Oksüdatiivse stressi roll Wolframi sündroom 1 ja hüpotermia korral

Väitekirja elektrooniline versioon ei sisalda publikatsiooneWolframi sündroom on haruldane autosomaalne retsessiivne haigus, mida iseloomustavad juveniilne diabeet (magediabeet, tüüp I suhkrudiabeet), nägemisnärvi kahjustus, kuulmishäired, progressiivne neurodegeneratsioon, endokriinsed kahjustused ja psühhiaatrilised probleemid. Wolframi sündroom on põhjustatud mõlemas alleelis esinevatest mutatsioonidest WFS1 geenis, mille tõttu geeni produkt, wolframiin, ei oma enam tavapärast funktsiooni. Mutantse wolframiini puhul kuhjuvad voltumata valgud endoplasmaatilise retiikulumi luumenisse ning põhjustavad endoplasmaatilise retiikulumi stressi (ka oksüdatiivset stressi) ja sealse homoöstaasi häirumist ja apoptootilise raja käivitumist. Antud töö peaeesmärgiks oli kirjeldada Wfs1-defektiga (KO) hiire metaboloomi ning oksüdatiivse stressi taset erinevates kudedes (maks, süda, neerud ja pankreas) ja biovedelikes (veri ja uriin) enne ja pärast antioksüdantide manustamist. Lisaks analüüsida missugune on hüpotermia mõju glutatiooni (GSH) süsteemile erinevates rakuliinides. Selgus, et nooremad KO hiired kasutavad energia saamiseks eelkõige glükoosi, glükoneogeneesi ja anaeroobset glükolüüsi, kuid hilisemas vanuses kui haigus rohkem progresseerunud, eelistatult lipolüüsi. Lisaks esines noorematel KO hiirtel glükosuuria, mis tüüpiliselt diabeedi varajases staadiumis ei esine. Näitasime, et redutseeritud GSH kontsentratsioon on üldiselt KO hiirte kudedes madalam kui metsiktüüpi (WT) liigikaaslastel. Maksakoes siiski täheldasime mõningast GSH taseme tõusu noortel hiirtel, mis viitab GSH sünteesi intensiivistumisele stressitingimustes. Antioksüdantse ensüümi GSH peroksüdaasi aktiivsus oli südames ja maksas KO hiirtel kõrgem ja GSH reduktaasi aktiivsus madalam võrreldes WT hiirtega. Neerukoes oli mõlema ensüümi aktiivsus KO hiirtel kõrgem. Antioksüdantite manustamine parandas eelkõige GSH taset südames ja maksakoes ning suurendas vanematel ja vähendas noorematel hiirtel GSH süsteemi ensüümide aktiivsust. Hüpotermia-indutseeritud rakkudes on kõrgem totaalse GSH kontsentratsioon metsik-tüüpi hiire embrüonaalsetes fibroblastides ja HeLa rakkudes, kusjuures esimestes vähenes oksüdeeritud GSH tase ja teises jäi see muutumatuks.The deficiency in WFS1 gene causes Wolfram syndrome (WS), which represents a valuable disease model currently available for identifying markers associated with endoplasmic reticulum (ER) stress, juvenile-onset diabetes and neurodegeneration. Another important factor is that WS arises from mutation of a single gene, which makes it a good model for teasing out the mechanisms of ER dysfunction than other multifactorial conditions like diabetes and oxidative stress. Studying oxidative stress and metabolic profiling of Wfs1-deficient mice under hyperglycemic conditions to find therapies aimed at reducing stress in patients or those at risk for developing diabetes. Also this might give new insight of the association between the Wfs1 and its functions. The metabolomic characterization of Wfs1-deficient mice (KO) revealed a broad spectrum of metabolic complications and affected glutathione redox status in the knock-out mice. At the whole organism level, the glucose use, gluconeogenesis and anaerobic glycolysis appear to be increased in the early stages of the disease, but later the energy demand is satisfied by intensified lipolysis. Furthermore, in the blood and liver tissue of KO mice, the progression of the WS exceeds hypouricemia into hyperuricemia. In the pancreas and heart tissue young mice, glycosuria preceded hyperglycemia, which implied to kidney dysfunction. The concentration of reduced glutathione (GSH) was generally decreased in Wfs1-deficient mice, but a slight upregulation of GSH in the liver is probably an attempt to control ER stress. In the liver and heart the activity of glutathione peroxidase was increased and the activity of glutathione reductase was decreased in KO mice compared to wild-type littermates. In the kidneys KO mice, the activity of both enzymes increased. The antioxidants had the highest effect improving the glutathione status in the liver and heart tissue of Wfs1-deficient mice. In the liver tissue, the studied antioxidants mainly reduced the acitivity of glutathione reductase and peroxidase in older mice and inversely in the younger littermates. Hypothermia induced the highest level of total glutathione in wild-type mouse embryonic fibroblasts and HeLa cells, whereas the concentration of oxidized glutathione was decreased or remained unchanged, respectively.https://www.ester.ee/record=b522645

