Structural and functional stabilization of protein entities

XI Reunião Regional Nordeste da SBBq | 4th International Symposium in Biochemistry of Macromolecules and BiotechnologyStabilization of protein and protein-like molecules translates into preservation of both structure and functionality during storage and/or targeting, and such stabilization is mostly attained through establishment of a thermodynamic equilibrium with the (micro)environment. The basic thermodynamic principles that govern protein structural transitions and the interactions of the protein and/or peptide molecule with its (micro)environment will, therefore, be tackled. Protein stabilization is based upon dampening the molecular motions and, therefore, eliminating conformational transitions while the molecule is still in the native 3D (folded) state. The 3D structure of a protein molecule depends mostly on two types of interactions: intramolecular interactions between aminoacid moieties and intermolecular interactions with solute and/or solvent molecules present in its microenvironment. Stabilizing a biomolecule (aiming at preserving its function) involves dampening its molecular motions, and this can be achieved by reducing the chemical activity of the water present in its microenvironment, thus stabilizing both its structure and functionality. Recently, the simultaneous entrapment-stabilization of proteins and enzymes based on nanoencapsulation in a nanoemulsion (W/O/W) matrix with an hydrophilic core has started to gain momentum. Similarly to the stabilization mechanism of osmolytes, in nanoencapsulation the water activity is altered thus affecting the molecular motions of the proteins. Highlights will also be given to structural and functional stabilization of protein entities (viz. enzymes, (macro)peptides, (recombinant) proteins, and bacteriophages) by chemical methodologies. Modification of the biomolecules microenvironment via multipoint covalent attachment onto a solid surface followed by hydrophylic polymer coimmobilization, are some of the (latest) strategies that will be discussed.info:eu-repo/semantics/publishedVersio

Quantitative reflectance spot test for the determination of acetylsalicylic acid in pharmaceutical preparations

This paper describes a quantitative reflectance spot test procedure for the determination of acetylsalicylic acid (ASA) in pharmaceutical preparations. The method is based on the reaction of salicylic acid, obtained from the hydrolysis of ASA, with Fe(III) forming a deep blue-violet compound. Medicines containing ASA can be easily analyzed by the proposed method as it is not necessary to do any separation. The final mixture is placed on a sheet of filter paper, and the reflectance is directly measured. Nine commercial medicines containing acetylsalicylic acid were analyzed with the proposed method. The mean RSD was 0.9%. Results were compared with those obtained with the United States Pharmacopoeia recommended procedure (RSD 0.6%). The quantitative detection limit is 0.6 mg ASA in the working solution. For a degree of freedom n = 4 (n = n1+ n2 - 2) and a confidence coefficient a = 0.05 all the results agree under the tabulated t-Student test value (2.78).Este trabalho descreve um método para a determinação quantitativa de ácido acetilsalicílico (ASA) utilizando procedimento spot test e reflectância difusa. O método é baseado na formação do complexo de cor roxa intensa entre o ácido salicílico, obtido a partir da hidrólise alcalina do ASA, e íons Fe(III). O procedimento proposto permite a análise de medicamentos contendo ASA de forma fácil e simples, uma vez que não é necessário fazer separações. A reflectância da mistura final, colocada sobre um disco de papel de filtro, é medida diretamente. Foram analisados nove medicamentos comerciais contendo ASA cujos resultados apresentaram um desvio padrão relativo médio de 0,9%. O limite para a determinação quantitativa é de 0,6 mg de ASA na solução de trabalho. As análises feitas com o método proposto foram comparadas com outras análises, das mesmas amostras, segundo o procedimento recomendado pela United States Pharmacopoeia, onde se observou um desvio padrão relativo de 0,6%. Compararam-se os dois métodos utilizando-se o teste t de Student. Para um grau de liberdade n = 4 (n = n1 + n2 - 2) e um limite de confiança a = 0.05, onde t = 2,78, todos os resultados foram concordantes.327330Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Molecular Characterization and Genome Mechanical Features of Two Newly Isolated Polyvalent Bacteriophages Infecting Pseudomonas syringae pv. garcae

