Implications of the mesophyll conductance to CO2 for photosynthesis and water-use efficiency during long-term water stress and recovery in two contrasting Eucalyptu species

Water stress (WS) slows growth and photosynthesis (An), but most knowledge comes from short-time studies that do not account for longer term acclimation processes that are especially relevant in tree species. Using two Eucalyptus species that contrast in drought tolerance, we induced moderate and severe water deficits by withholding water until stomatal conductance (gsw) decreased to two pre-defined values for 24 d, WS was maintained at the target gsw for 29 d and then plants were re-watered. Additionally, we developed new equations to simulate the effect on mesophyll conductance (gm) of accounting for the resistance to refixation of CO2. The diffusive limitations to CO2, dominated by the stomata, were the most important constraints to An. Full recovery of An was reached after re-watering, characterized by quick recovery of gm and even higher biochemical capacity, in contrast to the slower recovery of gsw. The acclimation to long-term WS led to decreased mesophyll and biochemical limitations, in contrast to studies in which stress was imposed more rapidly. Finally, we provide evidence that higher gm under WS contributes to higher intrinsic water-use efficiency (iWUE) and reduces the leaf oxidative stress, highlighting the importance of gm as a target for breeding/genetic engineering.

Differences in the oxidative balance of dispersing and non-dispersing individuals : an experimental approach in a passerine bird

Funding This work was supported by grants from the French Ministry of Research (PhD fellowship to CR), the University of Aberdeen (stipend to CR), the CNRS (PICS grant to BD), the L’Oréal Foundation-UNESCO “For Women in Science” program (fellowship to CR), the Région Rhône-Alpes (student mobility grant CMIRA Explora’doc to CR), the Rectors’ Conference of the Swiss Universities (mobility grant to CR), the Fédération de Recherche 41 BioEnvironnement et Santé (training grant to CR), and the Journal of Experimental Biology (travel grant to CR).

Oxidative damage during aging targets mitochondrial aconitase

The mechanisms that cause aging are not well understood. The oxidative stress hypothesis proposes that the changes associated with aging are a consequence of random oxidative damage to biomolecules. We hypothesized that oxidation of specific proteins is critical in controlling the rate of the aging process. Utilizing an immunochemical probe for oxidatively modified proteins, we show that mitochondrial aconitase, an enzyme in the citric acid cycle, is a specific target during aging of the housefly. The oxidative damage detected immunochemically was paralleled by a loss of catalytic activity of aconitase, an enzyme activity that is critical in energy metabolism. Experimental manipulations which decrease aconitase activity should therefore cause a decrease in life-span. This expected decrease was observed when flies were exposed to hyperoxia, which oxidizes aconitase, and when they were given fluoroacetate, an inhibitor of aconitase. The identification of a specific target of oxidative damage during aging allows for the assessment of the physiological age of a specific individual and provides a method for the evaluation of treatments designed to affect the aging process.

Heat Stress Activates Fission Yeast Spc1/StyI MAPK by a MEKK-Independent Mechanism

Fission yeast Spc1/StyI MAPK is activated by many environmental insults including high osmolarity, oxidative stress, and heat shock. Spc1/StyI is activated by Wis1, a MAPK kinase (MEK), which is itself activated by Wik1/Wak1/Wis4, a MEK kinase (MEKK). Spc1/StyI is inactivated by the tyrosine phosphatases Pyp1 and Pyp2. Inhibition of Pyp1 was recently reported to play a crucial role in the oxidative stress and heat shock responses. These conclusions were based on three findings: 1) osmotic, oxidative, and heat stresses activate Spc1/StyI in wis4 cells; 2) oxidative stress and heat shock activate Spc1/StyI in cells that express Wis1AA, in which MEKK consensus phosphorylation sites were replaced with alanine; and 3) Spc1/StyI is maximally activated in Δpyp1 cells. Contrary to these findings, we report: 1) Spc1/StyI activation by osmotic stress is greatly reduced in wis4 cells; 2) wis1-AA and Δwis1 cells have identical phenotypes; and 3) all forms of stress activate Spc1/StyI in Δpyp1 cells. We also report that heat shock, but not osmotic or oxidative stress, activate Spc1 in wis1-DD cells, which express Wis1 protein that has the MEKK consensus phosphorylation sites replaced with aspartic acid. Thus osmotic and oxidative stress activate Spc1/StyI by a MEKK-dependent process, whereas heat shock activates Spc1/StyI by a novel mechanism that does not require MEKK activation or Pyp1 inhibition.

