Mechanism Of Uncoupling Research Articles

Water-Saving Traits Can Protect Wheat Grain Number Under Progressive Soil Drying at the Meiotic Stage: A Phenotyping Approach

In wheat, water deficit during meiosis of pollen mother cells greatly reduces seed set and grain number. A promising option to avoid grain losses and maintain wheat productivity under water stress is to exploit conservative water-use strategies during reproduction. In this work, two cultivars known to be adapted to different environments were studied. Water stress, with or without a polymer spray known to reduce stomatal conductance, was applied to both cultivars just prior to meiosis. Two experiments were carried out in a phenotyping platform to (1) assess and validate daily non-destructive estimation of projected leaf area and to (2) evaluate different water-use (WU) strategies across the meiotic period and their effect on physiology and yield components. Gladius displays an elevated breakpoint (BP) in the regression of WU against fraction of transpirable soil water (FTSW) for both daily and night-time WU suggesting higher conservative whole-plant response when compared to Paragon. At the same time, Gladius maintained flag leaf gas-exchange with a significant reduction at ~ 0.2 FTSW only, suggesting an uncoupled mechanism of WU reduction that optimized the water resource available for flag leaf gas-exchange maintenance. Under progressive soil drying, seed set and grain number of tillers stressed at GS41 were significantly reduced in Paragon (p < 0.05) thus leading to lower grain yield and grain number at plant level than Gladius. Polymer-induced reduction of transpiration is potentially useful when applied to the non-conservative stressed Paragon, maintaining higher FTSW, water-use efficiency and RWC during the progressive soil drying treatment. This led to better seed set (p < 0.05) and grain number maintenance (p < 0.05) than in the stressed Paragon control. We conclude that the different conservative traits detected in this work, protect grain development around meiosis and therefore maintain grain number under water-limiting conditions. Additionally, non-conservative genotypes (often with a greater expected yield potential) can be protected at key stages by reducing their water use with a polymer spray. Thus, future efforts can integrate both crop breeding and management strategies to achieve drought-resilience during the early reproductive phase in wheat and potentially other cereals.

Mitochondrial uncoupling proteins UCP4 and UCP5 from the Pacific white shrimp Litopenaeus vannamei.

Mitochondrial uncoupling proteins (UCP) transport protons from the intermembrane space to the mitochondrial matrix uncoupling oxidative phosphorylation. In mammals, these proteins have been implicated in several cellular functions ranging from thermoregulation to antioxidant defense. In contrast, their invertebrate homologs have been much less studied despite the great diversity of species. In this study, two transcripts encoding mitochondrial uncoupling proteins were, for the first time, characterized in crustaceans. The white shrimp Litopenaeus vannamei transcript LvUCP4 is expressed in all tested shrimp tissues/organs, and its cDNA includes a coding region of 954bp long which encodes a deduced protein 318 residues long and a predicted molecular weight of 35.3kDa. The coding region of LvUCP5 transcript is 906bp long, encodes a protein of 302 residues with a calculated molecular weight of 33.17kDa. Both proteins share homology with insect UCPs, their predicted structures show the conserved motifs of the mitochondrial carrier proteins and were confirmed to be located in the mitochondria through a Western blot analysis. The genic expression of LvUCP4 and LvUCP5 was evaluated in shrimp at oxidative stress conditions and results were compared to some antioxidant enzymes to infer about their antioxidant role. LvUCP4 and LvUCP5 genes expression did not change during hypoxia/re-oxygenation, and no coordinated responses were detected with antioxidant enzymes at the transcriptional level. Results confirmed UCPs as the first uncoupling mechanism reported in this species, but their role in the oxidative stress response remains to be confirmed.

Using computer simulation to explore mechanisms of right ventriculo-arterial uncoupling after lung resection

