Biochemistry Commons^™

Novel And Predominant Pathogen Responsible For The Enterovirus-Associated Encephalitis In Eastern China, Lei Zhang, Jie Yan, David M. Ojcius, Huakun Lv, Ziping Miao, Yin Chen, Yanjun Zhang, Jvying Yan

All Dugoni School of Dentistry Faculty Articles

Enteroviruses (EV) have been increasingly identified as the causative agent for unknown etiological encephalitis in many parts of the world, but the long period surveillance for enterovirus-associated encephalitis (EAE) was not reported in China. From 2002-2012 in Zhejiang, Coxsackieviruses A9, B1, B2, B3, B4, B5; and echoviruses 3, 4, 6, 9, 14, 25, 30 were detected from the unknown etiological encephalitis cases, with coxsackievirus B4 been identified here for the first time. From 2002-2004 and 2010-2012, echovirus 30 was found to be the periodically predominant serotype for in the EAE. The molecular typing results showed that all the EV isolates …

Biochemical Assay Optimization And Computational Screening Efforts To Identify Potential Luxs Inhibitors, Keeshia Q. Wang

Master's Theses

Quorum sensing (QS) is a process of coordination of bacterial gene expression in response to cell population. System two QS is regulated by the small signaling molecule autoinducer-2 (AI-2) and is implicated in the infectious behaviors of various bacterial species. AI-2 is biosynthesized from S-ribosylhomocysteine (SRH) by the enzyme LuxS and induces interspecies cell-to-cell communication. Inhibition of LuxS would therefore inhibit interspecies QS. Herein, a search for novel molecular species that will competitively bind with SRH in the LuxS binding site is performed in silico. Computational screening results are then validated in vitro using an optimized LuxS inhibition …

Membrane Vesicles Nucleate Mineralo-Organic Nanoparticles And Induce Carbonate Apatite Precipitation In Human Body Fluids, Cheng-Yeu Wu, Jan Martel, Wei-Yun Cheng, Chao-Chih He, David M. Ojcius, John Ding-E. Young

All Dugoni School of Dentistry Faculty Articles

Recent studies indicate that membrane vesicles (MVs) secreted by various cells are associated with human diseases, including arthritis, atherosclerosis, cancer, and chronic kidney disease. The possibility that MVs may induce the formation of mineralo-organic nanoparticles (NPs) and ectopic calcification has not been investigated so far. Here, we isolated MVs ranging in size between 20 and 400 nm from human serum and FBS using ultracentrifugation and sucrose gradient centrifugation. The MV preparations consisted of phospholipid-bound vesicles containing the serum proteins albumin, fetuin-A, and apolipoprotein A1; the mineralization-associated enzyme alkaline phosphatase; and the exosome proteins TNFR1 and CD63. Notably, we observed that …

Identification Of Leptospira Interrogans Phospholipase C As A Novel Virulence Factor Responsible For Intracellular Free Calcium Ion Elevation During Macrophage Death, Jing-Fang Zhao, Hong-Hu Chen, David M. Ojcius, Xin Zhao, Dexter Sun, Yu-Mei Ge, Lin-Li Zheng, Xu'ai Lin, Lan-Juan Li, Jie Yan

All Dugoni School of Dentistry Faculty Articles

Background: Leptospira-induced macrophage death has been confirmed to play a crucial role in pathogenesis of leptospirosis, a worldwide zoonotic infectious disease. Intracellular free Ca²⁺ concentration ([Ca²⁺]i) elevation induced by infection can cause cell death, but [Ca²⁺]i changes and high [Ca²⁺]i-induced death of macrophages due to infection of Leptospira have not been previously reported.

Methodology/Principal Findings: We first used a Ca²⁺-specific fluorescence probe to confirm that the infection of L. interrogans strain Lai triggered a significant increase of [Ca²⁺]i in mouse J774A.1 or human THP-1 macrophages. Laser confocal microscopic examination …

Bioengineering The Expression Of Active Recombinant Human Cathepsin G, Enteropeptidase, Neutrophil Elastase, And C-Reactive Protein In Yeast, Eliot T. Smith

Electronic Theses and Dissertations

The yeasts Pichia pastoris and Kluyveromyces lactis were used to express several recombinant human proteins for further biochemical characterization. Two substitution variants of recombinant human enteropeptidase light chain (rhEPL) were engineered to modify the extended substrate specificity of this serine protease. Both were secreted as active enzymes in excess of 1.7 mg/L in P. pastoris fermentation broth. The substitution variant rhEPL R96Q showed significantly reduced specificities for the preferred substrate sequences DDDDK and DDDDR; however, the rhEPL Y174R variant displayed improved specificities for these substrate sequences relative to all other reported variants of this enzyme. The neutrophil serine proteases human …

