Thermodynamic characterization of the palm tree Roystonea regia peroxidase stability

The structural stability of a peroxidase, a dimeric protein from royal palm tree (Roystonea regia) leaves, has been characterized by high-sensitivity differential scanning calorimetry, circular dichroism, steady-state tryptophan fluorescence and analytical ultracentifugation under different solvent conditions. It is shown that the thermal and chemical (using guanidine hydrochloride (Gdn-HCl)) folding/unfolding of royal palm tree peroxidase (RPTP) at pH 7 is a reversible process involving a highly cooperative transition between the folded dimer and unfolded monomers, with a free stabilization energy of about 23 kcal per mol of monomer at 25 degrees C. The structural stability of RPTP is pH-dependent. At pH 3, where ion pairs have disappeared due to protonation, the thermally induced denaturation of RPTP is irreversible and strongly dependent upon the scan rate, suggesting that this process is under kinetic control. Moreover, thermally induced transitions at this pH value are dependent on the protein concentration, allowing it to be concluded that in solution RPTP behaves as dimer, which undergoes thermal denaturation coupled with dissociation. Analysis of the kinetic parameters of RPTP denaturation at pH 3 was accomplished on the basis of the simple kinetic scheme N ->(k) D, where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state, and thermodynamic information was obtained by extrapolation of the kinetic transition parameters to an infinite heating rate. Obtained in this way, the value of RPTP stability at 25 degrees C is ca. 8 kcal per mole of monomer lower than at pH 7. In all probability, this quantity reflects the contribution of ion pair interactions to the structural stability of RPTP. From a comparison of the stability of RPTP with other plant peroxidases it is proposed that one of the main factors responsible for the unusually high stability of RPTP which enhances its potential use for biotechnological purposes, is its dimerization. (c) 2008 Elsevier Masson SAS. All rights reserved.

Ligand dissociation from estrogen receptor is mediated by receptor dimerization: Evidence from molecular dynamics simulations

Estrogen Receptor (ER) is an important target for pharmaceutical design. Like other ligand-dependent transcription factors, hormone binding regulates ER transcriptional activity. Nevertheless, the mechanisms by which ligands enter and leave ERs and other nuclear receptors remain poorly understood. Here, we report results of locally enhanced sampling molecular dynamics simulations to identify dissociation pathways of two ER ligands [the natural hormone 17 beta-estradiol (E-2) and the selective ER modulator raloxifene (RAL)] from the human ER alpha ligand-binding domain in monomeric and dimeric forms. E-2 dissociation occurs via three different pathways in ER monomers. One resembles the mousetrap mechanism (Path I), involving repositioning of helix 12 (H12), others involve the separation of H8 and H11 (Path II), and a variant of this pathway at the bottom of the ligand-binding domain (Path II`). RAL leaves the receptor through Path I and a Path I variant in which the ligand leaves the receptor through the loop region between H11 and H12 (Path I`). Remarkably, ER dimerization strongly suppresses Paths II and II` for E-2 dissociation and modifies RAL escape routes. We propose that differences in ligand release pathways detected in the simulations for ER monomers and dimers provide an explanation for previously observed effects of ER quaternary state on ligand dissociation rates and suggest that dimerization may play an important, and hitherto unexpected, role in regulation of ligand dissociation rates throughout the nuclear receptor family.

Immobilization of lutetium bisphthalocyanine in nanostructured biomimetic sensors using the LbL technique for phenol detection

This study describes the development of amperometric sensors based on poly(allylamine hydrochloride) (PAH) and lutetium bisphthalocyanine (LuPc(2)) films assembled using the Layer-by-Layer (LbL) technique. The films have been used as modified electrodes for catechol quantification. Electrochemical measurements have been employed to investigate the catalytic properties of the LuPc(2) immobilized in the LbL films. By chronoamperometry, the sensors present excellent sensitivity (20 nA mu M(-1)) in a wide linear range (R(2) = 0.994) up to 900 mu M and limit of detection (s/n = 3) of 37.5 x 10(-8) M for catechol. The sensors have good reproducibility and can be used at least for ten times. The work potential is +0.3 V vs. saturated calomel electrode (SCE). In voltammetry measurements, the calibration curve shows a good linearity (R(2) = 0.992) in the range of catechol up to 500 mu M with a sensitivity of 90 nA mu M(-1) and LD of 8 mu M. (C) 2011 Elsevier B.V. All rights reserved.

