Biochemistry, Biophysics, and Structural Biology Commons^™

Plasma Membrane Voltage Changes During Nanosecond Pulsed Electric Field Exposure, W. Frey, R. O. Price, P. F. Blackmore, R. P. Joshi, R. Nuccitelli, S. J. Beebe, K. H. Schoenbach, J. F. Kolb

Bioelectrics Publications

The change in the membrane potential of Jurkat cells in response to nanosecond pulsed electric fields was studied for pulses with a duration of 60 ns and maximum field strengths of similar to 100 kV/cm (100 V/cell diameter). Membranes of Jurkat cells were stained with a fast voltage-sensitive dye, ANNINE-6, which has a subnanosecond voltage response time. A temporal resolution of 5 ns was achieved by the excitation of this dye with a tunable laser pulse. The laser pulse was synchronized with the applied electric field to record images at times before, during, and after exposure. When exposing the Jurkat …

Identification And Expansion Of Pancreatic Stem/Progenitor Cells., You-Qing Zhang, Marcie Kritzik, Nora Sarvetnick

Journal Articles: Regenerative Medicine

Pancreatic islet transplantation represents an attractive approach for the treatment of diabetes. However, the limited availability of donor islets has largely hampered this approach. In this respect, the use of alternative sources of islets such as the ex vivo expansion and differentiation of functional endocrine cells for treating diabetes has become the major focus of diabetes research. Adult pancreatic stem cells /progenitor cells have yet to be recognized because limited markers exist for their identification. While the pancreas has the capacity to regenerate under certain circumstances, questions where adult pancreatic stem/progenitor cells are localized, how they are regulated, and even …

Selective Field Effects On Intracellular Vacuoles And Vesicle Membranes With Nanosecond Electric Pulses, Ephrem Tekle, Hammou Oubrahim, Sergey M. Dzekunov, Juergen F. Kolb, Karl H. Schoenbach

Bioelectrics Publications

Electric pulses across intact vesicles and cells can lead to transient increase in permeability of their membranes. We studied the integrity of these membranes in response to external electric pulses of high amplitude and submicrosecond duration with a primary aim of achieving selective permeabilization. These effects were examined in two separate model systems comprising of 1), a mixed population of 1,2-di-oleoyl-sn-glycero-3-phosphocholine phospholipid vesicles and in 2), single COS-7 cells, in which large endosomal membrane vacuoles were induced by stimulated endocytosis. It has been shown that large and rapidly varying external electric fields, with pulses shorter than the charging time of …

Layer-By-Layer Self -Assembly For Enzyme And Dna Encapsulation And Delivery, Amish Patel

Doctoral Dissertations

Thin wall microcapsules were formed via Layer-by-Layer Self-Assembly of alternate adsorption of oppositely charged polyelectrolyte on microcores. After the core dissolution, empty polymeric shells with 20–25 nm thick walls were obtained. These microcapsules were loaded with Myoglobin, Hemoglobin and Glucose Oxidase by opening capsule pores at low pH and closing them at higher pH. The native structure of the enzyme was not affected due to different treatments. Biocompatible nanoshells were also prepared for encasing DNA. Using the same Layer-by-Layer Self-Assembly approach nanoparticle were constructed containing DNA as one of the layers. The nanoparticles of different architecture were used to deliver …

A High -Order Finite Difference Method For Solving Bioheat Transfer Equations In Three-Dimensional Triple -Layered Skin Structure, Haofeng Yu

Doctoral Dissertations

Investigations on instantaneous skin burns are useful for an accurate assessment of burn-evaluation and for establishing thermal protections for various purposes. Meanwhile, hyperthermia with radiation is important in the treatment of cancer, and it is essential for developers and users of hyperthermia systems to predict, and interpret correctly the biomass thermal and vascular response to heating. In this dissertation, we employ the well-known Pennes' bioheat transfer equation to predict the degree of skin burn and the temperature distribution in hyperthermia cancer treatment.

