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ScienceDirect Publication: Free Radical Biology and Medicine
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  • Caloric restriction induces H2O2 formation as a trigger of AMPK-eNOS-NO pathway in obese rats: Role for CAMKII

    Publication date: 1 August 2019

    Source: Free Radical Biology and Medicine, Volume 139

    Author(s): Concha F. García-Prieto, Marta Gil-Ortega, Adrián Plaza, F. Javier Manzano-Lista, Raquel González-Blázquez, Martín Alcalá, Pilar Rodríguez-Rodríguez, Marta Viana, Isabel Aránguez, Maik Gollasch, Beatriz Somoza, María S. Fernández-Alfonso

    Abstract

    Caloric restriction (CR) improves endothelial function through the upregulation of adenosine monophosphate-activated protein kinase (AMPK) and endothelial nitric oxide synthase (eNOS). Moreover, hydrogen peroxide (H2O2) is upregulated in yeast subjected to CR. Our aim was to assess if mild short-term CR increases vascular H2O2 formation as a link with AMPK and eNOS activation.

    Twelve-week old Zucker obese (fa/fa) and control Zucker lean male rats were fed a standard chow either ad libitum (AL, n=10) or with a 20% CR (CR, n=10) for two weeks. CR significantly improved relaxation to ACh in fa/fa rats because of an enhanced endogenous production of H2O2 in aortic rings (H2O2 levels fa/faAL=0.5 ± 0.05 nmol/mg vs. H2O2 levels fa/faCR=0.76 ± 0.07 nmol/mg protein; p<0.05). Expression of mitochondrial superoxide dismutase (Mn-SOD) and total SOD activity were increased in aorta from fa/fa animals after CR. In cultured aortic endothelial cells, serum deprivation or 2-deoxy-d-glucose induced a significant increase in: i) superoxide anion and H2O2 levels, ii) p-AMPK/AMPK and p-eNOS/eNOS expression and iii) nitric oxide levels. This effect was reduced by catalase and strongly inhibited by Ca2+/calmodulin-dependent kinase II (CamkII) silencing.

    In conclusion, we propose that mild short-term CR might be a trigger of mechanisms aimed at protecting the vascular wall by the increase of H2O2, which then activates AMPK and nitric oxide release, thus improving endothelium-dependent relaxation. In addition, we demonstrate that CAMKII plays a key role in mediating CR-induced AMPK activation through H2O2 increase.

    Graphical abstract

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  • p66Shc protein through a redox mechanism enhances the progression of prostate cancer cells towards castration-resistance

    Publication date: 1 August 2019

    Source: Free Radical Biology and Medicine, Volume 139

    Author(s): Dannah R. Miller, Matthew A. Ingersoll, Arpita Chatterjee, Brian Baker, Shashank Shrishrimal, Elizabeth A. Kosmacek, Yuxiang Zhu, Pi-Wan Cheng, Rebecca E. Oberley-Deegan, Ming-Fong Lin

    Abstract

    Prostate cancer (PCa) remains the second leading cause of cancer-related deaths in U.S. men due to the development of the castration-resistant (CR) PCa phenotype. A useful cell model for analysis of the molecular mechanism of PCa progression is required for developing targeted therapies toward CR PCa. In this study, we established a PCa cell progressive model in three separate cell lines, of which androgen-independent (AI) cells were derived from respective androgen-sensitive (AS) cells. Those AI PCa cells obtain the biochemical properties of the clinical CR phenotype, including AR and PSA expression as well as enhanced proliferation and tumorigenicity under androgen-deprived conditions. Thus, those AI cells recapitulate CR PCa and exhibit increased oxidant species levels as well as enhanced signaling of proliferation and survival pathways. H2O2 treatment directly enhanced AS cell growth and migration, which was counteracted by antioxidant N-acetyl cysteine (NAC). We further identified p66Shc protein enhances the production of oxidant species which contributes to phenotypic and cell signaling alterations from AS to AI PCa cells. H2O2-treated LNCaP-AS cells had a similar signaling profile to that of LNCaP-AI or p66Shc subclone cells. Conversely, the oxidant species-driven alterations of LNCaP-AI and p66Shc subclone cell signaling is mitigated by p66Shc knockdown. Moreover, LNCaP-AI cells and p66Shc subclones, but not LNCaP-AS cells, develop xenograft tumors with metastatic nodules, correlating with p66Shc protein levels. Together, the data shows that p66Shc enhances oxidant species production that plays a role in promoting PCa progression to the CR stage.

