Exogenous Cytochrome Research Articles

Effects of exogenous riboflavin or cytochrome addition on the cathodic reduction of Cr(VI) in microbial fuel cell with Shewanella putrefaciens.

Bioreduction of Cr(VI) is recognized as a cost-effective and environmentally friendly method, attracting widespread interest. However, the slow rate of Cr(VI) bioreduction remains a practical challenge. Additionally, the direct removal efficiency of microbes for high concentrations of Cr(VI) is not ideal due to the toxicity. Therefore, this study investigated the effects of exogenous riboflavin or cytochrome on the cathodic reduction of Cr(VI) in microbial fuel cells. The results demonstrated that the exogenous riboflavin or cytochrome effectively improved the voltage output of the cells, with riboflavin increasing the voltage by 52.08%. Within the first 24h, the Cr(VI) removal ratio in the normal, cytochrome, and riboflavin groups was 14.3%, 29.3%, and 53.8%, respectively. And the final removal ratio was 55.1%, 69.1%, and 98.0%, respectively. These results showed different enhancement effects of riboflavin and cytochrome on Cr(VI) removal. The analysis of riboflavin and cytochrome contents revealed that the additions did not have a significant impact on the autocrine riboflavin of S. putrefaciens, but affected the autocrine cytochrome. SEM, XPS, and FTIR results confirmed the presence of reduced Cr(III) on the cathode, which formed precipitate and adhered to the cathode surface. The EDS analysis showed that the amount of Cr on the cathode in normal, cytochrome, and riboflavin groups was 4.71%, 6.37%, 7.56%, respectively, which was consistent with the voltage and Cr(VI) removal data. These findings demonstrated the significant enhancement of exogenous riboflavin or cytochrome on Cr(VI) reduction, thereby providing data reference for the future bio-assisted remediation of Cr(VI) pollution.

Mechanistic complexities of sulfite oxidase: An enzyme with multiple domains, subunits, and cofactors

Sulfite oxidase (SO) deficiency, an inherited disease that causes severe neonatal neurological problems and early death, arises from defects in the biosynthesis of the molybdenum cofactor (Moco) (general sulfite oxidase deficiency) or from inborn errors in the SUOX gene for SO (isolated sulfite oxidase deficiency, ISOD). The X-ray structure of the highly homologous homonuclear dimeric chicken sulfite oxidase (cSO) provides a template for locating ISOD mutation sites in human sulfite oxidase (hSO). Catalysis occurs within an individual subunit of hSO, but mutations that disrupt the hSO dimer are pathological. The catalytic cycle of SO involves five metal oxidation states (MoVI, MoV, MoIV, FeIII, FeII), two intramolecular electron transfer (IET) steps, and couples a two-electron oxygen atom transfer reaction at the Mo center with two one-electron transfers from the integral b-type heme to exogenous cytochrome c, the physiological oxidant. Several ISOD examples are analyzed using steady-state, stopped-flow, and laser flash photolysis kinetics and physical measurements of recombinant variants of hSO and native cSO. In the structure of cSO, Mo…Fe = 32 Å, much too long for efficient IET through the protein. Interdomain motion that brings the Mo and heme centers closer together to facilitate IET is supported indirectly by decreasing the length of the interdomain tether, by changes in the charges of surface residues of the Mo and heme domains, as well as by preliminary molecular dynamics calculations. However, direct dynamic measurements of interdomain motion are in their infancy.

Influence of B16/F10 melanoma growth variant on the level of cytochrome C in mitochondria in various organs of female mice

