RESUMEN
A 24,118 Da (MALDI-TOF) cysteine peptidase (EC 3.4.22.16) was purified from the latex extract of Cryptostegia grandiflora by two chromatographic steps involving ion exchange and Reverse-phase HPLC. The purified protein (Cg24-I) exhibits a single band profile following reduced SDS-PAGE and a unique amino terminal sequence, 1VPASIDWREKGTVL14, that is similar to other plant cysteine peptidases. Cg24-I displayed optimal proteolytic activity at pH 8.0 with 25 mM phosphate buffer. The proteolytic activity was inhibited with 0.2 mM E-64 and 1 mM iodoacetamide and was stimulated with 1 mM DTT. Cg24-I fully inhibited spore germination of phytopathogenic fungi Fusarium solani at a dose of 28.1 µg/mL. Its toxicity involves the membrane permeabilization of spores as probed by propidium iodide uptake. These results show that latex peptidases are part of the plant's defensive strategy against phytopathogens and that they most likely act synergistically with other recognized defensive proteins.
Asunto(s)
Antifúngicos/química , Apocynaceae/enzimología , Apocynaceae/microbiología , Cisteína Endopeptidasas/química , Extractos Vegetales/química , Secuencia de Aminoácidos , Antifúngicos/aislamiento & purificación , Antifúngicos/metabolismo , Apocynaceae/química , Cisteína Endopeptidasas/aislamiento & purificación , Cisteína Endopeptidasas/metabolismo , Fusariosis/microbiología , Fusariosis/prevención & control , Fusarium/efectos de los fármacos , Fusarium/crecimiento & desarrollo , Datos de Secuencia Molecular , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/prevención & control , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/metabolismo , ProteolisisRESUMEN
Gelatinization and retrogradation of starch was followed in real time with an acoustic wave sensor. This study relies on the monitorization of the frequency of oscillation of a piezoelectric quartz crystal in contact with a 2.5% emulsion of a commercial maize starch, during heating and cooling. The technique showed to be very powerful and sensitive to most of the changes described in the literature, which have been elucidated by some other techniques. The value for the temperature of gelatinization found using the sensor was confirmed by the analysis of the same starch emulsion by polarized light microscopy. Temperatures of gelatinization were found to vary with the sample heating rate, as follows: 73.5 °C at 2.0 °C/min, 66.0 °C at 1.0 °C/min, and 65.0 °C at 0.5 °C/min. Hysteresis of the studied system was evidenced by the frequency shift before heating and after cooling till the initial temperature. Analysis performed on a 1.5% emulsion of a rice starch heated at 2.0 °C/min and cooled as before, evidenced no hysteresis and showed complete reversibility, in which concerns to the series frequency of the piezoelectric quartz crystal.
Asunto(s)
Oryza/química , Almidón/química , Agua/química , Zea mays/química , Rastreo Diferencial de Calorimetría , Elasticidad , Calor , Microscopía de Polarización , Transición de Fase , Sonido , ViscosidadRESUMEN
Members of the spider genus Lasiodora are widely distributed in Brazil, where they are commonly known as caranguejeiras. Lasiodora spider venom is slightly harmful to humans. The bite of this spider causes local pain, edema and erythema. However, Lasiodora sp. spider venom may be a source of important pharmacological tools. Our research group has described previously that Lasiodora sp. venom produces bradycardia in the isolated rat heart. In the present work, we sought to evaluate the vascular effect of Lasiodora sp. venom and to isolate the vasoactive compounds from the venom. The results showed that Lasiodora spider venom induced a concentration-dependent vasodilation in rat aortic rings, which was dependent on the presence of a functional endothelium and abolished by the nitric oxide synthase (NOS) inhibitor L-NAME. Western blot experiments revealed that the venom also increased endothelial NOS function by increasing phosphorylation of the Ser¹¹77 residue. Assay-directed fractionation isolated a vasoactive fraction from Lasiodora sp. venom. Mass spectrometry (MS) and nuclear magnetic resonance (NMR) assays identified a mixture of two compounds: adenosine diphosphate (ADP, approximately 90%) and adenosine monophosphate (AMP, approximately 10%). The vasodilator effects of Lasiodora sp. whole venom, as well as ADP, were significantly inhibited by suramin, which is a purinergic P2-receptor antagonist. Therefore, the results of the present work indicate that ADP is a main vasodilator component of Lasiodora sp. spider venom.
