Browsing by Subject "mitochondria"
Now showing 1 - 10 of 10
Results Per Page
Sort Options
Item Assaying protein import into mitochondria using fluorescence spectroscopy(Texas A&M University, 2006-08-16) Cargill, Holly BethMost proteins residing in the mitochondrial matrix are synthesized in the cytosol and post-translationally imported into the mitochondrial matrix. The matrix-targeted preproteins traverse the outer mitochondrial membrane (OM) via the Translocase of the Outer Membrane (TOM) complex, and the inner mitochondrial membrane (IM) via the Translocase of the Inner Membrane 23 (TIM23) complex. A novel system was set up to examine the import of matrix-targeted preproteins into mitochondria using fluorescence spectroscopy. The fluorescent probe 6-(7-nitrobenz-2-oxa-1,3-diazol-4- yl)aminohexanoic acid (NBD) was site-specifically incorporated into different positions along the model matrix protein Su9-DHFR. The fluorescent-labeled polypeptides were either fully imported into isolated mitochondria or were arrested along the translocation pathway by the binding of methotrexate (MTX) to the DHFR moiety, creating NBDSu9- DHFR??MTX import intermediates. The NBD-Su9-DHFR polypeptides were able to be fully imported into the mitochondrial matrix in the absence of MTX, and were inaccessible to externally-added iodide ion quenchers. Treatment of the mitochondria with the pore-forming antibiotic alamethicin allowed the iodide ion quenchers access to the matrix through pores in the inner membrane (IM). After Alamethicin treatment the fully-imported NBD-Su9-DHFR polypeptides were accessible to the externally-added iodide ions. The extent of collisional quenching of the NBD fluorophores by the iodide ions was measured as the Stern-Volmer quenching constant, Ksv. Ksv values were obtained for the NBD-Su9-DHFR polypeptides in the presence of MTX (import intermediates) or in the absence of MTX (fully-imported). The Ksv values for NBD-Su9- DHFR import intermediates were similar, despite the location of the NBD probe along the translocation pathway. These Ksv values were similar to those obtained for the fullyimported NBD-Su9-DHFR polypeptides (-MTX). The locations of the varying probe positions along the import pathway were addressed using chemical crosslinking of Su9- DHFR Cys mutants. The use of fluorescence spectroscopy, in association with chemical crosslinking, to analyze the mitochondrial protein import pathways will prove a useful tool to probe the environment of the nascent chain as it is crossing the import pathway (the TOM, TIM23 complexes).Item Biophysical Investigation of the 'Ironome' of Jurkat Cells and Saccharomyces cerevisiae(2013-08-30) Jhurry, NemaThe speciation of iron in intact Jurkat cells and their isolated mitochondria was assessed using biophysical methods. [Fe4S4]^(2+) clusters, low-spin (LS) Fe^(II) heme centers, non-heme high-spin (NHHS) FeII species, ferritin-like material and FeIII oxyhydroxide nanoparticles were detected, via M?ssbauer, in intact Jurkat cells and their isolated mitochondria. EPR spectroscopy was used to quantify Fe-containing species in the respiratory complexes. Contributions from heme a, b and c centers were quantified using electronic absorption spectroscopy. Results were collectively assessed to estimate the first ?ironome? profile of a human cell. The Fe content of Jurkat cells grown on transferrin-bound iron (TBI) and Fe^(III) citrate (FC), and of isolated mitochondria therefrom, was characterized. On average, only 400 ? 100 Fe?s loaded per ferritin complex, regardless of the medium Fe concentration. The extent of nanoparticle formation scaled nonlinearly with the concentration of FC in the medium. Nanoparticle formation was not strongly correlated with ROS damage. Cells could utilize nanoparticles Fe, converting them into essential Fe forms. Cells grown on galactose rather than glucose respired faster, grew slower, exhibited more ROS damage, and generally contained more nanoparticles. Cells grown with TBI rather than FC contained lower Fe concentrations, more ferritin and fewer nanoparticles. Frataxin-deficient cells contained more nanoparticles than comparable WT cells. Data were analyzed by a chemically-based mathematical model. Fermenting Saccharomyces cerevisiae cells grown with varying [Fe] were also studied. The high-affinity Fe import pathway was active only in Fe-deficient cells. Whether Fe-deficient cells were grown under fermenting or respirofermenting conditions had no effect on Fe content; such cells prioritized their use of Fe to essential forms devoid of nanoparticles and vacuolar Fe. Fermenting cells grown on Fe-sufficient and Fe-overloaded medium contained 400 ? 