Biochemistry, Biophysics, and Structural Biology Commons^™

Basic Features Of A Cell Electroporation Model: Illustrative Behavior For Two Very Different Pulses, Reuben S. Son, Kyle C. Smith, Thiruvallur R. Gowrishankar, P. Thomas Vernier, James C. Weaver

Bioelectrics Publications

Science increasingly involves complex modeling. Here we describe a model for cell electroporation in which membrane properties are dynamically modified by poration. Spatial scales range from cell membrane thickness (5 nm) to a typical mammalian cell radius (10 μm), and can be used with idealized and experimental pulse waveforms. The model consists of traditional passive components and additional active components representing nonequilibrium processes. Model responses include measurable quantities: transmembrane voltage, membrane electrical conductance, and solute transport rates and amounts for the representative "long" and "short" pulses. The long pulse-1.5 kV/cm, 100 μs-evolves two pore subpopulations with a valley at ~5 …

Cellular Regulation Of Extension And Retraction Of Pseudopod-Like Blebs Produced By Nanosecond Pulsed Electric Field, Mikhail A. Rassokhin, Andrei G. Pakhomov

Bioelectrics Publications

Recently we described a new phenomenon of anodotropic pseudopod-like blebbing in U937 cells exposed to nanosecond pulsed electric field (nsPEF). In Ca²⁺ -free buffer such exposure initiates formation of pseudopod-like blebs (PLBs), protrusive cylindrical cell extensions that are distinct from apoptotic and necrotic blebs. PLBs nucleate predominantly on anode-facing cell pole and extend toward anode during nsPEF exposure. Bleb extension depends on actin polymerization and availability of actin monomers. Inhibition of intracellular Ca²⁺ , cell contractility, and RhoA produced no effect on PLB initiation. Meanwhile, inhibition of WASP by wiskostatin causes dose-dependent suppression of PLB growth. Soon after …

Cancellation Of Cellular Responses To Nanoelectroporation By Reversing The Stimulus Polarity, Andrei G. Pakhomov, Iurii Semenov, Shu Xiao, Olga N. Pakhomova, Betsy Gregory, Karl H. Schoenbach

Bioelectrics Publications

Nanoelectroporation of biomembranes is an effect of high-voltage, nanosecond-duration electric pulses (nsEP). It occurs both in the plasma membrane and inside the cell, and nanoporated membranes are distinguished by ion-selective and potential-sensitive permeability. Here we report a novel phenomenon of bioeffects cancellation that puts nsEP cardinally apart from the conventional electroporation and electrostimulation by milli- and microsecond pulses. We compared the effects of 60- and 300-ns monopolar, nearly rectangular nsEP on intracellular Ca²⁺mobilization and cell survival with those of bipolar 60 + 60 and 300 + 300 ns pulses. For diverse endpoints, exposure conditions, pulse numbers (1-60), and …

Disassembly Of Actin Structures By Nanosecond Pulsed Electric Field Is A Downstream Effect Of Cell Swelling, Andrei G. Pakhomov, Shu Xiao, Olga N. Pakhomova, Iurii Semenov, Marjorie A. Kuipers, Bennett L. Ibey

Bioelectrics Publications

Disruption of the actin cytoskeleton structures was reported as one of the characteristic effects of nanosecond-duration pulsed electric field (nsPEF) in both mammalian and plant cells. We utilized CHO cells that expressed the monomeric fluorescent protein (mApple) tagged to actin to test if nsPEF modifies the cell actin directly or as a consequence of cell membrane permeabilization. A train of four 600-ns pulses at 19.2 kV/cm (2 Hz) caused immediate cell membrane poration manifested by YO-PRO-1 dye uptake, gradual cell rounding and swelling. Concurrently, bright actin features were replaced by dimmer and uniform fluorescence of diffuse actin. To block the …

Calcium-Mediated Pore Expansion And Cell Death Following Nanoelectroporation, Olga N. Pakhomova, Betsy Gregory, Iurii Semenov, Andrei G. Pakhomov

