Petr Ježek - Academia.edu (original) (raw)

Papers by Petr Ježek

Research paper thumbnail of Hydroperoxy Fatty Acid Cycling Mediated by Mitochondrial Uncoupling Protein UCP2

Journal of Biological Chemistry, 2004

Research paper thumbnail of Biochemical Background in Mitochondria Affects 2HG Production by IDH2 and ADHFE1 in Breast Carcinoma

Cancers

Mitochondrial production of 2-hydroxyglutarate (2HG) can be catalyzed by wild-type isocitrate deh... more Mitochondrial production of 2-hydroxyglutarate (2HG) can be catalyzed by wild-type isocitrate dehydrogenase 2 (IDH2) and alcohol dehydrogenase, iron-containing 1 (ADHFE1). We investigated whether biochemical background and substrate concentration in breast cancer cells promote 2HG production. To estimate its role in 2HG production, we quantified 2HG levels and its enantiomers in breast cancer cells using analytical approaches for metabolomics. By manipulation of mitochondrial substrate fluxes using genetic and pharmacological approaches, we demonstrated the existence of active competition between 2HG producing enzymes, i.e., IDH2 and ADHFE1. Moreover, we showed that distinct fractions of IDH2 enzyme molecules operate in distinct oxido-reductive modes, providing NADPH and producing 2HG simultaneously. We have also detected 2HG release in the urine of breast cancer patients undergoing adjuvant therapy and detected a correlation with stages of breast carcinoma development. In summary, ...

Research paper thumbnail of Colloidally Stable P(DMA-AGME)-Ale-Coated Gd(Tb)F3:Tb3+(Gd3+),Yb3+,Nd3+ Nanoparticles as a Multimodal Contrast Agent for Down- and Upconversion Luminescence, Magnetic Resonance Imaging, and Computed Tomography

Nanomaterials

Multimodal imaging, integrating several modalities including down- and up-conversion luminescence... more Multimodal imaging, integrating several modalities including down- and up-conversion luminescence, T1- and T2(T2*)-weighted MRI, and CT contrasting in one system, is very promising for improved diagnosis of severe medical disorders. To reach the goal, it is necessary to develop suitable nanoparticles that are highly colloidally stable in biologically relevant media. Here, hydrophilic poly(N,N-dimethylacrylamide-N-acryloylglycine methyl ester)-alendronate-[P(DMA-AGME)-Ale]-coated Gd(Tb)F3:Tb3+(Gd3+),Yb3+,Nd3+ nanoparticles were synthesized by a coprecipitation method in ethylene glycol (EG) followed by coating with the polymer. The particles were tho-roughly characterized by a dynamic light scattering (DLS), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), X-ray energy dispersive spectroscopy (EDAX), selected area electron diffraction (SAED), elemental ana-lysis and fluorescence spectroscopy. Aqueous particle di...

Research paper thumbnail of Mitochondrial Superoxide Production Decreases on Glucose-Stimulated Insulin Secretion in Pancreatic β Cells Due to Decreasing Mitochondrial Matrix NADH/NAD+ Ratio

Antioxidants & Redox Signaling

Aims: Glucose-stimulated insulin secretion (GSIS) in pancreatic b cells was expected to enhance m... more Aims: Glucose-stimulated insulin secretion (GSIS) in pancreatic b cells was expected to enhance mitochondrial superoxide formation. Hence, we elucidated relevant redox equilibria. Results: Unexpectedly, INS-1E cells at transitions from 3 (11 mM; pancreatic islets from 5 mM) to 25 mM glucose decreased matrix superoxide release rates (MitoSOX Red monitoring validated by MitoB) and H 2 O 2 (mitoHyPer, subtracting mitoSypHer emission). Novel double-channel fluorescence lifetime imaging, approximating free mitochondrial matrix NADH F, indicated its *20% decrease. Matrix NAD + F increased on GSIS, indicated by the FAD-emission lifetime decrease, reflecting higher quenching of FAD by NAD + F. The participation of pyruvate/malate and pyruvate/citrate redox shuttles, elevating cytosolic NADPH F (iNAP1 fluorescence monitoring) at the expense of matrix NADH F , was indicated, using citrate (2-oxoglutarate) carrier inhibitors and cytosolic malic enzyme silencing: All changes vanished on these manipulations. 13 Cincorporation from 13 C-L-glutamine into 13 C-citrate reflected the pyruvate/isocitrate shuttle. Matrix NADPH F (iNAP3 monitored) decreased. With decreasing glucose, the suppressor of Complex III site Q electron leak (S3QEL) suppressor caused a higher Complex I I F site contribution, but a lower superoxide fraction ascribed to the Complex III site III Qo. Thus, the diminished matrix NADH F /NAD + F decreased Complex I flavin site I F superoxide formation on GSIS. Innovation: Mutually validated methods showed decreasing superoxide release into the mitochondrial matrix in pancreatic b cells on GSIS, due to the decreasing matrix NADH F /NAD + F (NADPH F /NADP + F) at increasing cytosolic NADPH F levels. The developed innovative methods enable real-time NADH/NAD + and NADPH/ NADP + monitoring in any distinct cell compartment. Conclusion: The export of reducing equivalents from mitochondria adjusts lower mitochondrial superoxide production on GSIS, but it does not prevent oxidative stress in pancreatic b cells. Antioxid. Redox Signal. 33, 789-815.

