Haptoglobin Genotype-dependent Differences in Macrophage Lysosomal Oxidative Injury

The major function of the Haptoglobin (Hp) protein is to control trafficking of extracorpuscular hemoglobin (Hb) thru the macrophage CD163 receptor with degradation of the Hb in the lysosome. There is a common copy number polymorphism in the Hp gene (Hp 2 allele) that has been associated with a severalfold increased incidence of atherothrombosis in multiple longitudinal studies. Increased plaque oxidation and apoptotic markers have been observed in Hp 2-2 atherosclerotic plaques, but the mechanism responsible for this finding has not been determined. We proposed that the increased oxidative injury in Hp 2-2 plaques is due to an impaired processing of Hp 2-2-Hb complexes within macrophage lysosomes, thereby resulting in redox active iron accumulation, lysosomal membrane oxidative injury, and macrophage apoptosis. We sought to test this hypothesis in vitro using purified Hp-Hb complex and cells genetically manipulated to express CD163. CD163-mediated endocytosis and lysosomal degradation of Hp-Hb were decreased for Hp 2-2-Hb complexes. Confocal microscopy using lysotropic pH indicator dyes demonstrated that uptake of Hp 2-2-Hb complexes disrupted the lysosomal pH gradient. Cellular fractionation studies of lysosomes isolated from macrophages incubated with Hp 2-2-Hb complexes demonstrated increased lysosomal membrane oxidation and a loss of lysosomal membrane integrity leading to lysosomal enzyme leakage into the cytoplasm. Additionally, markers of apoptosis, DNA fragmentation, and active caspase 3 were increased in macrophages that had endocytosed Hp 2-2-Hb complexes. These data provide novel mechanistic insights into how the Hp genotype regulates lysosomal oxidative stress within macrophages after receptor-mediated endocytosis of Hb.     

Ascorbic acid prolongs the viability and stability of isolated perfused lungs: A mechanistic study using 31P and hyperpolarized 13C nuclear magnetic resonance

Ex vivo lung perfusion (EVLP) has recently shown promise as a means of more accurately gauging the health of lung grafts and improving graft performance post-transplant. However, reperfusion of ischemic lung promotes the depletion of high-energy compounds and a progressive loss of normal mitochondrial function, and it remains unclear how and to what extent the EVLP approach contributes to this metabolic decline. Although ascorbate has been used to mitigate the effects of ischemia–reperfusion injury, the nature of its effects during EVLP are also not clear. To address these uncertainties, this study monitored the energy status of lungs during EVLP and after the administration of ascorbate using ³¹P and hyperpolarized ¹³C NMR (nuclear magnetic resonance). Our experiments demonstrated that the oxidative phosphorylation capacity and pyruvate dehydrogenase flux of lungs decline during ex vivo perfusion. The addition of ascorbate to the perfusate prolonged lung viability by 80% and increased the hyperpolarized ¹³C bicarbonate signal by a factor of 2.7. The effect of ascorbate is apparently due not to its antioxidant quality but rather to its ability to energize cellular respiration given that it increased the lung’s energy charge significantly, whereas other antioxidants (glutathione and α-lipoic acid) did not alter energy metabolism. During ascorbate administration, inhibition of mitochondrial complex I with rotenone depressed energy charge and shifted the metabolic state of the lung toward glycolysis; reenergizing the electron transport chain with TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) recovered metabolic activity. This indicates that ascorbate slows the decline of the ex vivo perfused lung’s mitochondrial activity through an independent interaction with the electron transport chain complexes.     

Cytotoxic reactions of rainbow trout, Salmo gairdneri Richardson, macrophages for larvae of the eye fluke Diplostomum spathaceum (Digenea)

The killing ability of rainbrow trout macrophages for the infective larval stages of Diploslomum spathaceum , cercariae and diplostomules, was investigated. Isolated macrophages kill significant numbers of diplostomules at effector: target ratios of 150: 1 or greater. In vitro killing was not increased using antiserum-coated larvae or in vivo -activated macrophages individually, but when they were combined increased killed occurred. Diplostomules were capable of eliciting respiratory burst activity from macrophages in vitro , suggesting that reactive oxygen species may have a role to play in the killing mechanism. The importance of macrophage activation in the protection afforded by immunization against this parasite is discussed.     

