Mechanisms and regulation of monosaccharide transport in suspension cultured cells of Vitis Vinifera

Apresentação efectuada no "14th Congress of the Federation of European Societies of Plant Biology", em Cracow, Polland em 2004.Fundação para a Ciência e a Tecnologia (FCT) - grant SFRH/BD/10689/2002

Utilization and transport of mannitol in Olea europaea and their implications on salt stress tolerance

Comunicação em painel no congresso "14th Congress of the Federation of European Societies of Plant Biology". August 23-27. 2004. Cracow. Poland.Fundação para a Ciência e a Tecnologia (FCT

Activation of Type 1 Cannabinoid Receptor (CB1R) promotes neurogenesis in murine subventricular zone cell cultures

The endocannabinoid system has been implicated in the modulation of adult neurogenesis. Here, we describe the effect of type 1 cannabinoid receptor (CB1R) activation on self-renewal, proliferation and neuronal differentiation in mouse neonatal subventricular zone (SVZ) stem/progenitor cell cultures. Expression of CB1R was detected in SVZ-derived immature cells (Nestin-positive), neurons and astrocytes. Stimulation of the CB1R by (R)-(+)-Methanandamide (R-m-AEA) increased self-renewal of SVZ cells, as assessed by counting the number of secondary neurospheres and the number of Sox2+/+ cell pairs, an effect blocked by Notch pathway inhibition. Moreover, R-m-AEA treatment for 48 h, increased proliferation as assessed by BrdU incorporation assay, an effect mediated by activation of MAPK-ERK and AKT pathways. Surprisingly, stimulation of CB1R by R-m-AEA also promoted neuronal differentiation (without affecting glial differentiation), at 7 days, as shown by counting the number of NeuN-positive neurons in the cultures. Moreover, by monitoring intracellular calcium concentrations ([Ca2+](i)) in single cells following KCl and histamine stimuli, a method that allows the functional evaluation of neuronal differentiation, we observed an increase in neuronal-like cells. This proneurogenic effect was blocked when SVZ cells were co-incubated with R-m-AEA and the CB1R antagonist AM 251, for 7 days, thus indicating that this effect involves CB1R activation. In accordance with an effect on neuronal differentiation and maturation, R-m-AEA also increased neurite growth, as evaluated by quantifying and measuring the number of MAP2-positive processes. Taken together, these results demonstrate that CB1R activation induces proliferation, self-renewal and neuronal differentiation from mouse neonatal SVZ cell cultures.Fundacao para a Ciencia e a Tecnologia - Portugal [POCTI/SAU-NEU/68465/2006, PTDC/SAU-NEU/104415/2008, PTDC/SAU-NEU/101783/2008, POCTI/SAU-NEU/110838/2009]; Fundacao Calouste Gulbenkian [96542]; Fundacao para a Ciencia e Tecnologiainfo:eu-repo/semantics/publishedVersio

Mannitol transport and oxidation are synchronized in Olea europaea under salt and drought stresses

The intracellular accumulation of organic compatible solutes acting as osmoprotectants, such as polyols, is an essential response mechanism of several plants to drought and salinity. In Olea europaea a mannitol transport system (OeMaT1) has been previously characterised as a key player in plant response to salinity, and here, heterotrophic sink models, such as olive suspension-cultured cells and fruit tissues, and source leaves were used for analytical, biochemical and molecular studies focused on mannitol metabolism to better understand its involvement in drought and salt stress tolerance. The kinetic parameters of mannitol dehydrogenase (MTD) determined in mannitol-growing cells, at 25 ºC and pH 9.0, were as follows: Km, 54.5 mM mannitol and Vmax, 0.47 µmol h-1 mg-1 protein. The corresponding cDNA was cloned and subsequently named OeMTD1. OeMTD1 expression was correlated with MTD activity, OeMaT1 expression and carrier-mediated mannitol transport, in both mannitol- and sucrose-growing cells. Moreover, sucrose-growing cells showed only residual OeMTD activity, even though high levels of OeMTD1 transcription were observed. The results support that OeMTD is regulated at both transcriptional and post-transcriptional levels due to substrate influence. Remarkably, MTD activity and OeMTD1 expression were dramatically repressed after Na+, K+ and PEG treatments, both in mannitol- and sucrose-growing cells. In contrast, salt and drought significantly increased mannitol transport activity and OeMaT1 expression, thus allowing for the intracellular accumulation of this polyol. Altogether, the results strongly suggest that olive tree copes with drought and salinity by tightly coordinating mannitol transport with intracellular metabolism. Taking into account the protective role of mannitol in olive tree, it would be extremely interesting to investigate if a criterious exogenous addition of this polyol during usual agricultural practices could be beneficial for olive tree development and productivity, and consequently for the whole olive-based industry.This work was supported by the Fundação para a Ciência e Tecnologia (FCT) (research project ref. PTDC/AGR-ALI/100636/2008 and A. Conde grant ref. SFRH/BD/47699/2008

