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Articles 1 - 30 of 49
Full-Text Articles in Physical Sciences and Mathematics
Investigation Of Cofactor Activities Of Endothelial Microparticle- Thrombomodulin With Liposomal Surrogate, Valentinas Gruzdys, Lin Wang, Dan Wang, Rachel Huang, Xue-Long Sun
Investigation Of Cofactor Activities Of Endothelial Microparticle- Thrombomodulin With Liposomal Surrogate, Valentinas Gruzdys, Lin Wang, Dan Wang, Rachel Huang, Xue-Long Sun
Chemistry Faculty Publications
Thrombomodulin (TM) is a type I transmembrane glycoprotein mainly expressed on the endothelial cells, where it binds thrombin to form the thrombin-TM complex that can activate protein C and thrombin-activable fibrinolysis inhibitor (TAFI) and induce anticoagulant and anti-fibrinolytic reactions, respec-tively. Cell activation and injury often sheds microparticles that contain membrane TM, which circulate in biofluids like blood. However, the biological function of circulating microparticle-TM is still unknown even though it has been recognized as a biomarker of endothelial cell injury and damage. In comparison with cell membrane, different phospholipids are exposed on the microparticle surface due to cell membrane "flip-flop"upon …
Nucleobase-Modified Nucleosides And Nucleotides: Applications In Biochemistry, Synthetic Biology, And Drug Discovery, Anthony J. Berdis
Nucleobase-Modified Nucleosides And Nucleotides: Applications In Biochemistry, Synthetic Biology, And Drug Discovery, Anthony J. Berdis
Chemistry Faculty Publications
DNA is often referred to as the "molecule of life " since it contains the genetic blueprint for all forms of life on this planet. The core building blocks composing DNA are deoxynucleotides. While the deoxyribose sugar and phosphate group are ubiquitous, it is the composition and spatial arrangement of the four natural nucleobases, adenine (A), cytosine (C), guanine (G), and thymine (T), that provide diversity in the coding information present in DNA. The ability of DNA to function as the genetic blueprint has historically been attributed to the formation of proper hydrogen bonding interactions made between complementary nucleobases. However, …
A Non-Natural Nucleotide Uses A Specific Pocket To Selectively Inhibit Telomerase Activity, Wilnelly Hernandez-Sanchez, Wei Huang, Brian Plucinsky, Nelson Garcia-Vazquez, Nathaniel J. Robinson, William P. Schiemann, Anthony J. Berdis, Emmanuel Skordalakes, Derek J. Taylor
A Non-Natural Nucleotide Uses A Specific Pocket To Selectively Inhibit Telomerase Activity, Wilnelly Hernandez-Sanchez, Wei Huang, Brian Plucinsky, Nelson Garcia-Vazquez, Nathaniel J. Robinson, William P. Schiemann, Anthony J. Berdis, Emmanuel Skordalakes, Derek J. Taylor
Chemistry Faculty Publications
Telomerase, a unique reverse transcriptase that specifically extends the ends of linear chromosomes, is up-regulated in the vast majority of cancer cells. Here, we show that an indole nucleotide analog, 5-methylcarboxyl-indolyl-2′-deoxyriboside 5′-triphosphate (5-MeCITP), functions as an inhibitor of telomerase activity. The crystal structure of 5-MeCITP bound to the Tribolium castaneum telomerase reverse transcriptase reveals an atypical interaction, in which the nucleobase is flipped in the active site. In this orientation, the methoxy group of 5-MeCITP extends out of the canonical active site to interact with a telomerase-specific hydrophobic pocket formed by motifs 1 and 2 in the fingers domain and …
Analysis Of Oxygen-18 Labeled Phosphate To Study Positional Isotope Experiments Using Lc-Qtof-Ms, Sujatha Chilakala, Iteen Cheng, Ireen Lee, Yan Xu
Analysis Of Oxygen-18 Labeled Phosphate To Study Positional Isotope Experiments Using Lc-Qtof-Ms, Sujatha Chilakala, Iteen Cheng, Ireen Lee, Yan Xu
Chemistry Faculty Publications
