ABSTRACT
RESUMEN Introducción: Actualmente los péptidos sintéticos se han constituido en una novedosa alternativa para el tratamiento de la piel envejecida. Acetilhexapéptido-3 (Ac-EEMQRR-NH2) ha sido utilizado para inducir reducción de líneas de expresión de manera análoga a la toxina botulínica, pero sin efectos tóxicos. Objetivo: Sintetizar el acetilhexapéptido-3 y desarrollar un sistema liposomal para su encapsulación y favorecer su paso a través de una membrana modelo. Metodología: El péptido fue obtenido mediante síntesis en fase sólida (SFS) empleando la estrategia Fmoc/tBu, fue purificado y plenamente caracterizado. El sistema liposomal con acetilhexapéptido-3 encapsulado fue desarrollado mediante la formación de una emulsión con posterior inversión de fase por evaporación del solvente orgánico. Los sistemas fueron caracterizados en su tamaño, potencial zeta, eficiencia de encapsulación del neuropéptido y de manera preliminar se realizó un estudio de permeabilidad ex vivo. Resultados: Es posible sintetizar péptidos cortos con alto grado de pureza y buen rendimiento, utilizando la metodología de SFS Fmoc/tBu. De acuerdo con la caracterización, el sistema liposomal adelantado sugiere una buena estrategia para la encapsulación del acetilhexapéptido-3 y su potencial aplicación en el desarrollo un novedoso producto cosmecéutico.
SUMMARY Introduction: Currently synthetic peptides have become a novel alternative for the treatment of aging skin. Acetylhexapeptide-3 (Ac-EEMQRR-NH2) has been used to induce reduction of expression lines in a manner analogous to botulinum toxin, but without toxic effects. Aim: To synthesize acetylhexapeptide-3 and to develop a lipo-somal system for its encapsulation and favor its passage through a model membrane. Methodology: The peptide was obtained by solid phase synthesis (SFS) using the Fmoc / tBu strategy, it was purified and fully characterized. The liposomal system with encapsulated acetylhexapeptide-3 was developed by forming an emulsion with subsequent phase inversion by evaporation of the organic solvent. The systems were characterized in their size, zeta potential, neuropeptide encapsulation efficiency and a preliminary ex vivo permeability study was carried out. Results: It is possible to synthesize short peptides with a high degree of purity and good yield, using the SFS Fmoc / tBu methodology. According to the characterization, the advanced liposomal system suggests a good strategy for the encapsulation of acetylhexapeptide-3 and its potential application in the development of a novel cosmeceutical product.
RESUMO Introdução: Atualmente os peptídeos sintéticos têm se tornado uma nova alternativa para o tratamento do envelhecimento cutâneo. O acetilhexapeptídeo-3 (Ac-EEMQR-R-NH2) tem sido usado para induzir a redução das linhas de expressão de maneira análoga à toxina botulínica, mas sem efeitos tóxicos. Objetivo: Sintetizar acetilhe-xapeptídeo-3 e desenvolver um sistema lipossomal para seu encapsulamento e favorecer sua passagem por uma membrana modelo. Metodologia: O peptídeo foi obtido por síntese em fase sólida (SFS) utilizando a estratégia Fmoc / tBu, foi purificado e totalmente caracterizado. O sistema lipossomal com acetilhexapeptídeo-3 encapsulado foi desenvolvido pela formação de uma emulsão com subsequente inversão de fase por evaporação do solvente orgânico. Os sistemas foram caracterizados quanto ao tamanho, potencial zeta, eficiência de encapsulação de neuropeptídeos e um estudo preliminar de permeabilidade ex vivo foi realizado. Resultados: É possível sintetizar peptídeos curtos com alto grau de pureza e bom rendimento, utilizando a metodologia SFS Fmoc / tBu. De acordo com a caracterização, o sistema lipossomal avançado sugere uma boa estratégia para a encapsulação do acetilhexapeptídeo-3 e seu potencial aplicação no desenvolvimento de um novo produto cosmecêutico.
