Life Sciences Commons^™

Endoplasmic Reticulum Stress-Induced Hepatocellular Death Pathways Mediate Liver Injury And Fibrosis Via Stimulator Of Interferon Genes., Arvin Iracheta-Vellve, Jan Petrasek, Benedek Gyongyosi, Abhishek Satishchandran, Patrick Lowe, Karen Kodys, Donna Catalano, Charles D. Calenda, Evelyn A. Kurt-Jones, Kate A. Fitzgerald, Gyongyi Szabo

Katherine A. Fitzgerald

Fibrosis, driven by inflammation, marks the transition from benign to progressive stages of chronic liver diseases. Although inflammation promotes fibrogenesis, it is not known whether other events, such as hepatocyte death, are required for the development of fibrosis. Interferon Regulatory Factor 3 (IRF3) regulates hepatocyte apoptosis and production of Type-I interferons (IFNs). In the liver, IRF3 is activated via Toll-like receptor 4 (TLR4) signaling or the ER adapter, Stimulator of Interferon Genes (STING). We hypothesized that IRF3-mediated hepatocyte death is an independent determinant of chemically-induced liver fibrogenesis. To test this, we performed acute or chronic carbontetrachloride (CCl4) administration to WT, …

Endoplasmic Reticulum Stress-Induced Hepatocellular Death Pathways Mediate Liver Injury And Fibrosis Via Stimulator Of Interferon Genes., Arvin Iracheta-Vellve, Jan Petrasek, Benedek Gyongyosi, Abhishek Satishchandran, Patrick Lowe, Karen Kodys, Donna Catalano, Charles D. Calenda, Evelyn A. Kurt-Jones, Kate A. Fitzgerald, Gyongyi Szabo

Gyongyi Szabo

Fibrosis, driven by inflammation, marks the transition from benign to progressive stages of chronic liver diseases. Although inflammation promotes fibrogenesis, it is not known whether other events, such as hepatocyte death, are required for the development of fibrosis. Interferon Regulatory Factor 3 (IRF3) regulates hepatocyte apoptosis and production of Type-I interferons (IFNs). In the liver, IRF3 is activated via Toll-like receptor 4 (TLR4) signaling or the ER adapter, Stimulator of Interferon Genes (STING). We hypothesized that IRF3-mediated hepatocyte death is an independent determinant of chemically-induced liver fibrogenesis. To test this, we performed acute or chronic carbontetrachloride (CCl4) administration to WT, …

Jmh Dissertation 2016.Pdf, Jennifer Hayashi

Jennifer Hayashi

Characterization Of Wy 14,643 And Its Complex With Aldose Reductase, Michael R. Sawaya, Malkhey Verma, Vaishnavi Balendiran, Nigam P. Rath, Duilio Cascio, Ganesaratnam K. Balendiran

Nigam Rath

Rna Sequencing Analysis Of The Developing Chicken Retina, Christophe Langouet-Astrie*, Annamarie Meinsen*, Emily R. Grunwald*, Stephen Turner, Raymond A. Enke

Ray Enke Ph.D.

Involvement Of Reactive Oxygen Species In A Feed-Forward Mechanism Of Na/K-Atpase Mediated Signaling, Yanling Yan, Anna P. Shapiro, Steven Haller, Vinal Katragadda, Lijun Liu, Jiang Tian, Venkatesha Basrur, Deepak Malhotra, Zi-Jian Xie, Nader G. Abraham, Joseph I. Shapiro Md, Jiang Liu

Yanling Yan

Cardiotonic steroids (such as ouabain) signaling through Na/K-ATPase regulate sodium reabsorption in the renal proximal tubule. We report here that reactive oxygen species are required to initiate ouabain-stimulated Na/K-ATPase·c-Src signaling. Pretreatment with the antioxidant N-acetyl-L-cysteine prevented ouabain-stimulated Na/K-ATPase·c-Src signaling, protein carbonylation, redistribution of Na/K-ATPase and sodium/proton exchanger isoform 3, and inhibition of active transepithelial ²²Na⁺ transport. Disruption of the Na/K-ATPase·c-Src signaling complex attenuated ouabain-stimulated protein carbonylation. Ouabain-stimulated protein carbonylation is reversed after removal of ouabain, and this reversibility is largely independent of de novo protein synthesis and degradation by either the lysosome or the proteasome pathways. Furthermore, …

Connecting Common Genetic Polymorphisms To Protein Function: A Modular Project Sequence For Lecture Or Lab, Christopher E. Berndsen, Byron H. Young, Quinlin Mccormick*, Raymond A. Enke

Ray Enke Ph.D.

