Cytochrome-c Reductase Research Articles

Alpha-Mangostin and its nano-conjugates induced programmed cell death in Acanthamoeba castellanii belonging to the T4 genotype.

Acanthamoeba are free living amoebae that are the causative agent of keratitis and granulomatous amoebic encephalitis. Alpha-Mangostin (AMS) is a significant xanthone; that demonstrates a wide range of biological activities. Here, the anti-amoebic activity of α-Mangostin and its silver nano conjugates (AMS-AgNPs) were evaluated against pathogenic A. castellanii trophozoites and cysts in vitro. Amoebicidal assays showed that both AMS and AMS-AgNPs inhibited the viability of A. castellanii dose-dependently, with an IC50 of 88.5 ± 2.04 and 20.2 ± 2.17μM, respectively. Both formulations inhibited A. castellanii-mediated human keratinocyte cell cytopathogenicity. Functional assays showed that both samples caused apoptosis through the mitochondrial pathway and reduced mitochondrial membrane potential and ATP production, while increasing reactive oxygen species (ROS) and nicotinamide adenine dinucleotide phosphate (NADPH) cytochrome-c reductase in the cytosol. Whole transcriptome sequencing of A. castellanii showed the expression of 826 genes, with 447 genes being up-regulated and 379 genes being down-regulated post treatment. The Kyoto Encyclopedia of Genes and Genomes analysis showed that the majority of genes were linked to apoptosis, autophagy, RAP1, AGE-RAGE and oxytocin signalling pathways. Seven genes (PTEN, H3, ARIH1, SDR16C5, PFN, glnA GLUL, and SRX1) were identified as the most significant (Log2 (FC) value 4) for molecular mode of action in vitro. Future in vivo studies with AMS and nanoconjugates are needed to realize the clinical potential of this work.

Methoxol's Impact on the Hepatic Microsomal Mixed Function Oxidase System Induced by Phenobarbital and Pyrene in Rats

Insecticides like methomyl (Lannate) are commonly used to keep pests at bay in vineyards. The mixed-function oxidase system undergoes dramatic change after being exposed to methomyl. This research set out to determine if methomyl inhibits phenobarbital-induced CYP 2B1, 2B2, and 3A, as well as benzo[a]pyrene-induced CYP 2D6 (CYP 1A1). There were a total of eight groups, each including six adult male rats. Calcium precipitation was used to separate the microsomes. Extinction coefficients were used to quantify the amounts of the electron transport components cytochrome P450, cytochrome B5, and cytochrome c-reductase. Drug metabolizing enzymes were tested for their efficacy. Mixed function oxidase was significantly elevated in rats when exposed to inducers like phenobarbital and benzo[a]pyrene. Inducer-(Phenobarbital, benzop [a] pyrene) pretreated rats given 4mg/kg methomyl had significantly lower levels of electron transport components and drug-metabolizing enzyme activity compared to inducer treatment alone. The enzymes involved in mixed-function oxidation were likewise affected by methomyl's influence on the induction of phenobarbital. The levels of electron transport components and drug metabolizing enzymes in methomyl-pretreated rats treated with benzo[a]pyrene were much lower than in rats treated with benzo[a]pyrene alone. These findings show that phenobarbital and benzo[a]pyrene increased cytochrome P450 isoform susceptibility to methomyl (CYP 2B1, 2B2, 3A; and CYP 1A1) and the induction pattern of some inducers with respect to CYP 450 isoforms are both modified by methomyl

Sodium chloride-induced oxidation of bighead carp (Aristichthys nobilis) fillets: The role of mitochondria and underlying mechanisms

