Biochemistry Commons^™

Chemoenzymatic Studies To Enhance The Chemical Space Of Natural Products, Jhong-Min Chen

Theses and Dissertations--Pharmacy

Natural products provide some of the most potent anticancer agents and offer a template for new drug design or improvement with the advantage of an enormous chemical space. The overall goal of this thesis research is to enhance the chemical space of two natural products in order to generate novel drugs with better in vivo bioactivities than the original natural products.

Polycarcin V (PV) is a gilvocarcin-type antitumor agent with similar structure and comparable bioactivity with the principle compound of this group, gilvocarcin V (GV). Modest modifications of the polyketide-derived tetracyclic core of GV had been accomplished, but the most …

Imber- Research For Marine Sustainability: Synthesis And The Way Forward, Eileen Hofmann, Alida Bundy, Ken Drinkwater, Alberto R. Piola, Bernard Avril, Carol Robinson

CCPO Publications

The Integrated Marine Biogeochemistry and Ecosystem Research (IMBER) project aims at developing a comprehensive understanding of and accurate predictive capacity of ocean responses to accelerating global change and the consequent effects on the Earth system and human society. Understanding the changing ecology and biogeochemistry of marine ecosystems and their sensitivity and resilience to multiple drivers, pressures and stressors is critical to developing responses that will help reduce the vulnerability of marine-dependent human communities. This overview of the IMBER project provides a synthesis of project achievements and highlights the value of collaborative, interdisciplinary, integrated research approaches as developed and implemented through …

Sharp Rna Recognition Motif Optimizations, Extensions, And Mutations For Use In 2d And 3d Nmr Experiments, Shaun M. Christie

Williams Honors College, Honors Research Projects

SMRT/HDAC Associated Repressor Protein interacts with the long noncoding RNA, produced by SRA, by binding at the RRMs. Three projects were done to prepare the truncated proteins for use in 2D and 3D NMR experiments. The first focuses on RRM 3 and its optimization during the purification process. The second focuses on RRM 2-4, which was found to be missing two alpha helices that may be important for protein stability. These helices can also interact with RRM 3 as well due to the tight association of RRMs 3 and 4. The two step PCR extension of RRM 2-4 was assumed …

An Nmr-Guided Approach To The Isolation Of Secondary Metabolites From Nrps And Pks Gene Clusters, Connor Craig

Honors Theses

Secondary metabolites continue to offer a promising source of novel chemical entities with therapeutic bioactivities. Our research aims to isolate secondary metabolites produced by non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) constructs. A metagenomic library was created from the marine bacterium Pseudoalteromonas sp., which was isolated from the Chesapeake Bay sponge Halichondria bowerbanki. Using a functional screen for PPTase activity we were able to identify colonies that contained NRPS and PKS gene clusters. Cultures of this microbe have been produced and analyzed using a combination of chromatography and 1H, 13C, HMBC, HSQC, COSY, AND NOESY NMR spectra. …

Development Of A Genetic Over-Expression System For The Freshwater Sponge Ephydatia Muelleri, Joe Walsh

Honors Theses

The aim of this work is to develop an over-expression system for Ephydatia muelleri. Preliminary work by Klaske Schippers (Schippers, 2013) showed sponges were induced to transcribe reporter genes driven by the widely used cytomegalovirus promoter (CMV). Building off Schippers work, newly identified native Ephydatia promoters were identified from the genome and verified to drive expression ofreporter genes. Validated over-expression vectors and transfection technology will allow researchers around the world who are working with this organism to ask questions about cell specification, gene regulation and animal development.

