Redox Balance In Cells Research Articles

Proteome Response of Staphylococcus xylosus DSM 20266T to Anaerobiosis and Nitrite Exposure.

The viability and competitiveness of Staphylococcus xylosus in meat mostly depend on the ability to adapt itself to rapid oxygen and nutrients depletion during meat fermentation. The utilization of nitrite instead of oxygen becomes a successful strategy for this strain to improve its performance in anaerobiosis; however, metabolic pathways of this strain underlying this adaptation, are partially known. The aim of this study was to provide an overview on proteomic changes of S. xylosus DSM 20266T cultured under anaerobiosis and nitrite exposure. Thus, two different cultures of this strain, supplemented or not with nitrite, were in vitro incubated in aerobiosis and anaerobiosis monitoring cell viability, pH, oxidation reduction potential and nitrite content. Protein extracts, obtained from cells, collected as nitrite content was depleted, were analyzed by 2DE/MALDI-TOF/TOF-MS. Results showed that DSM 20266T growth was significantly sustained by nitrite in anaerobiosis, whereas no differences were found in aerobiosis. Accordingly, nitrite content was depleted after 13 h only in anaerobiosis. At this time of sampling, a comparative proteomic analysis showed 45 differentially expressed proteins. Most differences were found between aerobic and anaerobic cultures without nitrite; the induction of glycolytic enzymes and glyoxylate cycle, the reduction of TCA enzymes, and acetate fermentation were found in anaerobiosis to produce ATP and maintain the cell redox balance. In anaerobic cultures the nitrite supplementation partially restored TCA cycle, and reduced the amount of glycolytic enzymes. These results were confirmed by phenotypic microarray that, for the first time, was carried out on cell previously adapted at the different growth conditions. Overall, metabolic changes were similar between aerobiosis and anaerobiosis NO2-adapted cells, whilst cells grown under anaerobiosis showed different assimilation profiles by confirming proteomic data; indeed, these latter extensively assimilated substrates addressed at both supplying glucose for glycolysis or fueling alternative pathways to TCA cycle. In conclusion, metabolic pathways underlying the ability of S. xylosus to adapt itself to oxygen starvation were revealed; the addition of nitrite allowed S. xylosus to take advantage of nitrite to this condition, restoring some metabolic pathway underlying aerobic behavior of the strain.

Beyond the Antioxidant Activity of Dietary Polyphenols in Cancer: the Modulation of Estrogen Receptors (ERs) Signaling.

The potential “health benefits” of dietary polyphenols have been ascribed to their direct antioxidant activity and their impact on the regulation of cell and tissue redox balance. However, because of the relative poor bioavailability of many of these compounds, their effects could not be easily explained by the antioxidant action, which may occur only at high circulating and tissue concentrations. Therefore, many efforts have been put forward to clarify the molecular mechanisms underlining the biological effect of polyphenols in physiological and pathological conditions. Polyphenols’ bioavailability, metabolism, and their effects on enzyme, membrane, and/or nuclear receptors and intracellular transduction mechanisms may define the overall impact of these compounds on cancer risk and progression, which is still debated and not yet clarified. Polyphenols are able to bind to estrogen receptor α (ERα) and β (ERβ), and therefore induce biological effects in human cells through mimicking or inhibiting the action of endogenous estrogens, even at low concentrations. In this work, the role and effects of food-contained polyphenols in hormone-related cancers will be reviewed, mainly focusing on the different polyphenols’ mechanisms of action with particular attention on their estrogen receptor-based effects, and on the consequences of such processes on tumor progression and development.

Antioxidant intervention promotes cell survival and redox balance within the ovary and subsequent oocyte resulting in improved IVF outcomes

Antioxidant intervention promotes cell survival and redox balance within the ovary and subsequent oocyte resulting in improved IVF outcomes

The Effect of Sea Buckthorn (Hippophae rhamnoides L.) Seed Oil on UV-Induced Changes in Lipid Metabolism of Human Skin Cells.