The NaCl addition is associated closely with oxidation of meat, and mitochondria are the key regulator in oxidation process of proteins. This study was set out to find a potential mechanism of oxidation induced by different concentrations NaCl (0 M, 0.5 M, 1.0 M and 2.0 M) based on the mitochondria pathway in fish. With the increase in concentrations of NaCl, the oxidation levels in bighead carp fillets moved to a higher degree. Samples showed significant lower mitochondrial membrane potential and permeability than control after being treated with 1.0 M and 2.0 M NaCl for 3 h. The activities of succinate dehydrogenase and cytochrome-c reductase decreased significantly in 0.5 M, 1.0 M and 2.0 M NaCl-treated samples and reactive oxygen species content increased in 2.0 M NaCl-treated samples compared to 0 M group. NaCl also destroyed the structure of the mitochondrial membranes and together influenced the activities of peroxisomes. Overall, 2.0 M NaCl promotes the lipid or protein oxidation in fillets might due to its effects on structure, complexes and antioxidant enzymes activity in mitochondria.

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.

THE ANTIHYPOXIC ACTIVITY OF SIBERIAN FIR ESSENTIAL OIL FRACTIONS

The component composition and antihypoxic activity of Siberian fir (Abies sibirica) essential oil fractions were researched. The foliage of Siberian fir, collected on the territory of the Ilyinsky district of the Perm region in a dark coniferous forest was the raw material for obtaining the essential oil. Samples for the research were collected in December 2018 from trees 40–50 years old. The essential oil was obtained with the Clevenger apparatus. Determination of the component composition of Siberian fir essential oil fractions was made by using chromatography-mass-spectrometry. Experiments for determination of antihypoxic activity of Siberian fir essential oil fractions were made on a hypoxic hypoxia model with hypercapnia. According to the results of the research, it was found that the first fraction is more saturated with monoterpene compounds, but the proportion of sesquiterpenoids increases in following fractions. The main components of all fir oil fractions are: bornylacetate, limonene, ∆3-carene, karyophyllene. It was found that the fractions 1 and 2 have the highest antihypoxic activity in comparison with the reference sample. For further research, we offer fraction 1, because it contains the high level of bornylacetate, and the lowest content of ∆3-carene, which inhibits the ubiquinol cytochrome-C reductase complex, interrupting mitochondrial and cell respiration.

Trigonella stellata reduced the deleterious effects of diabetes mellitus through alleviation of oxidative stress, antioxidant- and drug-metabolizing enzymes activities

Ethnopharmacological relevanceGenus Trigonella has a history of folkloric medicinal uses in China, Japan, Egypt and India. There are a variety of therapeutic actions of Trigonella including hypocholesterolemia, hypoglycemia, antibacterial, antiviral, anti-inflammatory activities, antioxidants and appetite stimulant. Aim of the studyThe prevalence of diabetes mellitus is increasing annually. The present study aims at investigating the protective effects of Trigonella stellata against the adverse effects of diabetes mellitus through investigation of the changes in phase I & II drug-metabolizing enzyme activities, protein expression of cytochrome P450 isoenzymes [CYP2E1 & 3A4], oxidative stress, antioxidant enzymes as well as histopathology of both liver and kidney tissues. MethodsGC-MS and MALDI-TOF were used to analyze the main constituents of the aqueous and the ethanolic extract of Trigonella stellata. Western immunoblotting technique used to show the protein expression of CYP450 isozymes in different groups. Spectrophotometric- and fluorophotometric techniques were also used for assessment of different hepatic integrity enzymes. Histopathological techniques used to illustrate the changes in the tissues of both livers and kidneys after different treatments. ResultsTrigonelline was found to be the main constituent of both aqueous and ethanolic extract of Trigonella stellata. Administration of the aqueous and/or the ethanolic extracts of Trigonella stellata to the diabetic rats was found to decrease the blood glucose level, the biochemical markers of both liver (transaminases activities, Lactate dehydrogenase, gamma-glutamyl transferase) and the renal functions (urea, creatinine and bilirubin) which were increased in diabetic-treated rats relative to their normal levels. Diabetes mellitus potentially induced the oxidative stress, and also activities of dimethylnitrosamine N-demethylase I, cytochrome c-reductase, ethoxyresourfin O-deethylase, and the total hepatic content of cytochrome P450. On the other hand, the activity of catalase [CAT], superoxide dismutase [SOD], glutathione S-transferase [GST], glutathione reductase [GR], glutathione peroxidase [GPx] and levels of reduced glutathione [GSH] were potentially inhibited in diabetic rats compared to the control rats. However, treatments of diabetic rats with either aqueous and/or ethanolic extracts of Trigonella stellata restored such changes caused by diabetes almost nearly to their normal levels compared to the control group. Supporting the activity of dimethyl nitrosamine N-demethylase I activity, the protein expression of CYP2E1 was also induced in diabetic rats. However, the aqueous extract of Trigonella stellata was more effective than ethanolic extract in restoring the changes in the protein expression of CYP2E. On the other hand, the protein expression of CYP3A4 was markedly decreased in diabetic rats, and this decrease was partially restored to its normal level after treatment of diabetic rats with aqueous and/or ethanolic extracts. In addition, Trigonella stellata extracts alleviated the histopathological changes in livers and kidneys caused by diabetes mellitus. ConclusionIt is concluded that diabetes mellitus induced changes in oxidative stress, phase I & II drug-metabolizing enzymes, and antioxidant enzymes activities, whereas both extracts of Trigonella stellata alleviated such changes. Alterations in cytochrome P450 system should be considered when therapeutic drugs are administered to diabetic patients since most of xenobiotic are mainly metabolized by this system.