Probing conformational changes in orphan nuclear receptor: The NGFI-B intermediate is a partially unfolded dimer

Human nerve growth factor-induced B (NGFI-B) is a member of the NR4A subfamily of orphan nuclear receptors (NRs). Lacking identified ligands, orphan NRs show particular co-regulator proteins binding properties, different from other NRs, and they might have a non-classical quaternary organization. A body of evidence suggests that NRs recognition of and binding to ligands, DNA, homo- and heterodimerization partners and co-regulator proteins involve significant conformational changes of the NR ligand-binding domains (LBDs). To shed light on largely unknown biophysical properties of NGFI-B, here we studied structural organization and unfolding properties of NGFI-B ligand (like)-binding domain induced by chemical perturbation. Our results show that NGFI-B LBD undergoes a two-state guanidine hydrochloride (GndHCl) induced denaturation, as judged by changes in the a-helical content of the protein monitored by circular dichroism spectroscopy (CD). In contrast, changes in the tertiary structure of NGFI-B LBD, reported by intrinsic fluorescence, reveal a clear intermediate state. Additionally, SAXS results demonstrate that the intermediate observed by intrinsic fluorescence is a partially folded homodimeric structure, which further unfolds without dissociation at higher GndHCl concentrations. This partially unfolded dimeric assembly of NGFI-B LBD might resemble an intermediate that this domain access momentarily in the native state upon interactions with functional partners. (C) 2008 Elsevier B.V. All rights reserved.

Morphometric analysis of the urethra of castrated female rats treated with tamoxifen

Objectives: The aim of this study was to evaluate the effects of tamoxifen on the weight and thickness of the urethral epithelium of castrated female rats. Methods: Forty castrated adult female Wistar-Hannover rats were randomly divided into two groups: Group I (n = 20) in which the animals received only the vehicle (propylene glycol) and Group 11 (n = 20) in which the rats received tamoxifen 250 mu g/day by gavage. After 30 days of treatment, all animals were sacrificed and the urethra was immediately removed for weighing. Next, the urethra was divided into the proximal and distal segments, which were fixed in 10% formaldehyde and submitted to routine histological techniques for morphometric study. The data were analyzed using the weighted minimum mean-square error method and Student`s t-test for two independent samples (p < 0.05). Results: There was a significant increase in the mean weight of the urethra in the rats of Group 11 compared to the control group, 32.0 +/- 2.0 mg and 22.0 +/- 1.6 mg, respectively (p < 0.001). The mean thickness of the distal urethral epithelium of the animals treated with tamoxifen was significantly greater than that of the control group, 42.8 +/- 2.0 mu m and 36.6 +/- 1.5 mu m, respectively (p < 0.001). There was no statistically significant difference between the two groups with respect to the epithelial thickness of the proximal urethra (p = 0.514). Conclusion: Treating castrated adult rats with 250 mu g/day of tamoxifen for 30 days may increase the weight of the urethra and the thickness of the distal urethral epithelium. (c) 2008 Elsevier Ireland Ltd. All rights reserved.

Ki-67 and Bcl-2 Antigen Expression in Adenomatous Colorectal Polyps from Women with Breast Cancer

Background. The aim of this study was to evaluate Ki-67 and Bcl-2 antigen expression in colorectal polyps from women with breast cancer. Methods. A randomized, controlled study was carried out in 35 women, either with or without breast cancer, who had adenomatous colorectal polyps. The patients were divided into two groups: group A (without breast cancer; control group; n = 17) and group B (with breast cancer; study group; n = 18). Immunohistochemistry was performed on the colorectal polyps to evaluate Ki-67 and Bcl-2 antigen expression. Student`s t-test and the chi(2) test were used for the statistical analysis of Ki-67 and Bcl-2 expression, respectively. Statistical significance was established as P < 0.05. Results. The mean percentage of Ki-67-stained nuclei in groups A and B was 36.25 +/- 2.31 and 59.44 +/- 3.34 ( SEM), respectively (P < 0.0001), while the percentage of cases with cells expressing Bcl-2 in groups A and B was 23.5 and 77.8%, respectively (P < 0.001). Conclusions. In the present study, there was greater proliferative activity and greater expression of the antiapoptotic protein Bcl-2 in the colorectal polyps of women with breast cancer.

