Biochemistry & Molecular Biology.

Rapid Pacing of Weak Stimuli in the Heart. Deborah Janks and Bradley J. Roth, Oakland University, Department of Physics, Rochester, MI 48309

With the development of pacemakers and defibrillators comes an important question: what are the underlying mechanisms responsible for the induction of reentry within the heart? At least two mechanisms are known to lead to an arrhythmia (or "reentry"): inhomogeneities occurring within the tissue as well as a difference in electrical conductivity parallel and perpendicular to the fibers surrounding an electrode. Each of these mechanisms were modeled with the objective being to determine the dominant mechanism responsible for reentry. Our results are compared to experimental studies leading to the conclusion that the different electrical conductivity parallel and perpendicular to the fibers leads to results that agree more closely to those observed experimentally.

Theoretical Multisite Pacing Methods for Ventricular Fibrillation Utilizing Algorithms Formed by Nonlinear-Dynamics-Feedback. Victor D. Hosfeld and Bradley J. Roth, Oakland University, Department of Physics, Rochester, MI 48309

Ventricular fibrillation is one of the leading causes of death among cardiovascular disease patients worldwide. Gauthier et al. (Chaos, 2002, 952-961) experimentally tested a method to pace the heart utilizing algorithms formed by nonlinear-dynamics-feedback applied through a low-energy, single electrode stimulus. Pacing was applied to cardiac tissue of sheep as the tissue dynamics diverged from the desired periodic rhythm. This theoretical study applies the algorithms developed by Gauthier et al. to pace ventricular cardiac tissue models as the tissue dynamics enter a state of fibrillation. Low-energy stimuli are applied to ventricular tissue employing the use of multiple electrodes to terminate fibrillation. Multisite pacing algorithms have been shown to improve fibrillation termination both experimentally (Pak et al., Am. J. Physiol., 2003, 285:H2704-H2711) and theoretically (Puwal & Roth, Journal Biological Systems, in press). In this study, multisite pacing protocol is established by algorithms developed by nonlinear-dynamics-feedback response to terminate ventricular fibrillation.

Optical Mapping of Voltage in the Heart. Phillip Prior and Bradley J. Roth, Oakland University, Department of Physics, Rochester, MI 48309

An important question in cardiology is how electrical stimulation of the heart affects the transmembrane potential during ventricular defibrillation. One way of experimentally determining the potential is by optical mapping using voltage-sensitive fluorescent dyes. The fluorescent light of these dyes originates from a few millimeters below the tissue surface as well as at the surface, causing the optical signal to be averaged over a depth. In our study, the transmembrane potential is calculated due to a point source that injects current at the surface of the tissue. The average transmembrane potential is numerically integrated over a finite range of values, which is made possible by a change in variables. Numerical integration is performed using the trapezoidal rule, while values of the average transmembrane potential are investigated over a range of optical decay constant and electrode radii.

Using Experiments and Simulations to Understand the Role of Amyloid Fibers in Huntington's Disease. John M. Finke, Oakland University, Department of Chemistry, Rochester, MI 48309-4477

Despite many advances in the area of protein folding, much less is understood about the process of protein misfolding. This is especially notable concerning mechanisms and structures involved in the assembly of large fibers comprised of many miscoded proteins which have been shown to lead to neuron cell death in a number of aging diseases, including Alzheimer's, Parkinson's, and Lou Gehrig's Disease. Our work aims to develop both experimental and computational methods to capture molecular information on misfolded protein structures. Fluorescence Resonance Energy Transfer (FRET) experiments are used to measure molecular distances between parts of proteins before and after they participate in fibril assembly. This data is then used to help model realistic structures of these proteins in their fibrillized disease state using AMBER protein modeling software. Initial results will be presented for fibril assembly of polyglutamine peptides involved in Huntington's disease. Also, drug design strategies will be presented for identification of compounds which bind to neurotoxic polyglutamine fibrils and prevent their formation.

Identification of a Unique Alu-based Polymorphism and Its Use in Human Population Studies. Nicole Jamison, Melanie Mayberry, Eillen Tecle, and David H. Kass, Eastern Michigan University, Department of Biology, Ypsilanti, MI 48197

Alu elements are family of short interspersed DNA elements (SINEs) found in primate genomes. This is a member of a group of transposable elements that integrate into the genome by the process of retrotransposition. Recent integrations of Alu elements within the human genome have generated presence/absence variants useful as DNA markers in human population studies as well as in forensic and paternity analyses. Besides the ease of use, this type of marker is unique because the absence of the Alu represents the ancestral form. We have identified an Alu-based polymorphism that consists of four alleles in which we can predict the evolutionary order. Additionally, we have thus far analyzed DNA from a small set of samples comprising four different ethnic groups and have observed one allele to be significantly more prevalent within one group, demonstrating variability among populations. These features afford this marker as a unique tool in the study of both global and regional analyses of human populations.