Bioelectrics Publications

Opening of long-lived pores in the cell membrane is the principal primary effect of intense, nanosecond pulsed electric field (nsPEF). Here we demonstrate that the evolution of pores, cell survival, the time and the mode of cell death (necrotic or apoptotic) are determined by the level of external Ca²⁺ after nsPEF. We also introduce a novel, minimally disruptive technique for nsEP exposure of adherent cells on indium tin oxide (ITO)-coated glass coverslips, which does not require cell detachment and enables fast exchanges of bath media. Increasing the Ca²⁺ level from the nominal 2–5 μM to 2 mM for …

Introduction To Fourth Special Issue On Electroporation-Based Technologies And Treatments, Damijan Miklavčič, Lluis M. Mir, P. Thomas Vernier

Bioelectrics Publications

This fourth special electroporation-based technologies and treatments issue of the Journal of Membrane Biology contains reports on recent developments in the field of electroporation by participants in the 7th International Workshop and Postgraduate Course on electroporation based technologies and treatments (EBTT 2013) held in Ljubljana, November 17–23, 2013. The 65 participants included faculty members, invited lecturers, special guests, and young scientists, and students from 16 countries. In addition to lectures on the fundamentals, this year’s sessions included talks on microbial inactivation by pulsed electric fields, modeling of intracellular electroporation, electroporation in food processing, and electrotransfer-facilitated DNA vaccination.

Light Stimulates The Mouse Adrenal Through A Retinohypothalamic Pathway Independent Of An Effect On The Clock In The Suprachiasmatic Nucleus, Silke Kiessling, Patricia J. Sollars, Gary E. Pickard

School of Veterinary and Biomedical Sciences: Faculty Publications

The brain’s master circadian pacemaker resides within the hypothalamic suprachiasmatic nucleus (SCN). SCN clock neurons are entrained to the day/night cycle via the retinohypothalamic tract and the SCN provides temporal information to the central nervous system and to peripheral organs that function as secondary oscillators. The SCN clock-cell network is thought to be the hypothalamic link between the retina and descending autonomic circuits to peripheral organs such as the adrenal gland, thereby entraining those organs to the day/night cycle. However, there are at least three different routes or mechanisms by which retinal signals transmitted to the hypothalamus may be conveyed …

Dens Agenesis And Cervical Vertebral Malformation In A Labrador Retriever Puppy, Elisa Salas, Henry E. Cerny, Seth P. Harris

School of Veterinary and Biomedical Sciences: Faculty Publications

Cervical vertebral malformations can cause acute or progressive pain, paralysis, and potentially death. Herein we present a case report of dens agenesis; cervical vertebral malformations of C1, C2, C6, and C7; subluxation of C1 and C2 and C4 and C5; and associated degenerative myelopathy in a 5-month-old female Labrador Retriever puppy. We additionally review current knowledge regarding pathogenesis and treatment.

Pharmacokinetic And Biodistribution Assessment Of A Near Infrared-Labeled Psma-Specific Small Molecule In Tumor-Bearing Mice, Joy L. Kovar, Lael L. Cheung, Melanie A. Simpson, D. Michael Olive

Department of Biochemistry: Faculty Publications

Prostate cancer is themost frequently diagnosed cancer in men and often requires surgery. Use of near infrared (NIR) technologies to perform image-guided surgery may improve accurate delineation of tumor margins. To facilitate preclinical testing of such outcomes, here we developed and characterized a PSMA-targeted small molecule, YC-27. IRDye 800CW was conjugated to YC-27 or an anti-PSMA antibody used for reference. Human 22Rv1, PC3M-LN4, and/or LNCaP prostate tumor cells were exposed to the labeled compounds. In vivo targeting and clearance properties were determined in tumor-bearing mice. Organs and tumors were excised and imaged to assess probe localization. YC-27 exhibited a dose …

Preliminary Joint X-Ray And Neutron Protein Crystallographic Studies Of Ecdhfr Complexed With Folate And Nadp+, Qun Wan, Audrey Y. Kovalevsky, Mark A. Wilson, Brad C. Bennett, Paul Langan, Chris Dealwis

Department of Biochemistry: Faculty Publications

A crystal of Escherichia coli dihydrofolate reductase (ecDHFR) complexed with folate and NADP⁺ of 4 x 1.3 x 0.7 mm (3.6 mm³) in size was obtained by sequential application of microseeding and macroseeding. A neutron diffraction data set was collected to 2.0 A resolution using the IMAGINE diffractometer at the High Flux Isotope Reactor within Oak Ridge National Laboratory. A 1.6 A resolution X-ray data set was also collected from a smaller crystal at room temperature. The neutron and X-ray data were used together for joint refinement of the ecDHFR–folate–NADP⁺ ternary-complex structure in order to examine …

