Medical Biochemistry Commons^™

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 …

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 …

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. …

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. …

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.

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 …

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, …

Cerebral Amyloid Angiopathy And Transition Metals In Alzheimer's Disease, Matthew Schrag

Loma Linda University Electronic Theses, Dissertations & Projects

Alterations in brain metals homeostasis and particularly brain iron overload have been postulated to play a role in Alzheimer's disease, contributing to oxidative stress and neuronal injury; however, the source of this iron is not clear and may be due to metabolic derangement(s), failed iron clearance mechanisms or exogenous deposition such as through bleeding. This series of studies was designed to evaluate the extent of metals dyshomeostasis in the Alzheimer's disease brain and specifically whether microvascular bleeding is a major contributor to Alzheimer's disease-related iron overload. Cerebral amyloid angiopathy (CAA) is a vascular manifestation of Alzheimer's disease present to some …

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 …

Mechanism Of N-Methylation By The Trna M1g37 Methyltransferase Trm5., Thomas Christian, Georges Lahoud, Cuiping Liu, Katherine Hoffmann, John J Perona, Ya-Ming Hou

Department of Biochemistry and Molecular Biology Faculty Papers

Trm5 is a eukaryal and archaeal tRNA methyltransferase that catalyzes methyl transfer from S-adenosylmethionine (AdoMet) to the N(1) position of G37 directly 3' to the anticodon. While the biological role of m(1)G37 in enhancing translational fidelity is well established, the catalytic mechanism of Trm5 has remained obscure. To address the mechanism of Trm5 and more broadly the mechanism of N-methylation to nucleobases, we examined the pH-activity profile of an archaeal Trm5 enzyme, and performed structure-guided mutational analysis. The data reveal a marked dependence of enzyme-catalyzed methyl transfer on hydrogen ion equilibria: the single-turnover rate constant for methylation increases by one …

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 …

Interactions Of Francisella Tularensis With Components Of The Host Fibrinolytic System, Shawn Russell Clinton

Theses and Dissertations (ETD)

Francisella tularensis (FT) is a Gram-negative coccobacillus and causative agent of a life-threatening disease commonly referred to as tularemia. Due to the highly infectious nature of the organism, its previous development as a biowarfare agent and its potential use in acts of bioterrorism, this bacterium is listed as a Category A select agent by the Centers for Disease Control and Prevention (CDC). Efforts to understand the pathogenic mechanisms of FT within the host environment are vital for the development of safe and effective vaccines, as well as treatments, against tularemia. Though considered an intracellular pathogen, FT research of late has …

Self-Protecting Bactericidal Titanium Alloy Surface Formed By Covalent Bonding Of Daptomycin Bisphosphonates., Chang-Po Chen, Eric Wickstrom

Department of Biochemistry and Molecular Biology Faculty Papers

Infections are a devastating complication of titanium alloy orthopedic implants. Current therapy includes antibiotic-impregnated bone cement and antibiotic-containing coatings. We hypothesized that daptomycin, a Gram-positive peptide antibiotic, could prevent bacterial colonization on titanium alloy surfaces if covalently bonded via a flexible, hydrophilic spacer. We designed and synthesized a series of daptomycin conjugates for bonding to the surface of 1.0 cm² Ti6Al4V foils through bisphosphonate groups, reaching a maximum yield of 180 pmol/cm². Daptomycin-bonded foils killed 53 ± 5% of a high challenge dose of 3 × 10⁵ cfu Staphylococcus aureus ATCC 29213.

Nanosized Hydroxyapatite And Other Calcium Phosphates: Chemistry Of Formation And Application As Drug And Gene Delivery Agents, Vuk Uskoković, Dragan Uskoković

Pharmacy Faculty Articles and Research

The first part of this review looks at the fundamental properties of hydroxyapatite (HAP), the basic mineral constituent of mammalian hard tissues, including the physicochemical features that govern its formation by precipitation. A special emphasis is placed on the analysis of qualities of different methods of synthesis and of the phase transformations intrinsic to the formation of HAP following precipitation from aqueous solutions. This serves as an introduction to the second part and the main subject of this review, which relates to the discourse regarding the prospects of fabrication of ultrafine, nanosized particles based on calcium phosphate carriers with various …

Biosynthesis: A New (Old) Way Of Hijacking Trna., Georges Lahoud, Ya-Ming Hou

Department of Biochemistry and Molecular Biology Faculty Papers

Aminoacylation of tRNA is the cellular process for providing aminoacyl donors for the ribosome synthesis of polypeptides. New research highlights an unexpected structural overlap between enzymes involved in this process and those involved in the biosynthesis of cyclodipeptides, an important class of bioactive molecules.

Insulin Degrading Enzyme Assays For Treatment Of Alzheimer's Disease, Louis B. Hersh

Molecular and Cellular Biochemistry Faculty Patents

Estrogen has been shown to increase the expression and activity of amyloid peptide inactivating enzymes in the brain. Peptides have been shown to increase the activity of an amyloid peptide inactivating enzyme. Methods of identifying compounds for, and methods of treating patients with, Alzheimer's Disease is disclosed.

Morphological Study Of Emulsion-Assisted Cholesterol Precipitation Processes, Vuk Uskoković

Pharmacy Faculty Articles and Research

Crystallization of cholesterol in chemical conditions that involve the presence of emulsions of three different types of surface active agents—cationic, anionic, and nonionic—was investigated by means of scanning and transmission electron microscopic analyses. In contrast with the previous attempts to modify the typical plate- and needle-shaped character of biaxially grown cholesterol crystals, the present study indicates that spherical templates of fine emulsion droplets may impose spherical morphologies to cholesterol particles nucleated within.