Peptide:lipid ratio and membrane surface charge determine the mechanism of action of the antimicrobial peptide BP100. Conformational and functional studies

The cecropin-melittin hybrid antimicrobial peptide BP100 (H-KKLFKKILKYL-NH2) is selective for Gram-negative bacteria, negatively charged membranes, and weakly hemolytic. We studied BP100 conformational and functional properties upon interaction with large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs, containing variable proportions of phosphatidylcholine (PC) and negatively charged phosphatidylglycerol (PG). CD and NMR spectra showed that upon binding to PG-containing LUVs BP100 acquires a-helical conformation, the helix spanning residues 3-11. Theoretical analyses indicated that the helix is amphipathic and surface-seeking. CD and dynamic light scattering data evinced peptide and/or vesicle aggregation, modulated by peptide: lipid ratio and PG content. BP100 decreased the absolute value of the zeta potential () of LUVs with low PG contents; for higher PG, binding was analyzed as an ion-exchange process. At high salt, BP100-induced LUVS leakage requires higher peptide concentration, indicating that both electrostatic and hydrophobic interactions contribute to peptide binding. While a gradual release took place at low peptide:lipid ratios, instantaneous loss occurred at high ratios, suggesting vesicle disruption. Optical microscopy of GUVs confirmed BP100-promoted disruption of negatively charged membranes. the mechanism of action of BP100 is determined by both peptide:lipid ratio and negatively charged lipid content While gradual release results from membrane perturbation by a small number of peptide molecules giving rise to changes in acyl chain packing, lipid clustering (leading to membrane defects), and/or membrane thinning, membrane disruption results from a sequence of events large-scale peptide and lipid clustering, giving rise to peptide-lipid patches that eventually would leave the membrane in a carpet-like mechanism. (C) 2014 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Institut Nacional de Ciencia e Tecnologia de fluidos complexos (INCTFCx)Nude de Apoio Pesquisa de Fluidos Complexos (NAPFCx)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Univ São Paulo, Inst Chem, Dept Biochem, BR-05513970 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, BR-04044020 São Paulo, BrazilUniv Fed Rio de Janeiro, Inst Med Biochem, Nucl Magnet Resonance Natl Ctr, Rio de Janeiro, BrazilEmbrapa Recursos Genet & Biotecnol, BR-70770917 Brasilia, DF, BrazilUniversidade Federal de São Paulo, Dept Biophys, BR-04044020 São Paulo, BrazilFAPESP: 2007/50970-5FAPESP: 2013/08166-5Web of Scienc