Doxorubicin-induced alterations in kidney functioning,oxidative stress,DNA damage,and renal tissue morphology; Improvement by Acacia hydaspica tannin-rich ethyl acetate fraction

Doxorubicin (DOX) is an anthracycline drug used for cancer treatment. However, its treatment is contiguous with toxic effects. We examined the nephroprotective potential of A. hydaspica polyphenol-rich ethyl acetate extract (AHE) against DOX persuaded nephrotoxicity. 36 male Sprague Dawley rats were randomly assorted into 6 groups. Control group received saline; DOX group: 3 mg/kg b.w. dosage of DOX intraperitoneally for 6 weeks (single dose/week). In co-treatment groups, 200 and 400 mg/kg b.w AHE was given orally for 6 weeks in concomitant with DOX (3 mg/kg b.w, i.p. injection per week) respectively. Standard group received silymarin 400 mg/kg b.w daily + DOX (single dose/week). Biochemical kidney function tests, oxidative stress markers, genotoxicity, antioxidant enzyme status, and histopathological changes were examined. DOX caused significant body weight loss and decrease kidney weight. DOX-induced marked deterioration in renal function indicators in both urine and serum, i.e., PH, specific gravity, total protein, albumin, urea, creatinine, uric acid, globulin, blood urea nitrogen, etc. Also, DOX treatment increases renal tissue oxidative stress markers, while lower antioxidant enzymes in tissue along with degenerative alterations in the renal tissue compared to control rats. AHE co-treatment ameliorates DOX-prompted changes in serum and urine chemistry. Likewise, AHE treatment decreases sensitive markers of oxidative stress and prevented DNA damages by enhancing antioxidant enzyme levels. DOX induction in rats also caused DNA fragmentation which was restored by AHE co-treatment. Moreover, the histological observations evidenced that AHE effectively rescued the kidney tissue from DOX interceded oxidative damage. Our results suggest that co-treatment of AHE markedly improve DOX-induced deleterious effects in a dose-dependent manner. The potency of AHE co-treatment at 400 mg/kg dose is similar to silymarin. These outcomes revealed that A. hydaspica AHE extract might serve as a potential adjuvant that avoids DOX-induced nephrotoxicity.     

The mitochondrial small heat-shock protein protects NADH:ubiquinone oxidoreductase of the electron transport chain during heat stress in plants

Functional inactivation of the mitochondrial small heat-shock protein (lmw Hsp) in submitochondrial vesicles using protein-specific antibodies indicated that this protein protects NADH:ubiquinone oxidoreductase (complex I), and consequently electron transport from complex I to cytochrome c:O₂ oxidoreductase (complex IV). Lmw Hsp function completely accounted for heat acclimation of complex I electron transport in pre-heat-stressed plants. Addition of purified lmw Hsp to submitochondrial vesicles lacking this Hsp increased complex I electron transport rates 100% in submitochondrial vesicles assayed at high temperatures. These results indicate that production of the mitochondrial lmw Hsp is an important adaptation to heat stress in plants.     

MITOCHONDRIA: Are mitochondria accessory to metastasis?

Since the nineteenth century the importance of mitochondria in cellular physiology has been growing steadily. Not only the organelle harbors the main systems for ATP generation, but also buffers the redox potential in the cytosol and is one of the protagonists of the intrinsic pathway for apoptosis. In tumor cells, mitochondria went from being dysfunctional compartments to playing a supportive or perhaps even a triggering part in metastasis. This “Organelle In Focus” article discusses the classical metabolic events that occur in mitochondria and why these pathways could be essential for the onset of the malignant phenotype. Finally, we propose that the oxidative metabolism of tumor cells in conjunction with the inactivation of anoikis may have been coopted through a non-adaptive evolutionary process.     

A comparison of mechanisms of desiccation tolerance among three angiosperm resurrection plant species

