Fusicoccin Counteracts the n-Ethylmaleimide and Silver-Induced Stimulation of Oxygen Uptake in Egeria densa Leaves1

It was previously shown that a number of sulfhydryl [SH] group reagents (N-ethylmaleimide [NEM], iodoacetate, Ag+, HgCl2, etc.) can induce a marked, transitory stimulation of O2 uptake (QO2) in Egeria densa leaves, insensitive to CN− and salicylhydroxamic acid and inhibited by diphenylene iodonium and quinacrine. The phytotoxin fusicoccin (FC) also induces a marked increase in O2 consumption in E. densa leaves, apparently independent of the recognized stimulating action on the H+-ATPase. In this investigation we compared the FC-induced increase in O2 consumption with those induced by NEM and Ag+, and we tested for a possible interaction between FC and the two SH blockers in the activation of QO2. The results show (a) the different nature of the FC- and NEM- or Ag+-induced increases of QO2; (b) that FC counteracts the NEM- (and Ag+)-induced respiratory burst; and (c) that FC strongly reduces the damaging effects on plasma membrane permeability observed in E. densa leaves treated with the two SH reagents. Two alternative models of interpretation of the action of FC, in activating a CN−-sensitive respiratory pathway and in suppressing the SH blocker-induced respiratory burst, are proposed.

Identification of a Role for an Azide-Sensitive Factor in the Thylakoid Transport of the 17-Kilodalton Subunit of the Photosynthetic Oxygen-Evolving Complex1

We have examined the transport of the precursor of the 17-kD subunit of the photosynthetic O2-evolving complex (OE17) in intact chloroplasts in the presence of inhibitors that block two protein-translocation pathways in the thylakoid membrane. This precursor uses the transmembrane pH gradient-dependent pathway into the thylakoid lumen, and its transport across the thylakoid membrane is thought to be independent of ATP and the chloroplast SecA homolog, cpSecA. We unexpectedly found that azide, widely considered to be an inhibitor of cpSecA, had a profound effect on the targeting of the photosynthetic OE17 to the thylakoid lumen. By itself, azide caused a significant fraction of mature OE17 to accumulate in the stroma of intact chloroplasts. When added in conjunction with the protonophore nigericin, azide caused the maturation of a fraction of the stromal intermediate form of OE17, and this mature protein was found only in the stroma. Our data suggest that OE17 may use the sec-dependent pathway, especially when the transmembrane pH gradient-dependent pathway is inhibited. Under certain conditions, OE17 may be inserted across the thylakoid membrane far enough to allow removal of the transit peptide, but then may slip back out of the translocation machinery into the stromal compartment.

Oxygen Requirement and Inhibition of C4 Photosynthesis1 : An Analysis of C4 Plants Deficient in the C3 and C4 Cycles

The basis for O2 sensitivity of C4 photosynthesis was evaluated using a C4-cycle-limited mutant of Amaranthus edulis (a phosphoenolpyruvate carboxylase-deficient mutant), and a C3-cycle-limited transformant of Flaveria bidentis (an antisense ribulose-1,5-bisphosphate carboxylase/oxygenase [Rubisco] small subunit transformant). Data obtained with the C4-cycle-limited mutant showed that atmospheric levels of O2 (20 kPa) caused increased inhibition of photosynthesis as a result of higher levels of photorespiration. The optimal O2 partial pressure for photosynthesis was reduced from approximately 5 kPa O2 to 1 to 2 kPa O2, becoming similar to that of C3 plants. Therefore, the higher O2 requirement for optimal C4 photosynthesis is specifically associated with the C4 function. With the Rubisco-limited F. bidentis, there was less inhibition of photosynthesis by supraoptimal levels of O2 than in the wild type. When CO2 fixation by Rubisco is limited, an increase in the CO2 concentration in bundle-sheath cells via the C4 cycle may further reduce the oxygenase activity of Rubisco and decrease the inhibition of photosynthesis by high partial pressures of O2 while increasing CO2 leakage and overcycling of the C4 pathway. These results indicate that in C4 plants the investment in the C3 and C4 cycles must be balanced for maximum efficiency.