Protein kinase Cα mediates recovery of renal and mitochondrial functions following acute injury.

Previously, we have shown that active protein kinase Cα (PKCα) promotes recovery of mitochondrial function after injury invitro [Nowak G & Bakajsova D (2012) Am J Physiol Renal Physiol 303, F515-F526]. This study examined whether PKCα regulates recovery of mitochondrial and kidney functions after ischemia-induced acute injury (AKI) invivo. Markers of kidney injury were increased after bilateral ischemia and returned to normal levels in wild-type (WT) mice. Maximum mitochondrial respiration and activities of respiratory complexes and Fo F1 -ATPase decreased after ischemia and recovered in WT mice. Reperfusion after ischemia was accompanied by translocation of active PKCα to mitochondria. PKCα deletion reduced mitochondrial respiration and activities of respiratory complex I and Fo F1 -ATPase in noninjured kidneys, indicating that PKCα is essential in developing fully functional renal mitochondria. These changes in PKCα-deficient mice were accompanied by lower levels of complex I subunits (NDUFA9 and NDUFS3) and the γ-subunit of Fo F1 -ATPase. Also, lack of PKCα exacerbated ischemia-induced decreases in respiration, complex I and Fo F1 -ATPase activities, and blocked their recovery after injury, indicating a crucial role of PKCα in promoting mitochondrial recovery after AKI. Further, PKCα deletion exacerbated acetylation and succinylation of key mitochondrial proteins of energy metabolism after ischemia due to decreases in deacetylase and desuccinylase (sirtuin3 and sirtuin5) levels in renal mitochondria. Thus, our data show a novel role for PKCα in regulating levels of mitochondrial sirtuins and acetylation and succinylation of key mitochondrial proteins. We conclude that PKCα deletion: (a) affects renal physiology by decreasing mitochondrial capacity for maximum respiration; (b) blocks recovery of mitochondrial functions, renal morphology, and functions after AKI; and (c) decreases survival after AKI. ENZYMES: Protein kinase C: EC 2.7.11.13; NADH:ubiquinone reductase (H+ -translocating; complex I): EC 7.1.1.2; FoF1-ATPase (H+ -transporting two-sector ATPase): EC 7.1.2.2; Succinate:ubiquinone oxidoreductase (complex II): EC 1.3.5.1; Ubiquinol:cytochrome-c reductase (complex III): EC 7.1.1.8; Cytochrome c oxidase (complex IV): EC 1.9.3.1; NAD-dependent protein deacetylase sirtuin-3, mitochondrial: EC 2.3.1.286; NAD-dependent protein deacetylase sirtuin-5, mitochondrial: EC 3.5.1.-; Proteinase K (peptidase K): EC 3.4.21.64.