Lipids and proteins of skin cells are the most exposed to harmful ultraviolet (UV) radiation contained in sunlight. There is a growing need for natural compounds that will protect these sensitive molecules from damage, without harmful side effects. The aim of this study was to investigate the effect of sea buckthorn seed oil on the redox balance and lipid metabolism in UV irradiated cells formed different skin layers to examine whether it had a protective effect. Human keratinocytes and fibroblasts were subjected to UVA (ultraviolet type A; 30 J/cm2 and 20 J/cm2) or UVB (ultraviolet type B; 60 mJ/cm2 and 200 mJ/cm2, respectively) radiation and treated with sea buckthorn seed oil (500 ng/mL), and the redox activity was estimated by reactive oxygen species (ROS) generation and enzymatic/non-enzymatic antioxidants activity/level (using electron spin resonance (ESR), high-performance liquid chromatography (HPLC), and spectrophotometry). Lipid metabolism was measured by the level of fatty acids, lipid peroxidation products, endocannabinoids and phospholipase A2 activity (GC/MS (gas chromatography/mass spectrometry), LC/MS (liquid chromatography/mass spectrometry), and spectrophotometry). Also, transcription factor Nrf2 (nuclear erythroid 2-related factor) and its activators/inhibitors, peroxisome proliferator-activated receptors (PPAR) and cannabinoid receptor levels were measured (Western blot). Sea buckthorn oil partially prevents UV-induced ROS generation and enhances the level of non-enzymatic antioxidants such as glutathione (GSH), thioredoxin (Trx) and vitamins E and A. Moreover, it stimulates the activity of Nrf2 leading to enhanced antioxidant enzyme activity. As a result, decreases in lipid peroxidation products (4-hydroxynonenal, 8-isoprostaglandin) and increases in the endocannabinoid receptor levels were observed. Moreover, sea buckthorn oil treatment enhanced the level of phospholipid and free fatty acids, while simultaneously decreasing the cannabinoid receptor expression in UV irradiated keratinocytes and fibroblasts. The main differences in sea buckthorn oil on various skin cell types was observed in the case of PPARs—in keratinocytes following UV radiation PPAR expression was decreased by sea buckthorn oil treatment, while in fibroblasts the reverse effect was observed, indicating an anti-inflammatory effect. With these results, sea buckthorn seed oil exhibited prevention of UV-induced disturbances in redox balance as well as lipid metabolism in skin fibroblasts and keratinocytes, which indicates it is a promising natural compound in skin photo-protection.

GSH depletion, mitochondrial membrane breakdown, caspase-3/7 activation and DNA fragmentation in U87MG glioblastoma cells: New insight into the mechanism of cytotoxicity induced by fluoroquinolones

Fluoroquinolones are a known synthetic group of antibiotics that have been the subject of many research interests. This class of antibiotics was shown to be cytotoxic towards various cancer cell lines, thus representing a potentially important source of new anticancer agents. The present study was designed to examine the effect of ciprofloxacin and moxifloxacin on cell viability, redox balance and apoptosis in U87MG glioblastoma cells. Herein, we found that both fluoroquinolones decrease the viability and exert an anti-proliferative effect on U87MG cells. The EC50 values were found to be as 0.75 µmol/ml, 0.57 µmol/ml, 0.53 µmol/ml for ciprofloxacin and 24, 48, 72 h incubation time, respectively, and 0.48 µmol/ml, 0.22 µmol/ml, 0.15 µmol/ml for moxifloxacin and 24, 48, 72 h incubation time, respectively. Ciprofloxacin and moxifloxacin have also induced the intracellular GSH depletion and apoptosis as shown by externalization of phosphatidylserine, caspase-3/7 activation, S and sub-G1 cell cycle arrest, nuclear morphological changes induction and DNA fragmentation. The mechanism of apoptosis was related to the loss of mitochondrial membrane potential suggesting activation of the intrinsic mitochondrial pathway. This is the first study that may provide the basis for understanding potential cellular and molecular mechanism underlying ciprofloxacin and moxifloxacin cytotoxic and pro-apoptotic effect towards U87MG glioblastoma cells, suggesting that these fluoroquinolone derivatives may have value for the development as anti-glioma agents.

