Biochemistry, Biophysics, and Structural Biology Commons^™

The Crosstalk Between Human Fatty Acid Transport Protein 1 And Fatty Acid Transport Protein 4, Zhe Yuan

Department of Biochemistry: Dissertations, Theses, and Student Research

Fatty acid transport proteins (FATPs) provide pivotal roles in fatty acid transport and activation and thus are crucial for overall fatty acid homeostasis. Peroxisome Proliferators Activated Receptors (PPARs) are important transcription factors, which control many genes that govern lipid metabolism.

Using 293 T-REx cell lines that stably express FATP1 or FATP4 from a tetracycline-inducible promoter, this work evaluated gene expression of key genes involved in fatty acid metabolism using QPCR, protein expression of FATP1 and FATP4 using Western blots, and fatty acid transport to address the roles of these two FATP isoforms in fatty acid homeostasis. This work found that …

Electrosensory Ampullary Organs Are Derived From Lateral Line Placodes In Bony Fishes, Melissa S. Modrell, William E. Benis, R. Glenn Northcutt, Marcus C. Davis, Clare V.H. Baker

Faculty Articles

Electroreception is an ancient subdivision of the lateral line sensory system, found in all major vertebrate groups (though lost in frogs, amniotes and most ray-finned fishes). Electroreception is mediated by 'hair cells' in ampullary organs, distributed in fields flanking lines of mechanosensory hair cell-containing neuromasts that detect local water movement. Neuromasts, and afferent neurons for both neuromasts and ampullary organs, develop from lateral line placodes. Although ampullary organs in the axolotl (a representative of the lobe-finned clade of bony fishes) are lateral line placode-derived, non-placodal origins have been proposed for electroreceptors in other taxa. Here we show morphological and molecular …

Total Synthesis Of 4'-Ester Resveratrol Analogs And 8.9-Amido Geldanamycin Analog And Toward The Total Synthesis Of (-)-Englerin A, Yong Wang

Theses and Dissertations

Total Synthesis of 4'-ester Resveratrol Analogs and 8, 9-amido Geldanamycin Analog and toward the Total Synthesis of (-)-Englerin A Yong Wang Department of Chemistry and Biochemistry, BYU Doctor of Philosophy The phytoalexin resveratrol and its 4'-ester analogs have been prepared with a decarbonylative Heck reaction. The deprotecting step has been modified and improved to increase yield and avoid chromatography. A set of resveratrol analogs and resveratrol have been tested with melanoma and pancreatic cell assays. The 8, 9-amido Geldanamycin analog has been synthesized with a convergent route, involving 28 simplified steps in its longest linear sequence. Synthetic methodologies, such as …

Splice Variant–Specific Cellular Function Of The Formin Inf2 In Maintenance Of Golgi Architecture, Vinay Ramabhadran, Farida Korobova, Gilbert J. Rahme, Henry N. Higgs

Dartmouth Scholarship

INF2 is a unique formin that can both polymerize and depolymerize actin filaments. Mutations in INF2 cause the kidney disease focal and segmental glomerulosclerosis. INF2 can be expressed as two C-terminal splice variants: CAAX and non-CAAX. The CAAX isoform contains a C-terminal prenyl group and is tightly bound to endoplasmic reticulum (ER). The localization pattern and cellular function of the non-CAAX isoform have not been studied. Here we find that the two isoforms are expressed in a cell type-dependent manner, with CAAX predominant in 3T3 fibroblasts and non-CAAX predominant in U2OS, HeLa, and Jurkat cells. Although INF2-CAAX is ER localized …

Dopamine Regulation Of Cone-Cone Gap Junctions In Ground Squirrel Retina, Xiaoyu Li '12, Steven Devries

Student Publications & Research

Cone photoreceptors are electrically coupled such that when the electrical potential in one cell changes, it also changes in adjacent, coupled cells. Phosphorylation of the cone gap junction protein, connexin 36 (Cx36), determines whether a coupling channel is “open” or “closed”. Cx36- containing gap junctions are phosphorylated in the open state and dephosphorylated in the closed state. Cone-cone gap junction modulation has not been extensively studied, however, the presence of rod-cone gap junction modulation raises the possibility that the same is true for cone pairs. Pieces of thirteen-lined ground squirrel retina were removed from the eye and the tissue was …

The Rice Miniature Inverted Repeat Transposable Element Mping Is An Effective Insertional Mutagen In Soybean, C. Nathan Hancock, Feng Zhang, Kristen Floyd, Aaron O. Richardson, Peter Lafayette, Donna Tucker, Susan R. Wessler, Wayne A. Parrott

