Identification of chickpea seed proteins resistant to simulated in vitro human digestion.

Proteins and peptides able to resist gastrointestinal digestion and reach the intestinal mucosa have the potential to influence human health. Chickpea (Cicer arietinum L.) seed proteins are able to resist cooking (86.9% total protein) and/or in vitro simulated human digestion (15.9% total protein resists soaking, cooking and digestion with pepsin and pancreatin). To identify and characterize proteins resisting digestion we made use of different MS methodologies. The efficiency of several proteases (trypsin, AspN, chymotrypsin and LysC) was tested, and two technologies were employed (MALDI-MS/MS and LC-nESI-MS/MS). Digestion with trypsin and AspN were most successful for the identification of seed proteins. When analyzed by MALDI- MS/MS, trypsin allowed the identification of at least one protein in 60% of the polypeptide bands, while AspN allows the identification in 48%. The use of LC-nESI-MS/MS, allowed the identification of much more proteins/polypeptides from digested seeds (232 vs 17 using trypsin). The majority of the proteins found to be able to resist simulated digestion were members of the 7S vicilin and 11S legumin seed storage protein classes, which are reported to contain bio-active functions. In addition, we have found proteins that had not yet been described as potentially able to cause an impact on human health. SIGNIFICANCE: This is the first proteomic study to analyze the effect of processing and simulated human gastrointestinal digestion on the proteome of chickpea seed. Chickpea is reported to have anti-nutritional effects as well as nutraceutical properties, so the identification and characterization of the proteins able to resist digestion is crucial to understand the targets underlying such properties.

Controlling stomatal aperture in semi-arid regions-The dilemma of saving water or being cool?

Stomatal regulation of leaf gas exchange with the atmosphere is a key process in plant adaptation to the environment, particularly in semi-arid regions with high atmospheric evaporative demand. Development of stomata, integrating internal signaling and environmental cues sets the limit for maximum diffusive capacity of stomata, through size and density and is under a complex genetic control, thus providing multiple levels of regulation. Operational stomatal conductance to water vapor and CO2 results from feed-back and/or feed-forward mechanisms and is the end-result of a plethora of signals originated in leaves and/or in roots at each moment. CO2 assimilation versus water vapor loss, proposed to be the subject of optimal regulation, is species dependent and defines the water use efficiency (WUE). WUE has been a topic of intense research involving areas from genetics to physiology. In crop plants, especially in semi-arid regions, the question that arises is how the compromise of reducing transpiration to save water will impact on plant performance through leaf temperature. Indeed, plant transpiration by providing evaporative cooling, is a major component of the leaf energy balance. In this paper we discuss the dilemma of 'saving water or being cool' bringing about recent findings from molecular genetics, to development and physiology of stomata. The question of 'how relevant is screening for high/low WUE in crops for semi-arid regions, where drought and heat co-occur' is discussed.

Differential physiological response of the grapevine varieties Touriga Nacional and Trincadeira to combined heat, drought and light stresses.

Worldwide, extensive agricultural losses are attributed to drought, often in combination with heat in Mediterranean climate regions, where grapevine traditionally grows. The available scenarios for climate change suggest increases in aridity in these regions. Under natural conditions plants are affected by a combination of stresses, triggering synergistic or antagonistic physiological, metabolic or transcriptomic responses unique to the combination. However the study of such stresses in a controlled environment can elucidate important mechanisms by allowing the separation of the effects of individual stresses. To gather those effects, cuttings of two grapevine varieties, Touriga Nacional (TN) and Trincadeira (TR), were grown under controlled conditions and subjected to three abiotic stresses (drought - WS, heat - HS and high light - LS) individually and in combination two-by-two (WSHS, WSLS, HSLS) or all three (WSHSLS). Photosynthesis, water status, contents of H2 O2 , abscisic acid and metabolites of the ascorbate-glutathione cycle were measured in the leaves. Common and distinct response features were identified in the different stress combinations. Photosynthesis was not hindered in TN by LS, while even individual stresses severely affect photosynthesis in TR. Abscisic acid may be implicated in grapevine osmotic responses since it is correlated with tolerance parameters, especially in combined stresses involving drought. Overall, the responses to drought-including treatments were clearly distinct to those without drought. From the specific behaviours of the varieties, it can be concluded that TN shows a higher capacity for heat dissipation and for withstanding high light intensities, indicating better adjustment to warm conditions, provided that water supply is plentiful.

