A study of chymotrypsin immobilization conditions for improved peptide mapping

La cartographie peptidique est une technique de grande importance utilisée lors de l’identification des protéines et la caractérisation des modifications post-traductionnelles des protéines. Deux méthodes sont utilisées afin de couper les protéines en peptides pour la cartographie : les méthodes chimiques et les méthodes enzymatiques. Dans ce projet, l’enzyme chymotrypsine a été utilisée pour l’hydrolyse (la digestion) des liens peptidiques. Cependant, l’autoprotéolyse des enzymes peut augmenter la complexité des échantillons, rendant ainsi ardue l’obtention de pics résolus suite à l’apparition de pics non-désirés dans la carte peptidique. Par conséquent, nous avons utilisé la réticulation des enzymes protéolytiques par réaction avec le glutaraldéhyde (GA) donnant une enzyme insoluble afin de réduire l’autoprotéolyse. L’immobilisation de la chymotrypsine par GA a été effectuée selon une méthode rapportée précédemment par le groupe Waldron. L’électrophorèse capillaire (CE) couplée à l’absorption UV-visible a été utilisée pour la séparation et la détection de peptides et pour obtenir ainsi une cartographie peptidique. Deux tampons différents ont été évalués afin d’obtenir les meilleures conditions pour la digestion de substrats protéiques par la chymotrypsine libre (soluble) ou la GAchymotrypsine et l’analyse par CE. Les cartes des peptides autoprotéolytiques ont été comparées entre les deux formats de chymotrypsine. Afin d’améliorer la cartographie peptidique, nous avons évalué trois méthodes de conditionnement du capillaire CE et deux méthodes pour stopper la digestion. Le bicarbonate d’ammonium s’est avéré être le tampon optimal pour la digestion en solution et l’utilisation d’un bain d’acétone et de glace sèche s’est avérée être la méthode optimale pour stopper la digestion. Une solution de SDS, 25 mM, dans l’étape de rinçage a été utilisée après chaque analyse CE et a permis d’améliorer la résolution des cartes peptidiques. La comparaison entre l’autoprotéolyse de la chymotrypsine libre et de celle immobilisé par GA a été effectuée par des tests utilisant une gamme de six différentes combinaisons de conditions afin d’évaluer le temps (30 et 240 min) et la température de digestion (4, 24 et 37°C). Dans ces conditions, nos résultats ont confirmé que le GA-chymotrypsine réduit l’autoprotéolyse par rapport à l’enzyme libre. La digestion (à 37°C/240 min) de deux substrats modèles par la chymotrypsine libre et immobilisée en fonction de la température de dénaturation du substrat a été étudiée. iii Avant la digestion, les substrats (l’albumine de sérum bovine, BSA, et la myoglobine) ont été dénaturés par chauffage pendant 45 min à trois températures différentes (60, 75 et 90°C). Les résultats ont démontré que la dénaturation par chauffage du BSA et de la myoglobine n’a pas amélioré la cartographie peptidique pour la GA-chymotrypsine, tandis que la digestion de ceux-ci en présence de la chymotrypsine libre a amélioré de façon quantifiable à des températures élevées. Ainsi, le chauffage du substrat à 90°C avec l’enzyme soluble facilite le dépliement partiel du substrat et sa digestion limitée, ce qui a été mieux pour la myoglobine que pour la BSA.

Fédéralisme et concurrence interjuridictionnelle

Éditorial du 23 novembre 2015

The HUman MicroNucleus project on eXfoLiated buccal cells (HUMNXL): The role of life-style, host factors, occupational exposures, health status, and assay protocol