Biochemical changes throughout grape berry development and fruit and wine quality

Wine is made up of more than one thousand compounds, the majority of which, such as vitamins and minerals, come from the grapes, while others, like ethanol and glycerol, are products of the winemaking process. Although sugars are either partially or completely transformed, sugar import and accumulation into the ripening berry is a major parameter of wine quality. Sugar status is directly related to the final alcoholic content of wine, and regulates several genes responsible for the development of its aromatic and organoleptic properties. Physiological ripeness is reached when the grapes achieve sufficiently high sugar levels without loosing too much acidity; however, aromatic and phenolic compound content must also be taken into account. Softening and water content are other essential characteristics of a ripe berry. From a winemaker point of view, optimal grape maturity is essential for wine quality, but is difficult to assess because it is under multifactorial control, involving grapevine cultivar variety and environmental parameters such as soil, temperature, exposure to sun, and hormonal regulation. Continued study of the key control points in grape ripening is crucial if we ultimately hope to improve grape and wine quality.Fundação para a Ciência e a Tecnologia (FCT)(research project ref. POCI/AGR/56378/2004; to C.C., grant ref. SFRH/BD/10689/2002, to P.S. grant ref. SFRH/BD/13460/2003, to N.F. grant ref. SFRH/BD/23169/2005, and to A.A., grant ref. SFRH/PBD/17166/2004

Modulation of subventricular zone oligodendrogenesis: a role for hemopressin?

Neural stem cells (NSCs) from the subventricular zone (SVZ) have been indicated as a source of new oligodendrocytes to use in regenerative medicine for myelin pathologies. Indeed, NSCs are multipotent cells that can self-renew and differentiate into all neural cell types of the central nervous system. In normal conditions, SVZ cells are poorly oligodendrogenic, nevertheless their oligodendrogenic potential is boosted following demyelination. Importantly, progressive restriction into the oligodendrocyte fate is specified by extrinsic and intrinsic factors, endocannabinoids being one of these factors. Although a role for endocannabinoids in oligodendrogenesis has already been foreseen, selective agonists and antagonists of cannabinoids receptors produce severe adverse side effects. Herein, we show that hemopressin (Hp),a modulator of CB1 receptors, increased oligodendroglial differentiation in SVZ neural stem/progenitor cell cultures derived from neonatal mice. The original results presented in this work suggest that Hp and derivates may be of potential interest for the development of future strategies to treat demyelinating diseases

Mannitol transport and mannitol dehydrogenase activities are coordinated in olea europaea under salt and osmotic stresses

This is a pre-copy-editing, author-produced PDF of an article accepted for publication in Plant and Cell Physiology following peer review. The definitive publisher-authenticated version is available online at http://pcp.oxfordjournals.org/cgi/content/abstract/pcr121? ijkey=6orgUM5fkIjedYn&keytype=refThe intracellular accumulation of organic compatible solutes functioning as osmoprotectants, such as polyols, is an important response mechanism of several plants to drought and salinity. In Olea europaea a mannitol transport system (OeMaT1) was previously characterised as a key player in plant response to salinity. In the present study, heterotrophic sink models, such as olive cell suspensions and fruit tissues, and source leaves were used for analytical, biochemical and molecular studies. The kinetic parameters of mannitol dehydrogenase (MTD) determined in mannitol-growing cells, at 25 °C and pH 9.0, were as follows: Km, 54.5 mM mannitol and Vmax, 0.47 μmol h-1 mg-1 protein. The corresponding cDNA was cloned and named OeMTD1. OeMTD1 expression was correlated with MTD activity, OeMaT1 expression and carrier-mediated mannitol transport, in mannitol- and sucrose-growing cells. Furthermore, sucrosegrowing cells displayed only residual OeMTD activity, even though high levels of OeMTD1 transcription were observed. There is evidence OeMTD is regulated at both transcriptional and post-transcriptional levels. MTD activity and OeMTD1 expression were repressed after Na+, K+ and PEG treatments, both in mannitol- and sucrose-growing cells. In contrast, salt and drought significantly increased mannitol transport activity and OeMaT1 expression. Altogether, these studies support that olive tree copes with salinity and drought by coordinating mannitol transport with intracellular metabolism.This work was supported by the Portuguese Foundation for Science and Technology (FCT) (research project ref. PTDC/AGR-ALI/100636/2008; to A. Conde, grant ref. SFRH/BD/47699/2008; to C. Conde, grant ref. SFRH/BPD/34998/2007; to P. Silvagrant ref. SFRH/BD/13460/2003)