Cyclic adenosine monophosphate protects renal cell lines against amphotericin B toxicity in a PKA-independent manner.

Amphotericin B is the "gold standard" agent in the management of serious systemic fungal infections. However, this drug can cause nephrotoxicity, which contributes up to 25% of all acute kidney injuries in critically ill patients. Cyclic adenosine monophosphate can protect kidney cells from death due to injury or drug exposure in some cases. Hence, the objective of this work was to evaluate if cAMP could prevent cell death that occurs in renal cell lines subjected to AmB treatment and, if so, to assess the involvement of PKA in the transduction of this signal. Two different renal cell lines (LLC-PK1 and MDCK) were used in this study. MTT and flow cytometry assays showed increased cell survival when cells were exposed to cAMP in a PKA-independent manner, which was confirmed by western blot. This finding suggests that cAMP (db-cAMP) may prevent cell death caused by exposure to AmB. This is the first time this effect has been identified when renal cells are exposed to AmB's nephrotoxic potential.

Hemocytes of Rhipicephalus sanguineus (Acari: Ixodidae): Characterization, Population Abundance, and Ultrastructural Changes Following Challenge with Leishmania infantum.

Few studies have examined the cellular immune response of ticks, and further research on the characterization of the hemocytes of ticks is required, particularly on those of Rhipicephalus sanguineus (Latreille) because of the medical and veterinary importance of this tick. The aims of this study were to characterize the morphology and the ultrastructure of the different types of hemocytes of adult R. sanguineus and to determine the population abundance and the ultrastructural changes in the hemocytes of ticks infected with Leishmania infantum. The hemocytes were characterized through light and transmission electron microscopy. Within the variability of circulating cells in the hemolymph of adult R. sanguineus, five cell types were identified, which were the prohemocytes, plasmatocytes, granulocytes, spherulocytes, and adipohemocytes. The prohemocytes were the smallest cells found in the hemolymph. The plasmatocytes had polymorphic morphology with vesicles and cytoplasmic projections. The granulocytes had an elliptical shape with the cytoplasm filled with granules of different sizes and electrodensities. The spherulocytes were characterized by several spherules of uniform shapes and sizes that filled the entire cytoplasm, whereas the adipohemocytes had an irregular shape with multiple lipid inclusions that occupied almost the entire cytoplasmic space. The total counts of the hemocyte population increased in the group that was infected with L. infantum. Among the different cell types, the numbers increased and the ultrastructural changes occurred in the granulocytes and the plasmatocytes in the infected group of ticks.

Role of protein kinase A signaling pathway in cyclosporine nephrotoxicity.

Cyclosporine is an important immunosuppressive agent; however, nephrotoxicity is one of the main adverse effects. The purpose of this study was to evaluate the effect of inhibiting the protein kinase A (PKA) signaling pathway in nephrotoxicity caused by cyclosporine from the assessment of cell viability, pro-inflammatory cytokines, and nitric oxide (NO) production in LLC-PK1 and MDCK cell lines. Cyclosporine proved to be cytotoxic for both cell lines, as assessed by the mitochondrial enzyme activity assay (MTT), caused DNA fragmentation, determined by flow cytometry using the propidium iodide dye, and activated the PKA pathway (western blot assay). In MDCK cells, the inhibition of the PKA signaling pathway (H89 inhibitor) caused a significant reduction in DNA fragmentation. In both cell lines, the production of IL-6 proved to be a dependent PKA pathway, while TNF-α was not influenced by the inhibition of the PKA pathway. The NO production was increased when cells were pre-incubated with H89 followed by cyclosporine, and this production was dependent on the PKA pathway in LLC-PK1 and MDCK cells lines. Therefore, considering the present study's results, it can be concluded that the inhibition of PKA signaling pathway can aid in reducing the degree of nephrotoxicity caused by cyclosporine.