Research paper thumbnail of Overexpression of native IF1 downregulates glucose-stimulated insulin secretion by pancreatic INS-1E cells

Scientific Reports

We have previously reported that transient knock-down of ATPase inhibitory factor 1 (IF1) by siRN... more We have previously reported that transient knock-down of ATPase inhibitory factor 1 (IF1) by siRNA upregulates ATP levels and subsequently augments insulin secretion in model pancreatic β-cells INS-1E. Here we investigated how long-term IF1-overexpression impacts pancreatic β-cell bioenergetics and insulin secretion. We generated INS-1E cell line stably overexpressing native IF1. We revealed that IF1 overexpression leads to a substantial decrease in ATP levels and reduced glucose-stimulated insulin secretion. A decrease in total cellular ATP content was also reflected in decreased free ATP cytosolic and mitochondrial levels, as monitored with ATeam biosensor. Consistently, cellular respiration of IF1-overexpressing cells was decreased. 3D structured illumination microscopy (SIM) revealed a higher amount of insulin granules with higher volume in IF1-overexpressing cells. Similar effects occurred when cells were incubated at low glucose concentrations. Noteworthy, activation of PKA by...

Research paper thumbnail of Mitochondrial cristae narrowing upon higher 2-oxoglutarate load

Biochimica et Biophysica Acta (BBA) - Bioenergetics

Research paper thumbnail of Role of ATPase Inhibitory Factor 1 (IF1) in metabolic regulations of insulin secreting INS-1E cells

Biochimica et Biophysica Acta (BBA) - Bioenergetics

Research paper thumbnail of Mitochondrial Nucleoids: Superresolution microscopy analysis

The International Journal of Biochemistry & Cell Biology

Research paper thumbnail of 3D super-resolution microscopy reflects mitochondrial cristae alternations and mtDNA nucleoid size and distribution

Biochimica et Biophysica Acta (BBA) - Bioenergetics

Research paper thumbnail of Colocalization of inhibitor factor IF1 and mitochondrial ATP synthase in pancreatic INS1E cells by 3D STED super-resolution microscopy

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2016

Research paper thumbnail of STED and dSTORM super-resolution microscopy of mitochondrial ATP synthase in relation to metabolic state of pancreatic INS1E cells

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2016

Research paper thumbnail of Mitochondrial nucleoid clusters protect newly synthesized mtDNA during Doxorubicin- and Ethidium Bromide-induced mitochondrial stress

Toxicology and Applied Pharmacology, 2016

Mitochondrial DNA (mtDNA) is compacted in ribonucleoprotein complexes called nucleoids, which can... more Mitochondrial DNA (mtDNA) is compacted in ribonucleoprotein complexes called nucleoids, which can divide or move within the mitochondrial network. Mitochondrial nucleoids are able to aggregate into clusters upon reaction with intercalators such as the mtDNA depletion agent Ethidium Bromide (EB) or anticancer drug Doxorobicin (DXR). However, the exact mechanism of nucleoid clusters formation remains unknown. Resolving these processes may help to elucidate the mechanisms of DXR-induced cardiotoxicity. Therefore, we addressed the role of two key nucleoid proteins; mitochondrial transcription factor A (TFAM) and mitochondrial single-stranded binding protein (mtSSB); in the formation of mitochondrial nucleoid clusters during the action of intercalators. We found that both intercalators cause numerous aberrations due to perturbing their native status. By blocking mtDNA replication, both agents also prevented mtDNA association with TFAM, consequently causing nucleoid aggregation into large nucleoid clusters enriched with TFAM, co-existing with the normal nucleoid population. In the later stages of intercalation (>48h), TFAM levels were reduced to 25%. In contrast, mtSSB was released from mtDNA and freely distributed within the mitochondrial network. Nucleoid clusters mostly contained nucleoids with newly replicated mtDNA, however the nucleoid population which was not in replication mode remained outside the clusters. Moreover, the nucleoid clusters were enriched with p53, an anti-oncogenic gatekeeper. We suggest that mitochondrial nucleoid clustering is a mechanism for protecting nucleoids with newly replicated DNA against intercalators mediating genotoxic stress. These results provide new insight into the common mitochondrial response to mtDNA stress and can be implied also on DXR-induced mitochondrial cytotoxicity.

Research paper thumbnail of Division of Mitochondrial Nucleoids Visualized by Biplane FPALM/dSTORM

Biophysical Journal, 2016

Research paper thumbnail of Silica-coated upconversion lanthanide nanoparticles: The effect of crystal design on morphology, structure and optical properties

Beilstein journal of nanotechnology, 2015

NaYF4:Yb(3+)/Er(3+) nanoparticles were synthesized by thermal decomposition of lanthanide trifluo... more NaYF4:Yb(3+)/Er(3+) nanoparticles were synthesized by thermal decomposition of lanthanide trifluoroacetates using oleylamine (OM) as both solvent and surface binding ligand. The effect of reaction temperature and time on the properties of the particles was investigated. The nanoparticles were characterized by transmission electron microscopy (TEM), electron diffraction (ED), energy dispersive spectroscopy (EDX), dynamic light scattering (DLS), thermogravimetric analysis (TGA), elemental analysis and X-ray diffraction (XRD) to determine morphology, size, polydispersity, crystal structure and elemental composition of the nanocrystals. TEM microscopy revealed that the morphology of the nanoparticles could be fine-tuned by modifying of the synthetic conditions. A cubic-to-hexagonal phase transition of the NaYF4:Yb(3+)/Er(3+) nanoparticles at temperatures above 300 °C was confirmed by both ED and XRD. Upconversion luminescence under excitation at 980 nm was observed in the luminescence s...