450 ?M Fe. In these cells the concentration of nonmitochondrial NHHS Fe^(II) declined 3-fold, relative to in Fe-deficient cells, whereas the concentration of vacuolar NHHS Fe^(III) increased to a limiting cellular concentration of ~ 300 ?M. Isolated mitochondria contained more NHHS Fe^(II) ions and substantial amounts of Fe^(III) nanoparticles. The Fe contents of cells grown with excessive Fe in the medium were similar over a 250-fold change of nutrient Fe levels.Item Evaluation of oocyte competency in bovine and canine species via non-invasive assessment of oocyte quality(2009-05-15) Willingham-Rocky, Lauri A.Traditional methods of oocyte selection for in vitro studies have proven inefficient with respect to achieving a level of predictability for competency. In this study, a novel method of oocyte selection was implemented that identified a relationship between oocyte morphological parameters (as defined by a ratio of a shape factor (SF) to average fluorescence intensity (AFI) and AFI, followed by in vitro fertilization (IVF) and in vitro culture (IVC) using the Well of Well (WOW) method to evaluate oocyte competency. Specifically, we used non-cytotoxic fluorescent molecular probes and multiphoton microscopy to non-invasively characterize spatial localization and functional activity of mitochondria, mitochondrial membrane potential (??m), and intracellular calcium activity ([Ca2+]i) using rhodamine 123, JC-1 and Fluo-4, AM, respectively in bovine and canine in vitro matured (IVM) oocytes. Comparison of morphological grading with fluorescence intensity yielded similar trends between all grades of oocytes for both species with no visually obvious, distinct characteristic staining that would permit classification of each oocyte as a specific morphological grade. Our studies confirmed that oocyte mitochondria were homogeneously distributed but primarily localized to the peri- and sub-cortical regions of the oocyte at MII stage for both species. Further, heterogeneously polarized mitochondria were localized to the peri- and sub- cortical regions of the oocyte for both species. In bovine oocytes labeled with Fluo-4, AM, levels of [Ca2+]i were either unremarkable, or very low and limited to the peri-cortical areas, just beneath the oolema. For canine MII stage oocytes, levels of [Ca2+]i were within the same range of AFI as bovine. Ranges of fluorescence intensity compatible for optimal embryo development for bovine and optimal fertilization for canine oocytes were 30-300 and 20-35, and 20-30 and 20-25.5 for rhodamine 123 and Fluo-4, AM, respectively. The optimal range for bovine oocytes imaged with JC-1 was 1.25-2.25 and <6 for canine.Item Influence of Insulin Resistance on Contractile Activity-Induced Anabolic Response of Skeletal Muscle(2011-02-22) Nilsson, Mats I.Although the long-term therapeutic benefits of exercise are indisputable, contractile activity may induce divergent adaptations in insulin-resistant vs. insulin-sensitive skeletal muscle. The purpose of this study was to elucidate if the anabolic response following resistance exercise (RE) is altered in myocellular sub-fractions in the face of insulin resistance. Lean (Fa/?) and obese (fa/fa) Zucker rats were assigned to sedentary and RE groups and engaged in either cage rest or four lower-body RE sessions over an 8-d period. Despite obese Zucker rats having significantly smaller hindlimb muscles when compared to age-matched lean rats, basal 24-h fractional synthesis rates (FSR) of mixed protein pools were near normal in distally located muscle groups (gastrocnemius, plantaris, and soleus) and even augmented in those located more proximally (P<0.05; quadriceps). Although 2 x 2 ANOVA indicated a significant main effect of phenotype on mixed FSR in