Biochemistry Commons^™

Substrate And Regulation Of Mitochondrial Μ-Calpain, Aashish Joshi

University of Kentucky Doctoral Dissertations

μ -Calpain is localized to the mitochondrial intermembrane space. Apoptosisinducing factor (AIF), which executes caspase-independent cell death, is also localized to the mitochondrial intermembrane space. Following processing at the N-terminus, AIF becomes truncated (tAIF) and is released from mitochondria. The protease responsible for AIF processing has not been established. The same submitochondrial localization of mitochondrial μ-calpain and AIF gives support to the hypothesis that mitochondrial μ-calpain may be responsible for processing AIF. Atractyloside-induced tAIF release in rat liver mitochondria was inhibited by cysteine protease inhibitor MDL28170, but not by calpain inhibitors PD150606 or calpastatin. Moreover, μ-calpain immunoreactivity was difficult to …

Protection Of Macrophages J774a.1 By Purine Nucleoside Analogues From Bacillus Anthracis Mediated Necrosis, Zadkiel R. Alvarez

UNLV Theses, Dissertations, Professional Papers, and Capstones

Eight years after the lamentable anthrax attacks, major scientific effort continues to be done, in order to stop imminent acts of bioterrorism. Innovative ways of therapy against the anthrax disease are being investigated. B. anthracis, the etiological agent of the infection, has a dormant stage in its life cycle known as the endospore. When conditions become favorable spores germinate, transforming into vegetative bacteria. In inhalational anthrax, the most fatal manifestation of the disease, spores enter the organism through the respiratory track, and are phagocytosized by alveolar macrophages of the lungs. Spores are able to sense nutrient availability, activating their germination …

Characterization Of The Ompl1 Gene Of Pathogenic Leptospira Species In China And Cross-Immunogenicity Of The Ompl1 Protein, Haiyan Dong, Ye Hu, Feng Xue, Dexter Sun, David M. Ojcius, Yafei Mao, Jie Yan

All Dugoni School of Dentistry Faculty Articles

Background: The usefulness of available vaccine and serological tests for leptospirosis is limited by the low cross-reactivity of antigens from numerous serovars of pathogenic Leptospira spp. Identification of genus-specific protein antigens (GP-Ag) of Leptospira would be important for development of universal vaccines and serodiagnostic methods. OmpL1, a transmembrane porin of pathogenic leptospires, was identified as a possible GP-Ag, but its sequence diversity and immune cross-reactivity among different serovars of pathogenic leptospires remains largely unknown.

Results: PCR analysis demonstrated that the ompL1 gene existed in all 15 official Chinese standard strains as well as 163 clinical strains of pathogenic leptospires isolated …

Molecular Characterisation Of A Bovine-Like Rotavirus Detected From A Giraffe, Emily Mulherin, Jill Bryan, Marijke Beltman, Luke O'Grady, Eugene Pidgeon, Lucie Garon, Andrew Lloyd, John Bainbridge, Helen O'Shea, Paul Whyte, Séamus Fanning

Department of Biological Sciences Publications

Background

Rotavirus (RV), is a member of the Reoviridae family and an important etiological agent of acute viral gastroenteritis in the young. Rotaviruses have a wide host range infecting a broad range of animal species, however little is known about rotavirus infection in exotic animals. In this paper we report the first characterisation of a RV strain from a giraffe calf.

Results

This report describes the identification and detailed molecular characterisation of a rotavirus strain detected from a 14-day-old Giraffe (Giraffa camelopardalis), presenting with acute diarrhea. The RV strain detected from the giraffe was characterized molecularly as G10P[11]. …

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

Biochemistry and Microbiology

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 …

Aminoacyl-Trna Synthetase Complexes: Molecular Multitasking Revealed, Corinne D. Hausmann, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