Introduction. Cytochrome C in mitochondria transfers electrons from complex III to complex IV, and it is a signaling molecule in the apoptosis realization.The objective was to evaluate the level of cytochrome C in cell mitochondria in various organs of female mice with standard and stimulated growth of experimental B16/F10 melanoma.Methods and materials. The experiment was performed on female C57BL/6 mice (n=168). The groups were: intact animals (n=21); controls with a model of chronic neurogenic pain (CNP) (n = 21); group M — standard B16/F10 melanoma transplantation (n=63), group CNP + M — B16/F10 melanoma transplantation 3 weeks after CNP model creation (n=63). The level of cytochrome C (ng / mg protein) were measured by ELISA (Bioscience, Austria). Statistical analysis of results was performed using the «Statistica 10.0» program.Results. After 1 week of standard melanoma growth, an increase in the level of cytochrome C by 2.7 and 1.7 times was detected in mitochondria of the brain and liver; by the 3rd week, it decreased in the liver and skin by 1.7 times. In melanoma mitochondria, the level of cytochrome C was lower than in the skin of intact animals: by 2.5 times after week 1, by 4.5 times after week 2, and by 4.6 times after week 3. After 1 week of stimulated melanoma growth, the level of cytochrome C decreased compared control values: by 2.2 times in the brain, by 1.9 times in the liver, by 1.4 times in the skin; by week 3, it decreased by 4.8 times in mitochondria of the brain, by 4.7 times — in the liver, by 2.3 times — in the heart, by 1.9 times — in the skin. In melanoma mitochondria, the level of cytochrome C was lower than in the skin of intact animals: by 15.3 times after week 1, by 10.3 times after week 2, and by 8.8 times after week 3.Conclusion. Low level of cytochrome C were found in melanoma mitochondria in standard and stimulated tumor growth. The data can be used in the experiment and in clinic for using exogenous cytochrome C as an agent slowing down the malignant process.

The Mechanisms of Electrogenic Reactions in Bacterial Photosynthetic Reaction Centers: Studies in Collaboration with Alexander Konstantinov.

In this review, we discuss our studies conducted in 1985-1988 in collaboration with A. A. Konstantinov, one of the top scientists in the field of membrane bioenergetics. Studying fast kinetics of membrane potential generation in photosynthetic reaction centers (RCs) of purple bacteria in response to a laser flash has made it possible to examine in detail the mechanisms of electrogenic reactions at the donor and acceptor sides of RCs. Electrogenesis associated with the intraprotein electron transfer from the exogenous secondary donors, redox dyes, and soluble cytochrome (cyt) c to the photooxidized dimer of bacteriochlorophyll P870 was studied using proteoliposomes containing RCs from the non-sulfur purple bacterium Rhodospirillum rubrum. It was found that reduction of the secondary quinone electron acceptor QB accompanied by its protonation in the chromatophores from R. rubrum in response to every second light flash was electrogenic. Spectral characteristics and redox potentials of the four hemes in the tightly bound cyt c in the RC of Blastochloris viridis and electrogenic reactions associated with the electron transfer within the RC complex were identified. For the first time, relative amplitudes of the membrane potential generated in the course of individual electrogenic reactions were compared with the distances between the redox cofactors determined based on the three-dimensional structure of the Bl. viridis RC.

Acute Myocardial Infarction Reduces Respiration in Rat Cardiac Fibers, despite Adipose Tissue Mesenchymal Stromal Cell Transplant.

Adipose-derived mesenchymal stromal cell (AD-MSC) administration improves cardiac function after acute myocardial infarction (AMI). Although the mechanisms underlying this effect remain to be elucidated, the reversal of the mitochondrial dysfunction may be associated with AMI recovery. Here, we analyzed the alterations in the respiratory capacity of cardiomyocytes in the infarcted zone (IZ) and the border zone (BZ) and evaluated if mitochondrial function improved in cardiomyocytes after AD-MSC transplantation. Female rats were subjected to AMI by permanent left anterior descending coronary (LAD) ligation and were then treated with AD-MSCs or PBS in the border zone (BZ). Cardiac fibers were analyzed 24 hours (necrotic phase) and 8 days (fibrotic phase) after AMI for mitochondrial respiration, citrate synthase (CS) activity, F0F1-ATPase activity, and transmission electron microscopy (TEM). High-resolution respirometry of permeabilized cardiac fibers showed that AMI reduced numerous mitochondrial respiration parameters in cardiac tissue, including phosphorylating and nonphosphorylating conditions, respiration coupled to ATP synthesis, and maximal respiratory capacity. CS decreased in IZ and BZ at the necrotic phase, whereas it recovered in BZ and continued to drop in IZ over time when compared to Sham. Exogenous cytochrome c doubled respiration at the necrotic phase in IZ. F0F1-ATPase activity decreased in the BZ and, to more extent, in IZ in both phases. Transmission electron microscopy showed disorganized mitochondrial cristae structure, which was more accentuated in IZ but also important in BZ. All these alterations in mitochondrial respiration were still present in the group treated with AD-MSC. In conclusion, AMI led to mitochondrial dysfunction with oxidative phosphorylation disorders, and AD-MSC improved CS temporarily but was not able to avoid alterations in mitochondria function over time.