Asunto(s)
Adenosina Difosfato/farmacología , Venenos de Araña/química , Arañas/química , Vasodilatadores/farmacología , Adenosina Difosfato/química , Adenosina Monofosfato/química , Animales , Western Blotting , Fraccionamiento Químico , Endotelio/efectos de los fármacos , Técnicas In Vitro , Espectrometría de Masas , NG-Nitroarginina Metil Éster/metabolismo , Óxido Nítrico Sintasa/metabolismo , Resonancia Magnética Nuclear Biomolecular , Fosforilación/efectos de los fármacos , Ratas , Suramina/química , Vasodilatación/efectos de los fármacos , Vasodilatadores/químicaRESUMEN
We report the initial characterization of a leptospiral isolate, Leptospira interrogans, serogroup Sejroe, serovar Hardjo, genotype Hardjoprajitno, strain Norma, and its relatedness with L. interrogans, serogroup Sejroe, serovar Hardjo, genotype Hardjoprajitno, strain Hardjo and Leptospira borgpetersenii, serogroup Sejroe, serovar Hardjo, genotype Hardjobovis, strain Sponselee. The Norma strain singled out during a leptospirosis outbreak in cattle immunized with antigens from the reference strain Hardjoprajitno (OMS). By applying a microscopic agglutination serological test (MAT) to cattle (n = 2966) with symptoms of leptospirosis between 2003 and 2007, more than 50% of sera were found positive for one of the following serotypes: Hardjoprajitno (31-21%), Hardjo Norma (46-40%), Hardjo hardjobovis (18-10%), Mini (8-4%) and Wolffi (7-4%). In immunization trials using six isolates plus Norma isolate, the remission of MAT in these isolates was observed following 6 months of the initial vaccination. To provide molecular ground for the high MAT Norma frequency found in these isolates, a DNA polymorphic analysis was conducted by comparing the Norma isolate with reference strains Hardjoprajitno and Sponselee. The polymorphic analysis in secY showed five base changes in Norma relative to Hardjoprajitno strain, corresponding to 98% identity, while Sponselee displayed 49 polymorphic sites relative to the Hardjoprajitno strain, representing 80% identity. The alignment of secY translated sequences shows no differences between Hardjoprajitno and Norma, and eight polymorphisms between genotype hardjoprajttno and strain Sponselee. Three-dimensional modelling located these variations within the loop region connecting helices 7 and 8 from secY which is less conserved. DNA sequencing of 23S ribosomal conserved fragment revealed a single polymorphism between Hardjoprajitno and Norma, and 13 polymorphisms between strains Sponselee, Hardjoprajitno and Norma. The differences between Hardjo and Norma were confirmed by low stringency single-specific primer polymerase chain reaction (LSSP-PCR) signature experiments with the primer G2, using as template the 285 bp fragment initially amplified with G1/G2 primers.
Asunto(s)
Antígenos Bacterianos/inmunología , Enfermedades de los Bovinos/microbiología , Leptospira interrogans/clasificación , Leptospirosis/veterinaria , Polimorfismo Genético/genética , Pruebas de Aglutinación/veterinaria , Animales , Secuencia de Bases , Brasil , Bovinos , Enfermedades de los Bovinos/prevención & control , Cartilla de ADN/genética , ADN Bacteriano/genética , Femenino , Genotipo , Leptospira interrogans/genética , Leptospira interrogans/inmunología , Leptospira interrogans/aislamiento & purificación , Leptospirosis/microbiología , Leptospirosis/prevención & control , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Polimorfismo de Longitud del Fragmento de Restricción , Embarazo , Alineación de Secuencia/veterinaria , Análisis de Secuencia de ADN/veterinaria , Especificidad de la Especie , Vacunación/veterinariaRESUMEN
A series of the polymers imprinted with diphenylamine (DPA) and respective non imprinted polymers were synthesized using precipitation polymerization. Synthesized polymers were characterized by Fourier Transform Infra-Red spectroscopy with Total Attenuated Reflectance (FTIR-ATR), Scanning Electron Microscopy (SEM) and equilibrium batch re-binding experiments. Influence of the synthesis conditions, namely monomer/template ratio and reaction duration, on the polymer binding capacity and selectivity towards aromatic compounds was investigated. Binding behavior of MIP was described using Freundlich isotherm. Significance of the effects of the synthesis conditions on the polymer properties was evaluated using ANOVA. MIPs synthesized at different conditions, which displayed different properties (binding capacity and selectivity), and respective non-imprinted polymers were employed for the fabrication of the potentiometric sensors. While sensors prepared using imprinted polymers had higher sensitivity and selectivity compared to the ones containing non-imprinted polymer, no difference was observed between sensors containing different imprinted polymers. No correspondence between polymers' characteristics obtained in the equilibrium re-binding studies and potentiometric behavior of the sensors based on the same polymers was observed. Therefore, equilibrium re-binding studies cannot be used for predicting sensor behavior.
