Project Title: Mechanisms Leading to Enhanced Tolerance to Oxidative Stress and Increased Lifespan in Arabidopsis: Role of Mitochondrial, ER, and Chloroplastic Enzymes Involved in Ascorbate Biosynthesis

ABSTRACT

The metabolism of aerobic organisms leads to various risks for oxidative damage, due to the formation of reactive oxygen species (ROS). Although there is considerable evidence implicating oxidative stress in aging, there is a clear gap in our knowledge of the underlying biochemical mechanisms involved in this process. Vitamin C (ascorbate, AsA), a powerful non-enzymatic ROS scavenger, is a major contributor to the antioxidant cell capacity. Across kingdoms AsA is found at very high concentrations in multiple subcellular compartments including the mitochondria, cytoplasm, endoplasmic reticulum (ER), and chloroplast. A major objective of my group is to understand AsA metabolism and its role in conferring plants tolerance to oxidative stress and thus in delaying aging. In particular we focus on the study of the inositol pathway to AsA. Our preliminary data indicate that some of the isoforms of glucuronolactonase (GNL) and gulono-1,4-lactone oxidase (GLOase) are targeted to the mitochondria, ER, and chloroplast. The long-term goal of this project is to take advantage of the unique and powerful tools we have developed to investigate the role of subcellular AsA pools in the underlying biochemical mechanisms leading to tolerance to oxidative stress and delayed aging. We hypothesize that plants have evolved isoforms of GNL and GLOase that maintain AsA subcellular pools needed to protect essential and vulnerable molecules against oxidative stress, and that this protection is critical for the increased lifespan, enhanced growth, and extended reproductive activity displayed by the high-AsA Arabidopsis lines we have developed. Specific Aims 1−3 will test this hypothesis. Aim 1: Investigate the role of the putative mitochondrial GLOase At5g56470 in contributing to the mitochondrial AsA subcellular pool, to protection against oxidative stress, and to increased lifespan. Aim 2: Explore the role of ER-targeted GNLs and GLOases in maintaining a redox balance conducive to proper ER function, protection against oxidative stress, and increased lifespan. Aim 3: Evaluate the role of the putative chloroplastic GNL At1g56500 in contributing to the chloroplast AsA subcellular pool, to protection against oxidative stress, and to increased lifespan. Public Health Relevance: The results of the proposed research will have direct impact on our understanding of the mechanisms by which AsA delays aging, with clear implications for human and animal health. This project will also contribute to the growing pipeline of young scientists already involved in this research, making a clear contribution to the INBRE mission.

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Project Title:  Structure-Activity Studies of Novel Gold(III) Compounds for use in the Treatment of Cisplatin-Resistant Ovarian Cancer

Project Leader

Allyn Ontko, Ph.D.

Associate Professor, Biochemistry
Arkansas State University

Peter Crooks, Ph.D.

Professor, College of Pharmacy, Department of Pharmaceutical Sciences
University of Arkansas for Medical Sciences

ABSTRACT

Platinum-containing compounds such as cisplatin and the newer, second generation carbo- and oxaliplatin are widely used in the treatment of a variety of cancers. Second generation platinum agents were designed primarily to improve efficacy, particularly against cell lines that demonstrate second pass cisplatin resistance. Structurally, second generation platinum agents are little more than cisplatin with a slightly modified ligand environment.  While this ligand modification does enhance the selectivity and cellular uptake of platinum by cancer cells, these agents still exert their anticancer activity through a platinum-mediated mechanism.  To circumvent the issue of second pass cisplatin resistance, alternative treatment agents need to be explored.   

To alleviate the problem of cisplatin resistance, my group has evaluated gold(III)-based complexes as a potential alternative to cisplatin or adjuvant agent to be co-administered with cisplatin. Gold(III) complexes exhibit isoelectronic and isostructural features with platinum(II) and have similar uptake and DNA interference activity. In addition, the higher charge of gold(III) compared to platinum(II) offers several advantages.  These include enhanced DNA binding affinity, increased acid stability in the development of oral chemotherapeutic agents, and a more redox-active system capable of inducing oxidative stress in vivo.  While many studies report that gold(III) complexes are emerging as potential anticancer targets, few detailed mechanistic studies exist. To this end, this work seeks to evaluate the cytotoxic activity, cellular uptake, intracellular signaling cascades and DNA binding affinity of several well-defined gold(III) complexes in cisplatin-sensitive, cisplatin-resistant and multidrug resistant ovarian cancer cells.  