Epigenetics and Mitochondrial Stability in Diabetic Retinopathy

Mitochondria constantly fuse and divide to form a cellular network, and this dynamic fusion-fission controls their function and genome stability. Although both, mitofusin 1 and 2 (Mfn1 and 2) regulate outer mitochondrial membrane fusion, Mfn2 itself is sufficient to modulate mitochondrial metabolism. In diabetes, retinal mitochondria are swollen, respiration is impaired, Mfn2 is decreased and mtDNA is damaged. Diabetes also facilitates epigenetic modifications, altering gene expressions without changing their DNA sequences. This study aims to investigate the role of DNA methylation of Mfn2 in the development of diabetic retinopathy. Methods: Human retinal endothelial cells, incubated in normal (5mmols/L) or high glucose (20mmols/L) for four days, in the presence/absence of DNA methyltransferase (Dnmt) inhibitors (5-Azacytidine or Dnmt1-siRNA), were analyzed for methylated cytosine (5mC) at Mfn2 promoter by immunocapturing method. Mitochondrial integrity was evaluated by membrane permeability, coenzyme Q: cytochrome-c reductase (complex-III) activity, and transcripts of mtDNA-encoded cytochrome b (Cytb). Similar measurements were made in the retinal microvessels from streptozotocin-induced diabetic mice receiving 5-Aza (2.5mg/kg, i.p.) or Dnmt1-siRNA (2µg/2µl, intravitreal). Results: High glucose increased 5mC levels at Mfn2 promoter by over 2 fold, and decreased Mfn2 expression. Dnmt inhibition ameliorated glucose-induced increase in 5mC at Mfn2 and mitochondrial membrane permeability, and decrease in Mfn2 expression, complex III activity and Cytb transcripts. Similarly, in mouse retinal microvessels, inhibition of Dnmt prevented diabetes-induced increase in 5mC at Mfn2 promoter and decrease in Mfn2 expression. Conclusions: In diabetes, epigenetic modifications of Mfn2 promoter impair mitochondrial functional and genomic stability. Thus, targeting DNA methylation could help maintain mitochondrial homeostasis, and halt/retard the development of diabetic retinopathy. Disclosure R. Kowluru: None. M. Mishra: None.

Effect on oxidative stress, hepatic chemical metabolizing parameters, and genotoxic damage of mad honey intake in rats.

A total of 66 male Wistar rats were used and six groups (control: 10 animals and experimental: 12 animals) were formed. While a separate control group was established for each study period, mad honey application to the animals in the experimental group was carried out with a single dose (12.5 g kg-1 body weight (b.w.); acute stage), at a dose of 7.5 g kg-1 b.w. for 21 days (subacute stage), and at a dose of 5 g kg-1 b.w. for 60 days (chronic stage). Tissue and blood oxidative stress markers (malondialdehyde (MDA), nitric oxide (NO), 4-hydroxynonenal (HNE), superoxide dismutase, catalase, glutathione (GSH) peroxidase, and glucose-6-phosphate dehydrogenase), hepatic chemical metabolizing parameters in the liver (cytochrome P450 2E1, nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase, nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome c reductase (CYTC), GSH S-transferase (GST), and GSH), and micronucleus and comet test in some samples were examined. Findings from the study showed that single and repeated doses given over the period increased MDA, NO, and HNE levels while decreasing/increasing tissue and blood antioxidant enzyme activities. From hepatic chemical metabolizing parameters, GST activity increased in the subacute and chronic stages and CYTC activity increased in the acute period, whereas GSH level decreased in the subacute stage. Changes in tail and head intensities were found in most of the comet results. Mad honey caused oxidative stresses for each exposure period and made some significant changes on the comet test in certain periods for some samples obtained. In other words, according to the available research results obtained, careless consumption of mad honey for different medical purposes is not appropriate.