Right ventriculo-arterial (RV-VA) coupling expressed as a ratio of ventricular contractility to afterload (Ees/Ea) provides an integrated assessment of the efficiency of the pulmonary vascular—right ventricular unit. In previous work our group has demonstrated deterioration in cardiovascular magnetic resonance (CMR)-derived indices of VA coupling in patients undergoing lung resection. In patients, however, exploration of the mechanisms underlying uncoupling (i.e. the relative contributions of changes in contractility and afterload) is challenging, requiring expensive, invasive techniques. In this study we sought to use the Aplysia CardioVascular Lab haemodynamic simulator1 to explore in silico the mechanisms of uncoupling observed after lung resection. Demographic and haemodynamic data [including stroke volume (SV) and end-systolic volume (ESV)] were obtained from 15 patients enrolled in a CMR study examining RV-VA function before and on Day 2 after lung resection. Coupling was derived as the ratio of SV to ESV.2 Physiological adaptations were run for each patient within the simulator both before and after simulated lung resection (simulated by reducing the number of pulmonary blood vessels in the simulated patients in proportion to the volume of lung resected according to a 19-segment model of the pulmonary tree). The ratio of SV/ESV fell after both real and simulated lung resection implying deterioration in RV-VA coupling. The median SV/ESV fell by –0.14 [inter-quartile range (IQR), –0.34 to 0.01; P=0.02] in vivo and by –0.10 (IQR, –0.29 to –0.08; P<0.01) in silico; bias for the difference 0.01 (LOA –0.62 to 0.62), concordance 81.8%. Within the simulation this was attributable to a significant increase in Ea (P<0.01 for change; association between change in Ea and SV/ESV r=–0.9, P<0.01). There was, however, no association between changes in SV/ESV in real and simulated patients (r=0.32, P=0.24), and no association between simulated changes in Ea after lung resection and observed changes in SV/ESV in patients (r=–0.11, P=0.69). The mechanism of postoperative RV dysfunction after lung resection is widely believed to be the result of increased afterload, although this has not been demonstrated in clinical studies. The lack of association observed between simulated changes in Ea and changes in coupling seen in patients suggests that the mechanism of postoperative un-coupling is not simply related to the mechanical effect of a reduced pulmonary vasculature as would have been simulated according to the electrical analogue model within Aplysia. We hypothesise that an acute inflammatory injury to the RV impairs RV contractility and contributes to postoperative RV dysfunction, a mechanism which would not have been demonstrated by the computational model. 1.Broome M, Maksuti E, Bjallmark, et al. Biomed Eng Online 2013; 12: 692.Sanz J, Garcia-Alvarez A, Fernandez-Friere L. Heart 2012; 98: 238–43

Evaluation of Mn-Fe mixed oxide doped with TiO2 for the combustion with CO2 capture by Chemical Looping assisted by Oxygen Uncoupling

Bimetallic manganese-iron materials have been identified as suitable oxygen carriers for Chemical Looping Combustion (CLC) and Chemical Looping with Oxygen Uncoupling (CLOU) processes. These materials allow the combustion of a fuel with inherent CO2 capture according to two parallel mechanisms: reaction with lattice oxygen and oxygen uncoupling. This work is focused on the evaluation of the reactivity and physicochemical characterization of oxygen carrier particles consisting of a manganese-iron mixed oxide with manganese to iron molar ratio of 66:34 and doped with titanium (7 wt% TiO2) with the objective of determining suitable conditions to be used in CLC and CLOU processes. Particles were prepared by spray drying and the sintering procedure was optimized in order to achieve particles with high reactivity and mechanical strength. In addition, suitable magnetic properties were also sought in order to allow oxygen carrier recover and reuse in a chemical looping unit burning solid fuels. Optimum operating conditions for the fuel combustion and regeneration stages were determined in order to promote the oxygen uncoupling mechanism. Thus, temperature during the fuel combustion must be as high as possible to enhance the oxygen transference; but conditions for oxygen carrier regeneration by air must be carefully selected in order to take advantage of the oxygen uncoupling capability of this material. An oxidizing temperature interval of 1123–1173 K maximized the regeneration, while an air excess higher than 20% would be recommended in order to guarantee oxygen uncoupling capability.

Interpretation of the mechanism of action of antituberculosis drug bedaquiline based on a novel two-ion theory of energy coupling in ATP synthesis.