Faculty Publications

Insertional mutagenesis of legume genomes such as soybean (Glycine max) should aid in identifying genes responsible for key traits such as nitrogen fixation and seed quality. The relatively low throughput of soybean transformation necessitates the use of a transposon-tagging strategy where a single transformation event will produce many mutations over a number of generations. However, existing transposon-tagging tools being used in legumes are of limited utility because of restricted transposition (Ac/Ds: soybean) or the requirement for tissue culture activation (Tnt1: Medicago truncatula). A recently discovered transposable element from rice (Oryza …

Activation Of Mmp-9 By Human Lung Epithelial Cells In Response To The Cystic Fibrosis-Associated Pathogen Burkholderia Cenocepacia Reduced Wound Healing In Vitro, Ciara Wright ], Ruth Pilkington, Máire Callaghan, Siobhan Mcclean

Articles

Burkholderia cepacia complex is a group of bacterial pathogens that cause opportunistic infections in cystic fibrosis (CF). The most virulent of these is Burkholderia cenocepacia. Matrix metalloproteinases (MMPs) are upregulated in CF patients. The aim of this work was to examine the role of MMPs in the pathogenesis of B. cepacia complex, which has not been explored to date. Real-time PCR analysis showed that B. cenocepacia infection upregulated MMP-2 and MMP-9 genes in the CF lung cell line CFBE41o- within 1 h, whereas MMP-2, -7, and -9 genes were upregulated in the non-CF lung cell line 16HBE14o-. Conditioned media from …

Using Vesicular Stomatitis Virus Glycoprotein To Pseudotype Bacmam Viruses For Enhancement Of Transduction Efficiency, Lydia Sanchez

PCOM Biomedical Studies Student Scholarship

Baculovirus has been a common tool for protein production since the mid 1980s. The virus has an exclusive tropism only capable of infecting the larval stages of insects. Originally, baculovirus recombinant gene expression used Spodoptera frugiperda (Sf9) insect cells as the host cells for protein production. However, in 1995, it was discovered that baculoviruses could be modified to transduce mammalian cells with the insertion of a mammalian promoter and gene of interest. Due to its ease of generation and low biology safety hazards, this technology, known as BacMam (Baculovirus Mammalian) is popular in the research field for protein production and …

The Discovery And Exploration Of A Universal Targeting Mechanism In Eukaryotic Cells, Priyanka Sivadas

Dissertations (1934 -)

A wide range of eukaryotic organisms generate motile cilia and flagella. These slender organelles beat rhythmically to move the surrounding fluid or to propel cells in aqueous environment. Organisms use these powerful yet nimble organelles to forage, evade, adapt and mate. The machinery that drives this tightly controlled movement is the sophisticated microtubule-based axoneme. As it is critical for the survival of individual species, this machinery has largely been preserved to the molecular level throughout evolution. Proteomic studies have shown that most proteins in this biological machine consist of molecular modules commonly used in the cell body. But the usage …

Iron Insufficiency Compromises Motor Neurons And Their Mitochondrial Function In Irp2-Null Mice, Suh Young Jeong, Daniel R. Crooks, Hayden Wilson-Ollivierre, Manik C. Ghosh, Rachid Sougrat, Jaekwon Lee, Sharon Cooperman, James B. Mitchell, Carole Beaumont, Tracey A. Rouault

Department of Biochemistry: Faculty Publications

Genetic ablation of Iron Regulatory Protein 2 (Irp2, Ireb2), which post-transcriptionally regulates iron metabolism genes, causes a gait disorder in mice that progresses to hind-limb paralysis. Here we have demonstrated that misregulation of iron metabolism from loss of Irp2 causes lower motor neuronal degeneration with significant spinal cord axonopathy. Mitochondria in the lumbar spinal cord showed significantly decreased Complex I and II activities, and abnormal morphology. Lower motor neurons appeared to be the most adversely affected neurons, and we show that functional iron starvation due to misregulation of iron import and storage proteins, including transferrin receptor 1 …

Akap2 Anchors Pka With Aquaporin-0 To Support Ocular Lens Transparency, Matthew G. Gold, Steve Reichow, Susan E. O'Neill, Chad R. Weisbrod, Lorene K. Langeberg, James E. Bruce, Tamir Gonen, John D. Scott

