Aerobic Cells Research Articles

SR4 Uncouples Mitochondrial Oxidative Phosphorylation, Modulates AMP-dependent Kinase (AMPK)-Mammalian Target of Rapamycin (mTOR) Signaling, and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells

Mitochondrial oxidative phosphorylation produces most of the energy in aerobic cells by coupling respiration to the production of ATP. Mitochondrial uncouplers, which reduce the proton gradient across the mitochondrial inner membrane, create a futile cycle of nutrient oxidation without generating ATP. Regulation of mitochondrial dysfunction and associated cellular bioenergetics has been recently identified as a promising target for anticancer therapy. Here, we show that SR4 is a novel mitochondrial uncoupler that causes dose-dependent increase in mitochondrial respiration and dissipation of mitochondrial membrane potential in HepG2 hepatocarcinoma cells. These effects were reversed by the recoupling agent 6-ketocholestanol but not cyclosporin A and were nonexistent in mitochondrial DNA-depleted HepG2 cells. In isolated mouse liver mitochondria, SR4 similarly increased oxygen consumption independent of adenine nucleotide translocase and uncoupling proteins, decreased mitochondrial membrane potential, and promoted swelling of valinomycin-treated mitochondria in potassium acetate medium. Mitochondrial uncoupling in HepG2 cells by SR4 results in the reduction of cellular ATP production, increased ROS production, activation of the energy-sensing enzyme AMPK, and inhibition of acetyl-CoA carboxylase and mammalian target of rapamycin signaling pathways, leading to cell cycle arrest and apoptosis. Global analysis of SR4-associated differential gene expression confirms these observations, including significant induction of apoptotic genes and down-regulation of cell cycle, mitochondrial, and oxidative phosphorylation pathway transcripts at 24 h post-treatment. Collectively, our studies demonstrate that the previously reported indirect activation of AMPK and in vitro anticancer properties of SR4 as well as its beneficial effects in both animal xenograft and obese mice models could be a direct consequence of its mitochondrial uncoupling activity.

Quantum Calculations of Electron Tunneling in Respiratory Complex III.

The most detailed and comprehensive to date study of electron transfer reactions in the respiratory complex III of aerobic cells, also known as bc1 complex, is reported. In the framework of the tunneling current theory, electron tunneling rates and atomistic tunneling pathways between different redox centers were investigated for all electron transfer reactions comprising different stages of the proton-motive Q-cycle. The calculations reveal that complex III is a smart nanomachine, which under certain conditions undergoes conformational changes gating electron transfer, or channeling electrons to specific pathways. One-electron tunneling approximation was adopted in the tunneling calculations, which were performed using hybrid Broken-Symmetry (BS) unrestricted DFT/ZINDO levels of theory. The tunneling orbitals were determined using an exact biorthogonalization scheme that uniquely separates pairs of tunneling orbitals with small overlaps out of the remaining Franck-Condon orbitals with significant overlap. Electron transfer rates in different redox pairs show exponential distance dependence, in agreement with the reported experimental data; some reactions involve coupled proton transfer. Proper treatment of a concerted two-electron bifurcated tunneling reaction at the Q(o) site is given.

Mutagenesis of conserved active site residues of dihydrolipoamide succinyltransferase enhances the accumulation of α-ketoglutarate in Yarrowia lipolytica

α-Ketoglutarate (α-KG) is an important intermediate in the tricarboxylic acid cycle and has broad applications. The mitochondrial ketoglutarate dehydrogenase (KGDH) complex catalyzes the oxidation of α-KG to succinyl-CoA. Disruption of KGDH, which may enhance the accumulation of α-KG theoretically, was found to be lethal to obligate aerobic cells. In this study, individual overexpression of dihydrolipoamide succinyltransferase (DLST), which serves as the inner core of KGDH, decreased overall activity of the enzyme complex. Furthermore, two conserved active site residues of DLST, His419, and Asp423 were identified. In order to determine whether these residues are engaged in enzyme reaction or not, these two conserved residues were individually mutated. Analysis of the kinetic parameters of the enzyme variants provided evidence that the catalytic reaction of DLST depended on residues His419 and Asp423. Overexpression of mutated DLST not only impaired balanced assembly of KGDH, but also disrupted the catalytic integrity of the enzyme complex. Replacement of the Asp423 residue by glutamate increased extracellular α-KG by 40 % to 50 g L(-1) in mutant strain. These observations uncovered catalytic roles of two conserved active site residues of DLST and provided clues for effective metabolic strategies for rational carbon flux control for the enhanced production of α-KG and related bioproducts.