Coffee plants have been targeted by a devastating bacterial disease, a condition known as bacterial blight, caused by the phytopathogen Pseudomonas syringae pv. garcae (Psg). Conventional treatments of coffee plantations affected by the disease involve frequent spraying with copper- and kasugamycin-derived compounds, but they are both highly toxic to the environment and stimulate the appearance of bacterial resistance. Herein, we report the molecular characterization and mechanical features of the genome of two newly isolated (putative polyvalent) lytic phages for Psg. The isolated phages belong to class Caudoviricetes and present a myovirus-like morphotype belonging to the genuses Tequatrovirus (PsgM02F) and Phapecoctavirus (PsgM04F) of the subfamilies Straboviridae (PsgM02F) and Stephanstirmvirinae (PsgM04F), according to recent bacterial viruses' taxonomy, based on their complete genome sequences. The 165,282 bp (PsgM02F) and 151,205 bp (PsgM04F) genomes do not feature any lysogenic-related (integrase) genes and, hence, can safely be assumed to follow a lytic lifestyle. While phage PsgM02F produced a morphogenesis yield of 124 virions per host cell, phage PsgM04F produced only 12 virions per host cell, indicating that they replicate well in Psg with a 50 min latency period. Genome mechanical analyses established a relationship between genome bendability and virion morphogenesis yield within infected host cells

Influence of water and ultraviolet irradiation on the induction period of the oxidation of biodiesel

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Biodiesel degrades due to oxidative processes, causing a decrease in its quality. In the present work, it has been clearly shown that the incidence of ultraviolet radiation on biodiesels obtained from soy, canola, linseed and microalgae oils initiate oxidative processes which lead to the decrease in the induction period (IP) of the fuel. The influence of the residual water content of the same biodiesels on the oxidation process was also investigated with and without the incidence of ultraviolet radiation. Between 190 and 850 ppm of water in the biodiesel and without UV irradiation, no significant change in the IP values was observed under the experimental conditions.Biodiesel degrades due to oxidative processes, causing a decrease in its quality. In the present work, it has been clearly shown that the incidence of ultraviolet radiation on biodiesels obtained from soy, canola, linseed and microalgae oils initiate oxid284676680FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)sem informaçãosem informaçãoThe authors are grateful to CNPq for financial support and to Dr. Fabio Batista (EXTRAE-UNICAMP) who graciously provided the microalgae oi

Development of a water-in-oil-in-water multiple emulsion system integrating biomimetic aqueous-core lipid nanodroplets for protein entity stabilization. Part I: experimental factorial design

Lipid nanoballoons integrating multiple emulsions of the type water-in-oil-in-water enclose, at least in theory, a biomimetic aqueous-core suitable for housing hydrophilic biomolecules such as proteins, peptides and bacteriophage particles. The research effort entertained in this paper reports a full statistical 23x31 factorial design study (three variables at two levels and one variable at three levels) to optimize biomimetic aqueous-core lipid nanoballoons for housing hydrophilic protein entities. The concentrations of protein, lipophilic and hydrophilic emulsifiers, and homogenization speed were set as the four independent variables, whereas the mean particle hydrodynamic size (HS), zeta potential (ZP) and polydispersity index (PI) were set as the dependent variables. The V23x31 factorial design constructed led to optimization of the higher (+1) and lower (-1) levels, with triplicate testing for the central (0) level, thus producing thirty three experiments and leading to selection of the optimized processing parameters as 0.015% (w/w) protein entity, 0.75% (w/w) lipophilic emulsifier (soybean lecithin) and 0.50% (w/w) hydrophilic emulsifier (poloxamer 188). In the present research effort, statistical optimization and production of protein derivatives encompassing full stabilization of their three-dimensional structure, has been attempted via housing said molecular entities within biomimetic aqueous-core lipid nanoballoons integrating a multiple (W/O/W) emulsion

Characterization of a water-in-oil-in-water multiple emulsion integrating biomimetic aqueous-core lipid nanoballoons housing protein entities

Project funding by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, São Paulo, Brazil) ( FAP ESP Ref. No. 2013/03181 - 6, Project PneumoPhageKill

Development and characterization of a hydrogel containing nitrofurazone for antimicrobial topical applications

