Rate-limiting Step Preceding Cytochrome c Release in Cells Primed for Fas-mediated Apoptosis Revealed by Analysis of Cellular Mosaicism of Respiratory Changes

In the present work, Jurkat cells undergoing anti-Fas antibody (anti-Fas)-triggered apoptosis exhibited in increasing proportion a massive release of cytochrome c from mitochondria, as revealed by double-labeling confocal immunofluorescence microscopy. The cytochrome c release was followed by a progressive reduction in the respiratory activity of the last respiratory enzyme, cytochrome c oxidase (COX), and with a little delay, by a decrease in overall endogenous respiration rate, as measured in vivo in the whole cell population. Furthermore, in vivo titration experiments showed that an ~30% excess of COX capacity over that required to support endogenous respiration, found in naive cells, was maintained in anti-Fas-treated cells having lost ~40% of their COX respiratory activity. This observation strongly suggested that only a subpopulation of anti-Fas-treated cells, which maintained the excess of COX capacity, respired. Fractionation of cells on annexin V-coated paramagnetic beads did indeed separate a subpopulation of annexin V-binding apoptotic cells with fully released cytochrome c and completely lacking respiration, and a nonbound cell subpopulation exhibiting nearly intact respiration and in their great majority preserving the mitochondrial cytochrome c localization. The above findings showed a cellular mosaicism in cytochrome c release and respiration loss, and revealed the occurrence of a rate-limiting step preceding cytochrome c release in the apoptotic cascade. Furthermore, the striking observation that controlled digitonin treatment caused a massive and very rapid release of cytochrome c and complete loss of respiration in the still respiring anti-Fas-treated cells, but not in naive cells, indicated that the cells responding to digitonin had already been primed for apoptosis, and that this treatment bypassed or accelerated the rate-limiting step most probably at the level of the mitochondrial outer membrane

Flux control of cytochrome c oxidase in human skeletal muscle

In the present work, by titrating cytochrome c oxidase (COX) with the specific inhibitor KCN, the flux control coefficient and the metabolic reserve capacity of COX have been determined in human saponin-permeabilized muscle fibers. In the presence of the substrates glutamate and malate, a 2.3 ± 0.2-fold excess capacity of COX was observed in ADP-stimulated human skeletal muscle fibers. This value was found to be dependent on the mitochondrial substrate supply. In the combined presence of glutamate, malate, and succinate, which supported an approximately 1.4-fold higher rate of respiration, only a 1.4 ± 0.2-fold excess capacity of COX was determined. In agreement with these findings, the flux control of COX increased, in the presence of the three substrates, from 0.27 ± 0.03 to 0.36 ± 0.08. These results indicate a tight in vivo control of respiration by COX in human skeletal muscle. This tight control may have significant implications for mitochondrial myopathies. In support of this conclusion, the analysis of skeletal muscle fibers from two patients with chronic progressive external ophthalmoplegia, which carried deletions in 11 and 49% of their mitochondrial DNA, revealed a substantially lowered reserve capacity and increased flux control coefficient of COX, indicating severe rate limitations of oxidative phosphorylation by this enzyme

Analogs in the treatment of chronic hepatitis B: real life experience with tenofovir and entecavir

INTRODUCTION: Tenofovir and entecavir are potent antiviral agents. By suppressing viral replication, they induce histological improvement and finally delay the progression of chronic hepatitis B and the development of complications. They are rarely associated with serious side effects. Our data from a real life experience support data from the literature and suggest some minimal difference that may be useful in tailoring therapy.PATIENTS AND METHODS: We retrospectively analyzed 54 patients affected by chronic hepatitis B (31 and 23 treated by entecavir and tenofovir, respectively). Eight patients were cirrhotic. At baseline and 4-12 and 24 weeks after starting therapy, biochemical and virological analysis were performed in all patients. Renal function tests (serum creatinine, creatinine clearance and blood urea), serum (calcium and phosphate blood level) and urine electrolyte were also studied.RESULTS: All the patients reached virological control within 24 weeks. Only in the group treated by tenofovir we observed a complete viral suppression within 12 weeks. Some patients treated with tenofovir showed increased creatinine clearance without serum creatinine alteration. No significant side effects were reported with the exception of one case of persistent headache in the entecavir group for which the drug was suspended.CONCLUSIONS: Entecavir and tenofovir are effective in suppressing viral replication in patients with chronic hepatitis B. Tenofovir is more potent than entecavir and viral replication is blocked within 12 weeks of therapy. Tenofovir administration is associated with slight increase of creatinine clearance without alteration of serum creatinine levels. The choice of one or the other should be made according to target and specific patients characteristics. In patients with high serum viral load where the complete and quick control of viral replication is the main target, tenofovir may represent the best choice