Copolymers characterization and effect on the structure of biomimetic systems

Copolímeros, macromoléculas orgânicas ou inorgânicas com alta massa molar, consistem de unidades monômericas repetidas, unidas por ligações covalentes e que apresentam mais de um tipo de monômero. Neste trabalho estudamos três copolímeros constituídos por dois blocos distintos, isto é copolímeros dibloco, onde um dos blocos é constituído pelo polimetacrilato de metila, PMMA e o outro pelo polimetacrilato de N,N dimetilamino, PDMAEMA, e o homopolímero de PDMAEMA. A fórmula geral dos copolímeros dibloco estudados é PMMAm-b-PDMAEMAn onde o número de monômeros de cada bloco é representado pelos índices m e n. Os copolímeros PMMA1-b-PDMAEMA6,3 (MM, 54.200), PMMA1-b-PDMAEMA3,0 (MM 27.558), o PMMA1-b-PDMAEMA1,1 (MM 25.555) e o homopolímero PDMAEMA (MM 41.614,9) tiveram suas massas molares e a razão entre os monômeros de cada bloco caracterizados por cromatografia de gel permeação, GPC, e NMR. Determinamos a concentração micelar crítica (CMC) dos copolímeros e verificamos, por RMN e fluorescência, o efeito da concentração e do pH na sua agregação. A interação dos copolímeros com vesículas unilamelares grandes, LUVs, preparadas com misturas de fosfatidilcolina, PC, e fosfatidilglicerol, PG, foi estudada em diferentes condições de pH e força iônica. A interação dos polímeros com as LUVs também foi estudada medindo-se o diâmetro hidrodinâmico e potencial zeta das LUVs na presença dos polímeros. Observou-se que, quando a razão polímero/lipídio é alta, os polímeros se ligam às LUVs, neutralizando completamente a carga das vesículas. Quando a concentração de LUVs é próxima à dos polímeros, forma-se uma rede que conduz à agregação e precipitação dos complexos. Estudamos o efeito dos polímeros na permeabilização de LUVs e vesículas unilamelares gigantes (GUVs) preparadas com misturas de PC:PG. Os copolímeros permeabilizaram as LUVs, dependendo da razão polímero/lipídio, do pH, força iônica e das características hidrofóbicas e hidrofílicas de cada copolímero. Quanto maior a porcentagem de PG nas LUVs maior a interação com os copolímeros. A ligação dos copolímeros às LUVs deve produzir segregação dos fosfolipídios negativos na bicamada das LUVs (i.e., separação lateral de fases) facilitando a permeabilização das vesículas. Demonstramos que os copolímeros se ligam à superfície das GUVs modificando a sua forma e levando ao rompimento das vesículas. Estes efeitos foram modulados pelo pH e a força iônica do meio. O efeito de permeabilização dos copolímeros foi correlacionado com as razões entre os blocos hidrofóbicos e hidrofílicos. Quando o copolímero tem uma maior fração de PDMAEMA, o bloco mais hidrofílico do copolímero, este efeito é mais evidente. A porcentagem de permeabilização, após um tempo definido, para copolímeros de mesma massa molar, como o PMMA1-b-PDMAEMA3,0 (MM 27.558) e o PMMA1-b-PDMAEMA1,1 (MM 25.555), é muito maior com o copolímero com maior porção hidrofílica, o PMMA1-b-PDMAEMA3,0. O homopolímero DMAEMA também se mostrou eficiente na interação com as LUVs, porém menos que o PMMA1-b-PDMAEMA6,3.que possui maior numero de unidades DMAEMA e uma sequencia de monômeros de MMA. Demonstramos, nesta Tese, que o efeito de copolímeros sintéticos contendo regiões hidrofóbicas e hidrofílicas são bons modelos de peptídeos e proteínas permitindo avaliar quantitativamente o efeito dessas interações em modelos de membranas.Copolymers are organic or inorganic macromolecules with high molecular weight, consisting of repeated monomer units joined by covalent bonds and exhibit more than one type of monomer. Here, we studied three copolymers consisting of two different blocks, i.e. diblock copolymers, where one block is constituted by methyl polymethacrylate, PMMA and other by polymethacrylate N, N-dimethylamino, PDMAEMA and a homopolymer PDMAEMA. The general formula of the diblock copolymers studied was PMMAm-b-PDMAEMAn where the number of monomers of each block is represented by indexes m and n. The PMMA1-b-PDMAEMA6,3 copolymers (MW, 54,200), PMMA1 PDMAEMA3,0-b (MW 27,558), the PMMA1 PDMAEMA1,1-b (MW 25,555) and PDMAEMA homopolymer (MW 41614.9) had their molecular weights and the ratio between the monomers of each block characterized by gel permeation chromatography, GPC, and NMR. The critical micelle concentration (CMC) of the copolymers was determined by fluorescence and the aggregation of the copolymers verified by NMR. The effect pH on the copolymers CMC was also determined. The interaction of copolymers with large unilamellar vesicles, LUV, prepared with mixtures of phosphatidylcholine, PC, and phosphatidylglycerol, PG, was studied under different conditions of pH and ionic strength. The interaction of the polymers with the LUVs was also studied measuring the hydrodynamic diameter and zeta potential of the LUVs in the presence of the polymers. It was observed that when the ratios polymer / lipid are high, the polymers bind to the LUVs, completely neutralizing the charge of the vesicles. When the concentration of LUVs is close to that of the polymers, it forms a network that leads to aggregation and precipitation of the complexes. We studied the effect of polymers in permeabilization LUVs and giant unilamellar vesicles (GUVs) prepared with PC:PG. The copolymers permeabilized the LUVs, depending on the ratio polymer / lipid, pH, ionic strength and the hydrophobic and hydrophilic characteristics of each copolymer. The higher the percentage PG LUVs lead to greater interaction with the copolymers. The binding of the copolymers to the LUVs with negative charge induced phase separation of phospholipids in the bilayer of LUVs (ie, lateral phase separation) facilitating the permeability of the vesicles. We demonstrate that copolymers bind to the surface of GUVs changing its shape and leading to rupture of vesicles. These effects are modulated by pH and ionic strength of the media. The permeabilization effect of the copolymers was correlated with the ratios of the hydrophobic and hydrophilic blocks. When the copolymer has a larger fraction of PDMAEMA, the more hydrophilic block copolymer, this effect is more evident. The percentage of permeabilization, after a set time, for the same molar mass copolymers such as b-PDMAEMA3,0 PMMA1 (MW 27,558) and PMMA1 PDMAEMA1,1-b (MW 25,555), is much more efficient with copolymer with greater hydrophilic portion, the b-PMMA1 PDMAEMA3,0. The DMAEMA homopolymer also proved efficient in interacting with the LUVs. We demonstrated that synthetic copolymers containing hydrophobic and hydrophilic regions are good models of peptides and proteins allowing quantitatively evaluate the effects of these interactions on model membrane

