Biocompatible in-tube solid phase microextraction coupled with liquid chromatography-fluorescence detection for determination of interferon alpha in plasma samples

The present work demonstrates the successful application of automated biocompatible in-tube solid-phase microextraction coupled with liquid chromatography (in-tube SPME/LC) for determination of interferon alpha(2a) (IFN alpha(2a)) in plasma samples for therapeutic drug monitoring. A restricted access material (RAM, protein-coated silica) was employed for preparation of a lab-made biocompatible in-tube SPME capillary that enables the direct injection of biological fluids as well as the simultaneous exclusion of macromolecules by chemical diffusion barrier and drug pre-concentration. The in-tube SPME variables, such as sample volume, draw/eject volume, number of draw-eject cycles, and desorption mode were optimized, to improve the sensitivity of the proposed method. The IFN alpha(2a) analyses in plasma sample were carried out within 25 min (sample preparation and LC analyses). The response of the proposed method was linear over a dynamic range, from 0.06 to 3.0 MIU mL(-1), with correlation coefficient equal to 0.998. The interday precision of the method presented coefficient of variation lower than 8%. The proposed automated method has adequate analytical sensitivity and selectivity for determination of IFN alpha(2a) in plasma samples for therapeutic drug monitoring. (C) 2010 Elsevier B.V. All rights reserved.

Polydimethylsiloxane/polypyrrole stir bar sorptive extraction and liquid chromatography (SBSE/LC-UV) analysis of antidepressants in plasma samples

A new polymeric coating consisting of a dual-phase, polydimethylsiloxane (PDMS) and polypyrrole (PPY) was developed for the stir bar sorptive extraction (SBSE) of antidepressants (mirtazapine, citalopram, paroxetine, duloxetine, fluoxetine and sertraline) from plasma samples, followed by liquid chromatography analysis (SBSE/LC-UV). The extractions were based on both adsorption (PPY) and sorption (PDMS) mechanisms. SBSE variables, such as extraction time, temperature, pH of the matrix, and desorption time were optimized, in order to achieve suitable analytical sensitivity in a short time period. The PDMS/PPY coated stir bar showed high extraction efficiency (sensitivity and selectivity) toward the target analytes. The quantification limits (LOQ) of the SBSE/LC-UV method ranged from 20 ng mL(-1) to 50 ng mL(-1), and the linear range was from LOQ to 500 ng mL(-1), with a determination coefficient higher than 0.99. The inter-day precision of the SBSE/LC-UV method presented a variation coefficient lower than 15%. The efficiency of the SBSE/LC-UV method was proved by analysis of plasma samples from elderly depressed patients. (C) 2008 Elsevier B.V. All rights reserved.

Modificação estrutural de bentonitas nacionais : caracterização e estudos de adsorção

