Fluctuations in populations of subsurface methane oxidizers in coordination with changes in electron acceptor availability.

The concentrations of electron donors and acceptors in the terrestrial subsurface biosphere fluctuate due to migration and mixing of subsurface fluids, but the mechanisms and rates at which microbial communities respond to these changes are largely unknown. Subsurface microbial communities exhibit long cellular turnover times and are often considered relatively static-generating just enough ATP for cellular maintenance. Here, we investigated how subsurface populations of CH4 oxidizers respond to changes in electron acceptor availability by monitoring the biological and geochemical composition in a 1339 m-below-land-surface (mbls) fluid-filled fracture over the course of both longer (2.5 year) and shorter (2-week) time scales. Using a combination of metagenomic, metatranscriptomic, and metaproteomic analyses, we observe that the CH4 oxidizers within the subsurface microbial community change in coordination with electron acceptor availability over time. We then validate these findings through a series of 13C-CH4 laboratory incubation experiments, highlighting a connection between composition of subsurface CH4 oxidizing communities and electron acceptor availability.

Redox-active and redox-silent compounds: synergistic therapeutics in cancer.

Tumours exhibit higher basal levels of reactive oxygen species (ROS) and altered redox environment compared to normal cells. Excessive level of ROS can be toxic to these cells, thus they become more vulnerable to damage by further ROS insults induced by pharmacological agents. However, the upregulation of antioxidant capacity in adaptation to intrinsic oxidative stress in cancer cells can confer drug resistance. Therefore, abrogation of such drug-resistant mechanisms by redox modulation could have significant therapeutic implications. Many redox-modulating agents have been developed. The redox-active system epitomised by ascorbate-driven quinone redox cycling, and the group of redox-silent vitamin E analogues represented by α-tocopheryl succinate have been shown to induce selective cancer cell death in different types of cancer. These compounds synergistically act by destabilising organelles like mitochondria, unleashing their apoptogenic potential, which results in efficient death of malignant cells and suppression of tumour growth. Consistent with this notion, clinical trials that aim to examine the therapeutic performance of novel redox-modulating drugs in cancer patients are currently under way.

Alpha-tocopheryl succinate and TRAIL selectively synergise in induction of apoptosis in human malignant mesothelioma cells.

Malignant mesothelioma (MM) is a fatal type of neoplasia with poor therapeutic prognosis, largely due to resistance to apoptosis. We investigated the apoptotic effect of alpha-tocopheryl succinate (alpha-TOS), a strong proapoptotic agent, in combination with the immunological apoptogen TNF-related apoptosis-inducing ligand (TRAIL) on both MM and nonmalignant mesothelial cells, since MM cells show low susceptibility to the clinically intriguing TRAIL. All MM cell lines tested were sensitive to alpha-TOS-induced apoptosis, and exerted high sensitivity to TRAIL in the presence of subapoptotic doses of the vitamin E analogue. Neither TRAIL or alpha-TOS alone or in combination caused apoptosis in nonmalignant mesothelial cells. Isobologram analysis of the cytotoxicity assays revealed a synergistic interaction between the two agents in MM cells and their antagonistic effect in nonmalignant mesothelial cells. TRAIL-induced apoptosis and its augmentation by alpha-TOS were inhibited by the caspase-8 inhibitor Z-IETD-FMK and the pan-caspase inhibitor Z-VAD-FMK. Activation of caspase-8 was required to induce apoptosis, which was amplified by alpha-TOS via cytochrome c release following Bid cleavage, with ensuing activation of caspase-9. Enhancement of TRAIL-induced apoptosis in MM cells by alpha-TOS was also associated with upregulation of the TRAIL cognate death receptors DR4 and DR5. Our results show that alpha-TOS and TRAIL act in synergism to kill MM cells via mitochondrial pathway, and are nontoxic to nonmalignant mesothelial cells. These findings are indicative of a novel strategy for treatment of thus far fatal MM.

Coenzyme Q10 levels in idiopathic and varicocele-associated asthenozoospermia.