198 Acute Trimethylamine-N-oxide Depresses Cardiac Function and “stress” Mitochondria into Overdrive

Trimethylamine-N-oxide (TMAO) is a secondary gut metabolite recently linked to cardiovascular disease (CVD). However, whether TMAO concentrations are sufficient to directly drive dysfunction and CVD is unclear. Since detrimental effects on the heart and mitochondria are yet to be detailed, we investigated the impacts of acute TMAO on function and respiration in murine hearts. Isolated hearts from male C57Bl/6 mice were retrogradely perfused at either constant flow or pressure while infused with increasing TMAO concentrations to assess effects on contractile function and coronary flow respectively. Shredded left ventricular (LV) myocardium was also suspended in mitochondrial respiration medium ±10 μM or 100 μM TMAO, and analysed using an Oxygraph-2k instrument. A substrate-uncoupler-inhibitor-titration (SUIT) protocol assessed routine mitochondrial respiration. TMAO appears to be a 'cardiodepressant': LV developed pressure and coronary flow were significantly decreased with ≥3 μM TMAO (p<0.05), with flow and contractile changes correlated (R2=0.59). Unexpectedly, however, mitochondrial Complex I as well as complex I & II linked respiration was significantly increased in response to both 10 μM (p<0.01) and 100 μM (p<0.05) TMAO, as was maximum respiratory capacity (p<0.01). However, significant increases in exogenous cytochrome c dependent respiration with both TMAO concentrations (p<0.01), supports disruption of mitochondrial membrane integrity. Data indicate that acute TMAO, at concentrations reported in cardiac, renal and metabolic diseases, appears to decrease cardiac contractile function while potentially 'stressing' cardiac mitochondria into overdrive. This is congruent with the role of elevated TMAO in CVD pathogenesis.

Structure of a functional obligate complex III2IV2 respiratory supercomplex from Mycobacterium smegmatis.

In the mycobacterial electron-transport chain, respiratory complex III passes electrons from menaquinol to complex IV, which in turn reduces oxygen, the terminal acceptor. Electron transfer is coupled to transmembrane proton translocation, thus establishing the electrochemical proton gradient that drives ATP synthesis. We isolated, biochemically characterized, and determined the structure of the obligate III2IV2 supercomplex from Mycobacterium smegmatis, a model for Mycobacterium tuberculosis. The supercomplex has quinol:O2 oxidoreductase activity without exogenous cytochrome c and includes a superoxide dismutase subunit that may detoxify reactive oxygen species produced during respiration. We found menaquinone bound in both the Qo and Qi sites of complex III. The complex III-intrinsic diheme cytochrome cc subunit, which functionally replaces both cytochrome c1 and soluble cytochrome c in canonical electron-transport chains, displays two conformations: one in which it provides a direct electronic link to complex IV and another in which it serves as an electrical switch interrupting the connection.

Photooxidation and photoreduction of exogenous cytochrome c by photosystem II preparations after various modifications of the water-oxidizing complex

The redox interaction of exogenous cytochrome c550 (Cyt) with PSII isolated from spinach was studied. Illumination of PSII particles in the presence of Cyt led to: (1) Cyt photooxidation by PSII reaction center (demonstrated at the first time), (2) Cyt photoreduction via O2−• photoproduced on the acceptor side of PSII, and (3) Cyt photoreduction by reduced electron carriers of PSII. A step-by-step removal of components of water-oxidizing complex was accompanied by the appearance of Cyt photooxidation, an increase in the superoxide dismutase (SOD)-dependent Cyt photoreduction (related to O2−• formation), and a decrease in the SOD-independent Cyt photoreduction. Re-addition of PsbO protein diminished the Cyt-induced restoration of electron transfer in PSII. Addition of diuron led to inhibition of these photoprocesses, while exogenous Mn2+ inhibited only the Cyt c photooxidation. The results can be important for correct measurements of O2−• photoproduction in PSII and for elucidation of the role of cytochrome c550 in cyanobacterial PSII.

