Angiotensin II promotes erythroid proliferation in a three-stage erythroid culture system.

At present, the numbers of cultured erythroid cells obtained from culture systems are not on a scale that can be used for therapeutics since the cultured erythroid cells have limited proliferation capacity. Stromal cells are believed to play important roles during erythropoiesis. Our previous study shows that factors secreted by stromal cells enhance the proliferation capacity of adult erythroid cells in the culture system. Among the identified factors, angiotensinogen is one of the most abundant proteins secreted by the stromal cells. This study aims to investigate the effect of angiotensin II, an angiotensinogen derivative, on the proliferation of erythroid cells. •The receptor for angiotensin II was first checked by PCR analysis. It was expressed in erythroblasts at all stages during differentiation.•To study the effect of angiotensin II, CD34+ hematopoietic stem cells were cultured in a 3-stage erythroid culture system with and without angiotensin II. The addition of angiotensin II to the culture media, from day 0 to 8, significantly increases the numbers of cultured erythroid cells, whereas no difference in enucleation is observed.

Bioactive Compounds from Germinated Brown Rice Protect Cardiomyocytes Against Simulated Ischemic/Reperfusion Injury by Ameliorating Mitochondrial Dysfunction.

PURPOSE: Ischemic/reperfusion (I/R) injury is the principal mechanism during Ischemic Heart Disease (IHD). The key modulator of I/R injury is dysregulation of mitochondria function. Germinated Brown Rice (GBR) has been recommended as a bio-functional food and has clarified the potential properties in several effects. However, the effect of GBR mediated cardioprotective properties, focusing on mitochondrial function's role, remains unexplored. Thus, this study aims to investigate the cardioprotective effects of GBR pretreatment against simulated I/R injury. METHODS: H9c2 cardiomyocytes were incubated with GBR at a five ƞg/mL concentration for 24 hours and simulated I/R (sI/R) for 40 minutes. Cell viability and cell apoptosis were assessed by 7-AAD staining and Annexin V/PI staining, respectively. The mitochondrial membrane potential was determined by JC-1 staining and mitochondrial respiration represented by oxygen consumption rate (OCR) using Seahorse Flux analyzer. RESULTS: The results revealed that the administration of GBR before sI/R significantly decreased the percentage of cell death and total cell apoptosis in H9c2 during stimulation of ischemic/reperfusion. Besides, pretreatment of cardiomyocytes with GBR remarkably stabilized mitochondrial membrane potential and improved impaired mitochondrial respiration in simulated-H9c2 injury. CONCLUSION: The present research is the first study to report the effective cardioprotection of GBR. Pretreatment of GBR potentially protects H9c2 cardiomyocytes against sI/R injury through mitochondrial function. The underlying therapeutic activities are possibly associated with its bio-functional compounds. However, the underlying mechanism on the cardioprotective effects of GBR needs further studies.

Identification of a novel mitochondrial complex I assembly factor ACDH-12 in Caenorhabditis elegans.

Assembly of complex I of the mitochondrial respiratory chain (MRC) requires not only structural subunits for electron transport, but also assembly factors. In the nematode Caenorhabditis elegans, NUAF-1 and NUAF-3 are the only two assembly factors that have been characterized. In this study, we identify ACDH-12 as an assembly factor of the respiratory complex I. We demonstrate for the first time that a deficiency of ACDH-12 affects the formation and function of complex I. RNAi knockdown of acdh-12 also shortens lifespan and decreases fecundity. Although ACDH-12 has long been recognized as a very long-chain acyl-CoA dehydrogenase (VLCAD), the knockdown nematodes did not exhibit any change in body fat content. We suggested that in Caenorhabditis elegans, ACDH-12 is required for the assembly of the respiratory complex I, but may not be crucial to fatty acid oxidation. Interestingly, sequence analysis shows high homology between ACDH-12 and the human ACAD9, a protein that has initially been identified as a VLCAD, but later found to also be involved in the assembly of complex I in human.