A fourth-order compact finite difference scheme is developed to solve Pennes' bioheat transfer equation in a three-dimensional single …

Energy-Landscape-Model Analysis For Irreversibility And Its Pulse-Width Dependence In Cells Subjected To A High-Intensity Ultrashort Electric Pulse, R. P. Joshi, Q. Hu, Karl H. Schoenbach, Stephen J. Beebe

Bioelectrics Publications

We provide a simple, but physical analysis for cell irreversibility and apoptosis in response to an ultrashort (nanosecond), high-intensity electric pulse. Our approach is based on an energy landscape model for determining the temporal evolution of the configurational probability function p(q). The primary focus is on obtaining qualitative predictions of a pulse width dependence to apoptotic cell irreversibility that has been observed experimentally. The analysis couples a distributed electrical model for current flow with the Smoluchowski equation to provide self-consistent, time-dependent transmembrane voltages. The model captures the essence of the experimentally observed pulse-width dependence, and provides a possible physical picture …

Growth And Transport Properties Of Complementary Germanium Nanowire Field Effect Transistors, Andrew B. Greytak, Lincoln J. Lauhon, Mark S. Gudiksen, Charles M. Lieber

Faculty Publications

n- and p-type Ge nanowires were synthesized by a multistep process in which axial elongation, via vapor–liquid–solid (VLS) growth, and doping were accomplished in separate chemical vapor deposition steps. Intrinsic, single-crystal, Ge nanowires prepared by Au nanocluster-mediated VLS growth were surface-doped in situ using diborane or phosphine, and then radial growth of an epitaxial Ge shell was used to cap the dopant layer. Field-effect transistors prepared from these Ge nanowires exhibited on currents and transconductances up to 850 µA/µm and 4.9 µA/V, respectively, with device yields of >85%.

Stimulation Of Capacitative Calcium Entry In Hl-60 Cells By Nanosecond Pulsed Electric Fields, Jody A. White, Peter F. Blackmore, Karl H. Schoenbach, Stephen J. Beebe

Bioelectrics Publications

Nanosecond pulsed electric fields (nsPEFs) are hypothesized to affect intracellular structures in living cells providing a new means to modulate cell signal transduction mechanisms. The effects of nsPEFs on the release of internal calcium and activation of calcium influx in HL-60 cells were investigated by using real time fluorescent microscopy with Fluo-3 and fluorometry with Fura-2. nsPEFs induced an increase in intracellular calcium levels that was seen in all cells. With pulses of 60 ns duration and electric fields between 4 and 15 kV/cm, intracellular calcium increased 200-700 nM, respectively, above basal levels (similar to100 nM), while the uptake of …

The Stromal Cell-Derived Factor-1alpha/Cxcr4 Ligand-Receptor Axis Is Critical For Progenitor Survival And Migration In The Pancreas., Ayse G. Kayali, Kurt Van Gunst, Iain L. Campbell, Aleksandr Stotland, Marcie Kritzik, Guoxun Liu, Malin Flodström-Tullberg, You-Qing Zhang, Nora Sarvetnick

Journal Articles: Regenerative Medicine

The SDF-1alpha/CXCR4 ligand/chemokine receptor pair is required for appropriate patterning during ontogeny and stimulates the growth and differentiation of critical cell types. Here, we demonstrate SDF-1alpha and CXCR4 expression in fetal pancreas. We have found that SDF-1alpha and its receptor CXCR4 are expressed in islets, also CXCR4 is expressed in and around the proliferating duct epithelium of the regenerating pancreas of the interferon (IFN) gamma-nonobese diabetic mouse. We show that SDF-1alpha stimulates the phosphorylation of Akt, mitogen-activated protein kinase, and Src in pancreatic duct cells. Furthermore, migration assays indicate a stimulatory effect of SDF-1alpha on ductal cell migration. Importantly, blocking …

The Effects Of Intense Submicrosecond Electrical Pulses On Cells, Jingdong Deng, Karl H. Schoenbach, E. Stephen Buescher, Pamela S. Hair, Paula M. Fox, Stephen J. Beebe