    Graphical abstract

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  • MicroRNAs in cancer cell death pathways: Apoptosis and necroptosis

    Publication date: 1 August 2019

    Source: Free Radical Biology and Medicine, Volume 139

    Author(s): Solmaz Shirjang, Behzad Mansoori, Samira Asghari, Pascal H.G. Duijf, Ali Mohammadi, Morten Gjerstorff, Behzad Baradaran

    Abstract

    To protect tissues and the organism from disease, potentially harmful cells are removed through programmed cell death processes, including apoptosis and necroptosis. These types of cell death are critically controlled by microRNAs (miRNAs). MiRNAs are short RNA molecules that target and inhibit expression of many cellular regulators, including those controlling programmed cell death via the intrinsic (Bcl-2 and Mcl-1), extrinsic (TRAIL and Fas), p53-and endoplasmic reticulum (ER) stress-induced apoptotic pathways, as well as the necroptosis cell death pathway. In this review, we discuss the current knowledge of apoptosis and necroptosis pathways and how these are impaired in cancer cells. We focus on how miRNAs disrupt apoptosis and necroptosis, thereby critically contributing to malignancy. Understanding which and how miRNAs and their targets affect cell death pathways could open up novel therapeutic opportunities for cancer patients. Indeed, restoration of pro-apoptotic tumor suppressor miRNAs (apoptomiRs) or inhibition of oncogenic miRNAs (oncomiRs) represent strategies that are currently being trialed or are already applied as miRNA-based cancer therapies. Therefore, better understanding the cancer type-specific expression of apoptomiRs and oncomiRs and their underlying mechanisms in cell death pathways will not only advance our knowledge, but also continue to provide new opportunities to treat cancer.

    Graphical abstract

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  • The extracellular role of Ref-1 as anti-inflammatory function in lipopolysaccharide-induced septic mice

    Publication date: 1 August 2019

    Source: Free Radical Biology and Medicine, Volume 139

    Author(s): Hee Kyoung Joo, Yu Ran Lee, Eun-Ok Lee, Myoung Soo Park, Sunga Choi, Cuk-Seong Kim, Jin-Bong Park, Byeong Hwa Jeon

    Abstract

    Apurinic/apyrimidinic endonuclease/redox factor-1 (Ref-1), a multifunctional protein secreted from stimulated cells, has been identified as a new serological biomarker. Despite recent reports on the role of Ref-1 in inflammation, the biological function of secreted Ref-1 remains unknown, especially in vivo. This study aimed to evaluate the possible roles of secreted Ref-1 in lipopolysaccharide-induced systemic inflammation in vivo. We generated a secretory Ref-1 adenoviral vector system, AdPPT-LS-Ref-1, by conjugation of preprotrypsin leading sequence (PPT-LS) with full-length Ref-1 sequences. Expression of tumor necrosis factor-α (TNF-α)-induced vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells and lipopolysaccharide (LPS)-induced cyclooxygenase-2 in Raw264.7 cells was inhibited by secretory Ref-1, and this inhibitory effect was abrogated following neutralization of Ref-1 with anti-Ref-1 antibody. Plasma Ref-1 levels following administration of AdPPT-LS-Ref-1 (2 × 109 ifu, i.p.) for 24 h were substantially higher than those recorded following administration of Adβgal (84.6 ± 7.2 ng/ml vs. 4.4 ± 1.5 ng/ml). Treatment with LPS (10 mg/kg, i.v. for 6 h) markedly increased VCAM-1 expression, cathepsin or myeloperoxidase activity, which were significantly suppressed by treatment with AdPPT-LS-Ref-1. Furthermore, LPS-induced cytokines, such as TNF-α, interleukin (IL)-1β, IL-6, and monocyte chemoattractant protein 1, were significantly inhibited in AdPPT-LS-Ref-1-treated mice. However, LPS-induced myeloperoxidase activities were not suppressed by treatment with the redox mutant of secretory Ref-1, AdPPT-LS-Ref-1(C65A/C93A), or wild-type AdRef-1. Collectively, these results suggest that secreted Ref-1 has anti-inflammatory properties and that its redox cysteine residue is associated with the anti-inflammatory activity in vivo. Furthermore, our findings indicate that secretory Ref-1 may be useful as a therapeutic biomolecule against systemic inflammation.