Analysis of Ki-67 and Bcl-2 protein expression in normal colorectal mucosa of women with breast cancer

The aim of this study was to evaluate Ki-67 and Bcl-2 protein expression in the normal colorectal mucosa adjacent to adenomatous polyps in women with breast cancer. A cross-sectional, controlled study was conducted in 35 women with and without breast cancer who had adenomatous colorectal polyps. The patients were divided into two groups: Group A (a control group of women without breast cancer, n = 18) and Group B (a study group of women with breast cancer, n = 17). A sample of normal colonic mucosa was collected at a distance of 5 cm from the polypoid lesion to evaluate immunchistochemical expression of the Ki-67 and Bcl-2 proteins. Student`s t-test and the chi-square test were used to analyse Ki-67 and Bcl-2 expression, respectively. Statistical significance was established at p < 0.05. The mean percentage of Ki-67-stained nuclei in Groups A and B was 25.12 +/- 2.08 and 41.50 +/- 1.85, respectively (p < 0.001), whereas the percentage of cases with cells expressing Bcl-2 in Groups A and B was 17.6% and 82.4%, respectively (p < 0.003). In the present study, greater proliferative activity and greater expression of the antiapoptotic protein Bcl-2 was found in the normal colorectal mucosa of women with breast cancer. (C) 2009 Elsevier Ltd. All rights reserved.

Neuropsychological Assessment of Current and Past Crack Cocaine Users

Background: Cognitive changes due to crack cocaine consumption remain unclear Methods: For clarification, 55 subjects were assigned to three groups: control group, crack cocaine current users, and ex-users. Participants were submitted to Mini-Mental State Examination (MMSE) and tasks evaluating executive functioning and verbal memory Mood state was also measured. Intergroup comparisons were carried out. Results: Control group performance on the MMSE was better than that of users and ex-users. Verbal memory performance for logical memory of users was impaired. Ex-users scored lower on DSST and Trail Making Test (Part B). Conclusion: Chronic crack cocaine use seems to disrupt general cognitive functioning (MMSE), verbal memory, and attentional resources, but findings suggest that some of these effects could be reversed by abstinence.

Implementing stepwise solvent elution in sequential injection chromatography for fluorimetric determination of intracellular free amino acids in the microalgae Tetraselmis gracilis

The concept of sequential injection chromatography (SIC) was exploited to automate the fluorimetric determination of amino acids after pre-column derivatization with ophthaldialdehyde (OPA) in presence of 2-mercaptoethanol (2MCE) using a reverse phase monolithic C(18) stationary phase. The method is low-priced and based on five steps of isocratic elutions. The first step employs the mixture methanol: tetrahydrofuran: 10 mmol L(-1) phosphate buffer (pH 7.2) at the volumetric ratio of 8:1:91; the other steps use methanol: 10 mmol L-1 phosphate buffer (pH 7.2) at volumetric ratios of 20:80, 35:65, SO:SO and 65:35. At a flow rate of 10 mu L s(-1) a 25 mm long-column was able to separate aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), serine (Ser), glutamine (Gln), glycine (Gly), threonine (Thr), citruline (Ctr), arginine (Arg), alanine (Ala), tyrosine (Tyr), phenylalanine (Phe), ornithine (Orn) and lysine (Lys) with resolution >1.2 as well as methionine (Met) and valine (Val) with resolution of 0.6. Under these conditions isoleucine (Ile) and leucine (Leu) co-eluted. The entire cycle of amino acids derivatization, chromatographic separation and column conditioning at the end of separation lasted 25 min. At a flow rate of 40 mu L s(-1) such time was reduced to 10 min at the cost of resolution worsening for the pairs Ctr/Arg and Orn/Lys. The detection limits varied from 0.092 mu mol L(-1) for Tyr to 0.51 mu mol L(-1) for Orn. The method was successfully applied to the determination of intracellular free amino acids in the green alga Tetraselmis gracilis during a period of seven days of cultivation. Samples spiked with known amounts of amino acids resulted in recoveries between 94 and 112%. (C) 2008 Elsevier B.V. All rights reserved.

