Cytochrome C Oxidase Activity Research Articles

Photobiomodulation of Cytochrome c Oxidase by Chronic Transcranial Laser in Young and Aged Brains

In cellular bioenergetics, cytochrome c oxidase (CCO) is the enzyme responsible for oxygen consumption in the mitochondrial electron transport chain, which drives oxidative phosphorylation for adenosine triphosphate (ATP) production. CCO is also the major intracellular acceptor of photons in the light wavelengths used for photobiomodulation (PBM). Brain function is critically dependent on oxygen consumption by CCO for ATP production. Therefore, our objectives were (1) to conduct the first detailed brain mapping study of the effects of PBM on regional CCO activity, and (2) to compare the chronic effects of PBM on young and aged brains. Specifically, we used quantitative CCO histochemistry to map the differences in CCO activity of brain regions in healthy young (4 months old) and aged (20 months old) rats from control groups with sham stimulation and from treated groups with 58 consecutive days of transcranial laser PBM (810 nm wavelength and 100 mW power). We found that aging predominantly decreased regional brain CCO activity and systems-level functional connectivity, while the chronic laser stimulation predominantly reversed these age-related effects. We concluded that chronic PBM modified the effects of aging by causing the CCO activity on brain regions in laser-treated aged rats to reach levels similar to those found in young rats. Given the crucial role of CCO in bioenergetics, PBM may be used to augment brain and behavioral functions of older individuals by improving oxidative energy metabolism.

Cytochrome c Oxidase Activity as a Metabolic Regulator in Pancreatic Beta-Cells.

Pancreatic β-cells couple glucose-stimulated insulin secretion (GSIS) with oxidative phosphorylation via cytochrome c oxidase (COX), a mitochondrial respiratory-chain enzyme. The Cohen diabetic-sensitive (CDs) rats exhibit hyperglycemia when fed a diabetogenic diet but maintain normoglycemia on a regular diet. We have previously reported a decreased COX activity in CDs rats and explored its relevance for type 2 diabetes (T2D). In this study, we investigated the relation between COX activity in islets, peripheral-blood mononuclear cells (PBMCs), and GSIS during diabetes development in CDs rats fed a diabetogenic diet for 4, 11, 20, and 30 days and during reversion to normoglycemia in hyperglycemic CDs rats fed a reversion diet for 7, 11, and 20 days. An oral glucose-tolerance test was performed at different periods of the diets measuring blood glucose and insulin concentrations. COX activity was determined in islets and PBMCs isolated from rats at the different periods of the diets. We demonstrated a progressive reduction in COX activity in CDs-islets that correlated positively with the decreasing GSIS (R2 = 0.9691, p < 0.001) and inversely with the elevation in blood glucose levels (R2 = 0.8396, p < 0.001). Hyperglycemia was initiated when islet COX activity decreased below 46%. The reversion diet restored >46% of the islet COX activity and GSIS while re-establishing normoglycemia. Interestingly, COX activity in PBMCs correlated significantly with islet COX activity (R2 = 0.8944, p < 0.001). Our data support islet COX activity as a major metabolic regulator of β-cells function. The correlation between COX activity in PBMCs and islets may serve as a noninvasive biomarker to monitor β-cell dysfunction in diabetes.

Direct Interaction of Mitochondrial Cytochrome c Oxidase with Thyroid Hormones: Evidence for Two Binding Sites.

Thyroid hormones regulate tissue metabolism to establish an energy balance in the cell, in particular, by affecting oxidative phosphorylation. Their long-term impact is mainly associated with changes in gene expression, while the short-term effects may differ in their mechanisms. Our work was devoted to studying the short-term effects of hormones T2, T3 and T4 on mitochondrial cytochrome c oxidase (CcO) mediated by direct contact with the enzyme. The data obtained indicate the existence of two separate sites of CcO interaction with thyroid hormones, differing in their location, affinity and specificity to hormone binding. First, we show that T3 and T4 but not T2 inhibit the oxidase activity of CcO in solution and on membrane preparations with Ki ≈ 100–200 μM. In solution, T3 and T4 compete in a 1:1 ratio with the detergent dodecyl-maltoside to bind to the enzyme. The peroxidase and catalase partial activities of CcO are not sensitive to hormones, but electron transfer from heme a to the oxidized binuclear center is affected. We believe that T3 and T4 could be ligands of the bile acid-binding site found in the 3D structure of CcO by Ferguson-Miller’s group, and hormone-induced inhibition is associated with dysfunction of the K-proton channel. A possible role of this interaction in the physiological regulation of the enzyme is discussed. Second, we find that T2, T3, and T4 inhibit superoxide generation by oxidized CcO in the presence of excess H2O2. Inhibition is characterized by Ki values of 0.3–5 μM and apparently affects the formation of O2●− at the protein surface. The second binding site for thyroid hormones presumably coincides with the point of tight T2 binding on the Va subunit described in the literature.