The overall goal of this project is to examine the structure-activity relationships among a series of novel gold(III) compounds known to overcome cisplatin resistance in ovarian cancer cell lines.  We seek to further understand how an isostructural and isoelectronic system can overcome resistance.  We believe that gold(III) exerts a unique mechanism of anticancer action different from that of the currently employed Pt(II) agents. 

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Project Title: The role of host protein kinase C signaling in Coxiella burnetii infection

Project Leader
C. Joel Funk, Ph.D.
Assistant Professor, Division of Science, Biology
John Brown University

Dan Voth, Ph.D.

Assistant Professor, Department of Microbiology & Immunology

UAMS

ABSTRACT

Q fever is caused by the intracellular bacterium Coxiella burnetii. The infection of host cells by C. burnetii involves redirecting the host to provide a protected environment, within a parasitophorous vacuole (PV), where replication of the pathogen can take place.  Many of the events leading up to formation of the PV are not understood.  Previous studies using inhibitors have indicated that host cell Protein Kinase C (PKC) signaling is one of the key pathways that are critical for formation of the PV.  We plan to initially determine the specific PKC isoform(s) that are involved in this kinase signaling pathway using immunoblots to study their activation state. Confirmation of PKC isoform involvement will involve localization studies and siRNA knockdown approaches.  The finding of this research will be used to identify potential drug targets that could result in novel therapies for treating Q fever and the many symptoms that it causes.  

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Project Title: Effects of Prenatal Steroids on the Fatigue Properties of Breathing Muscles

Project Leader
Jennifer Dearolf, Ph.D.
Associate Professor, Department of Biology
Hendrix College

Mentor
Steven Post, Ph.D.
Professor, Department of Pathology
University of Arkansas for Medical Sciences

ABSTRACT

Prenatal steroids are routinely used in the care of women who show signs of or have risk factors for premature birth. These steroids stimulate lung development in premature infants, leading to decreased mortality rates. Our work has shown that exposure to a multicourse injection (2 injections/week at 65%, 75%, and 85% gestation) of betamethasone leads to increased oxidative capacities of the slow- and fast-twitch fibers of the fetal guinea pig (Cavia porcellus) scalenus, an accessory inspiratory muscle. Therefore, the purpose of this study is to examine the effects of prenatal steroid exposure, both single (2 injections at 70% gestation) and multicourse, on the metabolic and functional properties of guinea pig breathing muscles. We propose to test the hypotheses that prenatal steroids lead to changes in the (1) oxidative capacities and (2) fatigue resistance of five guinea pig ventilatory muscles. Pregnant guinea pigs will be injected intramuscularly with betamethasone (0.5 mg/kg of body weight) or sterile water according to the presented single and multicourse injection schedules. Fetuses will be delivered 24 hours after the final injection, and breathing muscle samples will be collected. Histo- and immunocytochemical methods will be used to compare the (1) NADH-tetrazolium reductase activities in slow- and fast-twitch fibers and (2) capillary densities (number of capillaries per fiber cross-sectional area) of the ventilatory muscles in steroid-treated and untreated fetuses to determine whether prenatal steroid exposure leads to increases in both of these measures of oxidative capacity. Whole-muscle citrate synthase (CS) activity will also be measured biochemically to assess whether prenatal steroids increase CS activity levels in treated muscles. Finally, the fatigue resistance of these muscles will be determined. If the treated muscles express more oxidative enzymes, greater capillary densities, and fatigue resistance, these muscles will be better prepared to support the ventilatory needs of premature infants. Public Health Relevance: Prenatal steroids, routinely given to women who show signs of or have risk factors for giving birth prematurely, are known to accelerate fetal lung development, allowing newborns to breathe more competently if they are born prematurely. Whereas corticosteroids have detrimental effects in mature skeletal muscle, prenatal steroids may accelerate the acquisition of the metabolic machinery necessary to sustain ventilation in these premature infants, better preparing them for life outside the womb.