Antioxidant-Enhancing Property of the Polar Fraction of Mangosteen Pericarp Extract and Evaluation of Its Safety in Humans.

Crude extract from the pericarp of the mangosteen (mangosteen extract [ME]) has exhibited several medicinal properties in both animal models and human cell lines. Interestingly, the cytotoxic activities were always observed in nonpolar fraction of the extract whereas the potent antioxidant was often found in polar fraction. Although it has been demonstrated that the polar fraction of ME exhibited the antioxidant activity, the safety of the polar fraction of ME has never been thoroughly investigated in humans. In this study, we investigated the safety of oral administration of the polar fraction of ME in 11 healthy Thai volunteers. During a 24-week period of the study, only minor and tolerable side effects were reported; no serious side effects were documented. Blood chemistry studies also showed no liver damage or kidney dysfunction in all subjects. We also demonstrated antioxidant property of the polar fraction of ME both in vitro and in vivo. Interestingly, oral administration of the polar fraction of ME enhanced the antioxidant capability of red blood cells and decreased oxidative damage to proteins within red blood cells and whole blood.

A Drosophila model of mitochondrial disease caused by a complex I mutation that uncouples proton pumping from electron transfer.

Mutations affecting mitochondrial complex I, a multi-subunit assembly that couples electron transfer to proton pumping, are the most frequent cause of heritable mitochondrial diseases. However, the mechanisms by which complex I dysfunction results in disease remain unclear. Here, we describe a Drosophila model of complex I deficiency caused by a homoplasmic mutation in the mitochondrial-DNA-encoded NADH dehydrogenase subunit 2 (ND2) gene. We show that ND2 mutants exhibit phenotypes that resemble symptoms of mitochondrial disease, including shortened lifespan, progressive neurodegeneration, diminished neural mitochondrial membrane potential and lower levels of neural ATP. Our biochemical studies of ND2 mutants reveal that complex I is unable to efficiently couple electron transfer to proton pumping. Thus, our study provides evidence that the ND2 subunit participates directly in the proton pumping mechanism of complex I. Together, our findings support the model that diminished respiratory chain activity, and consequent energy deficiency, are responsible for the pathogenesis of complex-I-associated neurodegeneration.

Mitochondrial complex I deficiency increases protein acetylation and accelerates heart failure.

Mitochondrial respiratory dysfunction is linked to the pathogenesis of multiple diseases, including heart failure, but the specific mechanisms for this link remain largely elusive. We modeled the impairment of mitochondrial respiration by the inactivation of the Ndufs4 gene, a protein critical for complex I assembly, in the mouse heart (cKO). Although complex I-supported respiration decreased by >40%, the cKO mice maintained normal cardiac function in vivo and high-energy phosphate content in isolated perfused hearts. However, the cKO mice developed accelerated heart failure after pressure overload or repeated pregnancy. Decreased NAD(+)/NADH ratio by complex I deficiency inhibited Sirt3 activity, leading to an increase in protein acetylation and sensitization of the permeability transition in mitochondria (mPTP). NAD(+) precursor supplementation to cKO mice partially normalized the NAD(+)/NADH ratio, protein acetylation, and mPTP sensitivity. These findings describe a mechanism connecting mitochondrial dysfunction to the susceptibility to diseases and propose a potential therapeutic target.

Novel interactions between mitochondrial superoxide dismutases and the electron transport chain.

The processes that control aging remain poorly understood. We have exploited mutants in the nematode, Caenorhabditis elegans, that compromise mitochondrial function and scavenging of reactive oxygen species (ROS) to understand their relation to lifespan. We discovered unanticipated roles and interactions of the mitochondrial superoxide dismutases (mtSODs): SOD-2 and SOD-3. Both SODs localize to mitochondrial supercomplex I:III:IV. Loss of SOD-2 specifically (i) decreases the activities of complexes I and II, complexes III and IV remain normal; (ii) increases the lifespan of animals with a complex I defect, but not the lifespan of animals with a complex II defect, and kills an animal with a complex III defect; (iii) induces a presumed pro-inflammatory response. Knockdown of a molecule that may be a pro-inflammatory mediator very markedly extends lifespan and health of certain mitochondrial mutants. The relationship between the electron transport chain, ROS, and lifespan is complex, and defects in mitochondrial function have specific interactions with ROS scavenging mechanisms. We conclude that mtSODs are embedded within the supercomplex I:III:IV and stabilize or locally protect it from reactive oxygen species (ROS) damage. The results call for a change in the usual paradigm for the interaction of electron transport chain function, ROS release, scavenging, and compensatory responses.