Morphological and physiological correlates of among- individual variation in basal metabolic rate in two passerine birds

Basal metabolic rate (BMR) has been shown to be a highly phenotypic flexibility trait within species. A significant proportion of an individual's energy budget is accounted for by BMR, hence among-individual variation in this trait may affect other energetic processes, as well as fitness. In this study, we measured BMR, organ mass, mitochondrial respiration capacities and cytochrome c oxidase (COX) activities in muscle and liver and circulating levels of plasma triiodothyronine (T3) in Chinese bulbuls (Pycnonotus sinensis) and Eurasian tree sparrows (Passer montanus). Our results showed that heart and kidney mass was positively correlated with BMR in Chinese bulbuls, whereas liver and kidney mass was positively correlated with BMR in Eurasian tree sparrows. Regarding metabolic biochemical markers of tissues, state 4 respiration and COX activity in the muscles of the Chinese bulbuls was correlated with BMR, while state 4 respiration in the muscle and liver was correlated with BMR in Eurasian tree sparrows. T3 was significantly and positively correlated with BMR in Chinese bulbuls and Eurasian tree sparrows. Consistent with the above results, our findings suggest that T3 levels play an important role in modulating BMR in Chinese bulbuls and Eurasian tree sparrows. Moreover, individual variation in BMR can be explained partly by morphological and physiological mechanisms.

Seasonal changes in mitochondrial bioenergetics and physiological performance of the bluegill sunfish, Lepomis macrochirus, from a shallow, Midwest river.

As global temperature shifts due to anthropogenic impacts, seasonal temperatures in shallow aquatic ecosystems are expected to increase. Previous studies on freshwater fishes that experience significant temperature changes during the annual seasons found pronounced physiological restructuring not observed in animals inhabiting more thermally stable environments. Studies evaluating mitochondrial bioenergetics in fish are often performed on animals acclimated to constant temperatures in the laboratory. However, natural habitats are much more complex. Fishes may experience substantial daily and seasonal variation in temperature, energy requirements and resource availability, which are impossible to emulate on acclimation studies. Our study explores the effects of these more complex natural environments on whole-organism thermal tolerance and mitochondrial bioenergetics in bluegill sunfish (Lepomis macrochirus), a native fish to the temperate zone of North America. Compensatory mechanisms and variations in physiological thresholds were observed in specimens acclimatized to the fall season compared to specimens acclimatized to spring and summer seasons. Somatic indices, such as relative weights and hepatosomatic indices, showed significant differences across seasons and critical thermal maxima significantly decreased in the cold acclimatized specimens. Liver mitochondria from L. macrochirus also showed significantly higher uncoupled proton conductance, cytochrome c oxidase (COX) activity, and reduced respiratory control ratios in individuals sampled in the colder season. These findings suggest that mechanisms regulating proton conductance and COX activity modulate mitochondrial function across seasons to sustain physiological fitness in ectotherms inhabiting shallow, inland aquatic habitats.

New approaches to the typology and prediction of the effectiveness of therapy for late-life depression