Involvement Of Reactive Oxygen Species In A Feed-Forward Mechanism Of Na/K-Atpase Mediated Signaling, Yanling Yan, Anna P. Shapiro, Steven Haller, Vinal Katragadda, Lijun Liu, Jiang Tian, Venkatesha Basrur, Deepak Malhotra, Zi-Jian Xie, Nader G. Abraham, Joseph I. Shapiro Md, Jiang Liu

Zijian Xie

Cardiotonic steroids (such as ouabain) signaling through Na/K-ATPase regulate sodium reabsorption in the renal proximal tubule. We report here that reactive oxygen species are required to initiate ouabain-stimulated Na/K-ATPase·c-Src signaling. Pretreatment with the antioxidant N-acetyl-L-cysteine prevented ouabain-stimulated Na/K-ATPase·c-Src signaling, protein carbonylation, redistribution of Na/K-ATPase and sodium/proton exchanger isoform 3, and inhibition of active transepithelial ²²Na⁺ transport. Disruption of the Na/K-ATPase·c-Src signaling complex attenuated ouabain-stimulated protein carbonylation. Ouabain-stimulated protein carbonylation is reversed after removal of ouabain, and this reversibility is largely independent of de novo protein synthesis and degradation by either the lysosome or the proteasome pathways. Furthermore, …

Stress-Induced Alternative Splice Forms Of Mdm2 And Mdmx Modulate The P53-Pathway In Distinct Ways, Matthew F. Rouhier, Aishwarya G. Jacob, Ravi K. Singh, Daniel F. Comiskey Jr, Fuad Mohammad, Thomas W. Bebee, Dawn S. Chandler

Matthew F Rouhier

MDM2 and MDMX are the chief negative regulators of the tumor-suppressor protein p53 and are essential for maintaining homeostasis within the cell. In response to genotoxic stress and also in several cancer types, MDM2 and MDMX are alternatively spliced. The splice variants MDM2-ALT1 and MDMX-ALT2 lack the p53-binding domain and are incapable of negatively regulating p53. However, they retain the RING domain that facilitates dimerization of the full-length MDM proteins. Concordantly, MDM2-ALT1 has been shown to lead to the stabilization of p53 through its interaction with and inactivation of full-length MDM2. The impact of MDM2-ALT1 expression on the p53 pathway …

Pharmacological Validation Of An Inward-Rectifier Potassium (Kir) Channel As An Insecticide Target In The Yellow Fever Mosquito Aedes Aegypti, Matthew F. Rouhier, Rene Raphemot, Jerod S. Denton, Peter M. Piermarini

Matthew F Rouhier

Mosquitoes are important disease vectors that transmit a wide variety of pathogens to humans, including those that cause malaria and dengue fever. Insecticides have traditionally been deployed to control populations of disease-causing mosquitoes, but the emergence of insecticide resistance has severely limited the number of active compounds that are used against mosquitoes. Thus, to improve the control of resistant mosquitoes there is a need to identify new insecticide targets and active compounds for insecticide development. Recently we demonstrated that inward rectifier potassium (Kir) channels and small molecule inhibitors of Kir channels offer promising new molecular targets and active compounds, respectively, …

Discovery And Characterization Of A Potent And Selective Inhibitory Of Aedes Aegypti Inward Rectifier Potassium Channels, Matthew F. Rouhier, Rene Raphemot, Daniel R. Swale, Emily Days, C. David Weaver, Kimberly M. Lovell, Leah C. Konkel, Darren W. Engers, Sean F. Bollinger, Corey Hopkins, Peter M. Piermarini, Jerod S. Denton