Molecular Mechanism Of Human Mismatch Repair Initiation, Sanghee Lee

Theses and Dissertations--Nutritional Sciences

DNA mismatch repair (MMR) is a highly conserved pathway that maintains genomic stability primarily by correcting mismatches generated during DNA replication. MMR deficiency leads to microsatellite instability (MSI), which is a hallmark of HNPCC (Hereditary Nonpolyposis Colorectal Cancer). Human mismatch repair is initiated by MutSα, a heterodimer of MSH2 and MSH6 subunits. Mismatch binding by MutSα triggers a series of downstream MMR events including interacting and communicating with other MMR proteins. The ATPase domain of MutSα is situated in the C-termini of its both subunits, and ATP binding is required for dissociation of MutSα from a mismatch. In eukaryotic cells, …

The Role Of Angiotensinogen In Atherosclerosis And Obesity, Congqing Wu

Theses and Dissertations--Nutritional Sciences

Angiotensinogen is the only known precursor in the renin-angiotensin system, a hormonal system best known as an essential regulator of blood pressure and fluid homeostasis. Angiotensinogen is sequentially cleaved by renin and angiotensin- converting enzyme to generate angiotensin II. As the major effector peptide, angiotensin II mainly function through angiotensin type 1 receptor.

Angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, and more recently renin inhibitors are widely known as the 3 classic renin-angiotensin system inhibitory drugs against hypertension and atherosclerosis. Here, we developed an array of regents to explore the effects of angiotensinogen inhibition. First, we demonstrated that genetic deficiency of …

Optimization Of A Chemical Genetic Screen To Identify Druggable Targets In U138 Cells Treated With Laromustine, Kathryn Coe

Honors Theses

Laromustine is an experimental sulfonylhydrazine prodrug used in late-stage clinical studies against acute myeloid leukemia (AML) and glioblastoma multiforme (GBM). Despite initial promise for both indications, clinical trials for GBM have not been as successful as those for AML. To investigate methods for improving the effectiveness of laromustine in GBM and to learn more about the mechanism of action of laromustine, a chemical genetic screen will be conducted to identify agents that sensitize GBM cells to the anti-proliferative effects of laromustine. The library, which will include approximately 450 FDA-approved drugs, will be screened using a newly optimized high throughput assay …

An Unexpected Particle Oscillation For Electrophoresis In Viscoelastic Fluids Through A Microchannel Constriction, Xinyu Lu, Saurin Patel, Meng Zhang, Sang Woo Joo, Shizhi Qian, Amod Ogale, Xiangchun Xuan

Mechanical & Aerospace Engineering Faculty Publications

Electrophoresis plays an important role in many applications, which, however, has so far been extensively studied in Newtonian fluids only. This work presents the first experimental investigation of particle electrophoresis in viscoelastic polyethylene oxide (PEO) solutions through a microchannel constriction under pure DC electric fields. An oscillatory particle motion is observed in the constriction region, which is distinctly different from the particle behavior in a polymer-free Newtonian fluid. This stream-wise particle oscillation continues until a sufficient number of particles form a chain to pass through the constriction completely. It is speculated that such an unexpected particle oscillating phenomenon is a …

Regulation And Mechanistic Functions Of Caspase-9 Rna Splicing, Ngoc T. Vu

Theses and Dissertations

Caspase-9 has two splice variants, pro-apoptotic caspase-9a and anti-apoptotic caspase-9b, and dysregulation of caspase-9 splice variant ratio or expression of caspase-9b isoform has been linked to augmentation of the anchorage-independent growth and tumorigenic capacity of non-small cell lung cancer (NSCLC) cells. This study focuses on cell signaling pathway(s) regulating the alternative splicing of caspase-9 pre-mRNA and mechanistic roles of caspase-9b in a certain oncogenic/survival pathway. In regards to the former, we have identified hnRNP U as a novel splice-enhancer associated with exon 3 of caspase-9 (C9/E3). Moreover, hnRNP U binds specifically to C9/E3 at an RNA cis-element previously reported …

Structural And Functional Characterization Of The Mbd2-Nurd Co-Repressor Complex, Megha Desai

Theses and Dissertations

The MBD2-NuRD co-repressor complex is an epigenetic regulator of the developmental silencing of embryonic and fetal β-type globin genes in adult erythroid cells as well as aberrant methylation-dependent silencing of tumor suppressor genes in neoplastic diseases. Biochemical characterization of the MBD2-NuRD complex in chicken erythroid cells identified RbAp46/48, HDAC1/2, MTA1/2/3, p66α/β, Mi2α/β and MBD2 to comprise this multi-protein complex.

