Biochemistry, Biophysics, and Structural Biology Commons^™

Molecular Epidemiology Of Antibiotic Resistant Salmonella Enterica Strains From Different Animal And Human Sources In Ireland, David George Lee

Theses

In this study, 176 isolates of Salmonella were collected from different laboratories in Ireland. All isolates were confirmed to be of the genus Salmonella by performing bacteriological and biochemical tests. Briefly, all isolates were cultured onto XLD agar after which pure cultures were verified and stocked at this institute. Definitive identification was carried out using the API 20-E system and a separate urease test was also performed on all isolates. After confirmation of the genus, all isolates were prepared for antibiotic susceptibility testing and r-types were determined using sixteen antibiotics. Depending on the antibiotic resistance profiles, isolates were selected for …

2-Aminoethoxydiphenyl Borate As A Prototype Drug For A Group Of Structurally Related Calcium Channel Blockers In Human Platelets, Yuliya Dobrydneva, Christopher J. Abelt, Beth Dovel, Celina M. Thadigiri, Roy L. Williams, Peter F. Blackmore

Chemistry & Biochemistry Faculty Publications

We have synthesized a series of 2-aminoethoxydiphenyl borate (2-APB, 2,2-diphenyl-1,3,2-oxazaborolidine) analogs and tested their ability to inhibit thrombin-induced Ca²⁺ influx in human platelets. The analogs were either synthesized by adding various substituents to the oxazaborolidine ring (methyl, dimethyl, tert-butyl, phenyl, methyl phenyl, and pyridyl) or increasing the size of the oxazaborolidine ring to seven- and nine-membered rings. NMR analysis of the boron-containing analogs suggests that each of them exist as a ring structure through the formation of an N -> B coordinate bond (except for the hexyl analog). The possibility that these boron-containing compounds formed dimers was also …

The Effects Of Varying The Timing Of Inputs On A Neural Oscillator, Christina Ambrosio-Mouser, Farzan Nadim, Amitabha Bose

Publications and Research

The gastric mill network of the stomatogastric ganglion of the crab Cancer borealis is comprised of a set of neurons that require modulatory input from outside the stomatogastric ganglion and input from the pyloric network of the animal in order to oscillate. Here we study how the frequency of the gastric mill network is determined when it receives rhythmic input from two different sources but where the timing of these inputs may differ. We find that over a certain range of the time difference one of the two rhythmic inputs plays no role what so ever in determining the network …

Intercellular Spreading Of Porphyromonas Gingivalis Infection In Primary Gingival Epithelial Cells, Özlem Yilmaz, Philippe Verbeke, Richard J. Lamont, David M. Ojcius

All Dugoni School of Dentistry Faculty Articles

Porphyromonas gingivalis, an important periodontal pathogen, is an effective colonizer of oral tissues. The organism successfully invades, multiplies in, and survives for extended periods in primary gingival epithelial cells (GECs). It is unknown whether P. gingivalis resides in the cytoplasm of infected cells throughout the infection or can spread to adjacent cells over time. We developed a technique based on flow cytofluorometry and fluorescence microscopy to study propagation of the organism at different stages of infection of GECs. Results showed that P. gingivalis spreads cell to cell and that the amount of spreading increases gradually over time. There was a …

Novel Cancer Therapeutics, The Generation Of Ros, And Cell Survival, Clint Mitchell

Theses and Dissertations

The impact of Ad.mda-7 on the survival of renal cell carcinoma lines (RCC), primary renal epithelial cells, glioblastoma multiforme lines (GBM), and primary rodent astrocytes is unknown. The present studies examine whether the GST fusion protein, GST-MDA-7, and the adenovirus, Ad.mda-7, altered the growth and survival of the A498 and UOK121N RCC lines or radiosensitized GBM, respectively. Due to previous findings that the RCC lines, but not primary renal epithelial cells, were resistant to type 5 adenoviral infection, we used purified GST-MDA-7 protein to show that GST-MDA-7, but not GST, caused a dose-dependent reduction in A498 and UOK121N proliferation but …

S-Adenosylmethionine Stabilizes Cystathionine Β-Synthase And Modulates Redox Capacity, Anna Prudova, Zachary Bauman, Aaron Braun, Victor Vitvitsky, Shelly C. Lu, Ruma Banerjee