ARM-microcontroller based portable nitrite electrochemical analyzer using cytochrome c reductase biofunctionalized onto screen printed carbon electrode

Nitrite (NO2−) supplementation limits hypoxia-induced oxidative stress and activates the alternate NO pathway which may partially account for the nitrite-mediated cardioprotection. So, sensitive and selective biosensors with point-of-care devices need to be explored to detect the physiological nitrite level due to its important role in human pathophysiology. In this work, cytochrome c reductase (CcR) biofunctionalized self assembled monolayer (SAM) functionalized on gold nanoparticles (GNPs) in polypyrrole (PPy) nanocomposite onto the screen printed carbon electrode (SPCE) was investigated as a biosensor for the detection of nitrite based on its electrochemical and catalytic properties. CcR was covalently coupled with SAM layers on GNPs by using EDC and NHS. Direct electrochemical response of CcR biofunctionalized electrodes showed a couple of well-defined and nearly reversible cyclic voltammetric peaks at −0.34 and −0.45 vs. Ag/AgCl. Under optimal conditions, the biosensor could be used for the determination of NO2− with a linear range from 0.1–1600µm and a detection limit of 60nM with a sensitivity of 0.172µAµM−1cm–2. Further, we have designed and developed a novel and cost effective portable electrochemical analyzer for the measurement of NO2− in hypoxia induced H9c2 cardiac cells using ARM microcontroller. The results obtained here using the developed portable electrochemical nitrite analyzer were also compared with the standard cyclic voltammetry instrument and found in agreement with each other.

A cost-effective volume miniaturized and microcontroller based cytochrome c assay

In this paper, a portable, cost-effective electrochemical assay is presented for rapid, sensitive, and quantitative detection of cytochrome c (cyt c) release. The developed cyt c assay consists of two parts: (i) a miniaturized electrochemical biosensor based on cytochrome c reductase (CcR) functionalized screen printed electrodes (SPE); (ii) a microcontroller based data acquisition unit integrated with potentiostat circuit capable of performing cyclic voltammetry technique for the analysis. The working electrode surface of SPE was integrated with polypyrrole (PPy)-carbon nanotubes (CNT) nanocomposite for an enhanced immobilization of the enzyme, CcR. The acquired biosensor data are processed into digital form by the microcontroller and further transferred to a PC through USB port for analysis. GUI based system implemented here makes the analyzer easy to operate. Under optimal conditions, the electroanalytical behavior of the CcR-CNT-PPy-SPE biosensor linearly responds to the cyt c concentration range from 10nM to 500μM with a detection limit of 10nM and a sensitivity of 0.102±0.005μAμM−1cm−2. The performance of the volume miniaturized SPE based biosensor coupled with the portable microcontroller based instrument was further evaluated by applying it for the measurement of mitochondrial cyt c release during cardiomyocytes apoptosis; the results are validated well with the commercial electrochemical analyzer and standard ELISA.

Curcumin supplementation improves mitochondrial and behavioral deficits in experimental model of chronic epilepsy

The present study was aimed to investigate the potential beneficial effect of curcumin, a polyphenol with pleiotropic properties, on mitochondrial dysfunctions, oxidative stress and cognitive deficits in a kindled model of epilepsy. Kindled epilepsy was induced in rats by administering a sub-convulsive dose of pentylenetetrazole (PTZ, 40mg/kg body weight) every alternate day for 30days. PTZ administered rats exhibited marked cognitive deficits assessed using active and passive avoidance tasks. This was accompanied by a significant decrease in NADH:cytochrome-c reductase (complex I) and cytochrome-c oxidase (complex IV) activities along with an increase in ROS, lipid peroxidation and protein carbonyls. The levels of glutathione also decreased in the cortex and hippocampus. Electron micrographs revealed disruption of mitochondrial membrane integrity with distorted cristae in PTZ treated animals. Histopathological examination showed pyknotic nuclei and cell loss in the hippocampus as well as in the cortex of PTZ treated animals. Curcumin administration at a dose of 100mg/kg, p.o. throughout the treatment paradigm was able to ameliorate cognitive deficits with no significant effect on seizure score. Curcumin was able to restore the activity of mitochondrial complexes. In addition, significant reduction in ROS generation, lipid peroxidation and protein carbonyls was observed in PTZ animals supplemented with curcumin. Moreover, glutathione levels were also restored in PTZ treated rats supplemented with curcumin. Curcumin protected mitochondria from seizure induced structural alterations. Further, the curcumin supplemented PTZ rats had normal cell morphology and reduced cell loss. These results suggest that curcumin supplementation has potential to prevent mitochondrial dysfunctions and oxidative stress with improved cognitive functions in a chronic model of epilepsy.