São apresentados os aspectos teóricos, práticos e bibliográficos envolvidos no desenvolvimento da tese de doutorado intitulada Modificação estrutural de bentonitas nacionais: caracterização e estudos de adsorção. O trabalho consistiu no desenvolvimento de um material adsorvente a partir de bentonitas, do tipo montmorilonitas, modificadas estruturalmente com o objetivo de aumentar sua capacidade de adsorção de poluentes, orgânicos e inorgânicos. O estudo visa incrementar o valor agregado deste recurso mineral e insere-se na área de tratamento de efluentes líquidos usando adsorventes não tradicionais, eficientes e de baixo custo em substituição ao carvão ativado ou às resinas de troca iônica. Foram estudadas as propriedades físicas e químicas; distribuição de tamanho de partículas, área superficial, potenciais eletrocinéticos, capacidade de troca catiônica, composição mineralógica, morfologia superficial e espaçamento basal, bem como as propriedades adsorptivas dos argilominerais não tratados e modificados, não modificadas e pilarizadas respectivamente. Também são discutidos os mecanismos de adsorção envolvidos e o desenvolvimento de um reator contínuo (adsorção em flocos) e de separação sólido/líquido. As modificações estruturais dos argilominerais foram realizadas via homoionização com cloreto de cálcio e posterior intercalação com compostos orgânicos com ação quelante de metais. A FENAN, bentonita obtida pela intercalação com Orto Fenantrolina (OF), foi a que apresentou melhor viabilidade técnica em termos de adsorção, adsorção/dessorção, floculação e de acumulação de poluentes na forma floculada e não floculada. Adicionalmente os estudos de reversibilidade da intercalação revelaram a alta estabilidade da OF na FENAN, em soluções fortemente ácidas, onde aproximadamente 90% da OF permanece ligada à superfície da argila. A quantidade de OF adsorvida na forma de unidades micelares foi de 112 mg por grama de bentonita a pH 8,5 ± 0,5. A caracterização das bentonitas, via difração de Raios X, análise térmica, microscopia eletrônica de varredura e por microscopia de força atômica, revelou que as FENAN possuem um comportamento estrutural muito estável ao longo da seqüência de adsorção/dessorção e que após a adsorção de poluentes inorgânicos, o quelato metálico formado apresenta alta estabilidade dentro da estrutura da organobentonita. A capacidade de acumulação alcançada nas FENAN foi de 110 mg de Cu/g de bentonita, valor superior à de diversos materiais adsorventes alternativos propostos em outros trabalhos similares. Os estudos de acumulação das FENAN floculadas – FENANFLOC, indicaram que a presença de floculante, na quantidade utilizada, não afeta significativamente a capacidade de remoção das bentonitas modificadas. Este comportamento apresentado, permitiu o desenvolvimento do Reator Expandido de Flocos Adsorventes (REFA), cujas características e parâmetros operacionais são discutidos em detalhe. Finalmente, os resultados são discutidos em termos dos fenômenos interfaciais envolvidos e dos potenciais práticos deste novo adsorvente e da nova técnica de adsorção em flocos no REFA.

Screening of volatile composition from portuguese multifloral honeys using headspace solid-phase microextraction-gas chromatography–quadrupole mass spectrometry

The volatile composition from four types of multifloral Portuguese (produced in Madeira Island) honeys was investigated by a suitable analytical procedure based on dynamic headspace solid-phase microextraction (HS-SPME) followed by thermal desorption gas chromatography–quadrupole mass spectrometry detection (GC–qMS). The performance of five commercially available SPME fibres: 100 μm polydimethylsiloxane, PDMS; 85 μm polyacrylate, PA; 50/30 μm divinylbenzene/carboxen on polydimethylsiloxane, DVB/CAR/PDMS (StableFlex); 75 μm carboxen/polydimethylsiloxane, CAR/PDMS, and 65 μm carbowax/divinylbenzene, CW/DVB; were evaluated and compared. The highest amounts of extract, in terms of the maximum signal obtained for the total volatile composition, were obtained with a DVB/CAR/PDMS coating fibre at 60 °C during an extraction time of 40 min with a constant stirring at 750 rpm, after saturating the sample with NaCl (30%). Using this methodology more than one hundred volatile compounds, belonging to different biosynthetic pathways were identified, including monoterpenols, C13-norisoprenoids, sesquiterpenes, higher alcohols, ethyl esters and fatty acids. The main components of the HS-SPME samples of honey were in average ethanol, hotrienol, benzeneacetaldehyde, furfural, trans-linalool oxide and 1,3-dihydroxy-2-propanone.

