Human interferon B1 ser17: Coding DNA synthesis, expression, purification and characterization of bioactive recombinant protein

A protocol to produce large amounts of bioactive homogeneous human interferon β1 expressed in Escherichia coli was developed. Human interferon β1 ser17 gene was constructed, cloned and subcloned, and the recombinant protein expressed in E. coli cells. Solubilization of recombinant human interferon β1 ser17 (rhIFN-β1 ser17) was accomplished by employing a brief shift to high alkaline pH in the presence of non-ionic detergent. The recombinant protein was purifi ed by three chromatographic steps. N-terminal amino acid sequencing and mass spectrometry analysis provided experimental evidence for the identity of the recombinant protein. Reverse phase liquid chromatography demonstrated that the content of deamidates and sulphoxides was similar to a commercial standard. Size exclusion chromatography demonstrated the absence of high molecular mass aggregates and dimers. The protocol represents an effi cient and high-yield method to obtain bioactive homogeneous monomeric rhIFN-β1 ser17 protein. It may thus represent an important step towards scaling up for rhIFN-β1 ser17 large-scale production. © 2010 Villela AD, et al.

Agelaia MP-I: A peptide isolated from the venom of the social wasp, Agelaia pallipes pallipes, enhances insulin secretion in mice pancreatic islets

Peptides isolated from animal venoms have shown the ability to regulate pancreatic beta cell function. Characterization of wasp venoms is important, since some components of these venoms present large molecular variability, and potential interactions with different signal transduction pathways. For example, the well studied mastoparan peptides interact with a diversity of cell types and cellular components and stimulate insulin secretion via the inhibition of ATP dependent K + (K ATP) channels, increasing intracellular Ca 2+ concentration. In this study, the insulin secretion of isolated pancreatic islets from adult Swiss mice was evaluated in the presence of synthetic Agelaia MP-I (AMP-I) peptide, and some mechanisms of action of this peptide on endocrine pancreatic function were characterized. AMP-I was manually synthesized using the Fmoc strategy, purified by RP-HPLC and analyzed using ESI-IT-TOF mass spectrometry. Isolated islets were incubated at increasing glucose concentrations (2.8, 11.1 and 22.2 mM) without (Control group: CTL) or with 10 μM AMP-I (AMP-I group). AMP-I increased insulin release at all tested glucose concentrations, when compared with CTL (P < 0.05). Since molecular analysis showed a potential role of the peptide interaction with ionic channels, insulin secretion was also analyzed in the presence of 250 μM diazoxide, a K ATP channel opener and 10 μM nifedipine, a Ca 2+ channel blocker. These drugs abolished insulin secretion in the CTL group in the presence of 2.8 and 11.1 mM glucose, whereas AMP-I also enhanced insulin secretory capacity, under these glucose conditions, when incubated with diazoxide and nifedipine. In conclusion, AMP-I increased beta cell secretion without interfering in K ATP and L-type Ca 2+ channel function, suggesting a different mechanism for this peptide, possibly by G protein interaction, due to the structural similarity of this peptide with Mastoparan-X, as obtained by modeling. © 2012 Elsevier Ltd.

Determination of Sudan II dye in ethanol fuel by chromatographic and electroanalytical methods

Chromatographic and electroanalytical methods were developed to detect and quantify Sudan II (SD-II) dye in fuel ethanol samples. Sudan II is reduced at +0.50 V vs. Ag/AgCl on a glassy carbon electrode using Britton-Robinson buffer (pH 4.0) and N,N-dimethylformamide (70:30, v/v) + sodium dioctyl sulfosuccinate surfactant as supporting electrolyte, due to the azo group. This is the basis for its determination by square-wave voltammetry (SWV). Using the optimized conditions, it is possible to get a linear calibration curve from 3.00×10-6 to 1.80×10-5 mol L-1 (r = 0.998) with limits of detection (LOD) and quantification (LOQ) of 2.05×10-6 and 6.76×10-6 mol L-1, respectively. In addition, the hydroxyl substituent in the SD-II dye is also oxidized at +0.85 V vs. Ag/AgCl, which was conveniently used for its determination by high-performance liquid chromatography coupled to electrochemical detection (HPLC-ED). Under the optimized condition, the SD-II dye was eluted and separated using a reversed-phase column (cyanopropyl, CN) using isocratic elution with the mobile phase containing acetonitrile and aqueous lithium chloride (5.00×10-4 mol L-1) at 70:30 (v/v) and a flow rate of 1.2 mL min-1. Linear calibration curves were obtained from 3.00×10-7 to 2.00×10-6 mol L-1 (r = 0.999) with LOD and LOQ of 3.10×10-8 and 1.05×10-7 mol L-1, respectively. Both methods were simple, fast and suitable to detect and quantify the dye in fuel ethanol samples at recovery values between 83.0 to 102% (SWV) and 88.0 to 112% (HPLC-ED) with satisfactory precision and accuracy.

