Trypsin-SBTI interaction in reverse micelles A slow intermicellar exchange-dependent binding

AbstractSolubilisate exchange between reverse micelles must take place before any reaction inside reverse micelles occurs if the reactants are confined to the aqueous micellar core. When the interacting species are 2 small molecules or one small molecule and one macromolecule, it has been shown that the exchange is faster than the typical turnover of an enzymatic reaction. The study of the interaction between 2 macromolecules (trypsin and soybean trypsin inhibitor) in reverse micelles carried out in this work reveals that the exchange between these macromolecule-containing reverse micelles slows down by a thousand times and the limiting-step in the exchange, the fusion, by 10* times. Both reverse micellar size (ωo = [water]/ [surfactant]) and temperature affected the rate of the fusion process. A hypothesis for the proposed active role of macromolecules in the exchange process is also given

Callus Induction and Cellular Suspensions from Murtilla (Ugni molinae Turcz.) for trans-resveratrol Production

The present study reports for the first time the quantification of resveratrol and the use of biotechnological techniques applied to stilbene productions in species from genus Ugni and calli production from adult explants (fruits). Resveratrol is synthesized by a series of families of higher plants, which has generated much interest in recent years for its antioxidant, anticancer and antitumor properties, which would allow longevity of cells to be prolonged. In this study, leaves and mature fruits were collected from three sites in Southern Chile and correspond to three ecotypes of the Chilean endemic species Ugni molinae Turz. (murtilla). These were established in vitro to prepare the callus and subsequent development of cellular suspensions for trans-resveratrol production. Our results showed that these stilbenes are present in murtilla and that their concentrations vary between ecotypes and tissues, reaching up to 553.5 µg g-1 of t-resveratrol produced in ecotype 3 callus. These values are relatively higher than those found in other plant species. Under optimum culture conditions, extraction of resveratrol from Ugni molinae is scalable to industrial levels, which makes it a viable alternative for obtaining stilbenes.This research was supported by a project of the Innova BíoBío, Corfo-Chile No.12.247

iTRAQ-based quantitative analysis of protein mixtures with large fold change and dynamic range

Comunicaciones a congreso

A Dige proteomic analysis of weat flag lea treated with terra-sorb foliar, a free amino acid high content biostimulator

Comunicaciones a congreso

A Focused Multiple Reaction Monitoring (MRM) Quantitative Method for Bioactive Grapevine Stilbenes by Ultra-High-Performance Liquid Chromatography Coupled to Triple-Quadrupole Mass Spectrometry (UHPLC-QqQ)

Grapevine stilbenes are a family of polyphenols which derive from trans-resveratrol having antifungal and antimicrobial properties, thus being considered as phytoalexins. In addition to their diverse bioactive properties in animal models, they highlight a strong potential in human health maintenance and promotion. Due to this relevance, highly-specific qualitative and quantitative methods of analysis are necessary to accurately analyze stilbenes in different matrices derived from grapevine. Here, we developed a rapid, sensitive, and specific analysis method using ultra-high-performance liquid chromatography coupled to triple-quadrupole mass spectrometry (UHPLC-QqQ) in MRM mode to detect and quantify five grapevine stilbenes, trans-resveratrol, trans-piceid, trans-piceatannol, trans-pterostilbene, and trans-ε-viniferin, whose interest in relation to human health is continuously growing. The method was optimized to minimize in-source fragmentation of piceid and to avoid co-elution of cis-piceid and trans-resveratrol, as both are detected with resveratrol transitions. The applicability of the developed method of stilbene analysis was tested successfully in different complex matrices including cellular extracts of Vitis vinifera cell cultures, reaction media of biotransformation assays, and red wine.A.M.-M. acknowledges a grant from Conselleria d’Educacio, Cultura I Sport de la Generalitat Valenciana (FPA/2013/A/074). This work has been supported by grants from University of Alicante (VIGROB-105), the Spanish Ministry of Economy and Competitiveness (BIO2014-51861-R), and European funds for Regional development (FEDER)

Development of MRM methods to quantify proteins isoforms of poliphenol oxidase

Comunicaciones a congreso

Comparación de métodos preparativos de tejidos para la extracción de proteínas de la mazorca de cacao (Theobroma cacao L.)

Cocoa, Theobroma cacao L. is one of the main tropical industrial crops. Cocoa has a very high level of interfering substances, such as polysaccharides and phenolic compounds that could prevent the isolation of suitable protein. Efficient methods of protein extraction are a priority to successfully apply proteomic analyses. We compared and evaluated two methods (A and B) of tissue preparation for total protein extract by phenol/SDS extraction protocol. The difference in the application of the two methods was that extensively washed dry powder of pod tissue were made in Method A, whereas that crude extract were prepared Method B. Extracted proteins were examined using one-dimensional electrophoresis (1-D). Results show that each extraction method isolated a unique subset of cocoa pod proteome. Principal component analysis showed little variation in the data obtained using Method A, while that in Methods B showed no low reproducibility, thus demonstrating that Method A is a reliable for preparing cocoa pod proteins. The protocol is expected to be applicable to other recalcitrant plant tissues and to be of interest to laboratories involved in plant proteomics analyses. A combination of extraction approaches is recommended for increasing proteome coverage when using gel-based isolation techniques.El cacao, Theobroma cacao L. es uno de los principales cultivos tropicales industriales. La mazorca de cacao tiene un nivel muy alto de sustancias interferentes, tales como polisacáridos y compuestos fenólicos, que podrían impedir el aislamiento adecuado de la proteína. El uso de métodos eficientes de extracción de proteínas es una prioridad para aplicar con éxito los análisis proteómicos. Nosotros comparamos y evaluamos dos métodos preparativos (A y B) de tejidos para la extracción de proteína total mediante el protocolo de extracción con fenol/SDS. La diferencia entre los dos métodos fue extensivos lavados del polvo seco, obtenido mediante trituración con nitrógeno, de la mazorca fueron realizados en el Método A, mientras que un extracto crudo se preparó en el Método B. Extracciones proteicas fueron examinadas utilizando electroforesis monodimensional (1-D). Los resultados muestran que cada método de extracción aisló un único subconjunto del proteoma de las mazorcas de cacao. El análisis de componentes principales mostró poca variación en los datos por el Método A, mientras que el Método B fue poco reproducible, lo que demuestra que el Método A de extracción es un método fiable para la preparación de proteínas de las mazorcas de cacao. Se espera que el protocolo sea aplicable a otros tejidos de plantas recalcitrantes y podría ser de interés para los laboratorios involucrados en análisis de proteómica de plantas. Se recomienda una combinación de los enfoques de extracción para aumentar la cobertura del proteoma utilizando las técnicas de separación a base de gel.This work has been supported by grants from the Senescyt-Government of Ecuador (UTEQ-Ambiental-9-FCAmb-IFOR-2014-FOCICYT002), AMM holds a grant MAEC-AECID (2014-2015) of Spain