gastrocnemius and soleus (P < 0.05), phenotypic differences were partially accounted for by an exercise effect in the lean phenotype. Interestingly, obese rats exhibited a significant suppression of myofibrillar FSR compared to their lean counterparts (P<0.05; gastrocnemius), while synthesis rates of mitochondrial and cytosolic proteins were normal (gastrocnemius and quadriceps), suggesting a mechanism whereby translation of specific mRNA pools encoding for metabolic enzymes may be favored over other transcripts (e.g., contractile proteins) to cope with nutrient excess in the insulin-resistant state. Immunoblotting of the cytosolic fraction in gastrocnemius muscle indicated an augmented phosporylation of eIF4EBP1 (+ 9%) and p70s6k (+85%) in obese vs. lean rats, but a more potent baseline inhibition of polypeptide-chain elongation as evidenced by an increased phospho/total ratio of eEF2 (+78%) in the obese phenotype. Resistance exercise did not improve synthesis rates of myofibrillar, cytosolic, or mitochondrial proteins to the same extent in obese vs. lean rats, suggesting a desensitization to contractile-induced anabolic stimuli in the insulin-resistant state. We conclude that insulin resistance has diverse effects on protein metabolism, which may vary between muscle groups depending on fiber type distribution, location along the proximodistal body axis, and myocellular sub-fraction, and may blunt the anabolic response to voluntary resistance exercise.Item Linking Sulfur Metabolism to the Cell Division Machinery in Yeast(2010-07-14) Blank, Heidi M.The longstanding view has been that metabolism allows for cell division to take place, but that metabolic processes do not actively promote cell division. I have recently challenged this notion by identifying a unique gain-of-function metabolic mutant in the budding yeast Saccharomyces cerevisiae. Moderate over-expression of Abf2p, a conserved mitochondrial DNA (mtDNA) maintenance protein, increases the amount of mtDNA by 100-150%. I have shown that cells moderately over-expressing Abf2p can out-proliferate their wild type (WT) counterparts, initiate DNA replication sooner, and increase in size faster than WT cells. Yeast grown under certain conditions in continuous cultures become synchronized with respect to their oxygen consumption, displaying distinctive oxidative and reductive phases. In cells over-expressing Abf2p, the reductive phase is expanded compared to that of WT cells. Since glutathione, the cell?s main redox buffer and sulfur containing metabolite, peaks during this phase, I asked if sulfur metabolism was altered in cells with more mtDNA. Sulfur metabolite levels are increased ~40% in cells over-expressing Abf2p. Furthermore, exogenous addition of various sulfur containing compounds, which is known to increase sulfur metabolic flux, caused WT cells to increase in size faster and initiate DNA replication sooner, mimicking the phenotype seen in cells moderately overexpressing Abf2p. I then investigated possible interactions between sulfur metabolism enzymes and the yeast Cdk, Cdc28p. Performing co-immunoprecipitation experiments, two enzymes of the sulfur metabolic pathway were found to bind Cdc28p. One of these, Cys4p, lies at the critical junction point between the pathways leading to the formation of glutathione versus one carbon metabolism. The interaction of the enzymes with Cdc28p appears to be dependent on progression through the cell cycle, and preliminary evidence suggests that Cdc28p/Cys4p binding may peak at the G1/S transition of the cell cycle. In summary, I have identified a unique gain-of-function metabolic mutant in S. cerevisiae that leads to accelerated initiation of DNA replication. Sulfur metabolic flux is up-regulated in cells over-expressing Abf2p, and exogenous sulfur sources added to WT cultures phenocopied cells over-expressing Abf2p. Most importantly, I have shown a physical interaction between sulfur metabolic enzymes and the Cdk driving the cell cycle in yeast.Item Mechanisms of methylenedianiline toxicity to rat biliary epithelial cells(2001-05-06) Vincente Santa Cruz; Mary F. Kanz, Ph.D.; Ronald A Faris, Ph.D.; Mary Treinen Moslen; Kenneth S. Ramos, Ph.D.; Guillermo