scholarly journals Nutrient Alteration Drives the Impacts of Seawater Acidification on the Bloom-Forming Dinoflagellate Karenia mikimotoi

2021 ◽  
Vol 12 ◽  
Author(s):  
Qian Liu ◽  
Yanqun Wang ◽  
Yuanyuan Li ◽  
Yijun Li ◽  
You Wang ◽  
...  
Keyword(s):  
Citrate Synthase ◽  
Experimental System ◽  
N:P Ratio ◽  
Toxin Production ◽  
Mitochondrial Pathway ◽  
Mitochondrial Metabolism ◽  
Dependent Manner ◽  
Karenia Mikimotoi ◽  
Seawater Acidification ◽  
P Limitation

Seawater acidification and nutrient alteration are two dominant environmental factors in coastal environments that influence the dynamics and succession of marine microalgae. However, the impacts of their combination have seldom been recorded. A simulated experimental system was set up to mimic the effects of elevated acidification on a bloom-forming dinoflagellate, Karenia mikimotoi, exposed to different nutrient conditions, and the possible mechanism was discussed. The results showed that acidification at different pH levels of 7.6 or 7.4 significantly influenced microalgal growth (p<0.05) compared with the control at pH 8.0. Mitochondria, the key sites of aerobic respiration and energy production, were impaired in a pH-dependent manner, and a simultaneous alteration of reactive oxygen species (ROS) production occurred. Cytochrome c oxidase (COX) and citrate synthase (CS), two mitochondrial metabolism-related enzymes, were actively induced with acidification exposure, suggesting the involvement of the mitochondrial pathway in coping with acidification. Moreover, different nutrient statuses indicated by various N:P ratios of 7:1 (N limitation) and 52:1 (P limitation) dramatically altered the impacts of acidification compared with those exposed to an N:P ratio of 17:1 (control), microalgal growth at pH 7.4 was obviously accelerated with the elevation of the nutrient ratio compared to that at pH 8.1 (p<0.05), and nutrient limitations seemed beneficial for growth in acidifying conditions. The production of alkaline phosphatase (AP) and acid phosphatase (AcP), an effective index indicating the microalgal growth status, significantly increased at the same time (p<0.05), which further supported this speculation. However, nitrate reductase (NR) was slightly inhibited. Hemolytic toxin production showed an obvious increase as the N:P ratio increased when exposed to acidification. Taken together, mitochondrial metabolism was suspected to be involved in the process of coping with acidification, and nutrient alterations, especially P limitation, could effectively alleviate the negative impacts induced by acidification. The obtained results might be a possible explanation for the competitive fitness of K. mikimotoi during bloom development.

scholarly journals 8a, a New Acridine Antiproliferative and Pro-Apoptotic Agent Targeting HDAC1/DNMT1

2021 ◽  
Vol 22 (11) ◽  
pp. 5516
Author(s):  
Qiting Zhang ◽  
Ziyan Wang ◽  
Xinyuan Chen ◽  
Haoxiang Qiu ◽  
Yifan Gu ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Dna Methyltransferase ◽  
Hdac Inhibitors ◽  
Mitochondrial Pathway ◽  
Epigenetic Therapy ◽  
Protein Interaction Data ◽  
Dependent Manner ◽  
Histone Deacetylase 1 ◽  
Histiocytic Lymphoma ◽  
Dose Dependent

