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The use of cyclic oligosaccharides like cyclodextrins (CDs), alone or combined with methyl jasmonate (MJ), as elicitors has proved very effective in stimulating the production of trans-resveratrol (trans-R) in Vitis vinifera suspension-cultured cells (SCC). Since elicitors can be used to increase trans-R production, understanding the molecular mechanisms involved would improve the management of grapevine cells as factories of this compound. The results obtained in this study provide evidence for a role of Ca(2+) in mediating elicitor-induced trans-R production in grapevine SCC. The Ca(2+) elevation was promoted by an uptake of Ca(2+) from the extracellular medium, and by Ca(2+) mobilization from intracellular organelles. Moreover, protein phosphorylation/dephosphorylation events seem to be involved in the signal transduction pathways triggered by CDs separately or in combination with MJ since trans-R production is dependent on both, the phosphorylation status of several proteins through mitogen-activated kinase pathway and the activity of tyrosine phosphatases. Our results also suggest that H(2)O(2) and NO participated in the production of trans-R triggered by both elicitors in grapevine SCC. Finally, a fast alkalinization of the extracellular medium is induced in the presence of CDs and/or MJ., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

trans-Resveratrol (trans-R) has been reported to be a potential cancer chemopreventive agent. Although its cytotoxic activity against different cancer cell lines has been tested, its effect on human acute leukemia cell lines has scarcely been investigated, and only a few in vitro studies were performed using human breast epithelial cell lines. Due to its potential value for human health, demand for trans-R has rapidly increased, and new biotechnological strategies to obtain it from natural edible sources have been developed. Thus, grapevine cell cultures represent a reliable system of trans-R production since they biosynthesize trans-R constitutively or in response to elicitation. In addition, there are no studies deepen on the inhibitory effect of trans-R, produced by elicited grapevine cell cultures, on growth of human tumor cell lines. In this work, the effect of trans-R extracted from the culture medium, after elicitation of grapevine cell cultures, was tested on two human acute lymphocytic and monocytic leukemia cell lines, and one human breast cancer cell line. The effect of trans-R on cell proliferation was not only dose- and time-dependent but also cell type-dependent, as seen from the different degrees of susceptibility of cancer cell lines tested. As regards the effect of trans-R on cell cycle distribution, low trans-R concentrations increased cells in the S phase whereas a high trans-R concentration increased G₀/G₁ phase in all cell lines. Perturbation of the cell cycle at low trans-R concentrations did not correlate with the induction of cell death, whereas a high trans-R concentration, cell proliferation decreased as a result of increasing apoptosis in the three cell lines. In leukemia cells, trans-R up-regulated the expression of caspase-3 while trans-R-induced apoptosis in breast cells occur through a caspase-3-independent mechanism mediated by a down-regulation of Bcl-2.

We analyze, for the first time, the early signal transduction pathways triggered by methyl jasmonate (MJ) and cyclodextrins (CDs) in tobacco (Nicotiana tabacum) cell cultures, paying particular attention to changes in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)), the production of hydrogen peroxide (H(2)O(2)) and nitric oxide (NO), and late events like the induction of capsidiol. Our data indicate that MJ and CDs trigger a [Ca(2+)](cyt) rise promoted by Ca(2+) influx through Ca(2+)-permeable channels. The joint presence of MJ and CDs provokes a first increase in [Ca(2+)](cyt) similar to that observed in MJ-treated cells, followed by a second peak similar to that found in the presence of CDs alone. Moreover, oxidative burst induced by MJ is more pronounced when tobacco cells are incubated with CDs alone or in combination with MJ. The presence of both elicitors provokes H(2)O(2) production similar to that found in CD-treated cells, and a sustained response similar to that found in MJ-treated cells. In all treatments, H(2)O(2) production is dependent on Ca(2+) influx and protein phosphorylation events. Similarly, the joint action of both elicitors provokes NO accumulation, although to a lesser extent that in MJ-treated cells because CDs alone do not trigger this accumulation. This NO production is dependent on Ca(2+) influx but independent of both H(2)O(2) production and staurosporine-sensitive phosphorylation events. Taken as a whole, these results suggest the existence of different intracellular signaling pathways for both elicitors. Likewise, CDs might act by regulating the signaling pathway triggered by MJ since, in the presence of both compounds, CDs neutralize the strong oxidative and nitrosative bursts triggered by MJ and therefore, they regulate both H(2)O(2) and NO levels., (Copyright © 2011. Published by Elsevier Masson SAS.)