The human buccal micronucleus cytome assay (BMCyt) is one of the most widely used techniques to measure genetic damage in human population studies. Reducing protocol variability, assessing the role of confounders, and estimating a range of reference values are research priorities that will be addressed by the HUMNXL, collaborative study. The HUMNXL, project evaluates the impact of host factors, occupation, life-style, disease status, and protocol features on the occurrence of MN in exfoliated buccal cells. In addition, the study will provide a range of reference values for all cytome endpoints. A database of 5424 subjects with buccal MN values obtained from 30 laboratories worldwide was compiled and analyzed to investigate the influence of several conditions affecting MN frequency. Random effects models were mostly used to investigate MN predictors. The estimated spontaneous MN frequency was 0.74 parts per thousand (95% CI 0.52-1.05). Only staining among technical features influenced MN frequency, with an abnormal increase for non-DNA-specific stains. No effect of gender was evident, while the trend for age was highly significant (p < 0.001). Most occupational exposures and a diagnosis of cancer significantly increased MN and other endpoints frequencies. MN frequency increased in heavy smoking (>= 40 cig/day. FR = 1.37:95% CI 1.03-.82) and decreased with daily fruit consumption (FR = 0.68; 95% CI 0.50-0.91). The results of the HUMNXL, project identified priorities for validation studies, increased the basic knowledge of the assay, and contributed to the creation of a laboratory network which in perspective may allow the evaluation of disease risk associated with MN frequency. (C) 2011 Elsevier B.V. All rights reserved.

Antiproliferative Effects of Fluoxetine on Colon Cancer Cells and in a Colonic Carcinogen Mouse Model

The antidepressant fluoxetine has been under discussion because of its potential influence on cancer risk. It was found to inhibit the development of carcinogen-induced preneoplastic lesions in colon tissue, but the mechanisms of action are not well understood. Therefore, we investigated anti-proliferative effects, and used HT29 colon tumor cells in vitro, as well as C57BL/6 mice exposed to intra-rectal treatment with the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as models. Fluoxetine increased the percentage of HT29 cells in the G(0)/G(1) phase of cell-cycle, and the expression of p27 protein. This was not related to an induction of apoptosis, reactive oxygen species or DNA damage. In vivo, fluoxetine reduced the development of MNNG-induced dysplasia and vascularization-related dysplasia in colon tissue, which was analyzed by histopathological techniques. An anti-proliferative potential of fluoxetine was observed in epithelial and stromal areas. It was accompanied by a reduction of VEGF expression and of the number of cells with angiogenic potential, such as CD133, CD34, and CD31-positive cell clusters. Taken together, our findings suggest that fluoxetine treatment targets steps of early colon carcinogenesis. This confirms its protective potential, explaining at least partially the lower colon cancer risk under antidepressant therapy.

High-fat diet causes an imbalance in the colonic serotonergic system promoting adipose tissue enlargement and dysplasia in rats

A high-fat (HF) diet, the serotonergic system and stromal elements have all been implicated in colon carcinogenesis. We investigated whether the colonic serotonergic system could play a main role in the development of colonic dysplasia and stromal reactivity in carcinogen-treated rats under HF diet. For this, dimethylhydrazine-treated rats were fed with standard diet and a HF diet. Fat distribution was quantified by computerized tomography exam, serotonergic activity was analyzed by high-performance liquid chromatography, gene expression, and immunohistochemistry, which along with histopathological technique enabled us to enumerate dysplasia, microvessels density, cell proliferation and COX-2 expression. We found that the HF diet induced an increase in the amount of viscera! adipose tissue, even without expressive changes in the average body weight. This was correlated with a loss of serotonergic balance in colon tissue. Moreover, the HF diet promoted dysplasia and microvessel density in association with increased proliferation and COX-2 expression within pericryptal colonic stroma. Our current findings suggest that a HF diet promotes the enlargement of adipose tissue via loss of control in colon serotonergic activity, which enhances colonic dysplasia by supporting microvessel development. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Luminescence Enhancement after Adding Stoppers to Europium(III) Nanozeolite L

Stopper molecules attached to nanozeolite L (NZL) boost the luminescence of confined Eu3+-β-diketonate complexes. The mechanism that is responsible was elucidated by comparing two diketonate ligands of different pKa and two aromatic imines, and by applying stationary and time resolved spectroscopy. The result is that the presence of the imidazolium based stopper is favorable to the sustainable formation of Eu3+-β-diketonate complexes with high coordination by decreasing the proton strength inside the channels of NZL. A consequence is that strongly luminescent transparent films can be prepared using aqueous suspension of the stopper modified composites.