Alteration in cellular viability, pro-inflammatory cytokines and nitric oxide production in nephrotoxicity generation by Amphotericin B: involvement of PKA pathway signaling.

Amphotericin B is one of the most effective antifungal agents; however, its use is often limited owing to adverse effects, especially nephrotoxicity. The purpose of this study was to evaluate the effect of inhibiting the PKA signaling pathway in nephrotoxicity using Amphotericin B from the assessment of cell viability, pro-inflammatory cytokines and nitric oxide (NO) production in LLC-PK1 and MDCK cell lines. Amphotericin B proved to be cytotoxic for both cell lines, as assessed by the mitochondrial enzyme activity (MTT) assay; caused DNA fragmentation, determined by flow cytometry using the propidium iodide (PI) dye; and activated the PKA pathway (western blot assay). In MDCK cells, the inhibition of the PKA signaling pathway (using the H89 inhibitor) caused a significant reduction in DNA fragmentation. In both cells lines the production of interleukin-6 (IL)-6 proved to be a dependent PKA pathway, whereas tumor necrosis factor-alpha (TNF-α) was not influenced by the inhibition of the PKA pathway. The NO production was increased when cells were pre-incubated with H89 followed by Amphotericin B, and this production produced a dependent PKA pathway in LLC-PK1 and MDCK cells lines. Therefore, considering the present study's results as a whole, it can be concluded that the inhibition of the PKA signaling pathway can aid in reducing the degree of nephrotoxicity caused by Amphotericin B.

New seminal plasma removal method for freezing stallion semen.

Seminal plasma removal, an indispensable step in equine semen cryopreservation, is usually done by centrifugation, but this might cause mechanical damage to sperm. A new method for seminal plasma removal from stallion semen, namely a filter composed of a synthetic hydrophilic membrane (Sperm Filter, BotuPharma, Botucatu, Sao Paulo, Brazil), was recently proposed. The objective of this study was to test the use of the Sperm Filter in the removal of seminal plasma before freezing stallion semen. Ejaculates from 31 stallions were divided into two groups and cryopreserved. In group 1 (G1), seminal plasma was removed with the Sperm Filter, and in group 2 (G2), seminal plasma was removed by centrifugation (600×g for 10 minutes). There were no differences (P < 0.05) between G1 and G2 in sperm kinetic parameters or plasma membrane integrity before or after cryopreservation. However, sperm recovery rate was higher (P < 0.05) for G1 versus G2 (mean ± SD, 89.4 ± 7.4% vs. 80.9 ± 5.5%). Therefore, the Sperm Filter was as efficient as centrifugation in removing seminal plasma from the stallion ejaculate. However, filtering was more practical and had significantly fewer sperm lost than the centrifugation technique.

TLR4 and RAGE: similar routes leading to inflammation in type 2 diabetic patients.