Effect of sprout extract from Tuscan black cabbage on xenobiotic-metabolizing and antioxidant enzymes in rat liver

In recent years, health protection by natural products has received considerable attention, and a multitude of nutraceuticals have been characterized and their use promoted. Dietary consumption of Cruciferous vegetables, rich in glucosinolates (GLs), and their myrosinase-mediated hydrolysis products isothiocyanates (ITCs), were associated with reductions in cancer risk. In this study, the chemo-preventive potential of sprout extract of Tuscan black cabbage (Brassica oleracea L. var. acephala subvar. Laciniata L.) (TBCSE), through modulation of the xenobiotic-metabolizing apparatus and antioxidant defenses, was investigated in Sprague-Dawley rat liver. TBCSE was administered either orally or intraperitoneally, at a dose of 15mg/kg b.w., daily for twenty-one consecutive days, in the absence or presence of exogenous myrosinase, β-thioglucoside glucohydrolase (MYR), to distinguish the effects of intact GLs and ITCs, in the context of the extract. A complex, mild modulation pattern of P450-related monooxygenases was observed, mainly regarding CYP content (up to 36% loss), NADPH cytochrome (P450) c-reductase (up to 26% loss), CYP1A1 (up to 23% loss), but no evident distinctions among the effects of the extracts containing GLs or ITCs, were noted. In contrast, significant inductions of phase-II enzymes (up to 107% for UDP-glucuronosyl-transferase, and up to 36% for glutathione S-transferase) were recorded only where the GLs to ITCs conversion had occurred. A boosting effect on catalase (up to 38%), NAD(P)H:quinone reductase (up to 70%), glutathione reductase and glutathione peroxidase (up to 10%) was also recorded, suggesting an indirect antioxidant capacity of the extracts. Overall, the general phase-I inhibition, together with the up-regulation of detoxifying phase-II and antioxidant enzymes, exerted by the TBCSE supplementation, seem to be in line with the classical chemopreventive theory, but whether the addition of exogenous MYR is relevant, still remains to be clarified. These results are in support of the potential health-promoting application of TBCSE, as a nutraceutical.

Nanomaterial-based electrochemical biosensors for cytochrome c using cytochrome c reductase

Emerging evidences have pointed out that the release of cytochrome c (cyt c) from mitochondria into cytosol is a critical step in the activation of apoptosis. This article presents a novel approach for the detection of mitochondrial cyt c release for the first time using cytochrome c reductase (CcR) immobilized on nanoparticles decorated electrodes. Two kinds of nanomaterial-based biosensor platforms were used: (a) carbon nanotubes (CNT) incorporated polypyrrole (PPy) matrix on Pt electrode and (b) self-assembled monolayer (SAM) functionalized gold nanoparticles (GNP) in PPy-Pt. Scanning electron microscope was used to characterize the surface morphologies of the nanomaterial modified electrodes. Cyclic voltammograms of both the biosensors showed reversible redox peaks at −0.45 and −0.34V vs Ag/AgCl, characteristic of CcR. In comparison, the CcR-CNT biosensor gave a detection limit of 0.5±0.03μM cyt c, which was 4-fold better than the CcR-GNP biosensor (2±0.03μM). Moreover, the CcR-CNT biosensor achieved a much larger linear range (1–1000μM) over the CcR-GNP biosensor (5–600μM) with 2-fold better sensitivity. The CcR-CNT-PPy-Pt biosensor was further applied to quantify the mitochondrial cyt c released in cytosol of A549 cells upon induction of apoptosis with doxorubicin, the results agreed well with standard western blot analysis.