Department of Biochemistry: Faculty Publications

The transsulfuration pathway converts homocysteine to cysteine and represents the metabolic link between antioxidant and methylation metabolism. The first and committing step in this pathway is catalyzed by cystathionine β-synthase (CBS), which is subject to complex regulation, including allosteric activation by the methyl donor, S-adenosylmethionine (AdoMet). In this study, we demonstrate that methionine restriction leads to a >10-fold decrease in CBS protein levels, and pulse proteolysis studies reveal that binding of AdoMet stabilizes the protein against degradation by ≈12 kcal/mol. These observations predict that under pathological conditions where AdoMet levels are diminished, CBS, and therefore glutathione levels, will be reduced. …

Microsatellite Genotyping To Distinguish Colonies And Intraspecies Genetic Variation In The Eastern Subterranean Termite, Reticulitermes Flavipes (Isoptera: Rhinotermitidae), Timothy J. Husen, Shripat T. Kamble, Julie M. Stone

Department of Biochemistry: Faculty Publications

Microsatellite markers and fingerprinting techniques have demonstrated great potential for assessing colony affiliation in Reticulitermes species. This study utilized microsatellite allele fingerprints and examined variation at eight micro satellite loci within five different R. flavipes collection sites in each of Lancaster and Madison counties in Nebraska. In the termite populations from Lancaster County, five of the eight microsatellite loci (Rf 1-3, Rf 3-1, Rf 5-1 0, Rf 6-1, and Rf 24-2) provided informative genotypic variability allowing colony distinction. DNA sequence data at locus Rf24-2 also provided conclusive evidence that populations sampled were distinct colonies. Five micro satellite loci (Rf 3-1, …

Purification And Initial Biochemical Characterization Of Atp:Cob(I)Alamin Adenosyltransferase (Eutt) Enzyme Of Salmonella Enterica, Nicole R. Baun, Jorge C. Escalante-Semerena

Department of Biochemistry: Faculty Publications

ATP:cob(I)alamin adenosyltransferase (EutT) of Salmonella

enterica was overproduced and enriched to ~70% homogeneity,

and its basic kinetic parameters were determined. Abundant

amounts of EutT protein were produced, but all of it remained

insoluble. Soluble active EutT protein (~70% homogeneous) was

obtained after treatment with detergent. Under conditions in which

cobalamin (Cbl) was saturating, _Km(ATP) = 10 µM, k_cat = 0.03 s^-1,

and V_max = 54.5 nM min^-1. Similarly, under conditions in which

MgATPwas saturating,K_m(Cbl) = 4.1µM, k_cat = 0.06 s^-1, andV_max= …

Microorganisms Pumping Iron: Anaerobic Microbial Iron Oxidation And Reduction, Karrie A. Weber, Laurie A. Achenbach, John D. Coates

School of Biological Sciences: Faculty Publications

Iron (Fe) has long been a recognized physiological requirement for life, yet for many microorganisms that persist in water, soils and sediments, its role extends well beyond that of a nutritional necessity. Fe(II) can function as an electron source for iron-oxidizing microorganisms under both oxic and anoxic conditions and Fe(III) can function as a terminal electron acceptor under anoxic conditions for iron-reducing microorganisms. Given that iron is the fourth most abundant element in the Earth’s crust, iron redox reactions have the potential to support substantial microbial populations in soil and sedimentary environments. As such, biological iron apportionment has been described …

Characterizing An Rna Aptamer And Binding Studies With Synthetic Porphyrazines, Melanie Bozza

Seton Hall University Dissertations and Theses (ETDs)

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Potential Of Vibrational Spectroscopy In The Diagnosis Of Human Tumours., Eoghan O'Faolain

Doctoral

Just fewer than 20,000 people are annually diagnosed with some form of cancer in Ireland and one in three people are likely to contract some form of cancer by age 74. With the number of cases increasing at an annual rate of 2%, the early detection and treatment of cancer is becoming increasingly important. Both IR and Raman spectroscopy offer the potential for real time, quantitative detection of cancer and even precancer. This study investigates the potential of Raman and Fourier transform infrared, both benchtop and synchrotron spectroscopies for the detection of cervical cancer. The tissue was classified and its …

I. Comparison Of Translesion Bypass Of Guanine–N2 Monoadducts Of Mitomycin C And Guanine-N7 Monoadducts Of 2,7-Diaminomitosene By T7 Exo-, Klenow Exo-, Eta And Klenow Exo+ Dna Polymerases. Ii. Structure-Based Design, Synthesis, Structure-Conformation And Structure-Activity Relationships Studies Of D-Phe-Pro-D-Arg-P1’-Conh2 Tetrapeptides With Inhibitory Activity For Thrombin., Cristina C. Clement