    Graphical abstract

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  • Tissue-specific role of Nrf2 in the treatment of diabetic foot ulcers during hyperbaric oxygen therapy

    Publication date: July 2019

    Source: Free Radical Biology and Medicine, Volume 138

    Author(s): Umapathy Dhamodharan, Amin Karan, Dornadula Sireesh, Alladi Vaishnavi, Arumugam Somasundar, Kesavan Rajesh, Kunka Mohanram Ramkumar

    Abstract

    Hyperbaric oxygen (HBO) therapy is proven to be very successful for diabetic foot ulcer (DFU) treatment due to its antimicrobial effect, increased angiogenesis and enhanced collagen synthesis. The molecular mechanism underlying HBO therapy particularly the involvement of Nrf2 in the wound healing process was investigated in the present study. In addition, we have studied the levels of angiogenic markers in ulcer tissues and their correlation with Nrf2 during HBO therapy compared with standard therapy (Non-HBO) for DFU. A total of 32 Patients were recruited and randomized to standard wound care procedure alone (n = 17) or HBO therapy in combination with standard wound care procedure (n = 15) for 20 days. Our results showed that the tissue levels of Nrf2 along with its downstream targets were significantly increased in patients who underwent HBO therapy when compared to Non-HBO therapy. Further, HBO therapy induced angiogenesis as assessed by increased levels of angiogenesis markers such as EGF, VEGF, PDGF, FGF-2 and CXCL10 in the tissue samples. The expressions of eNOS and nitrite concentrations were also significantly increased in HBO therapy when compared to Non-HBO therapy subjects. Moreover, HBO therapy sensitises the macrophages to release FGF-2 and EGF thereby promotes angiogenesis. Further, it increased the levels of neutrophil attractant CXCL-8 thereby promotes the release of chemokine CCL2, a well-known mediator of neovascularization. The Pearson correlation showed that Nrf2 has a positive correlation with EGF, VEGF and PDGF. In conclusion, the findings of the present study suggest that HBO therapy promotes wound healing by increasing oxygen supply and distribution to damaged tissues, stimulating angiogenesis, decreasing inflammation, and increasing the nitrite levels. Increased levels of Nrf2 transiently regulate the expression of angiogenic genes in wound biopsies, which may result in accelerated healing of chronic wounds.

    Graphical abstract

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  • The endogenous hydrogen sulfide generating system regulates ovulation

    Publication date: July 2019

    Source: Free Radical Biology and Medicine, Volume 138

    Author(s): Anthony Estienne, Valério M. Portela, Yohan Choi, Gustavo Zamberlam, Derek Boerboom, Vickie Roussel, Marie-Charlotte Meinsohn, Mats Brännström, Thomas E. Curry, Misung Jo, Christopher A. Price

    Abstract

    The generation of free-radicals such as nitric oxide has been implicated in the regulation of ovarian function, including ovulation. Tissues that generate nitric oxide typically generate another free-radical gas, hydrogen sulfide (H2S), although little is known about the role of H2S in ovarian function. The hypothesis of this study was that H2S regulates ovulation. Treatment with luteinizing hormone (LH) increased the levels of mRNA and protein of the H2S generating enzyme cystathionine γ-lyase (CTH) in granulosa cells of mice and humans in vivo and in vitro. Pharmacological inhibition of H2S generating enzymes reduced the number of follicles ovulating in mice in vivo and in vitro, and this inhibitory action was reversed by cotreatment with a H2S donor. Addition of a H2S donor to cultured mouse granulosa cells increased basal and LH-dependent abundance of mRNA encoding amphiregulin, betacellulin and tumor necrosis alpha induced protein 6, proteins important for cumulus expansion and follicle rupture. Inhibition of CTH activity reduced abundance of mRNA encoding matrix metalloproteinase-2 and -9 and tissue-type plasminogen activator, and cotreatment with the H2S donor increased the levels of these mRNA above those stimulated by LH alone. We conclude that the H2S generating system plays an important role in the propagation of the preovulatory cascade and rupture of the follicle at ovulation.

    Graphical abstract

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  • Early induction of senescence and immortalization in PGC-1α-deficient mouse embryonic fibroblasts

    Publication date: July 2019

    Source: Free Radical Biology and Medicine, Volume 138

    Author(s): Ignacio Prieto, Alberto Zambrano, Javier Laso, Ana Aranda, Enrique Samper, María Monsalve

    Abstract
    Aims

    Oxidative stress is known to induce early replicative senescence. Senescence has been proposed to work as a barrier to immortalization and tumor development. Here, we aimed to evaluate the impact of the loss of peroxisome proliferator activated receptor γ co-activator 1α (PGC-1α), a master regulator of oxidative metabolism and mitochondrial reactive oxygen species (ROS) generation, on replicative senescence and immortalization in mouse embryonic fibroblasts (MEFs).

