RESUMO
Essential oils (EOs) from Citrus are the main by-product of Citrus-processing industries. In addition to food/beverage and cosmetic applications, citrus EOs could also potentially be used as an alternative to antibiotics in food-producing animals. A commercial citrus EO-Brazilian Orange Terpenes (BOT)-was fractionated by vacuum fractional distillation to separate BOT into various fractions: F1, F2, F3, and F4. Next, the chemical composition and biological activities of BOT and its fractions were characterized. Results showed the three first fractions had a high relative amount of limonene (≥10.86), even higher than the whole BOT. Conversely, F4 presented a larger relative amount of BOT's minor compounds (carvone, cis-carveol, trans-carveol, cis-p-Mentha-2,8-dien-1-ol, and trans-p-Mentha-2,8-dien-1-ol) and a very low relative amount of limonene (0.08-0.13). Antibacterial activity results showed F4 was the only fraction exhibiting this activity, which was selective and higher activity on a pathogenic bacterium (E. coli) than on a beneficial bacterium (Lactobacillus sp.). However, F4 activity was lower than BOT. Similarly, F4 displayed the highest antioxidant activity among fractions (equivalent to BOT). These results indicated that probably those minor compounds that detected in F4 would be more involved in conferring the biological activities for this fraction and consequently for the whole BOT, instead of the major compound, limonene, playing this role exclusively.
Assuntos
Antibacterianos/farmacologia , Antioxidantes/farmacologia , Citrus/química , Óleos Voláteis/química , Fracionamento Químico , Análise Fatorial , Testes de Sensibilidade Microbiana , Terpenos/análiseRESUMO
Different strategies have been used to overcome the difficulties to produce antimicrobial peptides. Here we used Intein Mediated Purification with an Affinity Chitin-binding Tag (IMPACT-System, New England Biolabs) for the expression of the antimicrobial peptide cecropin to reduce its sensitivity to intracellular proteases and use its inducible self-cleaving capability to remove the carrier. Cecropin was cloned into suitable expression vector pTYB11, and expression induced by IPTG in Escherichia coli ER2566. The use of 22ºC induction allowed the expression of cecropin with its intein carrier in soluble form. Cell extracts were purified by chitin affinity chromatography and intein-mediated splicing of the target protein was achieved by thiol addition, obtaining a final yield of 2.5 mg cecropin/l. Cecropin cleaved from the intein had its proper biologically active form, showing a micromolar antimicrobial activity against Vibrio ordalii, Vibrio alginolyticus and Escherichia coli.
Assuntos
Humanos , Antibacterianos/metabolismo , Cecropinas/metabolismo , Escherichia coli , Western Blotting , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Fusão Gênica , Inteínas , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas RecombinantesRESUMO
Antimicrobial peptides (AMP) have been widely described in several organisms from different kingdoms. We recently designed and evaluated a synthetic version of an AMP isolated and characterized from Argopecten purpuratus hemocytes. This study describes the generation of a chimaeric gene encoding for Ap-S, the use of this construct to transform E. coli strain BL21, and the evaluation of the purified recombinant Ap-S (rApS) as an antifungal agent. The proposed gene coding for rAp-S consists of 93 nucleotides arranged downstream from the IPTG-inducible T7 promoter. The best synthesis conditions were obtained after E. coli cultivation at 26°C for 3h, which allowed for the production of an rAp-S-enriched fraction containing the peptide at 249µM. Mass spectrometry analysis of the purified rAp-S (3085.80Da) showed the addition of a glycine residue on its N-terminal end derived from vector design and peptide purification. The purified rApS fraction was assayed for antifungal activity by direct addition of purified rApS elution to potato dextrose agar media at a final concentration of 81nM. These assays showed important growth inhibitions of both biotrophic (Fusarium oxysporum, Trichoderma harzianum) and necrotrophic (Botrytis cinerea, Alternaria spp.) fungi in that the hyphae structures and spore count were affected in all cases. The strategy of cloning and expressing rAp-S in E. coli, the high yield obtained and its successful use for controlling pathogenic fungi suggest that this molecule could be applied to agricultural crops using various management strategies.