Modulation of the cell growth by Aloe Vera extract and hydrogen peroxide

Reactive oxygen species (ROS), reactive nitrogen species, and their counterpart antioxidant agents are essential for physiological signaling, while imbalances between oxidants and antioxidants may provoke pathological reactions causing a range of diseases including cancer. In addition, ROS can stimulate signal transduction pathways and lead to activation of key transcription factors such as Nrf2 and NF-κB modulating cell growth and redox balance [1]. The well-known medical plant Aloe vera contains an abundance of antioxidants, which are capable of neutralising ROS [2]. This work evaluates the effects of the genuine Aloe vera extract (AV) on human cells in vitro in respect to the toxic effects of hydrogen peroxide. After propagating Aloe vera for 1 year in 0.5 kg plastic pots, the first fully developed leaf, fourth from the top, was harvested and subjected to biochemical tests for determination of vitamin C, carotenoids and total soluble phenolic content, while total antioxidant capacity of the plant extract was analysed using Brand-Williams method [3]. Afterwards, four different human cell lines, notably HeLa (human cervical cancer), HMEC (human microvascular endothelial cells), HaCat (human keratinocytes) and HOS (human osteosarcoma) were treated with 2 concentrations of AV for 1 hour after one hour pre-treatment with ranging concentrations of H2O2, thus inducing oxidative stress. The viability of the cells was determined by an MTT-based vitality assay, EZ4U (Biomedica, Vienna, Austria). The plant extract expressed strong antioxidant capacities (1.1 mmol of Trolox eq./g FW), mostly likely due to the combined effects of its antioxidants ingredients. However, while H2O2 inhibited in a concentration dependent manner the growth of all cell lines except HOS, the extract of AV did not show any particular effects except a slight stimulation of the HeLa and HMEC cells. We may assume that antioxidants of the plant extract are not involved in the observed growth modifying effects on the different cell lines used. More likely, some components of the plant extract could interfere with the cellular antioxidants and redox signalling and its effects together with hydrogen peroxide. The above actions of AV confirmed that it can potentially act as relatively safe natural source of medical remedies since there were no toxic effects observed, while further studies on the mechanisms of its actions are needed.

Juglone-ascorbic acid synergy inhibits metastasis and induces apoptotic cell death in poorly differentiated thyroid carcinoma by perturbing SOD and catalase activities.

Anaplastic thyroid carcinoma (ATC) requires more innovative approaches as the current regimes for therapy are inadequate, also most anticancer drugs cause general suppression of physiological functions. However, therapy with limited nontarget tissue damage is desirable. In the present study, we show prooxidant ability of ascorbic acid, which enhances cytotoxicity induced by juglone. We decipher that juglone-ascorbate combination induces reactive oxygen species-mediated apoptosis leading to cell death in ARO cell line originated from ATC. This combination also affects enzyme activity of catalase, glutathione reductase, and superoxide dismutase destabilizing redox balance in cell and thereby making juglone effective at a lower dose. We also show that juglone-ascorbate combination suppresses cell migration, invasion, and expression of tumor-promoting, and angiogenic genes in ARO cell line, thereby disrupting epithelial-mesenchymal transition ability of the cells. Overall, we show that ascorbic acid increases cytotoxic potency of juglone through redox cycling when used in synergy.

Matrix metalloproteinase-2-induced epidermal growth factor receptor transactivation impairs redox balance in vascular smooth muscle cells and facilitates vascular contraction