In this work, the effect of different inducing factors on trans-resveratrol extracellular production in Monastrell grapevine suspension cultured cells is evaluated. A detailed analysis provides the optimal concentrations of cyclodextrins, methyljasmonate and UV irradiation dosage, optimal cell density, elicitation time and sucrose content in the culture media. The results indicate that trans-resveratrol production decreases as the initial cell density increases for a constant elicitor concentration in Monastrell suspension cultured cells treated with cyclodextrins individually or in combination with methyljasmonate; the decrease observed in cell cultures elicited with cyclodextrins alone is far more drastic than those observed in the combined treatment. trans-Resveratrol extracellular production observed by the joint use of cyclodextrins and methyljasmonate (1,447.8 ± 60.4 μmol trans-resveratrol g(-1) dry weight) is lower when these chemical compounds are combined with UV light short exposure (669.9 ± 45.2 μmol trans-resveratrol g(-1) dry weight). Likewise, trans-resveratrol production is dependent on levels of sucrose in the elicitation medium with the maximal levels observed with 20 g l(-1) sucrose and the joint action of cyclodextrins and 100 μM methyljasmonate. The sucrose concentration did not seem to limit the process although it affects significantly the specific productivity since the lowest sucrose concentration is 10 g l(-1), the highest productivity is reached (100.7 ± 5.8 μmol trans-resveratrol g(-1) dry weight g(-1) sucrose) using cyclodextrins and 25 μM methyljasmonate.

Introduction: Bronchogenic cyst is pathology of the respiratory track. It consists of a defect during the embryological development of the tracheobronchial tree. Most common presentation is as a solid or cystic mass located in mediastinum, and it is usually diagnosed in relation to respiratory problems or recurrent infections in children. In adulthood, it is a rare pathology, and its diagnosis is usually incidental., Case Report: We present a case of a patient with a paraesophageal cystic mass suggestive of intraabdominal esophageal duplication cyst but, after the histopathological examination, was discovered to be a bronchogenic cyst, something extremely rare as in most cases of subdiaphragmatic location; bronchogenic cysts appear as retroperitoneal lesions., Discussion: After we review the current literature, surgical extirpation appears to be the treatment of choice due to potential complications, and laparoscopic approach is a feasibily and safe procedure for this pathology up to date.

Introduction: Many risk scores for malignancy in gastrointestinal stromal tumors (GISTs) are based on the combination of size, mitotic index, and location in order to predict recurrence, appearance of metastases, or survival., Design: This is a prospective analysis of prognosis factors (size, mitotic index, Ki-67, and others) and malignancy risk scores (Fletcher's, modified NIH, Miettinen, NCCN, and A or B Goh's scores). This is a study of the sensitivity and specificity of the different malignancy risk scores in the prognosis of recurrence and survival., Materials and Methods: We operated on 52 patients from 1 January 2002 to 10 January 2008. Mean follow-up was 35 months. We used SPSS 13.0 for Windows for the statistical analysis. The differences between frequencies and means were calculated using the chi-square test, Student's t test, and analysis of variance. Kaplan-Meier and the Cox multiple regression methods were used to calculate overall and disease-free survival. Sensitivity and specificity were depicted graphically as receiver operating characteristic (ROC) curves., Results: Thirteen patients suffered recurrences (27.7%). Eleven died from disease-related causes. Two-year and 5-year actuarial survival was 80% and 76%, respectively, and the mean survival was 60 months (CI 95% 51.9-69.5). Two-year and 5-year actuarial disease-free survival was 76% and 72% respectively, and the mean disease-free survival was 52 months (CI 95% 43.5-61.1). The score proposed by Fletcher has the greatest sensitivity and specificity in our series to predict overall survival (AUROC = 0.761; SE 0.084, p = 0.009; CI 95% 0.597-0.926) and recurrence (AUROC RECURRENCE = 0.693; SE 0.087, p = 0.042; CI 95% 0.523-0.864) compared with other scores.