AIM: The present study investigates the interaction of TLR4 and RAGE with their respective ligands as inducers of the inflammatory markers IL-6 and TNF-α. Also, the reactivity of peripheral blood mononuclear cells (PBMNC) from type 2 diabetic (T2D) patients and non-diabetic healthy controls (ND) were comparatively studied. METHODS: Concentrations of IL-6 and TNF-α were measured by sandwich Elisa, using kits supplied by Assay Designs (Ann Arbor, MI, USA). PBMNC from T2D and ND were incubated in the presence or absence of LPS, anti-TLR4 or anti-RAGE for 72 hours at 37°C under 5% CO(2). The final volume was adjusted to 300 µL in DMEM supplemented with 10% fetal bovine serum. After incubation, the cells were centrifuged, the supernatant collected and the cytokines measured. RESULTS: PBMNC from T2D were more sensitive to innate immune stimulation with LPS and monoclonal agonist anti-TLR4 than were cells from ND. The actions of LPS, anti-TLR4 and anti-RAGE potentiated the production of IL-6 and TNF-α in both groups. The simultaneous activation of monoclonal anti-RAGE and anti-TLR4 suggests that both antibodies used different receptors on the cell surface, but converged on the same PBMNC signaling metabolic pathways. This simultaneous activation induced a higher production of IL-6 and TNF-α in PBMNC from the T2D patients than from the ND subjects. CONCLUSION: Our results clearly show an exacerbation of innate immunity in PBMNC with T2D that was possibly hyperglycaemia-induced. These data, when analyzed together, suggest the importance of innate immunity in the pathogenesis of T2D.

Colchicine inhibits cationic dye uptake induced by ATP in P2X2 and P2X7 receptor-expressing cells: implications for its therapeutic action.

BACKGROUND AND PURPOSE: The two longest C-termini of the purinergic P2X receptors occur in the P2X2 and P2X7 receptors and are thought to interact with multiple cytoplasmic proteins, among which are members of the cytoskeleton, including microtubules. In this work we asked whether disrupting the microtubule cytoskeleton might affect the functions of these receptors. EXPERIMENTAL APPROACH: Functions of heterologously expressed P2X2 and P2X7 receptors were evaluated with electrophysiology and dye uptake following ATP application. Permeabilization and secretion of pro-inflammatory agents were quantified from fresh or cultured peritoneal mouse macrophages, treated in vitro or in vivo with colchicine. KEY RESULTS: Disrupting the microtubule network with colchicine did not affect currents generated by ATP in P2X2 and P2X7 receptor-expressing cells but inhibited uptake of the dye Yo-Pro-1 in Xenopus oocytes and HEK293 cells expressing these channels. Peritoneal mouse macrophages showed less ATP-induced permeabilization to ethidium bromide in the presence of colchicine, and less reactive oxygen species (ROS) formation, nitric oxide (NO) and interleukin (IL)-1ß release. Colchicine treatment did not affect ATP-evoked currents in macrophages. Finally, in vivo assays with mice inoculated with lipopolysaccharide and ATP showed diminished ROS, IL-1ß, interferon-γ and NO production after colchicine treatment. CONCLUSIONS AND IMPLICATIONS: Colchicine has known anti-inflammatory actions and is used to treat several conditions involving innate immunity, including gout and familial Mediterranean fever. Here we propose a new mechanism of action - inhibition of pore formation induced by activation of P2X receptors - which could explain some of the anti-inflammatory effects of colchicine.

Photosynthesis and drought: can we make metabolic connections from available data?

Photosynthesis is one of the key processes to be affected by water deficits, via decreased CO2 diffusion to the chloroplast and metabolic constraints. The relative impact of those limitations varies with the intensity of the stress, the occurrence (or not) of superimposed stresses, and the species we are dealing with. Total plant carbon uptake is further reduced due to the concomitant or even earlier inhibition of growth. Leaf carbohydrate status, altered directly by water deficits or indirectly (via decreased growth), acts as a metabolic signal although its role is not totally clear. Other relevant signals acting under water deficits comprise: abscisic acid (ABA), with an impact on stomatal aperture and the regulation at the transcription level of a large number of genes related to plant stress response; other hormones that act either concurrently (brassinosteroids, jasmonates, and salycilic acid) or antagonistically (auxin, cytokinin, or ethylene) with ABA; and redox control of the energy balance of photosynthetic cells deprived of CO2 by stomatal closure. In an attempt to systematize current knowledge on the complex network of interactions and regulation of photosynthesis in plants subjected to water deficits, a meta-analysis has been performed covering >450 papers published in the last 15 years. This analysis shows the interplay of sugars, reactive oxygen species (ROS), and hormones with photosynthetic responses to drought, involving many metabolic events. However, more significantly it highlights (i) how fragmented and often non-comparable the results are and (ii) how hard it is to relate molecular events to plant physiological status, namely photosynthetic activity, and to stress intensity. Indeed, the same data set usually does not integrate these different levels of analysis. Considering these limitations, it was hard to find a general trend, particularly concerning molecular responses to drought, with the exception of the genes ABI1 and ABI3. These genes, irrespective of the stress type (acute versus chronic) and intensity, show a similar response to water shortage in the two plant systems analysed (Arabidopsis and barley). Both are associated with ABA-mediated metabolic responses to stress and the regulation of stomatal aperture. Under drought, ABI1 transcription is up-regulated while ABI3 is usually down-regulated. Recently ABI3 has been hypothesized to be essential for successful drought recovery.