A. Altenberg, M.D., Ph.D.; Gerald A Campbell, M.D., Ph.D.Methylenedianiline (4,4’-diaminodiphenylmethane, DAPM), a compound used to produce polyurethanes and epoxy resins, rapidly injures biliary epithelial cells (BEC) of rats in vivo. DAPM also increases biliary inorganic phosphate and glucose, suggesting that DAPM loosens hepatic tight junctions. Early ultrastructural alterations in BEC mitochondria, however, suggested a possible role of mitochondrial dysfunction in the ability of BEC to maintain glucose transport and tight junction integrity. The working hypothesis for this dissertation project was that DAPM alters tight junction integrity and/or glucose absorption by mechanisms involving mitochondrial injury. The proposed aims were to: 1). Assess tight junction integrity in vivo using a “sleeping” rat model and radioactive, non-electrolyte tracers. 2). Develop an in vitro model of DAPM injury using primary cultures of polarized rat BEC. 3). Determine if BEC tight junction integrity is altered by DAPM/ metabolite(s) in vitro using both electrophysiological and conventional tracer methods. 4). Determine if DAPM/ metabolite(s) induce mitochondrial dysfunction in BEC by assessing cellular ATP levels and mitochondrial membrane potentials. 5). Determine if BEC glucose uptake is altered following exposure to analog, alpha-methyl-D-glucopyranoside. Numerous in vivo and in vitro methods were combined to develop a model of polarized rat BEC monolayers that were assessed for changes in TJ integrity, glucose uptake and mitochondrial function after exposure to bile from untreated rats, rats treated with vehicle, or rats treated with DAPM. This model confirmed prior studies that demonstrated increased hepatobiliary tight junction permeability following DAPM treatment in vivo. Tight junctions between BEC in vitro showed both increased “leakiness” and decreased ion selectivity following exposure to bile from DAPM-treaded rats (DAPM-Bile). Furthermore, this model indicated that BEC ATP levels and mitochondrial membrane potentials were altered prior to any changes in tight junction integrity and glucose absorption. These data support the working hypothesis that mitochondria are the initial site of DAPM injury in BEC and that mitochondrial dysfunction may indirectly impair BEC glucose uptake and tight junction integrity.Item n-3 Polyunsaturated Fatty Acids Suppress Mitochondrial Translocation to the Immunological Synapse and Modulate Calcium Signaling in T Cells(2011-02-22) Yog, RajeshwariT helper (Th) cell activation is necessary for the adaptive immune response. Formation of an immunological synapse (IS) between Th cells and antigen-presenting cells is the first step in Th cell activation. In vitro studies indicate that formation of the IS induces cytoskeleton-dependent mitochondrial redistribution to the immediate vicinity of the IS. This redistribution of mitochondria to the IS in T cells is necessary to maintain Ca2 influx across the plasma membrane and Ca2 -dependent Th cell activation. Earlier studies have demonstrated that n-3 polyunsaturated fatty acids (PUFA) suppress the localization and activation of signaling proteins at the IS. Therefore, we hypothesized that n-3 PUFA suppress CD4 T cell mitochondrial translocation during the early stages of IS formation and down-modulate Ca2 dependent Th cell activation. CD4 cells derived from fat-1 mice, a transgenic model that synthesizes n-3 PUFA from n-6 PUFA, were co-cultured with anti-CD3-expressing hybridoma cells (145-2C11) for 15 min at 37 degrees C, and mitochondrial translocation to the IS was assessed by confocal microscopy. fat-1 mice exhibited a significantly (P< 0.05) reduced percentage of CD4 T cells with mitochondria which translocated to the IS; fat-1 (30 percent) versus wild type control (82 percent). With respect to an effect on the mitochondrial-to-cytosolic Ca2 ratio, wild type cells showed significant increases at the IS (71 percent) and total cell (60 percent) within 30 min of IS formation. In contrast, fat-1 CD4 T cells remained at basal levels following the IS formation. A similar blunting of the mitochondrial-to-cytosolic Ca2 ratio was observed in wild type cells co-incubated with inhibitors of the mitochondrial