Epigenetic therapy using histone deacetylase (HDAC) inhibitors has become an attractive project in new drug development. However, DNA methylation and histone acetylation are important epigenetic ways to regulate the occurrence and development of leukemia. Given previous studies, N-(2-aminophenyl)benzamide acridine (8a), as a histone deacetylase 1 (HDAC1) inhibitor, induces apoptosis and shows significant anti-proliferative activity against histiocytic lymphoma U937 cells. HDAC1 plays a role in the nucleus, which we confirmed by finding that 8a entered the nucleus. Subsequently, we verified that 8a mainly passes through the endogenous (mitochondrial) pathway to induce cell apoptosis. From the protein interaction data, we found that 8a also affected the expression of DNA methyltransferase 1 (DNMT1). Therefore, an experiment was performed to assess the binding of 8a to DNMT1 at the molecular and cellular levels. We found that the binding strength of 8a to DNMT1 enhanced in a dose-dependent manner. Additionally, 8a inhibits the expression of DNMT1 mRNA and its protein. These findings suggested that the anti-proliferative and pro-apoptotic activities of 8a against leukemia cells were achieved by targeting HDAC1 and DNMT1.

scholarly journals Atorvastatin impairs liver mitochondrial function in obese Göttingen Minipigs but heart and skeletal muscle are not affected

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Liselotte Bruun Christiansen ◽  
Tine Lovsø Dohlmann ◽  
Trine Pagh Ludvigsen ◽  
Ewa Parfieniuk ◽  
Michal Ciborowski ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Mitochondrial Function ◽  
Citrate Synthase ◽  
Obese Group ◽  
Control Group ◽  
Dependent Manner ◽  
Respiratory Capacity ◽  
Protein Carbonyl Content ◽  
Functional Changes ◽  
Mitochondrial Respiratory

AbstractStatins lower the risk of cardiovascular events but have been associated with mitochondrial functional changes in a tissue-dependent manner. We investigated tissue-specific modifications of mitochondrial function in liver, heart and skeletal muscle mediated by chronic statin therapy in a Göttingen Minipig model. We hypothesized that statins enhance the mitochondrial function in heart but impair skeletal muscle and liver mitochondria. Mitochondrial respiratory capacities, citrate synthase activity, coenzyme Q10 concentrations and protein carbonyl content (PCC) were analyzed in samples of liver, heart and skeletal muscle from three groups of Göttingen Minipigs: a lean control group (CON, n = 6), an obese group (HFD, n = 7) and an obese group treated with atorvastatin for 28 weeks (HFD + ATO, n = 7). Atorvastatin concentrations were analyzed in each of the three tissues and in plasma from the Göttingen Minipigs. In treated minipigs, atorvastatin was detected in the liver and in plasma. A significant reduction in complex I + II-supported mitochondrial respiratory capacity was seen in liver of HFD + ATO compared to HFD (P = 0.022). Opposite directed but insignificant modifications of mitochondrial respiratory capacity were seen in heart versus skeletal muscle in HFD + ATO compared to the HFD group. In heart muscle, the HFD + ATO had significantly higher PCC compared to the HFD group (P = 0.0323). In the HFD group relative to CON, liver mitochondrial respiration decreased whereas in skeletal muscle, respiration increased but these changes were insignificant when normalizing for mitochondrial content. Oral atorvastatin treatment in Göttingen Minipigs is associated with a reduced mitochondrial respiratory capacity in the liver that may be linked to increased content of atorvastatin in this organ.

scholarly journals Cardiac Remodeling Induced by All-Trans Retinoic Acid is Detrimental in Normal Rats

2017 ◽  
Vol 43 (4) ◽  
pp. 1449-1459 ◽  
Author(s):  
Renata A. C. Silva ◽  
Andréa F. Gonçalves ◽  
Priscila P. dos Santos ◽  
Bruna Rafacho ◽  
Renan F. T. Claro ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Retinoic Acid ◽  
Energy Metabolism ◽  
Cardiac Remodeling ◽  
Citrate Synthase ◽  
Isolated Heart ◽  
Lipid Hydroperoxide ◽  
Dependent Manner ◽  
Heart Study ◽  
Dose Dependent