Drought-induced photosynthetic inhibition and autumn recovery in two Mediterranean oak species (Quercus ilex and Quercus suber).

Responses of leaf water relations and photosynthesis to summer drought and autumn rewetting were studied in two evergreen Mediterranean oak species, Quercus ilex spp. rotundifolia and Quercus suber. The predawn leaf water potential (Ψ(lPD)), stomatal conductance (gs) and photosynthetic rate (A) at ambient conditions were measured seasonally over a 3-year period. We also measured the photosynthetic response to light and to intercellular CO2 (A/PPFD and A/C(i) response curves) under water stress (summer) and after recovery due to autumn rainfall. Photosynthetic parameters, Vc(max), J(max) and triose phosphate utilization (TPU) rate, were estimated using the Farquhar model. RuBisCo activity, leaf chlorophyll, leaf nitrogen concentration and leaf carbohydrate concentration were also measured. All measurements were performed in the spring leaves of the current year. In both species, the predawn leaf water potential, stomatal conductance and photosynthetic rate peaked in spring, progressively declined throughout the summer and recovered upon autumn rainfall. During the drought period, Q. ilex maintained a higher predawn leaf water potential and stomatal conductance than Q. suber. During this period, we found that photosynthesis was not only limited by stomatal closure, but was also downregulated as a consequence of a decrease in the maximum carboxylation rate (Vc(max)) and the light-saturated rate of photosynthetic electron transport (J(max)) in both species. The Vc(max) and J(max) increased after the first autumnal rains and this increase was related to RuBisCo activity, leaf nitrogen concentration and chlorophyll concentration. In addition, an increase in the TPU rate and in soluble leaf sugar concentration was observed in this period. The results obtained indicate a high resilience of the photosynthetic apparatus to summer drought as well as good recovery in the following autumn rains of these evergreen oak species.

Shifts in plant respiration and carbon use efficiency at a large-scale drought experiment in the eastern Amazon.

*The effects of drought on the Amazon rainforest are potentially large but remain poorly understood. Here, carbon (C) cycling after 5 yr of a large-scale through-fall exclusion (TFE) experiment excluding about 50% of incident rainfall from an eastern Amazon rainforest was compared with a nearby control plot. *Principal C stocks and fluxes were intensively measured in 2005. Additional minor components were either quantified in later site measurements or derived from the available literature. *Total ecosystem respiration (R(eco)) and total plant C expenditure (PCE, the sum of net primary productivity (NPP) and autotrophic respiration (R(auto))), were elevated on the TFE plot relative to the control. The increase in PCE and R(eco) was mainly caused by a rise in R(auto) from foliage and roots. Heterotrophic respiration did not differ substantially between plots. NPP was 2.4 +/- 1.4 t C ha(-1) yr(-1) lower on the TFE than the control. Ecosystem carbon use efficiency, the proportion of PCE invested in NPP, was lower in the TFE plot (0.24 +/- 0.04) than in the control (0.32 +/- 0.04). *Drought caused by the TFE treatment appeared to drive fundamental shifts in ecosystem C cycling with potentially important consequences for long-term forest C storage.