Isoflurane selectively inhibits distal mitochondrial complex I in Caenorhabditis elegans.

BACKGROUND: Complex I of the electron transport chain (ETC) is a possible target of volatile anesthetics (VAs). Complex I enzymatic activities are inhibited by VAs, and dysfunction of complex I can lead to hypersensitivity to VAs in worms and in people. Mutant analysis in Caenorhabditis (C.) elegans suggests that VAs may specifically interfere with complex I function at the binding site for its substrate ubiquinone. We hypothesized that isoflurane inhibits electron transport by competing with ubiquinone for binding to complex I. METHODS: Wildtype and mutant C. elegans were used to study the effects of isoflurane on isolated mitochondria. Enzymatic activities of the ETC were assayed and dose-response curves determined using established techniques. Two-dimensional native gels of mitochondrial proteins were performed after exposure of mitochondria to isoflurane. RESULTS: Complex I is the most sensitive component of the ETC to isoflurane inhibition; however, the proximal portion of complex I (the flavoprotein) is relatively insensitive to isoflurane. Isoflurane and quinone do not compete for a common binding site on complex I. The absolute rate of complex I enzymatic activity in vitro does not predict immobilization of the animal by isoflurane. Isoflurane had no measurable effect on stability of mitochondrial supercomplexes. Reduction of ubiquinone by complex I displayed positive cooperative kinetics not disrupted by isoflurane. CONCLUSIONS: Isoflurane directly inhibits complex I at a site distal to the flavoprotein subcomplex. However, we have excluded our original hypothesis that isoflurane and ubiquinone compete for a common hydrophobic binding site on complex I. In addition, immobilization of the nematode by isoflurane is not due to limiting absolute amounts of complex I electron transport as measured in isolated mitochondria.

Mutations in mitochondrial complex III uniquely affect complex I in Caenorhabditis elegans.

Mitochondrial supercomplexes containing complexes I, III, and IV of the electron transport chain are now regarded as an established entity. Supercomplex I·III·IV has been theorized to improve respiratory chain function by allowing quinone channeling between complexes I and III. Here, we show that the role of the supercomplexes extends beyond channeling. Mutant analysis in Caenorhabditis elegans reveals that complex III affects supercomplex I·III·IV formation by acting as an assembly or stabilizing factor. Also, a complex III mtDNA mutation, ctb-1, inhibits complex I function by weakening the interaction of complex IV in supercomplex I·III·IV. Other complex III mutations inhibit complex I function either by decreasing the amount of complex I (isp-1), or decreasing the amount of complex I in its most active form, the I·III·IV supercomplex (isp-1;ctb-1). ctb-1 suppresses a nuclear encoded complex III defect, isp-1, without improving complex III function. Allosteric interactions involve all three complexes within the supercomplex and are necessary for maximal enzymatic activities.

Genome-wide linkage scan and association study of PARL to the expression of LHON families in Thailand.