Increased reactive oxygen species (ROS) formation may enhance matrix metalloproteinase (MMP)-2 activity and promote cardiovascular dysfunction. We show for the first time that MMP-2 is upstream of increased ROS formation and activates signaling mechanisms impairing redox balance. Incubation of vascular smooth muscle cells (VSMC) with recombinant MMP-2 increased ROS formation assessed with dihydroethidium (DHE) by flow cytometry. This effect was blocked by the antioxidant apocynin or by polyethylene glycol-catalase (PEG-catalase), and by MMP inhibitors (doxycycline or GM6001). Next, we showed in HEK293 cells that MMP-2 transactivates heparin-binding epidermal growth factor (HB-EGF) leading to EGF receptor (EGFR) activation and increased ROS concentrations. This effect was prevented by the EGFR kinase inhibitor Ag1478, and by phospholipase C (PLC) or protein kinase C (PKC) inhibitors (A778 or chelerythrine, respectively), confirming the involvement of EGFR pathway in MMP-2-induce responses. Next, we showed that intraluminal exposure of aortas to MMP-2 increased vascular MMP-2 levels detected by immunofluorescence and gelatinolytic activity (by in situ zimography) in association with increased ROS formation. This effect was inhibited by MMP inhibitors (phenanthroline or doxycycline) and by apocynin or PEG-catalase. MMP-2 also increased aortic contractility to phenylephrine and this effect was prevented by MMP inhibitor GM6001 and by apocynin or PEG-catalase, showing again that increased ROS formation mediates functional effects of MMP-2. These results show that MMP-2 activates the EGFR and triggers downstream signaling pathways increasing ROS formation and promoting vasoconstriction. These findings may have various implications for cardiovascular diseases.

Salicylic acid alleviates salinity-caused damage to foliar functions, plant growth and antioxidant system in Ethiopian mustard (Brassica carinata A. Br.)

BackgroundSoil salinity is considered as one of the major environmental factors that has reduced plant productivity worldwide. This study investigates the impact of salinity on plant growth attributes, biochemical and physiological leaf characteristics in two cultivars (Adet and Merawi) of Brassica carinata and also explores the role of salicylic acid (SA) in mitigating the effect of salt stress.MethodsFour-week-old cultivars were treated with NaCl (50, 100 and 150 mM) and SA (0.5 mM) and watered regularly with 100% field capacity. Thus, they were grown under eight different treatments (T1 = no NaCl, no SA; T2 = 0 mM NaCl with 0.5 mM SA; T3 = 50 mM NaCl without SA; T4 = 50 mM NaCl with 0.5 mM SA; T5 = 100 mM NaCl without SA; T6 = 100 mM NaCl with 0.5 mM SA; T7 = 150 mM NaCl without SA; and T8 = 150 mM NaCl with 0.5 mM SA). Nine-week-old cultivars were sampled for analyzing the growth attributes, plant water status, nitrate reductase activity, proline accumulation, photosynthetic traits, lipid peroxidation level and activity of antioxidant enzymes.ResultsSalinity treatments hampered the overall plant growth performance in a dose-dependent manner. Salinity also reduced photosynthetic efficiency by inhibiting chlorophyll synthesis, nitrate reductase activity, chlorophyll fluorescence, stomatal conductance, net photosynthetic and transpiration rates and plant water status. On the other hand, SA application alleviated the adverse effects of salinity and improved the performance of the studied parameters in both the cultivars. Higher dose of salinity increased proline production, but SA application mitigates this impact in both the cultivars studied. The activity of antioxidant enzymes increased under salt stress in a dose-dependent manner. SA treatment to normal or salinity-stressed plants increased the enzymes activity, showing that SA has a crucial role in modulating the cell redox balance and protecting the plants from oxidative damage. SA significantly reduced the salinity-caused effects on the overall performance of plants and their antioxidant systems in both the cultivars. Of the two cultivars, Adet was more tolerant to salinity than Merawi.ConclusionsFoliar application of SA improved the performance of Ethiopian mustard cultivars and mitigated the damage caused by salt stress.

NF-kappaB Regulates Redox Status in Breast Cancer Subtypes.

Oxidative stress (OS) is an indispensable condition to ensure genomic instability in cancer cells. In breast cancer (BC), redox alterations have been widely characterized, but since this process results from a chain of inflammatory events, the causal molecular triggers remain to be identified. In this context, we used a microarray approach to investigate the role of the main pro-oxidant transcription factor, nuclear factor-kappa B (NF-κB), in gene profiles of BC subtypes. Our results showed that NF-κB knockdown in distinct BC subtypes led to differential expression of relevant factors involved in glutathione metabolism, prostaglandins, cytochrome P450 and cyclooxygenase, suggesting a relationship between the redox balance and NF-κB in such cells. In addition, we performed biochemical analyses to validate the microarray dataset focusing on OS and correlated these parameters with normal expression or NF-κB inhibition. Our data showed a distinct oxidative status pattern for each of the three studied BC subtype models, consistent with the intrinsic characteristics of each BC subtype. Thus, our findings suggest that NF-κB may represent an additional mechanism related to OS maintenance in BC, operating in various forms to mediate other important predominant signaling components of each BC subtype.