Chemistry Faculty Publications and Presentations

A decline in ocular lens transparency known as cataract afflicts 90% of individuals by the age 70. Chronic deterioration of lens tissue occurs as a pathophysiological consequence of defective water and nutrient circulation through channel and transporter proteins. A key component is the aquaporin-0 (AQP0) water channel whose permeability is tightly regulated in healthy lenses. Using a variety of cellular and biochemical approaches we have discovered that products of the A-kinase anchoring protein 2 gene (AKAP2/AKAP-KL) form a stable complex with AQP0 to sequester protein kinase A (PKA) with the channel. This permits PKA phosphorylation of serine 235 within a …

Advances In Structural And Functional Analysis Of Membrane Proteins By Electron Crystallography, Goragot Wisedchaisri, Steve Reichow, Tamir Gonen

Chemistry Faculty Publications and Presentations

Electron crystallography is a powerful technique for the study of membrane protein structure and function in the lipid environment. When well-ordered two-dimensional crystals are obtained the structure of both protein and lipid can be determined and lipid-protein interactions analyzed. Protons and ionic charges can be visualized by electron crystallography and the protein of interest can be captured for structural analysis in a variety of physiologically distinct states. This review highlights the strengths of electron crystallography and the momentum that is building up in automation and the development of high throughput tools and methods for structural and functional analysis of membrane …

Identification And Characterization Of A Novel Thermohalophilic Esterase From The Red Sea: Atlantis Ii Brine Pool, Yasmine Mustafa Mohamed

Theses and Dissertations

Industrial (white) biotechnology poses an increasing demand for novel biocatalysts that are robust under a wide range of conditions. Traditionally, biocatalysts were isolated from cultured isolates, however, less than 1% of microorganisms are culturable. Therefore, it became evident that the unculturable majority holds a great potential for the discovery of novel biocatalysts. Metagenomics is an invaluable tool for accessing the genomes of the uncultured majority and has led to the isolation of a large number of biocatalysts from various environments. Extreme environments such as hydrothermal vents, brine pools and glaciers, are an attractive source for biocatalysts. Biocatalysts from these environments …

The Pcdp1 Complex Coordinates The Activity Of Dynein Isoforms To Produce Wild-Type Ciliary Motility, Christen G. Dipetrillo, Elizabeth F. Smith

Dartmouth Scholarship

Generating the complex waveforms characteristic of beating cilia requires the coordinated activity of multiple dynein isoforms anchored to the axoneme. We previously identified a complex associated with the C1d projection of the central apparatus that includes primary ciliary dyskinesia protein 1 (Pcdp1). Reduced expression of complex members results in severe motility defects, indicating that C1d is essential for wild-type ciliary beating. To define a mechanism for Pcdp1/C1d regulation of motility, we took a functional and structural approach combined with mutants lacking C1d and distinct subsets of dynein arms. Unlike mutants completely lacking the central apparatus, dynein-driven microtubule sliding velocities are …

Differential Interactions Of The Formins Inf2, Mdia1, And Mdia2 With Microtubules, Jeremie Gaillard, Bvinay Ramabhadran, Emmanuelle Neumanne, Pinar Gurel, Laurent Blanchoin, Marylin Vantard, Henry N. Higgs

Dartmouth Scholarship

A number of cellular processes use both microtubules and actin filaments, but the molecular machinery linking these two cytoskeletal elements remains to be elucidated in detail. Formins are actin-binding proteins that have multiple effects on actin dynamics, and one formin, mDia2, has been shown to bind and stabilize microtubules through its formin homology 2 (FH2) domain. Here we show that three formins, INF2, mDia1, and mDia2, display important differences in their interactions with microtubules and actin. Constructs containing FH1, FH2, and C-terminal domains of all three formins bind microtubules with high affinity (K(d) < 100 nM). However, only mDia2 binds microtubules at 1:1 stoichiometry, with INF2 and mDia1 showing saturating binding at approximately 1:3 (formin dimer:tubulin dimer). INF2-FH1FH2C is a potent microtubule-bundling protein, an effect that results in a large reduction in catastrophe rate. In contrast, neither mDia1 nor mDia2 is a potent microtubule bundler. The C-termini of mDia2 and INF2 have different functions in microtubule interaction, with mDia2's C-terminus required for high-affinity binding and INF2's C-terminus required for bundling. mDia2's C-terminus directly binds microtubules with submicromolar affinity. These formins also differ in their abilities to bind actin and microtubules simultaneously. Microtubules strongly inhibit actin polymerization by mDia2, whereas they moderately inhibit mDia1 and have no effect on INF2. Conversely, actin monomers inhibit microtubule binding/bundling by INF2 but do not affect mDia1 or mDia2. These differences in interactions with microtubules and actin suggest differential function in cellular processes requiring both cytoskeletal elements.