To analyze clinical parameters and effectiveness of therapy in depressed patients of late age with different activity of enzymes of energy, glutamate metabolism and antioxidant glutathione system in platelets and red blood cells. The study included 53 hospitalized patients (41 women and 12 men), aged 60 to 86 years (median - 66 years), with a depressive episode of mild to marked severity within the framework of recurrent depressive disorder, bipolar affective disorder or a single depressive episode according to ICD-10. The patients were examined using clinical, psychometric, biochemical and statistical methods. Psychometric assessment of patients' condition was performed before the start of therapy and on the 28th day of treatment using HAMD-17 and HARS. Blood sampling was carried out to determine the activity of cytochrome c-oxidase (COX), glutathione reductase (GR), glutathione S-transferase (GST) and glutamate dehydrogenase (GDH). Cluster analysis revealed 3 clusters (Cl.1, Cl.2, Cl.3), which differed in biochemical indicators. In Cl.1 (n=24, 45.2%), there was a decrease in the activity of COX (p<0.05) and a decrease of GR activity in red blood cells and GST activity in platelets (p<0.001). Patients of Cl.2 (n=11, 20.8%) had increased COX activity (p<0.001) and decreased GDH activity (p<0.001). In patients of Cl.3 (n=18, 34%) the enzyme activity was similar to that in the controls. Later age at disease onset was observed in Cl.1 compared to Cl.2 (51 years [40; 60.5] and 32 years [19; 59], p=0.052). Compared with Cl.2 and Cl.3, patients of Cl.1 significantly more often (p<0.05) had apathetic depressions (41.7%), while complex depressions were observed almost 2 times less often than in Cl.2 (50% and 91.9%, p<0.05). The effectiveness and tolerability of psychopharmacotherapy was higher in Cl.3. There is a relationship between the nature of changes in metabolic parameters and differences in the phenomenology and course of late-age depression. The results of the study open up new directions in the field of predicting the effectiveness of therapy and the development of personalized therapeutic approaches to improve the effectiveness and safety of treatment of depressed elderly patients.

Chemical composition and actoprotective activity of extracts from the grass Lysimachia punctata L. (Verbena Pointness)

An important task of modern pharmacology and medical chemistry is to expand the range of effective and safe medicines. In this regard, the scientific community is paying more and more attention to objects of plant origin. The aim of the study was to study the chemical composition of the extraction of verbena pointness grass and to evaluate its actoprotective activity. The total content of flavonoids was determined by the spectrophotometric method. The amino acid and trace element composition in the extraction of verbena pointness grass was determined by capillary electrophoresis. The actoroprotective activity of the extraction of verbena pointness grass was evaluated on male mice of the Balb/c line in the forced swimming with load test. The effect of the studied extraction on the change in the activity of succinate dehydrogenase and cytochrome-c oxidase in muscle tissue was also determined. During the study, it was found that 12 free amino acids were found in the analyzed extract, of which 6 are essential (arginine, β-phenylalanine, leucine, methionine, threonine, valine), with a flavonoid content from 0.57% to 0.60%. The analyzed extraction is quantitatively dominated by potassium among trace elements. The use of the studied extract increased the level of efficiency of animals by 156.6% (p&lt;0.05) with an increase in the activity of cytochrome-c-oxidase and succinate dehydrogenase by 40.5% (p&lt;0.05) and 61.5% (p&lt;0.05), respectively. Thus, it was found that the extraction of verbena pointness grass is characterized by a rich chemical composition and the presence of actoprotective potential, which makes it a promising object for further study.

Prospective biomarker study in newly diagnosed glioblastoma: Cyto-C clinical trial.

BackgroundGlioblastoma (GBM) has a 5-year survival rate of 3%-5%. GBM treatment includes maximal resection followed by radiotherapy with concomitant and adjuvant temozolomide (TMZ). Cytochrome C oxidase (CcO) is a mitochondrial enzyme involved in the mechanism of resistance to TMZ. In a prior retrospective trial, CcO activity in GBMs inversely correlated with clinical outcome. The current Cyto-C study was designed to prospectively evaluate and validate the prognostic value of tumor CcO activity in patients with newly diagnosed primary GBM, and compared to the known prognostic value of MGMT promoter methylation status.MethodsThis multi-institutional, blinded, prospective biomarker study enrolled 152 patients with newly diagnosed GBM who were to undergo surgical resection and would be candidates for standard of care. The primary end point was overall survival (OS) time, and the secondary end point was progression-free survival (PFS) time. Tumor CcO activity and MGMT promoter methylation status were assayed in a centralized laboratory.ResultsOS and PFS did not differ by high or low tumor CcO activity, and the prognostic validity of MGMT promoter methylation was confirmed. Notably, a planned exploratory analysis suggested that the combination of low CcO activity and MGMT promoter methylation in tumors may be predictive of long-term survival.ConclusionsTumor CcO activity alone was not confirmed as a prognostic marker in GBM patients. However, the combination of low CcO activity and methylated MGMT promoter may reveal a subgroup of GBM patients with improved long-term survival that warrants further evaluation. Our work also demonstrates the importance of performing large, multi-institutional, prospective studies to validate biomarkers. We also discuss lessons learned in assembling such studies.