A method is proposed in this paper for the determination of oxygen-18 labeled phosphate so that positional isotope experiments using sensitive and rapid liquid chromatography–QTOF–mass spectrometry (LC-QTOF-MS) experiments can be carried out. The positional isotope exchange technique is a useful tool in understanding the mechanisms and kinetics of many enzyme-catalyzed reactions. Detection of the positions and concentration of these exchanged isotopes is the key. Gas chromatography–mass spectrometry (GC-MS) and nuclear magnetic resonance imaging are commonly used analytical techniques for measurement of 18O/16O, 31P and 15N isotope enrichment. Since these techniques either require a time-consuming derivatization …
Manganese Oxide/Hemin-Functionalized Graphene As A Platform For Peroxynitrite Sensing, Haitham F. Kalil, Shaimaa Maher, Tiyash Bose, Mekki Bayachou
Manganese Oxide/Hemin-Functionalized Graphene As A Platform For Peroxynitrite Sensing, Haitham F. Kalil, Shaimaa Maher, Tiyash Bose, Mekki Bayachou
Chemistry Faculty Publications
Peroxynitrite (ONOO−, PON) is a powerful oxidizing agent generated in vivo by the diffusion-limited reaction of nitric oxide (NO) and superoxide (O2˙−) radicals. Under oxidative stress, cumulated peroxynitrite levels are associated with chronic inflammatory disorders and other pathophysiological conditions. The accurate detection of peroxynitrite in biological systems is important, not only to understand the genesis and development of diseases, but also to explore and design potential therapeutics. Herein, a manganese oxide/hemin-modified graphene interface is explored as a platform for peroxynitrite amperometric detection. Hemin-functionalized reduced graphene oxide was further modified with manganese oxide nanoparticles to provide a …
N-Glycosylation In The Protease Domain Of Trypsin-Like Serine Proteases Mediates Calnexin-Assisted Protein Folding, Hao Wang, Shuo Li, Juejin Wang, Shenghan Chen, Xue-Long Sun, Qingyu Wu
N-Glycosylation In The Protease Domain Of Trypsin-Like Serine Proteases Mediates Calnexin-Assisted Protein Folding, Hao Wang, Shuo Li, Juejin Wang, Shenghan Chen, Xue-Long Sun, Qingyu Wu
Chemistry Faculty Publications
Trypsin-like serine proteases are essential in physiological processes. Studies have shown that N-glycans are important for serine protease expression and secretion, but the underlying mechanisms are poorly understood. Here, we report a common mechanism of N-glycosylation in the protease domains of corin, enteropeptidase and prothrombin in calnexin-mediated glycoprotein folding and extracellular expression. This mechanism, which is independent of calreticulin and operates in a domain-autonomous manner, involves two steps: direct calnexin binding to target proteins and subsequent calnexin binding to monoglucosylated N-glycans. Elimination of N-glycosylation sites in the protease domains of corin, enteropeptidase and prothrombin inhibits corin and enteropeptidase cell surface …
A Galactomannoglucan Derived From Agaricus Brasiliensis: Purification, Characterization And Macrophage Activation Via Mapk And Ikappab/Nfkappab Pathways, Yanqing Zhang, Danting Liu, Leilei Fang, Xiaotong Zhao, Aimin Zhou, Junbo Xie
A Galactomannoglucan Derived From Agaricus Brasiliensis: Purification, Characterization And Macrophage Activation Via Mapk And Ikappab/Nfkappab Pathways, Yanqing Zhang, Danting Liu, Leilei Fang, Xiaotong Zhao, Aimin Zhou, Junbo Xie
Chemistry Faculty Publications
In this study, a novel galactomannoglucan named as TJ2 was isolated from Agaricus brasiliensis with microwave extraction, macroporous resin, ion exchange resin and high resolution gel chromatography. TJ2 is composed of glucose, mannose and galactose in the ratio 99.2:0.2:0.6. Infrared spectra (IR), methylation analysis and nuclear magnetic resonance spectra indicated that TJ2 mainly contained a b-(1?3) – linked glucopyranosyl backbone. Interestingly, TJ2 significantly promoted RAW264.7 cell proliferation, and was able to activate the cells to engulf E. coli. In addition, TJ2 induced the expression of Interleukin 1b (IL-1b), Interleukin 6 (IL-6), tumor necrosis factor a (TNF-a) and cyclooxygenase-2 (Cox-2) in …