Leber hereditary optic neuropathy (LHON) is the most common mitochondrially inherited disease causing blindness, preferentially in young adult males. Most of the patients carry the G11778A mitochondrial DNA (mtDNA) mutation. However, the marked incomplete penetrance and the gender bias indicate some additional genetic and/or environmental factors to disease expression. Herein, we first conducted a genome-wide linkage scan with 400 microsatellite markers in 9 large Thai LHON G11778A pedigrees. Using an affecteds-only nonparametric linkage analysis, 4 regions on chromosomes 3, 12, 13 and 18 showed Zlr scores greater than 2 (P < 0.025), which is consistently significant across several linkage statistics. The most suggestive marker D3S1565 (Zlr > 2 in 10 of 16 allele sharing models tested) was then expanded to include the region 3q26.2-3q28 covering SLC7A14 (3q26.2), MFN1 (3q26.32), MRPL47 (3q26.33), MCCC1 (3q27.1), PARL (3q27.1) and OPA1 (3q28-q29). All of these candidate genes were selected from the Maestro database and had known to be localized in mitochondria. Sixty tag SNPs were genotyped in 86 cases, 211 of their relatives and 32 unrelated Thai controls, by multiplex-PCR-based Invader assay. Analyses using a powerful association testing tool that adjusts for relatedness (the M(QLS) statistic) showed the most evidence of association between two SNPs, rs3749446 and rs1402000 (located in PARL presenilins-associated rhomboid-like) and LHON expression (both P = 8.8 x 10(-5)). The mitochondrial PARL protease has been recently known to play a role with a dynamin-related OPA1 protein in preventing apoptotic events by slowing down the release of cytochrome c out of mitochondrial cristae junctions. Moreover, PARL is required to activate the intramembranous proteolyses resulting in the degradation of an accumulated pro-apoptotic protein in the outer mitochondrial membrane. Under these circumstances, variants of PARL are suggested to influence cell death by apoptosis which has long been believed to intrigue the neurodegeneration of LHON.

Subcomplex Ilambda specifically controls integrated mitochondrial functions in Caenorhabditis elegans.

Complex I dysfunction is a common, heterogeneous cause of human mitochondrial disease having poorly understood pathogenesis. The extensive conservation of complex I composition between humans and Caenorhabditis elegans permits analysis of individual subunit contribution to mitochondrial functions at both the whole animal and mitochondrial levels. We provide the first experimentally-verified compilation of complex I composition in C. elegans, demonstrating 84% conservation with human complex I. Individual subunit contribution to mitochondrial respiratory capacity, holocomplex I assembly, and animal anesthetic behavior was studied in C. elegans by RNA interference-generated knockdown of nuclear genes encoding 28 complex I structural subunits and 2 assembly factors. Not all complex I subunits directly impact respiratory capacity. Subcomplex Ilambda subunits along the electron transfer pathway specifically control whole animal anesthetic sensitivity and complex II upregulation, proportionate to their relative impairment of complex I-dependent oxidative capacity. Translational analysis of complex I dysfunction facilitates mechanistic understanding of individual gene contribution to mitochondrial disease. We demonstrate that functional consequences of complex I deficiency vary with the particular subunit that is defective.

Complex I function is defective in complex IV-deficient Caenorhabditis elegans.

Cytochrome c oxidase (COX) is hypothesized to be an important regulator of oxidative phosphorylation. However, no animal phenotypes have been described due to genetic defects in nuclear-encoded subunits of COX. We knocked down predicted homologues of COX IV and COX Va in the nematode Caenorhabditis elegans. Animals treated with W09C5.8 (COX IV) or Y37D8A.14 (COX Va) RNA interference had shortened lifespans and severe defects in mitochondrial respiratory chain function. Amount and activity of complex IV, as well as supercomplexes that included complex IV, were decreased in COX-deficient worms. The formation of supercomplex I:III was not dependent on COX. We found that COX deficiencies decreased intrinsic complex I enzymatic activity, as well as complex I-III enzymatic activity. However, overall amounts of complex I were not decreased in these animals. Surprisingly, intrinsic complex I enzymatic activity is dependent on the presence of complex IV, despite no overall decrease in the amount of complex I. Presumably the association of complex I with complex IV within the supercomplex I:III:IV enhances electron flow through complex I. Our results indicate that reduction of a single subunit within the electron transport chain can affect multiple enzymatic steps of electron transfer, including movement within a different protein complex. Patients presenting with multiple defects of electron transport may, in fact, harbor a single genetic defect.