Reactive oxygen species are downstream mediators of p53-dependent apoptosis.

Reactive oxygen species (ROS) have been implicated as potential modulators of apoptosis. Conversely, experiments under hypoxic conditions have suggested that apoptosis could occur in the absence of ROS. We sought to determine whether a central modulator of apoptosis, p53, regulates the levels of intracellular ROS and whether a rise in ROS levels is required for the induction of p53-dependent apoptosis. We transiently overexpressed wild-type p53, using adenoviral gene transfer, and identified cell types that were sensitive or resistant to p53-mediated apoptosis. Cells sensitive to p53-mediated apoptosis produced ROS concomitantly with p53 overexpression, whereas cells resistant to p53 failed to produce ROS. In sensitive cells, both ROS production and apoptosis were inhibited by antioxidant treatment. These results suggest that p53 acts to regulate the intracellular redox state and induces apoptosis by a pathway that is dependent on ROS production.

Unifying theory of hypoxia tolerance: molecular/metabolic defense and rescue mechanisms for surviving oxygen lack.

We develop a unifying theory of hypoxia tolerance based on information from two cell level models (brain cortical cells and isolated hepatocytes) from the highly anoxia tolerant aquatic turtle and from other more hypoxia sensitive systems. We propose that the response of hypoxia tolerant systems to oxygen lack occurs in two phases (defense and rescue). The first lines of defense against hypoxia include a balanced suppression of ATP-demand and ATP-supply pathways; this regulation stabilizes (adenylates) at new steady-state levels even while ATP turnover rates greatly decline. The ATP demands of ion pumping are down-regulated by generalized "channel" arrest in hepatocytes and by "spike" arrest in neurons. Hypoxic ATP demands of protein synthesis are down-regulated probably by translational arrest. In hypoxia sensitive cells this translational arrest seems irreversible, but hypoxia-tolerant systems activate "rescue" mechanisms if the period of oxygen lack is extended by preferentially regulating the expression of several proteins. In these cells, a cascade of processes underpinning hypoxia rescue and defense begins with an oxygen sensor (a heme protein) and a signal-transduction pathway, which leads to significant gene-based metabolic reprogramming-the rescue process-with maintained down-regulation of energy-demand and energy-supply pathways in metabolism throughout the hypoxic period. This recent work begins to clarify how normoxic maintenance ATP turnover rates can be drastically (10-fold) down-regulated to a new hypometabolic steady state, which is prerequisite for surviving prolonged hypoxia or anoxia. The implications of these developments are extensive in biology and medicine.

Generation of cell-to-cell signals in quorum sensing: acyl homoserine lactone synthase activity of a purified Vibrio fischeri LuxI protein.

Many bacteria use acyl homoserine lactone signals to monitor cell density in a type of gene regulation termed quorum sensing and response. Synthesis of these signals is directed by homologs of the luxi gene of Vibrio fischeri. This communication resolves two critical issues concerning the synthesis of the V. fischeri signal. (i) The luxI product is directly involved in signal synthesis-the protein is an acyl homoserine lactone synthase; and (ii) the substrates for acyl homoserine lactone synthesis are not amino acids from biosynthetic pathways or fatty acid degradation products, but rather they are S-adenosylmethionine (SAM) and an acylated acyl carrier protein (ACP) from the fatty acid biosynthesis pathway. We purified a maltose binding protein-LuxI fusion polypeptide and showed that, when provided with the appropriate substrates, it catalyzes the synthesis of an acyl homoserine lactone. In V. fischeri, luxi directs the synthesis of N-(3-oxohexanoyl) homoserine lactone and hexanoyl homoserine lactone. The purified maltose binding protein-LuxI fusion protein catalyzes the synthesis of hexanoyl homoserine lactone from hexanoyl-ACP and SAM. There is a high level of specificity for hexanoyl-ACP over ACPs with differing acyl group lengths, and hexanoyl homoserine lactone was not synthesized when SAM was replaced with other amino acids, such as methionine, S-adenosylhomocysteine, homoserine, or homoserine lactone, or when hexanoyl-SAM was provided as the substrate. This provides direct evidence that the LuxI protein is an auto-inducer synthase that catalyzes the formation of an amide bond between SAM and a fatty acyl-ACP and then catalyzes the formation of the acyl homoserine lactone from the acyl-SAM intermediate.