Schrödinger And Nietzsche On Life: The Eternal Recurrence Of The Same, Babette Babich

Working Papers

Schrödinger and Nietzsche on Life: The Eternal Recurrence of the Same

This essay explores Schrödinger’s reflections on measurement, consciousness, and personal identity. Schrödinger’s, What Is Life? is read together with Nietzsche’s own reflections on the same question, in his aphorism What is Life? together with Nietzsche’s teaching of the eternal return of the selfsame. Schrödinger’s own thinking is influenced as is Nietzsche’s by Schopenhauer but Schrödinger also has the Vedic tradition as this influenced Schopenhauer himself in view.

Exploring The Roles Of Nucleobase Desolvation And Shape Complementarity During The Misreplication Of O6-Methylguanine, Delia Chavarria, Andrea Ramos Serrano, Ichiro Hirao, Anthony J. Berdis

Chemistry Faculty Publications

O6-methylguanine is a miscoding DNA lesion arising from the alkylation of guanine. This report uses the bacteriophage T4 DNA polymerase as a model to probe the roles hydrogen-bonding interactions, shape/size, and nucleobase desolvation during the replication of this miscoding lesion. This was accomplished by using transient kinetic techniques to monitor the kinetic parameters for incorporating and extending natural and non-natural nucleotides. In general, the efficiency of nucleotide incorporation does not depend on the hydrogen-bonding potential of the incoming nucleotide. Instead, nucleobase hydrophobicity and shape complementarity appear to be the preeminent factors controlling nucleotide incorporation. In addition, shape complementarity plays a …

Rna Oxidation Adducts 8-Ohg And 8-Oha Change With Aβ42 Levels In Late-Stage Alzheimer's Disease, Adam M. Weidner, Melissa A. Bradley, Tina L. Beckett, Dana M. Niedowicz, Amy L.S. Dowling, Sergey V. Matveev, Harry Levine, Mark A. Lovell, M. Paul Murphy

Sanders-Brown Center on Aging Faculty Publications

While research supports amyloid-β (Aβ) as the etiologic agent of Alzheimer's disease (AD), the mechanism of action remains unclear. Evidence indicates that adducts of RNA caused by oxidation also represent an early phenomenon in AD. It is currently unknown what type of influence these two observations have on each other, if any. We quantified five RNA adducts by gas chromatography/mass spectroscopy across five brain regions from AD cases and age-matched controls. We then used a reductive directed analysis to compare the RNA adducts to common indices of AD neuropathology and various pools of Aβ. Using data from four disease-affected brain …

Beta-Lysine Discrimination By Lysyl-Trna Synthetase, Marla S. Gilreath, Hervé Roy, Tammy J. Bullwinkle, Assaf Katz, Michael Ibba, William Wiley Navarre

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Elongation factor P is modified with (R)‐β‐lysine by the lysyl‐tRNA synthetase (LysRS) paralog PoxA. PoxA specificity is orthogonal to LysRS, despite their high similarity. To investigate α‐ and β‐lysine recognition by LysRS and PoxA, amino acid replacements were made in the LysRS active site guided by the PoxA structure. A233S LysRS behaved as wild type with α‐lysine, while the G469A and A233S/G469A variants decreased stable α‐lysyl‐adenylate formation. A233S LysRS recognized β‐lysine better than wildtype, suggesting a role for this residue in discriminating α‐ and β‐amino acids. Both enantiomers of β‐lysine were substrates for tRNA aminoacylation by LysRS, which, together with …

Follicular Dendritic Cells, Resting Cd4+ T Cells And Human Immunodeficiency Virus Expression, Changna Wang

Theses and Dissertations

Many events associated with Human Immunodeficiency Virus (HIV) infection/replication occur in and around the germinal centers (GCs) of secondary lymphoid tissues where follicular dendritic cells (FDCs) reside, suggesting that this microenvironment may contribute unique signaling that is important to viral progression. My research focused on characterizing signaling, both positive and negative, contributed by FDCs that affects HIV infection and replication. Specifically, I determined if FDC signals could induce the expression of latent HIV in T cells and if so, to characterize the signaling pathways involved. Moreover, I also examined the ability of FDCs to produce inhibitory signals that might block …