Diversity of yeasts isolated from dairies in the dairy “Zona da Mata Mineira” by RAPD and PCR-RFLP

A diversidade genética de vinte e sete isolados de leveduras coletadas em laticínios, utilizando como referências dos gêneros Kluyveromyces e Debaryomyces, foi averiguada por meio de RAPD (Randomly Amplified Polymorphic DNA). A amplificação resultou em um total de oitenta e oito fragmentos polimórfico de DNA, utilizando treze oligonucleotídeos decâmeros aleatórios. As distâncias genéticas variaram de 6,5 a 71%, gerando na análise gráfica, cinco grupos geneticamente divergentes. Nas avaliações da região ITS do rDNA foi observado um polimorfismo de tamanho que variou de 380 a 710 pb. A análise de agrupamento utilizando valores da distância genética resultou na formação de oito grupos, sugerindo a existência de pelo menos oito espécies de leveduras. Na análise por PCR-RFLP da região ITS do rDNA, os produtos das amplificações foram hidrolisados com diferentes endonucleases de restrição evidenciando o padrão polimórfico, e os valores das distâncias genéticas foram utilizados para o agrupamento, resultando na formação de quinze grupos. Os agrupamentos obtidos com os marcadores moleculares possibilitaram a diferenciação genética dos isolados. Os resultados sugerem que quatro dos vinte e sete isolados pertencem à espécie Kluyveromyces lactis.The genetic diversity of twenty-seven yeasts collected at dairies, was evaluated by RAPD using Kluyveromyces and Debaryomyces reference genera. Amplification using thirteen random oligonucleotides resulted in eighty-eight DNA polymorphic fragments. The Genetic distances varied from 6,5 to 71%, and generated a Dendrogram with five different genetic groups. The amplification of the ITS 18SrDNA region from the yeast resulted in DNA fragments with length polymorphism (380 and 710 bp). The clustering analysis using the genetic distance value produced eight groups, reflecting at least eight yeasts species. The amplification products of the rDNA ITS region using PCR-RFLP analyses were cleaved with different restriction endonucleases showing polymorphic patterns. The values of the genetic distances were used for the clustering resulted in fifteen groups. The clusters obtained using the molecular markers showed genetic difference between you isolates. The results suggest that four of the twenty-seven isolates can be identified as the yeast Kluyveromyces lactis.Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