    Results

    We found that primary MEFs lacking PGC-1α showed higher levels of ROS than wild-type MEFs at all cell passages tested. The elevated production of ROS was associated with higher levels of oxidative DNA damage and the increased formation of DNA double-strand breaks. Evaluation of the induction of DNA repair systems in response to γ-radiation indicated that the loss of PGC-1α also resulted in a small but significant reduction in their activity. DNA damage induced the early activation of senescence markers, including an increase in the number of β-galactosidase-positive cells, the induction of p53 phosphorylation, and the increase in p16 and p19 protein. These changes were, however, not sufficient to reduce proliferation rates of PGC-1α-deficient MEFs at any cell passage tested. Moreover, PGC-1α-deficient cells escaped replicative senescence.

    Innovation & conclusion

    PGC-1α plays an important role in the control of cellular senescence and immortalization.

    Graphical abstract

    PGC-1α deficient MEFs showed an exacerbated production of ROS compared to PGC-1α+/+ when serially passaged, these increased ROS levels were associated with the early induction of senescence markers. However, PGC-1α deficient MEFs maintained similar proliferation rates to those of PGC-1α+/+ MEFs and immortalized earlier than PGC-1α+/+ MEFs.

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  • Effect of the additional cysteine 503 of vancomycin-resistant Enterococcus faecalis (V583) alkylhydroperoxide reductase subunit F (AhpF) and the mechanism of AhpF and subunit C assembling

    Publication date: July 2019

    Source: Free Radical Biology and Medicine, Volume 138

    Author(s): Yew Kwang Toh, Joon Shin, Asha Manikkoth Balakrishna, Neelagandan Kamariah, Ardina Grüber, Frank Eisenhaber, Birgit Eisenhaber, Gerhard Grüber

    Abstract

    The vancomycin-resistant Enterococcus faecalis alkyl hydroperoxide reductase complex (AhpR) with its subunits AhpC (EfAhpC) and AhpF (EfAhpF) is of paramount importance to restore redox homeostasis. Therefore, knowledge about this defense system is essential to understand its antibiotic-resistance and survival in hosts. Recently, we described the crystallographic structures of EfAhpC, the two-fold thioredoxin-like domain of EfAhpF, the novel phenomenon of swapping of the catalytic domains of EfAhpF as well as the unique linker length, connecting the catalytically active N-and C-terminal domains of EfAhpF. Here, using mutagenesis and enzymatic studies, we reveal the effect of an additional third cysteine (C503) in EfAhpF, which might optimize the functional adaptation of the E. faecalis enzyme under various physiological conditions. The crystal structure and solution NMR data of the engineered C503A mutant of the thioredoxin-like domain of EfAhpF were used to describe alterations in the environment of the additional cysteine residue during modulation of the redox-state. To glean insight into the epitope and mechanism of EfAhpF and -AhpC interaction as well as the electron transfer from the thioredoxin-like domain of EfAhpF to AhpC, NMR-titration experiments were performed, showing a coordinated disappearance of peaks in the thioredoxin-like domain of EfAhpF in the presence of full length EfAhpC, and indicating a stable EfAhpF-AhpC-complex. Combined with docking studies, the interacting residues of EfAhpF were identified and a mechanism of electron transfer of the EfAhpF donor to the electron acceptor EfAhpC is described.

    Graphical abstract

    Image 1



  • Increased serum strontium levels and altered oxidative stress status in early-onset preeclampsia

    Publication date: July 2019

    Source: Free Radical Biology and Medicine, Volume 138

    Author(s): Clara Barneo-Caragol, Eduardo Martínez-Morillo, Susana Rodríguez-González, Paloma Lequerica-Fernández, Ignacio Vega-Naredo, Francisco V. Álvarez

    Abstract
    Background

    Correctly distinguishing preeclampsia (PE), gestational hypertension (GH), and intrauterine growth retardation (IUGR) is a challenge for clinicians due to existing similarities. In our previous study, we showed that serum strontium (Sr) levels were elevated in preeclamptic women compared to healthy and GH pregnant women at the end of pregnancy. The main aim of this study was to evaluate Sr and oxidative stress in PE at the time of symptoms onset and before and compare it with IUGR/GH.