Tuberculosis (TB) claims the lives of 1.3 million people each year, more than any other bacterial infection. Hence great interest was generated in health communities upon the recent introduction of the new diarylquinoline anti‐TB drug, bedaquiline. Bedaquiline acts by binding to the c‐subunit in the membrane‐bound FO portion of the F1FO‐adenosine triphosphate (ATP) synthase, the universal enzyme that produces the ATP needed by cells. However, the mechanism of killing by bedaquiline is not fully understood. Recent observations related to the bactericidal effects of bedaquiline, which show that it is a potent uncoupler of respiration‐driven ATP synthesis in Mycobacterium smegmatis are summarized. These observations are then interpreted from the standpoint of Nath's two‐ion theory of energy coupling in ATP synthesis (Nath, Biophys. Chem. 2017; 230:45–52). Especial importance is given to the interpretation of biochemical fluorescence quenching data, and the differences between the uncoupling induced by bedaquiline from that by the classical anionic uncouplers of oxidative phosphorylation are highlighted. Suggestions for new experiments that could lead to a better understanding of the uncoupling mechanism are made. A model of uncoupling action by the drug is presented, and the biochemical basis underlying uncoupling of ATP synthesis and lethality in mycobacteria is elucidated. The major biological implications arising from these novel insights are discussed. It is hoped that the analysis will lead to a more fundamental understanding of biological energy coupling, uncoupling and transduction, and to an integrated view for the design of novel antimicrobials by future research in the field.

Molecular mechanistic insights into uncoupling of ion transport from ATP synthesis

A procedure is evolved to assess the maximum uncoupling activity of the classical unsubstituted phenolic uncouplers of mitochondrial oxidative phosphorylation (OX PHOS) 2,4-dinitrophenol and 2,6-dinitrophenol. The uncoupler concentrations, C, required for maximum uncoupling efficacy are found to be a strong function of the pH, and a linear relationship of pC with pH is obtained between pH 5 to pH 9. The slopes of the uncoupler concentrations in the aqueous and lipid phases as a function of pH have been estimated. It is shown that the experimental results can be derived from first principles by an enzyme kinetic model for uncoupling that is based on the same equations as formulated for the coupling of ion transport to ATP synthesis in a companion paper after imposition of the special conditions arising from the uncoupling process. The results reveal the catalysis of a reaction that involves both the anionic and protonated forms of the phenolic uncouplers in the vicinity of their binding sites in a non-aqueous region of the cristae membranes of mitochondria. The rate-limiting step in the overall process of uncoupling has been identified based on the uncoupling data. The data cannot be explained by a simple conduction of protons by uncouplers from one bulk aqueous phase to another as postulated by Mitchell's chemiosmotic theory. It is shown that Nath's two-ion theory of energy coupling/uncoupling in ATP synthase is consistent with the results. A molecular mechanism for uncoupling of ATP synthesis by the dinitrophenols is presented and the chief differences between coupling and uncoupling in ATP catalysis are summarized. The pharmacological consequences of our analysis of uncoupling are discussed, with particular reference to the mode of action of the anti-tuberculosis drug bedaquiline that specifically targets the c-subunit of the F1FO-ATP synthase and uncouples respiration from ATP synthesis in Mycobacterium tuberculosis. Hence the work is shown to be important both from the point of view of fundamental biology and is also pregnant with possibilities for practical pharmaceutical applications.

Sarcolipin, an Uncoupler of SERCA, Protects against Lipotoxicity and Insulin Resistance

Obesity is strongly associated with insulin resistance and type 2 diabetes, and increased consumption of high-caloric diet combined with decreased physical activity have made type 2 diabetes a worldwide epidemic. Excess accumulation of lipid and fatty acid metabolites has been shown to interfere with insulin signaling in skeletal muscle. Increasing energy expenditure and mitochondrial metabolism in muscle is an attractive strategy to protect against fat-induced insulin resistance. We have previously shown that sarcolipin (SLN), an uncoupler of SERCA pump, promotes futile cycling, increases ATP utilization and heat generation thereby contributing to muscle thermogenesis. When fed with high-fat diet (HFD), Sln-KO mice developed massive obesity whereas SLN overexpression (SlnOE) mice showed resistance to obesity. In this study, we investigated if SLN based uncoupling mechanism can be targeted to enhance muscle energy expenditure and protects against fat-induced lipotoxicity and insulin resistance in skeletal muscle. Here we show that SLN overexpression increases fatty acid oxidation in skeletal muscle by reprogramming mitochondria and upregulation of enzymes involved in fatty acid transport and oxidation. Interestingly, SlnOE mice fed on high-fat diet did not show intramuscular lipid accumulation and display improved insulin sensitivity. On the other hand, Sln-KO mice show excessive lipid accumulation in muscle and defects in insulin-stimulated glucose uptake, a sign of insulin resistance. Insulin resistant phenotype of Sln-KO muscle was associated with the increased intramuscular concentration of diacylglycerols (DAG), ceramides and acylcarnitines. These results suggest that sarcolipin mediated uncoupling of SERCA is a novel therapeutic target to increase energy expenditure and prevent insulin resistance in skeletal muscle. Disclosure S.K. Maurya: None.

