast cell degranulating activity and hemolysis. The Jelleines-I-III presented exclusively antimicrobial activities against yeast, Gram+ and Gram- bacteria; meanwhile, Jelleine-IV was not active in none of the assays performed. These peptides do not present any similarity with the other antimicrobial peptides from the honeybees; they are produced constitutively by the workers and secreted into Royal Jelly. (C) 2004 Elsevier B.V. All rights reserved.

Profiling the peptidome of the venom from the social wasp Agelaia pallipes pallipes

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

A systematic approach to identify STRE-binding proteins of the gsn glycogen synthase gene promoter in Neurospora crassa

The gene encoding glycogen synthase in Neurospora crassa (gsn) is transcriptionally down-regulated when mycelium is exposed to a heat shock from 30 to 45 degrees C. The gsn promoter has one stress response element (STRE) motif that is specifically bound by heat shock activated nuclear proteins. In this work, we used biochemical approaches together with mass spectrometric analysis to identify the proteins that bind to the STRE motif and could participate in the gsn transcription regulation during heat shock. Crude nuclear extract of heat-shocked mycelium was prepared and fractionated by affinity chromatography. The fractions exhibiting DNA-binding activity were identified by electrophoretic mobility shift assay (EMSA) using as probe a DNA fragment containing the STRE motif DNA-protein binding activity was confirmed by Southwestern analysis. The molecular mass (MM) of proteins was estimated by fractionating the crude nuclear extract by SDS-PAGE followed by EMSA analysis of the proteins corresponding to different MM intervals. Binding activity was detected at the 30-50 MM kDa interval. Fractionation of the crude nuclear proteins by IEF followed by EMSA analysis led to the identification of two active fractions belonging to the pIs intervals 3.54-4.08 and 6.77-7.31. The proteins comprising the MM and pI intervals previously identified were excised from a 2-DE gel, and subjected to mass spectrometric analysis (MALDI-TOF/TOF) after tryptic digestion. The proteins were identified by search against the MIPS and MIT N. crassa databases and five promising candidates were identified. Their structural characteristics and putative roles in the gsn transcription regulation are discussed.

Degradation of sulfamethoxazole in water by solar photo-Fenton. Chemical and toxicological evaluation

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Photodegradation of sulfamethoxazole in various aqueous media: Persistence, toxicity and photoproducts assessment

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Paracetamol degradation intermediates and toxicity during photo-Fenton treatment using different iron species

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

Conteúdo de peptídeos e avaliação morfofisiológica dos espermatozóides do epidídimo e ejaculado de bovinos

Objetivou-se com este estudo a identificação de alguns fatores protéicos envolvidos na qualidade funcional dos espermatozóides epididimais (SPZEP) e ejaculados (SPZEJ) de bovinos. Foram avaliadas as características morfofisiológicas e analisado o conteúdo peptídico destas estruturas de 11 animais mestiços Nelore, de 24 a 30 meses de idade. As avaliações morfofisiológicas foram motilidade progressiva (MOT, %), vigor, patologias espermáticas, integridade acrossômica e da cromatina. Foi observado que, os SPZEJ, na média, apresentaram MOT maior do que os SPZEP, 72,3 e 46,4%, respectivamente. Considerando as patologias espermáticas, taxas de defeitos maiores (DEFMAI), menores (DEFMEN) e totais (DEFTOT), houve diferença significativa entre as taxas dos DEFMEN e DEFTOT dos SPZEP e SPZEJ, sendo, em média, 91,1 e 8,5% e 95,4 e 11,8%, respectivamente. As taxas dos DEFMEN e DEFTOT dos SPZEP foram maiores em função da presença de espermatozóides com gotas citoplasmáticas distais. A análise das protéinas dos SPZEP e SPZEJ foi realizada por espectrometria de massa, método MALDI-TOF (matrix -assisted laser desorption/ionization - time of flight), e revelou presença de peptídeos de massa molecular variando de 1,1 a 26,3 kDa nos SPZEJ e de 1,1 a 11,6 kDa nos SPZEP. Foram identificados peptídeos de 10,6 e 13,4 kDa somente nos SPZEJ e de 6,8 kDa somente nos SPZEP. Foi observada relação do peptídeo de massa molecular de 7,4 kDa dos SPZEP e de 4,7 kDa dos SPZEJ, com a MOT Ê 80%, destas estruturas. Os resultados sugerem o envolvimento destes peptídeos nos processos funcionais das células espermáticas do epidídimo e ejaculado. O estudo utilizou o método MALDI/TOF para espectrômetro de massa, para identificar peptídeos em espermatozóides do epidídimo de bovinos, pela primeira vez no País.

High resolution ultra high pressure liquid chromatography-time-of-flight mass spectrometry dereplication strategy for the metabolite profiling of Brazilian Lippia species

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

Degradation of the antibiotic amoxicillin by photo-Fenton process - Chemical and toxicological assessment

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

Chemical and biological characterization of four new linear cationic α-helical peptides from the venoms of two solitary eumenine wasps

Four novel peptides were isolated from the venoms of the solitary eumenine wasps Eumenes rubrofemoratus and Eumenes fraterculus. Their sequences were determined by MALDI-TOF/TOF (matrix assisted laser desorption/ionization time-of-flight mass spectrometry) analysis, Edman degradation and solid-phase synthesis. Two of them, eumenitin-R (LNLKGLIKKVASLLN) and eumenitin-F (LNLKGLFKKVASLLT), are highly homologous to eumenitin, an antimicrobial peptide from a solitary eumenine wasp, whereas the other two, EMP-ER (FDIMGLIKKVAGAL-NH 2) and EMP-EF (FDVMGIIKKIAGAL-NH 2), are similar to eumenine mastoparan-AF (EMP-AF), a mast cell degranulating peptide from a solitary eumenine wasp. These sequences have the characteristic features of linear cationic cytolytic peptides; rich in hydrophobic and basic amino acids with no disulfide bond, and accordingly, they can be predicted to adopt an amphipathic α-helix secondary structure. In fact, the CD (circular dichroism) spectra of these peptides showed significant α-helical conformation content in the presence of TFE (trifluoroethanol), SDS (sodium dodecylsulfate) and asolectin vesicles. In the biological evaluation, all the peptides exhibited a significant broad-spectrum antimicrobial activity, and moderate mast cell degranulation and leishmanicidal activities, but showed virtually no hemolytic activity. © 2011 Elsevier Ltd.

Filamentous fungi from the Atlantic marine sponge Dragmacidon reticulatum

Dragmacidon reticulatum is a marine sponge of wide occurrence in the Eastern and Western Atlantic. Little is known about D. reticulatum fungal diversity. Filamentous fungi recovered from D. reticulatum were assessed in the present study using a polyphasic taxonomic approach, including classical morphology, molecular biology and MALDI-TOF ICMS. Ninety-eight fungal strains were isolated from two D. reticulatum samples by using six different culture media, which were identified up to the genus level. Sixty-four distinct fungal ribotypes were obtained, distributed among twenty-four different genera belonging to the Ascomycota and Zygomycota. Representatives of Penicillium and Trichoderma were the most diverse and abundant fungi isolated. Amongst Penicillium spp. three isolates belonged to the same ribotype can be considered as a putative new species. Data derived from the present study highlight the importance of using a polyphasic approach to get an accurate identification in order to structure a reliable culture collection. © 2012 Springer-Verlag Berlin Heidelberg.

Uma abordagem peptidômica do venedo do escorpião Tityus serrulatus

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