Biochemistry, Biophysics, and Structural Biology Commons^™

Plant Acyl-Coa:Lysophosphatidylcholine Acyltransferases (Lpcats) Have Different Specificities In Their Forward And Reverse Reactions, Ida Lager, Jenny Lindberg Yilmaz, Xue-Rong Zhou, Katarzyna Jasieniecka, Michael Kazachkov, Peng Wang, Jitao Zou, Randall Weselake, Mark A. Smith, Shen Bayon, John M. Dyer, Jay Shockey, Ernst Heinz, Allan Green, Antoni Banas, Sten Stymne

Department of Biochemistry: Faculty Publications

Background: Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) enzymes have central roles in acyl editing of phosphatidylcholine.

Results: Plant LPCATs were expressed in yeast and biochemically characterized.

Conclusion: LPCATs can edit acyl composition of phosphatidylcholine through their combined forward and reverse reactions.

Significance: Plant LPCATs play a role in editing both sn-positions of PC and remove ricinoleic acid with high selectivity from this lipid.

Emerging Regulatory Paradigms In Glutathione Metabolism, Yilin Liu, Annastasia S. Hyde, Melanie A. Simpson, Joseph J. Barycki

Department of Biochemistry: Faculty Publications

One of the hallmarks of cancer is the ability to generate and withstand unusual levels of oxidative stress. In part, this property of tumor cells is conferred by elevation of the cellular redox buffer glutathione. Though enzymes of the glutathione synthesis and salvage pathways have been characterized for several decades, we still lack a comprehensive understanding of their independent and coordinate regulatory mechanisms. Recent studies have further revealed that overall central metabolic pathways are frequently altered in various tumor types, resulting in significant increases in biosynthetic capacity, and feeding into glutathione synthesis. In this review, we will discuss the enzymes …

Emerging Roles For Hyaluronidase In Cancer Metastasis And Therapy, Caitlin O. Mcatee, Joseph J. Barycki, Melanie A. Simpson

Department of Biochemistry: Faculty Publications

Hyaluronidases are a family of five human enzymes that have been differentially implicated in the progression of many solid tumor types, both clinically and in functional studies. Advances in the past five years have clarified many apparent contradictions, (1) by demonstrating that specific hyaluronidases have alternative substrates to hyaluronan (HA) or do not exhibit any enzymatic activity, (2) that high molecular weight HA polymers elicit signaling effects that are opposite those of the hyaluronidase-digested HA oligomers, and (3) that it is actually the combined overexpression of HA synthesizing enzymes with hyaluronidases that confers tumorigenic potential. This review examines the literature …

Mechanisms Underlying The Exquisite Sensitivity Of Candida Albicans To Combinatorial Cationic And Oxidative Stress That Enhances The Potent Fungicidal Activity Of Phagocytes, Despoina Kaloriti, Mette Jacobsen, Zhikang Yin, Miranda Patterson, Anna Tillmann, Deborah A. Smith, Emily Cook, Tao You, Melissa J. Grimm, Iryna Bohovych, Celso Grebogi, Brahm H. Segal, Neil A.R. Gow, Ken Haynes, Janet Quinn, Alistair J.P. Brown

Department of Biochemistry: Faculty Publications

Immune cells exploit reactive oxygen species (ROS) and cationic fluxes to kill microbial pathogens, such as the fungus

Candida albicans. Yet, C. albicans is resistant to these stresses in vitro. Therefore, what accounts for the potent antifungal activity

of neutrophils? We show that simultaneous exposure to oxidative and cationic stresses is much more potent than the individual

stresses themselves and that this combinatorial stress kills C. albicans synergistically in vitro.We also show that the high

fungicidal activity of human neutrophils is dependent on the combinatorial effects of the oxidative burst and cationic fluxes, as

their pharmacological attenuation with …

New Clox Systems For Rapid And Efficient Gene Disruption In Candida Albicans, Shahida Shahana, Delma S. Childers, Elizabeth R. Ballou, Iryna Bohovych, Frank C. Odds, Neil A.R. Gow, Alistair J.P. Brown

Department of Biochemistry: Faculty Publications

Precise genome modification is essential for the molecular dissection of Candida albicans, and is yielding invaluable information about the roles of specific gene functions in this major fungal pathogen of humans. C. albicans is naturally diploid, unable to undergo meiosis, and utilizes a non-canonical genetic code. Hence, specialized tools have had to be developed for gene disruption in C. albicans that permit the deletion of both target alleles, and in some cases, the recycling of the Candida-specific selectable markers. Previously, we developed a tool based on the Cre recombinase, which recycles markers in C. albicans with 90–100% efficiency via …