Levels of coenzyme Q10 (CoQ10) and of its reduced and oxidized forms (ubiquinol, QH2, and ubiquinone, Qox) have been determined in sperm cells and seminal plasma of idiopathic (IDA) and varicocele-associated (VARA) asthenozoospermic patients and of controls. The results have shown significantly lower levels of coenzyme Q10 and of its reduced form, QH2, in semen samples from patients with asthenospermia; furthermore, the coenzyme Q10 content was mainly associated with spermatozoa. Interestingly, sperm cells from IDA patients exhibited significantly lower levels of CoQ10 and QH2 when compared to VARA ones. The QH2/Qox ratio was significantly lower in sperm cells from IDA patients and in seminal plasma from IDA and VARA patients when compared with the control group. The present data suggest that the QH2/Qox ratio may be an index of oxidative stress and its reduction, a risk factor for semen quality. Therefore, the present data could suggest that sperm cells, characterized by low motility and abnormal morphology, have low levels of coenzyme Q10. As a consequence, they could be less capable in dealing with oxidative stress which could lead to a reduced QH2/Qox ratio. Furthermore, the significantly lower levels of CoQ10 and QH2 levels in sperm cells from IDA patients, when compared to VARA ones, enable us to hypothesize a pathogenetic role of antioxidant impairment, at least as a cofactor, in idiopathic forms of asthenozoospermia.

Coenzyme Q blocks biochemical but not receptor-mediated apoptosis by increasing mitochondrial antioxidant protection.

Generation of free radicals is often associated with the induction and progression of apoptosis. Therefore, antioxidants can prove anti-apoptotic, and can help to elucidate specific apoptotic pathways. Here we studied whether coenzyme Q, present in membranes in reduced (ubiquinol) or oxidised (ubiquinone) forms, can affect apoptosis induced by various stimuli. Exposure of Jurkat cells to alpha-tocopheryl succinate (alpha-TOS), hydrogen peroxide, anti-Fas IgM or TRAIL led to induction of apoptosis. Cell death due to the chemical agents was suppressed in cells enriched with the reduced form of coenzyme Q. However, coenzyme Q did not block cell death induced by the immunological agents. Ubiquinol-10 inhibited reactive oxygen species (ROS) generation in cells exposed to alpha-TOS, and a mitochondrially targeted coenzyme Q analogue also blocked apoptosis triggered by alpha-TOS or hydrogen peroxide. Therefore, it is plausible that ubiquinol-10 protects cells from chemically-induced apoptosis by acting as an antioxidant in mitochondria. Our results also indicate that generation of free radicals may not be a critical step in induction of apoptosis by immunological agents.

Evaluation of ischemia-reperfusion damage during coronary angioplasty. Electrocardiographic assessment and biochemical modifications in blood from the coronary sinus.

BACKGROUND: Percutaneous transluminal coronary angioplasty (PTCA) constitutes a clinical model of reperfusion following a short period of ischemia connected to balloon inflation during the procedure. During the procedure some ischemic damage and oxidative injury related to free radical attack might occur. In the present study we investigated the extent of ischemic damage and some biochemical indexes of reperfusion damage in patients undergoing PTCA. METHODS: Twenty-five patients who underwent PTCA because of angiographically detected occlusion of the coronary artery were enrolled. Balloon inflation lasted from 30 to 60 s. ECG changes were monitored throughout the procedure and blood samples were taken from the coronary artery and coronary sinus before balloon inflation, and again from coronary sinus at the peak of ischemia, 2 and 10 min after reperfusion. RESULTS: During PTCA procedure angina pectoris appeared in 62.7% of patients, whereas ST-segment elevation was present in 87% of patients, regressing completely after balloon deflation. Plasma malonyldialdehyde, an index of lipid peroxidation, did not change; coenzyme Q10 (in its oxidized and reduced forms), vitamin E and beta-carotene were also unchanged. Total antioxidant capacity and uric acid decreased upon reperfusion. CONCLUSIONS: Myocardial ischemia occurring during balloon inflation is brief and regresses completely after balloon deflation. Reperfusion following a short period of acute ischemia such as in PTCA does not constitute an oxidative event detectable through a common marker of lipid peroxidation nor does it alter the concentration of lipophilic antioxidants. It only lowers hydrosoluble antioxidants therefore representing a mild oxidative insult.