Development of a dynamic headspace solid-phase microextraction procedure coupled to GC–qMSD for evaluation the chemical profile in alcoholic beverages

In the present study, a simple and sensitive methodology based on dynamic headspace solid-phase microextraction (HS-SPME) followed by thermal desorption gas chromatography with quadrupole mass detection (GC–qMSD), was developed and optimized for the determination of volatile (VOCs) and semi-volatile (SVOCs) compounds from different alcoholic beverages: wine, beer and whisky. Key experimental factors influencing the equilibrium of the VOCs and SVOCs between the sample and the SPME fibre, as the type of fibre coating, extraction time and temperature, sample stirring and ionic strength, were optimized. The performance of five commercially available SPME fibres was evaluated and compared, namely polydimethylsiloxane (PDMS, 100 μm); polyacrylate (PA, 85 μm); polydimethylsiloxane/divinylbenzene (PDMS/DVB, 65 μm); carboxen™/polydimethylsiloxane (CAR/PDMS, 75 μm) and the divinylbenzene/carboxen on polydimethylsiloxane (DVB/CAR/PDMS, 50/30 μm) (StableFlex). An objective comparison among different alcoholic beverages has been established in terms of qualitative and semi-quantitative differences on volatile and semi-volatile compounds. These compounds belong to several chemical families, including higher alcohols, ethyl esters, fatty acids, higher alcohol acetates, isoamyl esters, carbonyl compounds, furanic compounds, terpenoids, C13-norisoprenoids and volatile phenols. The optimized extraction conditions and GC–qMSD, lead to the successful identification of 44 compounds in white wines, 64 in beers and 104 in whiskys. Some of these compounds were found in all of the examined beverage samples. The main components of the HS-SPME found in white wines were ethyl octanoate (46.9%), ethyl decanoate (30.3%), ethyl 9-decenoate (10.7%), ethyl hexanoate (3.1%), and isoamyl octanoate (2.7%). As for beers, the major compounds were isoamyl alcohol (11.5%), ethyl octanoate (9.1%), isoamyl acetate (8.2%), 2-ethyl-1-hexanol (5.9%), and octanoic acid (5.5%). Ethyl decanoate (58.0%), ethyl octanoate (15.1%), ethyl dodecanoate (13.9%) followed by 3-methyl-1-butanol (1.8%) and isoamyl acetate (1.4%) were found to be the major VOCs in whisky samples.

Volatile flavour constituent patterns of terras madeirenses red wines extracted by dynamic headspace solid‐phase microextraction

A suitable analytical procedure based on static headspace solid-phase microextraction (SPME) followed by thermal desorption gas chromatography–ion trap mass spectrometry detection (GC–ITDMS), was developed and applied for the qualitative and semi-quantitative analysis of volatile components of Portuguese Terras Madeirenses red wines. The headspace SPME method was optimised in terms of fibre coating, extraction time, and extraction temperature. The performance of three commercially available SPME fibres, viz. 100 lm polydimethylsiloxane; 85 lm polyacrylate, PA; and 50/30 lm divinylbenzene/carboxen on polydimethylsiloxane, was evaluated and compared. The highest amounts extracted, in terms of the maximum signal recorded for the total volatile composition, were obtained with a PA coating fibre at 308C during an extraction time of 60 min with a constant stirring at 750 rpm, after saturation of the sample with NaCl (30%, w/v). More than sixty volatile compounds, belonging to different biosynthetic pathways, have been identified, including fatty acid ethyl esters, higher alcohols, fatty acids, higher alcohol acetates, isoamyl esters, carbonyl compounds, and monoterpenols/C13-norisoprenoids.

Effectiveness of different solid-phase microextraction fibres for differentiation of selected Madeira island fruits based on their volatile metabolite profile: identification of novel compounds