Asunto(s)
Difenilamina/análisis , Impresión Molecular/métodos , Polímeros/síntesis química , Adsorción , Cinética , Espectroscopía de Resonancia Magnética , Microscopía Electrónica de Rastreo , Polimerizacion , Potenciometría/métodos , Sensibilidad y Especificidad , Espectroscopía Infrarroja por Transformada de Fourier , TermodinámicaRESUMEN
A new fibrinogenolytic metalloproteinase (Bmoo FIBMP-I) was purified from Bothrops moojeni snake venom. This enzyme was isolated through a combination of three chromatographic steps (ion-exchange, molecular exclusion, and affinity chromatography). Analyses by reverse phase chromatography, followed by mass spectrometry, showed the presence of enzyme isoforms with average molecular mass of 22.8 kDa. The SDS-PAGE analyses showed a single chain of 27.6 kDa, in the presence and absence of reducing agent. The protein has a blocked N-terminal. One of the peptides obtained by enzymatic digestion of a reduced and S-alkylated isoform was completely sequenced by mass spectrometry (MS/MS). Bmoo FIBMP-I showed similarity with hemorrhagic factor and several metalloproteinases (MP). This enzyme degraded Aα-chain faster than the Bß-chain and did not affect the γ-chain of bovine fibrinogen. The absence of proteolytic activity after treatment with EDTA, together with the observed molecular mass, led us to suggest that Bmoo FIBMP-I is a member of the P-I class of the snake venom MP family. Bmoo FIBMP-I showed pH-dependent proteolytic activity on azocasein, but was devoid of coagulant, defibrinating, or hemorrhagic activities. The kinetic parameters of proteolytic activity in azocasein were determined (V max = 0.4596 Uh(-1)nmol(-1) ± 0.1031 and K m = 14.59 mg/mL ± 4.610).
RESUMEN
The progesterone receptor gene (PROGINS) has been identified as a risk modifier for benign and malignant gynecological diseases. The present case-control study is to evaluate the role of the PROGINS polymorphisms, as risk factor, for endometrial cancer development and to investigate the association between these genetics variants and clinical/pathologic variables of endometrial cancer. PROGINS polymorphism was examined in a total of 121 patients with endometrial cancer and 282 population-based control subjects, all located at the same area in São Paulo, SP, Brazil. The genotyping of PROGINS polymorphism was determined by polymerase chain reaction. The frequencies of PROGINS polymorphism T1/T1, T1/T2, and T2/T2 were 82.6%, 14.9%, and 2.5% in the endometrial cancer patients and 78.4%, 21.6%, and 0% in the controls, respectively. The chi(2) test showed a higher incidence of the T2/T2 genotype in the endometrial cancer group subjects, these results were statistically different (P= 0.012). However, due to the fact that there were no women in the control group showing homozygosis for the allele T2, the correct evaluation of odds ratio could not be properly calculated. Regarding the clinical and pathologic findings observed within the group of patients with endometrial cancer, there was significant correlation between T1/T2 genotype and the presence of myoma (P= 0.048). No correlations were observed among the other variables. These data suggest that the PROGINS polymorphism T2/T2 genotype might be associated with an increased risk of endometrial cancer.