Proliferative Hypothalamic Neurospheres Express NPY, AGRP, POMC, CART and Orexin-A and Differentiate to Functional Neurons

Some pathological conditions with feeding pattern alterations, including obesity and Huntington disease (HD) are associated with hypothalamic dysfunction and neuronal cell death. Additionally, the hypothalamus is a neurogenic region with the constitutive capacity to generate new cells of neuronal lineage, in adult rodents

Effect on comfort of administering bubble-humidified or dry oxygen: the Oxyrea non-inferiority randomized study.

The clinical interest of using bubble humidification of oxygen remains controversial. This study was designed to further explore whether delivering dry oxygen instead of bubble-moistened oxygen had an impact on discomfort of ICU patients. This randomized multicenter non-inferiority open trial included patients admitted in intensive care unit and receiving oxygen. Any patient receiving non-humidified oxygen (between 0 and 15 L/min) for less than 2 h could participate in the study. Randomization was stratified based on the flow rate at inclusion (less or more than 4 L/min). Discomfort was assessed 6-8 and 24 h after inclusion using a dedicated 15-item scale (quoted from 0 to 150). Three hundred and fifty-four ICU patients receiving non-humidified oxygen were randomized either in the humidified (HO) (n = 172), using bubble humidifiers, or in the non-humidified (NHO) (n = 182) arms. In modified intention-to-treat analysis at H6-H8, the 15-item score was 26.6 ± 19.4 and 29.8 ± 23.4 in the HO and NHO groups, respectively. The absolute difference between scores in both groups was 3.2 [90% CI 0.0; + 6.5] for a non-inferiority margin of 5.3, meaning that the non-inferiority analysis was not conclusive. This was also true for the subgroups of patients receiving either less or more than 4 L/min of oxygen. At H24, using NHO was not inferior compared to HO in the general population and in the subgroup of patients receiving 4 L/min or less of oxygen. However, for patients receiving more than 4 L/min, a post hoc superiority analysis suggested that patients receiving dry oxygen were less comfortable. Oxygen therapy-related discomfort was low. Dry oxygen could not be demonstrated as non-inferior compared to bubble-moistened oxygen after 6-8 h of oxygen administration. At 24 h, dry oxygen was non-inferior compared to bubble-humidified oxygen for flows below 4 L/min

Proteomic Analysis of Grape Berry Cell Cultures Reveals that Developmentally Regulated Ripening Related Processes Can Be Studied Using Cultured Cells

The original publication is available at http:/www.plosone.orgBackground: This work describes a proteomics profiling method, optimized and applied to berry cell suspensions to evaluate organ-specific cultures as a platform to study grape berry ripening. Variations in berry ripening within a cluster(s) on a vine and in a vineyard are a major impediment towards complete understanding of the functional processes that control ripening, specifically when a characterized and homogenous sample is required. Berry cell suspensions could overcome some of these problems, but their suitability as a model system for berry development and ripening needs to be established first. Methodology/Principal Findings: In this study we report on the proteomic evaluation of the cytosolic proteins obtained from synchronized cell suspension cultures that were established from callus lines originating from green, véraison and ripe Vitis vinifera berry explants. The proteins were separated using liquid phase IEF in a Microrotofor cell and SDS PAGE. This method proved superior to gel-based 2DE. Principal component analysis confirmed that biological and technical repeats grouped tightly and importantly, showed that the proteomes of berry cultures originating from the different growth/ripening stages were distinct. A total of twenty six common bands were selected after band matching between different growth stages and twenty two of these bands were positively identified. Thirty two % of the identified proteins are currently annotated as hypothetical. The differential expression profile of the identified proteins, when compared with published literature on grape berry ripening, suggested common trends in terms of relative abundance in the different developmental stages between real berries and cell suspensions. Conclusions: The advantages of having suspension cultures that accurately mimic specific developmental stages are profound and could significantly contribute to the study of the intricate regulatory and signaling networks responsible for berry development and ripening. © 2011 Sharathchandra et al.Publishers' Versio