In the work presented in Chapter 2, we have pursued biophysical and molecular studies to describe a previously uncharacterized domain of human MBD2 (MBD2_IDR). Biophysical analyses show that MBD2_IDR is an intrinsically disordered region (IDR). Despite this inherent …

Ycf1-Mediated Cadmium Resistance In Yeast Is Dependent On Copper Metabolism And Antioxidant Enzymes, Wenzhong Wei, Nathan Smith, Xiaobin Wu, Heejeong Kim, Javier Seravalli, Oleh Khalimonchuk, Jaekwon Lee

Department of Biochemistry: Faculty Publications

Aims: Acquisition and detoxification of metal ions are vital biological processes. Given the requirement of metallochaperones in cellular copper distribution and metallation of cuproproteins, this study investigates whether the metallochaperones also deliver metal ions for transporters functioning in metal detoxification. Results: Resistance to excess cadmium and copper of the yeast Saccharomyces cerevisiae, which is conferred by PCA1 and CaCRP1 metal efflux P-type ATPases, respectively, does not rely on known metallochaperones, Atx1p, Ccs1p, and Cox17p. Copper deficiency induced by the expression of CaCRP1 encoding a copper exporter occurs in the absence of Atx1p. Intriguingly, CCS1 encoding the copper chaperone for superoxide …

Characterization And Identification Of Novel Regulators Of The Synthesis Of Phospholipids, Cunqi Ye

Wayne State University Dissertations

Phospholipids are the most abundant lipids in cell membranes. The synthesis of phospholipids is crucial for cellular membrane biogenesis and nearly all aspects of cellular processes. Understanding the regulation of synthesis of phospholipids is beneficial to our fundamental knowledge of cell biology as well as human health.

Regulation of the synthesis of phospholipids is intensively studied in the yeast S. cerevisiae. Most notably, the synthesis of phospholipids is coordinated with the synthesis of inositol, a precursor of inositol-containing lipids, by controlling expression of the genes encoding phospholipid biosynthetic enzymes. In addition to this well-characterized regulatory circuit controlled by the …

Characterization Of Ns1 Sumoylation And Its Effect On Influenza A Viral Infection, Katherine Anne Meraz

Open Access Theses & Dissertations

Influenza virus is a contagious respiratory virus responsible for seasonal epidemics and several catastrophic pandemics in the last century. Its genome is comprised of negative sense, single-stranded RNA and, after entering the cell, it is capable of hijacking the host cellular machinery for reproducing its own genetic material. The activation of cellular defenses against influenza viral infection are triggered upon viral entry and help regulate the course of viral infection. This study focused on the interplay between the influenza A virus and the cellular SUMOylation system during viral infection. The first part of this Dissertation deals with the relevance of …

Vibrational Spectroscopic Analysis Of Body Fluids: Avoiding Molecular Contamination Using Centrifugal Filtration, Franck Bonnier, Matthew Baker, Hugh Byrne

Articles

The use of centrifugal filtration for protein purification and concentration represents an important improvement for the application of vibrational spectroscopy to analysis of bodily fluids based on protein fractions with specific molecular weight. Vibrational spectroscopic techniques are highly specific and can potentially detect small variations in the protein content indicating the presence of different diseases, therefore usable as markers for early diagnostic. However, due to the high sensitivity of the techniques, it is essential to verify that no molecular contamination occurs during the preparation of the samples. Concentration of the blood serum using commercially available centrifugal filters has been shown …

The Impact Of Down Syndrome And Folate Depletion On Genomic Stabilizing Pathways Of Lymphoblastoid Cells, Khadijah Ibrahim Alnabbat