P2x4 Assembles With P2x7 And Pannexin-1 In Gingival Epithelial Cells And Modulates Reactive Oxygen Species Production And Inflammasome Activation, Shu-Chen Hung, Chul Hee Choi, Najwane Said-Sadier, Larry Johnson, Kalina Rosenova Atanasova, Hanen Sellami, Özlem Yilmaz, David M. Ojcius

All Dugoni School of Dentistry Faculty Articles

We have previously reported that Porphyromonas gingivalis infection of gingival epithelial cells (GEC) requires an exogenous danger signal such as ATP to activate an inflammasome and caspase-1, thereby inducing secretion of interleukin (IL)-1β. Stimulation with extracellular ATP also stimulates production of reactive oxygen species (ROS) in GEC. However, the mechanism by which ROS is generated in response to ATP, and the role that different purinergic receptors may play in inflammasome activation, is still unclear. In this study, we revealed that the purinergic receptor P2X₄ is assembled with the receptor P2X₇ and its associated pore, pannexin-1. ATP induces ROS …

Genetic Approaches To Studying Complex Human Disease, Joseph B. Dube

Electronic Thesis and Dissertation Repository

Common, complex diseases such as cardiovascular disease (CVD) represent an intricate interaction between environmental and genetic factors and now account for the leading causes of mortality in western society. By investigating the genetic component of complex disease etiology, we have gained a better understanding of the biological pathways underlying complex disease and the heterogeneity of complex disease risk. However, the development of high throughput genomic technologies and large well-phenotyped multi-ethnic cohorts has opened the door towards more in-depth and trans-disciplinary approaches to studying the genetics of complex disease pathogenesis. Accordingly, we sought to investigate select complex traits and diseases using …

Global Analysis Of Gene Expression Changes During Retinoic Acid-Induced Growth Arrest And Differentiation Of Melanoma: Comparison To Differentially Expressed Genes In Melanocytes Vs Melanoma, Mary H. Estler, Goran Boskovic, James Denvir, Sarah Miles, Donald A. Primerano, Richard M. Niles

Goran Boskovic

BACKGROUND: The incidence of malignant melanoma has significantly increased over the last decade. Some of these malignancies are susceptible to the growth inhibitory and pro-differentiating effects of all-trans-retinoic acid (RA). The molecular changes responsible for the biological activity of RA in melanoma are not well understood. RESULTS: In an analysis of sequential global gene expression changes during a 4-48 h RA treatment of B16 mouse melanoma cells, we found that RA increased the expression of 757 genes and decreased the expression of 737 genes. We also compared the gene expression profile (no RA treatment) between non-malignant melan-a mouse melanocytes and …

Raman Spectroscopic Analysis Of Human Skin Tissue Sections Ex-Vivo: Evaluation Of The Effects Of Tissue Processing And Dewaxing, Syed Mehmood Ali, Franck Bonnier, Ali Tfayli, Helen Lambkin, Kathleen Flynn, Vincent Mcdonagh, Claragh Healy, Thomas Lee, Fiona Lyng, Hugh Byrne

Articles

Raman spectroscopy coupled with K-means clustering analysis (KMCA) is employed to elucidate the biochemical structure of human skin tissue sections, and the effects of tissue processing. Both hand and thigh sections of human cadavers were analysed in their unprocessed and formalin fixed paraffin processed (FFPP) and subsequently dewaxed forms. In unprocessed sections, KMCA reveals clear differentiation of the stratum corneum, intermediate underlying epithelium and dermal layers for sections from both anatomical sites. The stratum corneum is seen to be relatively rich in lipidic content; the spectrum of the subjacent layers is strongly influenced by the presence of melanin, while that …

A Novel Mechanism For Mechanosensing By Endothelial Cells, Jennifer Macdowell

Honors College

The formation of new vasculature is an essential process, but can also be utilized by cancerous cells. Angiogenesis requires the directed migration of the endothelial cells lining the nascent blood vessels. This process is largely mediated by integrin, which plays a key role in the interplay between sensing a force in the extracellular matrix (ECM) and transducing this signal, a process termed mechanotransduction. Through cell-ECM focal adhesions, integrin mediates the signaling both into and out of the cell, promoting growth of focal adhesions and subsequent cell spreading and migration. In order to study focal adhesion dynamics related to force, we …

Oxidation Of Thrombomodulin Methionine 388 In Cigarette Smokers, Samrat B. Thapa

Graduate Theses and Dissertations

This work tested the hypothesis that oxidation of methionine 388 in thrombomodulin is higher in cigarette smokers, and thus a likely contributor towards the hypercoagulable state in smokers. Thrombomodulin, a protein cofactor found on endothelial cell surfaces, regulates the activity of thrombin. Thrombin bound to thrombomodulin no longer converts fibrinogen to fibrin, but instead activates Protein C which, in turn, stops the coagulation cascade by inactivation of clotting factors. The oxidation of methionine 388 of thrombomodulin has been shown in vitro to dramatically decrease the anticoagulant cofactor activity of thrombomodulin. The blood of cigarette smokers is more prone to clot …