Matthew F Rouhier

Vector-borne diseases such as dengue fever and malaria, which are transmitted by infected female mosquitoes, affect nearly half of the world's population. The emergence of insecticide-resistant mosquito populations is reducing the effectiveness of conventional insecticides and threatening current vector control strategies, which has created an urgent need to identify new molecular targets against which novel classes of insecticides can be developed. We previously demonstrated that small molecule inhibitors of mammalian Kir channels represent promising chemicals for new mosquitocide development. In this study, high-throughput screening of approximately 30,000 chemically diverse small-molecules was employed to discover potent and selective inhibitors of Aedes …

Eliciting Renal Failure In Mosquitoes With A Small-Molecule Inhibitor Of Inward-Rectifying Potassium Channels, Matthew F. Rouhier, Rene Raphemot, Corey R. Hopkins, Rocco D. Gogliotti, Kimberly M. Lovel, Rebecca M. Hine, Dhairyasheel Ghosalkar, Anthony Longo, Klaus W. Beyenbach, Jerod S. Denton, Peter M. Piermarini

Matthew F Rouhier

Mosquito-borne diseases such as malaria and dengue fever take a large toll on global health. The primary chemical agents used for controlling mosquitoes are insecticides that target the nervous system. However, the emergence of resistance in mosquito populations is reducing the efficacy of available insecticides. The development of new insecticides is therefore urgent. Here we show that VU573, a small-molecule inhibitor of mammalian inward-rectifying potassium (Kir) channels, inhibits a Kir channel cloned from the renal (Malpighian) tubules of Aedes aegypti (AeKir1). Injection of VU573 into the hemolymph of adult female mosquitoes (Ae. aegypti) disrupts the production and excretion of urine …

Heterologous Expression Of A Rice Mir395 Gene In Nicotiana Tabacum Impairs Sulfate Homeostasis, Ning Yuan, Shuangrong Yuan, Zhigang Li, Dayong Li, Qian Hu, Hong Luo

Hong Luo

Sulfur participates in many important mechanisms and pathways of plant development. The most common source of sulfur in soil –SO42−– is absorbed into root tissue and distributed into aerial part through vasculature system, where it is reduced into sulfite and finally sulfide within the subcellular organs such as chloroplasts and mitochondria and used for cysteine and methionine biosynthesis. MicroRNAs are involved in many regulation pathways by repressing the expression of their target genes. MiR395 family in Arabidopsis thaliana has been reported to be an important regulator involved in sulfate transport and assimilation, and a high-affinity sulphate transporter and three ATP …

Ectopic Expression Of A Cyanobacterial Flavodoxin In Creeping Bentgrass Impacts Plant Development And Confers Broad Abiotic Stress Tolerance, Zhigang Li, Shuangrong Yuan, Haiyan Jia, Fangyuan Gao, Qian Hu, Dongfa Sun, Hong Luo

Hong Luo

Flavodoxin (Fld) plays a pivotal role in photosynthetic microorganisms as an alternative electron carrier flavoprotein under adverse environmental conditions. Cyanobacterial Fld has been demonstrated to be able to substitute ferredoxin (Fd) of higher plants in most electron transfer processes under stressful conditions. We have explored the potential of Fld for use in improving plant stress response in creeping bentgrass (Agrostis stolonifera L.). Overexpression of Fld altered plant growth and development. Most significantly, transgenic (TG) plants exhibited drastically enhanced performance under oxidative, drought and heat stress as well as nitrogen (N) starvation, which was associated with higher water retention and …

Snp-Based High Density Genetic Map And Mapping Of Btwd1 Dwarfing Gene In Barley, Xifeng Ren, Jibin Wang, Lipan Liu, Genlou Sun, Chengdao Li, Hong Luo, Dongfa Sun

Hong Luo

A high-density linkage map is a valuable tool for functional genomics and breeding. A newly developed sequence-based marker technology, restriction site associated DNA (RAD) sequencing, has been proven to be powerful for the rapid discovery and genotyping of genome-wide single nucleotide polymorphism (SNP) markers and for the high-density genetic map construction. The objective of this research was to construct a high-density genetic map of barley using RAD sequencing. 1894 high-quality SNP markers were developed and mapped onto all seven chromosomes together with 68 SSR markers. These 1962 markers constituted a total genetic length of 1375.8 cM and an average of …