Grapevine under deficit irrigation: hints from physiological and molecular data.

BACKGROUND: A large proportion of vineyards are located in regions with seasonal drought (e.g. Mediterranean-type climates) where soil and atmospheric water deficits, together with high temperatures, exert large constraints on yield and quality. The increasing demand for vineyard irrigation requires an improvement in the efficiency of water use. Deficit irrigation has emerged as a potential strategy to allow crops to withstand mild water stress with little or no decreases of yield, and potentially a positive impact on fruit quality. Understanding the physiological and molecular bases of grapevine responses to mild to moderate water deficits is fundamental to optimize deficit irrigation management and identify the most suitable varieties to those conditions. SCOPE: How the whole plant acclimatizes to water scarcity and how short- and long-distance chemical and hydraulic signals intervene are reviewed. Chemical compounds synthesized in drying roots are shown to act as long-distance signals inducing leaf stomatal closure and/or restricting leaf growth. This explains why some plants endure soil drying without significant changes in shoot water status. The control of plant water potential by stomatal aperture via feed-forward mechanisms is associated with 'isohydric' behaviour in contrast to 'anysohydric' behaviour in which lower plant water potentials are attained. This review discusses differences in this respect between grapevines varieties and experimental conditions. Mild water deficits also exert direct and/or indirect (via the light environment around grape clusters) effects on berry development and composition; a higher content of skin-based constituents (e.g. tannins and anthocyanins) has generally being reported. Regulation under water deficit of genes and proteins of the various metabolic pathways responsible for berry composition and therefore wine quality are reviewed.

Acclimation to short-term low temperatures in two Eucalyptus globulus clones with contrasting drought resistance.

We tested the hypothesis that Eucalyptus globulus Labill. genotypes that are more resistant to dry environments might also exhibit higher cold tolerances than drought-sensitive plants. The effect of low temperatures was evaluated in acclimated and unacclimated ramets of a drought-resistant clone (CN5) and a drought-sensitive clone (ST51) of E. globulus. We studied the plants' response via leaf gas exchanges, leaf water and osmotic potentials, concentrations of soluble sugars, several antioxidant enzymes and leaf electrolyte leakage. Progressively lowering air temperatures (from 24/16 to 10/-2 degrees C, day/night) led to acclimation of both clones. Acclimated ramets exhibited higher photosynthetic rates, stomatal conductances and lower membrane relative injuries when compared to unacclimated ramets. Moreover, low temperatures led to significant increases of soluble sugars and antioxidant enzymes activity (glutathione reductase, ascorbate peroxidase and superoxide dismutases) of both clones in comparison to plants grown at control temperature (24/16 degrees C). On the other hand, none of the clones, either acclimated or not, exhibited signs of photoinhibition under low temperatures and moderate light. The main differences in the responses to low temperatures between the two clones resulted mainly from differences in carbon metabolism, including a higher accumulation of soluble sugars in the drought-resistant clone CN5 as well as a higher capacity for osmotic regulation, as compared to the drought-sensitive clone ST51. Although membrane injury data suggested that both clones had the same inherent freezing tolerance before and after cold acclimation, the results also support the hypothesis that the drought-resistant clone had a greater cold tolerance at intermediate levels of acclimation than the drought-sensitive clone. A higher capacity to acclimate in a short period can allow a clone to maintain an undamaged leaf surface area along sudden frost events, increasing growth capacity. Moreover, it can enhance survival chances in frost-prone sites expanding the plantation range with more adaptive clones.

CO2 efflux, CO2 concentration and photosynthetic refixation in stems of Eucalyptus globulus (Labill.).