Research paper thumbnail of Constitutive Reprogramming of Fibroblast Mitochondrial Metabolism in Pulmonary Hypertension

American journal of respiratory cell and molecular biology, Jan 23, 2015

Remodeling of the distal pulmonary artery wall is a characteristic feature of pulmonary hypertens... more Remodeling of the distal pulmonary artery wall is a characteristic feature of pulmonary hypertension (PH). In hypoxic pulmonary hypertension, the most substantial pathologic changes occur in the adventitia. Here, there is marked fibroblast proliferation and profound macrophage accumulation. These fibroblasts (PH-Fibs) maintain a hyperproliferative, apoptotic resistant and pro-inflammatory phenotype in ex vivo culture. Considering that a similar phenotype is observed in cancer cells, where it has been associated at least in part with specific alterations in mitochondrial metabolism, we sought to define the state of mitochondrial metabolism in PH-Fibs. In PH-Fibs pyruvate dehydrogenase was markedly inhibited, resulting in metabolism of pyruvate to lactate, thus consistent with a Warburg-like phenotype. In addition, mitochondrial bioenergetics were suppressed and mitochondrial fragmentation was increased in PH-Fibs. Most importantly, Complex I activity was substantially decreased which...

Research paper thumbnail of 3D biplane PALM super-resolution microscopy: modeling biological structures & the system resolution

A unique 3D super-resolution microscope, based on a biplane detection scheme, enables single mole... more A unique 3D super-resolution microscope, based on a biplane detection scheme, enables single molecule localization in samples several µm thick with theoretical resolution 25 nm lateral and 50 nm axial [1]. Such resolution is achieved in a far-field optical setup through the use of special photo-switchable or photo-activated fluorescent dyes imaged under such conditions that only sparse subset of fluorophores is active at any given time so the position of individual molecules can be determined. To calculate axial position of the fluorophore, two parallel planes (about 600 nm apart) are imaged simultaneously, thus providing two sections of the Point Spread Function (PSF). The resolution is governed by the actual PSF achieved in the optical system and the number of photons captured from a single fluorophore activation event. The amount of information that can be gathered from the specimen also depends on the stability of the optical setup and durability of the sample [2]. The sample th...

Research paper thumbnail of Reductive carboxylation and 2-hydroxyglutarate formation by wild-type IDH2 in breast carcinoma cells

The International Journal of Biochemistry & Cell Biology, 2015

Mitochondrial NADPH-dependent isocitrate dehydrogenase, IDH2, and cytosolic IDH1, catalyze reduct... more Mitochondrial NADPH-dependent isocitrate dehydrogenase, IDH2, and cytosolic IDH1, catalyze reductive carboxylation of 2-oxoglutarate. Both idh2 and idh1 monoallelic mutations are harbored in grade 2/3 gliomas, secondary glioblastomas and acute myeloid leukemia. Mutant IDH1/IDH2 enzymes were reported to form an oncometabolite r-2-hydroxyglutarate (2HG), further strengthening malignancy. We quantified CO2-dependent reductive carboxylation glutaminolysis (RCG) and CO2-independent 2HG production in HTB-126 and MDA-MB-231 breast carcinoma cells by measuring (13)C incorporation from 1-(13)C-glutamine into citrate, malate, and 2HG. For HTB-126 cells, (13)C-citrate, (13)C-malate, and (13)C-2-hydroxyglutarate were enriched by 2-, 5-, and 15-fold at 5mM glucose (2-, 2.5-, and 13-fold at 25mM glucose), respectively, after 6h. Such enrichment decreased by 6% with IDH1 silencing, but by 30-50% upon IDH2 silencing while cell respiration and ATP levels rose up to 150%. Unlike 2HG production RCG declined at decreasing CO2. At hypoxia (5% O2), IDH2-related and unrelated (13)C-accumulation into citrate and malate increased 1.5-2.5-fold with unchanged IDH2 expression; whereas hypoxic 2HG formation did not. (13)C-2HG originated by ∼50% from other than IDH2 or IDH1 reactions, substantiating remaining activity in IDH1&2-silenced cells. Relatively high basal (12)C-2HG levels existed (5-fold higher vs. non-tumor HTB-125 cells) and (13)C-2HG was formed despite the absence of any idh2 and idh1 mutations in HTB-126 cells. Since RCG is enhanced at hypoxia (frequent in solid tumors) and 2HG can be formed without idh1/2 mutations, we suggest 2HG as an analytic marker (in serum, urine, or biopsies) predicting malignancy of breast cancer in all patients.