Activation-Triggered Subunit Exchange Between Camkii Holoenzymes Facilitates The Spread Of Kinase Activity, Margaret M. Stratton, I H. Lee, M Bhattacharyya, S M. Christensen, L H. Chao, H Schulman, J T. Groves, J Kuriyan

Margaret Stratton

The activation of the dodecameric Ca2+/calmodulin dependent kinase II (CaMKII) holoenzyme is critical for memory formation. We now report that CaMKII has a remarkable property, which is that activation of the holoenzyme triggers the exchange of subunits between holoenzymes, including unactivated ones, enabling the calcium-independent phosphorylation of new subunits. We show, using a single-molecule TIRF microscopy technique, that the exchange process is triggered by the activation of CaMKII, and that exchange is modulated by phosphorylation of two residues in the calmodulin-binding segment, Thr 305 and Thr 306. Based on these results, and on the analysis of molecular dynamics simulations, we …

A Simplified Direct Lipid Mixing Lipoplex Preparation: Comparison Of Liposomal-, Dimethylsulfoxide-, And Ethanol-Based Methods, Joseph W. Meisel, George W. Gokel

George Gokel

Characterization Of Ceriporiopsis Subvermispora Bicupin Oxalate Oxidase Expressed In Pichia Pastoris, Patricia Moussatche, Alexander Angerhofer, Witcha Imaram, Eric Hoffer, Kelsey Uberto, Christopher Brooks, Crystal Bruce, Daniel Sledge, Nigel G. J. Richards, Ellen W. Moomaw

Ellen Moomaw

Oxalate oxidase (E.C. 1.2.3.4) catalyzes the oxygen-dependent oxidation of oxalate to carbon dioxide in a reaction that is coupled with the formation of hydrogen peroxide. Although there is currently no structural information available for oxalate oxidase fromCeriporiopsis subvermispora (CsOxOx), sequence data and homology modeling indicate that it is the first manganese-containing bicupin enzyme identified that catalyzes this reaction. Interestingly, CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC). We show that CsOxOx activity directly correlates with Mn content and other metals do not appear to be able to support catalysis. EPR spectra indicate that the Mn is present …

Membrane Inlet Mass Spectrometry Reveals That Ceriporiopsis Subvermispora Bicupin Oxalate Oxidase Is Inhibited By Nitric Oxide, Ellen W. Moomaw, Richard Uberto, Tu Chingkuang

Ellen Moomaw

Membrane inlet mass spectrometry (MIMS) uses a semipermeable membrane as an inlet to a mass spectrometer for the measurement of the concentration of small uncharged molecules in solution. We report the use of MIMS to characterize the catalytic properties of oxalate oxidase (E.C. 1.2.3.4) from Ceriporiopsis subvermispora (CsOxOx). Oxalate oxidase is a manganese dependent enzyme that catalyzes the oxygen-dependent oxidation of oxalate to carbon dioxide in a reaction that is coupled with the formation of hydrogen peroxide. CsOxOx is the first bicupin enzyme identified that catalyzes this reaction. The MIMS method of measuring OxOx activity involves continuous, real-time direct detection …

Topographical Expression Of Class Ia And Class Ii Phosphoinositide 3-Kinase Enzymes In Normal Human Tissues Is Consistent With A Role In Differentiation, Soha Salama El Sheikh, Jan Domin, Prakitpunthu Tomtitchong, Paul Abel, Gordon Stamp, El-Nasir Lalani

El Nasir Lalani

Background: Growth factor, cytokine and chemokine-induced activation of PI3K enzymes constitutes the start of a complex signalling cascade, which ultimately mediates cellular activities such as proliferation, differentiation, chemotaxis, survival, trafficking, and glucose homeostasis. The PI3K enzyme family is divided into 3 classes; class I (subdivided into IA and IB), class II (PI3K-C2α, PI3K-C2β and PI3K-C2γ) and class III PI3K. Expression of these enzymes in human tissue has not been clearly defined.