Background: 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 trans-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 Vitis vinifera cv Monastrell albino cell suspension cultures., Findings: 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., Conclusion: 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.

Objective: To analyze the different factors predictive of survival associated with optimal R0-cytoreduction in c-kit-positive gastrointestinal stromal tumors., Methods: Thirty-five patients were operated on in our Oncological Surgery Department from January 2002 to February 2007 because of CD117/c-kit-positive gastrointestinal stromal tumors, and an optimal surgical cytoreduction was obtained without macroscopical residual disease. Demographic, anatomical, clinical, pathological, and immunohistochemical variables were analyzed from a specific database. Survival and multivariate analyses were developed using Kaplan-Meier and multiple Cox regression models, respectively., Results: Five-year overall survival was 77% with a mean survival of 52 months. Risk of malignant behaviour according to Fletcher s classification and tumor size higher than 10 cm had a significantly negative influence on overall survival in the univariate analysis (p < 0.05). Proliferative Ki-67 activity higher than 50% was the only statistically significant variable in the multivariate analysis. Twenty percent of tumors recurred. Only 3 patients with metastatic disease received adjuvant treatment with imatinib mesylate, all of them with Ki-67 > 50% and currently alive., Conclusions: The poliferative Ki-67 index could represent an excellent predictive factor for survival in patients with c-kit-positive stromal gastrointestinal tumors. Confirmation and an adequate cut-off level should be the main objectives for future prospective studies, mostly focused on the appropriate selection of optimal candidates to imatinib-mesylate-based treatment.

In grapevine (Vitis vinifera L.), defense responses after microbial infection or treatment with elicitors involve accumulation of phytoalexins, oxidative burst, and the synthesis of pathogenesis-related proteins. Oligosaccharide fractions from fungal or algal cell walls efficiently induce the defense responses, but a detailed analysis of the elicitor-plant cell surface interaction at the molecular level is precluded by the lack of chemically pure oligosaccharide elicitors. A grapevine liquid cell culture system was used to examine the properties of cyclodextrins (CDs) as inducers of defense responses. This work shows that the chemically pure heptakis(2,6-di-O-methyl)-betaCD caused a dramatic extracellular accumulation of the phytoalexin resveratrol and changes in peroxidase activity and isoenzymatic pattern. Other modified CDs tested on several grapevine cell lines resulted in different eliciting capacities of CDs and different sensibilities of the cell lines. The spent medium of elicited cultures was shown to disturb Botrytis cinerea growth in a plate assay.