Heat shock protein 70 (Hsp70) inhibits oxidative phosphorylation and compensates ATP balance through enhanced glycolytic activity

To address possible effects of heat shock protein 70 (Hsp70) on energy metabolism, we established a cell line expressing different levels of Hsp70 and evaluated changes in glucose and lactate metabolites, as well as ATP levels accordingly. In addition, activities of enzymes involved in glycolysis [phosphofructokinase (PFK) and lactate dehydrogenase (LDH)], Krebs cycle [citric synthase (CS)], and oxidative phosphorylation {NADH dehydrogenase [complex I (CI)] and ubiquinol:cytochrome-c reductase [complex III (CIII)]} were analyzed. The results show that both glucose consumption and lactate excretion were elevated significantly in cells expressing increased levels of Hsp70. Simultaneously, the activities of glycolytic enzymes PFK and LDH were increased markedly in cells overexpressing Hsp70. Activities of enzymes CI and CIII, both involved in oxidative phosphorylation, decreased upon increased expression of Hsp70. These findings were supported by nonsignificant reductions of CS activities in cells that overexpressed Hsp70, whereas intracellular ATP levels remained constant over a wide range of Hsp70 expression. In conclusion, overexpression of Hsp70 in HeLa cells results in downregulation of oxidative phosphorylation, in particular, multiprotein CIII, the main source of reactive oxygen species. In exchange, upregulation of the glycolytic pathway compensates for the homeostasis of cellular ATP supply.

Antioxidant activity of new aramide nanoparticles containing redox-active N-phthaloyl valine moieties in the hepatic cytochrome P450 system in male rats.

We report the synthesis of aramide nanoparticles containing a chiral N-phthaloyl valine moiety and their antioxidant activities on hepatic contents of cytochrome P450, amidopyrene N-demethylase, aniline-4-hyroxylase and induced the hepatic content of cytochrome b5 and nicotinamide adenine dinucleotide phosphate (NADPH) cytochrome C-reductase. Polymers were obtained as well-separated spherical nanoparticles while highly aggregated particles via H-bonding organization of the aramide-containing pyridine led to a thin layer formation. The effects of the nanoparticles and CCl4 on enzyme activities and thiobarbituric acid reactive substances (TBARS) levels of male rat liver were studied. Pretreatments of rats with the polyamides prior to the administration of CCl4 decreased the hepatic content of the tested enzymes. Doses reduced the toxic effects exerted by (•CCl3) upon the liver through inhibition of the cytochrome P450 system. Inhibition of such metabolizing enzymes could reduce the carcinogenic effects of chemical carcinogens.

A targeted mass spectrometry-based approach for the identification and characterization of proteins containing α-aminoadipic and γ-glutamic semialdehyde residues

The site-specific identification of α-aminoadipic semialdehyde (AAS) and γ-glutamic semialdehyde (GGS) residues in proteins is reported. Semialdehydic protein modifications result from the metal-catalyzed oxidation of Lys or Arg and Pro residues, respectively. Most of the analytical methods for the analysis of protein carbonylation measure change to the global level of carbonylation and fail to provide details regarding protein identity, site, and chemical nature of the carbonylation. In this work, we used a targeted approach, which combines chemical labeling, enrichment, and tandem mass spectrometric analysis, for the site-specific identification of AAS and GGS sites in proteins. The approach is applied to in vitro oxidized glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and an untreated biological sample, namely cardiac mitochondrial proteins. The analysis of GAPDH resulted in the site-specific identification of two AAA and four GGS residues. Computational evaluation of the identified AAS and GGS sites in GAPDH indicated that these sites are located in flexible regions, show high solvent accessibility values, and are in proximity with possible metal ion binding sites. The targeted proteomic analysis of semialdehydic modifications in cardiac mitochondria yielded nine AAS modification sites which were unambiguously assigned to distinct lysine residues in the following proteins: ATP/ATP translocase isoforms 1 and 2, ubiquinol cytochrome-c reductase core protein 2, and ATP synthase α-subunit.