Guiding protein delivery into live cells using DNA-programmed membrane fusion.

Intracellular delivery of proteins provides a direct means to manipulate cell function and probe the intracellular environment. However, direct cytoplasmic delivery of proteins suffers from limited availability of efficient toolsets, and thus remains challenging in research and therapeutic applications. Natural biological cargo delivery processes, like SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex mediated membrane fusion and other vesicle fusion in live cells, enable targeted delivery with high efficiency. A surrogate of SNARE machinery represents a new direction in intracellular protein delivery. Here, we report a DNA-programmed membrane fusion strategy for guiding the efficient intracellular delivery of proteins into live cells. The inherent programmability of DNA hybridization provides spatiotemporal control of the fusion between protein-encapsulated liposomes and cell membranes, enabling rapid release of proteins directly into the cytoplasm, while still remaining functional due to the bypassing of the endosomal trap. We further demonstrate that delivered exogenous Cytochrome c effectively regulates the cell fate. Hence, this DNA-mediated fusion strategy holds great potential for protein drug delivery, regenerative medicine and gene editing.

The effects of Caffeic Acid Phenethyl Ester (CAPE) on oxidative stress and hypoxia‐induced cell damage

Oxidative stress has been implicated in pathogenesis of hypoxia and ischemia/reperfusion (I/R) injury. In previous studies, we have shown that the antioxidant CAPE exerted cardioprotection in an isolated rat heart I (30 min)/R (60 min) injury model. In this study, we further evaluated the effects of CAPE on oxidative stress and hypoxia‐induced cell damage. We evaluated the inhibition of absorbance in the phorbol 12‐myristate 13‐acetate (30 nM) induced superoxide production spectrophotometrically in isolated rat neutrophils via reduction of exogenous cytochrome C. We found that CAPE (0.5 μM–40 μM; n=4–13) reduced phorbol 12‐myristate 13‐acetate induced neutrophil superoxide release dose‐dependently from 29±3% to 95±2%. In a rat hind limb I (30 min)/R (60 min) model, blood hydrogen peroxide levels serves as an indicator of blood oxidative stress and was measured in real‐time via a hydrogen peroxide microsensor (100 μm) inserted into both femoral veins (one served as sham, the other as I/R). We found that in the control group, I/R significantly increased blood hydrogen peroxide levels to 2.1±0.8 μM relative to the sham limb at 60 minutes reperfusion when saline was given at the beginning of reperfusion (n=5). By contrast, CAPE when given at reperfusion (40 μM, n=5) significantly reduced blood hydrogen peroxide levels from 30 min reperfusion and throughout the rest of experiment (p&lt;0.05). The relative change was −0.4±0.3 μM between the I/R and sham limbs at 60 minutes reperfusion. In H9C2 rat myoblast cells, cobalt chloride simulated hypoxia and induced cell damage. Tetrazolium, which can differentiate metabolically active and inactive cells/tissues, was used to measure cell damage by measuring absorbance at 450 nm. We found that incubation of cobalt chloride (800 μM, n=8) for 24 hr induced 55±5% cell death. By contrast, pretreatment of CAPE (0.5 μM–10 μM; n=1–3) for 24 hr dose‐dependently improved cell survival up to 30±9% relative to cobalt chloride treatment. In summary, this study suggests that CAPE attenuates oxidative stress from neutrophils and I/R, which may contribute to improvement in cell survival under hypoxia conditions.Support or Funding InformationThis study was supported by Division of Research and Department of Bio‐Medical Sciences at Philadelphia College of Osteopathic Medicine.

Abstract 3563: Ras-mediated regulation of cytochrome c-induced caspase activation is dependent on the status of extracellular matrix attachment

