Life Sciences 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.

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 …

Genetic Effect Of The Dwarfing Genes On Some Culm Characteristics Associatcd With Lodging Resistance In Bread Wheat, Md. Mahbub Hasan

Md. Mahbub Hasan

Due to the challenge of screening traits related to lodging resistance under natural field conditions, selection for lodging resistant varieties in wheat breeding programs is difficult. The identification of easily measurable culm anatomical traits related to lodging resistance would simplify the selection process. The present study was conducted to determine the effect of dwarfing genes on culm anatomical traits related to lodging resistance in our of basal internode 1. Field and laboratory study was conducted in Shahjalal University of Science and Technology, Sylhet, Bangladesh with eight wheat genotypes having Rhr1, Rht2 dwarfing genes in them and a local land race …

Applications Of Bret: The Detection And Visualization Of Protein Interactions And Intramolecular Conformational Changes, Lindsay Anne Staron

Masters Theses

Bioluminescence is a phenomenon in which chemical energy is converted into light energy. Here, the oxidation of a luciferin substrate, catalyzed by a luciferase enzyme, results in the emission of a photon. This biological process is exploited in a technology referred to as Bioluminescence Resonance Energy Transfer (BRET). As its name implies, BRET depends on a nonradiative energy transfer event that occurs between a donor luciferase and an acceptor fluorophore. Fusion of the donor and acceptor molecules to a protein(s) of-interest allows one to identify and monitor molecular events, such as protein interactions or hormone binding events, based solely on …

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 …

Light-Related Photosynthetic Gene Expression And Enzyme Activity In The Heterokont Alga Vaucheria Litorea And Its Symbiotic Partner The Sacoglossan Mollusc Elysia Chlorotica, Kara M. Soule

Electronic Theses and Dissertations

Photosynthesis is comprised of tightly coupled reactions and therefore requires strict matrices of regulation, particularly involving alterations in gene expression and enzyme activity within the nucleus and plastid. Extensive research has been carried out on these light-regulated mechanisms in plants and green algae, however, much less is known in the red algal lineage, including heterokonts. The goal of this study was to investigate the influence of light on photosynthetic gene expression and select enzyme activity in the heterokont alga Vaucheria litorea and its symbiotic partner Elysia chlorotica, a photosynthetic sacoglossan mollusc (sea slug). Elysia chlorotica harbors V. litorea plastids …

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 …

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 …

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

Dissertations & 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 …

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 …

Toc Receptor Dimerization Participates In The Initiation Of Membrane Translocation During Protein Import Into Chloroplasts, Jeonghwa Lee, Fei Wang Wang, Danny Schnell

Danny Schnell

The post-translational import of nucleus-encoded preproteins into chloroplasts occurs through multimeric translocons in the outer (Toc) and inner (Tic) membranes. The high fidelity of the protein import process is maintained by specific recognition of the transit peptide of preproteins by the coordinate activities of two homologous GTPase Toc receptors, Toc34 and Toc159. Structural and biochemical studies suggest that dimerization of the Toc receptors functions as a component of the mechanism to control access of preproteins to the membrane translocation channel of the translocon. We show that specific mutations that disrupted receptor dimerization in vitro reduced the rate of protein import …

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 …

Absolute Configuration And Biosynthesis Of Pahayokolide A From Lyngbya Sp. Strain 15-2 Of The Florida Everglades, Li Liu

FIU Electronic Theses and Dissertations

Pahayokolides A-D are cytotoxic cyclic polypeptides produced by the freshwater cyanobacterium Lyngbya sp. strain 15-2 that possess an unusual β-amino acid, 3-amino-2,5,7,8-tetrahydroxy-10-methylundecanoic acid (Athmu). The absolute configuration of pahayokolides A-D was determined using advanced Marfey’s method. It was also confirmed that a pendant N-acetyl-N-methyl leucine moiety in pahayokolide A was absent in pahayokolides B and pahayokolides C-D were conformers of pahayokolide A. Feeding experiments indicated that the biosynthesis of the Athmu sidechain arises from leucine or α-ketoisovalerate, however could not be further extended by three rounds of condensation with malonate units. Putative four peptide and one unique polyketide synthetases in …

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 and Research Publications

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 …

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 …

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 …

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.

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 …

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 …

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 …