Flow injection analysis using carbon film resistor electrodes for amperometric determination of ambroxol

Flow injection analysis (FIA) using a carbon film sensor for amperometric detection was explored for ambroxol analysis in pharmaceutical formulations. The specially designed flow cell designed in the lab generated sharp and reproducible current peaks, with a wide linear dynamic range from 5 x 10(-7) to 3.5 x 10(-4) mol L-1, in 0.1 mol L-1 sulfuric acid electrolyte, as well as high sensitivity, 0.110 A mol(-1) L cm(-2) at the optimized flow rate. A detection limit of 7.6 x 10(-8) mol L-1 and a sampling frequency of 50 determinations per hour were achieved, employing injected volumes of 100 mu L and a flow rate of 2.0 mL min(-1). The repeatability, expressed as R.S.D. for successive and alternated injections of 6.0 x 10(-6) and 6.0 x 10(-5) mol L-1 ambroxol solutions, was 3.0 and 1.5%, respectively, without any noticeable memory effect between injections. The proposed method was applied to the analysis of ambroxol in pharmaceutical samples and the results obtained were compared with UV spectrophotometric and acid-base titrimetric methods. Good agreement between the results utilizing the three methods and the labeled values was achieved, corroborating the good performance of the proposed electrochemical methodology for ambroxol analysis. (C) 2008 Elsevier B.V. All rights reserved.

Histological evaluation of chitosan-based biomaterials used for the correction of critical size defects in rat`s calvaria

Chitosan, a biopolymer obtained from chitin, and its derivates, such as chitosan hydrochloride, has been reported as wound healing accelerators and as possible bone substitutes for tissue engineering, and therefore these Substances could be relevant in dentistry and periodontology. The purpose of this investigation was to make a histological evaluation of chitosan and chitosan hydrochloride biomaterials (gels) used in the correction of critical size bone defects made in rat`s calvaria. Bone defects of 8 mm in diameter were surgically created in the calviria of 50 Holtzman (Rattus norvegicus) rats and filled with blood clot (control), low molecular weight chitosan, high molecular weight chitosan, low molecular weight chitosan hydrochloride, and high molecular weight chitosan hydrochloride, numbering 10 animals, divided into two experimental periods (15 and 60 days), for each biomaterial. The histological evaluation was made based on the morphology of the new-formed tissues in defect`s region, and the results indicated that there was no statistical difference between the groups when the new bone formation in the entire defect`s area were compared (p > 0.05) and, except in the control groups, assorted degrees of inflammation Could be Seen. In Conclusion, chitosan and chitosan hydrochloride biomaterials used in this study were not able to promote new bone formation in critical size defects made in rat`s calvaria. (C) 2009 Wiley Periodicals, Inc. J Biomed Mater Res 93A: 107-114, 2016

Thermal and spectrophotometric studies of new crosslinking method for collagen matrix with glutaraldehyde acetals

Despite the many existing crosslinking procedures, glutaraldehyde (GA) is still the method of choice used in the manufacture of bioprosthesis. The major problems with GA are: (a) uncontrolled reactivity due to the chemical complexity or GA solutions; (b) toxicity due to the release of GA from polymeric crosslinks; and (c) tissue impermeabilization due to polymeric and heterogeneous crosslinks formation, partially responsible for the undesirable calcification of the bioprosthesis. A new method of crosslinking glutaraldehyde acetals has been developed with GA in acid ethanolic solution, and after the distribution inside de matrix, GA is released to crosslinking. Concentrations of hydrochloride acid in ethanolic solutions between 0.1 and 0.001 mol/L with GA concentration between 0.1 and 1.0% were measured in an ultraviolet spectrophotometer to verify the presence of free aldehyde groups and polymeric compounds of GA. After these measurements, the solutions were used to crosslink bovine pericardium. The spectrophotometric results showed that GA was better protected in acetal forms for acid ethanolic solution with HCl at 0.003 mol/L and GA 1.0%(v/v). The shrinkage temperature results of bovine pericardium crosslinked with acetal solutions showed values near 85 C after the exposure to triethylamine vapors.