An Arginine Deprivation Response Pathway Is Induced in Leishmania during Macrophage Invasion

Amino acid sensing is an intracellular function that supports nutrient homeostasis, largely through controlled release of amino acids from lysosomal pools. The intracellular pathogen Leishmania resides and proliferates within human macrophage phagolysosomes. Here we describe a new pathway in Leishmania that specifically senses the extracellular levels of arginine, an amino acid that is essential for the parasite. During infection, the macrophage arginine pool is depleted due to its use to produce metabolites (NO and polyamines) that constitute part of the host defense response and its suppression, respectively. We found that parasites respond to this shortage of arginine by up-regulating expression and activity of the Leishmania arginine transporter (LdAAP3), as well as several other transporters. Our analysis indicates the parasite monitors arginine levels in the environment rather than the intracellular pools. Phosphoproteomics and genetic analysis indicates that the arginine-deprivation response is mediated through a mitogen-activated protein kinase-2-dependent signaling cascade.

Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to the development of pulmonary arterial hypertension

Aims: Pulmonary arterial hypertension [1] is a proliferative disorder associated with enhanced proliferation and suppressed apoptosis of pulmonary artery smooth muscle cells (PASMCs). Reactive oxygen species (ROS) is implicated in the development of PAH and regulates the vascular tone and functions. However, which cellular signaling mechanisms are triggered by ROS in PAH is still unknown. Hence, here we wished to characterize the signaling mechanisms triggered by ROS. Methods and Results: By Western blots, we showed that increased intracellular ROS caused inhibition of the glycolytic pyruvate kinase M2 (PKM2) activity through promoting the phosphorylation of PKM2. Monocrotaline (MCT)-induced rats developed severe PAH and right ventricular hypertrophy, with a significant increase in the P-PKM2 and decrease in pyruvate kinase activity which could be attenuated with the treatments of PKM2 activators, FBP and l-serine. The antioxidant NAC, apocynin and MnTBAP had the similar protective effects in the development of PAH. In vitro assays confirmed that inhibition of PKM2 activity could modulate the flux of glycolytic intermediates in support of cell proliferation through the increased pentose phosphate pathway (PPP). Increased ROS and decreased PKM2 activity also promoted the Cav1.2 expression and intracellular calcium. Conclusion: Our data provide new evidence that PKM2 makes a critical regulatory contribution to the PAHs for the first time. Decreased pyruvate kinase M2 activity confers additional advantages to rat PASMCs by allowing them to sustain anti-oxidant responses and thereby support cell survival in PAH. It may become a novel treatment strategy in PAH by using of PKM2 activators.

Reactions of copper macrocycles with antioxidants and HOCl:potential for biological redox sensing

A series of simple copper N(2)S(2) macrocycles were examined for their potential as biological redox sensors, following previous characterization of their redox potentials and crystal structures. The divalent species were reduced by glutathione or ascorbate at a biologically relevant pH in aqueous buffer. A less efficient reduction was also achieved by vitamin E in DMSO. Oxidation of the corresponding univalent copper species by sodium hypochlorite resulted in only partial (~65 %) recovery of the divalent form. This was concluded to be due to competition between metal oxidation and ligand oxidation, which is believed to contribute to macrocycle demetallation. Electrospray mass spectrometry confirmed that ligand oxidation had occurred. Moreover, the macrocyclic complexes could be demetallated by incubation with EDTA and bovine serum albumin, demonstrating that they would be inappropriate for use in biological systems. The susceptibility to oxidation and demetallation was hypothesized to be due to oxidation of the secondary amines. Consequently these were modified to incorporate additional oxygen donor atoms. This modification led to greater resistance to demetallation and ligand oxidation, providing a better platform for further development of copper macrocycles as redox sensors for use in biological systems.

