Biochemistry, Biophysics, and Structural Biology Commons^™

Familial Als-Associated Mutations Decrease The Thermal Stability Of Distinctly Metallated Species Of Human Copper/Zinc Superoxide Dismutase, Daryl K. Eggers, J. A. Rodriguez, J. S. Valentine, J. A. Roe, A. Tiwari, R. H. Brown, L. J. Hayward

Faculty Publications, Chemistry

We report the thermal stability of wild type (WT) and 14 different variants of human copper/zinc superoxide dismutase (SOD1) associated with familial amyotrophic lateral sclerosis (FALS). Multiple endothermic unfolding transitions were observed by differential scanning calorimetry for partially metallated SOD1 enzymes isolated from a baculovirus system. We correlated the metal ion contents of SOD1 variants with the occurrence of distinct melting transitions. Altered thermal stability upon reduction of copper with dithionite identified transitions resulting from the unfolding of copper-containing SOD1 species. We demonstrated that copper or zinc binding to a subset of “WT-like” FALS mutants (A4V, L38V, G41S, G72S, D76Y, …

Calorimetric Study Of Block-Copolymers Of Poly(N-Butyl Acrylate) And Gradient Poly(N-Butyl Acrylate-Co-Methyl Methacrylate), A I. Buzin, M Pyda, P Costanzo, K Matyjaszewski, B Wunderlich

Chemistry and Biochemistry

The nanophase separation in diblock and triblock copolymers consisting of immiscible poly(n-butyl acrylate) (block A) and gradient copolymers of methyl methacrylate (MMA) and n-butyl acrylate (n BA) (block M/A) were investigated by means of their heat capacity, C_p, as a function of the composition of the blocks M/A and temperature. In all copolymers studied, both blocks are represented by their C_p and glass transition temperature, T_g, as well as the broadening of the transition-temperature range. The low-temperature transition of the blocks A is always close to that of the …

Actin Dynamics Regulate Myosin Assembly In Muscle Cells, John Dylan Cook

Inquiry: The University of Arkansas Undergraduate Research Journal

During muscle cell (i.e. myocyte )development, organization of actin and myosin into the contractile unit (i.e. sarcomere) is required for proper muscle contraction. By disrupting the processes of muscle cell development, I am able to see which steps play important roles in proper maturation of myocytes. Elucidation of the key pathways in muscle development could lead to a better understanding of human cardiac hypertrophies and muscle myopathies. Since actin filament formation precedes myosin organization, I am using actin assembly inhibitors to determine if actin filaments are a necessary prerequisite for myosin organization. It is hypothesized that disruption of actin will …

Properties Of Modified Tryptophans In A Membrane- Spanning Channel, Erin M. Scherer

Inquiry: The University of Arkansas Undergraduate Research Journal

An emerging concept in biology assigns the amino acid tryptophan specific roles at the membrane/water interface that help to determine the conformation and biological function of membrane-spanning proteins. Previous studies involving the antibiotic model system gramicidin A (gA) have illustrated the importance of the indole ring of tryptophan (Trp) in anchoring proteins to a bilayer membrane and promoting ionic currents. To further investigate these phenomena, derivatives of Trp that have lost hydrogen-bonding ability (1-methyl-Trp ), have an altered dipole moment (7-aza-Trp), or both (1-methyl-7-azaTrp) were chosen for incorporation into gA. Gramicidin analogues that incorporate these modified Trps were then analyzed …

The Effect Of Cytosine Methylation On The Structure And Geometry Of The Holliday Junction: The Structure Of D(Ccggtacm5 Cgg) At 1.5 Å Resolution, Jeff Vargason, P. Shing Ho

Faculty Publications - Department of Biological & Molecular Science

No abstract provided.

Fluorescence In Situ Hybridization On Sperm Using Alkaline Denaturation, L. J. Aridgides, M. Stacey, L. Brihn, D. Scott, C. Osgood

Bioelectrics Publications

Fluorescence in situ hybridization (FISH) permits the detection of unique DNA sequences of target DNA within single cells. FISH allows the identifica­tion of DNA sequences within individ­ual cells and readily identifies genetic changes within a mixed population of cells. However, problems arise in main­taining nuclear morphology, particular­ly following harsh denaturation proce­dures where cells are heated to 80°C, causing the nuclear boundary to be­come blurred or the cell to completely burst. In an attempt to preserve cellular morphology and high hybridization ef­ficiency, particularly in fresh tissue samples, we evaluated a FISH protocol using alkaline denaturation in place of heat.