Research paper thumbnail of Delta Cell Hyperplasia in Adult Goto-Kakizaki (GK/MolTac) Diabetic Rats

Journal of Diabetes Research, 2015

Reduced beta cell mass in pancreatic islets (PI) of Goto-Kakizaki (GK) rats is frequently observe... more Reduced beta cell mass in pancreatic islets (PI) of Goto-Kakizaki (GK) rats is frequently observed in this diabetic model, but knowledge on delta cells is scarce. Aiming to compare delta cell physiology/pathology of GK to Wistar rats, we found that delta cell number increased over time as did somatostatin mRNA and delta cells distribution in PI is different in GK rats. Subtle changes in 6-week-old GK rats were found. With maturation and aging of GK rats, disturbed cytoarchitecture occurred with irregular beta cells accompanied by delta cell hyperplasia and loss of pancreatic polypeptide (PPY) positivity. Unlike the constant glucose-stimulation index for insulin PI release in Wistar rats, this index declined with GK age, whereas for somatostatin it increased with age. A decrease of GK rat PPY serum levels was found. GK rat body weight decreased with increasing hyperglycemia. Somatostatin analog octreotide completely blocked insulin secretion, impaired proliferation at low autocrine i...

Research paper thumbnail of Fibroblasts Derived from Distal Pulmonary Artery Wall of Chronic Hypoxia-Induced Pulmonary Hypertensive Neonatal Calves Exhibit Warburg-Like Energetic Metabolism with Pro-Oxidative Shift

Free Radical Biology and Medicine, 2013

Introduction: Pulmonary hypertension (PH) has been observed in patients suffering from both the i... more Introduction: Pulmonary hypertension (PH) has been observed in patients suffering from both the inherited and the acquired forms of hemolytic anemia. Despite the prevalence of PH with hemolytic anemia, the mechanism behind this process is unclear. Recently, our group and others have shown endothelial cell barrier dysfunction, in vitro and in vivo, with exposure to hemoglobin (Hb). According to basic physiology, endothelial cell barrier dysfunction would allow smooth muscle cells (SmC) to come into direct exposure to Hb, which may induce SmC proliferative phenotype. Hypothesis: Cell free Hb induces SMC proliferation by mechanisms associated with both direct exposure to SmC and indirectly by mediators released from Hb activated endothelial cells (i.g. cytokines). Methods: Quiescent rat smooth muscle cells were exposed to either Hb alone or, utilizing a transwell system, co-cultured with human pulmonary endothelial cell monolayers exposed to Hb. Colorimetric assays, cell counting, proliferation proliferating cell nuclear antigen (PCNA) and nuclear Cyclic AMP Response Element Binding (CREB), were used to assess SmC proliferation. Results: Hb incubated directly on smooth muscle cells increased PCNA at 4 h, but when Hb was incubated on EC monolayers situated over SmC this response was delayed to 8h. Compared to untreated cells, colormetric and cell counting analysis showed increased proliferation in SmC exposed directly to Hb or to conditioned media for 24 h. Finally, we observed loss of nuclear CREB in SmC directly exposed to Hb. Conclusion: Cell free Hb can affect the proliferation rate of SmC and nuclear CREB concentration by direct exposure or indirectly by mediators from activated endothelial cells.

Research paper thumbnail of Waves of gene regulation suppress and then restore oxidative phosphorylation in cancer cells

The International Journal of Biochemistry & Cell Biology, 2011

We posit the following hypothesis: Independently of whether malignant tumors are initiated by a f... more We posit the following hypothesis: Independently of whether malignant tumors are initiated by a fundamental reprogramming of gene expression or seeded by stem cells, "waves" of gene expression that promote metabolic changes occur during carcinogenesis, beginning with oncogene-mediated changes, followed by hypoxia-induced factor (HIF)-mediated gene expression, both resulting in the highly glycolytic "Warburg" phenotype and suppression of mitochondrial biogenesis. Because high proliferation rates in malignancies cause aglycemia and nutrient shortage, the third (second oncogene) "wave" of adaptation stimulates glutaminolysis, which in certain cases partially re-establishes oxidative phosphorylation; this involves the LKB1-AMPK-p53, PI3K-Akt-mTOR axes and MYC dysregulation. Oxidative glutaminolysis serves as an alternative pathway compensating for cellular ATP. Together with anoxic glutaminolysis it provides pyruvate, lactate, and the NADPH pool (alternatively to pentose phosphate pathway). Retrograde signaling from revitalized mitochondria might constitute the fourth "wave" of gene reprogramming. In turn, upon reversal of the two Krebs cycle enzymes, glutaminolysis may partially (transiently) function even during anoxia, thereby further promoting malignancy. The history of the carcinogenic process within each malignant tumor determines the final metabolic phenotype of the selected surviving cells, resulting in distinct cancer bioenergetic phenotypes ranging from the highly glycolytic "classic Warburg" to partial or enhanced oxidative phosphorylation. We discuss the bioenergetically relevant functions of oncogenes, the involvement of mitochondrial biogenesis/degradation in carcinogenesis, the yet unexplained Crabtree effect of instant glucose blockade of respiration, and metabolic signaling stemming from the accumulation of succinate, fumarate, pyruvate, lactate, and oxoglutarate by interfering with prolyl hydroxylase domain enzyme-mediated hydroxylation of HIFα prolines.