Saturated Free Fatty Acids Induce Cholangiocyte Lipoapoptosis, Sathish Kumar Natarajan, Sally A. Ingham, Ashley M. Mohr, Cody J. Wehrkamp, Anuttoma Ray, Sohini Roy, Sophie C. Cazanave, Mary Anne Phillippi, Justin L. Mott

Department of Biochemistry: Faculty Publications

Recent studies have identified a cholestatic variant of nonalcoholic fatty liver disease (NAFLD) with portal inflammation and ductular reaction. Based on reports of biliary damage, as well as increased circulating free fatty acids (FFAs) in NAFLD, we hypothesized the involvement of cholangiocyte lipoapoptosis as a mechanism of cellular injury. Here, we demonstrate that the saturated FFAs palmitate and stearate induced robust and rapid cell death in cholangiocytes. Palmitate and stearate induced cholangiocyte lipoapoptosis in a concentration-dependent manner in multiple cholangiocyte-derived cell lines. The mechanism of lipoapoptosis relied on the activation of caspase 3/7 activity. There was also a significant up-regulation …

Xiap Antagonist Embelin Inhibited Proliferation Of Cholangiocarcinoma Cells, Cody J. Wehrkamp, Ashley R. Gutwein, Sathish Kumar Natarajan, Mary Anne Phillippi, Justin L. Mott

Department of Biochemistry: Faculty Publications

Cholangiocarcinoma cells are dependent on antiapoptotic signaling for survival and resistance to death stimuli. Recent mechanistic studies have revealed that increased cellular expression of the E3 ubiquitin-protein ligase X-linked inhibitor of apoptosis (XIAP) impairs TRAIL- and chemotherapy-induced cytotoxicity, promoting survival of cholangiocarcinoma cells. This study was undertaken to determine if pharmacologic antagonism of XIAP protein was sufficient to sensitize cholangiocarcinoma cells to cell death. We employed malignant cholangiocarcinoma cell lines and used embelin to antagonize XIAP protein. Embelin treatment resulted in decreased XIAP protein levels by 8 hours of treatment with maximal effect at 16 hours in KMCH and Mz-ChA-1 …

Oxidative Stress, Redox Signaling, And Autophagy: Cell Death Versus Survival, Juliana Navarro-Yepes, Michaela Burns, Anandhan Annadurai, Oleh Khalimonchuk, Luz Maria Del Razo, Betzabet Quintanilla-Vega, Aglaia Pappa, Mihalis I. Panayiotidis, Rodrigo Franco

Department of Biochemistry: Faculty Publications

Significance: The molecular machinery regulating autophagy has started becoming elucidated, and a number of studies have undertaken the task to determine the role of autophagy in cell fate determination within the context of human disease progression. Oxidative stress and redox signaling are also largely involved in the etiology of human diseases, where both survival and cell death signaling cascades have been reported to be modulated by reactive oxygen species (ROS) and reactive nitrogen species (RNS). Recent Advances: To date, there is a good understanding of the signaling events regulating autophagy, as well as the signaling processes by which alterations in …

Kinetic And Structural Characterization Of Tunnel-Perturbing Mutants In Bradyrhizobium Japonicum Proline Utilization A, Benjamin W. Arentson, Min Luo, Travis A. Pemberton, John J. Tanner, Donald F. Becker

Department of Biochemistry: Faculty Publications

Proline utilization A from Bradyrhizobium japonicum (BjPutA) is a bifunctional flavoenzyme that catalyzes the oxidation of proline to glutamate using fused proline dehydrogenase (PRODH) and Δ¹-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. Recent crystal structures and kinetic data suggest an intramolecular channel connects the two active sites, promoting substrate channeling of the intermediate Δ¹-pyrroline-5-carboxylate/glutamate-γ-semialdehyde (P5C/GSA). In this work, the structure of the channel was explored by inserting large side chain residues at four positions along the channel in BjPutA. Kinetic analysis of the different mutants revealed replacement of D779 with Tyr (D779Y) or Trp (D779W) significantly decreased the overall …

Evidence That The C‑Terminal Domain Of A Type B Puta Protein Contributes To Aldehyde Dehydrogenase Activity And Substrate Channeling, Min Luo, Shelbi Christgen, Nikhilesh Sanyal, Benjamin W. Arentson, Donald F. Becker, John J. Tanner