Top-down and bottom-up quantitative proteomic approaches to characteriza the development of grape berry tissues

Comunicaciones a congreso

Synergistic effect of methyljasmonate and cyclodextrin on stilbene biosynthesis pathway gene expression and resveratrol production in Monastrell grapevine cell cultures

<p>Abstract</p> <p>Background</p> <p>Plant cell cultures have been shown as feasible systems for the production of secondary metabolites, being the elicitation with biotic or abiotic stimuli the most efficient strategy to increase the production of those metabolites. Vitaceae phytoalexins constitute a group of molecules belonging to the stilbene family which are derivatives of the <it>trans</it>-resveratrol structure and are produced by plants and cell cultures as a response to biotic and abiotic stresses. The potential benefits of resveratrol on human health have made it one of the most thoroughly studied phytochemical molecules. The aim of this study was to evaluate the elicitor effect of both cyclodextrin (CD) and methyljasmonate (MeJA) on grapevine cell cultures by carrying out a quantitative analysis of their role on resveratrol production and on the expression of stilbene biosynthetic genes in <it>Vitis vinifera </it>cv Monastrell albino cell suspension cultures.</p> <p>Findings</p> <p>MeJA and CD significantly but transiently induced the expression of stilbene biosynthetic genes when independently used to treat grapevine cells. This expression correlated with resveratrol production in CD-treated cells but not in MeJA-treated cells, which growth was drastically affected. In the combined treatment of CD and MeJA cell growth was similarly affected, however resveratrol production was almost one order of magnitude higher, in correlation with maximum expression values for stilbene biosynthetic genes.</p> <p>Conclusion</p> <p>The effect of MeJA on cell division combined with a true and strong elicitor like CD could be responsible for the observed synergistic effect of both compounds on resveratrol production and on the expression of genes in the stilbene pathway.</p

Production of highly bioactive resveratrol analogues pterostilbene and piceatannol in metabolically engineered grapevine cell cultures

Grapevine stilbenes, particularly trans-resveratrol, have a demonstrated pharmacological activity. Other natural stilbenes derived from resveratrol such as pterostilbene or piceatannol, display higher oral bioavailability and bioactivity than the parent compound, but are far less abundant in natural sources. Thus, to efficiently obtain these bioactive resveratrol derivatives, there is a need to develop new bioproduction systems. Grapevine cell cultures are able to produce large amounts of easily recoverable extracellular resveratrol when elicited with methylated cyclodextrins and methyl jasmonate. We devised this system as an interesting starting point of a metabolic engineering-based strategy to produce resveratrol derivatives using resveratrol-converting enzymes. Constitutive expression of either Vitis vinifera resveratrol O-methyltransferase (VvROMT) or human cytochrome P450 hydroxylase 1B1 (HsCYP1B1) led to pterostilbene or piceatannol, respectively, after the engineered cell cultures were treated with the aforementioned elicitors. Functionality of both gene products was first assessed in planta by Nicotiana benthamiana agroinfiltration assays, in which tobacco cells transiently expressed stilbene synthase and VvROMT or HsCYP1B1. Grapevine cell cultures transformed with VvROMT produced pterostilbene, which was detected in both intra- and extracellular compartments, at a level of micrograms per litre. Grapevine cell cultures transformed with HsCYP1B1 produced about 20 mg/L culture of piceatannol, displaying a sevenfold increase in relation to wild-type cultures, and reaching an extracellular distribution of up to 45% of total production. The results obtained demonstrate the feasibility of this novel system for the bioproduction of natural and more bioactive resveratrol derivatives and suggest new ways for the improvement of production yields.This work has been supported by grants from the Spanish Ministry of Science and Innovation (BIO2011-29856-C02-01, BIO2011-29856-C02-02 and BIO2014-51861-R), Generalitat de Catalunya (2014SGR215) and European Funds for Regional Development (FEDER) and Conselleria d’Educacio, Cultura i Sport de la Generalitat Valenciana (FPA/2013/A/074). J.M.C. acknowledges a postdoctoral and research grants from SENESCYT GOVERNMENT OF ECUADOR (006-IECE-SMG5-GPLR-2012 and Programa1-Senescyt-2014) and a grant from UTEQ (UTEQAmbiental-9-FCAmb-IFOR-2014-FOCICYT002)