Background/Aims: This study aimed to discern whether the cardiac alterations caused by retinoic acid (RA) in normal adult rats are physiologic or pathologic. Methods and Results: Wistar rats were assigned into four groups: control animals (C, n = 20) received a standard rat chow; animals fed a diet supplemented with 0.3 mg/kg/day all-trans-RA (AR1, n = 20); animals fed a diet supplemented with 5 mg/kg/day all-trans-RA (AR2, n = 20); and animals fed a diet supplemented with 10 mg/kg/day all-trans-RA (AR3, n = 20). After 2 months, the animals were submitted to echocardiogram, isolated heart study, histology, energy metabolism status, oxidative stress condition, and the signaling pathway involved in the cardiac remodeling induced by RA. RA increased myocyte cross-sectional area in a dose-dependent manner. The treatment did not change the morphological and functional variables, assessed by echocardiogram and isolated heart study. In contrast, RA changed catalases, superoxide dismutase, and glutathione peroxidases and was associated with increased values of lipid hydroperoxide, suggesting oxidative stress. RA also reduced citrate synthase, enzymatic mitochondrial complex II, ATP synthase, and enzymes of fatty acid metabolism and was associated with increased enzymes involved in glucose use. In addition, RA increased JNK 1/2 expression, without changes in TGF-β, PI3K, AKT, NFκB, S6K, and ERK. Conclusion: In normal rats, RA induces cardiac hypertrophy in a dose-dependent manner. The non-participation of the PI3K/Akt pathway, associated with the participation of the JNK pathway, oxidative stress, and changes in energy metabolism, suggests that cardiac remodeling induced by RA supplementation is deleterious.

scholarly journals A NewT. gondiiMouse Model of Gene-Environment Interaction Relevant to Psychiatric Disease

Scientifica ◽  
2018 ◽  
Vol 2018 ◽  
pp. 1-7 ◽  
Author(s):  
Geetha Kannan ◽  
Emese Prandovszky ◽  
Emily Severance ◽  
Robert H. Yolken ◽  
Mikhail V. Pletnikov
Keyword(s):  
Psychiatric Disorders ◽  
Acoustic Startle ◽  
Experimental System ◽  
Dominant Negative ◽  
Psychiatric Disease ◽  
Environment Interaction ◽  
Dependent Manner ◽  
Mechanistic Studies ◽  
Gene Environment Interaction ◽  
Gene Environment

Infection with the protozoan parasite,Toxoplasma gondii(T. gondii), was linked to several psychiatric disorders. The exact mechanisms of a hypothesized contribution ofT. gondiiinfection are poorly understood, and it appears that only a subset of seropositive individuals go on to develop a mental illness, suggesting genetic vulnerability. In order to stimulate mechanistic studies of how exposure toT. gondiicould interact with genetic predisposition to psychiatric disorders, we have generated and characterized a mouse model of chronicT. gondiiinfection in BALB/c mice with inducible forebrain neuronal expression of a C-terminus truncated dominant-negative form of disrupted-in-schizophrenia 1 (DN-DISC1). In this gene-environment interaction (GxE) model, exposing control and DN-DISC1 male and female mice toT. gondiiproduced sex-dependent abnormalities in locomotor activity and prepulse inhibition of the acoustic startle. No genotype- or sex-dependent effects were found on levels of anti-Toxoplasma IgG antibodies or anti-NMDAR or C1q antibodies. Our work demonstrates that a psychiatric genetic risk factor, DN-DISC1, modulates the neurobehavioral effects of chronicT. gondiiinfection in a sex-dependent manner. The presentT. gondiimodel of GxE provides a valuable experimental system for future mechanistic studies and evaluation of new treatments.

scholarly journals Exercise Training-Induced PPARβ Increases PGC-1α Protein Stability and Improves Insulin-Induced Glucose Uptake in Rodent Muscles

Nutrients ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 652 ◽  
Author(s):  
Ju-Sik Park ◽  
John O. Holloszy ◽  
Kijin Kim ◽  
Jin-Ho Koh
Keyword(s):  
Cytochrome C ◽  
Exercise Training ◽  
Protein Stability ◽  
Glucose Uptake ◽  
Citrate Synthase ◽  
Peroxisome Proliferator ◽  
Mitochondrial Enzymes ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Peroxisome Proliferator Activated Receptor