Annexin-like protein from Arabidopsis thaliana rescues delta oxyR mutant of Escherichia coli from H2O2 stress.

Reactive oxygen species are common causes of cellular damages in all aerobic organisms. In Escherichia coli, the oxyR gene product is a positive regulator of the oxyR regulon that is induced in response to H2O2 stress. To identify genes involved in counteracting oxidative stress in plants, we transformed a delta oxyR mutant of E. coli with an Arabidopsis thaliana cDNA library and selected for clones that restored the ability of the delta oxyR mutant to grow in the presence of H2O2. Using this approach, we isolated a cDNA that has strong homology with the annexin super-gene family. The complemented mutant showed higher catalase activity. mRNA expression of the annexin gene in A. thaliana was higher in roots as compared with other organs and was also increased when the plants were exposed to H2O2 stress or salicylic acid. Based on the results presented in this study, we propose a novel physiological role for annexin in counteracting H2O2 stress.

Glutathione-mediated destabilization in vitro of [2Fe-2S] centers in the SoxR regulatory protein.

SoxR is a transcription factor that governs a global defense against the oxidative stress caused by nitric oxide or excess superoxide in Escherichia coli. SoxR is a homodimer containing a pair of [2Fe-2S] clusters essential for its transcriptional activity, and changes in the stability of these metal centers could contribute to the activation or inactivation of SoxR in vivo. Herein we show that reduced glutathione (GSH) in aerobic solution disrupts the SoxR [2Fe-2S] clusters, releasing Fe from the protein and eliminating SoxR transcriptional activity. This disassembly process evidently involves oxygen-derived free radicals. The loss of [2Fe-2S] clusters does not occur in anaerobic solution and is blocked in aerobic solution by the addition of superoxide dismutase and catalase. Although H2O2 or xanthine oxidase and hypoxanthine (to generate superoxide) were insufficient on their own to cause [2Fe-2S] cluster loss, they did accelerate the rate of disassembly after GSH addition. Oxidized GSH alone was ineffective in disrupting the clusters, but the rate of [2Fe-2S] cluster disassembly was maximal when reduced and oxidized GSH were present at a ratio of approximately 1:3, which suggests the critical involvement of a GSH-based free radical in the disassembly process. Such a reaction might occur in vivo: we found that the induction by paraquat of SoxR-dependent soxS transcription was much higher in a GSH-deficient E. coli strain than in its GSH-containing parent. The results imply that GSH may play a significant role during the deactivation process of SoxR in vivo. Ironically, superoxide production seems both to activate SoxR and, in the GSH-dependent disassembly process, to switch off this transcription factor.

Age-related losses of cognitive function and motor skills in mice are associated with oxidative protein damage in the brain.

The hypothesis that age-associated impairment of cognitive and motor functions is due to oxidative molecular damage was tested in the mouse. In a blind study, senescent mice (aged 22 months) were subjected to a battery of behavioral tests for motor and cognitive functions and subsequently assayed for oxidative molecular damage as assessed by protein carbonyl concentration in different regions of the brain. The degree of age-related impairment in each mouse was determined by comparison to a reference group of young mice (aged 4 months) tested concurrently on the behavioral battery. The age-related loss of ability to perform a spatial swim maze task was found to be positively correlated with oxidative molecular damage in the cerebral cortex, whereas age-related loss of motor coordination was correlated with oxidative molecular damage within the cerebellum. These results support the view that oxidative stress is a causal factor in brain senescence. Furthermore, the findings suggest that age-related declines of cognitive and motor performance progress independently, and involve oxidative molecular damage within different regions of the brain.

A stationary-phase stress-response sigma factor from Mycobacterium tuberculosis.

Alternative RNA polymerase sigma factors are a common means of coordinating gene regulation in bacteria. Using PCR amplification with degenerate primers, we identified and cloned a sigma factor gene, sigF, from Mycobacterium tuberculosis. The deduced protein encoded by sigF shows significant similarity to SigF sporulation sigma factors from Streptomyces coelicolor and Bacillus subtilis and to SigB, a stress-response sigma factor, from B. subtilis. Southern blot surveys with a sigF-specific probe identified cross-hybridizing bands in other slow-growing mycobacteria, Mycobacterium bovis bacille Calmette-Guérin (BCG) and Mycobacterium avium, but not in the rapid-growers Mycobacterium smegmatis or Mycobacterium abscessus. RNase protection assays revealed that M. tuberculosis sigF mRNA is not present during exponential-phase growth in M. bovis BCG cultures but is strongly induced during stationary phase, nitrogen depletion, and cold shock. Weak expression of M. tuberculosis sigF was also detected during late-exponential phase, oxidative stress, anaerobiasis, and alcohol shock. The specific expression of M. tuberculosis sigF during stress or stationary phase suggests that it may play a role in the ability of tubercle bacilli to adapt to host defenses and persist during human infection.