Warfarin alters vitamin K metabolism: a surprising mechanism of VKORC1 uncoupling necessitates an additional reductase

AbstractThe anticoagulant warfarin inhibits the vitamin K oxidoreductase (VKORC1), which generates vitamin K hydroquinone (KH2) required for the carboxylation and consequent activation of vitamin K–dependent (VKD) proteins. VKORC1 produces KH2 in 2 reactions: reduction of vitamin K epoxide (KO) to quinone (K), and then KH2. Our dissection of full reduction vs the individual reactions revealed a surprising mechanism of warfarin inhibition. Warfarin inhibition of KO to K reduction and carboxylation that requires full reduction were compared in wild-type VKORC1 or mutants (Y139H, Y139F) that cause warfarin resistance. Carboxylation was much more strongly inhibited (∼400-fold) than KO reduction (two- to threefold). The K to KH2 reaction was analyzed using low K concentrations that result from inhibition of KO to K. Carboxylation that required only K to KH2 reduction was inhibited much less than observed with the KO substrate that requires full VKORC1 reduction (eg, 2.5-fold vs 70-fold, respectively, in cells expressing wild-type VKORC1 and factor IX). The results indicate that warfarin uncouples the 2 reactions that fully reduce KO. Uncoupling was revealed because a second activity, a warfarin-resistant quinone reductase, was not present. In contrast, 293 cells expressing factor IX and this reductase activity showed much less inhibition of carboxylation. This activity therefore appears to cooperate with VKORC1 to accomplish full KO reduction. Cooperation during warfarin therapy would have significant consequences, as VKD proteins function in numerous physiologies in many tissues, but may be poorly carboxylated and dysfunctional if the second activity is not ubiquitously expressed similar to VKORC1.

A novel mechanism of NO synthase uncoupling involving isolevuglandin adduction

We have previously shown that hypertension leads to modification of self‐proteins in antigen presenting cells by isolevuglandin (isoLG) lipid oxidation products. These modified proteins seem to be immunogenic. IsoLG‐modified peptides are presented in major histocompatibility complex (MHC) class 1 and drive CD8+ T cell proliferation. To understand alterations in the peptidome presented by MHC1 in hypertension, we created transgenic mice expressing a truncated form of MHC1 Kb lacking the transmembrane domain, driven by the CD11c promoter. The lack of a transmembrane domain causes MHC1 to be shed into the media when DCs are cultured. We treated these mice with either 2 weeks sham or angiotensin (ang) II infusion, isolated splenic DCs and placed these in culture for 72 hours. The shed soluble MHC1 Kb was harvested and associated peptides were analyzed by mass spectroscopy. One peptide identified in DCs from hypertensive mice was an identical match for a portion of the neuronal nitric oxide synthase (nNOS) containing an isoLG adduct at lysine 225. Because this lysine is positioned between the heme and arginine binding sites, we hypothesized that its modification would disrupt normal NO production and instead lead to superoxide production, i.e. NOS uncoupling. In subsequent studies, we found that nNOS expression is increased 5‐fold in DCs of ang II‐treated compared to sham‐treated mice (n = 6). Importantly, ang II infusion increased superoxide production of DCs from 37±3 to 111±5 pmol/mg protein (mean ± SE, p = 0.0001), and this was completely blocked by treatment of the cells with the selective nNOS inhibitor L‐vinyl NIO, suggesting that nNOS is a source of superoxide in DCs in hypertension. Next we expressed wild type nNOS and a mutant nNOS in which lysine 225 was mutated to an alanine (nNOS K225A) in human epithelial kidney (HEK) cells. Untransfected HEK cells failed to produce NO, as measured by electron spin resonance and the spin trap Fe[DETC]2. HEK cells transfected with either the WT or K225A nNOS produced 152 ± 5 vs 156 ± 6 arbitrary units of NO/mg protein (p = NS). However, exposure of the transfected HEK cells to tertbutyl hydroperoxide, which promotes isoLG formation, reduced NO production by cells expressing the wild‐type enzyme to 86 ± 6 units/mg protein, while having no effect on cells transfected with the mutant K225A (p = 0.003, n = 5 – 7), further implicating lysine 225 as a site of oxidative modification in nNOS. In summary, hypertension increases nNOS expression in DCs and this enzyme is altered by isoLG adduction at lysine 225. This leads to an increase in superoxide and reduced NO production in these immune cells. We conclude that isoLG adduction represents a novel mechanism of NOS uncoupling and a source of reactive oxygen species in dendritic cells.Support or Funding InformationAmerican Heart Association grants POST290900 and SFRN204200, and National Institutes of Health grants K01HL130497, R01HL125865, R01HL039006 and P01HL129941This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Alternative mitochondrial respiratory chains from two crustaceans: Artemia franciscana nauplii and the white shrimp, Litopenaeus vannamei