Research paper thumbnail of Hydroperoxy Fatty Acid Cycling Mediated by Mitochondrial Uncoupling Protein UCP2

Journal of Biological Chemistry, 2004

Research paper thumbnail of Biochemical Background in Mitochondria Affects 2HG Production by IDH2 and ADHFE1 in Breast Carcinoma

Cancers

Mitochondrial production of 2-hydroxyglutarate (2HG) can be catalyzed by wild-type isocitrate deh... more Mitochondrial production of 2-hydroxyglutarate (2HG) can be catalyzed by wild-type isocitrate dehydrogenase 2 (IDH2) and alcohol dehydrogenase, iron-containing 1 (ADHFE1). We investigated whether biochemical background and substrate concentration in breast cancer cells promote 2HG production. To estimate its role in 2HG production, we quantified 2HG levels and its enantiomers in breast cancer cells using analytical approaches for metabolomics. By manipulation of mitochondrial substrate fluxes using genetic and pharmacological approaches, we demonstrated the existence of active competition between 2HG producing enzymes, i.e., IDH2 and ADHFE1. Moreover, we showed that distinct fractions of IDH2 enzyme molecules operate in distinct oxido-reductive modes, providing NADPH and producing 2HG simultaneously. We have also detected 2HG release in the urine of breast cancer patients undergoing adjuvant therapy and detected a correlation with stages of breast carcinoma development. In summary, ...

Research paper thumbnail of Colloidally Stable P(DMA-AGME)-Ale-Coated Gd(Tb)F3:Tb3+(Gd3+),Yb3+,Nd3+ Nanoparticles as a Multimodal Contrast Agent for Down- and Upconversion Luminescence, Magnetic Resonance Imaging, and Computed Tomography

Nanomaterials

Multimodal imaging, integrating several modalities including down- and up-conversion luminescence... more Multimodal imaging, integrating several modalities including down- and up-conversion luminescence, T1- and T2(T2*)-weighted MRI, and CT contrasting in one system, is very promising for improved diagnosis of severe medical disorders. To reach the goal, it is necessary to develop suitable nanoparticles that are highly colloidally stable in biologically relevant media. Here, hydrophilic poly(N,N-dimethylacrylamide-N-acryloylglycine methyl ester)-alendronate-[P(DMA-AGME)-Ale]-coated Gd(Tb)F3:Tb3+(Gd3+),Yb3+,Nd3+ nanoparticles were synthesized by a coprecipitation method in ethylene glycol (EG) followed by coating with the polymer. The particles were tho-roughly characterized by a dynamic light scattering (DLS), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), X-ray energy dispersive spectroscopy (EDAX), selected area electron diffraction (SAED), elemental ana-lysis and fluorescence spectroscopy. Aqueous particle di...

Research paper thumbnail of Mitochondrial Superoxide Production Decreases on Glucose-Stimulated Insulin Secretion in Pancreatic β Cells Due to Decreasing Mitochondrial Matrix NADH/NAD+ Ratio

Antioxidants & Redox Signaling

Aims: Glucose-stimulated insulin secretion (GSIS) in pancreatic b cells was expected to enhance m... more Aims: Glucose-stimulated insulin secretion (GSIS) in pancreatic b cells was expected to enhance mitochondrial superoxide formation. Hence, we elucidated relevant redox equilibria. Results: Unexpectedly, INS-1E cells at transitions from 3 (11 mM; pancreatic islets from 5 mM) to 25 mM glucose decreased matrix superoxide release rates (MitoSOX Red monitoring validated by MitoB) and H 2 O 2 (mitoHyPer, subtracting mitoSypHer emission). Novel double-channel fluorescence lifetime imaging, approximating free mitochondrial matrix NADH F, indicated its *20% decrease. Matrix NAD + F increased on GSIS, indicated by the FAD-emission lifetime decrease, reflecting higher quenching of FAD by NAD + F. The participation of pyruvate/malate and pyruvate/citrate redox shuttles, elevating cytosolic NADPH F (iNAP1 fluorescence monitoring) at the expense of matrix NADH F , was indicated, using citrate (2-oxoglutarate) carrier inhibitors and cytosolic malic enzyme silencing: All changes vanished on these manipulations. 13 Cincorporation from 13 C-L-glutamine into 13 C-citrate reflected the pyruvate/isocitrate shuttle. Matrix NADPH F (iNAP3 monitored) decreased. With decreasing glucose, the suppressor of Complex III site Q electron leak (S3QEL) suppressor caused a higher Complex I I F site contribution, but a lower superoxide fraction ascribed to the Complex III site III Qo. Thus, the diminished matrix NADH F /NAD + F decreased Complex I flavin site I F superoxide formation on GSIS. Innovation: Mutually validated methods showed decreasing superoxide release into the mitochondrial matrix in pancreatic b cells on GSIS, due to the decreasing matrix NADH F /NAD + F (NADPH F /NADP + F) at increasing cytosolic NADPH F levels. The developed innovative methods enable real-time NADH/NAD + and NADPH/ NADP + monitoring in any distinct cell compartment. Conclusion: The export of reducing equivalents from mitochondria adjusts lower mitochondrial superoxide production on GSIS, but it does not prevent oxidative stress in pancreatic b cells. Antioxid. Redox Signal. 33, 789-815.