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Project Title: Role of Mitochondrial DNA Damage in Alcohol- and CYP2E1-dependent Toxicity

Project Leader
Andres Caro, Ph.D.
Assistant Professor, Department of Chemistry
Hendrix College

Mentor
Martin Ronis, Ph.D.
Professor, Department of Pharmacology and Toxicology
University of Arkansas for Medical Sciences

Alcoholic liver disease represents an important cause of death and disability in the United States. Acute and chronic ethanol consumption enhances oxidative stress in the liver, potentially leading to oxidative damage. Oxidative stress is characterized by an increased steady-state concentration of reactive oxygen species (ROS), which are oxidizing ions or molecules derived from the partial reduction of molecular oxygen. A central pathway by which ethanol generates a state of oxidative stress is the induction of the cytochrome P450 isoform 2E1 (CYP2E1), which metabolizes alcohol and generates ROS in the process. Ethanol causes liver mitochondrial DNA (mtDNA) oxidative damage and depletion, as well as mitochondrial dysfunction. In addition, prolonged alcohol administration induces a compensatory upregulation of mtDNA replication genes. However, the mechanisms of ethanol-induced mtDNA damage and imbalance and the extent to which mtDNA damage impairs mitochondrial function are currently unknown.
Our long-term goal is to decipher the molecular events affecting mitochondria that result in alcohol-induced liver injury. Our objective is to identify the mechanisms and effects of mtDNA damage in alcohol-induced hepatocyte injury. The central hypothesis of this proposal is that high levels of ROS produced as a result of CYP2E1 activity and alcohol administration result in acute oxidative liver mtDNA damage followed by mitochondrial dysfunction and liver injury. Prolonged CYP2E1-dependent oxidative stress may result in compensatory increases in mtDNA replication, which may contribute to adaptation to ethanol after chronic exposure in vivo. T

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Project Title: Nitroanisole Detoxification by CYP2E1

Project Leader
Martin Perry, Ph.D.
Professor, Department of Chemistry
Ouachita Baptist University

Mentor
Grover Miller, Ph.D.
Associate Professor, Department of Biochemistry and Molecular Biology
University of Arkansas for Medical Sciences

ABSTRACT

Nitroanisoles are environmental pollutants whose toxic potential depends on the efficiency of biological processes. While xanthine oxidase activates nitroanisoles to carcinogenic mutagens, cytochrome P450s, most notably CYP2E1, oxidize nitroanisoles to readily excreted metabolites, thereby detoxifying nitroanisoles. Thus, the efficiency of CYP2E1 activity toward nitroanisoles is a potentially important determinant of risk from exposure to those pollutants. Kinetic profiling for CYP2E1 has been an important tool in assessing the activation and detoxification of many small molecular weight compounds. Unlike traditional enzymes, CYP2E1 catalysis may involve multiple binding sites that alter the metabolism of compounds. Recently, we explained unusual non-hyperbolic kinetics for 4-nitrophenol oxidation through the presence of an effector site, which when occupied, suppressed the reaction. Lack of knowledge of this mechanism could lead to incorrect estimates for the clearance of pollutants from the body, leading to toxicity from exposure. In the proposed project, we hypothesize that the contribution of CYP2E1 to nitroanisole detoxification depends on the occupancy of an effector site by nitroanisoles or the corresponding metabolites. Specifically, we will test our hypothesis through the following aims: (1) determine the kinetic mechanisms for nitroanisole metabolism; (2) construct computer models for CYP2E1 complexes with substrates and effectors to predict non-hyperbolic reaction kinetics; (3) identify binding site residues for monocyclic molecules through photoaffinity labeling; and (4) confirm the functional role for effector site residues through site-directed mutagenesis. Public Health Relevance: Collectively, these findings will generate models to interpret and predict the efficiency of detoxification of nitroanisoles and provide tools for future toxicological studies. In addition, the combination of biophysical and computational techniques provides an excellent opportunity to cross-train students for successful careers in science.