Hypercapnia induces cleavage and nuclear localization of RelB protein, giving insight into CO2 sensing and signaling

Carbon dioxide (CO(2)) is increasingly being appreciated as an intracellular signaling molecule that affects inflammatory and immune responses. Elevated arterial CO(2) (hypercapnia) is encountered in a range of clinical conditions, including chronic obstructive pulmonary disease, and as a consequence of therapeutic ventilation in acute respiratory distress syndrome. In patients suffering from this syndrome, therapeutic hypoventilation strategy designed to reduce mechanical damage to the lungs is accompanied by systemic hypercapnia and associated acidosis, which are associated with improved patient outcome. However, the molecular mechanisms underlying the beneficial effects of hypercapnia and the relative contribution of elevated CO(2) or associated acidosis to this response remain poorly understood. Recently, a role for the non-canonical NF-?B pathway has been postulated to be important in signaling the cellular transcriptional response to CO(2). In this study, we demonstrate that in cells exposed to elevated CO(2), the NF-?B family member RelB was cleaved to a lower molecular weight form and translocated to the nucleus in both mouse embryonic fibroblasts and human pulmonary epithelial cells (A549). Furthermore, elevated nuclear RelB was observed in vivo and correlated with hypercapnia-induced protection against LPS-induced lung injury. Hypercapnia-induced RelB processing was sensitive to proteasomal inhibition by MG-132 but was independent of the activity of glycogen synthase kinase 3ß or MALT-1, both of which have been previously shown to mediate RelB processing. Taken together, these data demonstrate that RelB is a CO(2)-sensitive NF-?B family member that may contribute to the beneficial effects of hypercapnia in inflammatory diseases of the lung.

Role of reactive oxygen species in protein degradation in murine myotubes induced by proteolysis-inducing factor and angiotensin II

The antioxidants butylated hydroxytoluene (BHT, 1 mM) and d-α-tocopherol (10 μM) completely attenuated protein degradation in murine myotubes in response to both proteolysis-inducing factor (PIF) and angiotensin II (Ang II), suggesting that the formation of reactive oxygen species (ROS) plays an important role in this process. Both PIF and Ang II induced a rapid and transient increase in ROS formation in myotubes, which followed a parabolic dose-response curve, similar to that for total protein degradation. Antioxidant treatment attenuated the increase in expression and activity of the ubiquitin-proteasome proteolytic pathway by PIF and Ang II, by preventing the activation of the transcription factor nuclear factor-κB (NF-κB), through inhibition of phosphorylation of the NF-κB inhibitor protein (I-κB) and its subsequent degradation. ROS formation by both PIF and Ang II was attenuated by diphenyleneiodonium (10 μM), suggesting that it was mediated through the NADPH oxidase system. ROS formation was also attenuated by trifluoroacetyl arachidonic acid (10 μM), a specific inhibitor of cytosolic phospholipase A2, U-73122 (5 μM) and D609 (200 μM), inhibitors of phospholipase C and calphostin C (300 nM), a highly specific inhibitor of protein kinase C (PKC), all known activators of NADPH oxidase. Myotubes containing a dominant-negative mutant of PKC did not show an increase in ROS formation in response to either PIF or Ang II. The two Rac1 inhibitors W56 (200 μM) and NSC23766 (10 μM) also attenuated both ROS formation and protein degradation induced by both PIF and Ang II. Rac1 is known to mediate signalling between the phosphatidylinositol-3 kinase (PI-3K) product and NADPH oxidase, and treatment with LY24002 (10 μM), a highly selective inhibitor of PI-3K, completely attenuated ROS production in response to both PIF and Ang II, and inhibited total protein degradation, while the inactive analogue LY303511 (100 μM) had no effect. ROS formation appears to be important in muscle atrophy in cancer cachexia, since treatment of weight losing mice bearing the MAC16 tumour with d-α-tocopherol (1 mg kg- 1) attenuated protein degradation and increased protein synthesis in skeletal muscle. © 2007 Elsevier Inc. All rights reserved.