Abstract Hyperactivating mutations in Ras are found in a significant percentage of cancers, with a particularly high frequency in pancreatic and colon carcinomas. The hyperactivation of Ras drives a vast number of distinct downstream signaling pathways that play an important role in disease progression. One outcome of overactive oncogenic Ras signaling is the inhibition of anoikis, a form of apoptotic cell death caused by detachment from the extracellular matrix (ECM). It is well-established that one mechanism of anoikis inhibition by Ras involves impaired mitochondrial cytochrome c release. In addition, here, we have found that Ras-mediated anoikis inhibition can also occur downstream of mitochondrial cytochrome c release. Our data suggest that anoikis inhibition following cytochrome c release is not dependent on changes in the abundance of the Apaf-1, pro-caspase-9, or pro-caspase-3. Instead, our data suggest that Ras signaling leads to an inhibition in the ability of caspase-9 to bind Apaf-1 which thereby inhibits proper formation of the apoptosome and caspase activation. Interestingly, in stark contrast to the inhibition of cytochrome c-induced apoptosis in ECM-detached cells, the overexpression of oncogenic Ras in ECM-attached cells results in enhanced sensitivity to exogenous cytochrome c. This sensitization was found to be due to upregulation of apoptosomal proteins (e.g. Apaf-1, pro-caspase-9) in an ERK/MAPK dependent manner. In aggregate, our data suggest that in addition to inhibiting the release of cytochrome c in both attachment and detachment, oncogenic Ras drives additional mechanisms that prevent apoptosome formation and caspase activation in detachment. Furthermore, our data support a model whereby ECM-attached cells containing oncogenic Ras mutations could be selectively eliminated by cytochrome c or agents that mimic its action. Citation Format: Chelsea M. McCallister, Raju Rayavarapu, Nicholas Pagani, Brendan Heiden, Melissa Shaw, Sydeny Shuff, Zachary T. Schafer. Ras-mediated regulation of cytochrome c-induced caspase activation is dependent on the status of extracellular matrix attachment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3563.

Cannabinoids and Cytochrome P450 Interactions.

This review consists of three parts, representing three different possibilities of interactions between cannabinoid receptor ligands of both exogenous and endogenous origin and cytochrome P450 enzymes (CYPs). The first part deals with cannabinoids as CYP substrates, the second summarizes current knowledge on the influence of various cannabinoids on the metabolic activity of CYP, and the third outline a possible involvement of the endocannabinoid system and cannabinoid ligands in the regulation of CYP liver activity. We performed a structured search of bibliographic and drug databases for peer-reviewed literature using focused review questions. Biotransformation via a hydrolytic pathway is the major route of endocannabinoid metabolism and the deactivation of substrates is characteristic, in contrast to the minor oxidative pathway via CYP involved in the bioactivation reactions. Phytocannabinoids are extensively metabolized by CYPs. The enzymes CYP2C9, CYP2C19, and CYP3A4 catalyze most of their hydroxylations. Similarly, CYP represents a major metabolic pathway for both synthetic cannabinoids used therapeutically and drugs that are abused. In vitro experiments document the mostly CYP inhibitory activity of the major phytocannabinoids, with cannabidiol as the most potent inhibitor of many CYPs. The drug-drug interactions between cannabinoids and various drugs at the CYP level are reported, but their clinical relevance remains unclear. The direct activation/inhibition of nuclear receptors in the liver cells by cannabinoids may result in a change of CYP expression and activity. Finally, we hypothesize the interplay of central cannabinoid receptors with numerous nervous systems, resulting in a hormone-mediated signal towards nuclear receptors in hepatocytes.

Role of the salt bridge between glutamate 546 and arginine 907 in preservation of autoinhibited form of Apaf-1

Apaf-1, the key element of apoptotic mitochondrial pathway, normally exists in an auto-inhibited form inside the cytosol. WRD-domain of Apaf-1 has a critical role in the preservation of auto-inhibited form; however the underlying mechanism is unclear. It seems the salt bridges between WRD and NOD domains are involved in maintaining the inactive conformation of Apaf-1. At the present study, we have investigated the effect of E546-R907 salt bridge on the maintenance of auto-inhibited form of human Apaf-1. E546 is mutated to glutamine (Q) and arginine (R). Over-expression of wild type Apaf-1 and its E546Q and E546R variants in HEK293T cells does not induce apoptosis unlike – HL-60 cancer cell line. In vitro apoptosome formation assay showed that all variants are cytochrome c and dATP dependent to form apoptosome and activate endogenous procaspase-9 in Apaf-1-knockout MEF cell line. These results suggest that E546 is not a critical residue for preservation of auto-inhibited Apaf-1. Furthermore, the behavior of Apaf-1 variants for in vitro apoptosome formation in HEK293T cell is similar to exogenous wild type Apaf-1. Wild type and its variants can form apoptosome in HEK293T cell with different procaspase-3 processing pattern in the presence and absence of exogenous cytochrome c and dATP.