Collaborators at UAMS have proven immensely valuable to me as a faculty member in a small liberal arts university setting. The Arkansas INBRE program has allowed me to set up mentoring relationships with experienced UAMS investigators, who have been and will be integral in helping me establish the knowledge and skills base that will be critical to advance in the arena of biomedical research.

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Project Title: Cannabinoids and Inflammation: Relevance to Multiple Sclerosis

Project Leader
Lori Hensley, Ph.D.
Assistant Professor, Department of Biology
Ouachita Baptist University

Mentor
Paul Drew, Ph.D.
Associate Professor, Department of Neurobiology & Developmental Science
University of Arkansas for Medical Sciences

Steven Post, Ph.D.
Professor, Department of Pathology
University of Arkansas for Medical Sciences

ABSTRACT

An estimated 2.5 million people worldwide have been diagnosed with the neurodegenerative disease multiple sclerosis (MS). Though the number of patented drugs worldwide continues to rapidly increase, effective treatments for many neural diseases remain elusive because of the complex physiologic pathways involved. Cannabinoids, bioactive compounds from the Cannabis sativa plant, have recently emerged as promising therapeutic agents for neuroinflammatory conditions found in diseases such as MS. The long-term goal of our work is to characterize these natural compounds as potential therapeutic options for MS and elucidate pathways through which they mediate their effects. In this project’s Specific Aim 1 we use cultured glial cells (microglia and astrocytes) as a cellular model for testing the abilities of cannabinoid compounds—ajulemic acid and cannabidiol—to suppress markers of neuroinflammation such as nitric oxide and pro-inflammatory cytokines and chemokines. Drugs with such abilities are good candidates for treating diseases characterized by chronic neuroinflammation, such as MS. Since neurodegeneration often results from these inflammatory conditions, an ideal treatment would suppress the inflammatory response and protect surrounding neurons from damage induced by mediators of inflammation. In Specific Aim 2, we test whether our compounds provide such protection. We induce apoptosis in cortical neurons by treatment with 2 toxic inflammatory cytokines, interferon-gamma and tumor necrosis factor-alpha, and assess effectiveness of pre-treatment with our compounds in preventing subsequent neuronal death. In Specific Aim 3, we use cannabinoid receptor antagonists to determine whether observed anti-inflammatory and neuroprotective effects are mediated through classic CB1 and CB2 receptors.

Ouachita Baptist University provides an excellent research environment in which to complete these aims. The investigator has a 220 square foot lab, a tissue culture lab, a plate reader, small molecular biology equipment, a qRT-PCR machine, and a gel imaging system. All animal work will be completed at UAMS.

Through these proposed aims, we expect to provide the rationale for improved therapeutic options to address chronic neuroinflammation characterizing neurodegenerative diseases like MS. Characterization of the mechanisms by which the cannabinoid compounds mediate disease-modulating effects should aid the development of targeted drugs with less risk and fewer side effects than other classes of pharmaceuticals.

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Project Title: Prevention and Treatment of Cisplatin- and Rhabdomyolysis- Induced Nephrotoxicity Using Metal Complexes

Project Leader
Grant Wangila, Ph.D.
Associate Professor, Department of Chemistry
University of Arkansas at Pine Bluff

Mentor
Alexei Basnakian, M.D., Ph.D.
Associate Professor, Nephrology/Internal Medicine
University of Arkansas for Medical Sciences