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. …

Mechanisms Counteracting Swelling In Brain Cells During Hyponatremia, Herminia Pasantes-Morales, Rodrigo Franco, Benito Ordaz, Lenin D. Ochoa

School of Veterinary and Biomedical Sciences: Faculty Publications

Water gain in the brain consequent to hyponatremia is counteracted by mechanisms that initially include a compensatory displacement of liquid from the interstitial space to cerebrospinal fluid and systemic circulation and subsequently an active reduction in cell water accomplished by extrusion of intracellular osmolytes to reach osmotic equilibrium. Potassium (K⁺), chloride (Cl^–), amino acids, polyalcohols, and methylamines all contribute to volume regulation, with a major contribution of ions at the early phase and of organic osmolytes at the late phase of the regulatory process. Experimental models in vitro show that osmolyte fluxes occur via leak pathways …

The Atp:Co(I)Rrinoid Adenosyltransferase (Coba) Enzyme Of Salmonella Enterica Requires The 2’-Oh Group Of Atp For Function And Yields Inorganic Triphosphate As Its Reaction Byproduct, Maris V. Fonseca, Nicole R. Baun, Alexander R. Horswill, Ivan Rayment, Jorge C. Escalante-Semerena

Department of Biochemistry: Faculty Publications

The specificity of the ATP:corrinoid adenosyltransferase

(CobA) enzyme of Salmonella enterica serovar

Typhimurium LT2 for its nucleotide substrate was

tested using ATP analogs and alternative nucleotide donors.

The enzyme showed broad specificity for the nucleotide

base and required the 2’-OH group of the ribosyl

moiety of ATP for activity. ³¹P NMR spectroscopy

was used to identify inorganic triphosphate (PPP_i) as

the byproduct of the reaction catalyzed by the CobA

enzyme. Cleavage of triphosphate into pyrophosphate

and orthophosphate did not occur, indicating that

triphosphate cleavage was not required for release of

the adenosylcorrinoid product. Triphosphate was …

Impact Of Sea Grass Density On Carbonate Dissolution In Bahamian Sediments, David J. Burdige, Richard C. Zimmerman

OES Faculty Publications

Carbonate dissolution has been widely observed in shallow water tropical sediments. However, sediment budgets C have generally not been closed with respect to the amount of acid required to produce the observed carbonate dissolution. Recently it has been suggested that enhanced oxygen transport into sediments through the roots and rhizomes of sea grasses might play a role in resolving this mass balance problem. We conducted studies of sea grass-carbonate sediment interactions around Lee Stocking Island, Exuma Islands, Bahamas to further examine this problem. Our studies showed that alkalinity, total dissolved inorganic carbon (ΣCO₂) and Ca²⁺ increased with …

Development Of A Yeast Biosensor Strain For The Identification Of Genotoxic Compounds, Robin M. Reed

Inquiry: The University of Arkansas Undergraduate Research Journal

Advances in combinatorial chemistry have provided the pharmaceutical industry with innumerable lead compounds that could potentially serve as therapeutic agents. One of the challenges in the further development of such compounds is to rapidly, yet inexpensively, distinguish those that have undesirable effects such as genotoxicity. Thus, a simple biological assay that would permit the identification of potential DNA mutagens, and be adaptable to high-throughput technologies would be cost effective in screening such lead compounds. The current methods use the Ames and SOS tests involving prokaryotic organisms, while systems that utilize mammalian cell culture and/or animal testing are time-consuming and expensive. …

Polarized Fluorescence Depletion Reports Orientation Distribution And Rotational Dynamics Of Muscle Cross-Bridges, Marcus G. Bell, Robert E. Dale, Uulke A. Van Der Heide, Yale E. Goldman

PCOM Scholarly Works

The method of polarized fluorescence depletion (PFD) has been applied to enhance the resolution of orientational distributions and dynamics obtained from fluorescence polarization (FP) experiments on ordered systems, particularly in muscle fibers. Previous FP data from single fluorescent probes were limited to the 2nd- and 4th-rank order parameters, and , of the probe angular distribution (ß) relative to the fiber axis and , a coefficient describing the extent of rapid probe motions. We applied intense 12-µs polarized photoselection pulses to transiently populate the triplet state of rhodamine probes and measured the polarization of the ground-state depletion using a weak interrogation …

Characterization Of An Acyl-Coa Thioesterase That Functions As A Major Regulator Of Peroxisomal Lipid Metabolism, Mary Hunt, Karianne Solaas, Bengt F. Kase, Stefan E H Alexson