Bioelectrics Publications

A simple electrical model for living cells predicts an increasing probability for electric field interactions with intracellular substructures of both prokaryotic and eukaryotic cells when the electric pulse duration is reduced into the submicrosecond range. The validity of this hypothesis was verified experimentally by applying electrical pulses (durations 100 μs– 60 ns, electric field intensities 3–150 kV/cm) to Jurkat cells suspended in physiologic buffer containing propidium iodide. Effects on Jurkat cells were assessed by means of temporally resolved fluorescence and light microscopy. For the longest applied pulses, immediate uptake of propidium iodide occurred consistent with electroporation as the cause of …

Protocols For Disease Classification From Mass Spectrometry Data, Michael Wagner, Dayanand Naik, Alex Pothen

Mathematics & Statistics Faculty Publications

We report our results in classifying protein matrix-assisted laser desorption/ionizationtime of flight mass spectra obtained from serum samples into diseased and healthy groups. We discuss in detail five of the steps in preprocessing the mass spectral data for biomarker discovery, as well as our criterion for choosing a small set of peaks for classifying the samples. Cross-validation studies with four selected proteins yielded misclassification rates in the 10-15% range for all the classification methods. Three of these proteins or protein fragments are down-regulated and one up-regulated in lung cancer, the disease under consideration in this data set. When cross-validation studies …

Ccr4-Bearing T Cells Participate In Autoimmune Diabetes., Soon H. Kim, Mary M. Cleary, Howard S. Fox, Icos Coporation, Nora Sarvetnick

Journal Articles: Regenerative Medicine

Chemokine receptor expression is exquisitely regulated on T cell subsets during the course of their migration to inflammatory sites. In the present study we demonstrate that CCR4 expression marks a pathogenic population of autoimmune T cells. CCR4 was found exclusively on memory CD4(+) T cells during the progression of disease in NOD mice. Cells expressing the CCR4 ligand TARC (thymus- and activation-regulated chemokine) were detected within infiltrated islets from prediabetic mice. Interestingly, neutralization of macrophage-derived chemokine (MDC) with Ab caused a significant reduction of CCR4-positive T cells within the pancreatic infiltrates and inhibited the development of insulitis and diabetes. Furthermore, …

Presented Antigen From Damaged Pancreatic Beta Cells Activates Autoreactive T Cells In Virus-Mediated Autoimmune Diabetes., Marc S. Horwitz, Alex Ilic, Cody Fine, Enrique Rodriguez, Nora Sarvetnick

Journal Articles: Regenerative Medicine

The induction of autoimmunity by viruses has been attributed to numerous mechanisms. In mice, coxsackievirus B4 (CB4) induces insulin-dependent diabetes mellitus (IDDM) resembling the final step of disease progression in humans. The immune response following the viral insult clearly precipitates IDDM. However, the molecular pathway between viral infection and the subsequent activation of T cells specific for islet antigen has not been elucidated. These T cells could become activated through exposure to sequestered antigens released by damaged beta cells, or they could have responded to factors secreted by the inflammatory response itself. To distinguish between these possibilities, we treated mice …

Theoretical Predictions Of Electromechanical Deformation Of Cells Subjected To High Voltages For Membrane Electroporation, R. P. Joshi, Q. Hu, K. H. Schoenbach, H. P. Hjalmarson

Bioelectrics Publications

An electromechanical analysis based on thin-shell theory is presented to analyze cell shape changes in response to external electric fields. This approach can be extended to include osmotic-pressure changes. Our calculations demonstrate that at large fields, the spherical cell geometry can be significantly modified, and even ellipsoidal forms would be inappropriate to account for the deformation. Values of the surface forces obtained from our calculations are in very good agreement with the 1–10 mN/m range for membrane rupture reported in the literature. The results, in keeping with reports in the literature, demonstrate that the final shape depends on membrane thickness. …