Novel therapeutic strategy for cancer and autoimmune conditions: Modulating cell metabolism and redox capacity.

Dysregulation of cell metabolism and redox balance is implicated in the pathogenesis and progression of cancer and autoimmune diseases. Because the cell proliferation and apoptotic regulatory pathways are interconnected with metabolic and redox signalling pathways, the current mono-target treatment is ineffective, and multi-drug resistance remains common. Complex diseases are often implicated in a network-based context of pathology; therefore, a new holistic intervention approach is required to block multi-crosstalk in such complicated circumstances. The use of therapeutic agents isolated from herbs to holistically modulate metabolism and redox state has been shown to relieve carcinoma growth and the inflammatory response in autoimmune disorders. Multiple clinically applied or novel herbal chemicals with metabolic and redox modulatory capacity as well as low toxicity have recently been identified. Moreover, new metabolic targets and mechanisms of drug action have been discovered, leading to the exploration of new pathways for drug repositioning, clinical biomarker spectra, clinical treatment strategies and drug development. Taken together with multiple supporting examples, the modulation of cell metabolism and the redox capacity using herbal chemicals is emerging as a new, alternative strategy for the holistic treatment of cancer and autoimmune disorders. In the future, the development of new diagnostic tools based on the detection of metabolic and redox biomarkers, reformulation of optimized herbal compositions using artificial intelligence, and the combination of herbs with mono-targeting drugs will reveal new potential for clinical application.

Imaging of oxygen and hypoxia in cell and tissue samples.

Molecular oxygen (O2) is a key player in cell mitochondrial function, redox balance and oxidative stress, normal tissue function and many common disease states. Various chemical, physical and biological methods have been proposed for measurement, real-time monitoring and imaging of O2 concentration, state of decreased O2 (hypoxia) and related parameters in cells and tissue. Here, we review the established and emerging optical microscopy techniques allowing to visualize O2 levels in cells and tissue samples, mostly under in vitro and ex vivo, but also under in vivo settings. Particular examples include fluorescent hypoxia stains, fluorescent protein reporter systems, phosphorescent probes and nanosensors of different types. These techniques allow high-resolution mapping of O2 gradients in live or post-mortem tissue, in 2D or 3D, qualitatively or quantitatively. They enable control and monitoring of oxygenation conditions and their correlation with other biomarkers of cell and tissue function. Comparison of these techniques and corresponding imaging setups, their analytical capabilities and typical applications are given.

P-196 - Natural killer recognition of primary melanoma cells treated with BRAFV600E inhibitor is favored by heme oxygenase 1 down-regulation

Cell redox balance is crucially driven by heme oxygenase 1 (HO-1) activity. HO-1 up-regulation has been correlated with the gain of resistance to therapy in different types of cancers and its involvement in cancer immune-escape has been hypothesized. We have investigated the role of HO-1 expression in Vemurafenib/PLX4032 (PLX)-treated BRAFV600E melanoma cells (MeOV-1) in modulating natural killer (NK) degranulation and killing. MeOV-1 cell exposure to 1–10 µM PLX4032, which efficiently reduced ERK phosphorylation, was able to induce only a slight decrease of cell viability, while HO-1 expression was upregulated. Moreover, NK cells degranulation and killing activity was decreased upon interaction with MeOV-1 exposed to PLX4032. HO-1 silencing was able to induce a mild but significant reduction of PLX-treated MeOV-1 viability and to completely restore NK ability to degranulate and their cytolytic activity. We provided evidence that HO-1 up-regulation induced by PLX4032 down-regulated B7H6 and ULBP3 expression on MeOV-1 cell surface since HO-1 silencing was able to restore their expression. Our results indicate that HO-1 down-regulation can improve the efficacy of PLX4032 favoring the degranulation and increasing killing activity of NK cells.