The accurate synthesis of proteins, dictated by the corresponding nucleotide sequence encoded in mRNA, is essential for cell growth and survival. Central to this process are the aminoacyl-tRNA synthetases (aaRSs), which provide amino acid substrates for the growing polypeptide chain in the form of aminoacyl-tRNAs. The aaRSs are essential for coupling the correct amino acid and tRNA molecules, but are also known to associate in higher order complexes with proteins involved in processes beyond translation. Multiprotein complexes containing aaRSs are found in all three domains of life playing roles in splicing, apoptosis, viral assembly, and regulation of transcription and translation. …

Critical Involvement Of The Atm-Dependent Dna Damage Response In The Apoptotic Demise Of Hiv-1-Elicited Syncytia, Jean-Luc Perfettini, Roberta Nardacci, Mehdi Bourouba, Frederic Subra, Laurent Gros, Claire Séror, Gwenola Manic, Filippo Rosselli, Alessandra Amendola, Peggy Masdehors, Luciana Chessa, Giuseppe Novelli, David M. Ojcius, Jan Konrad Siwicki, Magdalena Chechlinska, Christian Auclair, Jose R. Regueiro, Hughes De Thé, Marie-Lise Gougeon, Mauro Piacentini, Guido Kroemer

All Dugoni School of Dentistry Faculty Articles

DNA damage can activate the oncosuppressor protein ataxia telangiectasia mutated (ATM), which phosphorylates the histone H2AX within characteristic DNA damage foci. Here, we show that ATM undergoes an activating phosphorylation in syncytia elicited by the envelope glycoprotein complex (Env) of human immunodeficiency virus-1 (HIV-1) in vitro. This was accompanied by aggregation of ATM in discrete nuclear foci that also contained phospho-histone H2AX. DNA damage foci containing phosphorylated ATM and H2AX were detectable in syncytia present in the brain or lymph nodes from patients with HIV-1 infection, as well as in a fraction of blood leukocytes, correlating with viral status. Knockdown …

Structural And Functional Mapping Of The Archaeal Multi-Aminoacyl-Trna Synthetase Complex, Corinne D. Hausmann, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Methanothermobacter thermautotrophicus contains a multi-aminoacyl-tRNA synthetase complex (MSC) of LysRS, LeuRS and ProRS. Elongation factor (EF) 1A also associates to the MSC, with LeuRS possibly acting as a core protein. Analysis of the MSC revealed that LysRS and ProRS specifically interact with the idiosyncratic N- and C- termini of LeuRS, respectively. EF-1A instead interacts with the inserted CP1 proofreading domain, consistent with models for post-transfer editing by class I synthetases such as LeuRS. Together with previous genetic data, these findings show that LeuRS plays a central role in mediating interactions within the archaeal MSC by acting as a core scaffolding …

Atf4 Is An Oxidative Stress–Inducible, Prodeath Transcription Factor In Neurons In Vitro And In Vivo, Philipp Lange, Juan Chavez, John T. Pinto, Giovanni Coppola, Chiao-Wang Sun, Tim Townes, Rajiv Ratan

NYMC Faculty Publications

Oxidative stress is pathogenic in neurological diseases, including stroke. The identity of oxidative stress-inducible transcription factors and their role in propagating the death cascade are not well known. In an in vitro model of oxidative stress, the expression of the bZip transcription factor activating transcription factor 4 (ATF4) was induced by glutathione depletion and localized to the promoter of a putative death gene in neurons. Germline deletion of ATF4 resulted in a profound reduction in oxidative stress-induced gene expression and resistance to oxidative death. In neurons, ATF4 modulates an early, upstream event in the death pathway, as resistance to oxidative …

Susceptibility Of Chlamydia Trachomatis To The Excipient Hydroxy-Ethylcellulose: Ph And Concentration Dependence Of Antimicrobial Activity, Ali A. Abdul-Sater, David M. Ojcius, M. P. Meyer

All Dugoni School of Dentistry Faculty Articles

Hydroxyethyl cellulose (HEC) is used as a neutral excipient in microbicides used against sexually transmitted pathogens. However, HEC inhibits the infection of cervical epithelial cells by Chlamydia trachomatis at pH 5 in a concentration-dependent manner. At pH 7, infection is inversely dependent on the concentration of HEC, possibly due to pH-dependent calcium sequestration.