Determination of the relative biological effectiveness and oxygen enhancement ratio for micronuclei formation using high-LET radiation in solid tumor cells: An in vitro and in vivo study

We determined the relative biological effectiveness (RBE) and oxygen enhancement ratio (OER) of micronuclei (MN) formation in clamped (hypoxic) and non-clamped (normoxic) solid tumors in mice legs following exposure to X-rays and heavy ions. Single-cell suspensions (aerobic) of non-irradiated tumors were prepared in parallel and used directly to determine the radiation response for aerobic cells. Squamous cell carcinoma (SCCVII) cells were transplanted into the right hind legs of syngeneic C3H/He male mice. Irradiation doses with either X-rays or heavy ions at a dose-averaged LET (linear energy transfer) of 14–192keV/μm were delivered to 5-mm diameter tumors and aerobic single-cells in sample-tubes. After irradiation, the tumors were excised and trypsinized to observe MN in single-cells. The single-cell suspensions were used for MN formation assays. The RBE values increased with increasing LET. The maximum RBE values for the three different oxygen conditions; hypoxic tumor, normoxic tumor, and aerobic cells, were 8.18, 5.30, and 3.76 at an LET of 192keV/μm, respectively. After X-irradiation, the OERh/n values (hypoxic tumor/normoxic tumor) were lower than the OERh/a (hypoxic tumor/aerobic cells), and were 1.73 and 2.58, respectively. We found that the OER for the in vivo studies were smaller in comparison to that for the in vitro studies. Both of the OER values at 192keV/μm were small in comparison to those of the X-ray irradiated samples. The OERh/n and OERh/a values at 192keV/μm were 1.12 and 1.19, respectively. Our results suggest that high LET radiation has a large biological effect even if a solid tumor includes substantial numbers of hypoxic cells. To conclude, we found that the RBE values under each oxygen state for non-MN fraction increased with increasing LET and that the OER values for both tumors in vivo and cells in vitro decreased with increasing LET.

Microbial Regulation of the Antagonistic Activity of Lactic Acid Bacteria Strains under the Influence of Transient Bacilli of the Bacillus Genus

Aim. To study the effect of metabolites and components of aerobic spore-forming bacilli cells (MCASB) of the Bacillus genus on the antagonistic activity of lactic acid bacteria. Methods. Lactic acid bacteria, sampled from rectal and oral swabs from calves, were used in the study. Strains of conditionally pathogenic Escherichia coli, 055k59 No. 3912, Staphylococcus aureus No. 906, Proteus mirabilis, No. 3177, obtained from the collection of the city sanitary-epidemiological inspection service, were used as test cultures. MCASB were prepared using autoclaved microbial mass of Bacillus subtilis production strain 44-p via treatment with lysozyme and ultra- sound. The antagonistic activity of lactic acid bacteria was determined by agar blocks method. Results. It was demonstrated that the addition of MCASB to the culture medium stimulates the antagonistic activity of lactic acid bacteria to pathogenic and conditionally pathogenic microorganisms of intestinal bacteria. The highest average stimulation index (2.56) of the antagonistic activity of lactic acid bacteria was found for E. coli 055k59 No. 3912. Conclusions. The results obtained may be used to improve the microbiological foundations of elaborating novel probiotic preparations.