Tca Cycle Inactivation In Staphylococcus Aureus Alters Nitric Oxide Production In Raw 264.7 Cells, Chandirasegaran Massilamany, Arunakumar Gangaplara, Donald Gardner, James Musser, David Steffen, Greg Somerville, Jay Reddy

Greg A. Somerville

Inactivation of the Staphylococcus aureus tricarboxylic acid (TCA) cycle delays the resolution of cutaneous ulcers in a mouse soft tissue infection model. In this study, it was observed that cutaneous lesions in mice infected with wild-type or isogenic aconitase mutant S. aureus strains contained comparable inflammatory infiltrates, suggesting the delayed resolution was independent of the recruitment of immune cells. These observations led us to hypothesize that staphylococcal metabolism can modulate the host immune response. Using an in vitro model system involving RAW 264.7 cells, the authors observed that cells cultured with S. aureus aconitase mutant strains produced significantly lower amounts …

Hirsutella Sinensis Mycelium Suppresses Interleukin-1b And Interleukin-18 Secretion By Inhibiting Both Canonical And Non-Canonical Inflammasomes, Tsung-Teng Huang, Kowit-Yu Chong, David M. Ojcius, Yi-Hui Wu, Yun-Fei Ko, Cheng-Yeu Wu, Jan Martel, Chia-Chen Lu, Hsin-Chih Lai, John Ding-E. Young

All Dugoni School of Dentistry Faculty Articles

Cordyceps sinensis is a medicinal mushroom used for centuries in Asian countries as a health supplement and tonic. Hirsutella sinensis—the anamorphic, mycelial form of C. sinensis—possesses similar properties, and is increasingly used as a health supplement. Recently, C. sinensis extracts were shown to inhibit the production of the pro-inflammatory cytokine IL-1β in lipopolysaccharide-treated macrophages. However, the molecular mechanism underlying this process has remained unclear. In addition, whether H. sinensis mycelium (HSM) extracts also inhibit the production of IL-1β has not been investigated. In the present study, the HSM extract suppresses IL-1β and IL-18 secretion, and ATP-induced activation of caspase-1. Notably, …

Inhibition Of Bacillus Cereus Growth By Bacteriocin Producing Bacillus Subtilis Isolated From Fermented Baobab Seeds (Maari) Is Substrate Dependent, Donatien Kaboré, Dennis S. Nielsen, Hagrétoui Sawadogo-Lingan, Bréhima Diawara, Mamoudou H. Dicko Prof., Mogens Jakobsen, Line Thorsen

Pr. Mamoudou H. DICKO, PhD

Cyclooxygenase-2 Dependent Metabolism Of 20-Hete Increases Adiposity And Adipocyte Enlargement In Mesenchymal Stem Cell-Derived Adipocytes, Dong Hyun Kim, Nitin Puri, Komal Sodhi, John R. Falck, Nader G. Abraham, Joseph I. Shapiro M.D., Michal L. Schwartzman

Biochemistry and Microbiology

Abstract 20-Hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE), a product of the cytochrome P450 (CYP)-catalyzed [1] -hydroxylation of arachidonic acid, induces oxidative stress and, in clinical studies, is associated with increased body mass index (BMI) and the metabolic syndrome. This study was designed to examine the effects of exogenous 20- HETE on mesenchymal stem cell (MSC)-derived adipocytes.

The expression levels of CYP4A11 and CYP4F2 (major 20-HETE synthases in humans) in MSCs decreased during adipocyte differentiation; however, exogenous administration of 20-HETE (0.1–1 M) increased adipogenesis in a dose dependent manner in these cells ( P < 0.05). The inability of a 20-HETE analog to reproduce these effects suggested the involvement of a metabolic product of 20-HETE in mediating its pro-adipogenic effects. A cyclooxygenase (COX)-1 selective inhibitor enhanced, whereas a COX-2 selective or a dual COX-1/2 inhibitor attenuated adipogenesis induced by 20-HETE. The COX-derived metabolite of 20-HETE, 20-OH-PGE 2 , enhanced adipogenesis and lipid accumulation in MSCs. The pro-adipogenic effects of 20-HETE and 20-OH-PGE 2 resulted in the increased expression of the adipogenic regulators PPAR and -catenin in MSC-derived adipocytes. Taken together we show for the fi rst time that 20-HETE-derived COX-2-dependent 20-OH-PGE 2 enhances mature infl amed adipocyte hypertrophy in MSC undergoing adipogenic differentiation. — Kim, D. H., N. Puri, K. Sodhi, J. R. Falck, N. G. Abraham, J. Shapiro, and M. L. Schwartzman. Cyclooxygenase-2 dependent metabolism of 20-HETE increasesadiposity and adipocyte enlargement in mesenchymal stem cell-derived adipocytes.

