Biochemistry, Biophysics, and Structural Biology Commons^™

Cell Permeabilization And Inhibition Of Voltage-Gated Ca²+ And Na+ Channel Currents By Nanosecond Pulsed Electric Fields, Vasyl Nesin, Angela M. Bowman, Shu Xiao, Andrei G. Pakhomov

Bioelectrics Publications

Previous studies have found that nanosecond pulsed electric field (nsPEF) exposure causes long-term permeabilization of the cell plasma membrane. In this study, we utilized the whole-cell patch-clamp method to study the nsPEF effect on currents of voltage-gated (VG) Ca²⁺ and Na⁺ channels (I_Ca and I_Na) in cultured GH3 and NG108 cells. We found that a single 300 or 600 ns pulse at or above 1.5-2 kV/cm caused prolonged inhibition of I_Ca and I_Na. Concurrently, nsPEF increased a non-inactivating leak current (I_leak), presumably due to the formation of nanoelectropores or larger …

Lesion-Specific Dna-Binding And Repair Activities Of Human O⁶-Alkylguanine Dna Alkyltransferase, Manana Melikishvili, Michael G. Fried

Center for Structural Biology Faculty Publications

Binding experiments with alkyl-transfer-active and -inactive mutants of human O⁶-alkylguanine DNA alkyltransferase (AGT) show that it forms an O⁶-methylguanine (6mG)-specific complex on duplex DNA that is distinct from non-specific assemblies previously studied. Specific complexes with duplex DNA have a 2:1 stoichiometry that is formed without accumulation of a 1:1 intermediate. This establishes a role for cooperative interactions in lesion binding. Similar specific complexes could not be detected with single-stranded DNA. The small difference between specific and non-specific binding affinities strongly limits the roles that specific binding can play in the lesion search process. Alkyl-transfer kinetics with …

Cell Wall Antibiotics Provoke Accumulation Of Anchored Mcherry In The Cross Wall Of Staphylococcus Aureus, Wenqi Yu, Friedrich Götz

Molecular Biosciences Faculty Publications

A fluorescence microscopy method to directly follow the localization of defined proteins in Staphylococcus was hampered by the unstable fluorescence of fluorescent proteins. Here, we constructed plasmid (pCX) encoded red fluorescence (RF) mCherry (mCh) hybrids, namely mCh-cyto (no signal peptide and no sorting sequence), mCh-sec (with signal peptide), and mCh-cw (with signal peptide and cell wall sorting sequence). The S. aureus clones targeted mCh-fusion proteins into the cytosol, the supernatant and the cell envelope respectively; in all cases mCherry exhibited bright fluorescence. In staphylococci two types of signal peptides (SP) can be distinguished: the +YSIRK motif SPlip and the −YSIRK …

Discovery Of Marinopyrrole A (Maritoclax) As A Selective Mcl-1 Antagonist That Overcomes Abt-737 Resistance By Binding To And Targeting Mcl-1 For Proteasomal Degradation*, Kenichiro Doi, Rongshi Li, Shen-Shu Sung, Hongwei Wu, Yan Liu, Wanda Manieri, Gowdahalli Krishnegowda, Andy Awwad, Alden Dewey, Xin Liu, Shantu Amin, Chunwei Cheng, Yong Qin, Ernst Schonbrunn, Gary W. Daughdrill, Thomas P. Loughran Jr., Said M. Sebti, Hong-Gang Wang

Molecular Biosciences Faculty Publications

The anti-apoptotic Bcl-2 family of proteins, including Bcl-2, Bcl-XL and Mcl-1, are well-validated drug targets for cancer treatment. Several small molecules have been designed to interfere with Bcl-2 and its fellow pro-survival family members. While ABT-737 and its orally active analog ABT-263 are the most potent and specific inhibitors to date that bind Bcl-2 and Bcl-XL with high affinity but have a much lower affinity for Mcl-1, they are not very effective as single agents in certain cancer types because of elevated levels of Mcl-1. Accordingly, compounds that specifically target Mcl-1 may overcome this resistance. In this study, we identified …

Effects Of Betel Nut On Cardiovascular Risk Factors In A Rat Model, Mohammad Perwaiz Iqbal, Naseema Mehboobali, Ghulam Haider, Shahid Pervez, Iqbal Azam