Articles

Peroxisomes function in b-oxidation of very long- and long-chain fatty acids, dicarboxylic fatty acids, bile acid intermediates, prostaglandins, leukotrienes, thromboxanes, pristanic acid and xenobiotic carboxylic acids. These lipids are mainly chain-shortened for excretion as the carboxylic acids or transported to mitochondria for further metabolism. Several of these carboxylic acids are slowly oxidized and may therefore sequester coenzyme A (CoASH). To prevent CoASH sequestration and to facilitate excretion of chain-shortened carboxylic acids, acyl-CoA thioesterases, which catalyze the hydrolysis of acyl-CoAs to the free acid and CoASH, may play important roles. We have here cloned and characterized a peroxisomal acyl-CoA thioesterase from …

16s Ribosomal Dna Sequencing Confirms The Synonymy Of Vibrio Harveyi And V. Carchariae, Eric J. Gauger, Marta Gomez-Chiarri

Fisheries, Animal and Veterinary Sciences Faculty Publications

Seventeen bacterial strains previously identified as Vibrio harveyi (Baumann et al. 1981) or V. carchariae (Grimes et al. 1984) and the type strains of V. harveyi, V. carchariae and V. campbellii were analyzed by 16S ribosomal DNA (rDNA) sequencing. Four clusters were identified in a phylogenetic analysis performed by comparing a 746 base pair fragment of the 16S rDNA and previously published sequences of other closely related Vibrio species. The type strains of V. harveyi and V. carchariae and about half of the strains identified as V. harveyi or V. carchariae formed a single, wellsupported cluster designed as ‘bona …

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), …

Stat6 Mediates Interleukin-4 Growth Inhibition In Human Breast Cancer Cells, Jennifer L. Gooch, B. Christy, D. Yee

PCOM Scholarly Works

In addition to acting as a hematopoietic growth factor, interleukin-4 (IL-4) inhibits growth of some transformed cells in vitro and in vivo. In this study, we show that insulin receptor substrate (IRS)-1, IRS-2, and signal transducer and activator of transcription 6 (STAT6) are phosphorylated following IL-4 treatment in MCF-7 breast cancer cells. STAT6 DNA binding is enhanced by IL-4 treatment. STAT6 activation occurs even after IRS-1 depletion, suggesting the two pathways are independent. To examine the role of STAT6 in IL-4-mediated growth inhibition and apoptosis, a full-length STAT6 cDNA was transfected into MCF-7 cells. Transient overexpression of STAT6 resulted in …

Role Of Bcl-2 Family Members In Caspase-Independent Apoptosis Due To Chlamydia Infection, Jean-Luc Perfettini, John C. Reed, Nicole Israel, Jean-Claude Martinou, Alice Dautry-Varsat, David M. Ojcius

All Dugoni School of Dentistry Faculty Articles

Infection with an obligate intracellular bacterium, the Chlamydia trachomatis lymphogranuloma venereum (LGV/L2) strain or the guinea pig inclusion conjunctivitis serovar of Chlamydia psittaci, leads to apoptosis of host cells. The apoptosis is not affected by a broad-spectrum caspase inhibitor, and caspase-3 is not activated in infected cells, suggesting that apoptosis mediated by these two strains of Chlamydia is independent of known caspases. Overexpression of the proapoptotic Bcl-2 family member, Bax, was previously shown to induce caspase-independent apoptosis, and we find that Bax is activated and translocates from the cytosol to the mitochondria in C. psittaci-infected cells. C. psittaci-induced apoptosis is …

Influence Of Protein Tyrosine Kinases On Cell Volume Change-Induced Taurine Release, Herminia Pasantes-Morales, Rodrigo Franco

School of Veterinary and Biomedical Sciences: Faculty Publications

Taurine efflux occurs in association with cell swelling in both hyposmotic and isosmotic conditions and during cell shrinkage in apoptotic death. Release occurs through a leak pathway, is largely Ca²⁺-independent and is sensitive to Cl^– channel blockers. Taurine efflux elicited by hyposmolarity is reduced or suppressed by tyrosine kinase blockers and increased by tyrosine phosphatase inhibitors. The specific kinases involved are still unknown and may be different in the various cell types. Non-receptor and scr-related protein kinases have been identified in some cells as elements that directly phosphorylate the taurine efflux pathway. Possible tyrosine kinase targets are …

Methods And Compositions For Protection Against Bovine Viral Diseases, Subramaniam Srikumaran

School of Veterinary and Biomedical Sciences: Faculty Publications

The present invention relates to methods and compositions for eliciting an immune response against bovine viral epitopes. The methods comprise combining at least one heat shock protein with at least one bovine viral epitope to form a purified epitope/heat shock protein complex and administration of an immune system stimulating amount of the purified epitope/heat shock protein complex. The compositions comprise, a purified epitope/heat shock protein complex comprising at least one bovine viral epitope complexed with at least one heat shock protein, and a pharmaceutically acceptable carrier, diluent or excipient.