Electrochemical Behavior of Verapamil at Graphite-Polyurethane Composite Electrodes: Determination of Release Profiles in Pharmaceutical Samples

A solid graphite-polyurethane composite electrode has been used to determine release profiles of verapamil, a calcium-channel blocker. The electro-oxidation process was characterized by cyclic voltammetry and electrochemical impedance spectroscopy and showed no adsorption of analyte or oxidation products, unlike at other carbon-based electrodes. Quantification gave linear ranges up to 40molL-1 with cyclic voltammetry and detection limits of 0.7molL-1 by differential pulse and square-wave voltammetry. Commercial product samples were successfully analyzed with results equal to those from spectrophotometry. Because no electrode surface renewal is needed, this electrode material has many advantages.

Desenvolvimento de redes de polímeros interpenetrantes (IPN) para a aplicação como eletrólito polimérico

Uma nova rede de polímeros interpenetrantes (IPN) baseada em poliuretana de óleo de mamona e poli(etileno glicol) e poli(metacrilato de metila) foi preparada para ser utilizada como eletrólito polimérico. Os seguintes parâmetros de polimerização foram avaliados: massa molecular do poli(etileno glicol) (PEG), concentração de PEG e concentração de metacrilato de metila. As membranas de IPN foram caracterizadas por calorimetria diferencial de varredura (DSC) e espectroscopia de infravermelho por transformada de Fourier (FT-IR). Os eletrólitos de redes de polímeros interpenetrantes (IPNE) foram preparados a partir da dopagem com sal de lítio através do inchamento numa solução de 10% em massa de LiClO4 na mistura de carbonato de etileno e carbonato de propileno na razão mássica de 50:50. As IPNEs foram caracterizadas por espectroscopia de impedância eletroquímica e Raman. As IPNEs foram testadas como eletrólito polimérico em supercapacitores. As células capacitivas foram preparadas utilizando eletrodos de polipirrol (PPy). Os valores de capacitância e eficiência foram calculados por impedância eletroquímica, voltametria cíclica e ciclos galvonostáticos de carga e descarga. Os valores de capacitância obtidos foram em torno de 90 F.g-1 e eficiência variou no intervalo de 88 a 99%. Os valores de densidade de potência foram superiores a 250 W.kg-1 enquanto que a densidade de energia variou de 10 a 33 W.h.kg-1, dependendo da composição da IPNE. As características eletroquímicas do eletrólito formado pela IPN-LiClO4 (IPNE) foram comparadas aos eletrólitos poliméricos convencionais, tais como poli(difluoreto de vinilideno)-(hexafluorpropileno) ((PVDF-HFP/LiClO4) e poliuretana comercial (Bayer desmopan 385) (PU385/LiClO4). As condutividades na temperatura ambiente foram da ordem de 10-3 S.cm-1. A capacitância da célula utilizando eletrodos de PPy com eletrólito de PVDFHFP foi de 115 F.g-1 (30 mF.cm-2) e 110 F.g-1 (25 mF.cm-2) para a célula com PU385 comparadas a 90 F.g-1 (20 mF.cm-2) para a IPNE. Os capacitores preparados com eletrólito de IPNE apresentaram valores de capacitância inferior aos demais, entretanto provaram ser mais estáveis e mais resistentes aos ciclos de carga/descarga. A interpenetração de duas redes poliméricas, PU e PMMA produziu um eletrólito com boa estabilidade mecânica e elétrica. Um protótipo de supercapacitor de estado sólido foi produzindo utilizando eletrodos impressos de carbono ativado (PCE) e o eletrólito polimérico de IPNE. A técnica de impressão de carbono possui várias vantagens em relação aos outros métodos de manufatura de eletrodos de carbono, pois a área do eletrodo, espessura e composição são variáveis que podem ser controladas experimentalmente. As células apresentaram uma larga janela eletroquímica (4V) e valores da capacitância da ordem de 113 mF.cm-2 (16 F.g-1). Métodos alternativos de preparação do PCE investigados incluem o uso de IPNE como polímero de ligação ao carbono ativado, estes eletrodos apresentaram valores de capacitância similares aos produzidos com PVDF. A influência do número de camadas de carbono usadas na produção do PCE também foi alvo de estudo. Em relação ao eletrólito polimérico, o plastificante e o sal de lítio foram adicionados durante a síntese, formando a IPNGel. As células apresentaram alta capacitância e boa estabilidade após 4000 ciclos de carga e descarga. As membranas de IPN foram testadas também como reservatório de medicamento em sistemas de transporte transdérmico por iontoforese. Os filmes, mecanicamente estáveis, formaram géis quando inchado em soluções saturadas de lidocaina.HCl, anestésico local, em propileno glicol (PG), poli(etileno glicol) (PEG400) e suas misturas. O grau de inchamento em PG foi de 15% e 35% em PEG400. Agentes químicos de penetração foram utilizados para diminuir a resistência da barreira causada pela pele, dentre eles o próprio PG, a 2-pirrolidinona (E1) e a 1-dodecil-2-pirrolidinona (E2). Os géis foram caracterizados por espectroscopia de impedância eletroquímica e transporte passivo e por iontoforese através de uma membrana artificial (celofane). O sistema IPN/ lidocaina.HCl apresentou uma correlação linear entre medicamento liberado e a corrente aplicada. Os melhores resultados de transporte de medicamento foram obtidos utilizando o PG como solvente.