The Influence Of Citrate, Maltolate And Fluoride On The Gastrointestinal Absorption Of Aluminum At A Drinking Water-Relevant Concentration: A 26Al And 14C Study, Yuzhao Zhou, Wesley R. Harris, Robert A. Yokel

Pharmaceutical Sciences Faculty Publications

The objectives were to test the null hypotheses that (1) citrate, maltolate, and fluoride do not significantly influence oral Al bioavailability, C_max or T_max at an Al dose relevant to drinking water exposure; and (2) Al citrate and maltolate are absorbed intact from the gastrointestinal tract. Male Fisher rats were given 1 ml of solution intra-gastrically containing 1 nCi ²⁶Al (65 nmol total Al) as the Al³⁺ ion, or as complexes with ¹⁴C-citrate, ¹⁴C-maltolate or fluoride, during concurrent ²⁷Al iv infusion. Blood was repeatedly collected for serum ²⁶Al, total Al and ^{14 …}

Treg Depletion Inhibits Efficacy Of Cancer Immunotherapy: Implications For Clinical Trials., James Curtin, Marianela Candolfi, Tamer Fakhouri, Chunyan Liu, Anderson Alden, Matthew Edwards, Pedro Lowenstein, Maria Castro

Articles

BACKGROUND: Regulatory T lymphocytes (Treg) infiltrate human glioblastoma (GBM); are involved in tumor progression and correlate with tumor grade. Transient elimination of Tregs using CD25 depleting antibodies (PC61) has been found to mediate GBM regression in preclinical models of brain tumors. Clinical trials that combine Treg depletion with tumor vaccination are underway to determine whether transient Treg depletion can enhance anti-tumor immune responses and improve long term survival in cancer patients. FINDINGS: Using a syngeneic intracrabial glioblastoma (GBM) mouse model we show that systemic depletion of Tregs 15 days after tumor implantation using PC61 resulted in a decrease in Tregs …

Retinoic Acid Decreases Atf-2 Phosphorylation And Sensitizes Melanoma Cells To Taxol-Mediated Growth Inhibition, Ying Huang, Jennifer Minigh, Sarah Miles, Richard N. Niles

Biochemistry and Microbiology

Cutaneous melanoma is often resistant to chemo- and radiotherapy. This resistance has recently been demonstrated to be due, at least in part, to high activating transcription factor 2 (ATF-2) activity in these tumors. In concordance with these reports, we found that B16 mouse melanoma cells had higher levels of ATF-2 than immortalized, but non-malignant mouse melanocytes. In addition, the melanoma cells had a much higher amount of phosphorylated (active) ATF-2 than the immortalized melanocytes. In the course of determining how retinoic acid (RA) stimulates activating protein-1 (AP-1) activity in B16 melanoma, we discovered that this retinoid decreased the phosphorylation of …

Novel Role Of Antioxidant-1 (Atox1) As A Copper-Dependent Transcription Factor Involved In Cell Proliferation, S. Itoh, H. W. Kim, O. Nakagawa, K. Ozumi, Susan M. Lessner, H. Aoki, K. Akram, R. D. Mckinney, M. Ushio-Fukai, T. Fukai

Faculty Publications

Copper plays a fundamental role in regulating cell growth. Many types of human cancer tissues have higher copper levels than normal tissues. Copper can also induce gene expression. However, transcription factors that mediate copper-induced cell proliferation have not been identified in mammals. Here we show that antioxidant-1 (Atox1), previously appreciated as a copper chaperone, represents a novel copper-dependent transcription factor that mediates copper-induced cell proliferation. Stimulation of mouse embryonic fibroblasts (MEFs) with copper markedly increased cell proliferation, cyclin D1 expression, and entry into S phase, which were completely abolished in Atox1^-/- MEFs. Promoter analysis and EMSA revealed that copper …