To Claim Growth Turf, mTOR Says SOD Off

Maintaining redox balance in cancer cells is essential for tumor development and progression. In this issue of Molecular Cell, Tsang etal. (2018) identify an evolutionarily conserved mTORC1-dependent mechanism by which cancer cells control redox homeostasis in ischemic tumor microenvironment.

The Glycerate and Phosphorylated Pathways of Serine Synthesis in Plants: The Branches of Plant Glycolysis Linking Carbon and Nitrogen Metabolism.

Serine metabolism in plants has been studied mostly in relation to photorespiration where serine is formed from two molecules of glycine. However, two other pathways of serine formation operate in plants and represent the branches of glycolysis diverging at the level of 3-phosphoglyceric acid. One branch (the glycerate – serine pathway) is initiated in the cytosol and involves glycerate formation from 3-phosphoglycerate, while the other (the phosphorylated serine pathway) operates in plastids and forms phosphohydroxypyruvate as an intermediate. Serine formed in these pathways becomes a precursor of glycine, formate and glycolate accumulating in stress conditions. The pathways can be linked to GABA shunt via transamination reactions and via participation of the same reductase for both glyoxylate and succinic semialdehyde. In this review paper we present a hypothesis of the regulation of redox balance in stressed plant cells via participation of the reactions associated with glycerate and phosphorylated serine pathways. We consider these pathways as important processes linking carbon and nitrogen metabolism and maintaining cellular redox and energy levels in stress conditions.

OZONE THERAPY IN LEG ULCERS OF PATIENTS WITH CHRONIC VENOUS INSUFFICIENCY

The term chronic venous insufficiency – CVI (due to a deep vein thrombosis, valvular insufficiency or both) indicates a pathologic condition characterized by alterations of the venous flow and is the main cause in the formation of ulcers in the lower limbs, creating an important socio-medical problem, which finally leads the patient to a total disability for the rest of his/her life. In healthy leg veins, one-way valves allow blood to move only in one direction: upstream towards the heart. This prevents the backward flow of blood, back down the legs. However, in disease leg veins, the valves are enlarged and retracted, in such way, that they become unable to block the retrograde venous flow being the main cause for the appearance of ulcers in the lower limbs. Taking into account the different therapeutic effects of ozone as: improvement of oxygen metabolism and blood rheologic properties, stimulation of the antioxidant defense system achieving the cell redox balance, modulation of the immunological system and nitric oxide, as well as its great germicide power, among others, the aim of this study was to evaluate the efficacy of ozone therapy on the healing process of chronic ulcers of the lower limb in comparison with conventional treatment. A controlled and randomized phase III clinical trial was performed in 90 hospitalized patients with ulcers (over 2 years of evolution, with a diameter of no less than 5 cm) of the lower limbs due to CVI. Efficacy was evaluated according to the least time of hospital stay, which was the necessary time for the lesions to heal or when they were aseptic or their granulation tissue was ready for skin graft. In the cases where the patient did not want to be skin grafted, he/she was discharged and continued treatment at home with topic OLEOZON® (sunflower ozonated oil). The sample was divided at random into 3 groups of 30 patients each: 1-Control group (conventional treatment): venous repose (leg elevation), hyposodic diet, analgesics, daily cure with saline solution and fomentation with antiseptic solutions, several times per day. 2-Ozone group (M-AHT): venous repose (leg elevation), hyposodic diet, analgesics and local ozone treatment (bagging) and topical OLEOZON®, once per day, combining with M-AHT (3 times per week), up to 10 sessions (50 mg/L and 50 mL in 100 mL of blood). 3-Ozone group (Rectal): As M-AHT group but using rectal ozone application up to 20 sessions, once per day (50 mg/L and 200 L). No systemic or local antibiotics were used in the study. Results indicated an average length hospital stay of (53.1 ± 13.9) d for the control group and of (31.5 ± 6.7) d for both ozone group, with significant differences between control and ozone groups. Therefore, hospital stay was reduced for patients with ulcers in the lower limbs treated by ozone therapy, with the subsequent economic saving. No significant differences were observed between the two ozone groups in length hospital stay. No adverse reactions were seen. Ozone therapy can be considered as another treatment within the therapeutical supply for this condition, which could provide a fast rehabilitation for patients as well as their incorporation to a useful life.