Department of Biological & Biomedical Sciences

Background: Areca nut (commonly known as betel nut) chewing has been shown to be associated with metabolic syndrome and cardiovascular disease (CVD). The mechanism by which betel nut ingestion could lead to development of CVD is not precisely known; however, dyslipidemia, hyperhomocysteinemia, hypertriglyceridemia and inflammation could be some of the potential risk factors. This study was undertaken to investigate the effects of two dosages of betel nut on homocysteinemia, inflammation and some of the components of metabolic syndrome, such as hypertriglyceridemia, low HDL-cholesterol, obesity and fasting hyperglycemia in a rat model.
Methods: Thirty-six adult female Sprague Dawley rats, aged 10–12 …

The Role Of Src Kinase In The Biology And Pathogenesis Of Acanthamoeba Castellanii, Ruqaiyyah Siddiqui, Junaid Iqbal, Marie-Josee Maugueret, Naveed Ahmed Khan

Department of Biological & Biomedical Sciences

Background: Acanthamoeba species are the causative agents of fatal granulomatous encephalitis in humans. Haematogenous spread is thought to be a primary step, followed by blood-brain barrier penetration, in the transmission of Acanthmaoeba into the central nervous system, but the associated molecular mechanisms remain unclear. Here, we evaluated the role of Src, a non-receptor protein tyrosine kinase in the biology and pathogenesis of Acanthamoeba.
Methods: Amoebistatic and amoebicidal assays were performed by incubating amoeba in the presence of Src kinase-selective inhibitor, PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and its inactive analog, PP3 (4-amino-7-phenylpyrazolo[3,4-d]pyrimidine). Using this inhibitor, the role of Src kinase in A. castellanii interactions …

Exploring Conformational Variability Of An Rna Domain In The Ribosome: From Structure And Function To Potential Antibiotic Targeting, Yogo Sakakibara

Wayne State University Dissertations

RNA in nature is modified at many specific sites to order to gain extra functions or to expand the genetic code. One of such RNAs is ribosomal RNA (rRNA), which contains several modified bases, particularly around the functionally significant sites. We have focused on understanding the influences of modified base on RNA structure and function by employing helix 69 (H69), which is a good region to evaluate the roles of modified bases since it contains three pseudouridines in the loop region and exists at the core of the ribosome.

Previous model studies using small hairpin H69 showed the conformational differences …

In Vivo Display: A Selection And Its Derivatives For Antimicrobial Peptide Lead Identification, Wesley David Colangelo

Wayne State University Dissertations

The rise of antibiotic resistance necessitates new approaches for the isolation of new antimicrobials with novel inhibitory mechanisms, bypassing the development of rapid resistance by modification of pre-existing resistance mechanisms. In response, we have developed a series of systems for the rapid isolation and identification of peptides that inhibit the growth of Escherichia coli and other bacteria, termed in vivo display (IVD).

IVD harnesses the cellular processes of E. coli for the expression of a library of random peptides at the terminus of a display protein. A library of 12-amino acid random peptide sequences was added to either the C- …

Molecular Mechanisms Regulating Chemokine Receptor Cxcr4 Signaling And Trafficking, Rohit Malik

Dissertations

CXCR4 is a G protein-coupled receptor (GPCR) that binds to the chemokine, stromal cell-derived factor-1 (SDF-1alpha; a.k.a. CXCL12). The SDF-1alpha/CXCR4 signaling axis plays an essential role during embryogenesis in the development of the heart, brain and vasculature and in the adult mediating immune cell trafficking and stem cell homing to the bone marrow. Dysregulation of SDF-1alpha/CXCR4 signaling is linked to several pathological conditions, including cardiovascular disease, immunological disorders as well as cancer growth and metastasis. However, the mechanisms that govern CXCR4 signaling remain poorly understood. In this dissertation project, we attempt to further our understanding of the molecular mechanisms that …

The Role Of Cyp33 In Mll Mediated Gene Repression, Steven D. Poppen

Dissertations

Mixed Lineage Leukemia (MLL) is a multidomain protein whose gene is translocated in a subset of AML leukemias. Translocation of the MLL gene is present in approximately five percent of adult acute leukemias and ten percent of pediatric leukemias (Daser, A 2004, Look, A 1997, Huret, J 2001) Patients presenting in the clinic at the time of diagnosis with an MLL fusion have been shown to respond poorly to treatment and have a worse prognosis than matched wild type MLL patients (Rubnitz, J 1994, Rubnitz, J 1999). Novel therapies therefore are needed in order to more effectively treat patients with …