A headspace solid-phase microextraction (HS-SPME) procedure based on five commercialised fibres (85 μm polyacrylate – PA, 100 μm polydimethylsiloxane – PDMS, 65 μm polydimethylsiloxane/divinylbenzene – PDMS/DVB, 70 μm carbowax/divinylbenzene – CW/DVB and 85 μm carboxen/polydimethylsiloxane – CAR/PDMS) is presented for the characterization of the volatile metabolite profile of four selected Madeira island fruit species, lemon (Citrus limon), kiwi (Actinidia deliciosa), papaya (Carica papaya L.) and Chickasaw plum (Prunus angustifolia). The isolation of metabolites was followed by thermal desorption gas chromatography–quadrupole mass spectrometry (GC–qMS) methodology. The performance of the target fibres was evaluated and compared. The SPME fibre coated with CW/DVB afforded the highest extraction efficiency in kiwi and papaya pulps, while in lemon and plum the same was achieved with PMDS/DVB fibre. This procedure allowed for the identification of 80 compounds, 41 in kiwi, 24 in plums, 23 in papaya and 20 in lemon. Considering the best extraction conditions, the most abundant volatiles identified in kiwi were the intense aldehydes and ethyl esters such as (E)-2-hexenal and ethyl butyrate, while in Chicasaw plum predominate 2-hexenal, 2-methyl-4-pentenal, hexanal, (Z)-3-hexenol and cyclohexylene oxide. The major compounds identified in the papaya pulp were benzyl isothiocyanate, linalool oxide, furfural, hydroxypropanone, linalool and acetic acid. Finally, lemon was shown to be the most divergent of the four fruits, being its aroma profile composed almost exclusively by terpens, namely limonene, γ-terpinene, o-cymene and α-terpinolene. Thirty two volatiles were identified for the first time in the fruit or close related species analysed and 14 volatiles are reported as novel volatile metabolites in fruits. This includes 5 new compounds in kiwi (2-cyclohexene-1,4-dione, furyl hydroxymethyl ketone, 4-hydroxydihydro-2(3H)-furanone, 5-acetoxymethyl-2-furaldehyde and ethanedioic acid), 4 in plum (4-hydroxydihydro-2(3H)-furanone, 5-methyl-2-pyrazinylmethanol, cyclohexylene oxide and 1-methylcyclohexene), 4 in papaya (octaethyleneglycol, 1,2-cyclopentanedione, 3-methyl-1,2-cyclopentanedione and 2-furyl methyl ketone) and 2 in lemon (geranyl farnesate and safranal). It is noteworthy that among the 15 volatile metabolites identified in papaya, 3-methyl-1,2-cyclopentanedione was previously described as a novel PPARγ (peroxisome proliferator-activated receptor γ) agonist, having a potential to minimize inflammation.

Headspace solid-phase microextraction-gas chromatography-quadrupole mass spectrometric methodology for the establishment of the volatile composition of Passiflora fruit species

Dynamic headspace solid-phase microextraction (HS-SPME) followed by thermal desorption gas chromatography-quadrupole mass spectrometry analysis (GC-qMS), was used to investigate the aroma profile of different species of passion fruit samples. The performance of five commercially available SPME fibres: 65 μm polydimethylsiloxane/divinylbenzene, PDMS/DVB; 100 μm polydimethylsiloxane, PDMS; 85 μm polyacrylate, PA; 50/30 μm divinylbenzene/carboxen on polydimethylsiloxane, DVB/CAR/PDMS (StableFlex); and 75 μm carboxen/polydimethylsiloxane, CAR/PDMS; was evaluated and compared. Several extraction times and temperature conditions were also tested to achieve optimum recovery. The SPME fibre coated with 65 μm PDMS/DVB afforded the highest extraction efficiency, when the samples were extracted at 50 °C for 40 min with a constant stirring velocity of 750 rpm, after saturating the sample with NaCl (17%, w/v — 0.2 g). A comparison among different passion fruit species has been established in terms of qualitative and semi-quantitative differences in volatile composition. By using the optimal extraction conditions and GC-qMS it was possible to tentatively identify seventy one different compounds in Passiflora species: 51 volatiles in Passiflora edulis Sims (purple passion fruit), 24 in P. edulis Sims f. flavicarpa (yellow passion fruit) and 21 compounds in Passiflora mollissima (banana passion fruit). It was found that the ethyl esters comprise the largest class of the passion fruit volatiles, including 82.8% in P. edulis variety, 77.4% in P. edulis Sims f. flavicarpa variety and 39.9% in P. mollissima. The semi-quantitative results were then submitted to principal component analysis (PCA) in order to establish relationships between the compounds and the different passion fruit species under investigation.