Mitochondrial ATP is synthesized by coupling between the electron transport chain and complex V. In contrast, physiological uncoupling of these processes allows mitochondria to consume oxygen at high rates without ATP synthesis. Such uncoupling mechanisms prevent reactive oxygen species overproduction. One of these mechanisms are the alternative redox enzymes from the mitochondrial respiratory chain, which may help cells to maintain homeostasis under stress independently of ATP synthesis. To date, no reports have been published on alternative redox enzymes in crustaceans mitochondria. Specific inhibitors were used to identify alternative redox enzymes in mitochondria isolated from Artemia franciscana nauplii, and the white shrimp, Litopenaeus vannamei. We report the presence of two alternative redox enzymes in the respiratory chain of A. franciscana nauplii, whose isolated mitochondria used glycerol-3-phosphate as a substrate, suggesting the existence of a glycerol-3-phosphate dehydrogenase. In addition, cyanide and octyl-gallate were necessary to fully inhibit this species' mitochondrial oxygen consumption, suggesting an alternative oxidase is present. The in-gel activity analysis confirmed that additional mitochondrial redox proteins exist in A. franciscana. A mitochondrial glycerol-3-phosphate dehydrogenase oxidase was identified by protein sequencing as part of a branched respiratory chain, and an alternative oxidase was also identified in this species by western blot. These results indicate different adaptive mechanisms from artemia to face environmental challenges related to the changing levels of oxygen concentration in seawater through their life cycles. No alternative redox enzymes were found in shrimp mitochondria, further efforts will determine the existence of an uncoupling mechanism such as uncoupling proteins.

The cytotoxic effects of VE-3N, a novel 1, 4-dihydropyridine derivative, involve the mitochondrial bioenergetic disruption via uncoupling mechanisms

The cytotoxic effects of VE-3N, a novel 1, 4-dihydropyridine derivative, involve the mitochondrial bioenergetic disruption via uncoupling mechanisms

The Incomplete Glutathione Puzzle: Just Guessing at Numbers and Figures?

Significance: Glutathione metabolism is comparable to a jigsaw puzzle with too many pieces. It is supposed to comprise (i) the reduction of disulfides, hydroperoxides, sulfenic acids, and nitrosothiols, (ii) the detoxification of aldehydes, xenobiotics, and heavy metals, and (iii) the synthesis of eicosanoids, steroids, and iron–sulfur clusters. In addition, glutathione affects oxidative protein folding and redox signaling. Here, I try to provide an overview on the relevance of glutathione-dependent pathways with an emphasis on quantitative data.Recent Advances: Intracellular redox measurements reveal that the cytosol, the nucleus, and mitochondria contain very little glutathione disulfide and that oxidative challenges are rapidly counterbalanced. Genetic approaches suggest that iron metabolism is the centerpiece of the glutathione puzzle in yeast. Furthermore, recent biochemical studies provide novel insights on glutathione transport processes and uncoupling mechanisms.Critical Issues: Which parts of the glutathione puzzle are most relevant? Does this explain the high intracellular concentrations of reduced glutathione? How can iron–sulfur cluster biogenesis, oxidative protein folding, or redox signaling occur at high glutathione concentrations? Answers to these questions not only seem to depend on the organism, cell type, and subcellular compartment but also on different ideologies among researchers.Future Directions: A rational approach to compare the relevance of glutathione-dependent pathways is to combine genetic and quantitative kinetic data. However, there are still many missing pieces and too little is known about the compartment-specific repertoire and concentration of numerous metabolites, substrates, enzymes, and transporters as well as rate constants and enzyme kinetic patterns. Gathering this information might require the development of novel tools but is crucial to address potential kinetic competitions and to decipher uncoupling mechanisms to solve the glutathione puzzle. Antioxid. Redox Signal. 27, 1130–1161.