ABSTRACT

The objective of this proposal is to synthesize, characterize, and test several new metal (copper and zinc) complexes of salicylic acid and aminothiol derivatives as catalytic antioxidants that scavenge a wide range of reactive oxygen species (ROS). Several compounds in this class have been shown to be efficacious in a variety of ways in in vitro and in vivo oxidative stress models of human diseases. Our preliminary data strongly indicate that these metal complexes satisfy many of the criteria for prevention and treatment of cisplatin-induced nephrotoxicity (an ROS-mediated injury), as they are active, stable, and nontoxic antioxidants. Since rhabdomyolysis-induced nephrotoxicity is also ROS-mediated, these compounds also satisfy many of the criteria for prevention and treatment of kidney injury due to this disease. We hypothesize that copper and zinc complexes can be synthesized that combine the cytoprotective effects of antioxidant ligands with the cytorecovery effects of the essential metals in order to protect kidney tubular epithelial cells from toxicity due to cisplatin administration or the development of rhabdomyolysis. The proposal has two specific aims: Specific Aim 1: Synthesize, characterize, and evaluate antioxidant properties of several copper and zinc complexes. The focus will be on ligands, which have or contribute to cytoprotective activity. We will examine ligands from the substituted salicylate and aminothiol classes. Several such compounds have been synthesized in our laboratory and have shown favorable activity in several in vitro models. This Specific Aim will focus on chemical studies of several new compounds, as well as some previously known but not fully characterized compounds. All new compounds will be tested for antioxidant properties using standard antioxidant assays and cyclic voltammetry. Specific Aim 2: Assess the nephroprotective activity of metal complexes in vitro and in vivo. The stable water-soluble compounds with the highest antioxidant activities will be tested for cytoprotection against cisplatin and rhabdomyolysis injuries using cultured tubular epithelial cells in vitro and in a mouse model. Experiments will be done to determine the effect of metal complexes on caspase activation and caspase-dependent or -independent endonuclease activation.

Public Health Relevance: Better agents for the prevention and treatment of cisplatin- and rhabdomyolysis-induced nephrotoxicities are greatly needed to reduce treatment-related and comorbid renal dysfunction. The compounds proposed in this study have molecular features that combine the cytoprotective effects of antioxidant ligands with the cytorecovery effects of the essential metals, zinc, and copper.

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Project Title: Targeted Drug Delivery of Anticancer Agents across the Blood–Brain Barrier

Project Leader
Antonie Rice, Ph.D.
Associate Professor, Department of Chemistry & Physics
University of Arkansas at Pine Bluff

Mentor
Howard P. Hendrickson, Ph.D.
Assistant Professor, Department of Pharmaceutical Sciences
University of Arkansas for Medical Sciences

Paul E. Gottschall, Ph.D.
Professor, Department of Pharmacology & Toxicology
University of Arkansas for Medical Sciences

ABSTRACT

The inadequate delivery of chemotherapeutics to the desired site of action in the brain results in a poor response to drug treatment for many neurological disorders (i.e., Alzheimer’s disease, brain tumors, HIV, and other genetic disorders). The blood–brain barrier (BBB) regulates the influx and efflux of a wide variety of substances and remains the major obstacle of delivery of pharmaceutics into the central nervous system (CNS). The development of BBB targeting strategies is a very active field of research and includes manipulation of the drug, modification of capillary permeability of the BBB, or attempts to increase the driving force for transport by increasing plasma concentrations of the drug (i.e., high-dose chemotherapy, intra-arterial injection). We have been successful in demonstrating that chemical modification of a highly lipophilic non-permeable p-glycoprotein (P-gp) substrate (i.e., paclitaxel [Taxol]) can be achieved, resulting in increased permeability through the BBB, and that targeting active drug transporter vector systems is viable. Our long-term objectives are to (1) demonstrate that through the utilization of combinatorial chemistry we can successfully synthesize new pharmacologically active derivatives of taxanes and other anticancer moieties with enhanced permeability, (2) characterize active transporters at the BBB with known vectors that may be useful in explaining the mechanistic pathways of the newly synthesized compounds, and (3) demonstrate an in vitro/in situ correlation to give further insight into the mechanistic pathways of a derivative's enhanced permeability.

This project focuses on elucidation of possible molecular mechanisms controlling permeation of the BBB by anticancer drugs. Knowledge of these mechanisms should lead to strategies that may be employed to enhance therapeutic delivery of pharmaceuticals to the brain. The specific aims of this proposal are a logical extension of the aims successfully carried out with current INBRE funding. Our original aims demonstrated the feasibility of making small chemical modifications to paclitaxel to generate new analogues with reduced affinity for P-gp, the major efflux pump restricting passage of molecules into the brain, while retaining pharmacological activity. We plan to apply this same method of generating new analogues to other anticancer agents or drugs with poor brain bioavailability. We hypothesize that taxane analogues and derivatives of other anticancer drugs can be prepared that elude the P-gp efflux pump by altering and/or deleting functional groups that are recognition elements for this transporter.