Monitoring Lys-TrnaLys Phosphatidylglycerol Transferase Activity, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

In some bacteria Lys-tRNA^Lys is used both in translation and for the specific addition of Lys to phosphatidylglycerol in the cytoplasmic membrane. This reaction is catalyzed by the membrane protein MprF, and the lysyl-phosphatidylglycerol formed contributes to the resistance of these bacteria to various cationic antibacterial molecules. Obtaining proteins and reconstituting an in vitro system mimicking membrane conditions is a major challenge to studying the function of membrane proteins, especially when labile substrates such as Lys-tRNA^Lys are required. Here we report methods to obtain a stable enriched membrane fraction containing MprF, and the techniques necessary to quantitatively monitor …

A Watershed Analysis Of Pattee Pond: Implications For Water Quality And Land Use Management, Problems In Environmental Science Course (Biology 493), Colby College, Colby Environmental Assessment Team, Colby College

Colby College Watershed Study: Pattee Pond (2008, 1992)

The Colby Environmental Assessment Team (CEAT) investigated the impact of land use patterns on the water quality of Pattee Pond in Winslow, Maine, during the summer and fall of 2008. Physical, chemical, and biological characteristics of water quality were analyzed to evaluate the current health of the lake. Data collected were compared with previous studies conducted by the Maine Department of Environmental Protection (DEP) and CEAT to examine changes in water quality and land use over time. Trends in Pattee Pond water quality suggest an improvement since the 1970s. However, the lake is still experiencing algal blooms resulting from phosphorus …

Rapid Enzyme-Linked Immunosorbent Assay For Detection Of The Algal Toxin Domoic Acid, R Wayne Litaker, Thomas N. Stewart, Bich-Thuy L. Eberhart, John C. Wekell, Vera L. Trainer, Raphael M. Kudela, Peter E. Miller, Alice Roberts, Cassandra Hertz, Tyler A. Johnson, Greg Frankfurter, G. Jason Smith, Astrid Schnetzer, Joe Schumacker, Jonnette L. Bastian, Anthony Odell, Patrick Gentien, Dominique Le Gal, D Ransom Hardison, Patricia A. Tester

Natural Sciences and Mathematics | Faculty Scholarship

Domoic acid (DA) is a potent toxin produced by bloom-forming phytoplankton in the genus Pseudo-nitzschia, which is responsible for causing amnesic shellfish poisoning (ASP) in humans. ASP symptoms include vomiting, diarrhea, and in more severe cases confusion, loss of memory, disorientation, and even coma or death. This paper describes the development and validation of a rapid, sensitive, enzyme linked immunosorbent assay test kit for detecting DA using a monoclonal antibody. The assay gives equivalent results to those obtained using standard high performance liquid chromatography, fluorenylmethoxycarbonyl high performance liquid chromatography, or liquid chromatography—mass spectrometry methods. It has a linear range …

Enzymatic Processing Of Amelogenin During Continuous Crystallization Of Apatite, Vuk Uskoković, M.-K. Kim, W. Li, S. Habelitz

Pharmacy Faculty Articles and Research

Dental enamel forms through a protein-controlled mineralization and enzymatic degradation with a nanoscale precision that new engineering technologies may be able to mimic. Recombinant fulllength human amelogenin (rH174) and a matrix-metalloprotease (MMP-20) were employed in a pHstat titration system that enabled a continuous supply of calcium and phosphate ions over several days, mimicking the initial stages of matrix processing and crystallization in enamel in-vitro. Effects on the self-assembly and crystal growth from a saturated aqueous solution containing 0.4 mg/ml rH174 and MMP-20 with the weight ratio of 1:1000 with respect to rH174 were investigated. A transition from nanospheres to fibrous …

Urine Protein Electrophoresis: Comparison Of Agarose Gel And Capillary Zone Electrophoretic Techniques., Anne-Marie Curtin