Concise Review: Conceptualizing Paralogous Stem-Cell Niches and Unfolding Bone Marrow Progenitor Cell Identities.

Lineage commitment and differentiation of skeletal stem cells/bone marrow stromal cells (SSCs/BMSCs, often called bone marrow-derived "mesenchymal stem/stromal" cells) offer an important opportunity to study skeletal and hematopoietic diseases, and for tissue engineering and regenerative medicine. Currently, many studies in this field have relied on cell lineage tracing methods in mouse models, which have provided a significant advancement in our knowledge of skeletal and hematopoietic stem-cell niches in bone marrow (BM). However, there is a lack of agreement in numerous fundamental areas, including origins of various BM stem-cell niches, cell identities, and their physiological roles in the BM. In order to resolve these issues, we propose a new hypothesis of "paralogous" stem-cell niches (PSNs); that is, progressively altered parallel niches within an individual species throughout the life span of the organism. A putative PSN code seems to be plausible based on analysis of transcriptional signatures in two representative genes that encode Nes-GFP and leptin receptors, which are frequently used to monitor SSC lineage development in BM. Furthermore, we suggest a dynamic paralogous BM niche (PBMN) model that elucidates the coupling and uncoupling mechanisms between BM stem-cell niches and their zones of active regeneration during different developmental stages. Elucidation of these PBMNs would enable us to resolve the existing controversies, thus paving the way to achieving precision regenerative medicine and pharmaceutical applications based on these BM cell resources. Stem Cells 2018;36:11-21.

Postnatal Hyperoxia Exposure Durably Impairs Right Ventricular Function and Mitochondrial Biogenesis.

Prematurity complicates 12% of births, and young adults with a history of prematurity are at risk to develop right ventricular (RV) hypertrophy and impairment. The long-term risk for pulmonary vascular disease, as well as mechanisms of RV dysfunction and ventricular-vascular uncoupling after prematurity, remain poorly defined. Using an established model of prematurity-related lung disease, pups from timed-pregnant Sprague Dawley rats were randomized to normoxia or hyperoxia (fraction of inspired oxygen, 0.85) exposure for the first 14 days of life. After aging to 1 year in standard conditions, rats underwent hemodynamic assessment followed by tissue harvest for biochemical and histological evaluation. Aged hyperoxia-exposed rats developed significantly greater RV hypertrophy, associated with a 40% increase in RV systolic pressures. Although cardiac index was similar, hyperoxia-exposed rats demonstrated a reduced RV ejection fraction and significant RV-pulmonary vascular uncoupling. Hyperoxia-exposed RV cardiomyocytes demonstrated evidence of mitochondrial dysregulation and mitochondrial DNA damage, suggesting potential mitochondrial dysfunction as a cause of RV dysfunction. Aged rats exposed to postnatal hyperoxia recapitulate many features of young adults born prematurely, including increased RV hypertrophy and decreased RV ejection fraction. Our data suggest that postnatal hyperoxia exposure results in mitochondrial dysregulation that persists into adulthood with eventual RV dysfunction. Further evaluation of long-term mitochondrial function is warranted in both animal models of premature lung disease and in human adults who were born preterm.

Increased Superoxide Dismutase‐2 in skeletal muscle in a rat model of preterm birth