The use of antioxidant medicine as an adjunctive therapy in late-onset schizophrenia spectrum disorders

BACKGROUND. Imbalance in the activity of neurotransmitter systems, mitochondrial impairment, and oxidative stress are potential neurobiological factors in the schizophrenia development.&#x0D; AIM. To test the hypothesis about the possibility of a subgroup selection from patients with late-onset schizophrenia spectrum disorders, for which the use of ethylmethylhydroxypyridine succinate as an adjunctive to antipsychotic therapy would be the most effective in relation to symptoms that are relatively more pronounced in patients of this subgroup.&#x0D; MATERIAL AND METHODS. 43 patients (women and men) aged 4578 years with late-onset (after 40 years) schizophrenia spectrum disorders were examined using clinical psychopathological, psychometric, biochemical and statistical research methods. Enzymatic activities of cytochrome C-oxidase, glutamate dehydrogenase, glutathione reductase and glutathione-S-transferase were assessed in blood cells twice (on a patient admission to the hospital and after a 28-day treatment course).&#x0D; RESULTS. Criteria for a patient assignment to the subgroup for ethylmethylhydroxypyridine succinate prescription were: more prominent side effects of pharmacotherapy, predominance of anxiety-hypochondriac symptoms and a lesser severity of psychosis. Before the treatment starting, the subgroup to which ethylmethylhydroxypyridine succinate was prescribed significantly differed in the activity of platelet glutamate dehydrogenase (p=0.031), glutathione-S-transferase (p=0.005), and erythrocyte glutathione reductase (p=0.045). As a result of the treatment course, the severity of symptoms by which the patients receiving ethylmethylhydroxypyridine succinate significantly differed before the starting the treatment, became undistinguished from those in the rest examined patients. After the treatment course, no significant differences in enzymatic activities were found in patients treated with ethylmethylhydroxypyridine succinate from those in other patients.&#x0D; CONCLUSION. This study confirmed the clinical validity of using a medicine with antioxidant properties as an adjunctive therapy to the main treatment in the selected subgroup of patients.

Hippocampal neuropeptide Y2 receptor blockade improves spatial memory retrieval and modulates limbic brain metabolism

IntroductionThe neuropeptide Y (NPY) is broadly distributed in the central nervous system (CNS), and it has been related to neuroprotective functions. NPY seems to be an important component to counteract brain damage and cognitive impairment mediated by drugs of abuse and neurodegenerative diseases, and both NPY and its Y2 receptor (Y2R) are highly expressed in the hippocampus, critical for learning and memory. We have recently demonstrated its influence on cognitive functions; however, the specific mechanism and involved brain regions where NPY modulates spatial memory by acting on Y2R remain unclear. MethodsHere, we examined the involvement of the hippocampal NPY Y2R in spatial memory and associated changes in brain metabolism by bilateral administration of the selective antagonist BIIE0246 into the rat dorsal hippocampus. To further evaluate the relationship between memory functions and neuronal activity, we analysed the regional expression of the mitochondrial enzyme cytochrome c oxidase (CCO) as an index of oxidative metabolic capacity in limbic and non-limbic brain regions. ResultsThe acute blockade of NPY Y2R significantly improved spatial memory recall in rats trained in the Morris water maze that matched metabolic activity changes in spatial memory processing regions. Specifically, CCO activity changes were found in the dentate gyrus of the dorsal hippocampus and CA1 subfield of the ventral hippocampus, the infralimbic region of the PFC and the mammillary bodies. ConclusionsThese findings suggest that the NPY hippocampal system, through its Y2R receptor, influences spatial memory recall (retrieval) and exerts control over patterns of brain activation that are relevant for associative learning, probably mediated by Y2R modulation of long-term potentiation and long-term depression.

Acclimations to Cold and Warm Conditions Differently Affect the Energy Metabolism of Diapausing Larvae of the European Corn Borer Ostrinia nubilalis (Hbn.).