ABSTRACT
PnPa11 and PnPa13 are synthetic peptides derived from Phoneutria nigriventer spider venom, which display antinociceptive and neuroprotective properties. In this work, we evaluated the safety of intravitreal use and the neuroprotective effect of these peptides. Methods: The cytotoxicity and the antiangiogenic activity of these peptides were evaluated by the sulforhodamine-B method and chicken chorioallantoic membrane (CAM) assay, respectively. The in vivo safety was analyzed in Wistar rats that were intravitreally injected with different doses (0.50; 1.25; 2.50; 3.75 and 5.00 µg/mL) of these peptides (right eye, n = 6). The retinal function was assessed by electroretinography exams (ERG), intraocular pressure (IOP), and histological analyzes. In order to investigate the neuroprotective effect, Wistar rats received intravitreal injections (right eye, n = 6) of peptides at 1.25 µg/mL and then were exposed to blue LED light. In addition, the visual function and the retinal microstructure were verified. Results: Cytotoxicity analyses demonstrated that the peptides did not present any toxicity over ARPE-19 (adult retinal pigmented epithelial) cell line and the antiangiogenic study highlighted that the peptides promoted the reduction of blood vessels. The intravitreal injection did not cause major changes, neither induced any irreversible damage. In the retinal degeneration assay, the ERG records demonstrated that the prior treatment with PnPa11 and PnPa13 protected the retina from damage. Morphological analyses confirmed the ERG findings. Immunoblotting analyses revealed that PnPa11 increased Erk1/2, NR2A, and NR2B retinal expression after the light stress model, but did not cause Akt1 activation, while PnPa13 prevented Erk1/2 and Akt1 dephosphorylation. Conclusions: The intraocular administration of these peptides was well tolerated and presented protective activity against retinal degeneration, suggesting the potential use of these peptides as neuroprotectors in the ophthalmological field.(AU)
Subject(s)
Animals , Peptides , Spider Venoms , Intravitreal Injections , Spiders , AnalgesicsABSTRACT
BACKGROUND: As a potential candidate that can be extracted from natural sources and used to fight antibiotic-resistant bacteria, antimicrobial peptides have attracted extensive attention of scientists. Familiarity with the antimicrobial mechanism of antimicrobial peptides is conducive to the clinical application of antimicrobial peptides. OBJECTIVE: To review the advance in research of antimicrobial mechanism of antimicrobial peptides. METHODS: The first author conducted a computer-based retrieval of PubMed, Springeriink, Web of Science. ScienceDirect databases for articles regarding the antimicrobial mechanism and research advance published from January 2013 to March 2019. RESULTS AND CONCLUSION: Antimicrobial peptides are a class of special molecules with broad-spectrum antimicrobial activity. In some organisms, antimicrobial peptides arc considered lo bo an important part of innate immune system. The antimicrobial mechanism of antimicrobial peptides can be divided into two main modes: direct killing and immune regulation, and direct killing mechanism can be further divided into membrane targeting and non-membrane targeting. At the same time, based on the extensive application of antimicrobial peptides, it is expected that many resistance strategies have been developed in microbial environments such as staphylococcus, oral bacteria (including streptococcus) and intestinal bacteria (including salmonella). These resistance strategies mainly include passive resistance and induction or adaptive resistance mechanisms. In the future research and application, cationic peptide is an effective choice to solve the increasing multidrug resistance. In addition to the obligation to design new methods to combat the resistance of antimicrobial peptides in bacteria, general preventive measures against the resistance of conventional antibiotics should also be paid attention to.
ABSTRACT
Resumen Introducción. La malaria continúa siendo una de las enfermedades que causa mayor morbi-mortalidad a nivel mundial. Por esta razón es importante desarrollar herramientas diagnósticas eficaces que se implementen como estrategias para el control de la enfermedad. Objetivos. Estandarizar las condiciones del inmunoensayo enzimático (ELISA), para la detección de IgG específica contra Plasmodium falciparum en sueros de pacientes diagnosticados por gota gruesa con malaria no complicada por P. falciparum, empleando como antígeno un extracto proteico obtenido a partir de cultivo de P. falciparum o un péptido sintético derivado de la proteína de superficie de merozoito GLURP (del inglés: glutamate rich protein). Materiales y métodos. Para la estandarización de la técnica, se utilizaron 22 sueros de pacientes positivos para malaria por P. falciparum y 11 diagnosticados positivos para malaria por P. vivax utilizando la técnica de gota gruesa. Como controles negativos se utilizaron 44 sueros de individuos sanos. Los sueros fueron probados contra extracto de proteínas del parásito y el péptido sintético IMT 94 derivado de la proteína GLURP, para evaluar las concentraciones y las diluciones óptimas de cada componente del sistema. Para la validación de la técnica se utilizaron 251 sueros de pacientes positivos para P. falciparum y 44 sueros de individuos sanos, diagnosticados utilizando la técnica de gota gruesa. Resultados. La técnica estandarizada con el péptido sintético permitió observar diferencia significativa en el reconocimiento de sueros de pacientes, controles positivos y negativos por los antígenos (extracto de proteínas y péptidos sintéticos). Conclusiones. La metodología usada permite identificar la respuestas inmune específica contra P. falciparum.