Proteomic analysis of mitochondrial proteins in a mouse model of type 2 diabetes

Impaired mitochondrial function may contribute to the onset of contractile dysfunction with insulin resistance/type 2 diabetes. Our aim was therefore to determine alterations in the mitochondrial proteome of a mouse model of obesity/type 2 diabetes. Mitochondrial proteins were isolated from hearts collected from 18- to 20-week-old female db/db mice and compared to matched controls. We performed two-dimensional polyacrylamide gel electrophoresis to determine differentially expressed proteins. Peptides of interest were further analysed by mass spectrometry and Mascot software was employed to identify protein matches. Our data showed that ATP synthase D chain, ubiquinol cytochrome-C reductase core protein 1 and electron transfer flavoprotein subunit alpha peptide levels were altered with obesity. Moreover, we found coordinate downregulation of contractile proteins in the obese heart, i.e. α-smooth muscle actin, α-cardiac actin, myosin heavy-chain α and myosin-binding protein C. We propose that decreased contractile protein levels may contribute to contractile dysfunction of hearts from diabetic mice.

Proteomic analysis of bovine omental, subcutaneous and intramuscular preadipocytes during in vitro adipogenic differentiation

Given the substantial rise in obesity, depot-specific fat accumulation and its associated diseases like diabetes, it is important to understand the molecular basis of depot-specific adipocyte differentiation. Many studies have successfully exploited the adipocyte differentiation, but most of them were not related to depot-specificity, particularly using freshly isolated primary preadipocytes. Using 2-dimensional polyacrylamide gel electrophoresis coupled with sequencing mass spectrometry, we searched and compared the proteins differentially expressed in undifferentiated and differentiated preadipocytes from bovine omental, subcutaneous and intramuscular adipose depots. Our proteome mapping strategy to identify differentially expressed intracellular proteins during adipogenic conversion revealed 65 different proteins that were found to be common for the three depots. Further, we validated the differential expression for a subset of proteins by immunoblotting analyses. The results demonstrated that many structural proteins were down-regulated during differentiation of preadipocytes from all the depots. Most up-regulated proteins like Ubiquinol–cytochrome-c reductase complex core protein I (UQCRC1), ATP synthase D chain, Superoxide dismutase (SOD), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Sulfotransferase 1A1 (SULT1A1), Carnitine O-palmitoyltransferase 2 (CPT2) and Heat-shock protein beta 1 (HSPB1) across the three depots were found to be associated with lipid metabolism and metabolic activity. Further, all the up-regulated proteins were found to have higher protein expression in omental than subcutaneous or intramuscular depots.

The role of individual lysine residues of horse cytochrome c in the formation of reactive complexes with components of the respiratory chain

A number of mutant forms of horse cytochrome-c with single or double substitutions of lysine residues near the heme cavity was prepared that provided an interaction of mitochondrial ubiquinone with cytochrome-c reductase (EC 1.10.2.2) (complex III) and cytochrome-c oxidase (EC 1.9.3.1) (complex IV). The succinate cytochrome-c reductase and cytochrome-c oxidase activities of mitoplasts of rat liver were measured in the presence of mutant forms of cytochrome-c. The lysine residues in positions 8, 27, 72, 86, and 87 were shown to be the main contribution to the formation of a reactive complex with ubiquinol-cytochrome-c reductase of the respiratory chain, whereas the lysine residues in positions 13, 79, 86, and 87 were predominantly responsible for the formation of a complex with cytochrome-c oxidase.