The Future Perspective: Metabolomics In Laboratory Medicine For Inborn Errors Of Metabolism, Yana Sandlers
The Future Perspective: Metabolomics In Laboratory Medicine For Inborn Errors Of Metabolism, Yana Sandlers
Chemistry Faculty Publications
Metabolomics can be described as a simultaneous and comprehensive analysis of small molecules in a biological sample. Recent technological and bioinformatics advances have facilitated large-scale metabolomic studies in many areas, including inborn errors of metabolism (IEMs). Despite significant improvements in the diagnosis and treatment of some IEMs, it is still challenging to understand how genetic variation affects disease progression and susceptibility. In addition, a search for new more personalized therapies and a growing demand for tools to monitor the long-term metabolic effects of existing therapies set the stage for metabolomics integration in preclinical and clinical studies. While targeted metabolomics approach …
Development And Validation Of A Novel Lc–Ms/Ms Method For Simultaneous Determination Of Abiraterone And Its Seven Steroidal Metabolites In Human Serum: Innovation In Separation Of Diastereoisomers Without Use Of A Chiral Column, Mohammad Alyamani, Zhenfei Li, Sunil K. Upadhyay, David J. Anderson, Richard J. Auchus, Nima Sharifi
Development And Validation Of A Novel Lc–Ms/Ms Method For Simultaneous Determination Of Abiraterone And Its Seven Steroidal Metabolites In Human Serum: Innovation In Separation Of Diastereoisomers Without Use Of A Chiral Column, Mohammad Alyamani, Zhenfei Li, Sunil K. Upadhyay, David J. Anderson, Richard J. Auchus, Nima Sharifi
Chemistry Faculty Publications
Abiraterone acetate (AA), the prodrug of abiraterone, is FDA-approved for the treatment of castration-resistant prostate cancer. Abiraterone is metabolized in patients to a more potent analogue, D4A. However, we have recently reported that this analogue is further metabolized to additional metabolites in patients treated with AA. Here, we present a liquid chromatography-tandem mass spectrometry method developed to resolve and detect abiraterone and its seven metabolites in human serum using an AB Sciex Qtrap 5500 mass analyzer coupled with a Shimadzu Nexera UPLC station. Analytes and the internal standard (abiraterone-d4) were extracted from human serum using the liquid–liquid extraction procedure. The …
Inhibiting Translesion Dna Synthesis As An Approach To Combat Drug Resistance To Dna Damaging Agents, Jung-Suk Choi, Seol Kim, Edward Motea, Anthony J. Berdis
Inhibiting Translesion Dna Synthesis As An Approach To Combat Drug Resistance To Dna Damaging Agents, Jung-Suk Choi, Seol Kim, Edward Motea, Anthony J. Berdis
Chemistry Faculty Publications
Anti-cancer agents exert therapeutic effects by damaging DNA. Unfortunately, DNA polymerases can effectively replicate the formed DNA lesions to cause drug resistance and create more aggressive cancers. To understand this process at the cellular level, we developed an artificial nucleoside that visualizes the replication of damaged DNA to identify cells that acquire drug resistance through this mechanism. Visualization is achieved using "click" chemistry to covalently attach azide-containing fluorophores to the ethynyl group present on the nucleoside analog after its incorporation opposite damaged DNA. Flow cytometry and microscopy techniques demonstrate that the extent of nucleotide incorporation into genomic DNA is enhanced …
Hmba Is A Putative Hsp70 Activator Stimulating Hexim1 Expression That Is Down-Regulated By Estrogen, Rati Lama, Chunfang Gan, Nethrie Idippily, Viharika Bobba, David Danielpour, Monica Montano, Bin Su Ph.D.
Hmba Is A Putative Hsp70 Activator Stimulating Hexim1 Expression That Is Down-Regulated By Estrogen, Rati Lama, Chunfang Gan, Nethrie Idippily, Viharika Bobba, David Danielpour, Monica Montano, Bin Su Ph.D.