Dissertations, Theses, and Capstone Projects

The guanine (G)-N² DNA monoadduct of mitomycin C (MC), a cytotoxic anticancer drug, inhibits translesion bypass by DNA polymerases. 2,7-Diaminomitosene (2,7-DAM) is the major metabolite of MC in tumor cells, generated by the reduction of MC. 2,7-DAM alkylates DNA in the cell in situ, forming an adduct at the N7 position of 2'-deoxyguanosine (2,7-DAM-dG) and is noncytotoxic. In part I of this study we tested a potential correlation between the lack of cytotoxicity of 2,7-DAM and the relative ease of bypass of this adduct as compared with the MC adduct. 24-mer and 27-mer templates, adducted at a single …

Dna Damage Responses In Progeroid Syndromes Arise From Defective Maturation Of Prelamin A, Michael Sinensky, Y. Liu, A. Rusinol, Y. Wang, Y. Zou

Faculty Publications, Biological Sciences

The genetic diseases Hutchinson-Gilford progeria syndrome (HGPS) and restrictive dermopathy (RD) arise from accumulation of farnesylated prelamin A because of defects in the lamin A maturation pathway. Both of these diseases exhibit symptoms that can be viewed as accelerated aging. The mechanism by which accumulation of farnesylated prelamin A leads to these accelerated aging phenotypes is not understood. Here we present evidence that in HGPS and RD fibroblasts, DNA damage checkpoints are persistently activated because of the compromise in genomic integrity. Inactivation of checkpoint kinases Ataxia-telangiectasia-mutated (ATM) and ATR (ATM- and Rad3-related) in these patient cells can partially overcome their …

Farnesylated Lamins, Progeroid Syndromes And Farnesyl Transferase Inhibitors, Michael Sinensky, A. E. Rusinol

Faculty Publications, Biological Sciences

Three mammalian nuclear lamin proteins, lamin B1, lamin B2 and the lamin A precursor, prelamin A, undergo canonical farnesylation and processing at CAAX motifs. In the case of prelamin A, there is an additional farnesylation-dependent endoproteolysis, which is defective in two congenital diseases: Hutchinson-Gilford progeria (HGPS) and restrictive dermopathy (RD). These two diseases arise respectively from defects in the prelamin A substrate and the enzyme (ZmpSte24) that processes it. Recent work has shed light on the roles of the lamin proteins and the enzymes involved in their farnesylation-dependent maturation. Other experimental work, including mouse model studies, have examined the possibility …

Characterization Of The Ftf/Hnf-4 Sites Within The 7alpha- And The 12alpha-Hydroxylase Promoters Involved In The Bile Acid-Mediated Transcription Of Their Regulation, Preeti Pramanik

Theses and Dissertations

Bile acids regulate their own synthesis through a feedback regulatory mechanism of mainly two enzymes in the classic pathway, the 7α-hydroxylase and the 12α-hydroxylase. In the early 1990's it was shown that the regulatory responses of 7α-hydroxylase are mediated at the transcriptional level and since then many positive and negative transcription factors that mediate regulatory response have been identified. An important finding was that the transcription factors regulating the expression of 7α- and 12α-hydroxylase genes are nuclear receptors.One of the first nuclear receptors identified to play a role in the transcription of the 7α-hydroxylase gene was HNF-4 since then many …

Role Of Mdm2 In Cell Growth Regulation, Rebecca Anne Frum

Theses and Dissertations

MDM2 has been shown to induce G0-Gl/S phase arrest. To determine the cell cycle step targeted by MDM2, flow cytometry was employed to detect induction of events during the G1-S phase transition in MDM2-arrested cells. MDM2 overexpression does not prevent expression of cyclin D, cyclin D-CDK mediated phosphorylation of Rb or cyclin E in normal, immortal or tumor-derived cells. However, MDM2 down-regulates cyclin A expression specifically in normal cells, which is associated with G1 arrest. The domain of MDM2 capable of this function is located within its N-terminal 58-109 amino acids. To down-regulate cyclin A, MDM2 requires a functional pl6/Brg …

Characterization Of The Rank Ligand Positive Giant Cell Found In The Interfacial Membrane, Patrick Emerson Jones