Department of Biochemistry: Faculty Publications

Proline utilization A (PutA) is a bifunctional enzyme that catalyzes the oxidation of proline to glutamate. Structures of type A PutAs have revealed the catalytic core consisting of proline dehydrogenase (PRODH) and Δ1- pyrroline-5-carboxylate dehydrogenase (P5CDH) modules connected by a substrate-channeling tunnel. Type B PutAs also have a C-terminal domain of unknown function (CTDUF) that is absent in type A PutAs. Small-angle X-ray scattering (SAXS), mutagenesis, and kinetics are used to determine the contributions of this domain to PutA structure and function. The 1127-residue Rhodobacter capsulatus PutA (RcPutA) is used as a representative CTDUF-containing type B PutA. The reaction progress …

Homogeneous Low-Molecular-Weight Heparins With Reversible Anticoagulant Activity, Yongmei Xu, Chao Cai, Kasemsiri Chandarajoti, Po-Hung Hsieh, Lingyun Li, Truong Q. Pham, Erica M. Sparkenbaugh, Juzheng Sheng, Nigel S. Key, Rafal Pawlinski, Edward N. Harris, Robert J. Linhardt, Jian Liu

Department of Biochemistry: Faculty Publications

Low-molecular-weight heparins (LMWHs) are carbohydrate-based anticoagulants clinically used to treat thrombotic disorders, but impurities, structural heterogeneity or functional irreversibility can limit treatment options. We report a series of synthetic LMWHs prepared by cost-effective chemoenzymatic methods. The high activity of one defined synthetic LMWH against human factor Xa (FXa) was reversible in vitro and in vivo using protamine, demonstrating that synthetically accessible constructs can have a critical role in the next generation of LMWHs.

Pharmacological Correction Of Obesity-Induced Autophagy Arrest Using Calcium Channel Blockers, Hwan-Woo Park, Haeli Park, Ian A. Semple, Insook Jang, Seung-Hyun Ro, Myungjin Kim, Victor A. Cazares, Edward L. Stuenkel, Jung-Jae Kim, Jeong Sig Kim, Jun Hee Lee

Department of Biochemistry: Faculty Publications

Autophagy deregulation during obesity contributes to the pathogenesis of diverse metabolic disorders. However, without understanding the molecular mechanism of obesity interference in autophagy, development of therapeutic strategies for correcting such defects in obese individuals is challenging. Here we show that a chronic increase of the cytosolic calcium concentration in hepatocytes during obesity and lipotoxicity attenuates autophagic flux by preventing the fusion between autophagosomes and lysosomes. As a pharmacological approach to restore cytosolic calcium homeostasis in vivo, we administered the clinically approved calcium channel blocker verapamil to obese mice. Such treatment successfully increases autophagosome–lysosome fusion in liver, preventing accumulation of …

Hepatoprotective Role Of Sestrin2 Against Chronic Er Stress, Hwan-Woo Park, Haeli Park, Seung-Hyun Ro, Insook Jang, Ian A. Semple, David N. Kim, Myungjin Kim, Myeongjin Nam, Deqiang Zhang, Lei Yin, Jun Hee Lee

Department of Biochemistry: Faculty Publications

Upon prolonged endoplasmic reticulum (ER) stress, cells attenuate protein translation to prevent accumulation of unfolded proteins. Here we show that Sestrin2 is critical for this process. Sestrin2 expression is induced by an ER stress-activated transcription factor CCAATenhancer- binding protein beta (c/EBPβ). Once induced, Sestrin2 halts protein synthesis by inhibiting mammalian target of rapamycin complex 1 (mTORC1). As Sestrin2-deficient cells continue to translate a large amount of proteins during ER stress, they are highly susceptible to ER stress-associated cell death. Accordingly, dietary or genetically induced obesity, which does not lead to any pathological indication other than simple fat accumulation in the …

In Vitro Characterization Of Unmodified And Pyroglutamylated Alzheimer's Amyloid Beta Peptide, Jason Matos

Electronic Theses and Dissertations

Plaques of amyloid β peptide (Aβ) are a hallmark trait of Alzheimer’s disease (AD). However, the precise role of Aβ aggregates is not well understood. Recent studies have identified that naturally occurring N-terminal truncation and pyroglutamylation of Aβ significantly increases its neurotoxicity by an unknown mechanism. Content of pyroglutamylated Aβ (pE-Aβ) in AD brains has been shown to reach up to 50% of total Aβ. Modified pE-Aβ co-aggregates with Aβ by a seeding mechanism and forms structurally distinct and highly toxic oligomers. We studied structural transitions of the full-length Aβ1-42, its pyroglutamylated form AβpE3-42, their 9:1 (Aβ1-42/AβpE3-42) and 1:1 molar …