Research paper thumbnail of Overexpression of native IF1 downregulates glucose-stimulated insulin secretion by pancreatic INS-1E cells

Scientific Reports

We have previously reported that transient knock-down of ATPase inhibitory factor 1 (IF1) by siRN... more We have previously reported that transient knock-down of ATPase inhibitory factor 1 (IF1) by siRNA upregulates ATP levels and subsequently augments insulin secretion in model pancreatic β-cells INS-1E. Here we investigated how long-term IF1-overexpression impacts pancreatic β-cell bioenergetics and insulin secretion. We generated INS-1E cell line stably overexpressing native IF1. We revealed that IF1 overexpression leads to a substantial decrease in ATP levels and reduced glucose-stimulated insulin secretion. A decrease in total cellular ATP content was also reflected in decreased free ATP cytosolic and mitochondrial levels, as monitored with ATeam biosensor. Consistently, cellular respiration of IF1-overexpressing cells was decreased. 3D structured illumination microscopy (SIM) revealed a higher amount of insulin granules with higher volume in IF1-overexpressing cells. Similar effects occurred when cells were incubated at low glucose concentrations. Noteworthy, activation of PKA by...

Research paper thumbnail of Mitochondrial cristae narrowing upon higher 2-oxoglutarate load

Biochimica et Biophysica Acta (BBA) - Bioenergetics

Research paper thumbnail of Role of ATPase Inhibitory Factor 1 (IF1) in metabolic regulations of insulin secreting INS-1E cells

Biochimica et Biophysica Acta (BBA) - Bioenergetics

Research paper thumbnail of Mitochondrial Nucleoids: Superresolution microscopy analysis

The International Journal of Biochemistry & Cell Biology

Research paper thumbnail of 3D super-resolution microscopy reflects mitochondrial cristae alternations and mtDNA nucleoid size and distribution

Biochimica et Biophysica Acta (BBA) - Bioenergetics

Research paper thumbnail of Colocalization of inhibitor factor IF1 and mitochondrial ATP synthase in pancreatic INS1E cells by 3D STED super-resolution microscopy

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2016

Research paper thumbnail of STED and dSTORM super-resolution microscopy of mitochondrial ATP synthase in relation to metabolic state of pancreatic INS1E cells

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2016

Research paper thumbnail of Mitochondrial nucleoid clusters protect newly synthesized mtDNA during Doxorubicin- and Ethidium Bromide-induced mitochondrial stress

Toxicology and Applied Pharmacology, 2016

Mitochondrial DNA (mtDNA) is compacted in ribonucleoprotein complexes called nucleoids, which can... more Mitochondrial DNA (mtDNA) is compacted in ribonucleoprotein complexes called nucleoids, which can divide or move within the mitochondrial network. Mitochondrial nucleoids are able to aggregate into clusters upon reaction with intercalators such as the mtDNA depletion agent Ethidium Bromide (EB) or anticancer drug Doxorobicin (DXR). However, the exact mechanism of nucleoid clusters formation remains unknown. Resolving these processes may help to elucidate the mechanisms of DXR-induced cardiotoxicity. Therefore, we addressed the role of two key nucleoid proteins; mitochondrial transcription factor A (TFAM) and mitochondrial single-stranded binding protein (mtSSB); in the formation of mitochondrial nucleoid clusters during the action of intercalators. We found that both intercalators cause numerous aberrations due to perturbing their native status. By blocking mtDNA replication, both agents also prevented mtDNA association with TFAM, consequently causing nucleoid aggregation into large nucleoid clusters enriched with TFAM, co-existing with the normal nucleoid population. In the later stages of intercalation (>48h), TFAM levels were reduced to 25%. In contrast, mtSSB was released from mtDNA and freely distributed within the mitochondrial network. Nucleoid clusters mostly contained nucleoids with newly replicated mtDNA, however the nucleoid population which was not in replication mode remained outside the clusters. Moreover, the nucleoid clusters were enriched with p53, an anti-oncogenic gatekeeper. We suggest that mitochondrial nucleoid clustering is a mechanism for protecting nucleoids with newly replicated DNA against intercalators mediating genotoxic stress. These results provide new insight into the common mitochondrial response to mtDNA stress and can be implied also on DXR-induced mitochondrial cytotoxicity.

Research paper thumbnail of Division of Mitochondrial Nucleoids Visualized by Biplane FPALM/dSTORM

Biophysical Journal, 2016

Research paper thumbnail of Silica-coated upconversion lanthanide nanoparticles: The effect of crystal design on morphology, structure and optical properties

Beilstein journal of nanotechnology, 2015

NaYF4:Yb(3+)/Er(3+) nanoparticles were synthesized by thermal decomposition of lanthanide trifluo... more NaYF4:Yb(3+)/Er(3+) nanoparticles were synthesized by thermal decomposition of lanthanide trifluoroacetates using oleylamine (OM) as both solvent and surface binding ligand. The effect of reaction temperature and time on the properties of the particles was investigated. The nanoparticles were characterized by transmission electron microscopy (TEM), electron diffraction (ED), energy dispersive spectroscopy (EDX), dynamic light scattering (DLS), thermogravimetric analysis (TGA), elemental analysis and X-ray diffraction (XRD) to determine morphology, size, polydispersity, crystal structure and elemental composition of the nanocrystals. TEM microscopy revealed that the morphology of the nanoparticles could be fine-tuned by modifying of the synthetic conditions. A cubic-to-hexagonal phase transition of the NaYF4:Yb(3+)/Er(3+) nanoparticles at temperatures above 300 °C was confirmed by both ED and XRD. Upconversion luminescence under excitation at 980 nm was observed in the luminescence s...