Chemistry Faculty Publications
Hexamethylene bis-acetamide inducible protein 1 (HEXIM1) is identified as a novel inhibitor of estrogen stimulated breast cell growth, and it suppresses estrogen receptor-a transcriptional activity. HEXIM1 protein level has been found to be downregulated by estrogens. Recently, HEXIM1 has been found to inhibit androgen receptor transcriptional activity as well. Researchers have used Hexamethylene bisacetamide (HMBA) for decades to stimulate HEXIM1 expression, which also inhibit estrogen stimulated breast cancer cell gene activation and androgen stimulated prostate cancer gene activation. However, the direct molecular targets of HMBA that modulate the induction of HEXIM1 expression in mammalian cells have not been identified. Based …
Myeloperoxidase-Mediated Protein Lysine Oxidation Generates 2- Aminoadipic Acid And Lysine Nitrile In Vivo, Hongqiao Lin, Bruce S. Levison, Jennifer A. Buffa, Ying Huang, Xiaoming Fu, Zeneng Wang, Valentin Gogonea, Joseph A. Didonato, Stanley L. Hazen
Myeloperoxidase-Mediated Protein Lysine Oxidation Generates 2- Aminoadipic Acid And Lysine Nitrile In Vivo, Hongqiao Lin, Bruce S. Levison, Jennifer A. Buffa, Ying Huang, Xiaoming Fu, Zeneng Wang, Valentin Gogonea, Joseph A. Didonato, Stanley L. Hazen
Chemistry Faculty Publications
Recent studies reveal 2-aminoadipic acid (2-AAA) is both elevated in subjects at risk for diabetes and mechanistically linked to glucose homeostasis. Prior studies also suggest enrichment of protein-bound 2-AAA as an oxidative post-translational modification of lysyl residues in tissues associated with degenerative diseases of aging. While in vitro studies suggest redox active transition metals or myeloperoxidase (MPO) generated hypochlorous acid (HOCl) may produce protein-bound 2-AAA, the mechanism(s) responsible for generation of 2- AAA during inflammatory diseases are unknown. In initial studies we observed that traditional acid- or basecatalyzed protein hydrolysis methods previously employed to measure tissue 2-AAA can artificially generate …
Oncogenic Pik3ca Mutations Reprogram Glutamine Metabolism In Colorectal Cancer, Yujun Hao, Yardena Samuels, Qingling Li, Dawid Krokowski, Bo-Jhih Guan, Chao Wang, Zhicheng Jin, Bohan Dong, Bo Cao, Xiujing Feng, Min Xiang, Claire Xu, Stephen Fink, Neal J. Meropol, Yan Xu
Oncogenic Pik3ca Mutations Reprogram Glutamine Metabolism In Colorectal Cancer, Yujun Hao, Yardena Samuels, Qingling Li, Dawid Krokowski, Bo-Jhih Guan, Chao Wang, Zhicheng Jin, Bohan Dong, Bo Cao, Xiujing Feng, Min Xiang, Claire Xu, Stephen Fink, Neal J. Meropol, Yan Xu
Chemistry Faculty Publications
Cancer cells often require glutamine for growth, thereby distinguishing them from most normal cells. Here we show that PIK3CA mutations reprogram glutamine metabolism by upregulating glutamate pyruvate transaminase 2 (GPT2) in colorectal cancer (CRC) cells, making them more dependent on glutamine. Compared with isogenic wild-type (WT) cells, PIK3CA mutant CRCs convert substantially more glutamine to alpha-ketoglutarate to replenish the tricarboxylic acid cycle and generate ATP. Mutant p110 alpha upregulates GPT2 gene expression through an AKT-independent, PDK1-RSK2-ATF4 signalling axis. Moreover, aminooxyacetate, which inhibits the enzymatic activity of aminotransferases including GPT2, suppresses xenograft tumour growth of CRCs with PIK3CA mutations, but not …
Metabolomics Reveals New Mechanisms For Pathogenesis In Barth Syndrome And Introduces Novel Roles For Cardiolipin In Cellular Function, Yana Sandlers, Kelly Mercier, Wimal Pathmasiri, Jim Carlson, Susan Mcritchie, Susan Sumner, Hilary J. Vernon
Metabolomics Reveals New Mechanisms For Pathogenesis In Barth Syndrome And Introduces Novel Roles For Cardiolipin In Cellular Function, Yana Sandlers, Kelly Mercier, Wimal Pathmasiri, Jim Carlson, Susan Mcritchie, Susan Sumner, Hilary J. Vernon
Chemistry Faculty Publications
Barth Syndrome is the only known Mendelian disorder of cardiolipin remodeling, with characteristic clinical features of cardiomyopathy, skeletal myopathy, and neutropenia. While the primary biochemical defects of reduced mature cardiolipin and increased monolysocardiolipin are well-described, much of the downstream biochemical dysregulation has not been uncovered, and biomarkers are limited. In order to further expand upon the knowledge of the biochemical abnormalities in Barth Syndrome, we analyzed metabolite profiles in plasma from a cohort of individuals with Barth Syndrome compared to age-matched controls via 1H nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry. A clear distinction between metabolite profiles of …