This study aimed to investigate the long-term effects of training intervention and resting on protein expression and stability of peroxisome proliferator-activated receptor β/δ (PPARβ), peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α), glucose transporter type 4 (GLUT4), and mitochondrial proteins, and determine whether glucose homeostasis can be regulated through stable expression of these proteins after training. Rats swam daily for 3, 6, 9, 14, or 28 days, and then allowed to rest for 5 days post-training. Protein and mRNA levels were measured in the skeletal muscles of these rats. PPARβ was overexpressed and knocked down in myotubes in the skeletal muscle to investigate the effects of swimming training on various signaling cascades of PGC-1α transcription, insulin signaling, and glucose uptake. Exercise training (Ext) upregulated PPARβ, PGC-1α, GLUT4, and mitochondrial enzymes, including NADH-ubiquinone oxidoreductase (NUO), cytochrome c oxidase subunit I (COX1), citrate synthase (CS), and cytochrome c (Cyto C) in a time-dependent manner and promoted the protein stability of PPARβ, PGC-1α, GLUT4, NUO, CS, and Cyto C, such that they were significantly upregulated 5 days after training cessation. PPARβ overexpression increased the PGC-1α protein levels post-translation and improved insulin-induced signaling responsiveness and glucose uptake. The present results indicate that Ext promotes the protein stability of key mitochondria enzymes GLUT4, PGC-1α, and PPARβ even after Ext cessation.

scholarly journals Suberoyl bis-hydroxamic acid reactivates Kaposi’s sarcoma-associated herpesvirus through histone acetylation and induces apoptosis in lymphoma cells

2020 ◽  
Author(s):  
Shun Iida ◽  
Sohtaro Mine ◽  
Keiji Ueda ◽  
Tadaki Suzuki ◽  
Hideki Hasegawa ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Hydroxamic Acid ◽  
Kaposi's Sarcoma ◽  
Primary Effusion Lymphoma ◽  
Kaposi’S Sarcoma ◽  
Mitochondrial Pathway ◽  
Dependent Manner ◽  
Transcription Activator ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an etiologic agent of Kaposi’s sarcoma as well as primary effusion lymphoma (PEL), an aggressive B-cell neoplasm which mostly arises in immunocompromised individuals. Lytic replication of KSHV is also associated with a subset of multicentric Castleman diseases. At present, there is no specific treatment available for PEL and its prognosis is poor. In this study, we found that the histone deacetylase inhibitor suberoyl bis-hydroxamic acid (SBHA) induced KSHV reactivation in PEL cells in a dose-dependent manner. Next-generation sequencing analysis showed that more than 40% of all transcripts expressed in SBHA-treated PEL cells originated from the KSHV genome compared with less than 1% in untreated cells. Chromatin immunoprecipitation assays demonstrated that SBHA induced histone acetylation targeting the promoter region of the KSHV replication and transcription activator gene. However, there was no significant change in methylation status of the promoter region of this gene. In addition to its effect of KSHV reactivation, this study revealed that SBHA induces apoptosis in PEL cells in a dose-dependent manner, inducing acetylation and phosphorylation of p53, cleavage of caspases, and expression of pro-apoptotic factors such as Bim and Bax. These findings suggest that SBHA reactivates KSHV from latency and induces apoptosis through the mitochondrial pathway in PEL cells. Therefore, SBHA can be considered a new tool for induction of KSHV reactivation, and could provide a novel therapeutic strategy against PEL. IMPORTANCE Kaposi’s sarcoma and primary effusion lymphoma cells are latently infected with Kaposi’s sarcoma-associated herpesvirus (KSHV), whereas KSHV replication is frequently observed in multicentric Castleman disease. Although KSHV replication can be induced by some chemical reagents (e.g. 12-O-tetradecanoylphorbol-13-acetate), the mechanism of KSHV replication is not fully understood. We found that the histone deacetylase inhibitor suberoyl bis-hydroxamic acid (SBHA) induced KSHV reactivation with high efficiency, through histone acetylation in the promoter of the replication and transcription activator gene, compared with 12-O-tetradecanoylphorbol-13-acetate. SBHA also induced apoptosis through the mitochondrial pathway in KSHV-infected cells, with a lower EC50 than measured for viral reactivation. SBHA could be used in a highly efficient replication system for KSHV in vitro, and as a tool to reveal the mechanism of replication and pathogenesis of KSHV. The ability of SBHA to induce apoptosis at lower levels than needed to stimulate KSHV reactivation, indicates its therapeutic potential.