Potentialities of two solventless extraction approaches—Stir bar sorptive extraction and headspace solid-phase microextraction for determination of higher alcohol acetates, isoamyl esters and ethyl esters in wines

A stir bar sorptive extraction with liquid desorption followed by large volume injection coupled to gas chromatography–quadrupole mass spectrometry (SBSE-LD/LVI-GC–qMS) was evaluated for the simultaneous determination of higher alcohol acetates (HAA), isoamyl esters (IsoE) and ethyl esters (EE) of fatty acids. The method performance was assessed and compared with other solventless technique, the solid-phase microextraction (SPME) in headspace mode (HS). For both techniques, influential experimental parameters were optimised to provide sensitive and robust methods. The SBSE-LD/LVI methodology was previously optimised in terms of extraction time, influence of ethanol in the matrix, liquid desorption (LD) conditions and instrumental settings. Higher extraction efficiency was obtained using 60 min of extraction time, 10% ethanol content, n-pentane as desorption solvent, 15 min for the back-extraction period, 10 mL min−1 for the solvent vent flow rate and 10 °C for the inlet temperature. For HS-SPME, the fibre coated with 50/30 μm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) afforded highest extraction efficiency, providing the best sensitivity for the target volatiles, particularly when the samples were extracted at 25 °C for 60 min under continuous stirring in the presence of sodium chloride (10% (w/v)). Both methodologies showed good linearity over the concentration range tested, with correlation coefficients higher than 0.984 for HS-SPME and 0.982 for SBES-LD approach, for all analytes. A good reproducibility was attained and low detection limits were achieved using both SBSE-LD (0.03–28.96 μg L−1) and HS-SPME (0.02–20.29 μg L−1) methodologies. The quantification limits for SBSE-LD approach ranging from 0.11 to 96.56 μg L−and from 0.06 to 67.63 μg L−1 for HS-SPME. Using the HS-SPME approach an average recovery of about 70% was obtained whilst by using SBSE-LD obtained average recovery were close to 80%. The analytical and procedural advantages and disadvantages of these two methods have been compared. Both analytical methods were used to determine the HAA, IsoE and EE fatty acids content in “Terras Madeirenses” table wines. A total of 16 esters were identified and quantified from the wine extracts by HS-SPME whereas by SBSE-LD technique were found 25 esters which include 2 higher alcohol acetates, 4 isoamyl esters and 19 ethyl esters of fatty acids. Generally SBSE-LD provided higher sensitivity with decreased analysis time.

Mesoestruturas porosas a partir de materiais naturais

The MCM-41 mesoporous synthesis was done using rice hulls ash and chrysotile as natural alternative silica sources. For the using of these sources, chemical and thermic treatments were done in both materials. After chemical and thermic treatments, these materials were employed on the MCM-41 mesoctructures synthesis. The natural materials treated and employed in the synthesis were characterized by several techniques such as X-ray diffraction, N2 adsorption and desorption, scanning electronic microscopy and thermogravimetric analysis. MCM-41 standart samples synthetized with aerosil 200 commercial sílica were used to evaluation. The formed material from rice hulls ash showed values from BET specific area about 468 m².g-1, N2 adsorption and desorption isotherms and loss mass similar to reference materials. The silica from chrysotile calcined and leached was employed to mesoporous materials synthesis. The BET specific area showed values about 700 m².g-1, N2 adsorption and desorption isotherms type IV and loss mass similar to mesoporous materials. The formed material from calcined and leached chrysotile, without calcination, applied to phenol remotion carried high performance liquid chromatography and evaluated with organophilic clays with different treatments. By the characterization techniques were proved that mesoporous materials with lesser order that reference samples. The material formed from rice hulls ash without the calcination step achieved better adsorption results than organophilic clays