The Dual Regulatory Role Of Amino Acids Leu480 And Gln481 Of Prothrombin, Joesph R. Wiencek, Jamila Hirbawi, Vivien C. Yee, Michael Kalafatis
The Dual Regulatory Role Of Amino Acids Leu480 And Gln481 Of Prothrombin, Joesph R. Wiencek, Jamila Hirbawi, Vivien C. Yee, Michael Kalafatis
Chemistry Faculty Publications
Prothrombin (FII) is activated to α-thrombin (IIa) by prothrombinase. Prothrombinase is composed of a catalytic subunit, factor Xa (fXa), and a regulatory subunit, factor Va (fVa), assembled on a membrane surface in the presence of divalent metal ions. We constructed, expressed, and purified several mutated recombinant FII (rFII) molecules within the previously determined fVa-dependent binding site for fXa (amino acid region 473–487 of FII). rFII molecules bearing overlapping deletions within this significant region first established the minimal stretch of amino acids required for the fVa-dependent recognition exosite for fXa in prothrombinase within the amino acid sequence Ser478–Val479 …
Multi-Dimensional Glycan Microarrays With Glyco-Macroligands, Satya Nandana Narla, Huan Nie, Yu Li, Xue-Long Sun
Multi-Dimensional Glycan Microarrays With Glyco-Macroligands, Satya Nandana Narla, Huan Nie, Yu Li, Xue-Long Sun
Chemistry Faculty Publications
Glycan microarray has become a powerful high-throughput tool for examining binding interactions of carbohydrates with the carbohydrate binding biomolecules like proteins, enzymes, antibodies etc. It has shown great potential for biomedical research and applications, such as antibody detection and profiling, vaccine development, biomarker discovery, and drug screening. Most glycan microarrays were made with monovalent glycans immobilized directly onto the array surface via either covalent or non-covalent bond, which afford a multivalent glycans in two dimensional (2D) displaying. A variety of glyco-macroligands have been developed to mimic multivalent carbohydrate-protein interactions for studying carbohydrate-protein interactions and biomedical research and applications. Recently, a …
Rnase L Interacts With Filamin A To Regulate Actin Dynamics And Barrier Function For Viral Entry, Krishnamurthy Malathi, Mohammad Adnan Siddiqui, Shubham Dayal, Merna Naji, Heather J, Ezelle, Chun Zeng, Aimin Zhou, Bret A. Hassel
Rnase L Interacts With Filamin A To Regulate Actin Dynamics And Barrier Function For Viral Entry, Krishnamurthy Malathi, Mohammad Adnan Siddiqui, Shubham Dayal, Merna Naji, Heather J, Ezelle, Chun Zeng, Aimin Zhou, Bret A. Hassel
Chemistry Faculty Publications
The actin cytoskeleton and its network of associated proteins constitute a physical barrier that viruses must circumvent to gain entry into cells for productive infection. The mechanisms by which the physical signals of infection are sensed by the host to activate an innate immune response are not well understood. The antiviral endoribonuclease RNase L is ubiquitously expressed in a latent form and activated upon binding 2-5A, a unique oligoadenylate produced during viral infections. We provide evidence that RNase L in its inactive form interacts with the actin-binding protein Filamin A to modulate the actin cytoskeleton and inhibit virus entry. Cells …
Reclaiming The Efficacy Of Β-Lactam–Β-Lactamase Inhibitor Combinations: Avibactam Restores The Susceptibility Of Cmy-2-Producing Escherichia Coli To Ceftazidime, Krisztina M. Papp-Wallace, Marisa L. Winkler, Julian A. Gatta, Magdalena A. Taracila, Sujatha Chilakala, Yan Xu, J. Kristie Johnson, Robert A. Bonomo
Reclaiming The Efficacy Of Β-Lactam–Β-Lactamase Inhibitor Combinations: Avibactam Restores The Susceptibility Of Cmy-2-Producing Escherichia Coli To Ceftazidime, Krisztina M. Papp-Wallace, Marisa L. Winkler, Julian A. Gatta, Magdalena A. Taracila, Sujatha Chilakala, Yan Xu, J. Kristie Johnson, Robert A. Bonomo
Chemistry Faculty Publications
CMY-2 is a plasmid-encoded Ambler class C cephalosporinase that is widely disseminated in Enterobacteriaceae and is responsible for expanded-spectrum cephalosporin resistance. As a result of resistance to both ceftazidime and β-lactamase inhibitors in strains carrying blaCMY, novel β-lactam–β-lactamase inhibitor combinations are sought to combat this significant threat to β-lactam therapy. Avibactam is a bridged diazabicyclo [3.2.1]octanone non-β-lactam β-lactamase inhibitor in clinical development that reversibly inactivates serine β-lactamases. To define the spectrum of activity of ceftazidime-avibactam, we tested the susceptibilities of Escherichia coli clinical isolates that carry blaCMY-2 or blaCMY-69 and investigated the inactivation kinetics of CMY-2. Our analysis showed that …