    Methods

    Samples collected at symptoms onset included 77 preeclamptic women and 72 women diagnosed with IUGR/GH divided into two groups according to the gestational extraction week (<34 and ≥ 34). Fifteen patients were also serialized until delivery. Samples collected before symptoms onset included 140 women who developed early-onset PE (E-PE, n = 9), late-onset PE (L-PE, n = 13), IUGR (n = 9), GH (n = 32) and no pathologies (n = 77). Strontium, placental growth factor (PlGF), soluble fms-like tyrosine kinase 1 (sFlt-1), uric acid (UA), creatinine, lipid peroxidation, and total antioxidant activity (TAA) were measured.

    Results

    Mean Sr, sFlt-1/PIGF ratio, UA, and lipid peroxidation/TAA ratio levels were significantly higher (p = 0.002, <0.0001, <0.0001 and = 0.03, respectively) and estimated glomerular filtration rate (eGFR) and TAA significantly lower (p = 0.0008 and < 0.0001, respectively) in E-PE vs other pathologies when gestational extraction week was <34. There was a significant correlation between Sr and eGFR (r = 0.43, p = 0.02), sFlt-1/PIGF ratio (r = 0.56, p = 0.002), TAA and gestational week of sampling (r = −0.45, p = 0.02) and UA (r = −0.82, p < 0.0001) in the E-PE serial samples. No differences were found in Sr levels before symptoms onset.

    Conclusion

    Serum Sr concentration and oxidative status are increased in E-PE when compared to other pathologies at the time of symptoms onset. More studies are needed to elucidate the causes of Sr levels elevation and its role in the pathophysiology of PE.

    Graphical abstract

    Image 1



  • Dietary intake of inorganic nitrate in vegetarians and omnivores and its impact on blood pressure, resting metabolic rate and the oral microbiome

    Publication date: July 2019

    Source: Free Radical Biology and Medicine, Volume 138

    Author(s): Ann Ashworth, Craig Cutler, Garry Farnham, Luke Liddle, Mia Burleigh, Ana Rodiles, Carla Sillitti, Michele Kiernan, Melanie Moore, Mary Hickson, Chris Easton, Raul Bescos

    Abstract

    Vegetarian diets are commonly associated with lower blood pressure levels. This has been related to greater consumption of inorganic nitrate, since vegetables are the main source of this anion. Dietary nitrate is reduced to nitrite by commensal bacteria in the mouth, which in turn leads to increased circulatory nitrite availability. Nitrite can form nitric oxide by several pathways promoting a reduction in the vascular tone and lower blood pressure. This study tested whether vegetarians have higher concentrations of nitrite in saliva and plasma, and lower blood pressure and resting metabolic rate (RMR), due to higher intakes of nitrate, compared to omnivores. Following a non-randomized, cross-over and single-blinded design we measured dietary nitrate intake, blood pressure and RMR in young and healthy vegetarians (n = 22) and omnivores (n = 19) with similar characteristics after using placebo or antibacterial mouthwash for a week to inhibit oral bacteria. Additionally, we analyzed salivary and plasma nitrate and nitrite concentrations, as well as the oral nitrate-reduction rate and oral microbiome in both groups. Dietary nitrate intake in vegetarians (97 ± 79 mg/day) was not statistically different (P > 0.05) to omnivores (78 ± 47 mg/day). Salivary and plasma nitrate and nitrite concentrations were similar after placebo mouthwash in both groups (P > 0.05). The oral nitrate-reducing capacity, abundance of oral bacterial species, blood pressure and RMR were also similar between vegetarians and omnivores (P > 0.05). Antibacterial mouthwash significantly decreased abundance of oral nitrate-reducing bacterial species in vegetarians (_16.9%; P < 0.001) and omnivores (_17.4%; P < 0.001), which in turn led to a significant reduction of the oral nitrate-reducing capacity in vegetarians (−78%; P < 0.001) and omnivores (−85%; P < 0.001). However, this did not lead to a significant increase in blood pressure and RMR in either groups (P > 0.05). These findings suggest that vegetarian diets may not alter nitrate and nitrite homeostasis, or the oral microbiome, compared to an omnivore diet. Additionally, inhibition of oral nitrite synthesis for a week with antibacterial mouthwash did not cause a significant raise in blood pressure and RMR in healthy, young individuals independent of diet.

    Graphical abstract

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