In spite of the importance of respiration in forest carbon budgets, the mechanisms by which physiological factors control stem respiration are unclear. An experiment was set up in a Eucalyptus globulus plantation in central Portugal with monoculture stands of 5-year-old and 10-year-old trees. CO(2) efflux from stems under shaded and unshaded conditions, as well as the concentration of CO(2) dissolved in sap [CO(2)(*)], stem temperature, and sap flow were measured with the objective of improving our understanding of the factors controlling CO(2) release from stems of E. globulus. CO(2) efflux was consistently higher in 5-year-old, compared with 10-year-old, stems, averaging 3.4 versus 1.3 mumol m(-2) s(-1), respectively. Temperature and [CO(2)(*)] both had important, and similar, influences on the rate of CO(2) efflux from the stems, but neither explained the difference in the magnitude of CO(2) efflux between trees of different age and size. No relationship was found between efflux and sap flow, and efflux was independent of tree volume, suggesting the presence of substantial barriers to the diffusion of CO(2) from the xylem to the atmosphere in this species. The rate of corticular photosynthesis was the same in trees of both ages and only reduced CO(2) efflux by 7%, probably due to the low irradiance at the stem surface below the canopy. The younger trees were growing at a much faster rate than the older trees. The difference between CO(2) efflux from the younger and older stems appears to have resulted from a difference in growth respiration rather than a difference in the rate of diffusion of xylem-transported CO(2).

Photosynthesis under drought and salt stress: regulation mechanisms from whole plant to cell.

BACKGROUND: Plants are often subjected to periods of soil and atmospheric water deficits during their life cycle as well as, in many areas of the globe, to high soil salinity. Understanding how plants respond to drought, salt and co-occurring stresses can play a major role in stabilizing crop performance under drought and saline conditions and in the protection of natural vegetation. Photosynthesis, together with cell growth, is among the primary processes to be affected by water or salt stress. SCOPE: The effects of drought and salt stresses on photosynthesis are either direct (as the diffusion limitations through the stomata and the mesophyll and the alterations in photosynthetic metabolism) or secondary, such as the oxidative stress arising from the superimposition of multiple stresses. The carbon balance of a plant during a period of salt/water stress and recovery may depend as much on the velocity and degree of photosynthetic recovery, as it depends on the degree and velocity of photosynthesis decline during water depletion. Current knowledge about physiological limitations to photosynthetic recovery after different intensities of water and salt stress is still scarce. From the large amount of data available on transcript-profiling studies in plants subjected to drought and salt it is becoming apparent that plants perceive and respond to these stresses by quickly altering gene expression in parallel with physiological and biochemical alterations; this occurs even under mild to moderate stress conditions. From a recent comprehensive study that compared salt and drought stress it is apparent that both stresses led to down-regulation of some photosynthetic genes, with most of the changes being small (ratio threshold lower than 1) possibly reflecting the mild stress imposed. When compared with drought, salt stress affected more genes and more intensely, possibly reflecting the combined effects of dehydration and osmotic stress in salt-stressed plants.

Inhibition of ROS production in peripheral blood mononuclear cells from type 2 diabetic patients by autologous plasma depends on Akt/PKB signaling pathway.

OBJECTIVE: To compare the role of Akt/PKB signaling pathway in the modulation of reactive oxygen species (ROS) production by autologous plasma in peripheral blood mononuclear cells (PBMNC) from type 2 diabetic patients and healthy subjects. MATERIALS AND METHODS: This study was approved by Santa Casa Ethical Committee and has included patients diagnosed with diabetes type 2 (DM2) and control group (non-diabetic) (ND). PBMNC were purified utilizing Ficoll-hypaque gradient. ROS was quantified by luminol-dependent chemiluminescence. The Akt/PKB phosphorylation was measured using a CASE kit. Statistical analyses were made with t Student test and chi-square (chi(2)). p<0.05 was considered significant. RESULTS: 12, 13-Phorbol dibutyrate (PDB) stimulated the production of higher levels of ROS in PBMNC from type 2 diabetic patients than that from healthy subjects. Autologous plasma, however, inhibited induced or not ROS production in PBMNC in both groups. The inhibition of PBMNC-ROS derived by autologous plasma from healthy subjects was higher than that from type 2 diabetic patients. Plasma phosphorylated (activated) Akt/PKB. The percentage of phosphorylation induced by autologous plasma in PBMNC from patients and healthy control were 14% and 93%, respectively. Inhibition of ROS production in PBMNC from DM2 were similar for PBMNC+plasma; PBMNC+Akti; and PBMNC+plasma+Akti. However, in ND control, plasma showed a higher ROS inhibition than Akti or plasma plus Akti. CONCLUSIONS: Our results suggest that the low antioxidant capacity observed in autologous plasma from DM2 patients in conjunction with the decreased activation of PKB may cause an imbalance in the oxidizing/reducing responses, possible inducing an oxidative stress state, which could be associated with tissular damage.