Molecular Characterization of Watermilfoil, An Invasive Aquatic Plant in Michigan Lakes. Nickole Hatley, Keith Williams and Ann Sturtevant, University of Michigan-Flint, Department of Biology, Flint, MI 48502-1950

Watermilfoil is an invasive, noxious aquatic plant that seriously impacts the water recreation and tourism industry of Michigan. The introduced, invasive form of watermilfoil can be devastating to aquatic ecosystems where it replaces native vegetation and negatively impacts species diversity among both plants and animals. Watermilfoil is difficult to control because of its rapid growth. We will identify the genotypes of the native northern milfoil (Myriophyllum sibiricum), the imported plant pest Eurasian watermilfoil (Myriophyllum spicatum) and the aggressive interspecies hybrid (M. spicatum X M. sibiricum) currently present in a number of Michigan lakes. Identification of watermilfoil species will be done by sequencing the ITS region of the ribosomal RNA genes. An extremely sensitive form of DNA fingerprinting, AFLP, will be used to characterize specific genotypes of each species of watermilfoil. There have also been reports of herbicide-resistant forms of watermilfoil in Michigan. The genetic markers produced by AFLP will be used to diagnose the presence of herbicide-resistant varieties of watermilfoil and determine its prevalence in Michigan lakes. These genetic markers can be used in the future to identify which lakes have the most aggressive form of watermilfoil, and to recommend the most appropriate herbicide to control this invasive aquatic plant.

Interleukin-1[beta]-Induced Growth Enhancement of Staphylococcus aureus Occurs in Biofilm But Not Planktonic Cultures. Arlene J. Hoogewerf, Renee A. McLaughlin, Nathan Vande Burgt, and Stacy Visser, Calvin College, Department of Biology, Grand Rapids, MI 49546-4403

Staphylococcus aureus causes recalcitrant infections and forms resistant biofilms. Mechanisms of biofilm resistance to host defenses may include changes in gene expression that confer responsiveness to chemical mediators. In earlier studies fresh clinical isolates responded to inflammatory cytokines, but responsiveness was lost after multiple in vitro passages (Meduri GU, Kanangat S, Stefan J, Tolley E, and Schaberg D. Cytokines IL-1[beta], IL-6, and TNF-[alpha] enhance the In Vitro growth of bacteria. Am. J. Respir. Crit. Care Med.1999; 160:961-7.) Since biofilms more closely resemble in vivo growth and are implicated in recalcitrant infections, we hypothesized that biofilms, but not planktonic cells, would respond to cytokines. Biofilms were induced by ethanol in S. aureus ATCC 12600. Biofilms treated with 2 ng/mL Interleukin-1[beta] (IL-1[beta]) for 6 hours contained 2.5-fold more cells than untreated biofilms, but no growth-enhancement occurred in planktonic cultures. As determined by flow cytometry, IL-[beta] bound to 63.1% of biofilm cells, but only 11.2% of planktonic cells. Our results provide evidence of a differential response of biofilm and planktonic bacteria to chemical mediators, and suggest that biofilm bacteria may evade host defenses by growing more rapidly in response to the inflammatory mediators released by activated host defense cells.

A Novel Analytical Method to Convert N-6 and N-3 Essential Fatty Acids to Their Methyl Esters by TMSD (Trimethyl silyl diazomethane). Steven J. Pernecky, Hemendra N. Basu, and Aditi Munmun Sengupta, Eastern Michigan University, Department of Chemistry, Ypsilanti, MI 48197