BackgroundIn the United States an estimated 12% of births are preterm, meaning a significant number of infants are at increased risk for hypertension, type‐2 diabetes, and insulin resistance later in life. After a preterm birth, a common intervention used is oxygen therapy in order to improve oxygen delivery to systemic tissues. Although acutely life sustaining, there may be adverse effects of oxygen therapy in the long term. Many metabolic alterations have been identified in adults born preterm, including increased risk of insulin resistance and metabolic syndrome. In these conditions, there have been alterations in uncoupling protein‐3 (UCP‐3) expression, a protein that aids in the release of energy from the mitochondria as heat, and superoxide dismutase one and two (SOD‐1 and SOD‐2) expression, which metabolize reactive oxygen species. We hypothesized that altered levels of uncoupling protein and antioxidants would be observed at one year of age in a rat model of prematurity.MethodsWithin 12 hours of their birth, we exposed Harlan Sprague‐Dawley (SD) rat pups for 14 days to either normoxia (NORM, 21% oxygen, n=12, 6 males) or hyperoxia (HYP, 85% oxygen, n=12, 6 males) in custom chambers. After the 14 days, the pups were aged out to one year of age in standard room air. The gastrocnemius muscles were then harvested and flash frozen in liquid nitrogen. Western blotting was used to observe the differences in UCP3, SOD1, and SOD2. Statistics were run by two way ANOVA with Tukey's post hoc analysis.ResultsThere were no differences observed in UCP3 protein expression or SOD1 protein expression (p=0.8). There is a trend towards higher expression of SOD2 expression in HYP rats (p=0.9). There was a trend towards higher expression of SOD2 in the HYP rats compared to NORM rats (38% higher, p=0.08).ConclusionsNo difference in UCP3 expression is indicative that the main mechanism of uncoupling in the skeletal muscle is unaltered, at least in expression, in this rat model of prematurity. SOD1 is the form of SOD present in the cytoplasm, and indicates that there may not be an increased need for antioxidants in the cytoplasm. However, the potentially higher level of SOD2, the mitochondrial form, in HYP rats indicates that there may be increased need for antioxidants in the mitochondria. Previous data has shown altered mitochondrial mass and function in the HYP rats, and this observation of greater expression of SOD2, a potent antioxidant, further supports the possibility of altered mitochondrial function in the HYP rats.Support or Funding InformationNHLBI, R01 HL115061‐03, Suppl (Eldridge) &amp; NHLBI, R01 HL115061 (Eldridge)

The cytotoxic effects of VE-3N, a novel 1,4-dihydropyridine derivative, involve the mitochondrial bioenergetic disruption via uncoupling mechanisms

Several 1,4-dihydropyridine derivatives overcome the multidrug resistance in tumors, but their intrinsic cytotoxic mechanisms remain unclear. Here we addressed if mitochondria are involved in the cytotoxicity of the novel 1,4-dihydropyridine derivative VE-3N [ethyl 6-chloro-5-formyl-2-methyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3-carboxylate] towards cancer cells by employing hepatic carcinoma (HepG2) cells and isolated rat liver mitochondria. In HepG2 cells, VE-3N induced mitochondrial membrane potential dissipation, ATP depletion, annexin V/propidium iodide double labeling, and Hoechst staining; events indicating apoptosis induction. In isolated rat liver mitochondria, VE-3N promoted mitochondrial uncoupling by exerting protonophoric actions and by increasing membrane fluidity. Mitochondrial uncoupling was evidenced by an increase in resting respiration, dissipation of mitochondrial membrane potential, inhibition of Ca2+ uptake, stimulation of Ca2+ release, decrease in ATP synthesis, and swelling of valinomycin-treated organelles in hyposmotic potassium acetate media. Furthermore, uncoupling concentrations of VE-3N in the presence of Ca2+ plus ruthenium red induced the mitochondrial permeability transition process. These results indicate that mitochondrial uncoupling is potentially involved in the VE-3N cytotoxic actions towards HepG2 cells. Considering that hepatocellular carcinoma is the most common form of liver cancer, our findings may open a new avenue for the development of VE-3N-based cancer therapies, and help to unravel the cytotoxic mechanisms of 1,4-dihydropyridines towards cancer cells.

Cytoplasmic Localization of HTLV-1 HBZ Protein: A Biomarker of HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP).

HTLV-1 is the causative agent of a severe form of adult T cell leukemia/Lymphoma (ATL), and of a chronic progressive neuromyelopathy designated HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). Two important HTLV-1-encoded proteins, Tax-1 and HBZ, play crucial roles in the generation and maintenance of the oncogenic process. Less information is instead available on the molecular and cellular mechanisms leading to HAM/TSP. More importantly, no single specific biomarker has been described that unambiguously define the status of HAM/TSP. Here we report for the first time the finding that HBZ, described until now as an exclusive nuclear protein both in chronically infected and in ATL cells, is instead exclusively localized in the cytoplasm of peripheral blood mononuclear cells (PBMC) from patients suffering of HAM/TSP. Interestingly, at the single cell level, HBZ and Tax-1 proteins are never found co-expressed in the same cell, suggesting the existence of mechanisms of expression uncoupling of these two important HTLV-1 viral products in HAM/TSP patients. Cells expressing cytoplasmic HBZ were almost exclusively found in the CD4+ T cell compartment that was not, at least in a representative HAM/TSP patient, expressing the CD25 marker. Less than 1 percent CD8+ T cells were fond positive for HBZ, while B cells and NK cells were found negative for HBZ in HAM/TSP patients. Our results identify the cytoplasmic localization of HBZ in HAM/TSP patient as a possible biomarker of this rather neglected tropical disease, and raise important hypotheses on the role of HBZ in the pathogenesis of the neuromyelopathy associated to HTLV-1 infection.