Bsa–Boronic Acid Conjugate As Lectin Mimetics, Satya Nandana Narla, Poornima Pinnamaneni, Huan Nie, Yu Li, Xue-Long Sun
Bsa–Boronic Acid Conjugate As Lectin Mimetics, Satya Nandana Narla, Poornima Pinnamaneni, Huan Nie, Yu Li, Xue-Long Sun
Chemistry Faculty Publications
We report bovine serum albumin (BSA)–boronic acid (BA) conjugates as lectin mimetics and their glyco-capturing capacity. The BSA–BA conjugates were synthesized by amidation of carboxylic acid groups in BSA with aminophenyl boronic acid in the presence of EDC, and were characterized by Alizarin Red S (ARS) assay and SDS–PAGE gel. The BSA–BA conjugates were immobilized onto maleimide-functionalized silica beads and their sugar capturing capacity and specificity were confirmed by ARS displacement assay. Further, surface plasmon resonance (SPR) analysis of the glyco-capturing activity of the BSA–BA conjugates was conducted by immobilizing BSA–BA onto SPR gold chip. Overall, we demonstrated a BSA–BA-based …
Quantification Of Free Sialic Acid In Human Plasma Through A Robust Quinoxalinone Derivatization And Lc–Ms/Ms Using Isotope-Labeled Standard Calibration, Dan Wang, Xiang Zhou, Lin Wang, Sihe Wang, Xue-Long Sun
Quantification Of Free Sialic Acid In Human Plasma Through A Robust Quinoxalinone Derivatization And Lc–Ms/Ms Using Isotope-Labeled Standard Calibration, Dan Wang, Xiang Zhou, Lin Wang, Sihe Wang, Xue-Long Sun
Chemistry Faculty Publications
We report an accurate quantification of free sialic acid (SA) in human plasma using LC–MS/MS method with isotope-labeled standard calibration (ILSC) and robust derivatization. Specifically, derivatization of SA with a stable and inexpensive 3,4-diaminotoluene (DAT) provides a stable product of SA with high MS response, proving a convenient and cost-effective LC–MS/MS analysis of free SA. In addition, the use of 13C3-SA as calibration standard ensured the accuracy for the measurement. This assay used ultra high performance liquid chromatography (UHPLC) for separation of native/labeled SA and IS from matrix interference, and employed mass spectrometry in multiple reaction monitoring …
Insights Into The Roles Of Desolvation And Π-Electron Interactions During Dna Polymerization, Edward A. Motea, Irene Lee, Anthony J. Berdis
Insights Into The Roles Of Desolvation And Π-Electron Interactions During Dna Polymerization, Edward A. Motea, Irene Lee, Anthony J. Berdis
Chemistry Faculty Publications
This report describes the use of several isosteric non-natural nucleotides as probes to evaluate the roles of nucleobase shape, size, solvation energies, and π-electron interactions as forces influencing key kinetic steps of the DNA polymerization cycle. Results are provided using representative high- and low-fidelity DNA polymerases. Results generated with the E. coli Klenow fragment reveal that this high-fidelity polymerase utilizes hydrophobic nucleotide analogues with higher catalytic efficiencies compared to hydrophilic analogues. These data support a major role for nucleobase desolvation during nucleotide selection and insertion. In contrast, the low-fidelity HIV-1 reverse transcriptase discriminates against hydrophobic analogues and only tolerates non-natural …
Spectroscopic Analysis Of Polymerization And Exonuclease Proofreading By A High-Fidelity Dna Polymerase During Translesion Dna Synthesis, Babho Devadoss, Irene Lee, Anthony J. Berdis
Spectroscopic Analysis Of Polymerization And Exonuclease Proofreading By A High-Fidelity Dna Polymerase During Translesion Dna Synthesis, Babho Devadoss, Irene Lee, Anthony J. Berdis
Chemistry Faculty Publications
High fidelity DNA polymerases maintain genomic fidelity through a series of kinetic steps that include nucleotide binding, conformational changes, phosphoryl transfer, polymerase translocation, and nucleotide excision. Developing a comprehensive understanding of how these steps are coordinated during correct and pro-mutagenic DNA synthesis has been hindered due to lack of spectroscopic nucleotides that function as efficient polymerase substrates. This report describes the application of a non-natural nucleotide designated 5-naphthyl-indole-2′-deoxyribose triphosphate which behaves as a fluorogenic substrate to monitor nucleotide incorporation and excision during the replication of normal DNA versus two distinct DNA lesions (cyclobutane thymine dimer and an abasic site). Transient …