The European corn borer Ostrinia nubilalis is a pest species, whose fifth instar larvae gradually develop cold hardiness during diapause. The physiological changes underlying diapause progression and cold hardiness development are still insufficiently understood in insects. Here, we follow a complex of changes related to energy metabolism during cold acclimation (5°C) of diapausing larvae and compare this to warm-acclimated (22°C) and non-diapause controls. Capillary electrophoresis of nucleotides and coenzymes has shown that in gradually cold-acclimated groups concentrations of ATP/ADP and, consequently, energy charge slowly decrease during diapause, while the concentration of AMP increases, especially in the first months of diapause. Also, the activity of cytochrome c oxidase (COX), as well as the concentrations of NAD+ and GMP, decline in cold-acclimated groups, until the latter part of diapause, when they recover. Relative expression of NADH dehydrogenase (nd1), coenzyme Q-cytochrome c reductase (uqcr), COX, ATP synthase (atp), ADP/ATP translocase (ant), and prohibitin 2 (phb2) is supressed in cold-acclimated larvae during the first months of diapause and gradually increases toward the termination of diapause. Contrary to this, NADP+ and UMP levels significantly increased in the first few months of diapause, after gradual cold acclimation, which is in connection with the biosynthesis of cryoprotective molecules, as well as regeneration of small antioxidants. Our findings evidence the existence of a cold-induced energy-saving program that facilitates long-term maintenance of larval diapause, as well as gradual development of cold hardiness. In contrast, warm acclimation induced faster depletion of ATP, ADP, UMP, NAD+, and NADP+, as well as higher activity of COX and generally higher expression of all energy-related genes in comparison to cold-acclimated larvae. Moreover, such unusually high metabolic activity, driven by high temperatures, lead to premature mortality in the warm-acclimated group after 2 months of diapause. Thus, our findings strongly support the importance of low temperature exposure in early diapause for gradual cold hardiness acquisition, successful maintenance of the resting state and return to active development. Moreover, they demonstrate potentially adverse effects of global climate changes and subsequent increase in winter temperatures on cold-adapted terrestrial organisms in temperate and subpolar regions.

W75. INVESTIGATING GENETIC ALLELES SHARED BETWEEN SCHIZOPHRENIA AND BLOOD METABOLITE CONCENTRATIONS

Ketamine is a noncompetitive antagonist of glutamatergic N-methyl-d-aspartate receptors. Its acute effects on healthy volunteers and schizophrenia patients mimic some acute psychotic, but also cognitive and negative symptoms of schizophrenia, and subchronic treatment with ketamine has been used as an animal model of psychotic disorders. Glutamatergic neurotransmission is tightly coupled to oxidative metabolism in the brain. Quantitative histochemical mapping of cytochrome c oxidase (COX) activity, which reflect long-term energy metabolism, was carried out in rats that received a daily subanaesthetic dose (30 mg/kg) of ketamine for 10 days. In total, COX activity was measured in 190 brain regions to map out metabolic adaptations to the subchronic administration of ketamine. Ketamine treatment was associated with elevated COX activity in nine brain sub-regions in sensory thalamus, basal ganglia, cortical areas, hippocampus and superior colliculi. Changes in pairwise correlations between brain regions were studied with differential correlation analysis. Ketamine treatment was associated with the reduction of positive association between brain regions in 66 % of the significant comparisons. Different layers of the superior colliculi showed the strongest effects. Changes in other visual and auditory brain centres were also of note. The locus coeruleus showed opposite pattern of increased coupling to mainly limbic brain regions in ketamine-treated rats. Our study replicated commonly observed activating effects of ketamine in the hippocampus, cingulate cortex, and basal ganglia. The current study is the first to extensively map the oxidative metabolism in the CNS in the ketamine model of schizophrenia. It shows that ketamine treatment leads to the re-organization of activity in sensory and memory-related brain circuits.

Seeing in the swamp: hydrogen sulfide inhibits eye metabolism and visual acuity in a sulfide-tolerant fish.

In fish, vision may be impaired when eye tissue is in direct contact with environmental conditions that limit aerobic ATP production. We hypothesized that the visual acuity of fishes exposed to hydrogen sulfide (H2S)-rich water would be altered owing to changes in cytochrome c oxidase (COX) activity. Using the H2S-tolerant mangrove rivulus (Kryptolebias marmoratus), we showed that a 10 min exposure to greater than or equal to 200 µM of H2S impaired visual acuity and COX activity in the eye. Visual acuity and COX activity were restored in fish allowed to recover in H2S-free water for up to 1 h. Since K. marmoratus are found in mangrove pools with H2S concentrations exceeding 1000 µM, visual impairment may impact predator avoidance, navigation and foraging behaviour in the wild.