TU-EF-304-10: Efficient Multiscale Simulation of the Proton Relative Biological Effectiveness (RBE) for DNA Double Strand Break (DSB) Induction and Bio-Effective Dose in the FLUKA Monte Carlo Radiation Transport Code

Purpose: One of the more critical initiating events for reproductive cell death is the creation of a DNA double strand break (DSB). In this study, we present a computationally efficient way to determine spatial variations in the relative biological effectiveness (RBE) of proton therapy beams within the FLUKA Monte Carlo (MC) code. Methods: We used the independently tested Monte Carlo Damage Simulation (MCDS) developed by Stewart and colleagues (Radiat. Res. 176, 587–602 2011) to estimate the RBE for DSB induction of monoenergetic protons, tritium, deuterium, hellium-3, hellium-4 ions and delta-electrons. The dose-weighted (RBE) coefficients were incorporated into FLUKA to determine the equivalent ⁶°60Co γ-ray dose for representative proton beams incident on cells in an aerobic and anoxic environment. Results: We found that the proton beam RBE for DSB induction at the tip of the Bragg peak, including primary and secondary particles, is close to 1.2. Furthermore, the RBE increases laterally to the beam axis at the area of Bragg peak. At the distal edge, the RBE is in the range from 1.3–1.4 for cells irradiated under aerobic conditions and may be as large as 1.5–1.8 for cells irradiated under anoxic conditions. Across the plateau region, the recorded RBE for DSB induction is 1.02 for aerobic cells and 1.05 for cells irradiated under anoxic conditions. The contribution to total effective dose from secondary heavy ions decreases with depth and is higher at shallow depths (e.g., at the surface of the skin). Conclusion: Multiscale simulation of the RBE for DSB induction provides useful insights into spatial variations in proton RBE within pristine Bragg peaks. This methodology is potentially useful for the biological optimization of proton therapy for the treatment of cancer. The study highlights the need to incorporate spatial variations in proton RBE into proton therapy treatment plans.

By reducing hexokinase 2, resveratrol induces apoptosis in HCC cells addicted to aerobic glycolysis and inhibits tumor growth in mice.

Cancer cells exhibit an altered metabolic phenotype known as the aerobic glycolysis. The expression of HK2 changes the metabolic phenotype of cells to support cancerous growth. In the present study, we investigated the inhibitory effect of resveratrol on HK2 expression and hepatocellular carcinoma (HCC) cell glycolysis. Aerobic glycolysis was observed in four HCC cell lines compared to the normal hepatic cells. Resveratrol sensitized aerobic glycolytic HCC cells to apoptosis, and this effect was attenuated by glycolytic inhibitors. The induction of mitochondrial apoptosis was associated with the decrease of HK2 expression by resveratrol in HCC cells. In addition, resveratrol enhanced sorafenib induced cell growth inhibition in aerobic glycolytic HCC cells. Combination treatment with both reagents inhibited the growth and promoted apoptosis of HCC-bearing mice. The reduction of HK2 by resveratrol provides a new dimension to clinical HCC therapies aimed at preventing disease progression.

Microbiota of coal pit waste heaps of Chervonograd Mining Region after coal ash application