Voltage-Controlled Enzyme-Catalyzed Glucose–Gluconolactone Conversion Using A Field-Effect Enzymatic Detector, Siu Tung Yau, Yan Xu, Yang Song, Ye Feng, Jiapeng Wang
Voltage-Controlled Enzyme-Catalyzed Glucose–Gluconolactone Conversion Using A Field-Effect Enzymatic Detector, Siu Tung Yau, Yan Xu, Yang Song, Ye Feng, Jiapeng Wang
Chemistry Faculty Publications
The field-effect enzymatic detection (FEED) technique was used to control the kinetics of the enzymatic conversion of glucose to gluconolactone. The glucose–gluconolactone conversion occurring at an enzyme-immobilized electrode, a well-studied process, was confirmed using mass spectrometry. Electrochemical studies showed that the glucose oxidation current depends on the gating voltage VG and the ion concentration of the sample solution. Additionally, the depletion of glucose in the sample also showed a dependence on VG. FEED was used to detect H2O2 on the zepto-molar level in order to show the ultrasensitive detection capability of the technique. These results, while providing evidence for the …
Exploring The Role Of A Conserved Class A Residue In The Ω-Loop Of Kpc-2 Β-Lactamase: A Mechanism For Ceftazidime Hydrolysis
Chemistry Faculty Publications
Gram-negative bacteria harboring KPC-2, a class A β-lactamase, are resistant to all β-lactam antibiotics and pose a major public health threat. Arg-164 is a conserved residue in all class A β-lactamases and is located in the solvent-exposed Ω-loop of KPC-2. To probe the role of this amino acid in KPC-2, we performed site-saturation mutagenesis. When compared with wild type, 11 of 19 variants at position Arg-164 in KPC-2 conferred increased resistance to the oxyimino-cephalosporin, ceftazidime (minimum inhibitory concentration; 32→128 mg/liter) when expressed in Escherichia coli. Using the R164S variant of KPC-2 as a representative β-lactamase for more detailed analysis, we …
Understanding The Molecular Determinants Of Substrate And Inhibitor Specificities In The Carbapenemase Kpc-2: Exploring The Roles Of Arg220 And Glu276
Chemistry Faculty Publications
β-Lactamases are important antibiotic resistance determinants expressed by bacteria. By studying the mechanistic properties of β-lactamases, we can identify opportunities to circumvent resistance through the design of novel inhibitors. Comparative amino acid sequence analysis of class A β-lactamases reveals that many enzymes possess a localized positively charged residue (e.g., R220, R244, or R276) that is critical for interactions with β-lactams and β-lactamase inhibitors. To better understand the contribution of these residues to the catalytic process, we explored the roles of R220 and E276 in KPC-2, a class A β-lactamase that inactivates carbapenems and β-lactamase inhibitors. Our study reveals that substitutions …
Development Of A ‘Clickable’ Non-Natural Nucleotide To Visualize The Replication Of Non-Instructional Dna Lesions, Edward A. Motea, Irene Lee, Anthony J. Berdis
Development Of A ‘Clickable’ Non-Natural Nucleotide To Visualize The Replication Of Non-Instructional Dna Lesions, Edward A. Motea, Irene Lee, Anthony J. Berdis
Chemistry Faculty Publications
The misreplication of damaged DNA is an important biological process that produces numerous adverse effects on human health. This report describes the synthesis and characterization of a non-natural nucleotide, designated 3-ethynyl-5-nitroindolyl-2′-deoxyriboside triphosphate (3-Eth-5-NITP), as a novel chemical reagent that can probe and quantify the misreplication of damaged DNA. We demonstrate that this non-natural nucleotide is efficiently inserted opposite an abasic site, a commonly formed and potentially mutagenic non-instructional DNA lesion. The strategic placement of the ethynyl moiety allows the incorporated nucleoside triphosphate to be selectively tagged with an azide-containing fluorophore using ‘click’ chemistry. This reaction provides a facile way to …
Removal Of Uracil By Uracil Dna Glycosylase Limits Pemetrexed Cytotoxicity: Overriding The Limit With Methoxyamine To Inhibit Base Excision Repair, A. D. Bulgar, L. D. Weeks, Y. Miao, S. Yang, Yan Xu, C. Guo, S. Markowitz, N. Oleinick, S. L. Gerson, Lili Liu