Wayne State University Theses

Understanding the role of nutrition plays in Down syndrome (DS) could help in the development and implementation of strategies that help overcome the negative consequences phenotypes of Down syndrome. Conserving genome integrity is crucial for cells to survive, and thus understanding how genetic defects induce damage to genomic DNA and impair subsequent repair of this damage is important. Evidence accumulated points to increased DNA damage and mutation accumulation associated with a decline in DNA repair capacity, Base Excision Repair (BER) in particular. Thus, the successful clinical management of DS resides in understanding the metabolic imbalance provoked by overexpression of genes …

Hiv-1 Rna Dimerization At Single Molecule Level, Hansini R. Mundigala

Wayne State University Dissertations

The Dimerization Initiation Sequence (DIS) is a conserved hairpin-loop motif on the 5' UTR of the HIV-1 genome. It plays an important role in genome dimerization through formation of a "kissing complex" intermediate between two homologous DIS sequences. This bimolecular kissing complex ultimately leads to the formation of an extended RNA duplex. Understanding the kinetics of this interaction is key to exploiting DIS as a possible drug target against HIV. We wish to report a novel study that makes an important contribution to understanding the dimerization mechanism of HIV-1 RNA in vitro. Our work has employed single-molecule fluorescence resonance energy …

Effects Of Statin Drugs And Tocotrienol Rich Fraction Supplementation In Chronic Hemodialysis Patients And Metabolomic Profile, Eno Latifi

Wayne State University Theses

Chronic kidney disease (CKD) is known as a heterogeneous disorder which currently is on the rise and lately has been classified as a public health issues in the United State and worldwide. CKD is an irreversible and progressive disease which can lead to kidney failure, and this is depicted by the advanced stage of the disorder when it reaches the point, that is classified as end stage of renal disease (ESRD) (Stage 5 of CKD) (eGRF <15 mL/min/ 1.73 m2 working capacity), where both organs are in a total or permanent kidney failure. End-Stage renal disease patients, on hemodialysis have been associated to experience an accelerated form of atherosclerosis, which is induced by inflammation, impairment of antioxidant system and elevated oxidative stress. Since the problem effecting ESRD patients is multifactorial, the objective of this investigation is to explore and look at the effects of supplementing with vitamin E-tocotrienol rich fraction (TRF), a micronutrient which has anti-inflammatory, antioxidant, and lipid lower capabilities into tackling these comorbid conditions experienced by this population. Therefore the aims of this investigation will be to explore changes in lipid profiles, inflammatory markers, and oxidative status, as well as look at any changes in metabolomic profiles. It was hypothesized that by supplementing with TRF a vitamin E, for 16 weeks in ESRD patients undergoing hemodialysis, it may help reverse and/or improve, oxidative status, inflammatory markers, increase antioxidants status and improve lipid profiles.

The study was double-blinded, randomized, parallel, placebo-controlled design trial, of 81 adult patients undergoing chronic hemodialysis at Great Lake Dialysis Clinic, Detroit MI, where …

Design And Study Of The Efflux Function Of The Egfp Fused Mexab-Oprm Membrane Transporter In Pseudomonas Aeruginosa Using Spectroscopy, Feng Ding, Kerry J. Lee, Ardeschir Vahedi-Faridi, Hiroshi Yoneyama, Christopher J. Osgood, Xiao-Hong Nancy Xu

Biological Sciences Faculty Publications

Multidrug membrane transporters (efflux pumps) can selectively extrude a variety of structurally and functionally diverse substrates (e.g., chemotoxics, antibiotics), leading to multidrug resistance (MDR) and ineffective treatment of a wide variety of diseases. In this study, we have designed and constructed a fusion gene (egfp-mexB) of N-terminal mexB with C-terminal egfp, inserted it into a plasmid vector (pMMB67EH), and successfully expressed it in the Δ MexB (MexB deletion) strain of Pseudomonas aeruginosato create a new strain that expresses MexA-(EGFP-MexB)-OprM. We characterized the fusion gene using gel electrophoresis and DNA sequencing, and determined its expression in live …

The Group Iva Cytosolic Phospholipase A2/C1p Interaction And Its Role In Eicosanoid Synthesis And Inflammation, Jennifer A. Mietla