Nonylphenol and Octylphenol Differently Affect Cell Redox Balance by Modulating the Nitric Oxide Signaling.

Nonylphenol (NP) and octylphenol (OP) are pervasive environmental contaminants belonging to the broader class of compounds known as alkylphenols, with potential human toxic effects. Classified as “xenoestrogens,” NP and OP are able to interfere with the cell endocrine physiology via a direct interaction with the estrogen receptors. Here, using HepG2 cells in culture, the changes of the cell redox balance and mitochondrial activity induced by OP and NP have been investigated at μM concentrations, largely below those provoking acute toxicity, as those typical of environmental contaminants. Following 24 h cell exposure to both OP and NP, ROS production appeared significantly increased (p ≤ 0.01), together with the production of higher NO oxides (p = 0.003) and peroxynitrated protein-derivatives (NP versus CTR, p = 0.003). The mitochondrial proton electrochemical potential gradient instead was decreased (p ≤ 0.05), as the oxygen consumption by complex IV, particularly following incubation with NP (NP versus CTR, p = 0.017). Consistently, the RT-PCR and Western blot analyses proved that the OP and NP can modulate to a different extent the expression of the inducible NOS (NP versus CTR, p ≤ 0.01) and the endothelial NOS (OP versus CTR, p ≤ 0.05), with a significant variation of the coupling efficiency of the latter (NP versus CTR, p ≤ 0.05), a finding that may provide a novel clue to understand the specific xenoestrogenic properties of OP and NP.

Proton-Translocating Nicotinamide Nucleotide Transhydrogenase: A Structural Perspective

Nicotinamide nucleotide transhydrogenase (TH) is an enzyme complex in animal mitochondria and bacteria that utilizes the electrochemical proton gradient across membranes to drive the production of NADPH. The enzyme plays an important role in maintaining the redox balance of cells with implications in aging and a number of human diseases. TH exists as a homodimer with each protomer containing a proton-translocating transmembrane domain and two soluble nucleotide binding domains that mediate hydride transfer between NAD(H) and NADP(H). The three-domain architecture of TH is conserved across species but polypeptide composition differs substantially. The complex domain coupling mechanism of TH is not fully understood despite extensive biochemical and structural characterizations. Herein the progress is reviewed, focusing mainly on structural findings from 3D crystallization of isolated soluble domains and more recently of the transmembrane domain and the holo-enzyme from Thermus thermophilus. A structural perspective and impeding challenges in further elucidating the mechanism of TH are discussed.