Abstract Introduction. Malaria continues being one of the diseases causing the greatest morbi-mortality around the world. For that reason, effective diagnostic tools must thus be developed which can be used in strategies for controlling the disease. Objectives. To standardise enzyme- linked immunosorbent assay (ELISA) conditions for detecting Plasmodium falciparum specific IgG in sera from patients diagnosed by thick smear as suffering non-complicated malaria caused by P. falciparum. A protein extract obtained from P. falciparum culture or a synthetic peptide derived from glutamate rich protein (GLURP) merozoite surface protein would be used as antigen. Materials and Methods. 22 serum samples from patients diagnosed as suffering from P. falciparum malaria, 11 serum samples from patients diagnosed as suffering from P. vivax and 44 from healthy donors, diagnosed by using the thick smear tecnique were used for standarising the technique. Serum samples were tested against parasite protein extract and GLURP- derived IMT 94 synthetic peptide for standardisign optimum dilutions and concentrations for each component in the system. 251 serum samples from patients diagnosed as suffering from P. falciparum malaria and 44 from healthy donors diagnosed by using the thick smear tecnique were used to validate the technique. Results. The technique led to significant differences being observed in antigens (protein extract and synthetic peptides) recognising serum from positive and negative patients and controls. Conclusions. The methodology used led to identifying specific immune response against P. falciparum.
Subject(s)
Humans , Malaria , Plasmodium falciparum , AntibodiesABSTRACT
Brucellosis is a zoonose produced by bacterial species from the Brucella genus. Its isolation and identification in food using classical microbiological techniques is not practical due to its slow growth rate. Therefore, it is necessary to establish fast and specific methods for the detection of the bacteria in food. The goal of this work was the production and characterization of monospecific polyclonal antibodies in chicken (IgY) against synthetic peptides from Brucella abortus OMP25 and BP26 proteins, suitable for an antigen-capture assay. Conformational as well as antigenic predictions were performed using the ANTHEPROT package. Chemical synthesis was carried out by the multiple manual synthesis using the t-boc strategy. The peptides were used as antigens for the preparation of polyclonal antibodies in chicken. Experimental animals produced specific antibodies against the OMP25 and BP26 peptides constructs determined by ELISA and MABA assays showing correspondence between the predictive study and the immunogenicity obtained in chicken. The IgY proved to be able to recognize B. abortus by MABA assays. The binding activity and specificity of antibodies was determined by Western blot with cell extract from B. abortus. In this study, we demonstrated that OMP25 and BP26 peptides constructs are good candidates for production of specific IgY antipeptide antibodies capable of recognizing proteins from sonicated B. abortus strain S19, indicating the potential usefulness of the IgY antibody for development of immunoassays for detection of Brucella abortus.
La brucelosis es una zoonosis producida por especies del género Brucella. El aislamiento e identificación de la bacteria en alimentos usando las técnicas clásicas de microbiología no es práctico debido a su lenta tasa de crecimiento. Por lo tanto, es necesario establecer métodos rápidos para la detección de la bacteria en alimentos. En el presente trabajo se desarrollaron y caracterizaron anticuerpos policlonales monoespecíficos en gallinas (IgY) contra péptidos sintéticos de las proteínas OMP25 y BP26 de Brucella abortus, que puedan ser utilizados en un ensayo de captura. Para ello, se realizaron estudios conformacionales y de predicción de epítopes en la selección de los péptidos, los cuales se utilizaron como antígenos para la producción de las IgY. Los animales desarrollaron anticuerpos específicos contra los péptidos, mostrando correspondencia entre los estudios predictivos y la inmunogenicidad obtenida. Las IgY reconocieron a B. abortus en un ensayo de MABA y la actividad de unión y especificidad fue determinada por western blot con extracto celular de B. abortus. En este estudio, demostramos que los péptidos de las proteínas OMP25 y BP26 de B. abortus son buenos candidatos para la producción de anticuerpos IgY especificos capaces de reconocer proteínas de extracto de B. abortus cepa S19, indicando el potencial uso de anticuerpos IgY para el desarrollo de inmunoensayos para la detección de Brucella abortus.