Methods: In this study, we analysed the immunohistochemical topographical expression profile of class IA (anti-p85 adaptor) and class II PI3K (PI3K-C2α and PI3K-C2β) enzymes in 104 formalin-fixed, paraffin embedded …

App Regulates Microglial Phenotype In A Mouse Model Of Alzheimer's Disease, Gunjan D. Manocha, Angela M. Floden, Keiko Rausch, Joshua A. Kulas, Brett A. Mcgregor, Lalida Rojanathammanee, Kelley R. Puig, Kendra L. Puig, Sanjib Karki, Michael R. Nichols, Diane C. Darland, James E. Porter, Colin K. Combs

Michael Nichols

Structural And Molecular Analysis Of A Protective Epitope Of Lyme Disease Antigen Ospa And Antibody Interactions, Shivender Shandilya, Nese Kurt Yilmaz, Ejemel Monir, Andrew Sadowski, William D. Thomas, Mark S. Klempner, Celia A. Schiffer, Yan Wang

Celia A. Schiffer

The murine monoclonal antibody LA-2 recognizes a clinically protective epitope on outer surface protein (OspA) of Borrelia burgdorferi, the causative agent of Lyme disease in North America. Human antibody equivalence to LA-2 is the best serologic correlate of protective antibody responses following OspA vaccination. Understanding the structural and functional basis of the LA-2 protective epitope is important for developing OspA-based vaccines and discovering prophylactic antibodies against Lyme disease. Here, we present a detailed structure-based analysis of the LA-2/OspA interaction interface and identification of residues mediating antibody recognition. Mutations were introduced into both OspA and LA-2 based on computational predictions on …

P-Rex1 Promotes Resistance To Vegf/Vegfr-Targeted Therapy In Prostate Cancer, Hira Lal Goel, Bryan M. Pursell, Leonard D. Shultz, Dale L. Greiner, Rolf A. Brekken, Craig W. Vander Kooi, Arthur M. Mercurio

Arthur M. Mercurio

Autocrine VEGF signaling is critical for sustaining prostate and other cancer stem cells (CSCs), and it is a potential therapeutic target, but we observed that CSCs isolated from prostate tumors are resistant to anti-VEGF (bevacizumab) and anti-VEGFR (sunitinib) therapy. Intriguingly, resistance is mediated by VEGF/neuropilin signaling, which is not inhibited by bevacizumab and sunitinib, and it involves the induction of P-Rex1, a Rac GEF, and consequent Rac1-mediated ERK activation. This induction of P-Rex1 is dependent on Myc. CSCs isolated from the PTEN(pc-/-) transgenic model of prostate cancer exhibit Rac1-dependent resistance to bevacizumab. Rac1 inhibition or P-Rex1 downregulation increases the sensitivity …

Isothermal Titration Calorimetry Uncovers Substrate Promiscuity Of Bicupin Oxalate Oxidase From Ceriporiopsis Subvermispora, Hassan Rana, Patricia Moussatche, Lis Souza Rocha, Ellen W. Moomaw

Ellen Moomaw

Isothermal titration calorimetry (ITC) may be used to determine the kinetic parameters of enzymecatalyzed reactions when neither products nor reactants are spectrophotometrically visible and when the reaction products are unknown. We report here the use of the multiple injection method of ITC to characterize the catalytic properties of oxalate oxidase (OxOx) from Ceriporiopsis subvermispora (CsOxOx), a manganese dependent enzyme that catalyzes the oxygen-dependent oxidation of oxalate to carbon dioxide in a reaction coupled with the formation of hydrogen peroxide. CsOxOx is the first bicupin enzyme identified that catalyzes this reaction. The multiple injection ITC method of measuring OxOx activity involves …

Real-Time Kinetic Studies Of Bacillus Subtilis Oxalate Decarboxylase And Ceriporiopsis Subvermispora Oxalate Oxidase Using Luminescent Oxygen Sensor, Laura Molina, Thomas Goodall, Umar Twahir, Ellen W. Moomaw

Ellen Moomaw

Oxalate decarboxylase (OxDC), an enzyme of the bicupin superfamily, catalyzes the decomposition of oxalate into carbon dioxide and formate at an optimal pH of 4.3 in the presence of oxygen. However, about 0.2% of all reactions occur through an oxidase mechanism that consumes oxygen while producing two equivalents of carbon dioxide and one equivalent of hydrogen peroxide. The kinetics of oxidase activity were studied by measuring the consumption of dissolved oxygen over time using a luminescent oxygen sensor. We describe the implementation of and improvements to the oxygen consumption assay. The oxidase activity of wild type OxDC was compared to …