Removal Of Uracil By Uracil Dna Glycosylase Limits Pemetrexed Cytotoxicity: Overriding The Limit With Methoxyamine To Inhibit Base Excision Repair, A. D. Bulgar, L. D. Weeks, Y. Miao, S. Yang, Yan Xu, C. Guo, S. Markowitz, N. Oleinick, S. L. Gerson, Lili Liu
Chemistry Faculty Publications
Uracil DNA glycosylase (UDG) specifically removes uracil bases from DNA, and its repair activity determines the sensitivity of the cell to anticancer agents that are capable of introducing uracil into DNA. In the present study, the participation of UDG in the response to pemetrexed-induced incorporation of uracil into DNA was studied using isogenic human tumor cell lines with or without UDG (UDG+/+/UDG−/−). UDG−/− cells were very sensitive to pemetrexed. Cell killing by pemetrexed was associated with genomic uracil accumulation, stalled DNA replication, and catastrophic DNA strand breaks. By contrast, UDG+/+ cells were >10 times more resistant to pemetrexed due to …
Exploring The Roles Of Nucleobase Desolvation And Shape Complementarity During The Misreplication Of O6-Methylguanine, Delia Chavarria, Andrea Ramos Serrano, Ichiro Hirao, Anthony J. Berdis
Exploring The Roles Of Nucleobase Desolvation And Shape Complementarity During The Misreplication Of O6-Methylguanine, Delia Chavarria, Andrea Ramos Serrano, Ichiro Hirao, Anthony J. Berdis
Chemistry Faculty Publications
O6-methylguanine is a miscoding DNA lesion arising from the alkylation of guanine. This report uses the bacteriophage T4 DNA polymerase as a model to probe the roles hydrogen-bonding interactions, shape/size, and nucleobase desolvation during the replication of this miscoding lesion. This was accomplished by using transient kinetic techniques to monitor the kinetic parameters for incorporating and extending natural and non-natural nucleotides. In general, the efficiency of nucleotide incorporation does not depend on the hydrogen-bonding potential of the incoming nucleotide. Instead, nucleobase hydrophobicity and shape complementarity appear to be the preeminent factors controlling nucleotide incorporation. In addition, shape complementarity plays a …
The Exonuclease Activity Of Hpmc2 Is Required For Transcriptional Regulation Of The Qr Gene And Repair Of Estrogen-Induced Abasic Sites, N. Krishnamurthy, C. R. Ngam, Anthony J. Berdis, M. M. Montano
The Exonuclease Activity Of Hpmc2 Is Required For Transcriptional Regulation Of The Qr Gene And Repair Of Estrogen-Induced Abasic Sites, N. Krishnamurthy, C. R. Ngam, Anthony J. Berdis, M. M. Montano
Chemistry Faculty Publications
We have previously reported that the expression of antioxidative stress enzymes is upregulated by trans-hydroxytamoxifen (TOT) in breast epithelial cell lines providing protection against estrogen-induced DNA damage. This regulation involves Estrogen Receptor β (ERβ) recruitment to the Electrophile Response Element (EpRE) and a novel protein, human homolog of Xenopus gene which Prevents Mitotic Catastrophe (hPMC2). We have also demonstrated that ERβ and hPMC2 are required for TOT-dependent recruitment of poly (ADP-ribose) polymerase 1 (PARP-1) and Topoisomerase IIβ (Topo IIβ) to the EpRE. Sequence analysis reveals that the C-terminus of hPMC2 encodes a putative exonuclease domain. Using in vitro kinetic assays, …
Epistatic Roles For Pseudomonas Aeruginosa Muts And Dinb (Dna Pol Iv) In Coping With Reactive Oxygen Species-Induced Dna Damage, Laurie H. Sanders, Babho Devadoss, Geraldine V. Raja, Jaime O'Connor, Shengchang Su, Daniel J. Wozniak, Daniel J. Hassett, Anthony J. Berdis, Mark D. Sutton
Epistatic Roles For Pseudomonas Aeruginosa Muts And Dinb (Dna Pol Iv) In Coping With Reactive Oxygen Species-Induced Dna Damage, Laurie H. Sanders, Babho Devadoss, Geraldine V. Raja, Jaime O'Connor, Shengchang Su, Daniel J. Wozniak, Daniel J. Hassett, Anthony J. Berdis, Mark D. Sutton
Chemistry Faculty Publications
Pseudomonas aeruginosa is especially adept at colonizing the airways of individuals afflicted with the autosomal recessive disease cystic fibrosis (CF). CF patients suffer from chronic airway inflammation, which contributes to lung deterioration. Once established in the airways, P. aeruginosa continuously adapts to the changing environment, in part through acquisition of beneficial mutations via a process termed pathoadaptation. MutS and DinB are proposed to play opposing roles in P. aeruginosa pathoadaptation: MutS acts in replication-coupled mismatch repair, which acts to limit spontaneous mutations; in contrast, DinB (DNA polymerase IV) catalyzes error-prone bypass of DNA lesions, contributing to mutations. As part of …