Rescuing Acetylcholinesterase From Nerve Agent Inhibition: Protein Dynamics Driven Drug Discovery, Aiyana M. Emigh, Brian Bennion

STAR Program Research Presentations

Severe morbidity and mortality consequences result from irreversible inhibition of human acetylcholinesterase by organophosphates (OPs). Oxime-based reactivators are currently the only available treatments but lack efficacy in the central nervous system (CNS) where the most damage occurs. Computational docking and molecular dynamics (MD) simulations reveal complex structural barriers that may reduce oxime efficacy. These results may guide future drug designs of more effective countermeasures.

Aβ Alters The Dna Methylation Status Of Cell-Fate Genes In An Alzheimer’S Disease Model, Gary D. Isaacs, Noor Taher, Courtney Mckenzie, Rebecca Garrett, Matthew Baker, Nena Fox

Faculty Publications and Presentations

Alzheimer’s disease (AD) is characterized by neurofibrillary tangles and extracellular amyloid-β plaques (Aβ). Despite ongoing research, some ambiguity remains surrounding the role of Aβ in the pathogenesis of this neurodegenerative disease. While several studies have focused on the mutations associated with AD, our understanding of the epigenetic contributions to the disease remains less clear. To that end, we determined the changes in DNA methylation in differentiated human neurons with and without Aβ treatment. We isolated the DNA from neurons treated with Aβ or vehicle, and digested the two samples with either a methylation-sensitive (HpaII) or a methylation-insensitive (MspI) restriction endonuclease. …

Enhanced Transmission Of Higher Order Plasmon Modes With Random Au Nanoparticles In Periodic Hole Arrays, Fang-Tzu Chuang

Fang-Tzu Chuang

The enhanced transmission of higher order plasmon modes with random gold nanoparticles embedded in periodic hole arrays using asymmetric pair aperture as a unit is investigated in the midinfrared regime. Different thicknesses of gold film were deposited inside holes and then annealed to form randomly sized and distributed nanoparticles. The holes deposited with thin gold film exhibit significantly enhanced transmission in higher order modes after thermal annealing. The enormous local electric field around the nanoparticles enhances the scattering effect that contributes to the enhanced infrared transmission. This unique design, which integrates localized and propagating surface plasmons, provides an easy way …

Electroporation-Mediated Gene Transfer Directly To The Swine Heart, Barbara Hargrave, Harre Downey, Cathryn Lundberg, Annelise Israel, Yeong-Jer Chen, Richard Heller

Bioelectrics Publications

In vivo gene transfer to the ischemic heart via electroporation holds promise as a potential therapeutic approach for the treatment of heart disease. In the current study, we investigated the use of in vivo electroporation for gene transfer using three different penetrating electrodes and one non-penetrating electrode. The hearts of adult male swine were exposed through a sternotomy. Eight electric pulses synchronized to the rising phase of the R wave of the electrocardiogram were administered at varying pulse widths and field strengths following an injection of either a plasmid encoding luciferase or one encoding green fluorescent protein. Four sites on …

Investigating Therapeutic Options For Lafora Disease Using Structural Biology And Translational Methods, Amanda R. Sherwood

Theses and Dissertations--Molecular and Cellular Biochemistry

Lafora disease (LD) is a rare yet invariably fatal form of epilepsy characterized by progressive degeneration of the central nervous and motor systems and accumulation of insoluble glucans within cells. LD results from mutation of either the phosphatase laforin, an enzyme that dephosphorylates cellular glycogen, or the E3 ubiquitin ligase malin, the binding partner of laforin. Currently, there are no therapeutic options for LD, or reported methods by which the specific activity of glucan phosphatases such as laforin can be easily measured. To facilitate our translational studies, we developed an assay with which the glucan phosphatase activity of laforin as …

Amalgamation Of Nucleosides And Amino Acids In Antibiotic Biosynthesis, Sandra H. Barnard

Theses and Dissertations--Pharmacy

The rapid increase in antibiotic resistance demands the identification of novel antibiotics with novel targets. One potential antibacterial target is the biosynthesis of peptidoglycan cell wall, which is both ubiquitous and necessary for bacterial survival. Both the caprazamycin-related compounds A-90289 and muraminomicin, as well as the capuramycin-related compounds A-503083 and A-102395 are potent inhibitors of the translocase I enzyme, one of the key enzymes required for cell wall biosynthesis. The caprazamycin-related compounds contain a core nonproteinogen b-hydroxy-a-amino acid referred to as 5’-C-glycyluridine (GlyU). Residing within the biosynthetic gene clusters of the aforementioned compounds is a shared open reading …