Assuntos
Antifúngicos/farmacologia , Escherichia coli/metabolismo , Pectinidae/química , Peptídeos/farmacologia , Alternaria/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Antifúngicos/isolamento & purificação , Sequência de Bases , Botrytis/efeitos dos fármacos , Clonagem Molecular , Contagem de Colônia Microbiana , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Fusarium/efeitos dos fármacos , Genes Sintéticos , Vetores Genéticos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Trichoderma/efeitos dos fármacosRESUMO
Knowledge of the 3D structure of the binding groove of major histocompatibility (MHC) molecules, which play a central role in the immune response, is crucial to shed light into the details of peptide recognition and polymorphism. This work reports molecular modeling studies aimed at providing 3D models for two class I and two class II MHC alleles from Salmo salar (Sasa), as the lack of experimental structures of fish MHC molecules represents a serious limitation to understand the specific preferences for peptide binding. The reliability of the structural models built up using bioinformatic tools was explored by means of molecular dynamics simulations of their complexes with representative peptides, and the energetics of the MHC-peptide interaction was determined by combining molecular mechanics interaction energies and implicit continuum solvation calculations. The structural models revealed the occurrence of notable differences in the nature of residues at specific positions in the binding groove not only between human and Sasa MHC proteins, but also between different Sasa alleles. Those differences lead to distinct trends in the structural features that mediate the binding of peptides to both class I and II MHC molecules, which are qualitatively reflected in the relative binding affinities. Overall, the structural models presented here are a valuable starting point to explore the interactions between MHC receptors and pathogen-specific interactions and to design vaccines against viral pathogens.
Assuntos
Epitopos/química , Complexo Principal de Histocompatibilidade/imunologia , Simulação de Dinâmica Molecular , Peptídeos/química , Salmo salar/imunologia , Alelos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Epitopos/imunologia , Epitopos/metabolismo , Humanos , Peptídeos/imunologia , Ligação Proteica/imunologia , Homologia de Sequência de AminoácidosRESUMO
We have isolated and purified a natural antimicrobial peptide from Argopecten purpuratus hemocytes. 47 residues were determined from its primary structure representing the N-terminal of the complete sequence. This peptide of 5100.78Da was chemically synthesized and named Ap. The peptide has 25% of hydrophobic amino acids with a net charge of +1, and partial homology with known active antimicrobial peptides. Based on that sequence, a new peptide was designed and modeled to increase hydrophobicity and cationicity. The designed 30-residue peptide was chemically synthesized resulting in a novel 38% hydrophobic molecule named peptide Ap-S, with a net charge of +5 and 3028Da. A secondary structure was shown by circular dichroism, thus exposing a hydrophobic epitope toward the N-terminus and a hydrophilic one toward the C-terminus, improving amphipathicity. Ap-S was much more active than the parental Ap. Ap-S up to 100microM has no cytotoxic effect against fish cell line CHSE-214. We demonstrated that the chemical modification of a natural peptide and the chemical synthesis of derived molecules may be a powerful tool for obtaining substitutes to conventional antibiotics, displaying the many advantages of antimicrobial peptides and overcoming the limitations of natural peptides for large-scale production and application, such as the low specific activity and the minute amounts recovered in vivo. This peptide may have a relevant application in aquaculture by controlling Saprolegna sp., a parasitic pathogen fungus that attacks the culture of fish in different stages of their growth, from egg to adult.
Assuntos
Anti-Infecciosos/isolamento & purificação , Antifúngicos/isolamento & purificação , Hemócitos/metabolismo , Pectinidae/metabolismo , Peptídeos/química , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Anti-Infecciosos/química , Antifúngicos/síntese química , Antifúngicos/química , Antifúngicos/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Dicroísmo Circular , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Peptídeos/síntese química , Salmonidae , Homologia de Sequência de AminoácidosRESUMO
Novel doublet molecules of cecropin A from Drosophila melanogaster were designed and constructed combining the regular (CECdir) with the inverted (CECret) coding sequence of the standard CEC A1 gene resulting in the following configurations: CECdir-CECret and CECret-CECdir. These two recombinant molecules were generated using a three-primer driven PCR reaction yielding composite single functional aminoacidic molecules with the coding sequences of CECdir linked in frame with the coding sequence of CECret and vice versa. In order to obtain these constructions, a retropeptide DNA-coding sequence was chemically synthesized to match the expected polarity of the newly generated CECret sequence. Both doublet antimicrobial peptides (drAMPs) were cloned in the T7 promoter driven expression plasmid pET27b+ and expressed in E. coli BL21 without any fusion protein. Only the former recombinant peptide was expressed and purified from cell extracts and its specific activity against two different bacteria showed to be higher than those displayed by their monomer parental counterparts.