Rabd, A Dictyostelium Rab14-Related Gtpase, Regulates Phagocytosis And Homotypic Phagosome And Lysosome Fusion, Ed Harris, James Cardelli

Department of Biochemistry: Faculty Publications

RabD, a Dictyostelium Rab14-related GTPase, localizes in the endo-lysosomal pathway and contractile vacuole system of membranes. Cell lines expressing dominant-negative RabD were defective in endocytosis, endosomal membrane flow and homotypic lysosome fusion. In support of a role for RabD in fusion, cells overexpressing constitutively active RabD^Q67L accumulated enlarged hydrolase-rich acidic vesicles ringed with GFP-RabD, consistent with RabD directly regulating lysosome fusion. To determine whether RabD also regulated phagocytosis and/or homotypic phagosome fusion (a process stimulated by many intracellular pathogens), cells overexpressing dominant-active (RabD^Q67L) or dominant-negative (Rab^N121I) RabD were analyzed microscopically and biochemically. The rate of …

Selenoprotein R Is A Zinc-Containing Stereo-Specific Methionine Sulfoxide Reductase, Gregory V. Kryukov, R. Abhilash Kumar, Ahmet Koc, Zhaohul Sun, Vadim N. Gladyshev

Department of Biochemistry: Faculty Publications

Selenoprotein R (SelR) is a mammalian selenocysteine-containing protein with no known function. Here we report that cysteine homologs of SelR are present in all organisms except certain parasites and hyperthermophiles, and this pattern of occurrence closely matches that of only one protein, peptide methionine sulfoxide reductase (MsrA). Moreover, in several genomes, SelR and MsrA genes are fused or clustered, and their expression patterns suggest a role of both proteins in protection against oxidative stress. Consistent with these computational screens, growth of Saccharomyces cerevisiae SelR and MsrA mutant strains was inhibited, and the strain lacking both genes could not grow, in …

Kinetic Properties Of Bifunctional 6-Phosphofructo-2-Kinase/Fructose-2,6-Bisphosphatase From Spinach Leaves, Jennifer E. Markham, Nicholas J. Kruger

Department of Biochemistry: Faculty Publications

A cDNA encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was isolated from a Spinacia oleracea leaf library and used to ex­press a recombinant enzyme in Escherichia coli and Spodoptera frugiperda cells. The insoluble protein expressed in E. coli was purified and used to raise antibodies. Western blot analysis of a protein extract from spinach leaf showed a single band of 90.8 kDa. Soluble protein was purified to homogeneity from S. frugiperda cells infected with recombinant baculovirus harboring the isolated cDNA. The soluble protein had a molecular mass of 320 kDa, estimated by gel filtration chromatography, and a subunit size of 90.8 kDa. The purified pro­tein …

Identification And Analysis Of A Conserved Immunoglobulin E-Binding Epitope In Soybean G1a And G2a And Peanut Ara H 3 Glycinins, Ping Ziang, Tom A. Beardslee, Michael G. Zeece, John P. Markwell, Gautam Sarath

Gautam Sarath Publications

To identify conserved immunoglobulin E (IgE)-binding epitopes among legume glycinins, we utilized recombinant soybean G2a and G2a-derived polypeptide fragments.

Purification And Characterization Of Acylation Stimulating Protein From Porcine Serum, Hong Zhang, Sheila K. Jacobi, Candace F. Toombs, Katherine H. Cianflone, Natalya Nersesian, Gautam Sarath, Jess L. Miner

Gautam Sarath Publications

A method for purifying acylation stimulating protein (ASP) from porcine serum is described. The mRNA encoding ASP was cloned by reverse transcriptase-polymerase chain reaction which predicted a 76 residue peptide. Based on this sequence, we generated antisera to a C-terminal peptide (ASP_1–20) which aided ASP purification. Identity of the purified protein was verified by N-terminal sequencing. The molecular mass of porcine ASP is 8926. Porcine ASP stimulated esterification of fatty acid into triacylglycerol in cultured human cells with potency similar to that of human ASP (twofold at 5 μM). Based on this evidence that ASP exists in porcine …

Development Of A Chromatographic Method For The Analysis Of Carbohydrates In Posidonia Coriacea In Artificial Cultures, Olivia J. Oldfield