Wolframi sündroom 1 geeni defektiga hiire metaboloomi kirjeldus

Ligipääs piiratud kuni 01.01.20162016-01-0

On the methodological limitations of detecting oxidative stress: effects of paraquat on measures of oxidative status in greenfinches

Oxidative stress (OS) is widely believed to be responsible for the generation of trade-offs in evolutionary ecology by means of constraining investment into a number of components of fitness. Yet, progress in understanding the true role of OS in ecology and evolution has remained elusive. Interpretation of current findings is particularly hampered by the scarcity of experiments demonstrating which of the many available parameters of oxidative status respond most sensitively to and are relevant for measuring OS. We addressed these questions in wild-caught captive greenfinches (Carduelis chloris) by experimental induction of OS by administration of the pro-oxidant compound paraquat with drinking water. Treatment induced 50% mortality, a significant drop in body mass and an increase in oxidative DNA damage and glutathione levels in erythrocytes among the survivors of the high paraquat (0.2 g l(-1) over 7 days) group. Samples taken 3 days after the end of paraquat treatment showed no effect on the peroxidation of lipids (plasma malondialdehyde), carbonylation of proteins (in erythrocytes), parameters of plasma antioxidant protection (total antioxidant capacity and oxygen radical absorbance), uric acid or carotenoids. Our findings of an increase in one marker of damage and one marker of protection from the multitude of measured variables indicate that detection of OS is difficult even under the most stringent experimental induction of oxidative insult. We hope that this study highlights the need for reconsideration of over-simplistic models of OS and draws attention to the limitations of detection of OS due to time-lagged and hormetic upregulation of protective mechanisms. This study also underpins the diagnostic value of measurement of oxidative damage to DNA bases and assessment of erythrocyte glutathione levels

<i>Pseudomonas putida</i> Responds to the Toxin GraT by Inducing Ribosome Biogenesis Factors and Repressing TCA Cycle Enzymes

The potentially self-poisonous toxin-antitoxin modules are widespread in bacterial chromosomes, but despite extensive studies, their biological importance remains poorly understood. Here, we used whole-cell proteomics to study the cellular effects of the Pseudomonas putida toxin GraT that is known to inhibit growth and ribosome maturation in a cold-dependent manner when the graA antitoxin gene is deleted from the genome. Proteomic analysis of P. putida wild-type and &#916;graA strains at 30 &#176;C and 25 &#176;C, where the growth is differently affected by GraT, revealed two major responses to GraT at both temperatures. First, ribosome biogenesis factors, including the RNA helicase DeaD and RNase III, are upregulated in &#916;graA. This likely serves to alleviate the ribosome biogenesis defect of the &#916;graA strain. Secondly, proteome data indicated that GraT induces downregulation of central carbon metabolism, as suggested by the decreased levels of TCA cycle enzymes isocitrate dehydrogenase Idh, &#945;-ketoglutarate dehydrogenase subunit SucA, and succinate-CoA ligase subunit SucD. Metabolomic analysis revealed remarkable GraT-dependent accumulation of oxaloacetate at 25 &#176;C and a reduced amount of malate, another TCA intermediate. The accumulation of oxaloacetate is likely due to decreased flux through the TCA cycle but also indicates inhibition of anabolic pathways in GraT-affected bacteria. Thus, proteomic and metabolomic analysis of the &#916;graA strain revealed that GraT-mediated stress triggers several responses that reprogram the cell physiology to alleviate the GraT-caused damage

E-Cigarette Affects the Metabolome of Primary Normal Human Bronchial Epithelial Cells

