Caracterização estrutural e funcional de fenilseptinas : peptídeos com atividades antimicrobiana e químico-sensorial presentes na secreção cutânea de Hypsiboas punctatus

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Pós-graduação em Biologia Molecular, 2011.As toxinas de origem vegetal, animal e microbiana há muito despertam o interesse do homem. Assim, a busca por moléculas bioativas que permitam uma ampla biblioteca informativa surge como alternativa na busca de soluções de problemas agrícolas e farmacológicos. A pele dos anfíbios é uma rica fonte destas moléculas, as quais são produzidas pelas glândulas serosas holócrinas do tegumento, as quais estão localizadas nos grânulos do lúmem, e são liberadas sob a presença de estímulos apropriados. Suas funções são variadas, atuando tanto na regulação das funções fisiológicas da pele, como na defesa contra predadores e microrganismos. As moléculas mais estudas são os peptídeos antimicrobianos (PAM), os quais atuam diretamente na defesa química contra patógenos. Neste trabalho, foi estudada uma nova classe de PAM presentes na secreção cutânea do anuro Hypsiboas punctatus, as fenilseptinas (Phes). Estas são peptídeos antimicrobianos de 18 resíduos de aminoácido (FFFDTLKNLAGKVIGALT-NH2), com massa molecular M+H+=1954.2, as quais ocorrem naturalmente na forma de uma mistura racêmica (L-/D-Phes), e apresentam propriedades sensoriais. A enantiomerização do resíduo de aminoácido Phe2 e a amidação C-terminal foram estudadas e confirmadas por uma combinação de técnicas incluindo RP-UFLC, espectrometria de massa combinada com mobilidade iônica, experimentos de MS/MS de alta resolução, sequenciamento de cDNA, síntese de peptídeos em fase sólida e sequenciamento N-terminal por degradação de Edman. Os estudos detalhados das 20 estruturas de menor energia obtidas por experimentos de 1H RMN demonstram que os peptídeos assumem uma conformação de α-hélice dos resíduos 5-18 (RMSD 0.22 ± 0.09), e existe em D-Phes uma de mudança de 90° na orientação da porção N-terminal 1-3. Os peptídeos foram ativos contra bactérias gram-positivas e gram-negativas em ensaios de determinação da atividade antimicrobiana in vitro (e.x. S. aureus -65,5 e 32,7 μM, L-Phes e D-Phes, respectivamente). E, as mesmas moléculas foram testadas em camundongos às propriedades gustativas, em um sistema de animais KO para um canal iônico essencial para a transdução do sabor amargo e doce (Trpm5). Os camundongos WT rejeitaram os peptídeos (p 0,3). Provavelmente, as divergências estruturais são mais relacionadas com as diferenças observadas em relação às atividades antimicrobianas diferenciadas entre as duas moléculas, do que com as propriedades sensoriais. Fenilseptinas representam o primeiro relato de moléculas antimicrobianas, com propriedades sensoriais e enorme plasticidade estrutural. _________________________________________________________________________________ ABSTRACTToxins from plant, animal and microorganism long light the interest of man. Thus, the search for bioactive molecules that allow a large databank appears as an alternative looking for solutions to agricultural problems and pharmacological. Amphibian skin secretions is a rich source of these molecules, which are produced by serous holocrine glands, which are located in the lumen of the granules and are released in the presence of appropriate stimuli. Their functions are diverse, acting both in the regulation of physiological functions of the skin, as in defense against predators and microorganisms. The molecules most studied are the naturally occurring antimicrobial peptides (AMP), which act directly on the chemical defense against pathogens. Here we report a new class of AMP present in the skin secretions of anuran Hypsiboas punctatus, the fenilseptina (Phes). These are antimicrobial peptides of 18 amino acid residues (FFFDTLKNLAGKVIGALT-NH2), with a molecular mass of M+H+ = 1954.2, that was purified as two naturally occurring D- and L-Phe analogs, with sensorial properties. The amino acid epimerazition and C-terminal amidation for both molecules (in short, D-Phes and L-Phes) were confirmed by a combination of techniques including reverse-phase UPLC, ion mobility mass spectrometry, high resolution MS/MS experiments, Edman degradation, cDNA sequencing and solid-phase peptide synthesis. RMSD analysis of the twenty best 1H NMR structures of each peptide revealed that the peptides assume an α-helical conformation of residues 5-18 (RMSD 0:22 ± 0.09), and there is D-Phes has a major 90° difference between the two backbones at the first four N-terminal residues and substantial orientation changes of the respective side chains. The peptides were active against gram-positive and gram-negative in vitro assays (S. aureus -65.5 and 32.7 mM, L-and D-Phes Phes, respectively). And the same molecules were tested in mice to evaluate the properties in a system of KO animals to a ion channel essential for transduction of bitter and sweet (Trpm5). The mice rejected the WT peptide (p 0.3). These structural divergences are most likely linked to the quantified differences in antimicrobial potencies determined for both molecules, but produced no detectable change in the gustatory aversive behavior. Perhaps the Phenylseptins are early examples of the amazing plasticity of small biomolecules yet to be understood