The aim of this work was to determine the impact of addition of coal ash from Dobrotvir TPP to waste heaps gangue (Chervonograd Mining Region) on the number of different groups of microorganisms. 20 samples from three waste heaps, from the black and red gangue, under the mosses and from bare substrate and also from terrace, top and base of each waste heap, were selected. Waste heaps gangues with coal ash from Dobrotir TPP were mixed in vitro and left for 10 days. We used proportion of coal ash to gangue as 1 to 5. Microorganisms were grown in Petri dishes containing 20–30 ml agar medium and in 22 ml tubes at temperature of 28 °C. Microscopic fungi were revealed on Mash-agar; oligonitrophilic bacteria – on Ashby medium; actinomycetes – on Chapek’s medium; cellulose decomposing aerobic bacteria – on Hetchenson medium; colorless sulfur oxidizing bacteria: neutrophilic – on Beyerinck medium, acidophilic – on Silverman and Lundgren 9К medium. The acidity value of waste heaps gangue samples was determined by рН meter рН-150М. We observed that samples collected under the mosses had lower acidity compared to samples from the bare substrate. We also revealed lower acidity of the overburn red gangue than the acidity of freshly deposited black gangue. To sum up, application of coal ash resulted in lowering of acidity value among all samples under study. Coal ash addition led to increase in number of microscopic fungi cells compared to the appropriate control samples. The highest quantity of microscopic fungi (16.2 ± 0.79) х 10 5 CFU/g of gangue) was revealed in sample from red rock of the main waste heap of Central Enrichment Plant (CEP). At the same time, we observed the highest cell number in the control sample under the mosses of “Nadija” coal pit waste heap, (6.1 ± 0.3) х 10 5 CFU/g of gangue. After coal ash addition, most samples featured 2–3 times higher quantities of colorless sulfur-oxidizing neutrophilic bacteria cells. The highest cell number of these microorganisms was observed in sample under the mosses of “Vizejska” coal pit waste heap. The same dominance of oligonitrophilic bacteria cell number in experimental samples over control samples was indicated. The highest cell quantity was recorded for the sample under the moss from terrace of waste heap of “Nadija” coal pit (25.6 ± 1.3) х 10 4 CFU/g of gangue and in sample from red rock of CEP main waste heap (21.1 ± 1.1) х 10 4 CFU/g of gangue, being 43.3 and 31.7 times higher than the appropriate controls. Meanwhile, the number of colorless sulfur-oxidizing acidophilic bacteria in control samples was higher than that after coal ash addition, particularly, on the bare substrate samples from the base and terrace of waste heap of “Nadija” coal pit. Higher cell number in control samples without coal ash was typical for actinomycetes with the greatest difference (8.6 times) before and after coal ash addition in a sample from red rock of CEP main waste heap. We detected 2 times lower number of cellulose-decomposing aerobic bacteria in the majority of experimental samples compared to appropriate control samples under study. In that way, we noticed that addition of coal ash from Dobrotvir TPP to waste heaps gangue (in proportion of coal ash to gangue as 1 to 5) caused reduction of substrate acidity value. Under these conditions the number of colorless sulfur-oxidizing neutrophilic bacteria, oligonitrophilic bacteria and microscopic fungi cells increased. But on the other hand, coal ash addition resulted in lowering of the number of colorless sulfur-oxidizing acidophilic bacteria, actinomycetes, and cellulose-decomposing aerobic bacteria cells.

Heme exporter FLVCR is required for T cell development and peripheral survival.

All aerobic cells and organisms must synthesize heme from the amino acid glycine and the tricarboxylic acid cycle intermediate succinyl CoA for incorporation into hemoproteins, such as the cytochromes needed for oxidative phosphorylation. Most studies on heme regulation have been done in erythroid cells or hepatocytes; however, much less is known about heme metabolism in other cell types. The feline leukemia virus subgroup C receptor (FLVCR) is a 12-transmembrane domain surface protein that exports heme from cells, and it was shown to be required for erythroid development. In this article, we show that deletion of Flvcr in murine hematopoietic precursors caused a complete block in αβ T cell development at the CD4(+)CD8(+) double-positive stage, although other lymphoid lineages were not affected. Moreover, FLVCR was required for the proliferation and survival of peripheral CD4(+) and CD8(+) T cells. These studies identify a novel and unexpected role for FLVCR, a major facilitator superfamily metabolite transporter, in T cell development and suggest that heme metabolism is particularly important in the T lineage.

