Medical Biochemistry Commons^™

Superparamagnetic Nanoparticle Capture Of Prions For Amplification, Michael B. Miller, Surachai Supattapone

Dartmouth Scholarship

Prion diseases are associated with the presence of PrP(Sc), a disease-associated misfolded conformer of the prion protein. We report that superparamagnetic nanoparticles bind PrP(Sc) molecules efficiently and specifically, permitting magnetic separation of prions from a sample mixture. Captured PrP(Sc) molecules retain the activity to seed protein misfolding cyclic amplification (PMCA) reactions, enabling the rapid concentration of dilute prions to improve detection. Furthermore, superparamagnetic nanoparticles clear contaminated solutions of PrP(Sc). Our findings suggest that coupling magnetic nanoparticle capture with PMCA could accelerate and improve prion detection. Magnetic nanoparticles may also be useful for developing a nontoxic prion decontamination method for biologically …

Genotoxin-Induced Acetylation Of The Werner Syndrome Protein (Wrn) And Effect On Its Dna Metabolic Function, Enerlyn Meliza Lozada Santiago

University of Kentucky Doctoral Dissertations

Loss of function of the WRN protein causes the genetic disorder Werner Syndrome that is characterized by increased cancer and premature aging. WRN belongs to the RecQ helicase family that plays key roles in preventing genome instability. In response to treatment with genotoxins, WRN is subject to post-translational modification. The relationship of post-translational modification of WRN with its function in DNA metabolism is unknown. There is accumulating evidence suggesting that WRN contributes to the maintenance of genomic integrity through its involvement in DNA replication. Consistent with this notion, WS cells are sensitive to DNA replication inhibitors and DNA damaging agents …

Mir-27b*, An Oxidative Stress-Responsive Microrna Modulates Nuclear Factor-Kb Pathway In Raw 264.7 Cells, Sivasubramani Thulasingam, Chandirasegaran Massilamany, Arunakumar Gangaplara, Hongjiu Dai, Shahlo Yarbaeva, Sakthivel Subramaniam, Jean-Jack Riethoven, James Eudy, Marjorie F. Lou, Jay Reddy

Jay Reddy Publications

Reactive oxygen species (ROS) produced in macrophages is critical for microbial killing, but they also take part in inflammation and antigen presentation functions. MicroRNAs (miRNAs) are endogenous regulators of gene expression, and they can control immune responses. To dissect the complex nature of ROS-mediated effects in macrophages, we sought to characterize miRNAs that are responsive to oxidative stress-induced with hydrogen peroxide (H₂O₂) in the mouse macrophage cell line, RAW 264.7. We have identified a set of unique miRNAs that are differentially expressed in response to H₂O₂. These include miR-27a*, miR-27b*, miR-29b*, miR-24-2*, …

Identification Of A Second Mimicry Epitope From Acanthamoeba Castellanii That Induces Cns Autoimmunity By Generating Cross-Reactive T Cells For Mbp 89–101 In Sjl Mice, Chandirasegaran Massilamany, Oluwatoyin A. Asojo, Arunakumar Gangaplara, David J. Steffen, Jay Reddy

Jay Reddy Publications

We had previously reported that Acanthamoeba castellanii (ACA) contains a mimicry epitope for proteolipid protein 139–151 capable of inducing central nervous system (CNS) autoimmunity in SJL/J mice. We now present evidence that ACA also contains a mimicry epitope for myelin basic protein (MBP) 89–101, a derivative from amoebic nicotinamide adenine dinucleotide dehydrogenase subunit 2 (NAD). The epitope, NAD 108–120, contains a discontinuous stretch of six amino acids in the core region (VVFFKNIILIGFL) sharing 46% identity with MBP 89–101 (VHFFKNIVTPRTP; identical residues are underlined). SJL mice immunized with NAD 108–120 develop encephalomyelitis similar to the disease induced by the cognate peptide. …

Cellular Inhibitor Of Apoptosis 1 (Ciap-1) Degradation By Caspase 8 During Tnf-Related Apoptosis-Inducing Ligand (Trail)-Induced Apoptosis., Maria Eugenia Guicciardi, Justin L. Mott, Steven F. Bronk, Satoshi Kurita, Christian D. Fingas, Gregory J. Gores

Journal Articles: Biochemistry & Molecular Biology

TNF-related apoptosis-inducing ligand (TRAIL) is a potential chemotherapeutic agent with high selectivity for malignant cells. Many tumors, however, are resistant to TRAIL cytotoxicity. Although cellular inhibitors of apoptosis 1 and 2 (cIAP-1 and -2) are often over-expressed in cancers, their role in mediating TRAIL resistance remains unclear. Here, we demonstrate that TRAIL-induced apoptosis of liver cancer cells is associated with degradation of cIAP-1 and X-linked IAP (XIAP), whereas cIAP-2 remains unchanged. Lower concentrations of TRAIL causing minimal or no apoptosis do not alter cIAP-1 or XIAP protein levels. Silencing of cIAP-1 expression, but not XIAP or cIAP-2, as well as …