Validation of a stability-indicating RP-LC method for the determination of tigecycline in lyophilized powder

A reversed-phase liquid chromatography (RP-LC) method was validated for the determination of tigecycline in lyophilized powder. The LC method was conducted on a Luna C18 column (250 × 4.6 mm i.d.), maintained at room temperature. The mobile phase consisted of buffer containing sodium phosphate monobasic (0.015M) and oxalic acid (0.015M) (pH 7.0)-acetonitrile (75:25, v/v), run at a flow rate of 1.0 mL/min and using ultraviolet detection at 280 nm. The chromatographic separation was obtained with a retention time of 8.6 min, and was linear in the range of 40-100 μg/mL (r2 = 0.9997). The specificity and stability-indicating capability of the method was proven through forced degradation studies, which also showed no interference of the excipients. The accuracy was 99.01% with a bias lower than 1.81%. The limits of detection and quantitation were 1.67 and 5.05 μg/mL, respectively. Moreover, method validation demonstrated satisfactory results for precision and robustness. The proposed method was applied for the analysis of the lyophilized powder formulation, contributing to improve the quality control and to assure the therapeutic efficacy. © The Author [2012]. Published by Oxford University Press. All rights reserved.

Evaluation of a propolis water extract using a reliable RP-HPLC methodology and in vitro and in vivo efficacy and safety characterisation

Since the beginning of propolis research, several groups have studied its antibacterial, antifungal, and antiviral properties. However, most of these studies have only employed propolis ethanolic extract (PEE) leading to little knowledge about the biological activities of propolis water extract (PWE). Based on this, in a previous study, we demonstrated the anti-inflammatory and immunomodulatory activities of PWE. In order to better understand the equilibrium between effectiveness and toxicity, which is essential for a new medicine, the characteristics of PWE were analyzed. We developed and validated an RP-HPLC method to chemically characterize PWE and PEE and evaluated the in vitro antioxidant/antimicrobial activity for both extracts and the safety of PWE via determining genotoxic potential using in vitro and in vivo mammalian micronucleus assays. We have concluded that the proposed analytical methodology was reliable, and both extracts showed similar chemical composition. The extracts presented antioxidant and antimicrobial effects, while PWE demonstrated higher antioxidant activity and more efficacious for the most of the microorganisms tested than PEE. Finally, PWE was shown to be safe using micronucleus assays. © 2013 Bruno Alves Rocha et al.

Influência das condições de cultivo e métodos de extração na produção de metabólitos antioxidantes por fungos isolados do litoral paulista

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Desenvolvimento de um método para a determinação de acilgliceróis em biodiesel utilizando cromatografia líquida de alta eficiência com detecção eletroquímica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Desenvolvimento de método para determinação de acetaldeído em álcool etílico hidratado combustível por cromatografia líquida de alta eficiência com detecção eletroquímica

Pós-graduação em Química - IQ

Avaliação química e biológica do fungo endofítico Schizophyllum commune isolado de Alchornea glandulosa

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Bioprospecção em Eugenia jambolana (Myrtaceae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Degradação de patulina por composto bioativo obtido por levedura

Pós-graduação em Engenharia e Ciência de Alimentos - IBILCE

Identificação dos princípios ativos antiulcerogênicos das folhas de Casearia sylvestris: contribuição para o desenvolvimento de um fitoterápico

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Avaliação de neurotoxicidade de formas enantioméricas de praguicidas organifosforados: estudos in vitro, in vivo e de neuroproteção

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Continuous vs. segmented second-dimension system gradients for comprehensive two-dimensional liquid chromatography of sugarcane (Saccharum spp.)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Validation of an HPLC Method for the Determination of Dibucaine Encapsulated in Solid Lipid Nanoparticles and Nanostructured Lipid Carriers

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)