Sestrin2, A Regulator Of Thermogenesis And Mitohormesis In Brown Adipose Tissue, Seung-Hyun Ro, Ian Semple, Allison Ho, Hwan-Woo Park, Jun Hee Lee

Department of Biochemistry: Faculty Publications

Sestrin2 is a stress-inducible protein that functions as an antioxidant and inhibitor of mTOR complex 1. In a recent study, we found that Sestrin2 overexpression in brown adipocytes interfered with normal metabolism by reducing mitochondrial respiration through the suppression of uncoupling protein 1 (UCP1) expression. The metabolic effects of Sestrin2 in brown adipocytes were dependent on its antioxidant activity, and chemical antioxidants produced similar effects in inhibiting UCP1-dependent thermogenesis. These observations suggest that low levels of reactive oxygen species (ROS) in brown adipocytes can actually be beneficial and necessary for proper metabolic homeostasis. In addition, considering that Sestrins are ROS …

On The Protective Properties Of Glycine Based Osmolytes In A Thiol Reducing Environment, John Michael Hausman

Dissertations, Master's Theses and Master's Reports - Open

The protective properties of osmolytes have been studied intently for decades. Originally used to aid in the crystallization of proteins in x-ray diffraction studies, these cosolvents have been shown to reverse protein denaturation and aggregation. Osmolytes aid extremophiles in surviving harsh environments by preferentially excluding themselves from the surface of the protein, thus directing water molecules to the protein’s surface. These osmolytes are naturally found in many health foods and also in many daily use products such as shampoo. Due to their osmoprotective effect their use in everyday consumer product is increasing. Consumers also supplement their diets with thiol-based antioxidants …

The Optimization Of Mitoneet Synthesis, Purification And Mutation Stability In The [2fe-2s] Cluster, Alexandria Defabio

Williams Honors College, Honors Research Projects

MitoNEET, a mitochondrial membrane protein with an unsymmetrical Fe-S cluster coordination, was compared to similar proteins within the Rieske and Ferredoxin families. Point mutations were introduced via site-directed mutagenesis to induce stability, greater retention of the cluster, and to uncover structural limitations of the protein. Mutations were made within the cluster coordination and within the hydrophobic core of the protein. Some of the cluster mutations displayed a natural affinity for nickel during immobilized metal-ion affinity chromatography. It is possible that these mutations aid in the formation of a chelate when exposed to nickel. Spectroscopic monitoring of changes in cluster stability …

Evaluation Of Small Molecules On The Excitatory Amino Acid Transporter 3 (Eaat3) For The Treatment Of Ocd, Emmanuel Zubia

Open Access Theses & Dissertations

We seek to discover new drug candidates for the excitatory amino acid transporter 3 (EAAT3) for the treatment of Obsessive Compulsive Disorder (OCD). We hypothesize that decreasing the glutamatergic transmission via EAAT3 intervention will create an amelioration of the symptoms of OCD. For this we have prepared in-silico binding calculations to identify a better compound that can stimulate EAAT3 and decrease glutamatergic transmission. This research will help us further understand and elucidate the role of glutamate in the neural mechanisms of this illness, which to date remain inconclusive. Those areas of study are significant because, until now, there is no …

Membrane Protein Spatial Organization In Mdck Cell Cultures, Rachel E. Neugebauer

Williams Honors College, Honors Research Projects

Studies on the organization of membrane proteins such as GPCRs in live cells help to draw important conclusions regarding cellular communication, and have become a significant topic in drug therapy development. Observing proteins in MDCK cells, which are able to form three-dimensional cultures, provides an intermediate step to better understanding the organization and behavior of these proteins in a tissue-like culture. In this study, fluorescent stains were used on fixed cells to show the morphological differences between MDCK cells in monolayer cultures and three-dimensional cultures, which form hollow, spherical cysts of tightly packed cells. Monolayer cells were transfected to view …

Evaluation Of The Signature Molecular Descriptor With Blosum62 And An All-Atom Description For Use In Sequence Alignment Of Proteins, Lindsay M. Aichinger

Williams Honors College, Honors Research Projects

This Honors Project focused on a few aspects of this topic. The second is comparing the molecular signature kernels to three of the BLOSUM matrices (30, 62, and 90) to test the accuracy of the mathematical model. The kernel matrix was manipulated in order to improve the relationship by focusing on side groups and also by changing how the structure was represented in the matrix by increasing the initial height distance from the central atom (Height 1 and Height 2 included).