Cadmium removal by Euglena gracilis is enhanced under anaerobic growth conditions

The facultative protist Euglena gracilis, a heavy metal hyper-accumulator, was grown under photo-heterotrophic and extreme conditions (acidic pH, anaerobiosis and with Cd2+) and biochemically characterized. High biomass (8.5×106cellsmL−1) was reached after 10 days of culture. Under anaerobiosis, photosynthetic activity built up a microaerophilic environment of 0.7% O2, which was sufficient to allow mitochondrial respiratory activity: glutamate and malate were fully consumed, whereas 25–33% of the added glucose was consumed. In anaerobic cells, photosynthesis but not respiration was activated by Cd2+ which induced higher oxidative stress. Malondialdehyde (MDA) levels were 20 times lower in control cells under anaerobiosis than in aerobiosis, although Cd2+ induced a higher MDA production. Cd2+ stress induced increased contents of chelating thiols (cysteine, glutathione and phytochelatins) and polyphosphate. Biosorption (90%) and intracellular accumulation (30%) were the mechanisms by which anaerobic cells removed Cd2+ from medium, which was 36% higher versus aerobic cells. The present study indicated that E. gracilis has the ability to remove Cd2+ under anaerobic conditions, which might be advantageous for metal removal in sediments from polluted water bodies or bioreactors, where the O2 concentration is particularly low.

The ADP/ATP carrier and its relationship to oxidative phosphorylation in ancestral protist trypanosoma brucei.

The highly conserved ADP/ATP carrier (AAC) is a key energetic link between the mitochondrial (mt) and cytosolic compartments of all aerobic eukaryotic cells, as it exchanges the ATP generated inside the organelle for the cytosolic ADP. Trypanosoma brucei, a parasitic protist of medical and veterinary importance, possesses a single functional AAC protein (TbAAC) that is related to the human and yeast ADP/ATP carriers. However, unlike previous studies performed with these model organisms, this study showed that TbAAC is most likely not a stable component of either the respiratory supercomplex III+IV or the ATP synthasome but rather functions as a physically separate entity in this highly diverged eukaryote. Therefore, TbAAC RNA interference (RNAi) ablation in the insect stage of T. brucei does not impair the activity or arrangement of the respiratory chain complexes. Nevertheless, RNAi silencing of TbAAC caused a severe growth defect that coincides with a significant reduction of mt ATP synthesis by both substrate and oxidative phosphorylation. Furthermore, TbAAC downregulation resulted in a decreased level of cytosolic ATP, a higher mt membrane potential, an elevated amount of reactive oxygen species, and a reduced consumption of oxygen in the mitochondria. Interestingly, while TbAAC has previously been demonstrated to serve as the sole ADP/ATP carrier for ADP influx into the mitochondria, our data suggest that a second carrier for ATP influx may be present and active in the T. brucei mitochondrion. Overall, this study provides more insight into the delicate balance of the functional relationship between TbAAC and the oxidative phosphorylation (OXPHOS) pathway in an early diverged eukaryote.

Channeling of Mitochondrial Energy in Cardiac and Cancer Cells by the Metabolically-Dependent Outer Membrane Potential

Mitochondria are the main source of energy in eukaryotic non-proliferating aerobic cells. In cancer cells, significant part of the energy metabolism is supported by the aerobic glycolysis. Oxidative phosphorylation metabolites cross the mitochondrial outer membrane through the voltage dependent anion channel (VDAC). The possibility of the voltage-gating functioning of VDAC under physiological conditions and proposed mechanisms of generation of the outer membrane potential (OMP) require further study. In this work, we present a computational thermodynamic analysis of a possible generation of OMP by the creatine kinase(CK)-VDAC and VDAC-hexokinase(HK) complexes, as well as by their intermembrane complexes with the adenine nucleotide translocator (ANT). The CK-VDAC and VDAC-HK complexes function as direct steady-state voltage generators, using the free energy of kinase reactions, while the ANT-CK-VDAC and ANT-VDAC-HK complexes allow the application of a part of the sum of the corresponding “kinase voltage” and the inner membrane potential to the outer membrane. The developed computational models demonstrate a high probability of generation of the metabolically-dependent OMP with magnitudes high enough to close free VDACs. We suggest that in case of cardiac cells, the CK-VDAC-mediated channeling of mitochondrial energy, earlier postulated by Saks and Wallimann, leads to the metabolically-dependent generation of a high OMP during systole, thus causing electrical closure of free VDACs and essentially avoiding the mitochondrial ATP usage by non-contraction system consumers. In case of cancer cells, that have a very high percentage of hexokinase attached to mitochondria, the electrical closure of free VDACs by OMP allows the preferential VDAC-HK-mediated channeling of the mitochondrial ATP to initiate the aerobic glycolysis by an anti-turbo mode. (Research grants #111852128625 and #520154531565, Colciencias).