Síntese, caracterização e aplicação de LaSBA-15 e como catalisador para obtenção de biodiesel de soja via rota etílica

Biodiesel is an alternative fuel, renewable, biodegradable and nontoxic. The transesterification of vegetable oils or animal fat with alcohol is most common form of production of this fuel. The procedure for production of biodiesel occurs most commonly through the transesterification reaction in which catalysts are used to accelerate and increase their income and may be basic, acid or enzyme. The use of homogeneous catalysis requires specific conditions and purification steps of the reaction products (alkyl ester and glycerol) and removal of the catalyst at the end of the reaction. As an alternative to improve the yield of the transesterification reaction, minimize the cost of production is that many studies are being conducted with the application of heterogeneous catalysis. The use of nano-structured materials as catalysts in the production of biodiesel is a biofuel alternative for a similar to mineral diesel. Although slower, can esterify transesterified triglycerides and free fatty acids and suffer little influence of water, which may be present in the raw material. This study aimed at the synthesis, characterization and application of nano-structured materials as catalysts in the transesterification reaction of soybean oil to produce biodiesel by ethylic route. The type material containing SBA-15 mesoporous lanthanum embedded within rightly Si / La = 50 was used catalyst. Solid samples were characterized by X-ray diffraction, thermogravimetric analysis, infrared spectroscopy, nitrogen adsorption and desorption. For the transesterification process, we used a molar ratio of 20:1 alcohol and oil with 0.250 g of catalyst at 60°C and times of 6 hours of reaction. It was determined the content of ethyl esters by H-NMR analysis and gas chromatography. It was found that the variable of conversion obtained was 80%, showing a good catalytic activity LaSBA-15 in the transesterification of vegetable oils via ethylic route

Pirólise térmica e catalítica de resíduos de vácuo gerados no refino de petróleo

In this study, was used a very promising technique called of pyrolysis, which can be used for obtaining products with higher added value. From oils and residues, since the contribution of heavier oils and residues has intensified to the world refining industry, due to the growing demand for fuel, for example, liquid hydrocarbons in the range of gasoline and diesel. The catalytic pyrolysis of vacuum residues was performed with the use of a mesoporous material belonging the M41S family, which was discovered in the early 90s by researchers Mobil Oil Corporation, allowing new perspectives in the field of catalysis. One of the most important members of this family is the MCM-41, which has a hexagonal arrangement of mesopores with pore diameters between 2 and 10 nm and a high specific surface area, making it very promising for use as a catalyst in petroleum refining for catalytic cracking, and their mesopores facilitate the access of large hydrocarbon molecules. The addition of aluminum in the structure of MCM-41 increases the acidity of the material, making it more positive for application in the petrochemical industry. The mesoporous material of the type Al-MCM41 (ratio Si / Al = 50) was synthesized by hydrothermal method starting from the silica gel, NaOH and distilled water added to the gel pseudobohemita synthesis. Driver was used as structural CTMABr. Removal of organic driver (CTMABr) was observed by TG / DTG and FTIR, but this material was characterized by XRD, which was observed the formation of the main peaks characteristic of mesoporous materials. The analysis of adsorption / desorption of nitrogen this material textural parameters were determined. The vacuum residues (VR's) that are products of the bottom of the vacuum distillation tower used in this study are different from oil fields (regions of Ceará and Rio de Janeiro). Previously characterized by various techniques such as FTIR, viscosity, density, SARA, elemental analysis and thermogravimetry, which was performed by thermal and catalytic degradation of vacuum residues. The effect of AlMCM-41 was satisfactory, since promoted a decrease in certain ranges of temperature required in the process of conversion of hydrocarbons, but also promoted a decrease in energy required in the process. Thus enabling lower costs related to energy expenditure from degradation during processing of the waste

Reforma a seco de metano com catalisadores Ni/MCM-41 sintetizados a partir de fontes alternativas de sílica