Theses and Dissertations

In the presented study, we demonstrate that the interaction of group IVA cytosolic phospholipase A2 and ceramide-1-phosphate is crucial for production of eicosanoid synthesis in inflammation. Inflammation is a critical component of many disease states including anaphylaxis, cancer, cardiovascular disease, rheumatoid arthritis, diabetes and asthma. Eicosanoids are well established mediators of inflammation, and the initial rate limiting step in the production of eicosanoids is the liberation of arachidonic acid (AA) from membrane phospholipids by a phospholipase A2 (PLA2). The major phospholipase involved in this liberation of AA during the inflammatory response is group IVA cytosolic phospholipase A2 (cPLA2α). Previous studies …

Monitoring Ligand-Induced Nucleic Acid Conformational Changes Using Ion Mobility Spectrometry-Mass Spectrometry, Bill Kenneth Redick

Legacy Theses & Dissertations (2009 - 2024)

Three-dimensional structures of biopolymers frequently dictate the biological role those molecules play. As such, investigation into structure of nucleic acids can provide important information pertaining to how those nucleic acids work. Many nucleic acid species, especially single-stranded RNA, fold into unique structures that allow them to function properly. Metals, and other cationic species, are often bound to the nucleic acid to make folding into the proper structure more favorable by neutralizing the negative charge on the nucleic acid imparted by the phosphate group. This investigation explores tertiary structure of nucleic acids that have been folded in the presence of ligands …

Regulation Of Ty1 Retrovirus-Like Transposon Rna Localization And Translation, Ryan Joseph Palumbo

Legacy Theses & Dissertations (2009 - 2024)

Replication of the Ty1 retrovirus-like transposon of the yeast Saccharomyces cerevisiae is stringently regulated to reduce the frequency of deleterious retrotransposition events. However, under stress conditions, Ty1 retrotransposition can lead to adaptive genomic alterations. To characterize host regulation of Ty1 retrotransposition, I analyzed ribosome profiling data and showed that Ty1 RNA is efficiently translated. Moreover, the ribosome biogenesis factors BUD21, DBP7, HCR1, LOC1, MRT4, and PUF6 are required for optimal expression of the Ty1 protein Gag.

Ph Regulation And The Assessment Of Renal Injury Biomarkers In A Warm Perfusion Renal Allograft Preservation System, Aaron Meyer

Legacy Theses & Dissertations (2009 - 2024)

A shortage of viable kidneys available for transplantation exists today, as the number of individuals waiting for a kidney transplant continues to grow while the number of kidneys available each year for transplantation has remained stagnant. The development of technology that will allow for transplantation of organs that currently may be considered too damaged for transplant will provide access to a large number of donors who have experienced traumatic injury deaths resulting in loss of cardiac function. These potential donors account for the majority of all traumatic injury deaths in intensive care units, however their organs have experienced a degree …

Mechanisms Of Dna Synthesis And Fidelity By Y-Family Translesion And C-Family Replicative Polymerases, Purba Mukherjee

Legacy Theses & Dissertations (2009 - 2024)

Since the discovery of the DNA polymerase by Arthur Kornberg nearly 60 years ago, there have been great advances in understanding the involvement of polymerases in replication and repair. Years of genetic, biochemical and structural studies have lead to the classification of DNA-dependent DNA polymerases into six families: A, B, C, D, X and Y. In this work, I have focussed on two different families, C and Y. Hence this work is divided into two parts. Part one discusses the studies on Y-family polymerases. All Y-family polymerases are involved in replicating past DNA lesions. The ability to tolerate unnatural nucleotides …

Modeling The Early, Aging-Related Changes In Human Recombinant Gammas-Crystallin In Vitro, Christiana Oluwaseun Salami

Legacy Theses & Dissertations (2009 - 2024)

Cataract is the leading cause of blindness in the world. Cataractous lenses scatter a significant part of the light entering the eye. Cataract can be of several types: Age-related cataract, secondary cataract, traumatic cataract, congenital cataract, and radiation cataract. Age-related cataract is the most common type of cataract. Several factors contribute to age-onset cataract; among them are the Post-Translational Modifications (PTMs) to the major lens proteins or crystallins, which constitute about 80-90% of the protein content of the lens. Predominant PTMs of the crystallins are oxidation, deamidation, phosphorylation, glycation, and disulfide crosslinking, which tend to increase as the lens ages.