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Project Title: Understanding Immune Cell Signaling: Effect of Retinoids on ADAM Shedding

Mentor
Jerry Ware, Ph.D.
Professor, College of Medicine, Department of Physiology & Biophysics
University of Arkansas for Medical Sciences

Atherosclerosis of the coronary arteries (i.e., coronary artery disease, CAD) is the number on killer of postmenopausal women and is considered an age-related disease.  the protective effects of estradiol (i.e., estrogen) against CAD may be due to the action of  its two primary metabolites, 2-hydroxyestradiol and 2-methoxyestradiol.  the overall lone-term goal of this project is to understand how the metabolites of estradiol protect against age-induced changes of the coronary arteries and thereby prevent CA.  These experiments will be conducted on the smooth muscle of coronary arteries from two different age groups, (9-month old verses 4 year old) of sexually mature female Yorkshire pigs.  the overall hypothe4sis of this project is that 2-methoxyestradiol is more efficacious that 2-hydroxyestradiol in protecting against CAD by reducing arterial tone and decreasing the proliferation of smooth muscle cells.  We predict that aging will eliminate these protective effects of the estradiol metabolites.  the specific aims are to investigate the impact of aging, and 2-hydroxyestradiol and 2-methoxyestradiol on (1) the intracellular Ca2+ induced smooth muscle contraction, (2) the Ca2+ activated big potassium (BKCa) channel mediated arterial relaxation, and (3) smooth muscle cell proliferation.  the methods for Aim 1 are to use fluorescent microscopy to measure intracellular Ca2+concentration ([Ca]i) changes in response to agonist-induced (K+ and endothelin-1, respectively) Ca2+ influx and Ca2+ release from the sarcoplasmic reticulum upon incubation of the smooth muscle cells in  2-hydroxyestradiol and 2-methoxyestradiol.  then it will be determined if the [Ca]i changes produce changes in isometric contraction of arterial rings.  Fro Aim # 2, whole cell patch clamp techniques will be used to measure BKCa channel current after exposing the isolated smooth muscle cells to 2-hydroxyestradiol or 2-methoxyestradiol.  Next, the isometric relaxation of arterial rings will be investigated in response to 2-hydroxyestradiol or 2-methoxyestradiol  in the presence of selective BKCa channel antagonists.  Fro Aim #3, bromodeoxyuridine (BrdU) incorporation during the cell cycle will be used as a measure of DNA synthesis.  A fluorescently tagged antibody to BrdU and FM will be used to measure BrdU incorporation into DNA.  the overall relevance of this project is that the metabolites of estrogen may protect against CAD without causing estrogen-induced tumor growth.

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Project Title: Estrogen Prevents the Subunit Association of Vascular Voltage-Gated Calcium Channels

Project Leader
Brent Hill, Ph.D.
Assistant Professor, Department of Biology
University of Central Arkansas

Mentor
Nancy Rusch, Ph.D.
Professor and Chair, Department of Pharmacology and Toxicology
University of Arkansas for Medical Sciences