Regulation of neovascularization by S-glutathionylation via the Wnt5a/sFlt-1 pathway

S-glutathionylation occurs when reactive oxygen or nitrogen species react with protein-cysteine thiols. Glutaredoxin-1 (Glrx) is a cytosolic enzyme which enzymatically catalyses the reduction in S-glutathionylation, conferring reversible signalling function to proteins with redox-sensitive thiols. Glrx can regulate vascular hypertrophy and inflammation by regulating the activity of nuclear factor κB (NF-κB) and actin polymerization. Vascular endothelial growth factor (VEGF)-induced endothelial cell (EC) migration is inhibited by Glrx overexpression. In mice overexpressing Glrx, blood flow recovery, exercise function and capillary density were significantly attenuated after hindlimb ischaemia (HLI). Wnt5a and soluble Fms-like tyrosine kinase-1 (sFlt-1) were enhanced in the ischaemic-limb muscle and plasma respectively from Glrx transgenic (TG) mice. A Wnt5a/sFlt-1 pathway had been described in myeloid cells controlling retinal blood vessel development. Interestingly, a Wnt5a/sFlt-1 pathway was found also to play a role in EC to inhibit network formation. S-glutathionylation of NF-κB components inhibits its activation. Up-regulated Glrx stimulated the Wnt5a/sFlt-1 pathway through enhancing NF-κB signalling. These studies show a novel role for Glrx in post-ischaemic neovascularization, which could define a potential target for therapy of impaired angiogenesis in pathological conditions including diabetes.

The 4-aza-S-ribosyl-L-homocysteine derivatives and the related gamma-lactam and azahemiacetal analogs: Synthesis, inhibition and quorum sensing activity

Quorum sensing (QS) is a population-dependent signaling process bacteria use to control multiple processes including virulence, critical for establishing infection. There are two major pathways of QS systems. Type 1 is species specific or intra-species communication in which N-acylhomoserine lactones (Gram-negative bacteria) or oligopeptides (Gram-positive bacteria) are employed as signaling molecules (autoinducer one). Type 2 is inter-species communication in which S-4,5-dihydroxy-2,3-pentanedione (DPD) or its borate esters are used as signaling molecules. The DPD is biosynthesized by LuxS enzyme from S-ribosylhomocysteine (SRH). Recent increase in prevalence of bacterial strains resistant to antibiotics emphasizes the need for the development of new generation of antibacterial agents. Interruption of QS by small molecules is one of the viable options as it does not affect bacterial growth but only virulence, leading to less incidence of microbial resistance. Thus, in this work, inhibitors of both N-acylhomoserine lactone (AHL) mediated intra-species and LuxS enzyme, involved in inter-species QS are targeted. The γ-lactam and their reduced cyclic azahemiacetal analogs, bearing the additional alkylthiomethyl substituent, were designed and synthesized targeting AHL mediated QS systems in P. aeruginosa and Vibrio harveyi. The γ-lactams with nonylthio or dodecylthio chains acted as inhibitors of las signaling in P. aeruginosa with moderate potency. The cyclic azahemiacetal with shorter propylthio or hexylthio substituent were found to strongly inhibit both las and rhl signaling in P. aeruginosa at higher concentrations. However, lactam and their azahemiacetal analogs were found to be inactive in V. harveyi QS systems. The 4-aza-S-ribosyl-L-homocysteine (4-aza-SRH) analogs and 2-deoxy-2-substituted-S-ribosyl-L-homocysteine analogs were designed and synthesized targeting Bacillus subtilis LuxS enzyme. The 4-aza-SRH analogs in which oxygen in ribose ring is replaced by nitrogen were further modified at anomeric position to produce pyrrolidine, lactam, nitrone, imine and hemiaminal analogs. Pyrrolidine and lactam analogs which lack anomeric hydroxyl, acted as competitive inhibitors of LuxS enzyme with KI value of 49 and 37 µM respectively. The 2,3-dideoxy lactam analogs were devoid of activity. Such findings attested the significance of hydroxyl groups for LuxS binding and activity. Hemiaminal analog of SRH was found to be a time-dependent inhibitor with IC50 value of 60 µM.