Protein Similarity Networks Reveal Relationships Among Sequence, Structure, And Function Within The Cupin Superfamily, Richard Uberto, Ellen W. Moomaw

Ellen Moomaw

The cupin superfamily is extremely diverse and includes catalytically inactive seed storage proteins, sugar-binding metal-independent epimerases, and metal-dependent enzymes possessing dioxygenase, decarboxylase, and other activities. Although numerous proteins of this superfamily have been structurally characterized, the functions of many of them have not been experimentally determined. We report the first use of protein similarity networks (PSNs) to visualize trends of sequence and structure in order to make functional inferences in this remarkably diverse superfamily. PSNs provide a way to visualize relatedness of structure and sequence among a given set of proteins. Structure- and sequence-based clustering of cupin members reflects functional …

Characterization Of Ceriporiopsis Subvermispora Bicupin Oxalate Oxidase Expressed In Pichia Pastoris, Patricia Moussatche, Eric Hoffer, Ellen W. Moomaw, Et Al.

Ellen Moomaw

Oxalate oxidase (E.C. 1.2.3.4) catalyzes the oxygen-dependent oxidation of oxalate to carbon dioxide in a reaction that is coupled with the formation of hydrogen peroxide. Although there is currently no structural information available for oxalate oxidase from Ceriporiopsis subvermispora (CsOxOx), sequence data and homology modeling indicate that it is the first manganese-containing bicupin enzyme identified that catalyzes this reaction. Interestingly, CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC). We show that CsOxOx activity directly correlates with Mn content and other metals do not appear to be able to support catalysis. EPR spectra indicate that the Mn is …

Fungal Oxalate Decarboxylase Activity Contributes To Sclerotinia Sclerotiorum Early Infection By Affecting Both Compound Appressoria Development And Function, Xiaofei Liang, Ellen W. Moomaw, Jeffrey A. Rollins

Ellen Moomaw

Sclerotinia sclerotiorum pathogenesis requires the accumulation of high levels of oxalic acid (OA). To better understand the factors affecting OA accumulation, two putative oxalate decarboxylase (OxDC) genes (Ss-odc1 and Ss-odc2) were characterized. Ss-odc1 transcripts exhibited significant accumulation in vegetative hyphae, apothecia, early stages of compound appressorium development and during plant colonization. Ss-odc2 transcripts, in contrast, accumulated significantly only during mid to late stages of compound appressorium development. Neither gene was induced by low pH or exogenous OA in vegetative hyphae. A loss-of-function mutant for Ss-odc1 (Δss-odc1) showed wild-type growth, morphogenesis and virulence, and was not characterized further. Δss-odc2 mutants hyperaccumulated …

Kinetic And Spectroscopic Studies Of Bicupin Oxalate Oxidase And Putative Active Site Mutants, Ellen W. Moomaw, Eric Hoffer, Patricia Moussatche, John C. Salerno

Ellen Moomaw

Ceriporiopsis subvermispora oxalate oxidase (CsOxOx) is the first bicupin enzyme identified that catalyzes manganese-dependent oxidation of oxalate. In previous work, we have shown that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated. CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC) and the 241-244DASN region of the N-terminal Mn binding domain of CsOxOx is analogous to the lid region of OxDC that has been shown to determine reaction specificity. We have prepared a series of CsOxOx …

Kinetic And Spectroscopic Studies Of Bicupin Oxalate Oxidase And Putative Active Site Mutants, Ellen W. Moomaw, Eric Hoffer, Patricia Moussatche, John C. Salerno

Ellen Moomaw

Ceriporiopsis subvermispora oxalate oxidase (CsOxOx) is the first bicupin enzyme identified that catalyzes manganese-dependent oxidation of oxalate. In previous work, we have shown that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated. CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC) and the 241-244DASN region of the N-terminal Mn binding domain of CsOxOx is analogous to the lid region of OxDC that has been shown to determine reaction specificity. We have prepared a series of CsOxOx …