Assuntos
Cecropinas , Drosophila melanogaster , Cromatografia Líquida de Alta Pressão , Escherichia coliRESUMO
CECdir-CECret is a novel non-toxic doublet 8.5 kDa peptide representing the natural coding sequence of the antimicrobial peptide Cecropin A from Drosophila melanogaster fused in-frame to its own inverted version. Expression of this cloned doublet peptide in Escherichia coli, yielded peptides that were mostly packaged into inclusion bodies. The new molecule was purified, solubilized and refolded, through a standard guanidine-based procedure. The recovered refolded peptides were then characterized by HPLC chromatography, MALDI-TOF-mass spectrometry and peptide sequencing, and finally evaluated for their antimicrobial potential. The novel doublet peptide CECdir-CECret, displays an enhanced in vitro antimicrobial activity and action spectrum in comparison to the monomer Cecropin A.
Assuntos
Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia , Animais , Anti-Infecciosos/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Corpos de Inclusão/metabolismo , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
A "defensin-like" antibacterial peptide from Mytilus edulis chilensis, was sub-cloned into a binary vector for expression in plant tissues. The resulting new clone was electroporated into A. tumefaciens to transform tobacco plants. The presence of the construct in transgenic tobacco lines was demonstrated through RT-PCR, Northern and Western blots. Transformed positive plants were selected and grown for challenging. Tobacco leaves were infiltrated with Pseudomonassyringae pv. syringae and visual lesions determined at different times post-exposure. Of seven plants exposed, four gave variable protection up to seven days post-infection while one of them appears to be fully protected. These results suggest that defensin-like antimicrobial peptides from molluscs are a good source to provide resistance of tobacco plants to Pseudomonassyringae pv. syringae.
RESUMO
Antimicrobial peptides are small-sized, cationic and amphipathic molecules able to neutralize pathogenic microorganisms. Their antimicrobial effects tie them to mechanisms of immune defense, which is why they have been normally purified from immune cells. We describe an apparently new group of antimicrobial peptides from gill tissues of the mussel Mytilus edulis chilensis. 20 specimens yielded 40 g of gills which produced 16 mg of an enriched fraction with antimicrobial activity as low as 0.045 µg/µl over reference strains. Considering the chemical nature of these molecules we used an acid extraction procedure followed by consecutive cationic exchange and hydrophobic interaction chromatography steps for peptide enrichment. The resulting post Sep-pak C-18® 20 percent acetonitrile (ACN) eluate was fractionated by reverse phase HPLC and all resulting fractions were the source for in vitro antimicrobial activity evaluation. Active fractions were characterized by SDS-containing protein gel electrophoresis. All fractions were particularly enriched with low molecular weight peptides displaying neutralizing growth activity against Gram positive and Gram negative bacteria and 10 times more efficient over fungal pathogens. Active fractions resulted to be thermostable and non cytotoxic to eukaryotic cells. Considering these results, industrial waste gills of bivalves arise as a new source for antimicrobial molecules.
Assuntos
Animais , Bivalves/química , Brânquias/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Antibacterianos/farmacologia , Bactérias Gram-Negativas , Bactérias Gram-Positivas , Cromatografia Líquida de Alta Pressão , Defensinas , Eletroforese , Interações Hidrofóbicas e Hidrofílicas , Imunidade Inata , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Temperatura , Extratos de Tecidos , Testes de ToxicidadeRESUMO
Se analiza la práctica de la enfermería en Colombia por medio de una investigación cualitativa que busca comprender el valor social del cuidado de enfermería. la información se obtuvo mediante entrevistas a profesionales y auxiliares que trabajaban en instituciones de salud con mayor desarrollo en el proceso de implementación de la reforma. Se expone la experiencia d enfermería desde la subjetividad de los actores entrevistados frente a lo que hacen, lo que saben, las cosas que hacen y el contexto en que trabajan. La naturaleza del estudio no permite generalizaciones, pero sí identifica tendencias de la práctica de la enfermería. Se constató que las condiciones de trabajo son precarias: existe distanciamiento del cuidador profesional con el sujeto de atención, idealización de roles, dilemas éticos, vacíos conceptuales para precisar la esencia de su quehacer, sufrimiento psíquico y burocratización de los servicios. Constituye un estudio de caso dentro de un trabajo multicéntrico orientado por la OPS realizado en Argentina, Colombia, Brasil, México y Estados Unidos donde se estudia, además, la regulación y la educación en enfermería.