The production of synthesis gas has received renewed attention due to demand for renewable energies to reduce the emissions of gases responsible for enhanced greenhouse effect. This work was carried out in order to synthesize, characterize and evaluate the implementation of nickel catalysts on MCM-41 in dry reforming reactions of methane. The mesoporous molecular sieves were synthesized using as silica sources the tetraethyl orthosilicate (TEOS) and residual glass powder (PV). The sieves were impregnated with 10% nickel to obtain the metallic catalysts (Ni/MCM-41). These materials were calcined and characterized by Thermogravimetric Analysis (TG), Infrared spectroscopy (FTIR), X-ray Diffraction (XRD), Temperature-Programmed Reduction (TPR) and N2 Adsorption/Desorption isotherms (BET/BJH). The catalytic properties of the samples were evaluated in methane dry reforming with CO2 in order to produce synthesis gas to be used in the petrochemical industry. The materials characterized showed hexagonal structure characteristic of mesoporous material MCM-41 type, being maintained after impregnation with nickel. The samples presented variations in the specific surface area, average volume and diameter of pores based on the type of interaction between the nickel and the mesoporous support. The result of the the catalytic tests showed conversions about 91% CO2, 86% CH4, yelds about 85% CO and 81% H2 to Ni/MCM-41_TEOS_C, and conversions about 87% CO2, 82% CH4, yelds about 70% CO and 59% H2 to Ni/MCM-41_PV_C. The similar performance confirms that the TEOS can be replaced by a less noble materials

Structure-function relationship of new crotamine isoform from the Crotalus durissus cascavella

In this work we isolated a novel crotamine like protein from the Crotalus durissus cascavella venom by combination of molecular exclusion and analytical reverse phase HPLC. Its primary structure was:YKRCHKKGGHCFPKEKICLPPSSDLGKMDCRWKRK-CCKKGS GK. This protein showed a molecular mass of 4892.89 da that was determined by Matrix Assisted Laser Desorption Ionization Time-of-flight (MALDI-TOF) mass spectrometry. The approximately pI value of this protein was determined in 9.9 by two-dimensional electrophoresis. This crotamine-like protein isolated here and that named as Cro 2 produced skeletal muscle spasm and spastic paralysis in mice similarly to other crotamines like proteins. Cro 2 did not modify the insulin secretion at low glucose concentration (2.8 and 5.6 mM), but at high glucose concentration (16.7 mM) we observed an insulin secretion increasing of 2.7-3.0-fold than to control. The Na+ channel antagonist tetrodoxin (6 mM) decreased glucose and Cro 2-induced insulin secretion. These results suggested that Na+ channel are involved in the insulin secretion. In this article, we also purified some peptide fragment from the treatment of reduced and carboxymethylated Cro 2 (RC-Cro 2) with cyanogen bromide and protease V8 from Staphylococcus aureus. The isolated pancreatic beta-cells were then treated with peptides only at high glucose concentration (16.7 mM), in this condition only two peptides induced insulin secretion. The amino acid sequence homology analysis of the whole crotamine as well as the biologically-active peptide allowed determining the consensus region of the biologically-active crotamine responsible for insulin secretion was KGGHCFPKE and DCRWKWKCCKKGSG.

Proteomic analysis of dog tears for potential cancer markers

The first reference map of the proteome of pooled normal dog tears was created using 2-dimensional polyacrylamide gel electrophoresis and the identity of a number of the major species determined using matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF) and peptide mass fingerprint matching on protein sequence databases. In order to understand the changes in protein expression in the tear film of dogs with cancer, tears from such animals were similarly examined. A number of differences were found between the tears of healthy dogs and the dogs with cancer. Differences were found in levels of actin and albumin and in an unidentified protein which may be analogous to human lacryglobulin. These findings suggest that it may be possible to develop tear film analysis to provide a simple non-invasive test for the diagnosis and/or management of canine cancers. (C) 2007 Elsevier Ltd. All rights reserved.