Overexpression of the Cell Death Suppressor Bcl-w in Ischemic Brain: Implications for a Neuroprotective Role via the Mitochondrial Pathway

2000 ◽  
Vol 20 (3) ◽  
pp. 620-630 ◽  
Author(s):  
Chaohua Yan ◽  
Jun Chen ◽  
Dexi Chen ◽  
Manabu Minami ◽  
Wei Pei ◽  
...  
Keyword(s):  
Cell Death ◽  
Cytochrome C ◽  
Permeability Transition ◽  
Brain Mitochondria ◽  
Mitochondrial Pathway ◽  
Border Zone ◽  
Dependent Manner ◽  
High Concentration ◽  
Ischemic Brain ◽  
Cell Death Suppressor

Bcl-w is a newly described cell death suppressor member of the Bcl-2 gene family. As these genes may have a role in the outcome of ischemic brain injury, the regional expression of Bcl-w protein in rat brain was examined at 6 to 72 hours after 90 minutes of transient middle cerebral artery occlusion. Bcl-w protein, although constitutively expressed at low levels in nonischemic brain, was found to be overexpressed in ischemic brain at all time points studied. Up-regulation of Bcl-w protein was particularly abundant in the penumbral region of the cortex and mainly in cells lacking DNA fragmentation. In the cortical penumbra, Bcl-w protein was detected predominantly in neurons and showed mitochondrial localization, as determined using double-label immunohistochemistry. Bcl-w expression was also detectable, to a lesser extent, in reactive astrocytes in the infarct border zone and in microvessel walls in the infarct regions. At the mechanistic level, incubation of isolated brain mitochondria with the addition of recombinant Bax or high concentration of calcium resulted in release of cytochrome c from the mitochondria. In the presence of recombinant Bcl-w protein, however, the release of cytochrome c induced by Bax or calcium was largely inhibited. Further, recombinant Bcl-w protein inhibited calcium-induced loss of mitochondrial transmembrane potential, indicative of permeability transition, in a dose-dependent manner. These results suggest that Bcl-w may be an endogenous neuroprotectant against ischemic neuronal death and that, like its analogues such as Bcl-2 and Bcl-x-long, Bcl-w may achieve this protection via the mitochondrial death-regulatory pathway.

Cytosolic Histone H1.2 Releasing under Different Apoptotic Stimuli in Chronic Lymphocytic Leukemia (CLL).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2108-2108
Author(s):  
Eva Gine ◽  
Marta Crespo ◽  
Ana Muntanola ◽  
Emili Montserrat ◽  
Francesc Bosch
Keyword(s):  
Functional Status ◽  
Annexin V ◽  
Histone H1 ◽  
Mitochondrial Pathway ◽  
Lymphocytic Leukemia ◽  
Fish Analysis ◽  
Dependent Manner ◽  
Immunofluorescence Analysis ◽  
Cytochrome C Release ◽  
Nuclear Histone