Coenzyme Q10 enrichment decreases oxidative DNA damage in human lymphocytes.

Ubiquinol-10, the reduced form of coenzyme Q10, is a powerful antioxidant in plasma and lipoproteins. It has been suggested that endogenous ubiquinol-10 also exerts a protective role even towards DNA oxidation mediated by lipid peroxidation. Even though the antioxidant activity of coenzyme Q10 is mainly ascribed to ubiquinol-10, a role for ubiquinone-10 (the oxidized form), has been suggested not only if appropriate reducing systems are present. To investigate whether the concentration of ubiquinol-10 or ubiquinone-10 affects the extent of DNA damage induced by H2O2, we supplemented in vitro human lymphocytes with both forms of coenzyme Q10 and evaluated the DNA strand breaks by Comet assay. The exposure of lymphocytes to 100 microM H2O2 resulted in rapid decrease of cellular ubiquinol-10 content both in ubiquinol-10-enriched and in control cells, whereas alpha-tocopherol and beta-carotene concentration were unchanged. After 30 min from H2O2 exposure, the amount of DNA strand breaks was lower and cells' viability was significantly higher in ubiquinol-10-enriched cells compared with control cells. A similar trend was observed in ubiquinone-10-enriched lymphocytes when compared with control cells. Our experiments suggest that coenzyme Q10 in vitro supplementation enhances DNA resistance towards H2O2-induced oxidation, but it doesn't inhibit directly DNA strand break formation.

Distribution of antioxidants among blood components and lipoproteins: significance of lipids/CoQ10 ratio as a possible marker of increased risk for atherosclerosis.

Total CoQ10 levels were evaluated in whole blood and in plasma obtained from a group of 83 healthy donors. Extraction with light petroleum ether/methanol was more efficient, for whole blood, than the extraction which is often used for plasma and serum, i.e., ethanol hexane. An excellent correlation was present between plasma CoQ10 and whole blood CoQ10. CoQ10 is mainly associated with plasma rather than with cellular components. Positive, significant correlations were found between the LDL-chol/CoQ10 ratio and the total-chol/HDL-chol ratio, which is usually considered a risk factor for atherosclerosis. The proportion of CoQ10 carried by LDL was 58 +/- 10%, while the amount carried by HDL was 26 +/- 8%. In VLDL + IDL CoQ10 was 16 +/- 8%. The content of CoQ10 in single classes of lipoproteins is strictly correlated with CoQ10 plasma concentration. In a parallel study conducted on a population of diabetic patients (one IDDM group and one NIDDM) CoQ10 plasma levels were generally higher compared to the control group, also when normalised to total cholesterol. In particular the LDL fraction showed a CoQ10/chol ratio higher in NIDDM but not in IDDM patients, compared to controls. The CoQ10/triglycerides ratio was lower in NIDDM respect to controls and even lower in IDDM patients.

Intensive predeposit-hemodilution autologous blood donation and IOBS increase in erythropoietic levels in patients undergoing hip arthroplasty.

Erythropoietin (EPO) plasma levels were monitored during the perioperative period in 61 consecutive patients (22 males - 39 females), aged 62.5 +/- 9.5 years, scheduled for hip arthroplasty. All patients underwent intraoperative blood salvage (IOBS) and were subdivided into three different groups according to their hemoglobin levels (Hb) 24 hours postoperatively (group A: Hb < 8 g/dl; group B: Hb between 8-9 g/dl; group C: HB > or = 9 g/dl). Seventy-two hours after surgery EPO levels were significantly different in group A (135 +/- 68) compared to group C (54.3 +/- 32), with a positive correlation (p < 0.01) between Hb and EPO levels. On the basis of these results we suggest that a programmed autologous red blood cell collection aimed at obtaining the lowest hemoglobin values during the first 24 hours after surgery, may be of clinical utility in preventing homologous blood needs.