Establishment Of Methods For Isolation Of Pnmt+ Cardiac Progenitor Cells, Namita Varudkar

Electronic Theses and Dissertations

Cardiovascular disease is the leading cause of death in the United States. Millions of patients suffer each year from endothelial dysfunction and/or debilitating myocardial damage resulting in decreased quality of life and increased risk of death or disablement. Current pharmacological approaches are only partly effective at treating cardiovascular disease, and hence, better strategies are needed to provide significant improvements in treatment options. Cardiac stem/progenitor cells have the potential to regenerate myocardial tissue and repair damaged heart muscle. There are many different types of cardiac progenitor cells, and each may have certain unique properties and characteristics that would likely be useful …

Cerium Oxide Nanoparticles: Their Phosphatase Activity And Its Control, Atul Dhall

Electronic Theses and Dissertations

Cerium oxide nanoparticles are established scavengers of reactive oxygen and nitrogen species. They have many potential biomedical applications that depend on their physicochemical properties and mode of preparation. Recent studies have found these nanoparticles possess phosphatase mimetic activity. Studying such catalytic activities will qualify their biomedical applications and render information on their bioavailability and potential toxicity. Two oxidation states of cerium exist in these nanoparticles (3+ or 4+). It is hypothesized that the oxidation state of cerium in the nanoparticles determines the amount of adsorbed water on the crystal lattices. This in turn governs their activity as phosphatases. Nanoparticles with …

Evaluations Of A Mechanistic Hypothesis For The Influence Of Extracellular Ions On Electroporation Due To High-Intensity, Nanosecond Pulsing, V. Sridhara, R. P. Joshi

Electrical & Computer Engineering Faculty Publications

The effect of ions present in the extracellular medium on electroporation by high-intensity, short-duration pulsing is studied through molecular dynamic simulations. Our simulation results indicate that mobile ions in the medium might play a role in creating stronger local electric fields across membranes that then reinforce and strengthen electroporation. Much faster pore formation is predicted in higher conductivity media. However, the impact of extracellular conductivity on cellular inflows, which depend on transport processes such as electrophoresis, could be different as discussed here. Our simulation results also show that interactions between cations (Na+ in this case) and the carbonyl oxygen of …

Anion-Peptide Adduct Formation And Decomposition As Studied By Fourier Transform Ion Cyclotron Resonance (Ft-Icr) Mass Spectrometry, Xiaohua Liu

LSU New Orleans Theses and Dissertations

A new “best match” match model has been developed to account for adduct formation on multiply charged peptides observed in negative ion electrospray mass spectrometry. To obtain a stable adduct, the model necessitates an approximate matching of apparent gas-phase basicity (GBapp) of a given proton bearing site on the peptide with the gas-phase basicity (GB) of the anion attaching at that site. Evidence supporting the model is derived from the fact that singly charged adducts were only observed for lower GB anions: HSO₄^-, I^-, CF₃COO^-. Ions that have medium GBs (NO …

The Effects Of Salinity, Ph, Temperature, And Dissolved Oxygen On Sensitivity Of Pcr Identification Of T4 Bacteriophage, Joesph F. Cannon, Nicholas A. Thurn, Paul E. Richardson

Journal of the South Carolina Academy of Science

Bacteriophages are used as indicators of pathogenic bacteria in drinking, and wastewaters. They also show potential in limiting aquatic bacterial populations through their lytic properties. The effect of different water characteristics (salinity, pH, dissolved oxygen, and temperature) on the sensitivity of the PCR identification of virus particles were analyzed to determine at what levels bacteriophage can be detected in environmental samples. Results from this preliminary study indicate that a PCR bacteriophage detection technique has potential as a relatively efficient and economical indicator of coliform contamination in multiple aquatic environments. While further evaluation is needed, the protocol appears to function in …

Biochemical Assay Optimization And Computational Screening Efforts To Identify Potential Luxs Inhibitors, Keeshia Q. Wang

Master's Theses

Quorum sensing (QS) is a process of coordination of bacterial gene expression in response to cell population. System two QS is regulated by the small signaling molecule autoinducer-2 (AI-2) and is implicated in the infectious behaviors of various bacterial species. AI-2 is biosynthesized from S-ribosylhomocysteine (SRH) by the enzyme LuxS and induces interspecies cell-to-cell communication. Inhibition of LuxS would therefore inhibit interspecies QS. Herein, a search for novel molecular species that will competitively bind with SRH in the LuxS binding site is performed in silico. Computational screening results are then validated in vitro using an optimized LuxS inhibition …