Water-use strategies in two co-occurring Mediterranean evergreen oaks: surviving the summer drought.

In the Mediterranean evergreen oak woodlands of southern Portugal, the main tree species are Quercus ilex ssp. rotundifolia Lam. (holm oak) and Quercus suber L. (cork oak). We studied a savannah-type woodland where these species coexist, with the aim of better understanding the mechanisms of tree adaptation to seasonal drought. In both species, seasonal variations in transpiration and predawn leaf water potential showed a maximum in spring followed by a decline through the rainless summer and a recovery with autumn rainfall. Although the observed decrease in predawn leaf water potential in summer indicates soil water depletion, trees maintained transpiration rates above 0.7 mm day(-1) during the summer drought. By that time, more than 70% of the transpired water was being taken from groundwater sources. The daily fluctuations in soil water content suggest that some root uptake of groundwater was mediated through the upper soil layers by hydraulic lift. During the dry season, Q. ilex maintained higher predawn leaf water potentials, canopy conductances and transpiration rates than Q. suber. The higher water status of Q. ilex was likely associated with their deeper root systems compared with Q. suber. Whole-tree hydraulic conductance and minimum midday leaf water potential were lower in Q. ilex, indicating that Q. ilex was more tolerant to drought than Q. suber. Overall, Q. ilex seemed to have more effective drought avoidance and drought tolerance mechanisms than Q. suber.

Modulation of the production of reactive oxygen species (ROS) by cAMP-elevating agents in granulocytes from diabetic patients: an Akt/PKB-dependent phenomenon.

BACKGROUND: Granulocytes from healthy subjects and from patients suffering from diabetes mellitus present differences in reactivity to stimulation with cyclic nucleotide-elevating agents. The production of reactive oxygen species (ROS) is inhibited in cells from non-diabetic subjects following such stimulation, but activated through a PKA-independent signaling pathway in granulocytes from type 1 and type 2 diabetic patients. The aim of the present study was to understand better the changes in signaling mechanisms induced by the disease. METHODS: ROS production in granulocytes from healthy subjects and from type 1 and type 2 diabetic patients was measured using a luminol-dependent chemiluminescence assay. Granulocytes were stimulated by the addition of the cAMP-elevating agent dibutyryl cAMP. In some experiments, granulocytes were pre-treated with an inhibitor of PKA or Akt/PKB prior to cAMP stimulation. RESULTS: Intracellular elevation of cAMP induced a PKA-dependent and Akt/PKB-independent inhibition of ROS production in granulocytes from healthy subjects, but a significant activation in cells from both type 1 and type 2 diabetic patients. Most significantly, activation of ROS generation in cells from diabetic patients was shown to be Akt/PKB-dependent and PKA-independent. CONCLUSIONS: These results suggest that chronic hyperglycaemia could induce metabolic adaptation in cAMP-related signaling mechanisms. Epac (exchange protein directly activated by cAMP) is a novel cAMP receptor besides PKA involved in different signaling pathways. The cAMP-stimulated inverse ROS response in granulocytes from type 1 and type 2 diabetic patients may be due to a change in signaling pathways from cAMP/PKA to cAMP/Epac/Akt/PKB. These preliminary results require further studies in order to evaluate their consequences on innate immunity and pathogenesis of diabetes mellitus.