Carvedilol inhibits mitochondrial complex I and induces resistance to H2O2-mediated oxidative insult in H9C2 myocardial cells

AbstractCarvedilol, a β-adrenoreceptor antagonist with strong antioxidant activity, produces a high degree of cardioprotection in a variety of experimental models of ischemic cardiac injury. Although growing evidences suggest specific effects on mitochondrial metabolism, how carvedilol would exert its overall activity has not been completely disclosed. In the present work we have investigated the impact of carvedilol-treatment on mitochondrial bioenergetic functions and ROS metabolism in H9C2 cells. This analysis has revealed a dose-dependent decrease in respiratory fluxes by NAD-dependent substrates associated with a consistent decline of mitochondrial complex I activity. These changes were associated with an increase in mitochondrial H2O2 production, total glutathione and protein thiols content. To evaluate the antioxidant activity of carvedilol, the effect of the exposure of control and carvedilol-pretreated H9C2 cells to H2O2 were investigated. The H2O2-mediated oxidative insult resulted in a significant decrease of mitochondrial respiration, glutathione and protein thiol content and in an increased level of GSSG. These changes were prevented by carvedilol-pretreatment. A similar protective effect on mitochondrial respiration could be obtained by pre-treatment of the cells with a sub-saturating amount of rotenone, a complex I inhibitor.We therefore suggest that carvedilol exerts its protective antioxidant action both by a direct antioxidant effect and by a preconditioning-like mechanism, via inhibition of mitochondrial complex I

Identification of toxigenic fungal species associated with maize ear rot: Calmodulin as single informative gene

Accurate identification of fungi occurring on agrofood products is the key aspect of any prevention and pest management program, offering valuable information in leading crop health and food safety. Fungal species misidentification can dramatically impact biodiversity assessment, ecological studies, management decisions, and, concerning toxigenic fungi, health risk assessment, since they can produce a wide range of toxic secondary metabolites, referred to as mycotoxins. Since each toxigenic fungal species can have its own mycotoxin profile, a correct species identification, hereby attempted with universal DNA barcoding approach, could have a key role in mycotoxins prevention strategies. Currently, identification of single marker for species resolution in fungi has not been achieved and the analysis of multiple genes is used, with the advantage of an accurate species identification and disadvantage of difficult setting up of PCR-based diagnostic assays. In the present paper, we describe our strategy to set up a DNA-based species identification of fungal species associated with maize ear rot, combining DNA barcoding approach and species-specific primers design for PCR based assays. We have (i) investigated the appropriate molecular marker for species identification, limited to mycobiota possibly occurring on maize, identifying calmodulin gene as single taxonomically informative entity; (ii) designed 17 sets of primers for rapid identification of 14 Fusarium, 10 Aspergillus, 2 Penicillium, and 2 Talaromyces species or species groups, and finally (iii) tested specificity of the 17 set of primers, in combination with 3 additional sets previously developed

Two-Dimensional Shear Wave Elastography Versus Transient Elastography: A Non-Invasive Comparison for the Assessment of Liver Fibrosis in Patients With Chronic Hepatitis C