Mutational Analysis Of The Rotavirus Nsp4 Enterotoxic Domain That Binds To Caveolin-1, Judith M. Ball, Megan E. Schroeder, Cecelia V. Williams, Friedhelm Schroeder, Rebecca D. Parr

Faculty Publications

Background: Rotavirus (RV) nonstructural protein 4 (NSP4) is the first described viral enterotoxin, which induces early secretory diarrhea in neonatal rodents. Our previous data show a direct interaction between RV NSP4 and the structural protein of caveolae, caveolin-1 (cav-1), in yeast and mammalian cells. The binding site of cav-1 mapped to the NSP4 amphipathic helix, and led us to examine which helical face was responsible for the interaction.

Methods: A panel of NSP4 mutants were prepared and tested for binding to cav-1 by yeast two hybrid and direct binding assays. The charged residues of the NSP4 amphipathic helix were changed …

Development Of A Metabolomic Method To Define The Phenylalanome In Arabidopsis Thaliana, Cole G. Wunderlich, Clint Chapple, Xu Li

The Summer Undergraduate Research Fellowship (SURF) Symposium

In the study of metabolomics, one of the greatest challenges can be accurately identifying compounds detected in biological extracts, especially when standards are not readily available. Current metabolomic methods are also limited in that they provide little to no information about a compound’s metabolic origin. In this study, we sought to address these issues by developing a novel metabolomic method that employs stable isotope feeding, LC-MS, Xcms, and an analytical software algorithm to study the ‘phenylalanome’ of Arabidopsis thaliana. Using this approach we were able to develop a method that, based on current results, is capable of detecting over …

The Creation Of Α2nc1 Transgenic Drosophila Melanogaster, Jordan Olberding

Mahurin Honors College Capstone Experience/Thesis Projects

The α2 isoform of collagen IV’s noncollagenous 1 domain (α2NC1 or canstatin) has shown promise as an anti-cancer molecule, possessing both angiostatic and pro-apoptotic properties. This work aims to further the knowledge of α2NC1 by conducting a truly in vivo study in which the Gal4-induced endogenous overexpression of α2NC1 in Drosophila melanogaster will be used to assess the effects of α2NC1 upon normal development as well as tumorigenesis. To this end, transgenic fly lines capable of overexpressing α2NC1 were created. Initial overexpression studies indicated no developmental phenotype was caused by abundance of α2NC1. However, future overexpression studies in which α2NC1 …

A Study On The Regulation Of Amino Acids And Glucose Sensing Pathways In Saccharomyces Cerevisiae, Mengying Chiang

LSU New Orleans Theses and Dissertations

Nutrient availability regulates eukaryotic cell growth. This study focuses on two signaling pathways, involved in sensing amino acids and carbon sources, which allow cells to respond appropriately to their presence. The first part of this study shows that Ssy1, a plasma membrane localized sensor in the Ssy1-Ptr3-Ssy5 (SPS) amino acid sensing pathway, can detect 19 common L-amino acids with different potencies and affinities based on the physiochemical structure of amino acids. Substituents around alpha carbon are critical for amino acid sensing by Ssy1. Furthermore, a high concentration of cysteine is toxic to cells. Inactivation of SPS signaling confers resistance to …

Preservation Of Biosignature Molecules In Potential Sample Return Container Of The Mars 2020 Mission, Kimberly E. Lykens, Fei Chen Ph.D

STAR Program Research Presentations

Preservation of Biosignature Molecules in Potential Sample Return Container of the Mars 2020 Mission

Kimberly Lykens¹ and Fei Chen²

¹Wittenberg University, Springfield, Ohio 45501 ²Jet Propulsion Laboratory, Pasadena, California, 91109

One requirement for sustainable life on terrestrial planets includes the presence of organic polymers, compounds that are essential for major biological functions such as replication and catalysis. An identified goal of the Mars mission in the year 2020 is to implement a sample-return to identify and validate signs of life on Mars through the discovery of biosignature molecules in Martian core samples. Martian core samples recovered …

Cytochrome B5 Reductase Encoded By Cbr1 Is Essential For A Functional Male Gametophyte In Arabidopsis, Laura L. Wayne, James G. Wallis, Rajesh Kumar, Jennifer E. Markham, John Browse