The mechanisms of protection against mechanical and oxidative stress were identified and compared in the angiosperm resurrection plants Craterostigma wilmsii, Myrothamnus flabellifolius and Xerophyta humilis. Drying-induced ultrastructural changes within mesophyll cells were followed to gain an understanding of the mechanisms of mechanical stabilisation. In all three species, water filled vacuoles present in hydrated cells were replaced by several smaller vacuoles filled with non-aqueous substances. In X. humilis, these occupied a large proportion of the cytoplasm, preventing plasmalemma withdrawal and cell wall collapse. In C. wilmsii, vacuoles were small but extensive cell wall folding occurred to prevent plasmalemma withdrawal. In M. flabellifolius, some degree of vacuolation and wall folding occurred, but neither were sufficient to prevent plasmalemma withdrawal. This membrane was not ruptured, possibly due to membrane repair at plasmodesmata junctions where tearing might have occurred. In addition, the extra-cytoplasmic compartment appeared to contain material (possibly similar to that in vacuoles) which could facilitate stabilisation of dry cells.Photosynthesis and respiration are particularly susceptible to oxidative stress during drying. Photosynthesis ceased at high water contents and it is proposed that a controlled shut down of this metabolism occurred in order to minimise the potential for photo-oxidation. The mechanisms whereby this was achieved varied among the species. In X. humilis, chlorophyll was degraded and thylakoid membranes dismantled during drying. In both C. wilmsii and M. flabellifolius, chlorophyll was retained, but photosynthesis was stopped due to chlorophyll shading from leaf folding and anthocyanin accumulation. Furthermore, in M. flabellifolius thylakoid membranes became unstacked during drying. All species continued respiration during drying to 10% relative water content, which is proposed to be necessary for energy to establish protection mechanisms. Activity of antioxidant enzymes increased during drying and remained high at low water contents in all species, ameliorating free radical damage from both photosynthesis and respiration. The nature and extent of antioxidant upregulation varied among the species. In C. wilmsii, only ascorbate peroxidise activity increased, but in M. flabellifolius and X. humilis ascorbate peroxidise, glutathione reductase and superoxide dismutase activity increased, to various extents, during drying. Anthocyanins accumulated in all species but this was more extensive in the homoiochlorophyllous types, possibly for protection against photo-oxidation.     

Errata

Human erythrocytes were exposed to oxidative stress by iodate and periodate. Oxidation causes a time- and concentration-dependent increase in membrane permeability for hydrophilic molecules and ions. The induced leak discriminates nonelectrolytes on the basis of molecular size and exhibits a very low activation energy (E_a = 1–4 kcal · mol^?1). These results are reconcilable with the formation of aqeous pores. The pore size was approximated to be between 0.45 and 0.6 nm. This increase in permeability is reversible upon treatment with dithioerythritol. Blocking of membrane thiol groups with N-ethylmaleimide protects the membranes against leak formation. The oxidation causes dithioerythritol-reversible modification of membrane proteins as indicated by the gel electrophoretic behavior. These modifications can also be suppressed by blocking the membrane thiol groups with N-ethylmaleimide. About half of the membrane methionine is oxidized to acid hydrolysis-stable derivatives. A fast saturating increase in diene conjugation was observed in whole cells but not in isolated membranes, with only minor degradation of fatty acid chains. The oxidation of cell membrane lipids as well as oxidation of cell surface carbohydrates are not involved in leak formation. Taken together with earlier data (Deuticke, B., Poster, B., Lütkemeier, P., and Haest, C.W.M. (1983) Biochim. Biophys. Acta 731, 196–210), these findings indicate that formation of disulfide bonds by different oxidative mechanisms results in leaks with similar properties.     

Control of Redox Balance by the Stringent Response Regulatory Protein Promotes Antioxidant Defenses of Salmonella

We report herein a critical role for the stringent response regulatory DnaK suppressor protein (DksA) in the coordination of antioxidant defenses. DksA helps fine-tune the expression of glutathione biosynthetic genes and discrete steps in the pentose phosphate pathway and tricarboxylic acid cycle that are associated with the generation of reducing power. Control of NAD(P)H/NAD(P)⁺ redox balance by DksA fuels downstream antioxidant enzymatic systems in nutritionally starving Salmonella. Conditional expression of the glucose-6-phosphate dehydrogenase-encoding gene zwf, shown here to be under DksA control, increases both the NADPH pool and antioxidant defenses of dksA mutant Salmonella. The DksA-mediated coordination of redox balance boosts the antioxidant defenses of stationary phase bacteria. Not only does DksA increase resistance of Salmonella against hydrogen peroxide (H₂O₂), but it also promotes fitness of this intracellular pathogen when exposed to oxyradicals produced by the NADPH phagocyte oxidase in an acute model of infection. Given the role of DksA in the adjustment of gene expression in most bacteria undergoing nutritional deprivation, our findings raise the possibility that the control of central metabolic pathways by this regulatory protein maintains redox homeostasis essential for antioxidant defenses in phylogenetically diverse bacterial species.     

M(en)TORship lessons on life and death by the integrated stress response

Cells employ pro-survival and pro-adaptive pathways to cope with different forms of environmental stress. When stress is excessive, and the damage caused by it is unsustainable, cells engage pro-death pathways, which are in place to protect the host from the deleterious effects of harmed cells. Two important pathways that determine the balance between survival and death of stressed cells are the integrated stress response (ISR) and the mammalian target of rapamycin (mTOR), both of which converge at the level of mRNA translation. The two pathways have established avenues of communication to control their activity and determine the fate of stressed cells in a context-dependent manner. The functional interplay between the ISR and mTOR may have significant ramifications in the development and treatment of human diseases such as diabetes, neurodegeneration and cancer.