Cereblon and Redox in Plasma Cells

The protein Cereblon (CRBN) has recently attracted considerable interest by virtue of its ability to mediate both anti-tumor as well as teratogenic activities of immunomodulatory drugs (IMiDs) 1-3 . Certain IMiDs have been shown to re-direct CRBN-associated ubiquitylation activity towards neo-substrates such as IKZF1/3 and CK1a, thereby promoting their proteasomal degradation 4-8. This mechanism has been implicated in the anti-proliferative effects of IMiDs in multiple myeloma (MM) and del(5q)-MDS. However, the pleiotropic anti-tumor effects of IMiDs, their adverse effects such as teratogenicity and neurotoxicity, and their synergy with proteasome inhibitors suggests further mechanisms of action. Previous research on CRBN has revealed novel insights that shed light on the physiological functions of this versatile protein in plasma cells. Indeed, next to its function as a ubiquitin ligase, CRBN has been shown to exist in ubiquitin-ligase independent complexes, where it functions as a chaperone for crucial trans-membrane receptors such as CD147 (also known as Basigin) and MCT1 (monocarboxylate transporter 1) 9. This functions links CRBN to the regulation of angiogenesis, cell invasion and particularly cell metabolism by governing lactate homeostasis. Likewise, CRBN has been implicated in the regulation of TrxR (thioredoxin reductase), thereby critically determining the redox balance in plasma cells 10. These findings have important implications for our understanding of IMiD biology and for the development of novel treatment approaches and predictive biomarkers for MM. Indeed, IMiDs inhibit the chaperone function of CRBN in a competitive manner and attenuate TrxR via CRBN, thereby preventing intracellular H2O2 decomposition. In turn, CRBN-dependent degradation of IKZF1 and IKZF3 have been shown to be the result high H2O2 levels. Against this background, CRBN-regulated proteins such as CD147 and MCT1 as well as TrxR may serve as new therapeutic target structures while functional read-outs as CD147 and MCT1 destabilization or antioxidative capacity may serve as predictive biomarkers for IMiD response.1. Ito T, Ando H, Suzuki T, et al. Identification of a primary target of thalidomide teratogenicity. Science . 2010;327(5971):1345- 1350.2. Zhu YX, Braggio E, Shi CX, et al. Cereblon expression is required for the antimyeloma activity of lenalidomide and pomalidomide. Blood . 2011;118(18):4771-4779.3. Lopez-Girona A, Mendy D, Ito T, et al. Cereblon is a direct protein target for immunomodulatory and antiproliferative activities of lenalidomide and pomalidomide. Leukemia . 2012;26(11):2326-2335.4. Kronke J, Udeshi ND, Narla A, et al. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science . 2014;343(6168):301-305.5. Lu G, Middleton RE, Sun H, et al. The myeloma drug lenalidomide promotes the cereblon-dependent destruction of Ikaros proteins. Science . 2014;343(6168):305-309.6. Gandhi AK, Kang J, Havens CG, et al. Immunomodulatory agents lenalidomide and pomalidomide co-stimulate T cells by inducing degradation of T cell repressors Ikaros and Aiolos via modulation of the E3 ubiquitin ligase complex CRL4(CRBN.). Br J Haematol . 2014;164(6):811-821.7. Zhu YX, Braggio E, Shi CX, et al. Identification of cereblon-binding proteins and relationship with response and survival after IMiDs in multiple myeloma. Blood . 2014;124(4):536-545.8. Kronke J, Fink EC, Hollenbach PW, et al. Lenalidomide induces ubiquitination and degradation of CK1alpha in del(5q) MDS. Nature . 2015;523(7559):183-188.9. Eichner R, Heider M, Fernandez-Saiz V, et al. Immunomodulatory drugs disrupt the cereblon-CD147-MCT1 axis to exert antitumor activity and teratogenicity. Nat Med . 2016;22(7):735-743.10. Sebastian S, Zhu YX, Braggio E, et al. Multiple myeloma cells' capacity to decompose H2O2 determines lenalidomide sensitivity. Blood . 2017;129(8):991-1007. DisclosuresBassermann:Celgene: Research Funding.

NADPH Oxidases and Their Roles in Skin Homeostasis and Carcinogenesis.

Skin protects the body from dehydration, pathogens, and external mutagens. NADPH oxidases are central components for regulating the cellular redox balance. There is increasing evidence indicating that reactive oxygen species (ROS) generated by members of this enzyme family play important roles in the physiology and pathophysiology of the skin. Recent Advances: NADPH oxidases are active producers of ROS such as superoxide and hydrogen peroxide. Different isoforms are found in virtually all tissues. They play pivotal roles in normal cell homeostasis and in the cellular responses to various stressors. In particular, these enzymes are integral parts of redox-sensitive prosurvival and proapoptotic signaling pathways, in which they act both as effectors and as modulators. However, continuous (re)activation of NADPH oxidases can disturb the redox balance of cells, in the worst-case scenario in a permanent manner. Abnormal NADPH oxidase activity has been associated with a wide spectrum of diseases, as well as with aging and carcinogenesis. Sunlight with its beneficial and deleterious effects induces the activation of NADPH oxidases in the skin. Evidence for the important roles of this enzyme family in skin cancer and skin aging, as well as in many chronic skin diseases, is now emerging. Understanding the precise roles of NADPH oxidases in normal skin homeostasis, in the cellular responses to solar radiation, and during carcinogenesis will pave the way for their validation as therapeutic targets not only for the prevention and treatment of skin cancers but also for many other skin-related disorders. Antioxid. Redox Signal. 28, 1238-1261.