Antiproliferative and proapoptotic effects of DODAC/synthetic phosphoethanolamine on hepatocellular carcinoma cells

Abstract Background Current studies have demonstrated that DODAC/PHO-S (Dioctadecyldimethylammonium Chloride/Synthetic phosphoethanolamine) liposomes induces cytotoxicity in Hepa1c1c7 and B16F10 murine tumor cells, with a higher proportion than PHO-S. Therefore, our aim was to evaluate the potential of DODAC/PHO-S to elucidate the mechanism of cell death whereby the liposomes induces cytotoxicity in hepatocellular carcinoma Hepa1c1c7, compared to the PHO-S alone. Methods Liposomes (DODAC/PHO-S) were prepared by ultrasonication. The cell cycle phases, protein expression and types of cell’s death on Hepa1c1c7 were analyzed by flow cytometry. The internalisation of liposomes, mitochondrial electrical potential and lysosomal stability were also evaluated by confocal laser scanning microscopy. Results After treatment with liposomes (DODAC/PHO-S), we observed a significant increase in the population of Hepa1c1c7 cells experiencing cell cycle arrest in the S and G2/M phases, and this treatment was significantly more effective to promote cell death by apoptosis. There also was a decrease in the mitochondrial electrical potential; changes in the lysosomes; nuclear fragmentation and catastrophic changes in Hepa1c1c7 cells. The liposomes additionally promoted increases in the expression of DR4 receptor, caspases 3 and 8, cytochrome c, p53, p21, p27 and Bax. There was also a decrease in the expression of Bcl-2, cyclin D1, CD90 and CD44 proteins. Conclusion The overall results showed that DODAC/PHO-S liposomes were more effective than PHO-S alone, in promoting cytotoxicity Hepa1c1c7 tumor cells, activating the intrinsic and extrinsic pathways of programmed cell death

Naphthalimide-Containing BP100 Leads to Higher Model Membranes Interactions and Antimicrobial Activity

In a large variety of organisms, antimicrobial peptides (AMPs) are primary defenses against pathogens. BP100 (KKLFKKILKYL-NH2), a short, synthetic, cationic AMP, is active against bacteria and displays low toxicity towards eukaryotic cells. BP100 acquires a α-helical conformation upon interaction with membranes and increases membrane permeability. Despite the volume of information available, the action mechanism of BP100, the selectivity of its biological effects, and possible applications are far from consensual. Our group synthesized a fluorescent BP100 analogue containing naphthalimide linked to its N-terminal end, NAPHT-BP100 (Naphthalimide-AAKKLFKKILKYL-NH2). The fluorescence properties of naphthalimides, especially their spectral sensitivity to microenvironment changes, are well established, and their biological activities against transformed cells and bacteria are known. Naphthalimide derived compounds are known to interact with DNA disturbing related processes as replication and transcription, and used as anticancer agents due to this property. A wide variety of techniques were used to demonstrate that NAPHT-BP100 bound to and permeabilized zwitterionic POPC and negatively charged POPC:POPG liposomes and, upon interaction, acquired a α-helical structure. Membrane surface high peptide/lipid ratios triggered complete permeabilization of the liposomes in a detergent-like manner. Membrane disruption was driven by charge neutralization, lipid aggregation, and bilayer destabilization. NAPHT-BP100 also interacted with double-stranded DNA, indicating that this peptide could also affect other cellular processes besides causing membrane destabilization. NAPHT-BP100 showed increased antibacterial and hemolytic activities, compared to BP100, and may constitute an efficient antimicrobial agent for dermatological use. By conjugating BP100 and naphthalimide DNA binding properties, NAPHT-BP100 bound to a large extent to the bacterial membrane and could more efficiently destabilize it. We also speculate that peptide could enter the bacteria cell and interact with its DNA in the cytoplasm