Toxoplasma gondii inhibits cytochrome c-induced caspase activation in its host cell by interference with holo-apoptosome assembly.

Inhibition of programmed cell death pathways of mammalian cells often facilitates the sustained survival of intracellular microorganisms. The apicomplexan parasite Toxoplasma gondii is a master regulator of host cell apoptotic pathways. Here, we have characterized a novel anti-apoptotic activity of T. gondii. Using a cell-free cytosolic extract model, we show that T. gondii interferes with the activities of caspase 9 and caspase 3/7 which have been induced by exogenous cytochrome c and dATP. Proteolytic cleavage of caspases 9 and 3 is also diminished suggesting inhibition of holo-apoptosome function. Parasite infection of Jurkat T cells and subsequent triggering of apoptosome formation by exogenous cytochrome c in vitro and in vivo indicated that T. gondii also interferes with caspase activation in infected cells. Importantly, parasite inhibition of cytochrome c-induced caspase activation considerably contributes to the overall anti-apoptotic activity of T. gondii as observed in staurosporine-treated cells. Co-immunoprecipitation showed that T. gondii abolishes binding of caspase 9 to Apaf-1 whereas the interaction of cytochrome c with Apaf-1 remains unchanged. Finally, T. gondii lysate mimics the effect of viable parasites and prevents holo-apoptosome functionality in a reconstituted in vitro system comprising recombinant Apaf-1 and caspase 9. Beside inhibition of cytochrome c release from host cell mitochondria, T. gondii thus also targets the holo-apoptosome assembly as a second mean to efficiently inhibit the caspase-dependent intrinsic cell death pathway.

Exogenous cytochrome c inhibits the expression of transforming growth factor-β1 in a mouse model of sepsis-induced myocardial dysfunction via the SMAD1/5/8 signaling pathway.

The current study investigated the role of exogenous cytochrome c in sepsis-induced myocardial dysfunction (SIMD) using a mouse model and aimed to elucidate its effect on transforming growth factor-β1 (TGF-β1) expression during this process. A total of 75 male Kunming mice were randomly divided into the following five group: Normal (N, n=15); sham-operation (SHAM, n=15); sepsis (CLP, n=15); normal saline (NS, n=15); and cytochrome c (Cytc, n=15). Animals were sacrificed at 0, 6 or 12 h and the samples were analyzed using transmission electron microscopy, histopathological examination, reverse transcription-quantitative polymerase chain reaction, ELISA, protein analysis by western blotting. The SIMD model was developed and a significant downregulation of TGF-β1 gene expression, in addition to a reduction in the plasma and protein levels of TGF-β1 as well as the protein levels of TGF-β1-activated SMAD 1/5/8 were observed in the CLP group. The data from the current study indicate that using exogenous cytochrome c as a therapeutic strategy for SIMD is feasible, and may function via the downregulation of TGF-β1 expression through the SMAD 1/5/8 signaling pathway.

Dual inhibition of Cdc2 protein kinase activation during apoptosis in Xenopus egg extracts.

When intracellular damage accumulates in proliferating somatic cells, the cell cycle usually arrests in G1 or G2 in a checkpoint-dependent manner, either to repair the damage or to die by apoptosis. In contrast, early embryonic cells lack checkpoint-mediated cell-cycle arrest, and it is not clear whether apoptosis in early embryonic cells occurs at a specific cell cycle stage or at random points. Here, we examined the functional molecular link between the embryonic cell cycle and apoptosis using Xenopus egg extracts. When apoptosis was induced in egg extracts by addition of exogenous cytochrome c during cell-cycle progression, cyclin B accumulation was inhibited, Cdc2 was not activated, and the cell cycle arrested at interphase. However, addition of recombinant cyclin B failed to activate Cdc2 due to the strong inhibitory phosphorylation of Cdc2 Tyr15 in apoptotic egg extracts. We found that endogenous Cdc25C, which activates the Cdc2-cyclin B complex by dephosphorylating Cdc2 Tyr15, was inactivated by caspase-mediated cleavage at two sites in the N-terminal regulatory domain. When the hyperactive Cdc25A catalytic fragment was added together with recombinant cyclin B to artificially dephosphorylate Cdc2 Tyr15, M-phase induction was restored in apoptotic egg extracts, indicating that the blockage of cyclin B accumulation and the caspase-mediated inactivation of Cdc25C dually inhibited Cdc2 activation. Apoptosis induction in cytostatic factor-arrested metaphase egg extracts resulted in inactivation of Cdc2 without cyclin B degradation. These results suggest that apoptotic inactivation of Cdc25C plays an important role in arresting the embryonic cell cycle at interphase during apoptosis.