A Low-Flow Self-Cleaning Drainage System for Fish Rearing Tanks

A float-stopper mechanism was designed to drain fish holding tanks directly from the bottom. Unlike traditional, top-drawn standpipe systems, it allows continuous flushing of settled solid waste. It also prevents the accumulation of these wastes between the two standpipes that are used in bottom-drawn, double-walled standpipe systems. When suspended solids are forced upward between the outer and inner standpipes of such systems, a minimum velocity must be maintained to prevent sediment accumulation. This minimum velocity determines the minimum flow rate through the tank. The system described in this report flushes well over a wide range of flow rates.

Efficient estimation of the total number of acini in adult rat lung

Pulmonary airways are subdivided into conducting and gas-exchanging airways. An acinus is defined as the small tree of gas-exchanging airways, which is fed by the most distal purely conducting airway. Until now a dissector of five consecutive sections or airway casts were used to count acini. We developed a faster method to estimate the number of acini in young adult rats. Right middle lung lobes were critical point dried or paraffin embedded after heavy metal staining and imaged by X-ray micro-CT or synchrotron radiation-based X-rays tomographic microscopy. The entrances of the acini were counted in three-dimensional (3D) stacks of images by scrolling through them and using morphological criteria (airway wall thickness and appearance of alveoli). Segmentation stopper were placed at the acinar entrances for 3D visualizations of the conducting airways. We observed that acinar airways start at various generations and that one transitional bronchiole may serve more than one acinus. A mean of 5612 (±547) acini per lung and a mean airspace volume of 0.907 (±0.108) μL per acinus were estimated. In 60-day-old rats neither the number of acini nor the mean acinar volume did correlate with the body weight or the lung volume.

Geophysical measurements on sediment cores from North Pond, Mid Atlantic Ridge

Die Porosität ist einer der wichtigsten gesteinsphysikalischen Parameter von Sedimenten. Daher werden in dieser Arbeit die Abhängigkeiten zwischen dem zentralen gesteinsphysikalischen Parameter Porosität und den Parametern Wärmeleitfähigkeit und spezifischer elektrischer Widerstand an Sedimenten von North Pond, eines Sedimentbeckens an der westlichen Flanke des Mittelatlantischen Rückens bei ca. 23°N, qualitativ und empirisch untersucht. Die Messung des spezifischen Widerstands wurde mittels eines miniaturisierten Wenner-Arrays mit einem Zentimeter Elektrodenabstand noch an Bord der FS Maria S. Merian auf der Reise MSM11/1 vom 10. Februar bis 13. März 2009 an Sedimentkernhälften durchgeführt, ebenso wie die Messung der Wärmeleitfähigkeit mit einer Nadelsonde. Die Messung der Gamma-Dichte an einem Multi Sensor Core Logger erfolgte im Rahmen dieser Arbeit und wurde wie die Messung der Porosität an Proben im Marum in Bremen durchgeführt. Die zugrundeliegende und zu untersuchende Beziehung zwischen spezifischem Widerstand und Porosität ist Archie's Law, die untersuchte Beziehung zwischen Wärmeleitfähigkeit und Porosität ist das Modell des geometrischen Mittels der Sedimentkomponenten. Die physikalische Beziehung zwischen der Porosität und den beiden Parametern spezifischer Widerstand und Wärmeleitfähigkeit konnte an den Sedimenten von North Pond bestätigt werden.

Experimental analysis of direct thermal methane cracking

The analysis of the viability of Hydrogen production without CO2 emissions is one of the most challenging activities that have been initiated for a sustainable energy supply. As one of the tracks to fulfil such objective, direct methane cracking has been analysed experimentally to assess the scientific viability and reaction characterization in a broad temperature range, from 875 to 1700 ?C. The effect of temperature, sweeping/carrier gas fraction proposed in some concepts, methane flow rate, residence time, and tube material and porosity has been analysed. The aggregation of carbon black particles to the reaction tube is the main technological show-stopper that has been identified.