Toxinas Cry : perspectivas para a obtenção de algodão transgênico brasileiro

Toxinas Cry provenientes de Bacillus thuringiensis, também denominadas de δ- endotoxinas, possuem ampla atividade inseticida e são específicas para insetos de diferentes ordens, não sendo nocivas a mamíferos, aves, anfíbios ou répteis. Devido a esta característica, seus genes vêm sendo utilizados no desenvolvimento de plantas transgênicas, visando o controle de insetos-praga de culturas de importância econômica. Dentre estas, destaca-se o algodoeiro que tem como principais pragas o bicudo-do-algodoeiro (Anthonomus grandis) e a lagarta-do-cartucho (Spodoptera frugiperda), ambas causadoras de grandes prejuízos econômicos. Assim, este trabalho objetivou: caracterizar uma estirpe de Bt (S811) tóxica contra S. frugiperda e A. grandis, identificar e clonar os genes codificadores de toxinas Cry potenciais para o controle dessas pragas, expressar os genes em sistema heterólogo Escherichia coli e testar a toxicidade das toxinas contra as pragas em questão. Os resultados obtidos nas análises da estirpe por microscopia eletrônica revelaram a presença de cristais esféricos e bipiramidais, enquanto que a análise de SDS-PAGE detectou proteínas de massa molecular com cerca de 140 kDa e 80 kDa. Posteriormente, uma caracterização gênica, utilizando oligonucleotídeos específicos para genes cry, identificou três genes, cry1Ab, cry1Ia e cry8, sendo estes dois últimos de maior interesse. O gene cry8, que foi isolado pela técnica TAIL-PCR, codifica uma proteína com 58% de identidade de aminoácidos com outras toxinas Cry8, enquanto que o gene cry1Ia12 (isolado com oligonucleotídeos específicos) codifica uma proteína que possui 99% de identidade com as outras proteínas do tipo Cry1Ia descritas na literatura. As porções tóxicas dos genes foram clonadas e expressadas em E. coli produzindo as proteínas recombinantes Cry8 e Cry1Ia12 de aproximadamente 70 kDa (cada uma). As proteínas recombinantes causaram mortalidade de 50% para larvas de A. grandis com 230 μg/ml e 160 μg/ml de Cry1Ia12 e Cry8, respectivamente, enquanto que para larvas de S. frugiperda, apenas Cry1Ia12 foi tóxica causando mortalidade de 50% com 5 μg/ml. Todos estes estudos contribuirão para o desenvolvimento de plantas transgênicas de algodão, bem como para a geração de novas moléculas com maior toxicidade e especificidade utilizando a evolução molecular in vitro (DNA shuffling) destes genes.Cry toxins or δ-endotoxins, produced by Bacillus thuringiensis, show broad activity against several orders of insects. In addition, they are harmless to mammals, birds, amphibian, and reptiles. Because of these characteristics, several genes have been used in the development of transgenic plants to control pests in important crops. Among them, the cotton crop is very important in Brazil and it has the cotton boll weevil, Anthonomus grandis, and the fall armyworm, Spodoptera frugiperda, as the major pests in this crop, which cause several economic damages. The objective of this work was: to characterize a Bt strain (S811), which is toxic towards S. frugiperda and A. grandis, to identify and clone the coding genes of the Cry toxins with potential to control these pests, to express the genes in Escherichia coli heterologous system, and to test the toxicity of the recombinant proteins towards the reported pests. Electronic microscopic analysis revealed the presence of bipyramidal and spherical crystals and SDS-PAGE analysis showed proteins with 140 kDa and 80 kDa of molecular weight mass. Further gene characterization, using specific primer sets to detect cry genes, identified three genes, cry1Ab, cry1Ia e cry8, although the cry1Ia e cry8 were the genes of major interest. The cry8 gene was isolated through TAIL-PCR techniques and encodes a protein 58% identical to other Cry8 toxins, while the protein deduced from cry1Ia12 gene showed 99% identity with other Cry1Ia toxins previously described. The cry1A12 and cry8 gene toxic fragments were cloned and expressed in E. coli, and the 70 kDa recombinant proteins of were tested against A. grandis and S. frugiperda. Cry1Ia12 toxin caused 50% mortality at 230 μg/ml and 5 μg/ml towards both A. grandis and S. frugiperda, respectively. Cry8 toxin caused 50% mortality only against A. grandis at 160 μg/ml. In addition to the transgenic cotton development program, these results will permit the generation of new molecules with higher toxicity and specificity for these pests through in vitro molecular evolution (DNA shuffling) of these genes