Department of Biochemistry: Faculty Publications

In all eukaryotes, NADH:cytochrome b5 reductase provides electrons, via cytochrome b5, for a range of biochemical reactions in cellular metabolism, including for fatty acid desaturation in the endoplasmic reticulum. Studies in mammals, yeast, and in vitro plant systems have shown that cytochrome b5 can, at least in some circumstances, also accept electrons from NADPH:cytochrome P450 reductase, potentially allowing for redundancy in reductase function. Here, we report characterization of three T-DNA insertional mutants of the gene encoding cytochrome b5 reductase in Arabidopsis thaliana, CBR1. The progeny of plants heterozygous for the cbr1-2 allele segregated 6% homozygous mutants, while cbr1-3 and cbr1-4 …

Influence Of Media Composition On The Growth Rate Of Chlorella Vulgaris And Scenedesmus Acutus Utilized For Co2 Mitigation, Czarena L. Crofcheck, Xinyi E, Aubrey Shea, Michael D. Montross, Mark Crocker, Rodney Andrews

Biosystems and Agricultural Engineering Faculty Publications

Atmospheric carbon dioxide levels have increased since the industrial revolution due to increasing combustion of fossil fuels. One possible CO₂ mitigation strategy is the use of microalgae for mitigation of CO₂. This paper focuses on the influence of media composition on the growth rate of two microalgae strains, Chlorella vulgaris and Scenedesmus actus. A KNO₃ based medium was found to work better for Chlorella, while a urea based equivalent worked better for Scenedesmus. The urea based media investigated here resulted in growth similar to that found with previously reported KNO₃ based media. …

Diversity Of Plant Methionine Sulfoxide Reductases B And Evolution Of A Form Specific For Free Methionine Sulfoxide, Dung Tien Le, Lionel Tarrago, Yasuko Watanabe, Alaattin Kaya, Byung Cheon Lee, Uyen Tran, Rie Nishiyama, Dmitri E. Fomenko, Vadim N. Gladyshev, Lam-Son Phan Tran

Department of Biochemistry: Faculty Publications

Methionine can be reversibly oxidized to methionine sulfoxide (MetO) under physiological conditions. Organisms evolved two distinct methionine sulfoxide reductase families (MSRA & MSRB) to repair oxidized methionine residues. We found that 5 MSRB genes exist in the soybean genome, including GmMSRB1 and two segmentally duplicated gene pairs (GmMSRB2 and GmMSRB5, GmMSRB3 and GmMSRB4). GmMSRB2 and GmMSRB4 proteins showed MSRB activity toward protein-based MetO with either DTT or thioredoxin (TRX) as reductants, whereas GmMSRB1 was active only with DTT. GmMSRB2 had a typical MSRB mechanism with Cys121 and Cys 68 as catalytic and resolving residues, respectively. Surprisingly, this enzyme also possessed …

Validation Of Antibodies Used To Study Hypoxia Inducible Factors In Two Species Of Fundulus, Jenna D. Hill

LSU New Orleans Theses and Dissertations

Hypoxia inducible factors (HIFs) are transcription factors and the master regulators of oxygen-dependent gene expression in animals. The focus of this thesis is the distribution of HIF protein in tissues of the fish Fundulus heteroclitus and F. grandis, two widespread species that occur in naturally hypoxic waters. Polyclonal antibodies against HIF-1α, HIF-2α, and HIF-3α were tested on proteins made in vitro and on extracts made from several tissues of normoxic and hypoxic fish. Antibodies against HIF-1α and 3α bound specifically to full length protein made in vitro, and produced bands on western blots of nuclear extracts of near …

Regulation Of The Target Of Rapamycin Signaling Pathway In Saccharomyces Cerevisiae, Tammy Pracheil

LSU New Orleans Theses and Dissertations

An integrative, biochemical, genetic, and molecular biology approach utilizing gene manipulation, gene knock outs, plasmid based protein expression, and in vivo protein localization of fluorescence tagged proteins was employed to determine the function of an essential protein, Lst8, in TORC1 and TORC2 signaling and a previously uncharacterized complex, the Far3-7-8-9-10-11 complex (Far complex) in the budding yeast, Saccharomyces cerevisiae. Mutations in SAC7 and FAR11 suppressed lethality of both lst8 and tor2-21 mutations but not TORC1 inactivation, suggesting that the essential function of Lst8 is linked only to TORC2.

Far11, a component of a six-member complex, was found to interact …