Research paper thumbnail of Constitutive Reprogramming of Fibroblast Mitochondrial Metabolism in Pulmonary Hypertension

American journal of respiratory cell and molecular biology, Jan 23, 2015

Remodeling of the distal pulmonary artery wall is a characteristic feature of pulmonary hypertens... more Remodeling of the distal pulmonary artery wall is a characteristic feature of pulmonary hypertension (PH). In hypoxic pulmonary hypertension, the most substantial pathologic changes occur in the adventitia. Here, there is marked fibroblast proliferation and profound macrophage accumulation. These fibroblasts (PH-Fibs) maintain a hyperproliferative, apoptotic resistant and pro-inflammatory phenotype in ex vivo culture. Considering that a similar phenotype is observed in cancer cells, where it has been associated at least in part with specific alterations in mitochondrial metabolism, we sought to define the state of mitochondrial metabolism in PH-Fibs. In PH-Fibs pyruvate dehydrogenase was markedly inhibited, resulting in metabolism of pyruvate to lactate, thus consistent with a Warburg-like phenotype. In addition, mitochondrial bioenergetics were suppressed and mitochondrial fragmentation was increased in PH-Fibs. Most importantly, Complex I activity was substantially decreased which...

Research paper thumbnail of 3D biplane PALM super-resolution microscopy: modeling biological structures & the system resolution

A unique 3D super-resolution microscope, based on a biplane detection scheme, enables single mole... more A unique 3D super-resolution microscope, based on a biplane detection scheme, enables single molecule localization in samples several µm thick with theoretical resolution 25 nm lateral and 50 nm axial [1]. Such resolution is achieved in a far-field optical setup through the use of special photo-switchable or photo-activated fluorescent dyes imaged under such conditions that only sparse subset of fluorophores is active at any given time so the position of individual molecules can be determined. To calculate axial position of the fluorophore, two parallel planes (about 600 nm apart) are imaged simultaneously, thus providing two sections of the Point Spread Function (PSF). The resolution is governed by the actual PSF achieved in the optical system and the number of photons captured from a single fluorophore activation event. The amount of information that can be gathered from the specimen also depends on the stability of the optical setup and durability of the sample [2]. The sample th...

Research paper thumbnail of Reductive carboxylation and 2-hydroxyglutarate formation by wild-type IDH2 in breast carcinoma cells

The International Journal of Biochemistry & Cell Biology, 2015

Mitochondrial NADPH-dependent isocitrate dehydrogenase, IDH2, and cytosolic IDH1, catalyze reduct... more Mitochondrial NADPH-dependent isocitrate dehydrogenase, IDH2, and cytosolic IDH1, catalyze reductive carboxylation of 2-oxoglutarate. Both idh2 and idh1 monoallelic mutations are harbored in grade 2/3 gliomas, secondary glioblastomas and acute myeloid leukemia. Mutant IDH1/IDH2 enzymes were reported to form an oncometabolite r-2-hydroxyglutarate (2HG), further strengthening malignancy. We quantified CO2-dependent reductive carboxylation glutaminolysis (RCG) and CO2-independent 2HG production in HTB-126 and MDA-MB-231 breast carcinoma cells by measuring (13)C incorporation from 1-(13)C-glutamine into citrate, malate, and 2HG. For HTB-126 cells, (13)C-citrate, (13)C-malate, and (13)C-2-hydroxyglutarate were enriched by 2-, 5-, and 15-fold at 5mM glucose (2-, 2.5-, and 13-fold at 25mM glucose), respectively, after 6h. Such enrichment decreased by 6% with IDH1 silencing, but by 30-50% upon IDH2 silencing while cell respiration and ATP levels rose up to 150%. Unlike 2HG production RCG declined at decreasing CO2. At hypoxia (5% O2), IDH2-related and unrelated (13)C-accumulation into citrate and malate increased 1.5-2.5-fold with unchanged IDH2 expression; whereas hypoxic 2HG formation did not. (13)C-2HG originated by ∼50% from other than IDH2 or IDH1 reactions, substantiating remaining activity in IDH1&2-silenced cells. Relatively high basal (12)C-2HG levels existed (5-fold higher vs. non-tumor HTB-125 cells) and (13)C-2HG was formed despite the absence of any idh2 and idh1 mutations in HTB-126 cells. Since RCG is enhanced at hypoxia (frequent in solid tumors) and 2HG can be formed without idh1/2 mutations, we suggest 2HG as an analytic marker (in serum, urine, or biopsies) predicting malignancy of breast cancer in all patients.