There were multiple design constraints for this project. The first was the comparison with the BLOSUM matrices (30, 62, …

Analyzing The Interactions Of Kdm5/Lid And Sin3 In Drosophila Melanogaster, Ambikai Gajan

Wayne State University Dissertations

SIN3, the scaffold protein of a histone modifying complex is conserved from yeast to mammals. Drosophila SIN3 associates with both a histone deactylase RPD3 and a histone demethylase dKDM5/LID. Immunopurification of dKDM5/LID verifies a previously observed interaction with SIN3 and RPD3. Furthermore, deficiency of dKDM5/LID phenocopies deficiency of SIN3 in many cellular and developmental processes. Knockdown of both Sin3A and lid hinder cell proliferation in Drosophila cultured cells and developing flies. Knockdown of these genes also results in a curved wing phenotype implicating a role in wing development. Analysis of underlying gene expression changes upon decreased expression of SIN3, dKDM5/LID …

Inhibition Of Toxic Iapp Amyloid By Extracts Of Common Fruits, David A. Moffet, Pei-Yu Kao, Evangeline Green, Catalina Pereirab, Shauna Ekimura, Dennis Juarez, Travis Whyte, Taylor Arhar, Bianca Malaspina, Luiza A. Nogaj

Chemistry and Biochemistry Faculty Works

The aggregation of the 37-amino acid polypeptide islet amyloid polypeptide (IAPP, amylin), as either insoluble amyloid or as small oligomers, appears to play a direct role in the death of pancreatic β-islet cells in type 2 diabetes. It is believed that inhibiting the aggregation of IAPP may slow down, if not prevent entirely, the progression of this disease. Extracts of thirteen different common fruits were analyzed for their ability to prevent the aggregation of amyloidogenic IAPP. Thioflavin T binding, immuno-detection and circular dichroism assays were performed to test the in vitro inhibitory potential of each extract. Atomic force microscopy was …

Dynamic Motions Of The Hiv-1 Frameshift Site Rna, Kathryn Mouzakis

Chemistry and Biochemistry Faculty Works

The HIV-1 frameshift site (FS) plays a critical role in viral replication. During translation, the HIV-1 FS transitions from a 3-helix to a 2-helix junction RNA secondary structure. The 2-helix junction structure contains a GGA bulge, and purine-rich bulges are common motifs in RNA secondary structure. Here, we investigate the dynamics of the HIV-1 FS 2-helix junction RNA. Interhelical motions were studied under different ionic conditions using NMR order tensor analysis of residual dipolar couplings. In 150 mM potassium, the RNA adopts a 43°(±4°) interhelical bend angle (β) and displays large amplitude, anisotropic interhelical motions characterized by a 0.52(±0.04) internal …

Global Shape Mimicry Of Trna Within A Viral Internal Ribosome Entry Site Mediates Translational Reading Frame Selection, Kathryn Mouzakis

Chemistry and Biochemistry Faculty Works

The dicistrovirus intergenic region internal ribosome entry site (IRES) adopts a triple-pseudoknotted RNA structure and occupies the core ribosomal E, P, and A sites to directly recruit the ribosome and initiate translation at a non-AUG codon. A subset of dicistrovirus IRESs directs translation in the 0 and +1 frames to produce the viral structural proteins and a +1 overlapping open reading frame called ORFx, respectively. Here we show that specific mutations of two unpaired adenosines located at the core of the threehelical junction of the honey bee dicistrovirus Israeli acute paralysis virus (IAPV) IRES PKI domain can uncouple 0 and …

Human Anatomy And Physiology Preparatory Course (1st Edition), Carlos Liachovitzky

Open Educational Resources

The overall purpose of this preparatory course textbook is to help students familiarize with some terms and some basic concepts they will find later in the Human Anatomy and Physiology I course.

The organization and functioning of the human organism generally is discussed in terms of different levels of increasing complexity, from the smallest building blocks to the entire body. This Anatomy and Physiology preparatory course covers the foundations on the chemical level, and a basic introduction to cellular level, organ level, and organ system levels. There is also an introduction to homeostasis at the beginning.

Characterization Of The Nodamura Virus Rna Dependent Rna Polymerase And Formation Of Rna Replication Complexes In Mammalian Cells, Vincent Ulysses Gant

Open Access Theses & Dissertations

Positive-strand RNA viruses amplify their genomes in membrane-bound structures associated with intracellular membranes and organelles called replication complexes (RCs). Here, we begin to elucidate mechanisms of Nodamura virus (NoV; family Nodaviridae) RC assembly. The literature reports that NoV-infected muscle tissue exhibits mitochondrial aggregation and rearrangement of mitochondrial structure, leading to disorganization of the muscle fibrils. However, the molecular basis for this pathogenesis and the role of mitochondria in NoV infection remained unclear until now. We tested the hypoThesis that NoV establishes RCs in association with mitochondria in cultured mammalian cells at physiological temperature. We used immunofluorescence confocal microscopy and biochemical …

Adp-Ribosylation Factor 6 (Arf6) Regulates Integrin Αiibβ3 Trafficking, Platelet Spreading, And Clot Retraction, Yunjie Huang

Theses and Dissertations--Molecular and Cellular Biochemistry

Endocytic trafficking of platelet surface receptors plays a role in the accumulation of granule cargo (i.e. fibrinogen and VEGF) and thus could contribute to hemostasis, angiogenesis, or inflammation. However, the mechanisms of platelet endocytosis are poorly understood. The small GTP-binding protein, ADP-ribosylation factor 6 (Arf6), regulates integrin trafficking in nucleated cells; therefore, we posited that Arf6 functions similarly in platelets. To address this, we generated platelet-specific, Arf6 knockout mice. Arf6^-/- platelets had a storage defect for fibrinogen but not other cargo, implying Arf6’s role in integrin αIIbβ3 trafficking. Additionally, platelets from Arf6^-/- mice injected with biotinylated-fibrinogen, showed …

The Role Of Phlpp In Pancreatic Cancer, Alena J. Smith

Theses and Dissertations--Molecular and Cellular Biochemistry

Medicine has come a long way in recent years with reliable treatments for many cancers. Pancreatic ductal adenocarcinoma (PDAC) has very few treatment options available. PDAC has a dismal 5 year survival rate of 4% and a median survival span of 6 months from point of diagnosis; with a high rate of chemotherapy and radiation resistance. A better understanding of the molecular events leading to cancer progression is needed in order to improve the treatment and prognosis of PDAC patients. We begin to elucidate the functional importance of PHLPP on suppressing progression and metastasis of PDAC. PHLPP belongs to a …

Regional-Scale Simulations Of Fungal Spore Aerosols Using An Emission Parameterization Adapted To Local Measurements Of Fluorescent Biological Aerosol Particles, Matthias Hummel, Corinna Hoose, M. Gallagher, D. A. Healy, J. Alex Huffman, D. O'Connor, U. Pöschl, C. Pöhlker, N. H. Robinson, M. Schnaiter, J. R. Sodeau, M. Stengel, E. Toprak, H. Vogel

Chemistry and Biochemistry: Faculty Scholarship

Fungal spores as a prominent type of primary biological aerosol particles (PBAP) have been incorporated into the COSMO-ART (Consortium for Small-scale Modelling- Aerosols and Reactive Trace gases) regional atmospheric model. Two literature-based emission rates for fungal spores derived from fungal spore colony counts and chemical tracer measurements were used as a parameterization baseline for this study. A third, new emission parameterization for fluorescent biological aerosol particles (FBAP) was adapted to field measurements from four locations across Europe. FBAP concentrations can be regarded as a lower estimate of total PBAP concentrations. Size distributions of FBAP often show a distinct mode at …

Fluorescence Imaging Of Analyte Profiles In An Inductively Coupled Plasma With Laser Ablation As A Sample Introduction Source, Lance Moses

Theses and Dissertations

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has risen to among the top tier techniques for the direct analysis of solid samples. However, significant problems remain that must be solved to achieve the full analytical potential of LA-ICP-MS. Inefficient conversion of aerosol to ions within the ICP or transmission through the MS interface may decrease precision, sensitivity, and/or accuracy. Although fundamental mechanisms that govern ion production and transmission have been studied extensively in solution-nebulization (SN) ICP-MS instruments, significant gaps in our understanding remain. Furthermore, it is unclear to what extent differences between the aerosols generated during SN and LA …

Physiological Function Of Fus: An Rna Binding Protein In Motor Neuron Disease, Liuqing Yang

Theses and Dissertations--Molecular and Cellular Biochemistry

FUS is an RNA binding protein implicated in the motor neuron disease— amyotrophic lateral sclerosis (ALS, also called Lou Gehrig’s disease). ALS is a fatal neurodegenerative disease characterized by progressive motor neuron death. Mutations in the FUS gene cause about 4% of familial ALS (FUS ALS). Mutated FUS protein mislocalizes from the motor neuron nucleus to the cytoplasm and forms inclusions in the cytoplasm. It is unclear how FUS mislocalization induces motor neuron dysfunction and degeneration. This dissertation research was designed to investigate the physiological functions of FUS in the nucleus, with a purpose to shed light on the pathogenesis …

Physical Interactions Between Neuropilin And Vegfrs, Integrins In Regulating Endothelial Cell Functions, Xiaobo Li

Theses and Dissertations--Molecular and Cellular Biochemistry

The neuropilin (Nrp) family consists of multifunctional cell surface receptors with critical roles in a number of different cell and tissue types. A core aspect of Nrp function is ligand-dependent cellular adhesion and migration, where it controls the multistep process of cellular motility through integration of ligand binding, receptor coupling and signaling via the coordinated action of its extracellular and intracellular domains. While Nrp regulates cellular adhesion and motility in the cardiovascular and nervous systems under physiological conditions, the emerging pathological role of Nrp in tumor cell migration and metastasis has been identified and provides motivation for continued efforts toward …

How Do Free-Living, Lerp-Forming, And Gall-Inducing Aphalaridae (Hemiptera: Psylloidea) Affect The Nutritional Quality Of Eucalyptus Leaves?, Anamika Sharma, James Allen, Soundararajan Madhavan, Anantanarayanan Raman, Gary S. Taylor, Murray J. Fletcher

Department of Biochemistry: Faculty Publications

About 380 described species of Psylloidea occur on Eucalyptus in Australia. These show differences in diversification, feeding behavior, and apparent patterns of development. We analyzed the quality of nutrients used by three species of Aphalaridae belonging to different feeding guilds on three species of Eucalyptus. We evaluated the quantity and quality of total nonstructural carbohydrates (TNC), δ¹³C and δ¹⁵N isotope ratios, amino acids, and fatty acids. In general, TNC levels were greater in infested leaves than in uninfested leaves of the three species of Eucalyptus. TNC levels in the leaves of E. macrorhyncha F. …

Overaccumulation Of Γ-Glutamylcysteine In A Jasmonate-Hypersensitive Arabidopsis Mutant Causes Jasmonate-Dependent Growth Inhibition, Hsin-Ho Wei, Martha Rowe, Jean-Jack M. Riethoven, Ryan Grove, Jiri Adamec, Yusuke Jikumaru, Paul E. Staswick

Department of Biochemistry: Faculty Publications

Glutathione (GSH) is essential for many aspects of plant biology and is associated with jasmonate signaling in stress responses. We characterized an Arabidopsis (Arabidopsis thaliana) jasmonate-hypersensitive mutant (jah2) with seedling root growth 100-fold more sensitive to inhibition by the hormone jasmonyl-isoleucine than the wild type. Genetic mapping and genome sequencing determined that the mutation is in intron 6 of GLUTATHIONE SYNTHETASE2, encoding the enzyme that converts γ-glutamylcysteine (γ-EC) to GSH. The level of GSH in jah2 was 71% of the wild type, while the phytoalexin-deficient2-1 (pad2-1) mutant, defective in GSH1 and …

First Evidence For Substrate Channeling Between Proline Catabolic Enzymes A Validation Of Domain Fusion Analysis For Predicting Protein-Protein Interactions, Nikhilesh Sanyal, Benjamin W. Arentson, John J. Tanner, Donald F. Becker

Department of Biochemistry: Faculty Publications

Background: PRODH and P5CDH from Thermus thermophilus are monofunctional enzymes in proline catabolism.