Abstract Recently, it has been shown that nuclear histone H1.2 is released into cytoplasm when apoptosis is induced by DNA double-strand breaks (DSB’s), this process being dependent on p53 functional status. In addition, cytosolic histone H1.2 induces cytochrome C release in a Bak-dependent manner. Thus, cytosolic histone H1.2 release represents a new mechanism that links DSB’s with activation of the apoptotic mitochondrial pathway. Against this background, we analyzed the release of histone H1.2 in the cytosol of purified CLL cells during apoptosis induced by fludarabine (F), mitoxantrone (M), etoposide, or ionizing radiation. In addition, the presence of histone H1 was correlated with p53 functional status. Cell viability and analysis of apoptosis were investigated by annexin V/PI staining and FACscan analysis. The presence of histone H1 and H1.2 in the cytosolic fraction was assessed by Western Blott using the anti-histone H1 (Upstate) and anti-histone H1.2 (Abcam) antibodies. Histone H1 traffic was also evaluated by using immunofluorescence analysis in CLL cells suspensions. FISH analysis was used to select samples with (n=3) or without (n=6) p53 deletion, and activation of p53 after treatment was assessed by Western Blot. In cases without p53 deletion, increased apoptosis was observed under all stimuli, the FM combination being the most effective. In such cases, histone H1.2 release was apparent 6 hours after the onset of irradiation or pharmacologic treatments, progressively increasing up to 24 hours. In contrast, cases with p53 deletion displayed a low cytotoxic effect upon different treatments. Interestingly, no p53 activation or histone H1.2 release into cytosol was observed. These results were also confirmed by immunofluorescence analysis, in which histone H1.2 was only visible in the cytosol of non-deleted p53 cases. These results demonstrate that, upon drug or irradiation exposure nuclear histone H1.2 is released into the cytoplasm of CLL cells in a p53-dependent manner. This suggests that, in CLL, histone H1.2 traffic contributes to the apoptosis induced by DSB’s and to drug resistance in cases with p53 deletion.

Histone H1.2 Releasing under Different Apoptotic Stimuli in Chronic Lymphocytic Leukemia (CLL).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1141-1141
Author(s):  
Eva Gine ◽  
Marta Crespo ◽  
Ana Muntañola ◽  
Eva Calpe ◽  
Maria Joao Gomes ◽  
...  
Keyword(s):  
Annexin V ◽  
Genetic Alterations ◽  
Mitochondrial Pathway ◽  
Lymphocytic Leukemia ◽  
Fish Analysis ◽  
Dependent Manner ◽  
Cytochrome C Release ◽  
Dna Damaging Agents ◽  
Genetic Abnormalities ◽  
P53 Status

Abstract Cytosolic release of histone H1.2 has been described as a new apoptogenic mechanism induced by DNA damage that results in cytochrome C release and activation of the apoptotic mitochondrial pathway. Primary tumoral CLL cells from 25 patients were investigated for histone H1.2 cytosolic release after treatment with genotoxic (fludarabine, mitoxantrone, etoposide, or X-ray radiation) and non-genotoxic (dexamethasone) agents. Cases were analyzed for the presence of poor-risk genetic alterations, particularly deletions at 17p13 and 11q22. Histone H1.2 release was correlated with the presence of genetic abnormalities and with the best clinical response obtained with standard treatments. FISH analysis, cell viability measured by annexin V binding, Western Blot studies and inmunofluorescence techniques with confocal spectral microscopy were also employed. DNA-damaging agents induced H1.2 release in a p53-dependent manner, which was confirmed by the lack of H1.2 release in p53-deleted cases. Non DNA-damaging agents induced release of H1.2 in both p53-deleted and non-deleted CLL cases. Moreover, nuclear H1.2 release was observed after genotoxic and non-genotoxic treatment independently of ATM function. From the clinical standpoint, the lack of histone H1.2 release correlated with resistance to genotoxic treatment. In CLL cells, histone H1.2 traffic was dependent on the p53-status after genotoxic treatment, but was also inducible after treatments acting independently of p53. In contrast, histone H1.2 release seemed not to be dependent on ATM function. Nuclear histone H1.2 release appears to be an important element in apoptosis induction in CLL, particularly in cases with abnormal p53 function resistant to conventional treatment.