The capability of cyclodextrins (CDs) to greatly enhance the solubility in water of poorly water-soluble substances makes them an ideal alternative system for studying the expression of enzyme activity with such substrates in aqueous solution. In order to evaluate the behaviour of the enzymes in presence of CDs a study of the lipoxygenase (LOX)-catalyzed oxidation of polyunsaturated fatty acids (PUFA) as model reaction has been carried out. This was done by using LOX from two different sources (soybean and potato tuber), at two pH values (6.3 and 9.0), with two substrates (linoleic acid and arachidonic acid) and three types of CD (beta-CD, methyl-beta-CD and monoglucosyl-beta-CD). PUFA have been shown to form inclusion complexes of 1:2 stoichiometry which are in equilibrium with free PUFA and free CD, thus complexation is governed by two equilibrium constants, K1 and K2 (J.M. López-Nicolás et al., Biochem. J. 308 (1995) 151-154; R. Bru et al., Colloids Surf. 97 (1995) 263-269). For the oxidation of PUFA by LOX in the presence of beta-CD we propose a model in which free PUFA is the only effective substrate, thus the oxidation of the complexed substrate requires the previous dissociation of the complex. The equilibrium constants of complex formation are determined by both a physico-chemical and an enzymatic method. In spite of giving quite similar results, the second was proven to be more accurate so it was employed in further studies. CD was shown to slow down the reaction rate of LOX, specifically due to the increase of Km, Vmax remaining unchanged. That apparent inhibition is due to removal of effective (free) substrate in the form of inclusion complexes. This 'sequestered' substrate can, however, be converted since it is in equilibrium with the free. The feasibility of realizing a CD-mediated accurate control over the conversion rate is demonstrated in the experiment called 'cyclodextrin assay' in which the concentration of the free substrate is calculated by using the equilibrium constants of complex formation and setting the initial concentrations of total substrate and total CD. From the observation of the reaction progress curves in the conditions of the CD assay, we have studied some characteristic parameters of the oxidation of PUFA by LOX in this new medium, such as enzymatic activity, duration of linear product accumulation and the lag phase.

Using the detergent octaethylene glycol monododecyl ether (C12E8), a spectrophotometric mixed micellar assay for lipoxygenas (LOX) activity was designed. Potato LOX was able to use the linoleic acid (LA) solubilized in C12E8 micelles, displaying the characteristic induction period of LOX-catalyzed LA peroxidation. In the mixed micellar system, LOX responds to the LA surface concentration expressed as mol% (=[lipid]*100/([detergent]-cmc)) and not to the molarity of the LA. For both potato and soybean LOX, Vmax was independent of the mixed micelle concentration, while Km was independent as well when expressed as mol% but was dependent on C12E8 concentration when expressed in molar. In mixed micelles, H2O2 shortened the induction period, while 13-hydroperoxylinoleic acid and t-butyl hydroperoxide completely removed it. C12E8/LA proved to be a reliable system for assaying LOX activity at pH values around neutrality. Like Tween 20, this system avoided the turbidity problems arising from the protonated fatty acid and did not interfere with the uv-absorption band of the hydroperoxide product. However, this system is superior to the commonly used Tween 20 because it permits investigation of the lipid requirements of LOX since the concentration-independent Km can be determined both in mol% and as the absolute number of lipids per micelle. In addition, the detergent did not affect the enzyme through any side effects.

The advantage of aqueous two-phase systems based on polyoxyethylene detergents over other liquid-liquid two-phase systems lies in their capacity to fractionate membrane proteins simply by heating the solution over a biocompatible range of temperatures (20 to 37 degrees C). This permits the peripheral membrane proteins to be effectively separated from the integral membrane proteins, which remain in the detergent-rich phase due to the interaction of their hydrophobic domains with detergent micelles. Since the first reports of this special characteristic of polyoxyethylene glycol detergents in 1981, numerous reports have consolidated this procedure as a fundamental technique in membrane biochemistry and molecular biology. As examples of their use in these two fields, this review summarizes the studies carried out on the topology, diversity, and anomalous behavior of transmembrane proteins on the distribution of glycosyl-phosphatidylinositol-anchored membrane proteins, and on a mechanism to describe the pH-induced translocation of viruses, bacterial endotoxins, and soluble cytoplasmic proteins related to membrane fusion. In addition, the phase separation capacity of these polyoxyethylene glycol detergents has been used to develop quick fractionation methods with high recoveries, on both a micro- and macroscale, and to speed up or increase the efficiency of bioanalytical assays.