Theses

Proteinuria is the excess excretion of protein in the urine and is found in a variety of conditions including renal diseases and neoplastic conditions such as multiple myeloma (MM). The investigation of proteinuria type through the use of electrophoresis is a useful and widely used non-invasive diagnostic tool. The aim of this study was to compare various agarose gel and capillary zone electrophoresis techniques in order to establish a method choice for the separation of protein in routine laboratories. Comparisons were made based on urine monoclonal peak quantification and protein fractions detected. An examination of a capillary zone electrophoresis (CZE) …

Gender Bias Of Cardiovascular Risk In Caucasian Type Ii Diabetics: A Haemostatic Explanation?, William Quirke

Theses

People with type II diabetes are disproportionately affected by cardiovascular disease compared to those without diabetes. The relative risk of cardiovascular disease caused by type II diabetes is higher in females than in males. This risk has not been fully explained by traditional risk factors and attention has turned to the possible role of haemostatic abnormalities in explaining the excess cardiovascular morbidity and mortality among diabetic females. We examined specific parameters of the coagulation (factor VII, factor VIII, von Willebrand factor, fibrinogen) and fibrinolytic (plasminogen activator inhibitor- 1, thrombin-activatable fibrinolysis inhibitor) system in 113 Irish Caucasian type II diabetics to …

Electrical Detection Of The Temperature Induced Melting Transition Of A Dna Hairpin Covalently Attached To Gold Interdigitated Microelectrodes, Greg P. Brewood, Yaswanth Rangineni, Daniel J. Fish, Ashwini Bhandiwad, David R. Evans, Raj Solanki, Albert S. Benight

Chemistry Faculty Publications and Presentations

The temperature induced melting transition of a self-complementary DNA strand covalently attached at the 5' end to the surface of a gold interdigitated microelectrode (GIME) was monitored in a novel, label-free, manner. The structural state of the hairpin was assessed by measuring four different electronic properties of the GIME (capacitance, impedance, dissipation factor and phase angle) as a function of temperature from 25 degrees C to 80 degrees C. Consistent changes in all four electronic properties of the GIME were observed over this temperature range, and attributed to the transition of the attached single-stranded DNA (ssDNA) from an intramolecular, folded …

Novel Functions Of Acyl-Coa Thioesterases And Acyltransferases As Auxiliary Enzymes In Peroxisomal Lipid Metabolism., Mary Hunt, Stefan Alexson

Articles

Peroxisomes are single membrane bound organelles present in almost all eukaryotic cells, and to date have been shown to contain approximately 60 identified enzymes involved in various metabolic pathways, including the oxidation of a variety of lipids. These lipids include very long-chain fatty acids, methyl branched fatty acids, prostaglandins, bile acid precursors, and xenobiotics that are either β-oxidized or α-oxidized in peroxisomes. The recent identification of several acyl-CoA thioesterases and acyltransferases in peroxisomes has revealed their various functions in acting as auxiliary enzymes in α- and β-oxidation in this organelle. To date, 9 functional acyl-CoA thioesterases and acyltransferases have been …

Discovery Of Platelet-Type 12-Human Lipoxygenase Selective Inhibitors By High-Throughput Screening Of Structurally Diverse Libraries., Joshua D. Deschamps, Jeffrey T. Gautschi, Stephanie Whitman, Tyler A. Johnson, Nadine C. Gassner, Phillip Crews, Theodore R. Holman

Natural Sciences and Mathematics | Faculty Scholarship

Human lipoxygenases (hLO) have been implicated in a variety of diseases and cancers and each hLO isozyme appears to have distinct roles in cellular biology. This fact emphasizes the need for discovering selective hLO inhibitors for both understanding the role of specific lipoxygenases in the cell and developing pharmaceutical therapeutics. To this end, we have modified a known lipoxygenase assay for high-throughput (HTP) screening of both the National Cancer Institute (NCI) and the UC Santa Cruz marine extract library (UCSC-MEL) in search of platelet-type 12-hLO (12-hLO) selective inhibitors. The HTP screen led to the characterization of five novel 12-hLO inhibitors …

A Role For The Forebrain In Mediating Time-Of-Day Differences In Glucocorticoid Counterregulatory Responses To Hypoglycemia In Rats, Lori Gorton, Arshad Khan, Maryann Bohland, Graciela Sanchez-Watts, Casey Donovan, Alan Watts

Selected Works Temporary Series

No abstract provided.