The formation of metabolites of n-6 fatty acids has been associated with chronic heart disease and cancer. It has been suggested that a substantial change in the polyunsaturated fatty acid composition from one right in n-6 fatty acids to one rich in n-3 fatty acids may suppress the tendency to form these compounds, and, thus, improve the prognosis and/or risk factors associated with these diseases. The overall objective of this research project is to prepare methyl esters of n-6 fatty acids and n-3 fatty acids for gas chromatographic analysis by treatment with acetone-methanol (350 micro-liters of 20% methanol in acetone) and TMSD (trimethyl silyl diazomethane). The reagent system is stable, easy to handle, quick, and convenient. Reaction conditions were investigated for n-6 fatty acids like arachidonic acid, [gamma]-linolenic acid, and linolenic acid, n-3 fatty acids like EPA (Eicosapentanoic acid), DHA (Docosahexanoic acid), and [alpha]-linolenic acid, as well as those that are contained in phosphatidyl chlorines and phosphatidyl ethanolamines. The fatty acid methyl esters were readily purified by thin layer chromatography and the yields were quantitative. There were a few side products, but they did not affect the validity of the method. The procedure developed is simple, rapid, and generally applicable to the lipids.

Effects of Picrotoxin (PTX) on Phonotaxis in Cricket. Michael T. Scalfan and Gordon Atkins, Andrews University, Department of Biology, Berrien Springs, MI 49104

Earlier studies of phonotaxis (the ability to approach sound sources) in female crickets, has shown a selective behavioral response as being very important in their choice of which males to mate with. This experiment was performed on female Acheta domesticus demonstrating a substantially greater plasticity in the behavioral choices made after intra-cellular injection of picrotoxin (PTX) into the left ventral portion of the prothoracic ganglia. Behavior tests were performed pre- and post-injection, by using a sand arena with a central speaker playing syllable periods (SPs) ranging from 30-90ms. A positive phonotaxis response was determined if the cricket reached the center speaker and a negative response if they did not. Prior to injection of PTX, some females were very unselective (responded to all SP's) and some were very selective (only responding to 1-2 SPs). Post-injection, the unselective females became more selective in the range of SPs that they responded to (range of 50-70ms), while the selective group continued to stay selective. These results suggest that the auditory area is located in the prothoracic ganglia and that PTX is able to increase female Acheta domesticus ability to selectively respond to SPs.

Effects of a Soy Protein, Genistein, on Tumor Suppressor Protein p53 in T47D Breast Cancer Cells. Seema Shah, Amelita Sanchez and V.K. Moudgil,, Oakland University, Department of Biological Sciences, Rochester, MI 48309

The growth and development of the human breast is influenced by the sex steroid 17-[beta]-estradiol ([E.sub.2]). Presence of estrogen in post-menopausal women has been suggested to increase the risk of cancer. Cancer arises from loss of balance between tumor suppressor genes and tumor promoting oncogenes. The tumor suppressor protein p53 is one of the most frequently mutated proteins in cancer. Genistein, an isoflavone, induces cell differentiation, and inhibits angiogenesis and cell proliferation. It also has estrogenic and anti-estrogenic properties. This study describes the influence of genistein on p53 levels in T47D cells, which express detectable levels of receptors for [E.sub.2] and progesterone and represent an [E.sub.2] responsive ductal carcinoma. Cells were cultured in media containing charcoal-treated serum to deplete the level of endogenous steroids. Upon confluency, the cells were treated with different concentrations of genistein (1 nM-1 [micro]M) for 24 h. The proteins were extracted, quantified and subjected to SDS-PAGE and Western blot analyses. Our observations suggest that at low concentrations (10-100 nM), genistein increases p53 expression, but higher concentrations (1 [micro]M) down- regulate the expression of this protein. Exploring the relationship between isoflavones and tumor suppressors could advance our understanding of the molecular mechanism of tumor suppression in cancer.

Purification and Characterization of Recombinant Human Paired Basic Amino Acid Cleaving Enzyme 4 (PACE4). Andrea MacFadden, Marcella Luercio, Jeremy Lynd, and Joseph F. Sucic, University of Michigan--Flint, Department of Biology, Flint, MI 48502

Paired Basic Amino Acid Cleaving Enzyme 4 (PACE4) is a eukaryotic endoprotease in the subtilisin-like proprotein convertase (SPC) family. SPCs play important roles in development, homeostasis, and pathology. However, specific roles for PACE4 in these processes have been difficult to determine, in part because of overlapping substrate specificities and activities of the SPCs. RPE.40 cells, an SPC-deficient strain of CHO-K1 cells, were stably transfected with a mammalian expression plasmid carrying the cDNA encoding human PACE4. These cells were propagated in an experimental protein-free medium, where they secreted the recombinant PACE4. PACE4 was purified from this medium using three steps: size exclusion, anion exchange chromatography, and affinity chromatography. Purity was examined with SDS-PAGE, and PACE4 activity was assayed during the purification scheme through the use of a synthetic peptide in which the PACE4 cleavage recognition sequence was conjugated to a fluorophore; cleavage of the fluorophore from the peptide was monitored via fluorescence spectroscopy. Key biochemical parameters were determined for PACE4, including km and vmax.