Methamphetamine Administration Targets Multiple Immune Subsets And Induces Phenotypic Alterations Suggestive Of Immunosuppression., Robert Z. Harms, Brenda M. Morsey, Craig W. Boyer, Howard S. Fox, Nora E. Sarvetnick

Journal Articles: Regenerative Medicine

Methamphetamine (Meth) is a widely abused stimulant and its users are at increased risk for multiple infectious diseases. To determine the impact of meth on the immune system, we utilized a murine model that simulates the process of meth consumption in a typical addict. Our phenotypic analysis of leukocytes from this dose escalation model revealed that meth affected key immune subsets. Meth administration led to a decrease in abundance of natural killer (NK) cells and the remaining NK cells possessed a phenotype suggesting reduced responsiveness. Dendritic cells (DCs) and Gr-1(high) monocytes/macrophages were also decreased in abundance while Gr-1(low) monocytes/macrophages appear …

An Ims/Atp Assay For The Detection Of Mycobacterium Tuberculosis In Urine, Dawn M. Hunter, Daniel V. Lim

Molecular Biosciences Faculty Publications

Background. Although sputum smears are the gold standard for diagnosis of tuberculosis, sensitivity in HIV/TB coinfection cases is low, indicating a need for alternative methods. Urine is being increasingly evaluated. Materials and Methods. A novel method for detecting Mycobacterium tuberculosis (MTB) in synthetic urine using a combined IMS/ATP assay was evaluated. Preliminary work established standard ATP conditions and the sensitivity and specificity of the MTB antibody. Eighty-four blinded samples in four replicate assays were evaluated for the presence of MTB using labeled immunomagnetic beads for capture. Beads were separated, washed, and resuspended in broth and added to a …

Identification Of New Cell Size Control Genes In S. Cerevisiae, Huzefa Dungrawala, Hui Hua, Jill Wright, Lesley Abraham, Thivakorn Kasemsri, Anthony Mcdowell, Jessica Stilwell, Brandt L. Schneider

Molecular Biosciences Faculty Publications

Cell size homeostasis is a conserved attribute in many eukaryotic species involving a tight regulation between the processes of growth and proliferation. In budding yeast S. cerevisiae, growth to a “critical cell size” must be achieved before a cell can progress past START and commit to cell division. Numerous studies have shown that progression past START is actively regulated by cell size control genes, many of which have implications in cell cycle control and cancer. Two initial screens identified genes that strongly modulate cell size in yeast. Since a second generation yeast gene knockout collection has been generated, we screened …

The Two-Component Signal Transduction System Arlrs Regulates Staphylococcus Epidermidis Biofilm Formation In An Ica-Dependent Manner, Yang Wu, Jiaxue Wang, Tao Xu, Jingran Liu, Wenqi Yu, Qiang Lou, Tao Zhu, Nianan He, Haijing Ben, Jian Hu, Friedrich Götz, Di Qu

Molecular Biosciences Faculty Publications

Due to its ability to form biofilms on medical devices, Staphylococcus epidermidis has emerged as a major pathogen of nosocomial infections. In this study, we investigated the role of the two-component signal transduction system ArlRS in regulating S. epidermidis biofilm formation. An ArlRS-deficient mutant, WW06, was constructed using S. epidermidis strain 1457 as a parental strain. Although the growth curve of WW06 was similar to that of SE1457, the mutant strain was unable to form biofilms in vitro. In a rabbit subcutaneous infection model, sterile disks made of polymeric materials were implanted subcutaneously followed with inoculation of WW06 or SE1457. …

Wipi-1 Positive Autophagosome-Like Vesicles Entrap Pathogenic Staphylococcus Aureus For Lysosomal Degradation, Mario Mauthe, Wenqi Yu, Oleg Krut, Martin Krönke, Friedrich Götz, Horst Robenek, Tassula Proikas-Cezanne