Encapsulation of single hMSCs in polyelectrolyte shells

The main objective of this Thesis was to encapsulate single viable cells within polyelectrolyte films using the Layer-by-Layer (LbL) technique. Most of the experiments used human mesenchymal stem cells (MSCs) whose characteristics (capacity of selfrenewal and potential to differentiate into several types of cells) make them particularly interesting to be used in biomedical applications. Also, most of the experiments used alginate (ALG) as the anionic polyelectrolyte and chitosan (CHI) or poly(allylamine hydrochloride) (PAH) as the cationic polyelectrolyte. Hyaluronic acid (HA) was also tested as an anionic polyelectrolyte. At the beginning of the work, the experimental conditions necessary to obtain the encapsulation of individual cells were studied and established. Through fluorescence microscopy visualization by staining the cell nucleus and using polyelectrolytes conjugated to fluorescent dyes, it was possible to prove the obtainment of capsules containing one single cell inside. Capsules aggregation was an observed problem which, despite the efforts to design an experimental process to avoid this situation (namely, by playing with cell concentration and different means of re-suspending and stirring the cells), was not completely overcome. In a second part of the project, single cells were encapsulated within polyelectrolyte layers made of CHI/ALG, PAH/ALG and PAH/HA and their viability was evaluated through the resazurin reduction assay and the Live/Dead assay. In these experiments, during the LbL process, polyelectrolyte solutions were used at a concentration of 1mg/mL based on literature. In general, the viability of the encapsulated cells was shown to be very low/absent. Then, as a consequence of the lack of viability of cells encapsulated within polyelectrolyte layers, the LbL technique was applied in cells growing adherent to the surface of cell culture plates. The cells were cultured like in a sandwich, between the surface of the cell culture dish and the polyelectrolyte layers. Also here, the polyelectrolyte solutions were used at a concentration of 1mg/mL during the LbL process. Surprisingly, cell viability was also absent in these systems. A systematic study (dose-effect study) was performed to evaluate the effect of the concentration of the individual polyelectrolytes (ALG, CHI and PAH were studied) in cell viability. Experiments were performed using cells growing adherent to the surface of cell culture plates. The results pointed out that a very high (cytotoxic) concentration of polyelectrolytes had been in use. Also, in general, PAH was much more cytotoxic than CHI, whereas ALG was the less cytotoxic polyelectrolyte. Finally, using alginate and chitosan solutions with adequate concentrations (low concentrations: 50ng/mL and 1μg/mL), the encapsulation of single viable cells was again attempted. Once again, the encapsulated cells were not shown to be viable. In conclusion, the viability of the encapsulated cells is not only dependent on the cytotoxic characteristics (or combined cytotoxic characteristics) of the polyelectrolytes but it seems that, when detached from the culture plates, the cells become too fragile and lose their viability very easily.