Dysglycemia Induces Abnormal Circadian Blood Pressure Variability, Sivarajan Kumarasamy, Kathirvel Gopalakrishnan, Dong Hyun Kim, Nader G. Abraham, William D. Johnson, Bina Joe, Alok K. Gupta

Biochemistry and Microbiology

Background: Prediabetes (PreDM) in asymptomatic adults is associated with abnormal circadian blood pressure variability (abnormal CBPV).

Hypothesis: Systemic inflammation and glycemia influence circadian blood pressure variability.

Methods: Dahl salt-sensitive (S) rats (n = 19) after weaning were fed either an American (AD) or a standard (SD) diet. The AD (high-glycemic-index, high-fat) simulated customary human diet, provided daily overabundant calories which over time lead to body weight gain. The SD (low-glycemic-index, low-fat) mirrored desirable balanced human diet for maintaining body weight. Body weight and serum concentrations for fasting glucose (FG), adipokines (leptin and adiponectin), and proinflammatory cytokines [monocyte chemoattractant protein-1 (MCP-1) …

Protein Modification By Arginylation, Hideko Kaji, Akira Kaji

Department of Biochemistry and Molecular Biology Faculty Papers

The modification of protein by arginine catalyzed by arginyltransferases (ATE1) described by the Kashina group in this issue shows that arginylation of protein occurs widely in biology and is being recognized as a key regulatory reaction such as phosphorylation of proteins (Wang et al., 2011).

Mitochondrial Genome Sequence Analysis: A Custom Bioinformatics Pipeline Substantially Improves Affymetrix Mitochip V2.0 Call Rate And Accuracy., Hongbo M Xie, Juan C Perin, Theodore G Schurr, Matthew C Dulik, Sergey I Zhadanov, Joseph A Baur, Michael P King, Emily Place, Colleen Clarke, Michael Grauer, Jonathan Schug, Avni Santani, Anthony Albano, Cecilia Kim, Vincent Procaccio, Hakon Hakonarson, Xiaowu Gai, Marni J Falk

Department of Biochemistry and Molecular Biology Faculty Papers

BACKGROUND: Mitochondrial genome sequence analysis is critical to the diagnostic evaluation of mitochondrial disease. Existing methodologies differ widely in throughput, complexity, cost efficiency, and sensitivity of heteroplasmy detection. Affymetrix MitoChip v2.0, which uses a sequencing-by-genotyping technology, allows potentially accurate and high-throughput sequencing of the entire human mitochondrial genome to be completed in a cost-effective fashion. However, the relatively low call rate achieved using existing software tools has limited the wide adoption of this platform for either clinical or research applications. Here, we report the design and development of a custom bioinformatics software pipeline that achieves a much improved call rate …

Rit Gtpase Signaling Mediates Oxidative Stress Resistance And Survival Of Adult Newborn Neurons After Traumatic Brain Injury, Weikang Cai

Theses and Dissertations--Molecular and Cellular Biochemistry

The small GTPases function as molecular switches to control diverse signaling cascades. The mammalian Rit and Rin, along with Drosophila Ric, comprise an evolutionarily conserved subfamily of the Ras-related GTPases. Previous studies using cultured cell models suggested that Rit was involved in the control of cell proliferation, transformation, neuronal differentiation, morphogenesis, and cell survival, but the principal physiological function of Rit remained uncharacterized.

To address this outstanding question, we employed a genetic approach, engineering a Rit knockout mouse. Using this animal model, we demonstrate a central role of Rit in governing cell survival in a p38-dependent fashion. Primary mouse embryonic …

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

Jay Reddy Publications

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 …

Detection Of Autoreactive Cd4 T Cells Using Major Histocompatibility Complex Class Ii Dextramers, Chandirasegaran Massilimany, Bijaya Upadhyaya, Arunakumar Gangaplara, Charles Kuszynski, Jay Reddy

Jay Reddy Publications

Background: Tetramers are useful tools to enumerate the frequencies of antigen-specific T cells. However, unlike CD8 T cells, CD4 T cells - especially self-reactive cells - are challenging to detect with major histocompatibility complex (MHC) class II tetramers because of low frequencies and low affinities of their T cell receptors to MHCpeptide complexes. Here, we report the use of fluorescent multimers, designated MHC dextramers that contain a large number of peptide-MHC complexes per reagent.