Asunto(s)
Neoplasias Endometriales/genética , Receptores de Progesterona/genética , Anciano , Alelos , Estudios de Casos y Controles , Neoplasias Endometriales/patología , Femenino , Predisposición Genética a la Enfermedad , Humanos , Persona de Mediana Edad , Estadificación de Neoplasias , Polimorfismo GenéticoRESUMEN
Latex from Caricaceae contains a number of proteins believed to be part of a defense mechanism that protects these plants from wounding. Prior evidence suggests that some components in Carica papaya improve healing of ulcerous wounds in mammals. This study shows the chromatographic isolation of a protein fraction from C. candamarcensis that stimulates cell proliferation of mammalian cells by measuring MTT reduction and thymidine incorporation. The effect appears to be cell specific as L929, MDA-MB231 and BHK-21 cells are stimulated while no effect is seen on CHO cells. The maximal stimulatory effect reaches 2.2-fold 72 h after addition of the active fraction to L929, 1.8-fold in MDA-MB231 cells and 1.6-fold in BHK cells. Proteolytic inactivation of the active fraction suggests that a protein is responsible for the proliferative activity and its size is estimated between 10 and 25 kDa. A potential candidate for this function is a 23 kDa protein found in the fraction that reacts with human EGF antibody.
Asunto(s)
Carica , Látex/química , Mitógenos/farmacología , Fitoterapia , Animales , Células CHO/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular/efectos de los fármacos , Cricetinae , Cisteína Endopeptidasas/metabolismo , Relación Dosis-Respuesta a Droga , Frutas , Mitógenos/administración & dosificación , Mitógenos/uso terapéutico , Proteínas de Plantas/administración & dosificación , Proteínas de Plantas/farmacología , Proteínas de Plantas/uso terapéuticoRESUMEN
An acoustic wave methodology was developed to quantify alcohols in aqueous solutions. The frequency at minimum impedance of a bare quartz crystal in contact with ethanol solutions was found to be a suitable parameter to quantify ethanol. Ethanol in several Portuguese white wines was analyzed both by the proposed methodology and by the usual areometric method with no statistically significant differences (alpha = 0.05) in precision or accuracy of the results.
Asunto(s)
Etanol/análisis , Vino/análisis , Electroquímica/métodos , Portugal , Reproducibilidad de los ResultadosRESUMEN
A new sensor for calcium based on a piezoelectric quartz crystal is presented. The selectivity depends on the ratio lipophilic salt/ionophore of the composition of the coating of the quartz crystal. A crystal coated with a THF solution of PVC (34.5% w/w), DOS (62.1% w/w) and 10,19-bis[(octadecylcarbamoyl) methoxyacetyl]-1,4,7,13,16-pentaoxa-10,19-diaza cycloheneicosane (3.4% w/w) and a salt/ionophore molar proportion of 60%, corresponding to a frequency decrease of the dry crystal of 6.0 kHz, showed a detection limit to calcium of 2.2 mg/L. Both a standard calcium chloride solution and a commercially balanced salt solution for tissue culture were analyzed subsequent to ion chromatographic separation. The results obtained with the sensor developed were compared with those obtained with a conductivity detector. Although the results from both detectors agreed for the standard solution, only the quartz crystal sensor was able to give reliable results for the tissue culture solution.
Asunto(s)
Calcio/análisis , Medios de Cultivo/química , Cloruro de Calcio , Técnicas de Cultivo , Ácido Edético , Electroquímica/métodos , Cuarzo , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
A new sensor based on oxidative combustion of nitroaromatics to NO(2) and its detection with a quartz crystal microbalance (QCM) coated with copper phthalocyanine (CuPc) was developed for determination of nitroaromatics in landfill gas. An alternative method based on gas chromatography-mass spectrometry (GC-MS) was also used in order to assess the performance of the proposed method. The results show that the analytical apparatus based on the QCM is less expensive than the GC-MS, and that the analytical error is 0.8% for both methods.