Oxidation of free fatty acids in low density lipoprotein by 15-lipoxygenase stimulates nonenzymic, alpha-tocopherol-mediated peroxidation of cholesteryl esters.

15-Lipoxygenase has been implicated in the in vivo oxidation of low density lipoprotein (LDL) a process thought to be important in the origin and/or progression of human atherogenesis. We have suggested previously that oxidation of LDL's cholesteryl esters (CE) and phospholipids by soybean (SLO) or human recombinant 15-lipoxygenase (rhLO) can be ascribed largely to alpha-tocopherol (alpha-TOH)-mediated peroxidation (TMP). In this study we demonstrate that addition to LDL of unesterified linoleate (18:2), other free fatty acid (FFA) substrates, or phospholipase A2 (PLA2) significantly enhanced the accumulation of CE hydro(pero)xides (CE-O(O)H) induced by rhLO, whereas the corresponding CE and nonsubstrate FFA were without effect. The enhanced CE-O(O)H accumulation showed a dependence on the concentration of free 18:2 in LDL. In contrast, addition of 18:2 had little effect on LDL oxidation induced by aqueous peroxyl radicals or Cu2+ ions. Analyses of the regio- and stereoisomers of oxidized 18:2 in SLO-treated native LDL demonstrated that the small amounts of 18:2 associated with the lipoprotein were oxidized enzymically and within minutes, whereas cholesteryl linoleate (Ch18:2) was oxidized nonenzymically and continuously over hours. alpha-Tocopheroxyl radical (alpha-TO.) formed in LDL exposed to SLO was enhanced by addition of 18:2 or PLA2. With rhLO and 18:2-supplemented LDL, oxidation of 18:2 was entirely enzymic, whereas that of Ch18:2 was largely, though not completely, nonenzymic. The small extent of enzymic Ch18:2 oxidation increased with increasing enzyme to LDL ratios. Ascorbate and the reduced form of coenzyme Q, ubiquinol-10, which are both capable of reducing alpha-TO. and thereby preventing TMP, inhibited nonenzymic Ch18:2 oxidation induced by rhLO. Trolox and ascorbyl palmitate, which also inhibit TMP, ameliorated both enzymic and nonenzymic oxidation of LDL's lipids, whereas probucol, a radical scavenger not capable of preventing TMP, was ineffective. These results demonstrate that rhLO-induced oxidation of CE is largely nonenzymic and increases with LDL's content of FFA substrates. We propose that conditions which increase LDL's FFA content, such as the presence of lipases, increase 15-LO-induced LDL lipid peroxidation and that this process requires only an initial, transient enzymic activity.

Oxidation of LDL and their subfractions: kinetic aspects and CoQ10 content.

Coenzyme Q10 in its reduced form, ubiquinol-10, although present in LDL at concentrations considerably lower than that of alpha-tocopherol, exerts a potent antioxidant action in this class of lipoproteins. Previous studies indicated that the content of CoQ10 is the lowest in the densest subfraction of LDL, i.e. LDL3, which is commonly regarded as the most peroxidizable and atherogenic one. These levels were associated with the highest levels of hydroperoxides detectable in the three subclasses. Enrichment of LDL with CoQ10, by means of exogenous supplementation, resulted in a significant increase of CoQ10 in LDL, mainly in LDL3, and in a lower extent of peroxidizability. Spontaneous oxidation of ubiquinol was monitored in plasma and in LDL of unsupplemented and of supplemented subjects and the time-course of oxidation was found considerably slower in CoQ10-enriched LDL. The lagphase of conjugated dienes formation upon induced oxidation was significantly correlated with the absolute content of ubiquinol-10. Distribution of CoQ10 among different classes of plasma lipoproteins was also studied: about 60% of plasma CoQ10 was found associated with LDL.

The protective role of ubiquinol-10 against formation of lipid hydroperoxides in human seminal fluid.