Molecular Biosciences Faculty Publications

Invading pathogens provoke the autophagic machinery and, in a process termed xenophagy, the host cell survives because autophagy is employed as a safeguard for pathogens that escaped phagosomes. However, some pathogens can manipulate the autophagic pathway and replicate within the niche of generated autophagosome-like vesicles. By automated fluorescence-based high content analyses, we demonstrate that Staphylococcus aureus strains (USA300, HG001, SA113) stimulate autophagy and become entrapped in intracellular PtdIns(3)P-enriched vesicles that are decorated with human WIPI-1, an essential PtdIns(3)P effector of canonical autophagy and membrane protein of both phagophores and autophagosomes. Further, agr-positive S. aureus (USA300, HG001) strains were more efficiently …

Early Events Of Human Metapneumovirus Infection, Andres Chang

Theses and Dissertations--Molecular and Cellular Biochemistry

Human metapneumovirus (HMPV) is a worldwide respiratory pathogen that belongs to the paramyxovirus family of enveloped viruses and affects primarily the pediatric, geriatric, and immunocompromised populations. Despite its prevalence and importance to human health, no therapies are available against this pathogen. For paramyxoviruses, it is believed that infection starts by attachment of the virus to the surface of the cell through the viral attachment protein followed by fusion between the viral and cellular membranes, a process mediated by the fusion (F) protein at the plasma membrane and at neutral pH. Previous work showed that HMPV infection can occur in the …

Thoughts On Quorum Sensing And Fungal Dimorphism, Kenneth W. Nickerson, Audrey L. Atkin, Jessica C. Hargarten, Ruvini U. Pathirana, Sahar Hasim

School of Biological Sciences: Faculty Publications

Farnesol has been best studied for its role in regulating fungal dimorphism. However, farnesol is also a lipid and in this review we analyze data relevant to farnesol’s function and synthesis from the perspective of farnesol and bacterial endotoxins acting as membrane active compounds. This analysis implicates the possible roles of: (1) endotoxins in the regulation of farnesol production by C. albicans; (2) farnesol in the interactions between C. albicans and the host during disseminated infections; and (3) ubiquinones in the mechanisms for unusually high resistance to farnesol by some C. albicans cell types. Finally we discuss the implications …

Theoretical Justification For Bond Valence -- Bond Length Empirical Correlations, F. D. Hardcastle, S. Laffoon

Journal of the Arkansas Academy of Science

Bond valence – bond length empirical correlations are of great interest in chemistry, biology, geology and materials science because they offer a quick and convenient way of checking and evaluating molecular structures. Linus Pauling’s relationship is the most commonly used, but is a two-parameter fit where R0 and b must be optimized. In this study, a simplified quantum-mechanical approach was used to derive Pauling’s empirical bond valence – bond length relationship. A covalency factor was also introduced to account for the difference in “softness” between cation and anion (resulting in increased orbital overlap). An expression for the b parameter was …

Development Of Mass Spectrometric Methods For The Analysis Of Serotonin And Related Compounds In Biological Matrices, Merisa Moriarty

Theses

Serotonin is a major neurotransmitter in the brain and affects various functions both in the brain and in the rest of the body. It has been linked to depression, migraines, and aggression, as well as many psychiatric disorders including schizophrenia, dementia and anorexia nervosa. It also has been associated with conditions like Alzheimer’s disease along with other neurotransmitters such as dopamine.

The aim of this research project is to develop various mass spectrometric methods for the analysis of serotonin, dopamine and similar indoles in both urine and serum matrices. This thesis provides extensive reviews detailing a general overview of serotonin …

Inhibition Of Voltage-Gated Na+ Current By Nanosecond Pulsed Electric Field (Nspef) Is Not Mediated By Na+ Influx Or Ca²+ Signaling, Vasyl Nesin, Andrei G. Pakhomov

Bioelectrics Publications

In earlier studies, we found that permeabilization of mammalian cells with nsPEF was accompanied by prolonged inhibition of voltage-gated (VG) currents through the plasma membrane. This study explored if the inhibition of VG Na⁺ current (I_Na) resulted from (i) reduction of the transmembrane Na⁺ gradient due to its influx via nsPEF-opened pores, and/or (ii) downregulation of the VG channels by a Ca²⁺ -dependent mechanism. We found that a single 300?ns electric pulse at 1.65.3?kV/cm triggered sustained Na⁺ influx in exposed NG108 cells and in primary chromaffin cells, as detected by increased fluorescence of a …