Results: The utility of MHC dextramers was evaluated in three autoimmune disease models: 1) proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis in SJL/J (H-2^s) …

Identification Of A Second Mimicry Epitope From Acanthamoeba Castellanii That Induces Cns Autoimmunity By Generating Cross-Reactive T Cells For Mbp 89–101 In Sjl Mice, Chandirasegaran Massilamany, Oluwatoyin A. Asojo, Arunakumar Gangaplara, David J. Steffen, Jay Reddy

Jay Reddy Publications

We had previously reported that Acanthamoeba castellanii (ACA) contains a mimicry epitope for proteolipid protein 139–151 capable of inducing central nervous system (CNS) autoimmunity in SJL/J mice. We now present evidence that ACA also contains a mimicry epitope for myelin basic protein (MBP) 89–101, a derivative from amoebic nicotinamide adenine dinucleotide dehydrogenase subunit 2 (NAD). The epitope, NAD 108–120, contains a discontinuous stretch of six amino acids in the core region (VVFFKNIILIGFL) sharing 46% identity with MBP 89–101 (VHFFKNIVTPRTP; identical residues are underlined). SJL mice immunized with NAD 108–120 develop encephalomyelitis similar to the disease induced by the cognate peptide. …

Click Chemistry Inspired One-Pot Synthesis Of 1, 4-Disubstituted 1, 2, 3-Triazoles And Their Src Kinase Inhibitory Activity, Dalip Kumar, V. Buchi Reddy, Anil Kumar, Deendayal Mandal, Rakesh Tiwari, Keykavous Parang

Pharmacy Faculty Articles and Research

Two classes of 1,4-disubstituted 1,2,3-triazoles were synthesized using one-pot reaction of α-tosyloxy ketones/α-halo ketones, sodium azide, and terminal alkynes in the presence of aq. PEG (1:1, v/v) using the click chemistry approach and evaluated for Src kinase inhibitory activity. Structure-activity relationship analysis demonstrated that insertion of C6H5- and 4-CH3C6H4- at position 4 for both classes and less bulkier aromatic group at position 1 in class 1 contribute critically to the modest Src inhibition activity (IC50 = 32-43 µM) of 1,4-disubstituted 1,2,3-triazoles.

Thiazolyl N-Benzyl-Substituted Acetamide Derivatives: Synthesis, Src Kinase Inhibitory And Anticancer Activities, Asal Fallah-Tafti, Alireza Foroumadi, Rakesh Tiwari, Amir Nasrolahi Shirazi, David G. Hangauer, Yahao Bu, Tahmineh Akbarzadeh, Keykavous Parang, Abbas Shafiee

Pharmacy Faculty Articles and Research

KX2-391 (KX-01/Kinex Pharmaceuticals), N-benzyl-2-(5-(4-(2-morpholinoethoxy)phenyl)pyridin-2-yl)acetamide, is a highly selective Src substrate binding site inhibitor. To understand better the role of pyridine ring and N-benzylsubstitution in KX2-391 and establish the structure-activity relationship, a number of N-benzyl substituted (2-morpholinoethoxy)phenyl)thiazol-4-yl)acetamide derivatives containing thiazole instead of pyridine were synthesized and evaluated for Src kinase inhibitory activities. The unsubstituted N-benzyl derivative (8a) showed the inhibition of c-Src kinase with GI50 values of 1.34 μM and 2.30 M in NIH3T3/c-Src527F and SYF/c-Src527F cells, respectively. All the synthesized compounds were evaluated for inhibition of cell proliferation of human colon carcinoma (HT-29), breast carcinoma (BT-20), and leukemia (CCRF-CEM) cells. …

3-Substitued Indoles: One Pot Synthesis And Evaluation Of Anticancer And Src Kinase Inhibitory Activities, V. Kameshwara Rao, Bhupender S. Chhikara, Amir Nasrolahi Shirazi, Rakesh Tiwari, Keykavous Parang, Anil Kumar

Pharmacy Faculty Articles and Research

An efficient and economical method was developed for the synthesis of 3-substituted indoles by one pot three-component coupling reaction of a substituted or unsubstituted benzaldehyde, N-methylaniline, and indole or N-methylindole using Yb(OTf)3-SiO2 as a catalyst. All the synthesized compounds were evaluated for inhibition of cell proliferation of human colon carcinoma (HT-29), human ovarian adenocarcinoma (SK-OV-3), and c-Src kinase activity. The 4-methylphenyl (4o and 4p) and 4-methoxyphenyl (4q) indole derivatives inhibited the cell proliferation of SK-OV-3 and HT-29 cells by 7077% at a concentration of 50 μM. The unsubstituted phenyl (4d) and 3-nitrophenyl (4l) derivatives showed the inhibition of c-Src kinase …