Variation in oxygen isotope fractionation during cellulose synthesis: intramolecular and biosynthetic effects

The oxygen isotopic composition of plant cellulose is commonly used for the interpretations of climate, ecophysiology and dendrochronology in both modern and palaeoenvironments. Further applications of this analytical tool depends on our in-depth knowledge of the isotopic fractionations associated with the biochemical pathways leading to cellulose. Here, we test two important assumptions regarding isotopic effects resulting from the location of oxygen in the carbohydrate moiety and the biosynthetic pathway towards cellulose synthesis. We show that the oxygen isotopic fractionation of the oxygen attached to carbon 2 of the glucose moieties differs from the average fractionation of the oxygens attached to carbons 3–6 from cellulose by at least 9%, for cellulose synthesized within seedlings of two different species (Triticum aestivum L. and Ricinus communis L.). The fractionation for a given oxygen in cellulose synthesized by the Triticum seedlings, which have starch as their primary carbon source, is different than the corresponding fractionation in Ricinus seedlings, within which lipids are the primary carbon source. This observation shows that the biosynthetic pathway towards cellulose affects oxygen isotope partitioning, a fact heretofore undemonstrated. Our findings may explain the species-dependent variability in the overall oxygen isotope fractionation during cellulose synthesis, and may provide much-needed insight for palaeoclimate reconstruction using fossil cellulose.

Structural flexibility and oxygen diffusion pathways in monomeric fluorescent proteins

Fluorescent proteins are valuable tools as biochemical markers for studying cellular processes. Red fluorescent proteins (RFPs) are highly desirable for in vivo applications because they absorb and emit light in the red region of the spectrum where cellular autofluorescence is low. The naturally occurring fluorescent proteins with emission peaks in this region of the spectrum occur in dimeric or tetrameric forms. The development of mutant monomeric variants of RFPs has resulted in several novel FPs known as mFruits. Though oxygen is required for maturation of the chromophore, it is known that photobleaching of FPs is oxygen sensitive, and oxygen-free conditions result in improved photostabilities. Therefore, understanding oxygen diffusion pathways in FPs is important for both photostabilites and maturation of the chromophores. We used molecular dynamics calculations to investigate the protein barrel fluctuations in mCherry, which is one of the most useful monomeric mFruit variants, and its GFP homolog citrine. We employed implicit ligand sampling and locally enhanced sampling to determine oxygen pathways from the bulk solvent into the mCherry chromophore in the interior of the protein. The pathway contains several oxygen hosting pockets, which were identified by the amino acid residues that form the pocket. We calculated the free-energy of an oxygen molecule at points along the path. We also investigated an RFP variant known to be significantly less photostable than mCherry and find much easier oxygen access in this variant. We showed that oxygen pathways can be blocked or altered, and barrel fluctuations can be reduced by strategic amino acid substitutions. The results provide a better understanding of the mechanism of molecular oxygen access into the fully folded mCherry protein barrel and provide insight into the photobleaching process in these proteins.