uniporter, RU360 or calcium release-activated Ca2 (CRAC) channels, BTP2. Together, these observations provide evidence that n-3 PUFA modulate Th cell activation by limiting mitochondrial translocation to the IS and reducing Ca2 entry.Item Proteomics of Oxidative Stress Using Inducible CYP2E1 Expressing HepG2 Cells and 3T3-L1 Adipocytes as Model Systems(2012-07-16) Newton, Billy WalkerThe overall goal of this research was to investigate oxidative stress related changes to the proteomes of 3T3-L1 adipocytes and an inducible CYP2E1 expressing HepG2 cells. Enhanced oxidative stress in hypertrophic adipocytes is associated with metabolic dysregulation and insulin resistance. Because mitochondria generate reactive oxygen species (ROS), we monitored changes to the adipocyte mitochondrial proteome during differentiation and enlargement. We labeled mitochondrial extracts from 3T3-L1 cells that were 0, 4, 7, 10, 14, and 18 days post differentiation with iTRAQ, followed by MS based identification. We found citric acid cycle proteins such as pyruvate carboxylase, citrate synthase, as well as beta-oxidation enzymes; cartinine acyl transferase and long-chain enoyl-CoA hydratase up-regulated from 7 through 18 days post differentiation onset. These data indicate TCA up-regulation for enhanced metabolic and citrate output necessary for lipid synthesis in adipocytes. Paradoxically, the data also show the simultaneous increase in the fatty acid oxidation, indicating a metabolic overdrive state. Biochemical assays showing peaks in ATP and ROS generation in 3 day old adipocytes provide further evidence of this overdrive state. A second peak in ROS generation occurred in 10 day old adipocytes; concurrent ATP generation reduced to near pre-adipocyte levels and this may indicate a metabolic shift that may be responsible for increased oxidative stress in hypertrophic adipocytes. We developed a doxycycline inducible CYP2E1 expressing HepG2 cell line using the pTet-On/pRevTRE expression system to allow greater control and sensitivity in the generation CYP2E1 mediated oxidative stress. Our cell line (RD12) demonstrated stability and tight expression control. After induction, RD12 cells showed 30 percent higher CYP2E1 activity when compared to the constitutive E47 cell line. RD12 cells showed 30 percent greater toxicity than E47 cells and 25 percent less free glutathione when exposed to 20 mM acetaminophen, indicating RD12 cells are more sensitive to the effects reactive intermediates and oxidative stress generated by CYP2E1. We conducted a survey of the toxicity of dietary fatty acids (oleic, linoleic, and palmitic) on HepG2 cells to determine fatty acid doses that induced metabolic changes, but did not cause excessive cell death. The dose of 0.20 mM linoleic and palmitic acid for 48 hours produced low toxicity, but oleic acid actually produced lower toxicity than untreated cells. After exposure cells were treated with a pro-oxidant to determine which fatty acid increased the susceptibility to protein carbonylation. The carbonylated protein isolation procedure indicated the palmitic acid may induce more carbonylation than oleic acid, but greater efficiency in the isolation procedure is required for a confidant determination.Item Resolution of Phylogenetic Relationships and Characterization of Y-Linked Microsatellites within the Big Cats, Panthera(2010-10-12) Davis, Brian W.The pantherine lineage of cats diverged from the remainder of modern Felidae less than 11 million years ago. This clade consists of the five big cats of the genus Panthera, the lion, tiger, jaguar, leopard, and snow leopard, as well as the closely related clouded leopard, which diverged from Panthera approximately 6 million years ago. A significant problem exists with respect to the precise phylogeny of these highly threatened great cats. Within the past four years, despite multiple publications on the subject, no two studies have reconstructed the phylogeny of Panthera with the same topology, showing particular discordance with respect to sister-taxa relationships to the lion and the position of the enigmatic snow leopard. The evolutionary relationship among these cats remains unresolved partially due to their recent