A Role For The Forebrain In Mediating Time-Of-Day Differences In Glucocorticoid Counterregulatory Responses To Hypoglycemia In Rats, Gorton, Arshad Khan, Maryann Bohland, Graciela Sanchez-Watts, Casey Donovan, Alan Watts

Departmental Papers (Biology)

No abstract provided.

Phenylalanyl-Trna Synthetase Editing Defects Result In Efficient Mistranslation Of Phenylalanine Codons As Tyrosine, Jiqiang Ling, Srujana S. Yadavalli, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Translational quality control is monitored at several steps, including substrate selection by aminoacyl-tRNA synthetases (aaRSs), and discrimination of aminoacyl-tRNAs by elongation factor Tu (EF-Tu) and the ribosome. Phenylalanyl-tRNA synthetase (PheRS) misactivates Tyr but is able to correct the mistake using a proofreading activity named editing. Previously we found that overproduction of editing-defective PheRS resulted in Tyr incorporation at Phe-encoded positions in vivo , although the misreading efficiency could not be estimated. This raised the question as to whether or not EF-Tu and the ribosome provide further proofreading mechanisms to prevent mistranslation of Phe codons by Tyr. Here we show that, …

An Aminoacyl-Trna Synthetase: Elongation Factor Complex For Substrate Channeling In Archaeal Translation, Corinne D. Hausmann, Mette Praetorius-Ibba, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Translation requires the specific attachment of amino acids to tRNAs by aminoacyl-tRNA synthetases (aaRSs) and the subsequent delivery of aminoacyl-tRNAs to the ribosome by elongation factor 1 alpha (EF-1α). Interactions between EF-1α and various aaRSs have been described in eukaryotes, but the role of these complexes remains unclear. To investigate possible interactions between EF-1α and other cellular components, a yeast two-hybrid screen was performed for the archaeon Methanothermobacter thermautotrophicus. EF-1α was found to form a stable complex with leucyl-tRNA synthetase (LeuRS; KD = 0.7 μ M). Complex formation had little effect on EF-1α activity, but increased the k_{cat …}

Catecholaminergic Control Of Mitogen-Activated Protein Kinase Signaling In Paraventricular Neuroendocrine Neurons In Vivo And In Vitro: A Proposed Role During Glycemic Challenges, Arshad Khan, Todd Ponzio, Graciela Sanchez-Watts, B. Stanley, Glenn Hatton, Alan Watts

Selected Works Temporary Series

No abstract provided.

Catecholaminergic Control Of Mitogen-Activated Protein Kinase Signaling In Paraventricular Neuroendocrine Neurons In Vivo And In Vitro: A Proposed Role During Glycemic Challenges, Khan, Todd Ponzio, Graciela Sanchez-Watts, B. Stanley, Glenn Hatton, Alan Watts

Departmental Papers (Biology)

No abstract provided.

Immunoadjuvanticity Of Chlorophyll, Jocelyn Helen Parks Ramos

All-Inclusive List of Electronic Theses and Dissertations

Background: Vaccines are formulations containing antigenic determinants (from non-self organic matters) and immuno-enhancers called adjuvants. Together, they evoke protective immune responses in hosts. Adjuvants are substances that further vaccines' capability to induce a robust immune response. Common side effects with conventional adjuvants used in most vaccines are toxicity, inflammation and granulomas. In this study we investigated the pigment chlorophyll (Chl) for immunoadjuvanticity. The selection of chlorophyll is based on the fact that it is a component in foods such as spinach, and that it contains an isoprenoid structure that is a feature of many conventional adjuvants, Pristane and Squalene. Chl …