Is redox signaling a feasible target for overcoming multidrug resistance in cancer chemotherapy?

Under physiological conditions, a balance between oxidants and antioxidants exists. Reactive oxygen species (ROS), are continuously generated by aerobic cells and eliminated through scavenging systems to maintain redox homeostasis. The two main sources of ROS are mitochondria and the NADPH oxidases family, but ROS are produced also by the cytochrome P450 system, xanthine oxidase and nitric oxide synthase (Holmstrom and Finkel, 2014). Because of ROS reactivity toward lipids, proteins and DNA, spatial and temporal regulatory strategies exist to regulate their intracellular levels. Excessive ROS levels are controlled by specific intracellular enzymes, such as superoxide dismutase (SOD), glutathione peroxidase, catalase, thioredoxin reductase, and glutathione S-transferase (Glasauer and Chandel, 2014).

A microscale mathematical model for metabolic symbiosis: Investigating the effects of metabolic inhibition on ATP turnover in tumors.

Cancer cells are notorious for their metabolic adaptations to hypoxic and acidic conditions, and especially for highly elevated glycolytic rates in tumor tissues. An end product of glycolysis is lactate, a molecule that cells can utilize instead of glucose to fuel respiration in the presence of oxygen. This could be beneficial to those cells that do not have sufficient oxygen as it conserves glucose for glycolysis. To better quantify this phenomenon we develop a diffusion-reaction mathematical model for nutrient concentrations in cancerous tissue surrounding a single cylindrical microvessel. We use our model to analyze the interdependence between cell populations' metabolic behaviors on a microscopic scale, specifically the emerging paradigm of metabolic symbiosis that exists between aerobic and glycolytic cells. The ATP turnover rates are calculated as a function of distance from the blood vessel, which exhibit a lactate-consuming population at intermediate distances from the vessel. We also consider the ramifications of the Warburg effect where cells utilize aerobic glycolysis along with this lactate-consuming respiration. We also investigate the effect of inhibiting metabolic pathways on cancer cells since insufficient ATP can trigger cell apoptosis. Effects that could be induced by metabolic inhibitors are analyzed by calculating the total ATP turnover in a unit tissue annulus in various parameter regimes that correspond to treatment conditions where specific metabolic pathways are knocked out. We conclude that therapies that target glycolysis, e.g. lactate dehydrogenase inhibitors or glycolytic enzyme inhibition, are the keys to successful metabolic repression.

Impact of the addition of a nitrifying activated sludge on ammonia oxidation during composting of residual household wastes.

To investigate the nitrogen-microbial community dynamics during composting of a mixture of nitrifying waste activated sludge (WAS) and fine organic fraction of residual household waste (RHW). To examine whether the addition of nitrifying sludge could promote ammonia oxidation and reduce ammonia emissions. The fine organic fraction of RHW was mixed with the WAS and homogenized. The mixture and each waste alone were loaded in aerobic cells under controlled conditions, respectively. Both nitrogen and microbial community dynamics were monitored during 50days of composting. The ammonia oxidizers were quantified and identified in the sludge and compost. The changes in ammonia-oxidizing bacteria (AOB) concentrations corresponded to the ammonia oxidation rates calculated from nitrogen balance. The addition of WAS did not efficiently reduce ammonia emissions because the Nitrosomonas oligotropha-like AOB introduced declined during the active stage of composting. Ammonia oxidation was probably limited by the intense heterotrophic activities at the active stage. Nitrosomonas europaea/eutropha and Nitrosomonas nitrosa-like AOB were established only during the maturation stage. They were the main contributors to ammonia oxidation during composting. The mixing of nitrifying WAS with the RHW during the early stages of composting does not promote ammonia oxidation nor reduce ammonia emissions because of limiting biologic factors during the active stage of composting. The mixing of activated sludge with RHW before composting is a common practice on composting plants. This study proved the limitation of this practice to reduce ammonia emissions during composting via bioaugmentation of ammonia-oxidizing organisms. It correlated successfully the ammonia oxidation rate with different groups of ammonia oxidizers and explains the fail of promoting ammonia oxidation during the early stages of composting. It suggests Nit.europaea/eutropha and Nit.nitrosa-like AOB were the main contributors to ammonia oxidation during composting.