Defective sperm function in infertile men has been associated with increased lipid peroxidation and impaired function of antioxidant defenses in spermatozoa. Evidence strongly suggests that CoQ10, a lipid-soluble component of the respiratory chain acts, in its reduced form (ubiquinol), as a potent antioxidant in various biological systems, such as lipoproteins and membranes. In this study we assayed CoQ10 content in both the reduced and oxidized form (ubiquinol/ubiquinone), and hydroperoxide levels in seminal plasma and seminal fluid from 32 subjects with a history of infertility. Our results showed a significant correlation between ubiquinol content and sperm count (r = 0.62; P < 0.05) in seminal plasma. An inverse correlation between ubiquinol content and hydroperoxide levels both in seminal plasma and in seminal fluid (r = -0.56; P = 0.01) was found. Using multiple regression analysis we also found a strong correlation among sperm count, motility and ubiquinol-10 content (P < 0.01) in seminal fluid. An inverse correlation between ubiquinol/ubiquinone ratio and percentage of abnormal morphology was also observed in total fluid. These results suggest that ubiquinol-10 inhibits hydroperoxide formation in seminal fluid and in seminal plasma. Since peroxidation in sperm cells is an important factor affecting male infertility, ubiquinol could assume a diagnostic and/or a therapeutic role in these patients.

The roles of coenzyme Q10 and vitamin E on the peroxidation of human low density lipoprotein subfractions.

The aim of our study was to investigate the relationships between the levels of coenzyme Q10 (CoQ10) and vitamin E and the levels of hydroperoxide in three subfractions of low density lipoproteins (LDL) that were isolated from healthy donors. LDL3, the densest of the three subfractions, has shown statistically significant lower levels of CoQ10 and vitamin E, which were associated with higher hydroperoxide levels when compared with the lighter counterparts. After CoQ10 supplementation, all three LDL subfractions had significantly increased CoQ10 levels. In particular, LDL3 showed the highest CoQ10 increase when compared with LDL1 and LDL2 and was associated with a significant decrease in hydroperoxide level. These results support the hypothesis that the CoQ10 endowment in subfractions of LDL affects their oxidizability, and they have important implications for the treatment of disease.

Physico-chemical properties of membranes of recovered erythrocytes in blood autologous transfusion: a study using fluorescence technique.

Using fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and its derivative trimethylammonium-1,6-diphenyl-1,3,5-hexatriene (TMA-DPH), we have investigated the fluidity in erythrocyte membranes isolated from intraoperatively and postoperatively recovered erythrocytes, compared to membranes isolated from untreated erythrocytes or stored red blood cells (RBCs). Fluorescence polarization (Pf) of DPH and TMA-DPH was not significantly modified in membranes isolated from intraoperatively and postoperatively recovered RBCs compared to membranes isolated from untreated fresh RBCs or stored RBCs. However, using 2-dimethyl-amino-6-lauroyl-naphtalene (Laurdan), a very sensitive molecule, a significant increase in polarity in membranes from stored RBCs compared to untreated, intraoperatively and postoperatively recovered RBCs was observed. Moreover, there was significantly higher susceptibility to oxidative stress in membranes isolated from stored RBCs, compared to untreated and intraoperatively or postoperatively recovered RBC membranes, both at baseline and after oxidative stress. Our results suggest that intraoperative and postoperative recovered RBCs are less damaged than stored RBCs.

Abnormalities of plasma lipoprotein composition and fluidity in psoriasis.

We have studied the lipoprotein chemical composition and fluidity, using the fluorescence polarization of the probe 1,6-diphenyl-1,3,5-hexatriene (DPH) in two age- and sex-matched groups of subjects (14 healthy normolipidemic children and 15 psoriatic children). The compositional study has shown a significant increase of the percentage content in triacylglycerol and a significant decrease of the apo-protein content in all lipoprotein fractions of psoriatic children compared to the controls. A significant increase of the percentage content in total cholesterol and of the cholesterol/protein ratio has also been observed in low-density lipoproteins (LDL) and in high-density lipoproteins (HDL) of psoriatic children. The compositional changes were associated with alterations of fluidity in LDL and HDL of psoriatic patients. The modifications of lipoprotein composition and fluidity observed in psoriatic patients could be of physiopathological and clinical relevance in relation to the pathogenesis of the disease.