The goal of the research work entertained herein was the development and characterization of a poly-(vinyl alcohol) (PVA) hydrogel cross-linked with glutaraldehyde and impregnated with 0.2% (w/w) nitrofurazone (NTZ), for topical applications. To verify the active principle release capability, one has determined (i) swelling profile, (ii) in vitro release of NTZ via UV-VIS spectrophotometry, and (iii) antimicrobial activity via exposure to the hydrogel of ATCC strains of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. The optimized hydrogel was further characterized via scanning electron microscopy (SEM), infrared spectroscopy with Fourier transform, moisture content determinations and thermal analyses via thermal gravimetry (TGA). Swelling tests revealed a mass increase from 100±5% up to 350±11%. Incorporated NTZ displayed bactericidal activity, as expected, being released in a linearly controlled fashion above 6 μg/mL during experiment timeframes of 14 h. SEM analyses allowed verification of a homogeneous surface morphology, while infrared spectra showed that NTZ did not bind strongly to the cross-linked polymer. Furthermore, results from thermal analyses suggested a loss of thermal stability arising from incorporation of NTZ in the hydrogel. The optimized hydrogel exhibited characteristics with high potential for (antimicrobial) treatment of skin lesions.Financial support to Victor M. Balcao, via an Invited Research Scientist fellowship (FAPESP Ref. No. 2011/51077-8) by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP, Sao Paulo, Brazil), is hereby gratefully acknowledged. Sebastiao Coelho de Lima gratefully acknowledges financial support from the Centro Universitario da Barra Mansa (Rio de Janeiro/RJ, Brazil)

Zidovudine-poly(l-lactic acid) solid dispersions with improved intestinal permeability prepared by supercritical antisolvent process

A supercritical antisolvent (SAS) process for obtaining zidovudine-poly(l-lactic acid) (PLLA) solid dispersions (SDs) was used to attain a better intestinal permeation of this drug. A 32 factorial design was used, having as independent variables the ratio 3-azido-23-dideoxythymidine (AZT)PLLA and temperature/pressure conditions, as dependent variables the process yield and particle macroscopic morphology. AZTPLLA production batches were carried out by the SAS process, and the resulting products evaluated via scanning electron microscope, X-ray diffraction, differential scanning calorimetry, and Fourier transform infrared analyses. From the nine possible combinations of tests performed experimentally, only one combination did not produced a solid. The L3 batch of SD, produced with 1:2 (AZTPLLA) ratio, resulted in a 91.54% yield, with 40% AZT content. Intestinal permeability studies using the AZTPLLA from L3 batch led to an AZT permeability of approximately 9.87%, which was higher than that of pure AZT (3.84%). AZT remained in crystalline form, whereas PLLA remained in semicrystalline form. AZT release is controlled by a diffusion mechanism. It has been demonstrated that it is possible to use PLLA carrier and SAS process to obtain SD, in a single step.Cristalia (Itapira, Brazil) for the kind supply of the reference substance used throughout the research work. Financial support from Fundação de Amparo á Pesquisa do Estado de São Paulo (2013-19300-4; 2012/01333-0; 2011/21219-5), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (PROSUP/CAPES) and Finep Inovação e Pesquisa (Finep, Brazil; 01.13.0286.00)

Comparative study of two spectrophotometric reagents for catechol analysis in guaraná seeds powder

In this article are described and compared two spectrophotometric methods for the analysis of catechol (o-hydroxyphenol) in Paullinea cupana var. sorbilis, popularly known as guaraná. The pounded seed samples were extracted with ethanol 97% v/v and the solutions were treated with p-aminophenol in alkaline ethanolic medium or m-aminophenol and sodium metaperiodate in buffered aqueous solution (pH 3.0). The absorbance was measured at 586 nm and 520 nm respectively. Four different commercial products were analyzed and the results were compared with those obtained with the m-aminophenol method. Despite the good quality of the calibration curves, in the two methods the interference of the matrix was observed. This problem was settled using the standard addition method. Comparison between the results obtained by the two methods, using the statistical Student's t-test, showed that there is no significant difference at the 95% confidence level.Neste trabalho são descritos e comparados dois métodos espectrofotométricos para análise de catecol (o-hidroxifenol) em Paullinea cupana var. sorbilis, popularmente conhecido como guaraná. As amostras de sementes trituradas foram extraídas com etanol 97% v/v e as soluções tratadas com p-aminofenol em meio etanólico alcalino ou m-aminofenol e metaperiodato de sódio em solução tampão (pH 3.0). A absorbância foi medida a 586 nm e 520 nm respectivamente. Quatro produtos comerciais diferentes foram analisados e os resultados foram comparados com aqueles obtidos com m-aminofenol. Apesar da boa qualidade das curvas de calibração, nos dois métodos houve interferência da matriz, o que foi contornado usando-se o método da adição de padrão. A comparação entre os resultados obtidos pelos dois métodos, usando o teste estatístico t de Student, mostrou que não há diferenças significantes para um nível de confiança de 95%.129132Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq