Biochemistry Commons^™

The Danger Signal Adenosine Induces Persistence Of Chlamydial Infection Through Stimulation Of A2b Receptors, Matthew A. Pettengill, Verissa W. Lam, David M. Ojcius

All Dugoni School of Dentistry Faculty Articles

Infections with intracellular bacteria such as chlamydiae affect the majority of the world population. Infected tissue inflammation and granuloma formation help contain the short-term expansion of the invading pathogen, leading also to local tissue damage and hypoxia. However, the effects of key aspects of damaged inflamed tissues and hypoxia on continued infection with intracellular bacteria remain unknown. We find that development of Chlamydia trachomatis is reversibly retarded by prolonged exposure of infected cells to extracellular adenosine, a hallmark of hypoxia and advanced inflammation. In epithelial cells, this effect was mediated by the A2b adenosine receptor, unique in the adenosine receptor …

Progress Toward The Total Synthesis Of Lyconadin A, Yu Zhang

Theses and Dissertations

Lyconadin A is a pentacyclic Lycopodium alkaloid isolated from the club moss Lycopodium complanatum with antitumor properties. We have developed a novel 7-exo/6-exo acyl radical cascade cyclization as a method of making the bicyclo[5.4.0]undecane ring system of lyconadin A. The model products are trans-fused ring systems, while a cis-fused ring system is needed in lyconadin A. We have discovered a method to convert the trans-fused model cascade cyclization product into the desired cis isomer. Based on Donohoe's pyridone synthesis, we developed a method for the construction of 5-alkyl and 3,5-dialkyl-6-carbomethoxy-2-pyridones, the former of which …

Overexpression And Partial Purification Of Serine Hydroxymethyltransferase, Kimberly Rose Stevens

Chemistry and Biochemistry

No abstract provided.

Cloning Of "Animal Cryptochrome" Cdna From The Model Organism Chlamydomonas Reinhardtii For Functional Analysis Of Its Protein Product, Shobha Lavanya Silparasetty

Masters Theses & Specialist Projects

reinhardtii, a unicellular green alga, is a model organism to study the circadian clock. Cryptochromes are the blue light photoreceptors that entrain the clock in some organisms. The CPH1 protein of C. reinhardtii resembles the cryptochromes of the plant model Arabidopsis, but whether CPH1 entrains the circadian clock in C. reinhardtii is not yet known. Recent reports have suggested the existence of one more cryptochrome in C. reinhardtii, which resembles the cryptochromes of animals. However, the amino acid sequence of this protein shows even higher sequence similarity with the 6-4 DNA photolyase of Arabidopsis. DNA photolyases …

Pot1 Proteins In Green Algae And Land Plants: Dna-Binding Properties And Evidence Of Co-Evolution With Telomeric Dna, Eugene V. Shakirov, Xiangyu Song, Jessica A. Joseph, Dorothy E. Shippen

Biological Sciences Faculty Research

Telomeric DNA terminates with a single-stranded 3′ G-overhang that in vertebrates and fission yeast is bound by POT1 (Protection Of Telomeres). However, no in vitro telomeric DNA binding is associated with Arabidopsis POT1 paralogs. To further investigate POT1–DNA interaction in plants, we cloned POT1 genes from 11 plant species representing major branches of plant kingdom. Telomeric DNA binding was associated with POT1 proteins from the green alga Ostreococcus lucimarinus and two flowering plants, maize and Asparagus. Site-directed mutagenesis revealed that several residues critical for telomeric DNA recognition in vertebrates are functionally conserved in plant POT1 proteins. However, the plant proteins …

Inactivation Of The Fliy Gene Encoding A Flagellar Motor Switch Protein Attenuates Mobility And Virulence Of Leptospira Interrogans Strain Lai, Sumei Liao, Ai-Hua Sun, David M. Ojcius, Senlin Wu, Jinfang Zhao, Jie Yan

All Dugoni School of Dentistry Faculty Articles

Background: Pathogenic Leptospira species cause leptospirosis, a zoonotic disease of global importance. The spirochete displays active rotative mobility which may contribute to invasion and diffusion of the pathogen in hosts. FliY is a flagellar motor switch protein that controls flagellar motor direction in other microbes, but its role in Leptospira, and paricularly in pathogenicity remains unknown.

Results: A suicide plasmid for the fliY gene of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai that was disrupted by inserting the ampicillin resistance gene (bla) was constructed, and the inactivation of fliY gene in a mutant (fliY-) was confirmed by PCR and …

The Physiological And Biochemical Understanding Of 5’-Amp Induced Deep Hypometabolism, William G. O'Brien

Dissertations and Theses (Open Access)

Our laboratory’s ability to induce deep hypometabolism (DH) via an injection of adenosine monophosphate (5’-AMP) followed by placement in an ambient temperature (Ta) around 15oC currently can last about 3-9 hours. While we have insight into how 5’-AMP induced hypometabolism is initiated, it remains unclear how arousal from hypometabolism is controlled. Other laboratory members have been unable to prolong this process safely and effectively with previous attempts of re-injecting a dose of 5’-AMP upon arousal or by decreasing the Ta. While these methods worked in suppressing arousal, the mortality rate is also increased. To gather a better understanding of the …

Characterization And Functional Regulation Of Bioactive Peptides In Avian Macrophages And Heterophils, Lakshmi Kannan

Graduate Theses and Dissertations

Oligopeptides and low molecular weight polypeptides play central roles as effectors and signal transducers acting as hormones, neurotransmitters, growth factors, toxins, and antimicrobial factors that are important for the survival of the organism. Owing to the ubiquitous involvement of peptides in many key regulatory processes, we have been interested to identify native peptides in different cells and tissues and understand their functions. To conduct our studies, we used avian macrophages and heterophils as models of specialized cells which constitute central components of innate immunity. These studies involved (a) qualitative identification and characterization of the peptides associated with high intensity mass …

Subcutaneous Administration Of Tc007 Reduces Disease Severity In An Animal Model Of Sma, Virginia B. Mattis, Marina Y. Fosso, Cheng-Wei Tom Chang, Christian L. Lorson

Chemistry and Biochemistry Faculty Publications

Background Spinal Muscular Atrophy (SMA) is the leading genetic cause of infantile death. It is caused by the loss of functional Survival Motor Neuron 1 (SMN1). There is a nearly identical copy gene, SMN2, but it is unable to rescue from disease due to an alternative splicing event that excises a necessary exon (exon 7) from the majority of SMN2-derived transcripts. While SMNΔ7 protein has severely reduced functionality, the exon 7 sequences may not be specifically required for all activities. Therefore, aminoglycoside antibiotics previously shown to suppress stop codon recognition and promote translation read-through have been examined to increase the …

Synthesis And Evaluation Of N6,5'-Bis-Ureido-5'-Amino-5'-Deoxyadenosine Derivatives: Novel Nucleosides With Antiproliferative And Protein Kinase Binding Activities, Marcelio Oliveira

Theses and Dissertations

A new series of N6,5'-bis-ureido-5'-amino-5'-deoxyadenosine derivatives was prepared and evaluated for anticancer activities using the NCI 60 panel of human cancers. Certain of the derivatives showed promising activities (low micromolar GI50's) against several of the representative cancers. These included cell lines from the following general cell types in the NCI 60: Leukemia, Breast, Central Nervous System, Non-Small Cell Lung, Ovarian, Prostate, Renal, and Colon cancers. Select compounds were also screened for their affinities for protein kinases. The synthesis of the compounds was straightforward and involved N6 acylation with arylisocyanates, preceded by activation and nucleophilic substitution of the 5'-position to give …

Expression And Function Of Hypoxia Inducible Factor-1 Alpha In Human Melanoma Under Non-Hypoxic Conditions, Caroline N. Mills, Sandeep S. Joshi, Richard M. Niles

Biochemistry and Microbiology

Background

Hypoxia inducible factor-1 alpha (HIF-1α) protein is rapidly degraded under normoxic conditions. When oxygen tensions fall HIF-1α protein stabilizes and transactivates genes involved in adaptation to hypoxic conditions. We have examined the normoxic expression of HIF-1α RNA and protein in normal human melanocytes and a series of human melanoma cell lines isolated from radial growth phase (RGP), vertical growth phase (VGP) and metastatic (MET) melanomas.

Results

HIF-1α mRNA and protein was increased in RGP vs melanocytes, VGP vs RGP and MET vs VGP melanoma cell lines. We also detected expression of a HIF-1α mRNA splice variant that lacks part …

Identification Of Functional Lsrb-Like Autoinducer-2 Receptors, C. S. Pereira, Anna Katherine De Regt , '09, P. H. Brito, Stephen T. Miller, K. B. Xavier

Chemistry & Biochemistry Faculty Works

Although a variety of bacterial species have been reported to use the interspecies communication signal autoinducer-2 (AI-2) to regulate multiple behaviors, the molecular mechanisms of AI-2 recognition and signal transduction remain poorly understood. To date, two types of AI-2 receptors have been identified: LuxP, present in Vibrio spp., and LsrB, first identified in Salmonella enterica serovar Typhimurium. In S. Typhimurium, LsrB is the ligand binding protein of a transport system that enables the internalization of AI-2. Here, using both sequence analysis and structure prediction, we establish a set of criteria for identifying functional AI-2 receptors. We test our predictions experimentally, …

Trnas: Cellular Barcodes For Amino Acids, Ranat Banerjee, Shawn Chen, Kiley Dare, Marla Gilreath, Mette Praetorius-Ibba, Medha Raina, Noah M. Reynolds, Theresa E. Rogers, Hervé Roy, Srujana S. Yadavalli, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

The role of tRNA in translating the genetic code has received considerable attention over the last 50 years, and we now know in great detail how particular amino acids are specifically selected and brought to the ribosome in response to the corresponding mRNA codon. Over the same period, it has also become increasingly clear that the ribosome is not the only destination to which tRNAs deliver amino acids, with processes ranging from lipid modification to antibiotic biosynthesis all using aminoacyl‐tRNAs as substrates. Here we review examples of alternative functions for tRNA beyond translation, which together suggest that the role of …

Yeast Ste23p Shares Functional Similarities With Mammalian Insulin-Degrading Enzymes, Benjamin Alper, Jarrad W. Rowse, Walter K. Schmidt

Chemistry & Physics Faculty Publications

The S. cerevisiae genome encodes two M16A enzymes: Axl1p and Ste23p. Of the two, Ste23p shares significantly higher sequence identity with M16A enzymes from other species, including mammalian insulin-degrading enzymes (IDEs). In this study, recombinant Ste23p and R. norvegicus IDE (RnIDE) were isolated from E. coli, and their enzymatic properties compared. Ste23p was found to cleave established RnIDE substrates including the amyloid-β peptide (Aβ 1-40) and insulin B chain. A novel internally quenched fluorogenic substrate (Abz-SEKKDNYIIKGVnitroY-OH) based on the polypeptide sequence of the yeast P2 a-factor mating propheromone was determined to be a suitable substrate for …

Integral Membrane Proteins Brr6 And Apq12 Link Assembly Of The Nuclear Pore Complex To Lipid Homeostasis In The Endoplasmic Reticulum, Christine A. Hodge, Vineet Choudhary, Michael J. Wolyniak, John J. Scarcelli, Roger Schneiter, Charles N. Cole

Dartmouth Scholarship

Cells of Saccharomyces cerevisiae lacking Apq12, a nuclear envelope (NE)-endoplasmic reticulum (ER) integral membrane protein, are defective in assembly of nuclear pore complexes (NPCs), possibly because of defects in regulating membrane fluidity. We identified BRR6, which encodes an essential integral membrane protein of the NE-ER, as a dosage suppressor of apq12 Delta. Cells carrying the temperature-sensitive brr6-1 allele have been shown to have defects in nucleoporin localization, mRNA metabolism and nuclear transport. Electron microscopy revealed that brr6-1 cells have gross NE abnormalities and proliferation of the ER. brr6-1 cells were hypersensitive to compounds that affect membrane biophysical properties and to …

Flagellar Formation In C-Ring-Defective Mutants By Overproduction Of Flii, The Atpase Specific For Flagellar Type Iii Secretion, Manabu Konishi, Masaomi Kanbe, Jonathan L. Mcmurry, Shin-Ichi Aizawa

Faculty Articles

The flagellar cytoplasmic ring (C ring), which consists of three proteins, FliG, FliM, and FliN, is located on the cytoplasmic side of the flagellum. The C ring is a multifunctional structure necessary for flagellar protein secretion, torque generation, and switching of the rotational direction of the motor. The deletion of any one of the fliG, fliM, and fliN genes results in a Fla - phenotype. Here, we show that the overproduction of the flagellum-specific ATPase FliI overcomes the inability of basal bodies with partial C-ring structures to produce complete flagella. Flagella made upon FliI overproduction were paralyzed, indicating that an …

Pore Size Characterization Of Monolithic Capillary Columns Using Capillary Flow Porometry, Yan Fang

Theses and Dissertations

A simple capillary flow porometer (CFP) was assembled for pore structure characterization of monolithic capillary liquid chromatography columns based on ASTM standard F316-86. Determination of differential pressures and flow rates through dry and wet samples provided the necessary information to determine the through-pore throat diameter, bubble point pore diameter, mean flow pore diameter, and pore distribution. Unlike measurements in bulk using traditional techniques to provide indirect information about the pore properties of monolithic columns, monoliths can be characterized in their original chromatographic forms with this system. The performance of the new CFP was first evaluated by characterizing the pore size …

Inflammasome-Dependent Caspase-1 Activation In Cervical Epithelial Cells Stimulates Growth Of The Intracellular Pathogen Chlamydia Trachomatis, Ali A. Abdul-Sater, Evonne Koo, Georg Hacker, David M. Ojcius

All Dugoni School of Dentistry Faculty Articles

Inflammasomes have been extensively characterized in monocytes and macrophages, but not in epithelial cells, which are the preferred host cells for many pathogens. Here we show that cervical epithelial cells express a functional inflammasome. Infection of the cells by Chlamydia trachomatis leads to activation of caspase-1, through a process requiring the NOD-like receptor family member NLRP3 and the inflammasome adaptor protein ASC. Secretion of newly synthesized virulence proteins from the chlamydial vacuole through a type III secretion apparatus results in efflux of K+ through glibenclamide-sensitive K+ channels, which in turn stimulates production of reactive oxygen species. Elevated levels of reactive …

Gas Phase Chiral Recognition, Characterization Of Porous Polymer Monolith Nanospray Ionization, And The Negative Mode Crafti Method Using Fourier Transform Ion Cyclotron Resonance Mass Spectrometry, Nannan Fang

Theses and Dissertations

Our group has been studying chiral recognition in gas phase using mass spectrometry for more than 10 years. We are interested in gas phase studies of fundamental interactions because the gas phase avoids complications and masking effects that may arise upon solvation. Therefore, the results of gas phase experiments can be directly compared with those of high-level computational studies. In chapter 2, I studied the roles of hydrogen bonding and pi stacking in gas phase chiral recognition between aromatic crown molecules and aromatic amines. High affinity between host and guest doesn't necessarily result in better recognition. If the affinity is …

Broad Range Amino Acid Specificity Of Rna-Dependent Lipid Remodelling By Multiple Peptide Resistance Factors, Hervé Roy, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Aminoacylphosphatidylglycerol synthases (aaPGSs) are multiple peptide resistance factors that transfer amino acids from aminoacyl-tRNAs to phosphatidylglycerol (PG) in the cytoplasmic membrane. Aminoacylation of PG is used by bacteria to decrease the net negative charge of the cell envelope, diminishing affinity for charged molecules and allowing for adaptation to environmental changes. Lys-PGS, which transfers lysine to PG, is essential for the virulence of certain pathogens, providing resistance to both host cationic antimicrobial peptides and therapeutic antibiotics. Ala-PGS was also recently described, but little is known about the possible activities of other members of the highly diverse aaPGS family of proteins. Systematic …

Self-Assembly And Disassembly Of The Snare Complex: Examined Using Circular Dichroism And Atomic Force Microscopy, Jeremy D. Cook, Won Jin Cho, Timothy L. Stemmler, Bhanu P. Jena

Biochemistry and Molecular Biology Faculty Publications

In this study, we report for the first time that both t-SNAREs and v-SNARE and their complexes in buffered suspension, exhibit defined peaks at CD signals of 208 and 222 nm wavelengths, consistent with a higher degree of helical secondary structure. Surprisingly, when incorporated in lipid membrane, both SNAREs and their complexes exhibit reduced folding. In presence of NSF-ATP, the SNARE complex disassembles, as reflected from the CD signals demonstrating elimination of α-helices within the structure.

The Cca Anticodon Specifies Separate Functions Inside And Outside Translation In Bacillus Cereus, Sandro F. Ataide, Theresa E. Rogers, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Bacillus cereus 14579 encodes two tRNAs with the CCA anticodon, tRNA^Trp and tRNA^Other. tRNA^Trp was separately aminoacylated by two enzymes, TrpRS1 and TrpRS2, which share only 34% similarity and display different catalytic capacities and specificities. TrpRS1 was 18-fold more proficient at aminoacylating tRNA^Trp with Trp, while TrpRS2 more efficiently utilizes the Trp analog 5-hydroxy Trp. tRNA^Other was not aminoacylated by either TrpRS but instead by the combined activity of LysRS1 and LysRS2, which recognized sequence elements absent from tRNA^Trp. Polysomes were found to contain tRNA^Trp, consistent with its role in …

Microbial Nad Metabolism: Lessons From Comparative Genomics, Francesca Gazzaniga, Rebecca Stebbins, Sheila Z. Chang, Mark A. Mcpeek, Charles Brenner

Dartmouth Scholarship

NAD is a coenzyme for redox reactions and a substrate of NAD-consuming enzymes, including ADP-ribose transferases, Sir2-related protein lysine deacetylases, and bacterial DNA ligases. Microorganisms that synthesize NAD from as few as one to as many as five of the six identified biosynthetic precursors have been identified. De novo NAD synthesis from aspartate or tryptophan is neither universal nor strictly aerobic. Salvage NAD synthesis from nicotinamide, nicotinic acid, nicotinamide riboside, and nicotinic acid riboside occurs via modules of different genes. Nicotinamide salvage genes nadV and pncA, found in distinct bacteria, appear to have spread throughout the tree of life …

Theoretical Studies Of 2,3 -Sigmatropic Rearrangements Of Allylic Selenoxides And Selenimides, Craig A. Bayse, Sonia Antony

Chemistry & Biochemistry Faculty Publications

Density-functional theory is used to model the endo and exo transition states for [2,3]-sigmatropic rearrangement of allylic aryl-selenoxides and -selenimides. The endo transition state is generally preferred for selenoxides if there is no substitution at the 2 position of the allyl group. Based upon the relative energies of the endo and exo transition states, enantioselectivity of rearrangements is expected to be greatest for molecules with substitutions at the 1- or (E)-3- position of the allyl group. Ortho substitution of a nitro group on the ancillary selenoxide phenyl ring reduces the activation barriers, increases the difference between the endo and exo …

The Crystal Structure Of The Escherichia Coli Autoinducer-2 Processing Protein Lsrf, Zamia Caridad Diaz , '09, K. B. Xavier, Stephen T. Miller

Chemistry & Biochemistry Faculty Works

Many bacteria produce and respond to the quorum sensing signal autoinducer-2 (AI-2). Escherichia coli and Salmonella typhimurium are among the species with the lsr operon, an operon containing AI-2 transport and processing genes that are up regulated in response to AI-2. One of the Lsr proteins, LsrF, has been implicated in processing the phosphorylated form of AI-2. Here, we present the structure of LsrF, unliganded and in complex with two phospho-AI-2 analogues, ribose-5-phosphate and ribulose-5-phosphate. The crystal structure shows that LsrF is a decamer of (alpha beta)(8)-barrels that exhibit a previously unseen N-terminal domain swap and have high structural homology …

Total Synthesis Of (-)-Acutumine, Fang Li

Theses and Dissertations

Acutumine is a tetracyclic alkaloid isolated from the Asian vine Menispermum dauricum with selective T-cell cytotoxicity and antiamnestic properties. We have developed a total synthetic route to this congested alkaloid, during which we also found a novel, stereoselective radical-crossover reaction that combines an intramolecular radical conjugate addition with a subsequent enolate hydroxylation. Key features of this synthesis also include a reagent-controlled diastereoselective ketone allylation, an anionic oxy-Cope rearrangement to form a congested quaternary sterocenter, a pyridine-mediated selective ozonolysis, and a Lewis acid promoted Michael-type cyclization.

Development Of Biocompatible Polymer Monoliths For The Analysis Of Proteins And Peptides, Yun Li

Theses and Dissertations

Biocompatibility is an important issue for the development of chromatographic stationary phases for the analysis of biomolecules (including proteins and peptides). A biocompatible stationary phase material is a material that resists nonspecific adsorption of biomolecules and does not interact with them in a way that would alter or destroy their structures or biochemical functions. The monolithic column format is a good alternative to typical spherical particle packed columns for capillary liquid chromatography of biomacromolecules. Several novel anion-exchange polymer monoliths for the analysis of proteins were synthesized for improved biocompatibility. Two novel polymeric monoliths were prepared in a single step by …

Real-Time Virus Analysis Via Image Charge Detection Surface Induced Dissociation Tandem Mass Spectrometry, Seth T. Call

Theses and Dissertations

This thesis reports on the development of a novel mass spectrometer combining image charge detection with surface induced dissociation for real-time analysis of intact viruses. Protonated viruses produced using electrospray are accelerated and subsequently impact on a solid surface. Capsid peptides released during the impact are analyzed using time-of-flight mass spectrometry. Image charge detection is used to measure the mass and charge states of structurally intact, electrosprayed viruses prior to impact. Since virus capsids are composed of loosely-bound proteins, collision of viruses with surfaces at moderate impact energies could release intact proteins. The masses and numbers of different protein types …

Design And Characterization Of A Human Exposure Chamber And Inversion Episodes In Salt Lake City, Utah In January/February Of 2009, Roman Yuri Kuprov

Theses and Dissertations

Research on health effects of particulate matter (PM) has been a very active area in the last two decades. One plausible mechanism by which exposure to PM affects human health includes modification of autonomic endothelium function. Decreased endothelium activity causes heightened risks of cardiovascular disease. A human exposure chamber designed to conduct experiments to quantify diminished function of endothelium from short term exposure to PM is described. The chamber consists of two stages for containment and pre-treatment of PM and exposure of human subjects. Concentrations of CO, CO2, NO, NO2, O3, and PM2.5, are monitored and controlled in the exposure …

The Characterization Of Two Diverse Magnetotactic Bacteria: Lems And Mms-1, Ulysses C. Pickard, Paul A. Howse, Dennis A. Bazylinski

Undergraduate Research Opportunities Program (UROP)

Magnetotactic bacteria (MTB) are a diverse group of prokaryotes that biomineralize membrane-bound magnetic crystals known as magnetosomes. The magnetosomes are aligned within the cell and consist of either magnetite (Fe3O4) or greigite (Fe3S4). The biomineralization of magnetosomes consists of several processes including: invagination of the cytoplasmic membrane, iron uptake into the cell and then into the magnetosome membrane vesicle, and crystallization of the mineral phase inside the vesicle. Mam genes control magnetosome biomineralization with most of the genes present in an island called a magnetosome island. Many of the mam genes are conserved between different species of MTB. The genes …