Implication of eNOS Uncoupling in Cardiovascular Disease

Under physiological conditions, nitric oxide (NO) is produced in the vasculature mainly by the endothelial nitric oxide synthase (eNOS). Endothelial NO relaxes blood vessels, inhibits platelet activity, and protects against atherosclerosis. Under pathological conditions such as hypertension, diabetes, and hypercholesterolemia, eNOS may become uncoupled. Uncoupled eNOS generates superoxide at the expense of NO and contributes substantially to oxidative stress and endothelial dysfunction. Major mechanisms of eNOS uncoupling include deficiency of the eNOS cofactor tetrahydrobiopterin, deficiency of the eNOS substrate L-arginine, and eNOS S-glutathionylation. Reversal of eNOS uncoupling may represent a feasible strategy for the prevention and treatment of cardiovascular diseases.

Natural Endogenous Human Matriptase and Prostasin Undergo Zymogen Activation via Independent Mechanisms in an Uncoupled Manner.

The membrane-associated serine proteases matriptase and prostasin are believed to function in close partnership. Their zymogen activation has been reported to be tightly coupled, either as a matriptase-initiated proteolytic cascade or through a mutually dependent mechanism involving the formation of a reciprocal zymogen activation complex. Here we show that this putative relationship may not apply in the context of human matriptase and prostasin. First, the tightly coupled proteolytic cascade between matriptase and prostasin might not occur when modest matriptase activation is induced by sphingosine 1-phospahte in human mammary epithelial cells. Second, prostasin is not required and/or involved in matriptase autoactivation because matriptase can undergo zymogen activation in cells that do not endogenously express prostasin. Third, matriptase is not required for and/or involved in prostasin activation, since activated prostasin can be detected in cells expressing no endogenous matriptase. Finally, matriptase and prostasin both undergo zymogen activation through an apparently un-coupled mechanism in cells endogenously expressing both proteases, such as in Caco-2 cells. In these human enterocytes, matriptase is detected primarily in the zymogen form and prostasin predominantly as the activated form, either in complexes with protease inhibitors or as the free active form. The negligible levels of prostasin zymogen with high levels of matriptase zymogen suggests that the reciprocal zymogen activation complex is likely not the mechanism for matriptase zymogen activation. Furthermore, high level prostasin activation still occurs in Caco-2 variants with reduced or absent matriptase expression, indicating that matriptase is not required and/or involved in prostasin zymogen activation. Collectively, these data suggest that any functional relationship between natural endogenous human matriptase and prostasin does not occur at the level of zymogen activation.

Small-Molecule Uncoupling Protein Mimics: Synthetic Anion Receptors as Fatty Acid-Activated Proton Transporters.

Uncoupling proteins (UCPs) regulate energy expenditure in living cells by inducing proton leakage across the mitochondrial inner membrane, thereby uncoupling adenosine diphosphate phosphorylation from nutrient oxidation. The proton transport activity of UCP1 and UCP2 requires activation by fatty acids. We report here the first examples of synthetic neutral anion receptors performing this biologically important fatty acid-activated function in phospholipid bilayers. We have shown that a tripodal thiourea possesses poor H+/OH- transport activity without fatty acids, but in the presence of long-chain fatty acids is "switched on" as a proton transporter with an activity close to that of a commonly used protonophore. The fatty acid-enhanced proton transport was also observed for other hydrogen and halogen bond-based synthetic anion transporters. We propose that these compounds induce proton permeability by catalyzing transbilayer movement ("flip-flop") of anionic forms of fatty acids, so allowing the fatty acids to complete a proton transport cycle. Several lines of evidence have been provided to support such a fatty acid cycling mechanism. Our findings open up new applications of anion receptor chemistry and provide important clues for understanding biological activities of synthetic anion transporters and potentially the uncoupling mechanism of naturally occurring membrane proteins.