Atherosclerosis of the coronary arteries (i.e., coronary artery disease, CAD) is the number on killer of postmenopausal women and is considered an age-related disease.  the protective effects of estradiol (i.e., estrogen) against CAD may be due to the action of  its two primary metabolites, 2-hydroxyestradiol and 2-methoxyestradiol.  the overall lone-term goal of this project is to understand how the metabolites of estradiol protect against age-induced changes of the coronary arteries and thereby prevent CA.  These experiments will be conducted on the smooth muscle of coronary arteries from two different age groups, (9-month old verses 4 year old) of sexually mature female Yorkshire pigs.  the overall hypothe4sis of this project is that 2-methoxyestradiol is more efficacious that 2-hydroxyestradiol in protecting against CAD by reducing arterial tone and decreasing the proliferation of smooth muscle cells.  We predict that aging will eliminate these protective effects of the estradiol metabolites.  the specific aims are to investigate the impact of aging, and 2-hydroxyestradiol and 2-methoxyestradiol on (1) the intracellular Ca2+ induced smooth muscle contraction, (2) the Ca2+ activated big potassium (BKCa) channel mediated arterial relaxation, and (3) smooth muscle cell proliferation.  the methods for Aim 1 are to use fluorescent microscopy to measure intracellular Ca2+concentration ([Ca]i) changes in response to agonist-induced (K+ and endothelin-1, respectively) Ca2+ influx and Ca2+ release from the sarcoplasmic reticulum upon incubation of the smooth muscle cells in  2-hydroxyestradiol and 2-methoxyestradiol.  then it will be determined if the [Ca]i changes produce changes in isometric contraction of arterial rings.  Fro Aim # 2, whole cell patch clamp techniques will be used to measure BKCa channel current after exposing the isolated smooth muscle cells to 2-hydroxyestradiol or 2-methoxyestradiol.  Next, the isometric relaxation of arterial rings will be investigated in response to 2-hydroxyestradiol or 2-methoxyestradiol  in the presence of selective BKCa channel antagonists.  Fro Aim #3, bromodeoxyuridine (BrdU) incorporation during the cell cycle will be used as a measure of DNA synthesis.  A fluorescently tagged antibody to BrdU and FM will be used to measure BrdU incorporation into DNA.  the overall relevance of this project is that the metabolites of estrogen may protect against CAD without causing estrogen-induced tumor growth.

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Project Title: Studying FszA to Elucidate the Link between Prokaryotes and Mitochondria

Project Leader
Kari Naylor, Ph.D.
Assistant Professor, Department of Biology
University of Central Arkansas

Mentor
Vladimir Lupashin, Ph.D.
Associate Professor, Department of Physiology & Biophysics
University of Arkansas for Medical Sciences

ABSTRACT

Mitochondria are dynamic organelles that continually undergo membrane remodeling by fission and fusion events that create and maintain the tubular structure, allowing the mitochondrial compartment to efficiently respond to metabolic needs of the cell. The proposed study focuses on understanding the mechanisms of mitochondrial fission, which appears to be an essential process in humans. During the evolution of mitochondria from prokaryotic ancestors, several primitive eukaryotic organisms, including Dictyostelium discoideum, retained the ancestral mechanism to divide their mitochondria. Our overall goal is to uncover the fission mechanism in D. discoideum and trace the evolutionary link between higher eukaryotic mitochondria and their prokaryotic ancestors. Prokaryotic cell division is mediated by FtsZ, which assembles into a ring that constricts and splits the cell into two daughter cells. Eukaryotes, such as yeast, that have apparently lost FtsZ proteins use dynamin-related proteins (DRPs) to mediate mitochondrial division. D. discoideum expresses at least two FtsZ orthologs (FszA and FszB) and appears to mediate division of its mitochondrial tubules by an FtsZ-type mechanism, possibly in conjunction with a DRP-type mechanism. A thorough understanding of eukaryotic mitochondrial fission requires knowledge of all fission mechanisms, including the rudimentary mechanisms used in D. discoideum. We hypothesize that FszA, together with its protein partners, plays a direct role in mitochondrial fission in D. discoideum by constricting and dividing the mitochondrial tubule. To test this hypothesis, we propose to develop a novel in vivo microscopy-based system to investigate fission events in D. discoideum in real time (Specific Aim 1) and to identify components of fission machinery (Specific Aim 2).

Ultimately, the results of the proposed study will contribute to our understanding of mitochondrial fission, a process necessary for maintaining the tubular mitochondrial structure. Disruption of this structure has been shown to cause developmental defects, lead to neurodegenerative diseases, and affect regulation of programmed cell death. Understanding the molecular mechanisms of mitochondrial fission in eukaryotes will help us to understand the cellular process of apoptosis and develop treatments for a variety of mitochondrial diseases. Thus, understanding the processes that maintain mitochondrial structure is important to human health.