Research paper thumbnail of Delta Cell Hyperplasia in Adult Goto-Kakizaki (GK/MolTac) Diabetic Rats

Journal of Diabetes Research, 2015

Reduced beta cell mass in pancreatic islets (PI) of Goto-Kakizaki (GK) rats is frequently observe... more Reduced beta cell mass in pancreatic islets (PI) of Goto-Kakizaki (GK) rats is frequently observed in this diabetic model, but knowledge on delta cells is scarce. Aiming to compare delta cell physiology/pathology of GK to Wistar rats, we found that delta cell number increased over time as did somatostatin mRNA and delta cells distribution in PI is different in GK rats. Subtle changes in 6-week-old GK rats were found. With maturation and aging of GK rats, disturbed cytoarchitecture occurred with irregular beta cells accompanied by delta cell hyperplasia and loss of pancreatic polypeptide (PPY) positivity. Unlike the constant glucose-stimulation index for insulin PI release in Wistar rats, this index declined with GK age, whereas for somatostatin it increased with age. A decrease of GK rat PPY serum levels was found. GK rat body weight decreased with increasing hyperglycemia. Somatostatin analog octreotide completely blocked insulin secretion, impaired proliferation at low autocrine i...

Research paper thumbnail of Fibroblasts Derived from Distal Pulmonary Artery Wall of Chronic Hypoxia-Induced Pulmonary Hypertensive Neonatal Calves Exhibit Warburg-Like Energetic Metabolism with Pro-Oxidative Shift

Free Radical Biology and Medicine, 2013

Introduction: Pulmonary hypertension (PH) has been observed in patients suffering from both the i... more Introduction: Pulmonary hypertension (PH) has been observed in patients suffering from both the inherited and the acquired forms of hemolytic anemia. Despite the prevalence of PH with hemolytic anemia, the mechanism behind this process is unclear. Recently, our group and others have shown endothelial cell barrier dysfunction, in vitro and in vivo, with exposure to hemoglobin (Hb). According to basic physiology, endothelial cell barrier dysfunction would allow smooth muscle cells (SmC) to come into direct exposure to Hb, which may induce SmC proliferative phenotype. Hypothesis: Cell free Hb induces SMC proliferation by mechanisms associated with both direct exposure to SmC and indirectly by mediators released from Hb activated endothelial cells (i.g. cytokines). Methods: Quiescent rat smooth muscle cells were exposed to either Hb alone or, utilizing a transwell system, co-cultured with human pulmonary endothelial cell monolayers exposed to Hb. Colorimetric assays, cell counting, proliferation proliferating cell nuclear antigen (PCNA) and nuclear Cyclic AMP Response Element Binding (CREB), were used to assess SmC proliferation. Results: Hb incubated directly on smooth muscle cells increased PCNA at 4 h, but when Hb was incubated on EC monolayers situated over SmC this response was delayed to 8h. Compared to untreated cells, colormetric and cell counting analysis showed increased proliferation in SmC exposed directly to Hb or to conditioned media for 24 h. Finally, we observed loss of nuclear CREB in SmC directly exposed to Hb. Conclusion: Cell free Hb can affect the proliferation rate of SmC and nuclear CREB concentration by direct exposure or indirectly by mediators from activated endothelial cells.

Research paper thumbnail of Waves of gene regulation suppress and then restore oxidative phosphorylation in cancer cells

The International Journal of Biochemistry & Cell Biology, 2011

We posit the following hypothesis: Independently of whether malignant tumors are initiated by a f... more We posit the following hypothesis: Independently of whether malignant tumors are initiated by a fundamental reprogramming of gene expression or seeded by stem cells, "waves" of gene expression that promote metabolic changes occur during carcinogenesis, beginning with oncogene-mediated changes, followed by hypoxia-induced factor (HIF)-mediated gene expression, both resulting in the highly glycolytic "Warburg" phenotype and suppression of mitochondrial biogenesis. Because high proliferation rates in malignancies cause aglycemia and nutrient shortage, the third (second oncogene) "wave" of adaptation stimulates glutaminolysis, which in certain cases partially re-establishes oxidative phosphorylation; this involves the LKB1-AMPK-p53, PI3K-Akt-mTOR axes and MYC dysregulation. Oxidative glutaminolysis serves as an alternative pathway compensating for cellular ATP. Together with anoxic glutaminolysis it provides pyruvate, lactate, and the NADPH pool (alternatively to pentose phosphate pathway). Retrograde signaling from revitalized mitochondria might constitute the fourth "wave" of gene reprogramming. In turn, upon reversal of the two Krebs cycle enzymes, glutaminolysis may partially (transiently) function even during anoxia, thereby further promoting malignancy. The history of the carcinogenic process within each malignant tumor determines the final metabolic phenotype of the selected surviving cells, resulting in distinct cancer bioenergetic phenotypes ranging from the highly glycolytic "classic Warburg" to partial or enhanced oxidative phosphorylation. We discuss the bioenergetically relevant functions of oncogenes, the involvement of mitochondrial biogenesis/degradation in carcinogenesis, the yet unexplained Crabtree effect of instant glucose blockade of respiration, and metabolic signaling stemming from the accumulation of succinate, fumarate, pyruvate, lactate, and oxoglutarate by interfering with prolyl hydroxylase domain enzyme-mediated hydroxylation of HIFα prolines.