Substrate Specificity of the Mammalian Endoprotease PACE4. Alexis Sanders, Jeremy Lynd, and Joseph F. Sucic, University of Michigan--Flint, Department of Biology, Flint, MI 48502

Endoproteases of the Subtilisin-like Proprotein Convertase (SPC) family cleave substrate proproteins on the carboxy terminal side of a conserved sequence described as N-Arginine-X-X-Arginine-C. Each residue in this sequence is designated with specific terminology: N-P4-P3-P2-P1-C. While the P1 and P4 arginine residues are largely invariant among SPC substrates, considerable variation is seen at the P2 and P3 positions; the amino acid diversity at these positions may mediate unique substrate preferences among the SPCs. Paired Basic Amino Acid Cleaving Enzyme 4 (PACE4) is a member of the SPC family that has been implicated in a number of proprotein processing events important in mammalian development, homeostasis, and pathology. Several reports have suggested that PACE4 requires a basic residue (lysine or arginine) in the P2 position. However, Bone Morphogenetic Protein 4 (BMP4), which harbors a serine at the P2 residue position, can be activated by PACE4. We have therefore more thoroughly investigated the P2 sequence requirements of PACE4, using purified PACE4 and a series of synthetic peptides conjugated to fluorescent dyes; each synthetic peptide harbored a different P2 residue. Results suggest that the P2 requirements of PACE4 are less stringent than previously reported.

Substrate Specificity in the Subtilisin-like Proprotein Convertases: A Role for Aromatic Amino Acids Adjacent to the Site of Endoproteolytic Cleavage. Manosha Dasanayaka, Natalie Duckett, and Joseph F. Sucic, University of Michigan--Flint, Department of Biology, Flint MI 48502

Endoproteases of the Subtilisin-like Proprotein Convertase (SPC) family are important in development and homeostasis. These enzymes also play important roles in the development of pathological conditions such as cancer and Alzheimer's Disease. SPCs cleave substrate proproteins on the carboxy terminal side of a conserved sequence described as N-Arginine-X-X-Arginine-C. However, each member of the SPC family has unique substrate preferences and can typically act upon only a subset all possible SPC substrates. Amino acids flanking the cleavage sequence also interact with the SPC active site, and the chemical properties of these amino acids are highly variable among proproteins. We have examined the possible role of amino acids with aromatic side chains in mediating substrate specificity in the SPCs. Site directed mutagenesis was used to alter amino acids adjacent to the cleavage sequence in pro-von Willebrand's (pro-vWF) factor to phenylalanine and tryptophan. The mutant versions of pro-vWF were expressed in an SPC-deficient cell strain, and were used for in vitro processing reactions with different SPCs. The ability of the SPCs to process pro-vWF was assessed by western blotting. Results of these experiments may suggest strategies for using SPCs as therapeutic targets for novel anticancer or Alzheimer's drugs.

Reversible Covalent Inhibition of a Phenol Sulfotransferase by Coenzyme A. Joe D. Beckmann, Alma College, Department of Biochemistry, Alma, MI 48801

Phenol sulfotransferases (SULTs) catalyze sulfuryl group transfer from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to acceptor substrates to form sulfate products generally destined for excretion. CoA and its thioesters, being structurally homologous to PAPS, are reversible SULT inhibitors under reducing conditions. Under non-reducing conditions, however, the inhibition of bovine SULT1A1 by CoA is time-dependent. CoA inactivation rates indicate an initial reversible SULT:CoA complex with a [K.sub.d] of 2.8 [micro]M and an inactivation rate constant of 0.023 [min.sup.-1]. Covalent attachment of CoA to the enzyme is observed by complete conversion of the protein to a slightly decreased electrophoretic mobility. Binding stoichiometry of 1:1 is suggested by titration inactivation. 2-Mercaptoethanol restores both activity and normal electrophoretic migration, suggesting a disulfide bond. The two bSULT1A1 cysteine residues were mutated to serines, and the results reveal that Cys-168 is the site of CoA adduction. Molecular models based on the human SULT1A1 structure nicely accomodate the binding of CoA and disulfide formation. A previously unrecognized molecular dynamic for this family of enzymes is also suggested and was tested by measuring cysteine reactivity with Ellman's reagent before and after binding with PAPS. A significant conformational change is consistent with proposals that SULTs undergo a "disorder-to-order" transition during substrate binding.