Hypoxia provokes base excision repair changes and a repair-deficient, mutator phenotype in colorectal cancer cells.

Regions of acute and chronic hypoxia exist within solid tumors and can lead to increased rates of mutagenesis and/or altered DNA damage and repair protein expression. Base excision repair (BER) is responsible for resolving small, non-helix-distorting lesions from the genome that potentially cause mutations by mispairing or promoting DNA breaks during replication. Germline and somatic mutations in BER genes, such as MutY Homolog (MUTYH/MYH) and DNA-directed polymerase (POLB), are associated with increased risk of colorectal cancer. However, very little is known about the expression and function of BER proteins under hypoxic stress. Using conditions of chronic hypoxia, decreased expression of BER proteins was observed because of a mechanism involving suppressed BER protein synthesis in multiple colorectal cancer cell lines. Functional BER was impaired as determined by MYH- and 8-oxoguanine (OGG1)-specific glycosylase assays. A formamidopyrimidine-DNA glycosylase (Fpg) Comet assay revealed elevated residual DNA base damage in hypoxic cells 24 hours after H2O2 treatment as compared with normoxic controls. Similarly, high-performance liquid chromatography analysis demonstrated that 8-oxo-2'-deoxyguanosine lesions were elevated in hypoxic cells 3 and 24 hours after potassium bromate (KBrO3) treatment when compared with aerobic cells. Correspondingly, decreased clonogenic survival was observed following exposure to the DNA base damaging agents H2O2 and MMS, but not to the microtubule interfering agent paclitaxel. Thus, a persistent downregulation of BER components by the microenvironment modifies and facilitates a mutator phenotype, driving genetic instability and cancer progression. Aberrant BER is a contributing factor for the observed genetic instability in hypoxic tumor cells.

Mitochondrial dysfunctions in brain of bulbectomized mice — Model of sporadic Alzheimer's disease

Oxygen is a highly toxic molecule that accumulated massively some 2,400000000 years ago as a byproduct of photosynthesis. It nearly extinguished life on Earth. Surviving organisms had to learn to deal with high atmospheric oxygen. Aerobic cells use it as the final electron acceptor of their respiratory chains, coupling the highly exergonic reduction of oxygen to the synthesis of ATP. Still, the toxicity problem had to be dealt with and a large number of strategies evolved. Active oxygen consumption in the absence of ATP synthesis that is, physiological uncoupling of oxidative phosphorylation, was achieved through different mechanisms that may be classified in proton sinks and non-productive redox proteins. Proton sinks: Proton gradients may be dissipated by opening of the permeability transition pore or activating uncoupling proteins. Non-productive redox enzymes are not proton pumps: The branched respiratory chains of plants, yeast and bacteria express varying amounts of alternative redox enzymes that do not pump protons. Expression varies with aging when subjected to different types of stress. Physiological uncoupling mechanisms are needed to cope with oxygen.