Expression of lactoferrin receptors is increased in the mesencephalon of patients with Parkinson disease.

The degeneration of nigral dopaminergic neurons in Parkinson disease is believed to be associated with oxidative stress. Since iron levels are increased in the substantia nigra of parkinsonian patients and this metal catalyzes the formation of free radicals, it may be involved in the mechanisms of nerve cell death. The cause of nigral iron increase is not understood. Iron acquisition by neurons may occur from iron-transferrin complexes with a direct interaction with specific membrane receptors, but recent results have shown a low density of transferrin receptors in the substantia nigra. To investigate whether neuronal death in Parkinson disease may be associated with changes in a pathway supplementary to that of transferrin, lactoferrin (lactotransferrin) receptor expression was studied in the mesencephalon. In this report we present evidence from immunohistochemical staining of postmortem human brain tissue that lactoferrin receptors are localized on neurons (perikarya, dendrites, axons), cerebral microvasculature, and, in some cases, glial cells. In parkinsonian patients, lactoferrin receptor immunoreactivity on neurons and microvessels was increased and more pronounced in those regions of the mesencephalon where the loss of dopaminergic neurons is severe. Moreover, in the substantia nigra, the intensity of immunoreactivity on neurons and microvessels was higher for patients with higher nigral dopaminergic loss. These data suggest that lactoferrin receptors on vulnerable neurons may increase intraneuronal iron levels and contribute to the degeneration of nigral dopaminergic neurons in Parkinson disease.

Mecanismos celulares, stress oxidativo e balanço protease-antiprotease na DPOC : revisão de literatura

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Testing the 'photoinhibition' model of coral bleaching using chemical inhibitors

Explants of the hard coral Seriatopora hystrix were exposed to sublethal concentrations of the herbicide diuron DCMU (N'-(3,4-dichlorophenyl,-N,N-dimethylurea)) and the heavy metal copper. Pulse amplitude modulated (PAM) chlorophyll fluorescence techniques were used to assess the effects on the photosynthetic efficiency of the algal symbionts in the tissue (in Symbio), and chlorophyll fluorescence and counts of symbiotic algae (normalised to surface area) were used to assess the extent of coral bleaching. At 30 mug DCMU l(-1), there was a reduction in both the maximum effective quantum yield (DeltaF/F-m') and maximum potential quantum yield (F-v/F-m) of the algal symbionts in symbio. Corals subsequently lost their algal symbionts and discoloured (bleached), especially on their upper sunlight-exposed surfaces. At the same DCMU concentration but under low light (5% of growth irradiance), there was a marked reduction in DeltaF/F-m' but only a slight reduction in F-v/F-m and slight loss of algae. Loss of algal symbionts was also noted after a 7 d exposure to concentrations as low as 10 mug DCMU l(-1) under normal growth irradiance, and after 14 d exposure to 10 mug DCMU l(-1) under reduced irradiance. Collectively the results indicate that DCMU-induced bleaching is caused by a light-dependent photoinactivation of algal symbionts, and that bleaching occurs when F-v/F-n, (measured 2 h after sunset) is reduced to a value of less than or equal to 0.6. Elevated copper concentrations (60 mug Cu l(-1) for 10 h) also induced a rapid bleaching in S. hystrix but without affecting the quantum yield of the algae in symbio. Tests with isolated algae indicated that substantially higher concentrations (300 mug Cu l(-1) for 8 h) were needed to significantly reduce the quantum yield. Thus, copper-induced bleaching occurs without affecting the algal photosynthesis and may be related to effects on the host (animal). It is argued that warm-water bleaching of corals resembles both types of chemically induced bleaching, suggesting the need for an integrated model of coral bleaching involving the effect of temperature on both host (coral) and algal symbionts.