In recent years, several non-invasive methods have been developed for staging liver fibrosis in patients with chronic hepatitis C. A 2D-Shear wave elastography (SWE) technique has been recently introduced on the EPIQ 7 US system (ElastQ), but its accuracy has not been validated in patients with chronic hepatitis C virus (HCV) infection. We enrolled 178 HCV patients to assess their liver fibrosis stage with ElastQ software using transient elastography as a reference standard. The best cut-off values to diagnose ≥ F2, ≥ F3, and F4 were 8.15, 10.31, and 12.65 KPa, respectively. Liver stiffness values had a positive correlation with transient elastography (r = 0.57; p < 0.001). The area under the receiver operating characteristics (AUROC) was 0.899 for ≥ F2 (moderate fibrosis), 0.900 for ≥ F3 (severe fibrosis), and 0.899 for cirrhosis. 2D-SWE has excellent accuracy in assessing liver fibrosis in patients with chronic hepatitis C and an excellent correlation with transient elastograph

Synergistic interaction of fatty acids and oxysterols impairs mitochondrial function and limits liver adaptation during nafld progression

The complete mechanism accounting for the progression from simple steatosis to steatohepatitis in nonalcoholic fatty liver disease (NAFLD) has not been elucidated. Lipotoxicity refers to cellular injury caused by hepatic free fatty acids (FFAs) and cholesterol accumulation. Excess cholesterol autoxidizes to oxysterols during oxidative stress conditions. We hypothesize that interaction of FAs and cholesterol derivatives may primarily impair mitochondrial function and affect biogenesis adaptation during NAFLD progression. We demonstrated that the accumulation of specific non-enzymatic oxysterols in the liver of animals fed high-fat+high-cholesterol diet induces mitochondrial damage and depletion of proteins of the respiratory chain complexes. When tested in vitro, 5α-cholestane-3β,5,6β-triol (triol) combined to FFAs was able to reduce respiration in isolated liver mitochondria, induced apoptosis in primary hepatocytes, and down-regulated transcription factors involved in mitochondrial biogenesis. Finally, a lower protein content in the mitochondrial respiratory chain complexes was observed in human non-alcoholic steatohepatitis. In conclusion, hepatic accumulation of FFAs and non-enzymatic oxysterols synergistically facilitates development and progression of NAFLD by impairing mitochondrial function, energy balance and biogenesis adaptation to chronic injury

Direct‐acting antivirals for HCV treatment in older patients: A systematic review and meta‐analysis

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Low reserve of cytochrome c oxidase capacity in vivo in the respiratory chain of a variety of human cell types

The question of whether and to what extent the in vivo cytochrome c oxidase (COX) capacity in mammalian cells exceeds that required to support respiration is still unresolved. In the present work, to address this question, a newly developed approach for measuring the rate of COX activity, either as an isolated step or as a respiratory chain-integrated step, has been applied to a variety of human cell types, including several tumor-derived semidifferentiated cell lines, as well as specialized cells removed from the organism. KCN titration assays, carried out on intact uncoupled cells, have clearly shown that the COX capacity is in low excess (16-40%) with respect to that required to support the endogenous respiration rate. Furthermore, measurements of O2 consumption rate supported by 0.4 mM tetramethyl-p-phenylenediamine in antimycin-inhibited uncoupled intact cells have given results that are fully consistent with those obtained in the KCN titration experiments. Similarly, KCN titration assays on digitonin-permeabilized cells have revealed a COX capacity that is nearly limiting (7-22% excess) for ADP + glutamate/malate-dependent respiration. The present observations, therefore, substantiate the conclusion that the in vivo control of respiration by COX is much tighter than has been generally assumed on the basis of experiments carried out on isolated mitochondria. This conclusion has important implications for understanding the role of physiological or pathological factors in affecting the COX threshold

Hepatic microRNA Expression by PGC-1α and PGC-1β in the Mouse.

The fine-tuning of liver metabolism is essential to maintain the whole-body homeostasis and to prevent the onset of diseases. The peroxisome proliferator-activated receptor-γ coactivators (PGC-1s) are transcriptional key players of liver metabolism, able to regulate mitochondrial function, gluconeogenesis and lipid metabolism. Their activity is accurately modulated by post-translational modifications. Here, we showed that specific PGC-1s expression can lead to the upregulation of different microRNAs widely implicated in liver physiology and diseases development and progression, thus offering a new layer of complexity in the control of hepatic metabolism