Interconnections of Reactive Oxygen Species Homeostasis and Circadian Rhythm inNeurospora crassa

Both circadian rhythm and the production of reactive oxygen species (ROS) are fundamental features of aerobic eukaryotic cells. The circadian clock enhances the fitness of organisms by enabling them to anticipate cycling changes in the surroundings. ROS generation in the cell is often altered in response to environmental changes, but oscillations in ROS levels may also reflect endogenous metabolic fluctuations governed by the circadian clock. On the other hand, an effective regulation and timing of antioxidant mechanisms may be crucial in the defense of cellular integrity. Thus, an interaction between the circadian timekeeping machinery and ROS homeostasis or signaling in both directions may be of advantage at all phylogenetic levels. The Frequency-White Collar-1 and White Collar-2 oscillator (FWO) of the filamentous fungus Neurospora crassa is well characterized at the molecular level. Several members of the ROS homeostasis were found to be controlled by the circadian clock, and ROS levels display circadian rhythm in Neurospora. On the other hand, multiple data indicate that ROS affect the molecular oscillator. Increasing evidence suggests the interplay between ROS homeostasis and oscillators that may be partially or fully independent of the FWO. In addition, ROS may be part of a complex cellular network synchronizing non-transcriptional oscillators with timekeeping machineries based on the classical transcription-translation feedback mechanism. Further investigations are needed to clarify how the different layers of the bidirectional interactions between ROS homeostasis and circadian regulation are interconnected.

S9-6 Elemental sulfur reduction by eukaryotic cytoplasm consistent with an ancient sulfur symbiosis

An accepted theory for the origin of eukaryotic cells postulates a methanogenic ancestor, although no evidence of methanogenesis exists in modern eukaryotic cells. Instead, sulfur reactions are ubiquitous. The seminal event in eukaryotic evolution might have been a sulfur-based symbiosis between a S°-reducing host and a sulfide-oxidizing bacterium. A vestige of those activities might remain in modern cells. To test this, Tetrahymena thermophila was examined as a representative eukaryotic cell. Aerobic cells consumed H 2 S at a rate of 140 nmol (g protein) −1 s −1 . Anoxic cells produced H 2 S at 1.1 nmol g −1 s −1 , but H 2 S production was suppressed by AOAA, an inhibitor of cysteine catabolism. When aerobic cells were fed H 2 S slowly so that all the H 2 S was oxidized, and then made anoxic, H 2 S was produced at up to 3.6 nmol g −1 s −1 , and not inhibited by AOAA. Evidently, H 2 S is oxidized to a compound that is available for H 2 S production. When aerobic incubation was continued after H 2 S addition had stopped, subsequent H 2 S production decreased with t 1/2 = 11 min. When S° was added to 7 mg cells at 85 pmol s −1 , H 2 S production was 4.4 nmol g −1 s −1 and not inhibited by AOAA. When cells were lysed and fractionated, there was NADH-stimulated S° reductase activity in the membrane fraction, distinguishing it from NADPH disulfide oxidoreductases and linking S° reduction to glycolysis. Thus, there is evidence of a sulfur cycle in which S° is reduced in the cytoplasm and H 2 S is oxidized in mitochondria.

Heme oxygenase 1 is expressed in murine erythroid cells where it controls the level of regulatory heme

AbstractHeme is essential for the function of all aerobic cells. However, it can be toxic when it occurs in a non–protein-bound form; cells maintain a fine balance between heme synthesis and catabolism. The only physiological mechanism of heme degradation is by heme oxygenases (HOs). The heme-inducible isoform, HO-1, has been extensively studied in numerous nonerythroid cells, but virtually nothing is known about the expression and potential significance of HO-1 in developing red blood cells. We have demonstrated that HO-1 is present in erythroid cells and that its expression is upregulated during erythroid differentiation. Overexpression of HO-1 in erythroid cells impairs hemoglobin synthesis, whereas HO-1 absence enhances hemoglobinization in cultured erythroid cells. Based on these results, we conclude that HO-1 controls the regulatory heme pool at appropriate levels for any given stage of erythroid differentiation. In summary, our study brings to light the importance of HO-1 expression for erythroid development and expands our knowledge about the fine regulation of hemoglobin synthesis in erythroid cells. Our results indicate that HO-1 plays an important role as a coregulator of the erythroid differentiation process. Moreover, HO-1 expression must be tightly regulated during red blood cell development.