Amino Acid Substitutions in the Non-Ordered Ω-Loop 70–85 Affect Electron Transfer Function and Secondary Structure of Mitochondrial Cytochrome c

The secondary structure of horse cytochrome c with mutations in the P76GTKMIFA83 site of the Ω-loop, exhibiting reduced efficiency of electron transfer, were studied. CD spectroscopy studies showed that the ordering of mutant structure increases by 3–6% compared to that of the WT molecules due to the higher content of β-structural elements. The IR spectroscopy data are consistent with the CD results and demonstrate that some α-helical elements change into β-structures, and the amount of the non-structured elements is decreased. The analysis of the 1H-NMR spectra demonstrated that cytochrome c mutants have a well-determined secondary structure with some specific features related to changes in the heme microenvironment. The observed changes in the structure of cytochrome c mutants are likely to be responsible for the decrease in the conformational mobility of the P76GTKMIFA83 sequence carrying mutations and for the decline in succinate:cytochrome c-reductase and cytochrome c-oxidase activities in the mitoplast system in the presence of these cytochromes c. We suggest that the decreased efficiency of the electron transfer of the studied cytochromes c may arise due to: (1) the change in the protein conformation in sites responsible for the interaction of cytochrome c with complexes III and IV and (2) the change in the heme conformation that deteriorates its optimal orientation towards donor and acceptor in complexes III and IV therefore slows down electron transfer. The results obtained are consistent with the previously proposed model of mitochondrial cytochrome c functioning associated with the deterministic mobility of protein globule parts.

Protective effects of vitexin on cadmium-induced renal toxicity in rats.

Cadmium (Cd) is an industrial contaminant that poses severe threats to human and animal health. Vitexin (VIT) is a polyphenolic flavonoid of characteristic pharmacological properties. We explored the curative role of vitexin on Cd-induced mitochondrial-dysfunction in rat renal tissues. Twenty-four rats were equally divided into four groups and designated as control, Cd, Cd + vitexin and vitexin treated groups. The results showed that Cd exposure increased urea and creatinine levels while decreased creatinine clearance. Cd reduced the activities of antioxidant enzymes, i.e., catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione content in the Cd exposed group. Cd exposure significantly (p < 0.05) elevated the reactive oxygen species (ROS) and Thiobarbituric acid reactive substances (TBARS) levels in rat kidney. Cd also caused a significant (p < 0.05) reduction in the mitochondrial TCA-cycle enzymes, including isocitrate dehydrogenase, succinate dehydrogenase, alpha-ketoglutarate dehydrogenase, and malate-dehydrogenase activities. Besides, mitochondrial respiratory chain enzymes, including NADH-dehydrogenase, coenzyme Q-cytochrome reductase, succinic-coenzyme Q, and cytochrome c-oxidase activities were also decreased under Cd exposure. Cd exposure also damaged the mitochondrial membrane potential (MMP). However, VIT treatment potentially reduced the detrimental effects of Cd in the kidney of rats. In conclusion, our study indicated that the VIT could attenuate the Cd-induced renal toxicity in rats.

Effects of waterborne cadmium exposure on Spinibarbus sinensis hepatopancreas and kidney: Mitochondrial cadmium accumulation and respiratory metabolism

To examine the relationship between heavy metal accumulation in mitochondria and their respiration function in fish during in vivo exposure, juvenile Spinibarbus sinensis were exposed to different waterborne cadmium (Cd) concentrations for up to 28 days. We measured the state III respiration rate and cytochrome c oxidase (CCO) activity of mitochondria in hepatopancreas and kidney and the accumulated Cd concentrations in mitochondria and heat-stable protein (HSP) fractions. Dose- and time-dependent Cd accumulation occurred at different levels in both organs, but was lower in hepatopancreas. When hepatopancreas mitochondrial Cd concentrations in Cd-exposed groups were > 5.5 μg/g dwt, their state III respiration rates were significantly lower than the control. CCO activity of hepatopancreas mitochondria exhibited decreasing dose- and time-dependent trends. However, kidney mitochondria respiratory activities were not affected significantly by Cd exposure. Cd concentrations in kidney HSP fraction were 2–5 times higher than in hepatopancreas under all exposure conditions, and were mainly present as non-deleterious metallothionein (MT)-Cd complexes. These results suggest that Cd accumulation occurred in hepatopancreas and kidney mitochondria of S. sinensis following waterborne Cd exposure, which significantly inhibited the respiration function of hepatopancreas mitochondria but did not have a deleterious effect on kidney mitochondria. The inhibitory pattern of hepatopancreas mitochondrial Cd concentrations related to function exhibited threshold and saturation effects, suggesting the capacity of S. sinensis to manage Cd toxicity. The difference in the relative proportion of Cd occurring as MT-Cd complexes in organs likely causes the organ-specific effects of Cd on hepatopancreas and kidney mitochondrial function.