Acute hepatic ischemic-reperfusion injury induces a renal cortical "stress response," renal "cytoresistance," and an endotoxin hyperresponsive state.

Hepatic ischemic-reperfusion injury (HIRI) is considered a risk factor for clinical acute kidney injury (AKI). However, HIRI's impact on renal tubular cell homeostasis and subsequent injury responses remain ill-defined. To explore this issue, 30-45 min of partial HIRI was induced in CD-1 mice. Sham-operated or normal mice served as controls. Renal changes and superimposed injury responses (glycerol-induced AKI; endotoxemia) were assessed 2-18 h later. HIRI induced mild azotemia (blood urea nitrogen ∼45 mg/dl) in the absence of renal histologic injury or proteinuria, implying a "prerenal" state. However, marked renal cortical, and isolated proximal tubule, cytoprotective "stress protein" gene induction (neutrophil gelatinase-associated lipocalin, heme oxygenase-1, hemopexin, hepcidin), and increased Toll-like receptor 4 (TLR4) expression resulted (protein/mRNA levels). Ischemia caused release of hepatic heme-based proteins (e.g., cytochrome c) into the circulation. This corresponded with renal cortical oxidant stress (malondialdehyde increases). That hepatic derived factors can evoke redox-sensitive "stress protein" induction was implied by the following: peritoneal dialysate from HIRI mice, soluble hepatic extract, or exogenous cytochrome c each induced the above stress protein(s) either in vivo or in cultured tubule cells. Functional significance of HIRI-induced renal "preconditioning" was indicated by the following: 1) HIRI conferred virtually complete morphologic protection against glycerol-induced AKI (in the absence of hyperbilirubinemia) and 2) HIRI-induced TLR4 upregulation led to a renal endotoxin hyperresponsive state (excess TNF-α/MCP-1 gene induction). In conclusion, HIRI can evoke "renal preconditioning," likely due, in part, to hepatic release of pro-oxidant factors (e.g., cytochrome c) into the systemic circulation. The resulting renal changes can impact subsequent AKI susceptibility and TLR4 pathway-mediated stress.

External mitochondrial NADH-dependent reductase of redox cyclers: VDAC1 or Cyb5R3?

It was reported that VDAC1 possesses an NADH oxidoreductase activity and plays an important role in the activation of xenobiotics in the outer mitochondrial membrane. In the present work, we evaluated the participation of VDAC1 and Cyb5R3 in the NADH-dependent activation of various redox cyclers in mitochondria. We show that external NADH oxidoreductase caused the redox cycling of menadione ≫ lucigenin>nitrofurantoin. Paraquat was predominantly activated by internal mitochondria oxidoreductases. An increase in the ionic strength stimulated and suppressed the redox cycling of negatively and positively charged acceptors, as was expected for the Cyb5R3-mediated reduction. Antibodies against Cyb5R3 but not VDAC substantially inhibited the NADH-related oxidoreductase activities. The specific VDAC blockers G3139 and erastin, separately or in combination, in concentrations sufficient for the inhibition of substrate transport, exhibited minimal effects on the redox cycler-dependent NADH oxidation, ROS generation, and reduction of exogenous cytochrome c. In contrast, Cyb5R3 inhibitors (6-propyl-2-thiouracil, p-chloromercuriobenzoate, quercetin, mersalyl, and ebselen) showed similar patterns of inhibition of ROS generation and cytochrome c reduction. The analysis of the spectra of the endogenous cytochromes b5 and c in the presence of nitrofurantoin and the inhibitors of VDAC and Cyb5R3 demonstrated that the redox cycler can transfer electrons from Cyb5R3 to endogenous cytochrome c. This caused the oxidation of outer membrane-bound cytochrome b5, which is in redox balance with Cyb5R3. The data obtained argue against VDAC1 and in favor of Cyb5R3 involvement in the activation of redox cyclers in the outer mitochondrial membrane.