Mechanism For Membrane Electroporation Irreversibility Under High-Intensity, Ultrashort Electrical Pulse Conditions, R. P. Joshi, K. H. Schoenbach

Bioelectrics Publications

An improved electroporation model is used to address membrane irreversibility under ultrashort electric pulse conditions. It is shown that membranes can survive a strong electric pulse and recover provided the pore distribution has a relatively large spread. If, however, the population consists predominantly of larger radii pores, then irreversibility can result. Physically, such a distribution could arise if pores at adjacent sites coalesce. The requirement of close proximity among the pore sites is more easily satisfied in smaller organelles than in outer cell membranes. Model predictions are in keeping with recent observations of cell damage to intracellular organelles (e.g., mitochondria), …

Studies On The Formation Of Dna-Cationic Lipid Composite Films And Dna Hybridization In The Composites, Murali Sastry, Vidya Ramakrishnan, Mrunalini Pattarkine, Krishna N. Ganesh

Faculty Works

The formation of composite films of double-stranded DNA and cationic lipid molecules (octadecylamine, ODA) and the hybridization of complementary single-stranded DNA molecules in such composite films are demonstrated. The immobilization of DNA is accomplished by simple immersion of a thermally evaporated ODA film in the DNA solution at close to physiological pH. The entrapment of the DNA molecules in the cationic lipid film is dominated by attractive electrostatic interaction between the negatively charged phosphate backbone of the DNA molecules and the protonated amine molecules in the thermally evaporated film and has been quantified using quartz crystal microgravimetry (QCM). Fluorescence studies …

Cationic Surfactant Mediated Hybridization And Hydrophobization Of Dna Molecules At The Liquid/Liquid Interface And Their Phase Transfer, Murali Sastry, Ashavani Kumar, Mrunalini Pattarkine, Vidya Ramakrishnan, Krishna N. Ganesh

Faculty Works

Hybridization of complementary oligonucleotides mediated by a cationic surfactant at the water/hexane interface leads to hydrophobic, double-helical DNA which may be readily phase transferred to the organic phase and cast into thin films on solid substrates.

Immobolisation And Biochemical Characterisation Of A Selected Commercially Available Laccase, Chandra Sundaralingam

Student Works (2000-2009)

ln this study, laccase from Novo Nordisk was chosen for biochemical characterization and immobilization investigation. The specific aims of this work was to develop optimum conditions for immobilization of laccase by entrapment method using copper alginate and characterize the laccase biochemically. The pH optimum of the crude enzyme extract was 5.5 and was found to be stable over a wide range of pH values. The apparent molecular weight of this enzyme was around 79,000 Daltons. Three peaks of activity (isoenzymes) were obtained from isoelectric focusing of pH values 4.7, 5.2 and 6.0. The optimum pH of the immobilized laccase by …

Characterization Of The Biological Activities Of Recombinant Fusion Protein Green Fluorescent Protein/Human Zona Pellucida Protein 3 (Gfp/Hzp3), Zhiyong Lin

Theses and Dissertations in Biomedical Sciences

Despite numerous reports indicating the successful production of bioactive recombinant ZP3, no report has shown the rhZP3 having direct binding activity with human sperm. Recombinant ZP3 generated from our previous study displayed binding activity with human sperm through indirect evidence from hemizona assay (HZA).

This present study focused on the production of recombinant ZP3 with direct binding activity with human sperm. Through the application of a pEGFP expression vector, fusion protein GFP/ZP3 was successfully generated and expressed. The expression of GFP/ZP3 was evidenced by RT-PCR and western blot. The fusion protein was partially purified by Ni-NTA affinity column from cell …

Hybridization Of Dna By Sequential Immobilization Of Oligonucleotides At The Air-Water Interface, Murali Sastry, Vidya Ramakrishnan, Mrunalini Pattarkine, Anand Gole, K. N. Ganesh