Roles of HIF‐1 on ventilatory, metabolic and mitochondrial cox responses to hypoxia in rats

The lack of oxygen at high altitudes presents constraints for mammals. One of the mechanisms involved in the response to hypoxia is the stabilization of HIF-1 (Hypoxia Inducible Factor 1) which contributes to ventilatory and metabolic responses, reduces mitochondrial respiration and stimulates anaerobic glycolysis. Upon acute hypoxia exposure, the expression of HIF-1 reaches peak levels after a few hours of hypoxic exposure. In rats, however, the expression of HIF-1 in hypoxia appears to be limited, as are the ventilatory and metabolic responses in comparison to other species better adapted to altitude. The purpose of this study is to better understand the interactions between hypoxia and HIF-1 and to determine if there are other mechanisms possibly related to hypoxic responses. To this end, we measured ventilation and metabolic rate under resting conditions (whole body plethysmography) and mitochondrial respiration of the cerebral cortex and liver (oxygraphy) in adult male SD rats exposed in normoxia (21% O2 - 6h), in hypoxia (10% O2 - 6h), in normoxia after injection of a HIF-1 stabilizer (deferoxamine; 100mg/kg), and in hypoxia after injection of a HIF-1 inhibitor (2-methoxyestradiol; 5mg/kg). Hypoxia reduces metabolism, increases minute ventilation and respiratory rate by 171 and 59% respectively and increases cytochrome c oxidase (COX) activity by an average of 72% in the cortex. Stabilization of HIF-1 in normoxia doesn't result in any of these responses. However, inhibition of HIF-1 in hypoxia decreases ventilatory responses by 30% for minute ventilation and 23% for respiratory rate and completely blocks the increase in COX activity. HIF-1 would therefore be totally responsible for the IV complex responses observed in hypoxia, but would only be partially involved in the ventilatory response. There thus appears to be an interaction between HIF-1 and other factors induced in hypoxia to establish ventilatory responses.

Reactive oxygen species production and the influence of exogenous copper in yeast mitochondria

Mitochondria are essential organelles within eukaryotic cells due to their role in metabolism, ATP production via the oxidative phosphorylation (OXPHOS) pathway, and other regulatory cellular processes. Our aim is to understand how copper in the mitochondria of yeast affects the production of reactive oxygen species due to its influence on the electron transport chain (ETC) and the localization of reactive oxygen species production during yeast lifespan. Reactive oxygen species (ROS) provide signaling in the cells of organisms which is beneficial for cells to maintain homeostasis. However, when there is a surplus of ROS this can lead to harmful and/or lethal results for the cell. To detect ROS, yeast cultures were grown for one to three days and treated with the fluorescent ROS indicator dihydroethidium (DHE), as well as the fluorescent indicators SYTO and DAPI which stain mitochondrial DNA and adenine-thymine rich regions in the DNA respectively. The ROS generated from the yeast was then analyzed via a fluorescence plate reader, fluorescent activated cell sorting (FACS), and fluorescent microscopy to compare methods of determining ROS in the samples. To investigate mitochondrial ROS production, cells were grown in the presence of antimycin A (a Complex III inhibitor), oligomycin (a Complex V inhibitor), or menadione (a futile redox cycling molecule). Detectable increases in ROS were observed through the inhibition of Complex III and in the presence of menadione. We are now using these methods in yeast with a superoxide dismutase 1 (SOD1) gene deletion, to understand the influence of exogenous copper on the generation of ROS. Copper is worth investigating in conjunction with ROS because the mitochondria in mammals and yeast cells contain a labile copper pool located in the matrix, which is used in the metalation of the copper containing enzymes Complex IV, Cytochrome C Oxidase(CcO), and superoxide dismutase (Sod1). It has been established that supplementing cultures with copper increases CcO and Sod1 activity, supporting previous results that exogenous copper contributes to a labile copper pool within the mitochondria to increase enzyme activity. Our results show that small amounts exogenous copper (0.25 mM copper sulfate in restricted nutrient media) extend yeast chronological lifespan, but a dramatic reduction in lifespan is observed when copper levels are increased to 2.0 mM copper sulfate or higher. These data indicates that low levels of exogenous copper in the media is beneficial for yeast in restricted media conditions. Our current work assesses how exogenous copper changes ETC protein complex expression, specifically looking at CcO subunits, and the production of ROS under these conditions.