RESUMEN
A new method, based on separation with gas chromatography and detection with a quartz crystal microbalance, was used for the quantification of ethane-1-thiol, propane-1-thiol, ethyl methyl sulfide, propane-2-thiol, butane-2-thiol and butane-1-thiol. The analytical error, the analysis time and the general analytical performance are identical for gas chromatography-mass spectrometry (GC-MS) and gas chromatography-quartz crystal microbalance (GC-QCM). However, the GC-QCM method constitutes a low cost alternative to GC-MS for monitoring individual sulfur compounds in landfill gas. It is an easily assembled apparatus made with an injection device, a furnace, a column, a crystal cell and a quartz crystal.
Asunto(s)
Contaminantes Atmosféricos/análisis , Monitoreo del Ambiente/métodos , Compuestos de Azufre/análisis , Cromatografía de Gases/métodos , Cristalización , Gases/química , Cuarzo , Eliminación de ResiduosRESUMEN
A quartz crystal microbalance has been used to check the purity of glycerol. The experimental procedure involves no derivatization or other preliminary step except determination of water content. The protocol consists of the direct reading of the frequency of a quartz crystal with one face in contact with an aqueous solution of the glycerol to be tested. The method is inexpensive, simple, rapid, and highly sensitive; diethylene glycol contamination at the 3.61% level could be detected.
Asunto(s)
Técnicas Biosensibles/métodos , Glicoles de Etileno/análisis , Glicerol/aislamiento & purificación , Técnicas Biosensibles/instrumentación , Química Farmacéutica , Glicerol/análisis , CuarzoRESUMEN
The selection of the compound to be used as a coating for a piezoelectric quartz crystal is of utmost importance in the development of a chemical sensor. The relevant parameters to be evaluated (stability, sensitivity, reversibility, response time, reproducibility, and selectivity), and the main variables affecting the results and influencing the choice of coatings are discussed and illustrated with experiments performed during the evaluation of coatings to detect carbon dioxide.
RESUMEN
Response surface and simplex methods were used for optimizing the determination of total sulfur present in gaseous samples using a quartz crystal microbalance. The optimization methodology increased the analytical signal from three to five times. The simplex method showed the highest efficiency in the search for the maximum sensor response. However, the response surface allows ranking the variables according to their effects and highlights the existence of a plateau in the optimal region.
RESUMEN
A new method based on a quartz crystal microbalance was developed for the determination of cyanide. As the sensitivity depends on pH, temperature and nitrogen flow rate, a modified simplex was used to optimise these experimental parameters. Two different versions of the proposed method were optimised. For the first version a sensitivity increase of 1.5 was observed after 27 runs, whereas for the second version a sensitivity increase of 1.7 was observed after 12 runs.
Asunto(s)
Cianuros/análisis , Cuarzo , Nitrógeno/administración & dosificación , Sensibilidad y Especificidad , Contaminantes Químicos del Agua/análisisRESUMEN
Foi determinado o Coeficiente de Digestibilidade Aparente da Matéria Seca, em raçöes para animais silvestres. Foram utilizados 16 animais, oito (8) saguis de tufo preto (Callithrix penicilatta) e oito (8) macacos-prego (Cebus apella). Os animais foram mantidos em gaiolas de digestibilidade e receberam a raçäo básica durante 10 dias. Após o período inicial, as amostras de fezes foram colhidas durante 5 dias para posterior análise. Os Coeficientes de Digestibilidade para o Callithrix estiveram entre 84,9 e 97,7 por cento, enquanto para o Cebus oscilaram entre 75,4 e 86,2 por cento. A pesquisa concluiu que as raçöes utilizadas foram muito bem aproveitadas pelos animais
Asunto(s)
Animales , Fenómenos Fisiológicos Nutricionales de los Animales , Digestión , Haplorrinos/fisiologíaRESUMEN
A new method for the analysis of both total and bound SO(2) in wine is proposed, based on a quartz crystal microbalance (QCM), and it is compared with the widely used Ripper method. The proposed method is faster than the Ripper's, and the instrumentation is either home-made or widely available. When both methods are applied to the same sample, the results obtained using the QCM method are bracketed in an interval less than one-tenth the size of that obtained using the Ripper method. Although the SO(2) concentrations found using the QCM method correlate well with the ones obtained with the Ripper method, the results are systematically higher, which can be explained as due to the absence of interferences known to affect the Ripper method.