The activity of porcine pancreatic elastase has been studied in reverse micelles formed by AOT (sodium bis(2-ethylhexyl) sulfosuccinate) in isooctane. For the two substrates succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide and succinyl-L-Ala-L-Ala-L-Pro-L-Leu-p-nitroanilide, the catalytic constant, kcat, in reverse micelles increases with increasing wo until, at high wo, the value of kcat measured in bulk buffer solution is approached (wo = [H2O/]AOT]). In analogy to alpha-chymotrypsin--and in apparent contrast to many other enzymes--elastase does not show a maximum in the kcat-wo profile. Within the wo range of 8 to 35, for both substrates, the Michaelis constant Km (as expressed relative to the total volume of the solution, Km,overall) increases with increasing wo.

The alpha-chymotrypsin-catalyzed hydrolysis of succinyl-L-alanyl-L-alanyl- L-prolyl-L-phenylalanyl p-nitroanilide has been studied in reverse micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in isooctane. It has been found that alpha-chymotrypsin is strongly inhibited competitively by the acidic peptide product which is formed during the course of the reaction. It has also been shown that the application of the integrated form of the Michaelis-Menten equation can be useful to detect possible inhibition effects and abnormal kinetic behavior of enzymes in reverse micelles. Furthermore, it has been shown that the turnover number (kcat) at low water content is lower than in water and increases as the water content in the system is lower than in water and increases as the water content in the system (wo = [H2O]/[AOT]) increases, kcat reaching the value in water at high wo. If however, initial velocity data, as obtained under conditions where the enzyme is not saturated with substrate, are plotted against wo, the curves are bell-shaped, with a maximum around wo = 15.

Solubilisate 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(6) times. Both reverse micellar size (omega 0 = [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.

Triton X-114 was used to partially purify broad bean polyphenol oxidase, a thylakoid membrane-bound enzyme, in latent form, free of phenolic compounds and chlorophylls, with a high recovery rate. The activation of the latent enzyme by detergents or trypsin was 10 times higher than that obtained when the enzyme was purified by other methods used in plant biochemistry, such as acetone powders and ammonium sulfate fractionation. The kinetic parameters of the latent and activated enzyme are also given.

Polyphenoloxidase from grape berries is extracted only by nonionic detergents with a hydrophilic-lipophilic balance between 12.4 and 13.5. The enzyme was partially purified in latent form, free of phenolics and chlorophylls, by using temperature phase partitioning in a solution of Triton X-114. This method permits the purification of the enzyme with the same fold purification as the commonly used method, but with a yield three times higher and a 90% reduction in time needed. The latent enzyme can be activated by different treatments, including trypsin and cationic and anionic detergents. Cetyltrimethylamonium bromide was found to be the most effective detergent activator, followed by sodium dodecyl sulfate. Polyphenoloxidase in grape berries, in spite of being an integral membrane protein, had an anomalous interaction with Triton X-114, remaining in the detergent-poor phase after phase separation. This could be explained by its having a short hydrophobic tail that anchors it to the membrane.

Isooctane-AOT-H(2)O is a suitable system for studying enzyme behavior in organic solvents. Tyrosinase was able to catalyze a well-known reaction in aqueous medium: oxidation of 4-methylcatechol to yield 4-methyl-o-benzoquinone. This reaction was studied using the preceding ternary system with adequate amounts of each component to make up reverse micelles. 4-Methyl-o-benzoquinone stability was demonstrated in isooctane even at alkaline pH values. Apparent K(m) and V(max) were similar to those in water, but substrate inhibition was more evident. The pH and temperature appear to be shifted toward high and low values, respectively. Characteristic parameters of reverse micelles, omega(0) (= H(2)O/AOT) and percentage of H(2)O (v/v), were investigated. The results obtained showed that the steady-state rate varies either with omega(0) or with percentage of H(2)O. The variation observed with omega(0) showed an optimal value while an increase in percentage of H(2)O can lead to decreased or increased activity depending on substrate concentration.