Transcriptome sequencing and analysis reveals the molecular mechanism of skeletal muscle atrophy induced by denervation.

BackgroundThe molecular mechanism of denervated muscle atrophy is very complex and has not been elucidated to date. In this study, we aimed to use transcriptome sequencing technology to systematically analyze the molecular mechanism of denervated muscle atrophy in order to eventually develop effective strategies or drugs to prevent muscle atrophy.MethodsTranscriptome sequencing technology was used to analyze the differentially expressed genes (DEGs) in denervated skeletal muscles. Unsupervised hierarchical clustering of DEGs was performed. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was used to analyze the DEGs.ResultsResults showed that 2,749 transcripts were up-regulated, and 2,941 transcripts were down-regulated in denervated tibialis anterior (TA) muscles after 14 days of denervation. The up-regulated expressed genes were analyzed through GO and the results demonstrated that biological processes of the up-regulated expressed genes included apoptotic process, cellular response to DNA damage stimulus, aging, and protein ubiquitination; the cellular component of the up-regulated expressed genes included cytoplasm, cytoskeleton, and nucleus; and the molecular function of the up-regulated expressed genes included ubiquitin-protein transferase activity and hydrolase activity. The KEGG pathway of the up-regulated expressed genes included ubiquitin mediated proteolysis, Fc gamma R-mediated phagocytosis, and transforming growth factor-beta (TGF-β) signaling pathway. The biological processes of the down-regulated expressed genes included angiogenesis, tricarboxylic acid cycle, adenosine triphosphate (ATP) biosynthetic process, muscle contraction, gluconeogenesis; the cellular component of the down-regulated expressed genes included mitochondrion, cytoskeleton, and myofibril; and the molecular function of the down-regulated expressed genes included nicotinamide adenine dinucleotide plus hydrogen (NADH) dehydrogenase (ubiquinone) activity, proton-transporting ATP synthase activity, ATP binding, electron carrier activity, cytochrome-c oxidase activity, and oxidoreductase activity. The KEGG pathway of the down-regulated expressed genes included oxidative phosphorylation, tricarboxylic acid cycle, glycolysis/gluconeogenesis, and the PI3K-Akt signaling pathway.ConclusionsA huge number of DEGs were identified in TA muscles after denervation. The up-regulated expressed genes mainly involve in proteolysis, apoptosis, and ageing. The down-regulated expressed genes mainly involve in energy metabolism, angiogenesis, and protein synthesis. This study further enriched the molecular mechanism of denervation-induced muscle atrophy.

CPPU-induced changes in energy status and respiration metabolism of grape young berry development in relation to Berry setting

The energy status, the level of respiration metabolism are improtant index reflecting plant growth state, participates in many growth processes. However, their relationship to berry setting remain unclear. In this study, the grape of ‘kyoho’ of CPPU [CPPU, N-(2-coloro-4-pyridinyl)-N-phenylurea] treated were used to measurement physiological indicators. The results showed that CPPU treatment reduced respiratory rate, reduced Cytochrome C oxidase (CCO) and ascorbic acid oxidase (AAO) activity, reduced NAD kinase (NADK) activity, maintained low nicotinamide adenine denucleotie phosphate (NADP) and nicotinamide adenine denucleotie phosphate reduced form (NADPH) levels, reduced nicotinamide adenine denucleotie (NADH) levels, and slowed down the reduction of energy charge. These results provided strong evidence that CPPU treatment promoted grape berry setting due to increased energy status, increased Embden-Meyerhof pathway (EMP) and the tricarboxylic acid (TCA) cycle respiratory metabolic pathways, decreased pentose phosphate pathway (PPP), and decreased CCO and AAO activity of respiratory terminal oxidase.