Results: Steady-state kinetics and intermediate trapping data show the PRODH and P5CDH reactions are coupled by a channeling step.

Conclusion: Substrate channeling in monofunctional enzymes is achieved via weak interactions.

Significance: Evidence for substrate channeling between monofunctional proline catabolic enzymes is shown and confirms the Rosetta Stone hypothesis.

Drosophila Muller F Elements Maintain A Distinct Set Of Genomic Properties Over 40 Million Years Of Evolution, Wilson Leung, Cheryl Bailey, Participating Students And Faculty Of The Genomics Education Partnership

Department of Biochemistry: Faculty Publications

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25–50%) than euchromatic reference regions (3–11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11–27%). F …

Overexpression Of Alpha-Synuclein At Non-Toxic Levels Increases Dopaminergic Cell Death Induced By Copper Exposure Via Modulation Of Protein Degradation Pathways, Annadurai Anandhan, Humberto Rodriguez-Rocha, Iryna Bohovych, Amy M. Griggs, Laura Zavala-Flores, Elsa M. Reyes-Reyes, Javier Seravalli, Lia A. Stanciu, Jaekwon Lee, Jean-Christophe Rochet, Oleh Khalimonchuk, Rodrigo Franco

Department of Biochemistry: Faculty Publications

Gene multiplications or point mutations in alpha (α)-synuclein are associated with familial and sporadic Parkinson’s disease (PD). An increase in copper (Cu) levels has been reported in the cerebrospinal fluid and blood of PD patients, while occupational exposure to Cu has been suggested to augment the risk to develop PD. We aimed to elucidate the mechanisms by which α-synuclein and Cu regulate dopaminergic cell death. Short-term overexpression of WT or A53T α-synuclein had no toxic effect in human dopaminergic cells and primary midbrain cultures, but it exerted a synergistic effect on Cu-induced cell death. Cell death induced by Cu was …

Triacylglycerol Synthesis During Nitrogen Stress Involves The Prokaryotic Lipid Synthesis Pathway And Acyl Chain Remodeling In The Microalgae Coccomyxa Subellipsoidea, James W. Allen, Concetta C. Dirusso, Paul N. Black

Department of Biochemistry: Faculty Publications

Triglyceride (TAG) synthesis during nitrogen starvation and recovery was addressed using Coccomyxa subellipsoidea by analyzing acylchain composition and redistribution using a bioreactor-controlled time course. Galactolipids, phospholipids and TAGs were profiled using liquid chromatography tandem mass spectroscopy (LC–MS/MS). TAG levels increased linearly through 10 days of N starvation to a final concentration of 12.6% dry weight (DW), while chloroplast membrane lipids decreased from 5% to 1.5% DW. The relative quantities of TAG molecular species, differing in acyl chain length and glycerol backbone position, remained unchanged from 3 to 10 days of N starvation. Six TAG species comprised approximately half the TAG …

Metalloprotease Oma1 Fine-Tunes Mitochondrial Bioenergetic Function And Respiratory Supercomplex Stability, Iryna Bohovych, Mario R. Fernandez, Jennifer J. Rahn, Krista D. Stackley, Jennifer E. Bestman, Annadurai Anandhan, Rodrigo Franco, Steven M. Claypool, Robert E. Lewis, Sherine S.L. Chan, Oleh Khalimonchuk

Department of Biochemistry: Faculty Publications

Mitochondria are involved in key cellular functions including energy production, metabolic homeostasis, and apoptosis. Normal mitochondrial function is preserved by several interrelated mechanisms. One mechanism – intramitochondrial quality control (IMQC) – is represented by conserved proteases distributed across mitochondrial compartments. Many aspects and physiological roles of IMQC components remain unclear. Here, we show that the IMQC protease Oma₁ is required for the stability of the respiratory supercomplexes and thus balanced and tunable bioenergetic function. Loss of Oma1 activity leads to a specific destabilization of respiratory supercomplexes and consequently to unbalanced respiration and progressive respiratory decline in yeast. Similarly, experiments …