<div><p>E-cigarettes are widely believed to be safer than conventional cigarettes and have been even suggested as aids for smoking cessation. However, while reasonable with some regards, this judgment is not yet supported by adequate biomedical research data. Since bronchial epithelial cells are the immediate target of inhaled toxicants, we hypothesized that exposure to e-cigarettes may affect the metabolome of human bronchial epithelial cells (HBEC) and that the changes are, at least in part, induced by oxidant-driven mechanisms. Therefore, we evaluated the effect of e-cigarette liquid (ECL) on the metabolome of HBEC and examined the potency of antioxidants to protect the cells. We assessed the changes of the intracellular metabolome upon treatment with ECL in comparison of the effect of cigarette smoke condensate (CSC) with mass spectrometry and principal component analysis on air-liquid interface model of normal HBEC. Thereafter, we evaluated the capability of the novel antioxidant tetrapeptide O-methyl-l-tyrosinyl-γ-l-glutamyl-l-cysteinylglycine (UPF1) to attenuate the effect of ECL. ECL caused a significant shift in the metabolome that gradually gained its maximum by the 5^th hour and receded by the 7^th hour. A second alteration followed at the 13^th hour. Treatment with CSC caused a significant initial shift already by the 1^st hour. ECL, but not CSC, significantly increased the concentrations of arginine, histidine, and xanthine. ECL, in parallel with CSC, increased the content of adenosine diphosphate and decreased that of three lipid species from the phosphatidylcholine family. UPF1 partially counteracted the ECL-induced deviations, UPF1’s maximum effect occurred at the 5^th hour. The data support our hypothesis that ECL profoundly alters the metabolome of HBEC in a manner, which is comparable and partially overlapping with the effect of CSC. Hence, our results do not support the concept of harmlessness of e-cigarettes.</p></div

The numbers of mass spectrometry signals (mass-to-charge ratios) from the lysate of primary normal human bronchial epithelial cells cultured in air-liquid interface significantly (p<i><</i>0.05) altered by e-cigarette liquid (ECL) (100 μM by nicotine) or 10 μg/mL cigarette smoke condensate (CSC) at the respective time points.

<p>The numbers of signals in case of which 10 μM O-methyl-l-tyrosinyl-γ-l-glutamyl-l-cysteinylglycine (UPF1)/2 mM N-acetylcysteine (NAC) significantly reduced the effect of the respective stimuli are shown in parentheses.</p><p>The numbers of mass spectrometry signals (mass-to-charge ratios) from the lysate of primary normal human bronchial epithelial cells cultured in air-liquid interface significantly (p<i><</i>0.05) altered by e-cigarette liquid (ECL) (100 μM by nicotine) or 10 μg/mL cigarette smoke condensate (CSC) at the respective time points.</p

Time dynamics of the expression of the metabolites of primary normal human bronchial epithelial cells (HBEC) cultured in air-liquid interface being affected by e-cigarette liquid (ECL) (100 μM by nicotine) (black lines) and 10 μg/mL cigarette smoke condensate (CSC) (grey lines) for 13 h.

<p>Solid lines: HBEC treated with ECL (solid black lines) or with CSC (solid grey lines). Long black dashes: HBEC treated with ECL and 10 μM O-methyl-l-tyrosinyl-γ-l-glutamyl-l-cysteinylglycine (UPF1) (added at 1h). Long grey dashes: HBEC treated with CSC and 10 μM UPF1 (added at 1h). Short black dashes: HBEC treated with ECL and 2 mM N-acetylcysteine (NAC) (added at 1h). Short grey dashes: HBEC treated with CSC and 2 mM NAC (added at 1h). For each treatment and for any time point, n = 3. Error bars indicate standard errors of means. (A) arginine ([M+H]⁺ = 175); (B) adenosine diphosphate ([M-H]^- = 426); (C) phosphatidylcholine (18:0/20:4) ([M+H]⁺ = 811); (D) α-ketoglutarate ([M-H]^- = 145). *p < 0.05, ECL-exposed cells versus untreated cells; #p < 0.05, ECL- and UPF1-exposed cells versus untreated cells; ^p < 0.05, ECL- and NAC-exposed cells versus untreated cells; ¤p < 0.05, CSC-exposed cells versus untreated cells; ~p < 0.05, CSC- and UPF1-exposed cells versus untreated cells; “p < 0.05, CSC- and NAC-exposed cells versus untreated cells.</p

Mass spectrum of primary normal human bronchial epithelial cells cultivated in air-liquid interface after exposure to e-cigarette liquid (ECL) (100 μM by nicotine) and 10 μg/mL cigarette smoke condensate (CSC) consisting of 1,822 distinct mass-to-charge signals.

<p>The figure represents the proportions of the spectrum that were significantly (p<i><</i>0.05) affected by addition of ECL (392 signals) or CSC (569 signals) during the first 7 h. There were 138 signals that were significantly affected by both stimuli.</p