Synthesis, docking studies, pharmacological activity and toxicity of a novel pyrazole derivative (LQFM 021) - possible effects on phosphodiesterase

Submitted by Liliane Ferreira ([email protected]) on 2018-08-23T13:40:13Z No. of bitstreams: 2 Artigo - Daniella Ramos Martins - 2013.pdf: 1568399 bytes, checksum: 5c6f84859e286fa8622ce63354ca2ed5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)Rejected by Luciana Ferreira ([email protected]), reason: Olhe a citação: MARTINS, Daniella Ramos et al. Synthesis, docking studies, pharmacological activity and toxicity of a novel pyrazole derivative (LQFM 021)-possible effects on phosphodiesterase. Chemical and Pharmaceutical Bulletin, Tokyo, v. 61, n. 5, p. 524-531, May, 2013. Não há vírgula depois do mês. on 2018-08-24T12:44:20Z (GMT)Submitted by Liliane Ferreira ([email protected]) on 2018-08-24T14:14:42Z No. of bitstreams: 2 license_rdf: 9 bytes, checksum: 42dd12a06de379d3ffa39b67dc9c7aff (MD5) Artigo - Daniella Ramos Martins - 2013.pdf: 1568399 bytes, checksum: 5c6f84859e286fa8622ce63354ca2ed5 (MD5)Approved for entry into archive by Luciana Ferreira ([email protected]) on 2018-08-27T11:23:32Z (GMT) No. of bitstreams: 2 license_rdf: 9 bytes, checksum: 42dd12a06de379d3ffa39b67dc9c7aff (MD5) Artigo - Daniella Ramos Martins - 2013.pdf: 1568399 bytes, checksum: 5c6f84859e286fa8622ce63354ca2ed5 (MD5)Made available in DSpace on 2018-08-27T11:23:32Z (GMT). No. of bitstreams: 2 license_rdf: 9 bytes, checksum: 42dd12a06de379d3ffa39b67dc9c7aff (MD5) Artigo - Daniella Ramos Martins - 2013.pdf: 1568399 bytes, checksum: 5c6f84859e286fa8622ce63354ca2ed5 (MD5) Previous issue date: 2013-05This study describes the synthetic route and molecular computational docking of LQFM 021, as well as examines its biological effects and toxicity. The docking studies revealed strong interaction of LQFM 021 to phosphodiesterase-3 (PDE-3). In isolated arteries, the presence of endothelium potentiates the relaxation for LQFM 021 and the inhibition cyclic nucleotides reduced the relaxation. Pre-contraction with KCl (45 mm), the treatment with tetraethylammonium (TEA) (5 mm) and inhibition of reticular Ca2+-ATPase showed an inhibitory effect on relaxation. Moreover, the compound reduced the contraction evoked by the Ca2+ influx. Acute toxicity tests revealed that the compound was practically nontoxic. In conclusion, this study showed that a new synthetic derivative of pyrazole is a possible PDE-3 inhibitor and has vasorelaxant activity and low toxicit

Characterization of novel Brazilian Bacillus thuringiensis strains active against Spodoptera frugiperda and other insect pests

Abstract: Brazilian strains of Bacillus thuringiensis, namely S701, S764 and S1265 were analysed regarding their cry gene and protein contents, crystal type, and activity against larvae of the lepidopteran fall armyworm (Spodoptera frugiperda Smith), the velvet caterpillar (Anticarsia gemmatalis), the dipterans (Culex quinquefasciatus and Aedes aegypti) and the coleopteran (Tenebrio molitor). The LC50 of the strains against second instar larvae of S. frugiperda or A. gemmatalis revealed a high potency against those insect species. The spore–crystal mixtures of the isolates were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and showed similar protein pattern as the B. thuringiensis subsp. kurstaki strain HD-1 (proteins approximately 130 and 65 kDa) for isolates S701 and S764, respectively, and only one major protein of approximately 130 kDa for isolate S1265. The polymerase chain reaction (PCR) using total DNA of the isolates and general and specific primers showed the presence of cry1Aa, cry1Ac, cry1Ia and cry2Ab genes in the two isolates serotyped as B. thuringiensis kurstaki (S701 and S764) and the presence of cry1D and cry2Ad in B. thuringiensis morrisoni S1265 strain. Scanning electron microscopy of strains S701 and S764, showed the presence of bipyramidal, cuboidal and round crystals, like in strain HD-1 and bipyramidal and round crystals like in strain S1265