and rapid radiation 3-5 million years ago, individual speciation events occurring within less than 1 million years, and probable introgression between lineages following their divergence. We assembled a 47.6 kb dataset using novel and published DNA sequence data from the autosomes, both sex chromosomes and the mitochondrial genome. This dataset was analyzed both as a supermatrix and with respect to individual partitions using maximum likelihood and Bayesian phylogeny inference. Since discord may exist among gene segments in a multilocus dataset due to their unique evolutionary histories, inference was also performed using Bayesian estimation of species trees (BEST) to form a robust consensus topology. Incongruent topologies for autosomal loci indicated phylogenetic signal conflict within the corresponding segments. We resequenced four mitochondrial and three nuclear gene segments used in recent attempts to reconstruct felid phylogeny. The newly generated data was combined with available GenBank sequence data from all published studies to highlight phylogenetic disparities stemming either from the amplification of a mitochondrial to nuclear translocation event, or errors in species identification. We provide an alternative, highly supported interpretation of the evolutionary history of the pantherine lineage using 39 single-copy regions of the felid Y chromosome and supportive phylogenetic evidence from a revised mitochondrial partition. These efforts result in a highly corroborated set of species relationships that open up new avenues for the study of speciation genomics and understanding the historical events surrounding the origin of the members of this lineage.Item Spectroscopic and analytical characterization of the distribution of iron in intact mitochondria from Saccharomyces cerevisiae(Texas A&M University, 2006-10-30) Hudder, Brandon NealElectron paramagnetic resonance (EPR) and M????ssbauer spectroscopy were used to examine the distribution of iron in mitochondria from Saccharomyces cerevisiae. These organelles were packed into EPR and M????ssbauer cuvettes, affording spectra with unprecedented signal/noise ratios. EPR spectra of as-isolated intact mitochondria exhibited fourteen distinct signals, some of which were assigned according to previously reported g-values obtained using isolated proteins. Signals from adventitious manganese (II) and iron (III) were largely removed when mitochondria were isolated in buffers supplemented with the metal chelators EDTA or EGTA. Signals were simulated and intensities were quantified to afford spin concentrations and estimates of the concentration of EPR-active species in mitochondria. The effects of treating samples with chemical modifiers were examined. Packed samples were analyzed for protein and metal content, affording averaged values of 50 mg/mL [protein], 590 ????M [Fe], 340 ????M [Cu], and 17 ????M [Mn]. 57Fe-enriched intact mitochondria isolated in the presence of metal chelators exhibited M????ssbauer spectra dominated by three components. Approximately 60% of the 57Fe in the sample gave rise to a quadrupole doublet, most of which was diamagnetic. The parameters of this doublet are typical of S = 0 [4Fe-4S]2+ clusters and S = 0 ferrous heme groups. Spectra of samples reduced with dithionite, pH 8.5, suggested that at least half of this doublet arose from [4Fe-4S]2+ clusters. The second major component exhibited in the M????ssbauer spectra arose from high-spin ferrous ions (10%-30%). The third major component (15%) came from iron exhibiting magnetic hyperfine interactions and is likely reflected in the Fe-containing species observed by EPR. The results presented here suggest that mitochondria contain ~ 600 ????M of Fe overall, ~ 200 ?????? 400 ????M organized as [4Fe-4S]2+ clusters, with about 25 ????M due to the [4Fe-4S]2+ cluster of aconitase. Approximately 60 ????M ?????? 200 ????M of the Fe in mitochondria is high-spin ferrous ions, ~ 40 ????M as the Rieske S = 1/2 [2Fe-2S]+ cluster of cytochrome bc1, and ~20 ????M as the S = 1/2 [2Fe-2S]+ cluster of succinate dehydrogenase. The high-spin ferric hemes of the a3:CuB site of cytochrome oxidase and cytochrome c peroxidase each account for ~ 4 ????M of Fe.