Faculty Works

The hybridization of DNA by sequential electrostatic and hydrogen-bonding immobilization of single-stranded complementary oligonucleotides at the air-water interface with cationic Langmuir monolayers is demonstrated. The complexation of the single-stranded DNA molecules with octadecylamine (ODA) Langmuir monolayers was followed in time by monitoring the pressure-area isotherms. A large (and slow) expansion of the ODA monolayer was observed during each stage of complexation in the following sequence: primary single-stranded DNA followed by complementary single-stranded DNA followed by the intercalator, ethidium bromide. Langmuir-Blodgett (LB) films of the ODA-DNA complex were formed on different substrates and characterized using quartz-crystal microgravimetry (QCM), Fourier transform infrared …

Urea Hydrolysis Using Immobilized Urease, Anita Arullan

Student Works (2000-2009)

An inexpensive carrier matrix. vermiculite was used for immobilizing urease. A cost effective and simple method of coupling was developed using vermiculite. Immobilization of urease on Amberlite MB-land Nylon-6 tube were also studied. Activities of free and immobilized urease were studied and compared: Among all methods used for immobilization of urease. the method of coupling the enzyme to vermiculite gave the highest retained activity of 82% (Method I) and 89% (Method 2) at pH 6.0 and 5.5 respectively. Urease immobilized by Method I retained 69% and 30% of its original activity and Method 2 (using glutaraldehyde as coupling agent) retained …

A Defect In Interleukin 12-Induced Activation And Interferon Gamma Secretion Of Peripheral Natural Killer T Cells In Nonobese Diabetic Mice Suggests New Pathogenic Mechanisms For Insulin-Dependent Diabetes Mellitus., Marika Falcone, Brian Yeung, Lee Tucker, Enrique Rodriguez, Nora Sarvetnick

Journal Articles: Regenerative Medicine

The function of natural killer T (NKT) cells in the immune system has yet to be determined. There is some evidence that their defect is associated with autoimmunity, but it is still unclear how they play a role in regulating the pathogenesis of T cell-mediated autoimmune diseases. It was originally proposed that NKT cells could control autoimmunity by shifting the cytokine profile of autoimmune T cells toward a protective T helper 2 cell (Th2) type. However, it is now clear that the major function of NKT cells in the immune system is not related to their interleukin (IL)-4 secretion. In …

Ibpp Research Associates: Space And Extreme Environments, M. Ephimia Morphew - Society For Human Performance In Extreme Environments

International Bulletin of Political Psychology

Per M. Ephimia Morphew. President of the Society for Human Performance in Extreme Environments previously online at http://www.hpee.org, colleagues at the Institute of Biomedical Problems in Russia (IBMP) are engaged in an isolation experiment pertaining to space exploration that is two months underway and preliminary findings are reported [in the article entitled First Two Months of Simulated Isolation Passed.]

IBPP commentary includes a discussion of human factors and political psychology as parameters of space.

Evaluation Of Toxicity, Bioavailability And Speciation Of Lead, Zinc And Cadmium In Mine/Mill Wastewaters, Mujde Erten-Unal, Bobby G. Wixson, Nord Gale, Jerry L. Pitt

Civil & Environmental Engineering Faculty Publications

The toxicity of common compounds of lead, cadmium and zinc was evaluated in waters similar to that found in the world's largest lead producing area in Missouri. Static, acute toxicity tests were performed using fathead minnows (Pimephales promelas) and water fleas (Daphnia magna) respectively. Test organisms were subjected to varying amounts of sulfide, carbonate, chloride and sulfate salts of lead, zinc and cadmium mixed in hard, alkaline waters typical to this region. Median lethal concentrations were calculated using nominal versus measured metal concentrations. Measured metal concentrations included four different metal fractionation (extraction/filtration) techniques at different pH …

Interferon Gamma (Ifn-Gamma) Is Necessary For The Genesis Of Acetylcholine Receptor-Induced Clinical Experimental Autoimmune Myasthenia Gravis In Mice., Balaji Balasa, Caishu Deng, Jae Lee, Linda M. Bradley, Dyanna K. Dalton, Premkumar Christadoss, Nora Sarvetnick