Synthesis Of 3-Phenylpyrazolopyrimidine-1,2,3-Triazole Conjugates And Evaluation Of Their Src Kinase Inhibitory And Anticancer Activities, Anil Kumar, Israr Ahmad, Bhupender S. Chhikara, Rakesh Tiwari, Deendayal Mandal, Keykavous Parang

Pharmacy Faculty Articles and Research

A series of two classes of 3-phenylpyrazolopyrimidine-1,2,3-triazole conjugates were synthesized using click chemistry approach. All compounds were evaluated for inhibition of Src kinase and human ovarian adenocarcinoma (SK-Ov-3), breast carcinoma (MDA-MB-361), and colon adenocarcinoma (HT-29). Hexyl triazolyl-substituted 3-phenylpyrazolopyrimidine exhibited inhibition of Src kinase with an IC50 value of 5.6 µM. 4-Methoxyphenyl triazolyl-substituted 3-phenylpyrazolopyrimidine inhibited the cell proliferation of HT-29 and SK-Ov-3 by 73% and 58%, respectively, at a concentration of 50 µM.

Inhibition Of Multi-Drug Resistant Hiv-1 Reverse Transcriptase By Nucleoside Beta-Triphosphates, Chandravanu Dash, Yousef Ahmadibeni, Michael J. Hanley, Jui Pandhare, Mathias Gotte, Stuart F. J. Le Grice, Keykavous Parang

Pharmacy Faculty Articles and Research

Despite the success of potent reverse transcriptase (RT) inhibitors against human immunodeficiency virus type 1 (HIV-1) in combination regimens, the development of drug resistant RTs constitutes a major hurdle for the long-term efficacy of current antiretroviral therapy. Nucleoside β-triphosphate analogs of adenosine and nucleoside reverse transcriptase inhibitors (NRTIs) (3′-azido-2′,3′-dideoxythymidine (AZT), 3′-fluoro-2′,3′-dideoxythymidine (FLT), and 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T)) were synthesized and their inhibitory activities were evaluated against wild-type and multidrug resistant HIV-1 RTs. Adenosine β-triphosphate (1) and AZT β-triphosphate (2) completely inhibited the DNA polymerase activity of wild type, the NRTI multi resistant, and nonnucleoside RT inhibitors (NNRTI) resistant HIV-1 RT at 10 …

Phosphorylation Meets Nuclear Import: A Review., Jonathan D Nardozzi, Kaylen Lott, Gino Cingolani

Department of Biochemistry and Molecular Biology Faculty Papers

Phosphorylation is the most common and pleiotropic modification in biology, which plays a vital role in regulating and finely tuning a multitude of biological pathways. Transport across the nuclear envelope is also an essential cellular function and is intimately linked to many degeneration processes that lead to disease. It is therefore not surprising that phosphorylation of cargos trafficking between the cytoplasm and nucleus is emerging as an important step to regulate nuclear availability, which directly affects gene expression, cell growth and proliferation. However, the literature on phosphorylation of nucleocytoplasmic trafficking cargos is often confusing. Phosphorylation, and its mirror process dephosphorylation, …

Role Of Extracellular Signal-Regulated Kinase (Erk) In Regulation Of Intestinal Epithelial Tight Junctions, Sudhir Aggarwal

Theses and Dissertations (ETD)

Evidence indicates that MAP kinase (ERK1/2) is involved in regulation of epithelial tight junctions. There are different opinions expressed by investigators as to whether ERK disrupts the junctions or protects them. ERK has also been demonstrated to mediate the EGF-caused protection of the intestinal epithelial tight junctions (TJ) from hydrogen peroxide. Studies using pharmacological inhibitors have shown that EGF increases Thr-phosphorylation of occludin by a MAP kinase-dependent mechanism. This study aimed at looking at the role of ERK in regulation of tight junctions using pharmacological and molecular techniques.

Hypothesis: ERK protects tight junctions in differentiated Caco-2 cells, while it is …

Development Of Novel Biomarkers In Cancer: Detection Of Circulating Mir-141 As A Potential Prognostic Marker For Prostate Cancer, Jason Cadaoas Gonzales

UNLV Theses, Dissertations, Professional Papers, and Capstones

Prostate cancer (CAP) is the most common epithelial malignancy and the second leading cause of cancer deaths in American men. The identification of predictive and prognostic biomarkers in CAP patients is critical for improving clinical outcomes. Although the measurement of prostate-specific antigen (PSA) and radiographic studies are clinically approved to predict response to therapy, these tests can oftentimes prove to be inadequate in certain patients. Thus, it is important to discover new biomarkers to improve chances of survivability. We and others have shown that longitudinal measurements of circulating tumor cells (CTC) and lactate dehydrogenase (LDH) may aid in predicting response …