Short-term peaks in glucose promote renal fibrogenesis independently of total glucose exposure

Postprandial hyperglycemia is implicated as a risk factor predisposing to vascular complications. This study was designed to assess recurrent short-term increases in glucose on markers of renal fibrogenesis. Human renal cortical fibroblasts were exposed to fluctuating short-term (2 h) increases to 15 mM D-glucose, three times a day over 72 h, on a background of 5 mM D-glucose. To determine whether observed changes were due to fluctuating osmolality, identical experiments were undertaken with cells exposed to L-glucose. Parallel experiments were performed in cells exposed to 5 mM D-glucose and constant exposure to either 15 or 7.5 mM D-glucose. Fluctuating D-glucose increased extracellular matrix, as measured by proline incorporation ( P < 0.05), collagen IV ( P < 0.005), and fibronectin production ( P < 0.001), in association with increased tissue inhibitor of matrix metalloproteinase (MMP) ( P < 0.05). Sustained exposure to 15 mM D-glucose increased fibronectin ( P < 0.001), in association with increased MMP-2 ( P = 0.01) and MMP-9 activity ( P < 0.05), suggestive of a protective effect on collagen matrix accumulation. Transforming growth factor-beta(1) (TGF-beta(1)) mRNA was increased after short-term (90 min) exposure to 15 mM glucose (P < 0.05) and after 24-h exposure to 7.5 mM ? ( P < 0.05). Normalization of TGF-beta(1) secretion occurred within 48 h of constant exposure to an elevated glucose. Fluctuating L-glucose also induced TGF-beta(1) mRNA and a profibrotic profile, however, to a lesser extent than observed with exposure to fluctuating D-glucose. The results suggest that exposure to fluctuating glucose concentrations increases renal interstitial fibrosis compared with stable elevations in D-glucose. The effects are, in part, due to the inherent osmotic changes.

Renal and hepatic accumulation of cadmium and lead in the expression of CYP4F2 and CYP2E1

The present study examined accumulation of the metal toxins cadmium (Cd) and lead (Pb) in relation to the abundance of cytochrome P450 4F2 (CYP4F2), CYP2E1 and concentrations of zinc and copper in liver and kidney samples using immunoblotting coupled with metal analysis. The post mortem liver and kidney cortex samples were from 23 males and 8 females aged 3-89 years. All were Caucasians who had not been exposed to metals in the workplace. Average kidney cortex Cd load of 17.4 mu g/g w.w. was 17 times greater than average liver Cd load (1.1 mu g/g w.w.). In contrast, average kidney cortex Ph load of 0.09 mu g/g w.w. was two times lower than liver Pb load of 0.19 mu g/g w.w. Average Zn and Cu concentrations in the kidney cortex samples were 67% and 33% lower than those in the liver. Liver and kidney Cd loads, but not liver or kidney Ph loads, correlated positively with donors' age. After controlling for liver Cd load, an inverse correlation was seen between Zn and age (partial r = -0.39, P = 0.02), suggesting reduction in liver Zn levels in old age. Liver CYP2E1 protein abundance correlated with age-adjusted Cd load (partial r = 0.37, P = 0.02) whereas kidney CYP4172 protein abundance showed a positive correlation with age-adjusted Cd loads (partial r = 0.40, P = 0.02). These findings suggest that Cd may be an inducer of renal CYP4172 and hepatic CYP2E1 and that increased renal CYP4172 expression may implicate in Cd-linked renal tubular dysfunction and high blood pressure, involving CYP4F2-dependent arachidonic acid metabolism. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Rho and vascular disease

The Rho family GTPases are regulatory molecules that link surface receptors to organisation of the actin cytoskeleton and play major roles in fundamental cellular processes. In the vasculature Rho signalling pathways are intimately involved in the regulation of endothelial barrier function, inflammation and transendothelial leukocyte migration, platelet activation, thrombosis and oxidative stress, as well as smooth muscle contraction, migration, proliferation and differentiation, and are thus implicated in many of the changes associated with atherogenesis. Indeed, it is believed that many of the beneficial, non-lipid lowering effects of statins occur as a result of their ability to inhibit Rho protein activation. Conversely, the Rho proteins can have beneficial effects on the vasculature, including the promotion of endothelial repair and the maintenance of SMC differentiation. Further identification of the mechanisms by which these proteins and their effectors act in the vasculature should lead to therapies that specifically target only the adverse effects of Rho signalling. (c) 2005 Elsevier Ireland Ltd. All rights reserved.