Retrograde Axonal Transport of Neurotrophins: What Molecules Move in the Same Vesicles as Neurotrophins? Laura Hill and Robert Stach, University of Michigan--Flint, Department of Chemistry, Flint MI 48502

In order for nerve cells to survive and then mature, they must receive information from the contact organ. From the cell body to the terminal through the axon, nerve cells can be over a meter in length. "There is a major problem for signal transduction for molecules acting on receptors at the nerve terminal that need to convey the survival signal to the cell body" (Hendry website 2005). During the development of the nervous system, there are both insoluble and soluble neurotrophic factors present. The neurons survival is dependent on these signals from the nerve terminal to the cell body. In order to understand neuronal development, it is necessary to comprehend the mechanisms "that control the flow of information from the nerve terminal to the cell body" (Hendry website 2005). This knowledge can then be applied to nerve regeneration (Hendry website 2005). The present paper is based on research carried out at Australian National University in the laboratory of Dr. Ian Hendry focusing on what molecules are transported with neurotrophins in vesicles. "The results of this study will suggest alternate ways to enhance neuronal regeneration by perturbing the second messenger cascades promoting axonal transport" (Hendry website 2005).

Analysis of RNP Aggregates in Oocytes of C. elegans. Jeremy Bickel, Emily Petty, Jennifer Mason, and Jennifer Schisa, Central Michigan University, Mount Pleasant, MI 48859

The genetic model system C. elegans can be used to elucidate the role of specific protein interactions required for germline development. P granules are cytoplasmic RNA/protein complexes found associated with C. elegans germ cells. When old-aged adult hermaphrodites are depleted of sperm and oogenesis arrests, P granules and several cytoplasmic RNAs and proteins aggregate into large foci in the oocytes. When sperm are introduced via mating, the RNP aggregates dissociate reversibly. The biological function aggregate formation is unknown but we hypothesize they may preserve the maternal mRNA and protein in oocytes when fertilization is delayed. Using the MEX-3 protein as a marker for aggregates, we have established timelines for aggregate formation and dissociation at 72 hours post-L4 stage and 50-70 minutes post-mating, respectively. We also have preliminarily identified a mutation, kgb-1, in which a failure to form aggregates in oocytes is correlated with embryonic lethality. kgb-1 is a member of a JNK-like MAPK pathway with mek-1 and mlk-1 as upstream signaling components. We are currently generating a double mutant strain of kgb-1 and fog-2 in order to confirm the correlation between a failure to produce RNP aggregates in oocytes and high embryonic.

Effects of Topiramate on Pituitary Glucocorticoid Receptor (GR), Proopioidmelanocortin (POMC), and CRH-receptor-1 (CRH-R1) mRNA Levels in a Rodent Model of PTSD. Rory Daniel Schwan, University of Michigan--Flint, Department of Psychology, Flint, MI 48502

Posttraumatic stress disorder (PTSD) is associated with neuroendocrine abnormalities including enhanced cortisol negative feedback and elevated levels of cerebrospinal fluid CRH. The single prolonged stress (SPS) paradigm is a validated rodent model of PTSD that replicates PTSD symptoms, which can be reversed by the anticonvulsant drug Topiramate, and also shows enhanced cortisol negative feedback and altered expression of glucocorticoid (GR) and mineralocorticoid (MR) receptors in hippocampus. However, the effects of SPS or Topiramate on pituitary gene expression are not known; in the present paper, we examine the effects of SPS and Topiramate on mRNAs for GR, CRH-R1, or POMC (involved with production of ACTH). Male Sprague-Dawley rats (n=24) received SPS (two hours of restraint, followed by forced swim and ether exposure) or handling only, followed by a 7-day undisturbed period, during which they received vehicle or 30 mg/kg Topiramate twice daily by oral ingestion, Animals were sacrificed on day 10, with pituitaries dissected and immediately frozen at -80C. Relative levels of pituitary expression of GR, CRH-R1, and POMC mRNAs were determined using TaqMan quantitative RT-PCR, with GAPDH or GHRH-R1 as reference genes. This research demonstrates that elevated pituitary GR expression may play a role in the therapeutic action of Topiramate.