Journal Articles: Regenerative Medicine

Experimental autoimmune myasthenia gravis (EAMG) is an animal model of human myasthenia gravis (MG). In mice, EAMG is induced by immunization with Torpedo californica acetylcholine receptor (AChR) in complete Freund's adjuvant (CFA). However, the role of cytokines in the pathogenesis of EAMG is not clear. Because EAMG is an antibody-mediated disease, it is of the prevailing notion that Th2 but not Th1 cytokines play a role in the pathogenesis of this disease. To test the hypothesis that the Th1 cytokine, interferon (IFN)-gamma, plays a role in the development of EAMG, we immunized IFN-gamma knockout (IFN-gko) (-/-) mice and wild-type (WT) …

Local Delivery Of Interleukin 4 By Retrovirus-Transduced T Lymphocytes Ameliorates Experimental Autoimmune Encephalomyelitis., Michael K. Shaw, James B. Lorens, Archana Dhawan, Richard Dalcanto, Harley Y. Tse, Alyssa B. Tran, Colleen Bonpane, Shanti L. Eswaran, Stefan Brocke, Nora Sarvetnick, Lawrence Steinman, Garry P. Nolan, C. Garrison Fathman

Journal Articles: Regenerative Medicine

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory autoimmune disease of the central nervous system which serves as a model for the human disease multiple sclerosis. We demonstrate here that encephalitogenic T cells, transduced with a retroviral gene, construct to express interleukin 4, and can delay the onset and reduce the severity of EAE when adoptively transferred to myelin basic protein-immunized mice. Thus, T lymphocytes transduced with retroviral vectors can deliver "regulatory cytokines" in a site-specific manner and may represent a viable therapeutic strategy for the treatment of autoimmune disease.

Ifn-Gamma, Igif, And Iddm., Nora Sarvetnick

Journal Articles: Regenerative Medicine

No abstract provided.

Mechanisms Of Cytokine-Mediated Localized Immunoprotection., Nora Sarvetnick

Journal Articles: Regenerative Medicine

No abstract provided.

Pancreatic Expression Of Interleukin-4 Abrogates Insulitis And Autoimmune Diabetes In Nonobese Diabetic (Nod) Mice., Regula Mueller, Troy Krahl, Nora Sarvetnick

Journal Articles: Regenerative Medicine

Diabetes in nonobese diabetic (NOD) mice is a T cell-dependent autoimmune disease. The destructive activities of autoreactive T cells have been shown to be tightly regulated by effector molecules. In particular, T helper (Th) 1 cytokines have been linked to diabetes pathogenesis, whereas Th2 cytokines and the cells that release them have been postulated to be protective from disease. To test this hypothesis, we generated transgenic NOD mice that express interleukin (IL) 4 in their pancreatic beta cells under the control of the human insulin promoter. We found that transgenic NOD-IL-4 mice, both females and males, were completely protected from …

Il-10 Is Necessary And Sufficient For Autoimmune Diabetes In Conjunction With Nod Mhc Homozygosity., Myung-Shik Lee, Regula Mueller, Linda S. Wicker, Laurence B. Peterson, Nora Sarvetnick

Journal Articles: Regenerative Medicine

Contrary to expectations based on in vitro experiments, we previously found that pancreatic IL-10 did not inhibit autoimmune diabetes but accelerated it in an MHC-dependent manner. Therefore, the ability of IL-10 to overcome the absence of all non-MHC diabetes susceptibility (Idd) alleles was studied in transgenic mice expressing pancreatic IL-10 backcrossed to B10.H2g7 congenic mice, which have no Idd alleles other than NOD MHC (H2g7). IL-10 transgenic backcross 1 (BC1) mice with H2g7/g7 haplotype developed clear-cut insulitis and diabetes, but neither transgenic mice with the H2g/b haplotype nor nontransgenic BC1 mice did so. Further implicating IL-10 in autoimmune diabetes, anti-IL-10 …