Theses : Honours

Carbohydrates in P. coriacea leaves were analysed to determine if, when in artificial culture, they were unable to replenish their carbohydrate stores once the seed starch reserve was consumed. While no evidence was found to suggest that this was happening, other findings were made. Soluble carbohydrates determined in P. coriacea were sucrose, fructose, glucose, trehalose, myo-inositol and mannitol. However, mannitol was only found in in situ plants sampled in June. This may be due to high epiphyte coverage elevating plant stress and therefore mannitol levels. P. coriacea grown in tissue culture has soluble carbohydrate levels up to 20-fold higher than …

Flexibility Of Biv Tar-Tat: Models Of Peptide Binding, M Hsieh, E D. Collins, T Blomquist, Brooke Lustig

Faculty Publications, Chemistry

No abstract provided.

Behavioral And Reproductive Aspects Of Captive Maxwell's Duiker (Cephalophus Mawellii) Husbandry, Janet Mcneill Mackinnon

College of Graduate Studies: Theses & Dissertations (1964–2006)

Female Maxwell's duikers (Cephalophus maxwellii) were studied at the Wildlife Survival Center on St. Catherine's Island, Georgia. The objectives were to quantify the effects of housing on activity budgets and progesterone cycling. Four animals were studied across two housing situations: housed individually versus housed in a grouped setting. Behavioral observations revealed no significant change in access to resources, rumination, or repetitive behaviors, once animals were housed together. Fecal samples were collected in order to determine levels of progesterone through radioimmunoassay (RIA). Fluctuations in progesterone levels revealed no clear estrous cycles. Animals showed less variability in progesterone levels and …

Glass Capillary Tube Feeding: A Method For Infecting Nymphal Ixodes Scapularis (Acari: Ixodidae) With The Lyme Disease Spirochete Borrelia Burgdorferi, Anne H. Broadwater, Daniel E. Sonenshine, Wayne L. Hynes, Shane Ceraul, Aravinda M. De Silva

Biological Sciences Faculty Publications

We evaluated an artificial capillary feeding method to infect nymphal Ixodes scapularis (Say) ticks with Borrelia burgdorferi, the causative agent of Lyme disease. Thirty to 70% of the nymphs were infected after feeding for 2.5 h from glass capillary tubes filled with a solution of spirochetes. Capillary infection was stable and persisted in the nymphs for at least 10 d after feeding. Capillary feeding also maintained natural vector competence patterns because I. scapularis ticks acquired infection unlike Dermacentor variablis (Say), which did not become infected. Capillary infected I. scapularis nymphs were capable of transmitting the infection to naive mice …

Molecular Mechanisms Involved In Human Platelet Aggregation By Synergistic Interaction Of Platelet-Activating Factor And 5-Hydroxytryptamine, B H. Shah, H Rasheed, I H. Rahman, A H. Shariff, F L. Khan, H B. Rahman, S Hanif, S A. Saeed

Department of Biological & Biomedical Sciences

Our recent studies have shown that co-activation of Gq and Gi proteins by 5-hydroxytryptamine (5-HT) and adrenaline show synergism in human platelet aggregation. This study was conducted to examine the mechanism(s) of synergistic interaction of 5-HT and platelet activating factor (PAF) in human platelets. We show that PAF, but not 5-HT, increased platelet aggregation in a concentration-dependent manner. However, low concentrations of 5-HT (2 microM) potentiated platelet aggregation induced by subthreshold concentration of PAF (40 nM) indicating a synergistic interaction between the two agonists and this synergism was blocked by receptor antagonists to either 5-HT or PAF. 5-HT also potentiated …

Aerobic Biodegradation Of Methyl Tert-Butyl Ether By Aquifer Bacteria From Leaking Underground Storage Tank Sites, S. R. Kane, H. R. Beller, T. C. Legler, C. J. Koester, Holly C. Pinkart, R. U. Halden, A. M. Happel

All Faculty Scholarship for the College of the Sciences

The potential for aerobic methyl tert-butyl ether (MTBE) degradation was investigated with microcosms containing aquifer sediment and groundwater from four MTBE-contaminated sites characterized by oxygen-limited in situ conditions. MTBE depletion was observed for sediments from two sites (e.g., 4.5 mg/liter degraded in 15 days after a 4-day lag period), whereas no consumption of MTBE was observed for sediments from the other sites after 75 days. For sediments in which MTBE was consumed, 43 to 54% of added [U-¹⁴C]MTBE was mineralized to¹⁴CO₂. Molecular phylogenetic analyses of these sediments indicated the enrichment of species closely …