ABSTRACT
yielded an average particle size of 120 nm with 70% encapsulation-efficiency. In vitro release profile of NP-OP showed sustained release of OP for 21 days. In vivo anti-fertility studies were conducted in marmosets. Results indicated that control animals conceived in the same cycle while two of three treated animals failed to conceive in treatment cycle. The in vivo studies thus corroborate with in vitro release of OP, demonstrating its anti-fertility activity in 66% of animals.
Subject(s)
Animals , Callithrix/physiology , Carrier Proteins/administration & dosage , Carrier Proteins/chemistry , Contraception , Female , Humans , Nanoparticles/administration & dosage , Nanoparticles/chemistry , Ovarian Follicle/chemistry , Particle Size , Peptide Fragments/administration & dosage , Peptide Fragments/chemistry , Polymers/administration & dosage , Polymers/chemistryABSTRACT
Considering the scarcity of defined antigens, actually useful and reliable for use in the field studies, we propose an alternative method for selection of cDNA clones with potential use in the diagnosis of schistosomiasis. Human antibodies specific to a protein fraction of 31/32 kDa (Sm31/32), dissociated from immune complexes, are used for screening of clones from an adult worm cDNA library. Partial sequencing of five clones, selected through this strategy, showed to be related to Schistosoma mansoni: two were identified as homologous to heat shock protein 70, one to glutathione S-transferase, one to homeodomain protein, and one to a previously described EST (expressed sequence tag) of S. mansoni. This last clone was the most consistently reactive during the screening process with the anti-Sm31/32 antibodies dissociated from the immune complexes. The complete sequence of this clone was obtained and the translation data yielded only one ORF (open reading frame) that code for a protein with 57 amino acids. Based on this amino acid sequence two peptides were chemically synthesized and evaluated separately against a pool of serum samples from schistosomiasis patients and non-schistosomiasis individuals. Both peptides showed strong reactivity only against the positive pool, suggesting that these peptides may be useful as antigens for the diagnosis of schistosomiasis mansoni.
Considerando a escassez de antígenos quimicamente definidos, realmente úteis e confiáveis para aplicação na soroepidemiologia da esquistossomose em larga escala, foi proposto, neste trabalho, um método alternativo para a seleção de clones de cDNA que expressam proteínas com putativo potencial diagnóstico na esquistossomose. Empregando anticorpos específicos contra uma fração proteica de 31/32 kDa (Sm31/32), purificados através da dissociação de imunocomplexos, foram selecionados cinco clones de cDNA a partir de genoteca de verme adulto de Schistosoma mansoni. O seqüenciamento parcial destes clones demonstrou que todos eram relacionados ao S. mansoni: dois apresentaram homologia com a proteína de choque térmico de 70 kDa e os demais com glutationa S-transferase, "homeodomain protein" e uma etiqueta de seqüência expressa (EST). Este último foi o clone que melhor reagiu, durante o processo de seleção, com os anticorpos anti-Sm31/32 dissociados de imunocomplexos. Baseado na seqüência de aminoácidos deste clone, dois peptídeos foram quimicamente sintetizados e analisados separadamente frente a misturas de soros de indivíduos normais e de pacientes com esquistossomose mansoni. Ambos os peptídeos demonstraram uma intensa reatividade somente contra a mistura de soros positivos, sugerindo que estes peptídeos podem ser úteis como antígenos para o diagnóstico da esquistossomose mansoni.
Subject(s)
Humans , Animals , DNA, Complementary/genetics , Peptide Library , Schistosoma mansoni/genetics , Schistosoma mansoni/immunology , Schistosomiasis mansoni/diagnosis , Antibodies, Helminth/genetics , Antibodies, Helminth/immunology , Antigens, Helminth/genetics , Antigens, Helminth/immunology , Cloning, Molecular/methods , DNA, Complementary/immunology , Expressed Sequence Tags , Gene Library , /immunology , Open Reading FramesABSTRACT
The importance and the use of synthetic peptides and recombinant proteins in immunohematology and the identification of antibodies are presented and discussed in this report.
No relato é apresentada e discutida a importância e o emprego dos peptideos sintéticos a proteinas recombinantes em imunohematologia e identificação de anticorpos.