Theses and Dissertations

Aseptic osteolysis is a major complication to total joint arthroplasty requiring several thousand people a year to have to undergo revisions of their joint prosthesis. The formation of the interfacial membrane has been associated with aseptic osteolysis leading to the failure of all types of total joints. Recent evidence suggests that RANKL, a potent activator of bone reabsorption, is present in the interfacial membrane. Prior research in this laboratory to determine the source of RANKL in the interfacial membrane has revealed the presence of intense areas of RANKL concentration in the membrane. These areas of RANKL concentration correspond to multiple …

The Use Of Proteomic Technologies To Identify Serum Glycoproteins For The Early Detection Of Liver And Prostate Cancers, Elizabeth Ellen Schwegler

Theses and Dissertations in Biomedical Sciences

The application of proteomic technologies to identify serum glycoproteins is an emerging technique to identify new biomarkers indicative of disease severity. Many of these newly evolving protein-profiling methodologies have evolved from previous global protein expression profiling studies such as those involving SELDI-TOF-MS technologies. Though the SELDI approach could distinguish disease from normal by utilizing protein patterns as shown herein with the HCC study of chapter II, it was unable to offer sequence information on the selected peaks, and did not have the ability to analyze the entire dynamic range of the serum/plasma proteome. To address these deficiencies, new strategies that …

Analysis Of The Mouse And Human Acyl-Coa Thioesterase (Acot) Gene Clusters Shows That Convergent, Functional Evolution Results In A Reduced Number Of Human Peroxisomal Acots., Mary Hunt, Anna Rautanen, Maria Westin, Thomas Svensson, Stefan Alexson

Articles

The maintenance of cellular levels of free fatty acids and acyl-CoAs, the activated form of free fatty acids, is extremely important as imbalances in lipid metabolism have serious consequences for human health. Acyl-CoA thioesterases (ACOTs) hydrolyze acyl-CoAs to the free fatty acid and CoASH, and thereby have the potential to regulate intracellular levels of these compounds. We have previously identified and characterized a mouse ACOT gene cluster, comprised of six genes that apparently arose by gene duplications, encoding acyl- CoA thioesterases with localizations in cytosol (ACOT1), mitochondria (ACOT2) and peroxisomes (ACOT3-6). However, the corresponding human gene cluster contains only three …

Development Of Single Nanoparticle Optical Assays For Imaging Single Living Cells, William John Brownlow

Chemistry & Biochemistry Theses & Dissertations

Multi-drug resistance (MDR) has been reported in both prokaryotes and eukaryotes; the pathogenic gram-negative bacteria Pseudomonas aeruginosa can extrude a variety of structurally and functionally diverse substrates via a number of membrane transport systems leading to MDR. We have developed a novel nanoparticle assay to characterize both the membrane transport system composed of the MexAB-OprM efflux pump and the membrane permeability induced by antibiotics. Gold (Au) and silver (Ag) nanoparticles were investigated for use as probes to explore membrane transport in P. aeruginosa.

The surface plasmon absorption (color) of Au nanoparticle solutions was found to change in the presence …

Structure-Function Studies Of The Camp-Dependent Protein Kinase In Vitro And In Intact Cells, Gary Z. Morris

Theses and Dissertations in Biomedical Sciences

There are 518 protein kinase genes in the human genome; this constitutes about 1.7% of all human genes. The cAMP-dependent protein kinase (PKA) serves as the prototypic model for the study of kinases because it contains a conserved catalytic core shared with all eukaryotic kinases, it is the simplest kinase, and it is one of the best-characterized serine/threonine kinases. PKA is ubiquitous in mammals and regulates multiple physiological mechanisms such as the cell cycle, apoptosis, cell motility, energy metabolism, and gene transcription through a well-defined intracellular signaling pathway. While PKA clearly has a central physiological role it is still unclear …

Isolation And Functional Mapping Of Human T-Cell Leukemia Virus Type 1 Tax Oncoprotein Dna-Damage Complexes, Sarah Saionz Durkin

Theses and Dissertations in Biomedical Sciences

Human T-cell Leukemia Virus Type 1 (HTLV-1) is a transforming retrovirus which causes Adult T-cell Leukemia (ATL) and HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). Cellular transformation can be caused by a single viral trans-activating protein, Tax. Tax may contribute to transformation through interaction with components of the DNA damage response pathway, promoting cellular genomic instability. We examined cellular Tax complexes in an effort to elucidate potential protein-protein interactions that can model the Tax-induced molecular events.

We also investigated the role of post-translational modification in regulating Tax function. We employed a direct physical analysis of Tax complexes isolated from mammalian …