Microcirculatory, mitochondrial, and histological changes following cerebral ischemia in swine

BackgroundIschemic brain injury due to stroke and/or cardiac arrest is a major health issue in modern society requiring urgent development of new effective therapies. The aim of this study was to evaluate mitochondrial, microcirculatory, and histological changes in a swine model of global cerebral ischemia.ResultsIn our model, significant microcirculatory changes, but only negligible histological cell alterations, were observed 3 h after bilateral carotid occlusion, and were more pronounced if the vascular occlusion was combined with systemic hypotension. Analysis of mitochondrial function showed that LEAK respiration (measured in the presence of pyruvate + malate but without ADP) was not affected in any model of global cerebral ischemia in pigs. The OXPHOS capacity with pyruvate + malate as substrates decreased compared with the control levels after bilateral carotid artery occlusion, and bilateral carotid artery occlusion + hypotension by 20% and 79%, respectively, resulting in decreases in the respiratory control index of 14% and 73%, respectively. OXPHOS capacity with succinate as a substrate remained constant after unilateral carotid artery occlusion or bilateral carotid artery occlusion, but decreased by 53% after bilateral carotid artery occlusion and hypotension compared with controls (p < 0.05, n = 3–6). Addition of exogenous cytochrome c to mitochondria isolated from ischemia brains had no effect on respiration in all models used in this study.ConclusionsWe found a decrease in microcirculation and mitochondrial oxidative phosphorylation activity, but insignificant neuronal death, after 3 h ischemia in all our pig models of global cerebral ischemia. Dysfunction of the mitochondrial oxidative phosphorylation system, particularly damage to complex I of the respiratory chain, may be the primary target of the ischemic insult, and occurs before signs of neuronal death can be detected.

Reperfusion-induced mitochondrial dysfunction in the porcine heart is reduced by TRO40303 in the area at risk predominantly through preservation of outer mitochondrial membrane intactness

Mitochondria are considered to play critical roles in cell death pathways following ischemia-reperfusion injury. The objective of the present study was to perform a functional assessment of mitochondria following ischemia-reperfusion injury of the porcine heart as well as to evaluate mitochondrial effects of hypothermia and the outer membrane translocator protein (TSPO) ligand TRO40303. Pigs were subjected to 40 min occlusion of the left anterior descending artery followed by 4 hours of reperfusion. Three groups of pigs were treated either by administration of 15 mg/kg TRO40303 or the same volume of saline (1 ml/kg) at normothermia 5 min before reperfusion, or by hypothermia (32°C) initiated prior to occlusion, n=8 for all groups. Transmural needle biopsies were taken from the non-ischemic area in the left lateral wall, from the area at risk in the midventricular anterior wall and from the ischemic core area in the apical anterior wall. Mitochondrial respiratory function was evaluated polarographically in skinned heart fibers using specific substrates and inhibitors. In the control group, heart fibers from both ischemic areas demonstrated a general reduction of respiratory states. However, respiration linked to respiratory complex I was more affected than that to complex II indicating loss of soluble matrix components such as NAD (H). Addition of exogenous cytochrome c (CytC) increased the level of respiration several fold in both ischemic areas indicating increased permeability of the outer membrane and that CytC loss contributed to the reduced levels of respiration. These changes were diminished by hypothermia in both ischemic areas. TRO40303 attenuated inhibition of respiration involving complex II and reduced the stimulatory effects of CytC in the area at risk, but did not significantly reduce the altered ratio of complex I- and II-mediated respiration, and was without effect in the ischemic core area. It is concluded that mitochondria in both the ischemic core area and the area at risk undergo significant alterations in respiratory function following ischemia-reperfusion injury consistent with both inner and outer mitochondrial membrane permeabilization which can be inhibited by hypothermia initiated prior to occlusion. Administration of TRO40303 prior to reperfusion appears to reduce reperfusion-induced mitochondrial dysfunction in the area at risk mainly by preserving outer membrane intactness and limiting CytC release.