Aconitase: An Iron Sensing Regulator of Mitochondrial Oxidative Metabolism and Erythropoiesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 74-74 ◽  
Author(s):  
Naomi D Gunawardena ◽  
Valerie Schrott ◽  
Chanté Richardson ◽  
Teague Nolan Cole ◽  
Catherine G Corey ◽  
...  
Keyword(s):  
Cell Lines ◽  
Citrate Synthase ◽  
Conflicts Of Interest ◽  
Mitochondrial Metabolism ◽  
Ros Generation ◽  
Atp Citrate Lyase ◽  
Complex Activity ◽  
Direct Role ◽  
Iron Restriction ◽  
Mitochondrial Aconitase

Abstract Anemia is a global health problem that decreases quality of life for billions of people. Previous studies have concluded that there is an iron-regulated checkpoint in erythropoiesis that suppresses red blood cell production. This results in anemia and the conservation of iron for use in other vital processes. We have shown that the aconitase enzymes are key to this pathway and that inhibition of aconitase enzyme activity by iron restriction or pharmacologic inhibitors blocks erythropoiesis in primary human hematopoietic progenitor cells (HPCs). Mitochondrial aconitase (ACO2) functions as an isomerase within the tricarboxylic acid (TCA) cycle to convert citrate into isocitrate, which contributes to ATP and heme synthesis. During iron restriction, there is a significant decrease of intracellular isocitrate with only a slight increase in intracellular citrate. A corresponding increase in ATP-citrate lyase activity suggests that excess citrate is shunted into acetyl-CoA production. The addition of exogenous isocitrate to iron-deprived HPCs abrogates the block in erythropoiesis and protects iron-deprived mice and chronically-inflamed rats from anemia. These results suggest that ACO2 regulates mitochondrial metabolism and erythropoiesis. Recent unpublished data shows that ACO2 inhibition by iron deprivation or by treatment with an ACO2 inhibitor decreases mitochondrial respiratory rates (RR) and increases mitochondrial reactive oxygen species (mito-ROS). Isocitrate normalizes RR and mito-ROS and restores erythropoiesis. Importantly, disruption of ROS generation with a variety of anti-oxidants blocks erythropoiesis, while surprisingly, treatment of iron restricted HPCs with oxidant generators or ROS promotes erythropoiesis. These data inform our overarchinghypothesis that iron-restriction inhibits ACO2, thereby inhibiting mitochondrial metabolism, resulting in the loss of a mitochondrial ROS signal that is required for erythropoiesis. We have recently extended these studies to ACO2 knock down (ACO2-KD) K562 cell lines which provide more material for biochemical assessment of mitochondrial function. New pilot data shows that a 70% decrease in ACO2 expression significantly reduces the induction of erythroid specific genes during hydroxyurea or hemin treatment. This confirms that ACO2 plays a direct role in erythropoiesis. Extracellular flux (XF, Seahorse Bioscience) experiments show a decreased RR in ACO2-KD cells. Mitochondrial complex activity assays show no differences in complex IV or citrate synthase activity between control and ACO2-KD cell lines. These studies also demonstrate that the shRNA-ACO2 lentiviral constructs are effectively targeting ACO2 and can be used in our HPC model system. We now have evidence in two different human cell culture models of erythropoiesis that mitochondrial aconitase is an iron-sensing regulator of both mitochondrial respiration and erythropoiesis. Our long term goals are to identify novel therapeutic targets in this iron dependent metabolic regulatory pathway that enhance or suppress erythropoiesis and have potential clinical application in the treatment of anemia or polycythemia. We are also investigating the role of the mitochondrion in the differentiation of other hematopoietic cell lineages. Disclosures No relevant conflicts of interest to declare.

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