Control by ATR-FTIR of surface treatment of cork stoppers and its effect on their mechanical performance

Fourier transform infrared (FTIR) spectroscopy was applied to determine the type of surface treatment and dose used on cork stoppers and to predict the friction between stopper and bottleneck. Agglomerated cork stoppers were finished with two different doses and using two surface treatments: P (paraffin and silicone), 15 and 25 mg/stopper, and S (only silicone), 10 and 15 mg/stopper. FTIR spectra were recorded at five points for each stopper by attenuated total reflectance (ATR). Absorbances at 1,010, 2,916, and 2,963 cm -1 were obtained in each spectrum. Discriminant analysis techniques allowed the treatment, and dose applied to each stopper to be identified from the absorbance values. 91.2% success rates were obtained from individual values and 96.0% from the mean values of each stopper. Spectrometric data also allowed treatment homogeneity to be determined on the stopper surface, and a multiple regression model was used to predict the friction index (If = Fe/Fc) (R 2 = 0.93)

Quality grading of cork stoppers based on porosity, density and elasticity.

An objective control method for grading cork stoppers is presented using a cork stopper quality index based on porosity, density and elasticity, these being the properties which have the greatest influence on the closure capacity of the stopper. The elasticity of the cork stopper is measured through the relaxation ratio, which is defined by the relationship between the relaxation force exerted by the cork in the bottleneck and the compressive force exerted by a caliper to fit the stopper in the bottle. The relaxation ratio, defined in this way, represents the part of the compression force which is applied to the stopper on insertion and which is recovered in the form of the relaxation force to achieve closure. The calculation of the relaxation ratio involves the measurement of the relaxation force of the fitted stopper. This force has been measured rigorously and precisely using a device developed in the Cork Laboratory at the INIA-CIFOR and which is presented for the first time in this paper.

Snuff bottle

Glass, Chinese, Qing; H: 1 3/8 in. (with stopper); overlaid glass, metal, jade

Tratamentos de superfície em rolhas de cortiça

Este trabalho foi desenvolvido na empresa Amorim & Irmãos, SA e teve dois objectivos fundamentais. O primeiro centrou-se na análise do processo de tratamento de superfície das rolhas, procurando um produto alternativo ao actualmente implementado na empresa e a sua optimização. O segundo objectivo foi a elaboração de um novo método de determinação da absorção em garrafa, que permitisse a sua determinação sem o conhecimento da massa inicial da rolha. Para a concretização do primeiro objectivo foram estudados doze produtos químicos em comparação com o actualmente utilizado, em que o objectivo foi obter-se forças de extracção entre 15 e 20 daN. Após realização do tratamento de superfície para cada produto foram realizados vários testes laboratoriais, nomeadamente: forças de extracção, vedação em tubo, absorção em garrafa, capilaridade, análise de risco à quantidade de produto adicionado e análise de risco ao tempo de distribuição do tratamento. Após análise global dos resultados obtidos verificou-se que o produto T13, embora apresente forças de extracção no limite inferior ao desejado possui uma boa estabilização. Os produtos T5 e T6 são bons alternativos ao produto actualmente implementado (T8), embora seja necessário ter alguns cuidados no seu manuseamento. O produto T5 como foi considerado mau no teste da absorção em garrafa não poderá ser utilizado para mercados mais distantes (EUA, Austrália e África do Sul) devido ao risco de ocorrência de migração de vinho através da rolha de cortiça. O produto T6 como apresentou um comportamento irregular na análise de risco à quantidade de produto adicionado, e na análise de risco à distribuição de produto, deve-se ter muita atenção à quantidade inserida no tambor assim como o tempo de distribuição. Para a concretização do segundo objectivo foi determinada a absorção em garrafa pelo método actual e comparou-se com o novo método. Apesar do desvio padrão ser de aproximadamente 0,85, pode-se afirmar que o novo método de determinação da absorção em garrafa é um método eficaz que pode ser aprovado pela empresa. Desta forma, foi possível solucionar esta questão e permitir ao laboratório de controlo de qualidade determinar a absorção em garrafa de garrafas de vinho provenientes de clientes.