Phi Zeta Day - Student Research Paper Presentations

Megan Brace, V'08 - Dr. Gary Smith, Sponsor
POPULATION DYNAMICS OF HEPATITIS B VIRUS IN WILD CHIMPANZEES

Pathogen transmission from humans to non-human primates (NHP) places both captive and wild NHPs at risk for zoonoses such as measles, tuberculosis, poliomyelitis, Ebola and viral hepatitis. Of particular interest are the dynamics of Hepatitis B virus (HBV) infection in wild chimpanzees. We describe a differential equation model of HBV transmission in wild chimpanzee populations and use the model to compare strategies for controlling HBV transmission. Analytical and numerical methods were used to investigate the behavior of the model and, in particular, to evaluate the feasibility vaccination as a method for control HBV transmission in wild chimpanzee populations. The basic reproduction number, R o, for HBV infection in chimpanzees was 1.74. This indicates that levels of vaccination coverage greater than 43% should be sufficient to provide the levels of herd immunity needed to protect wild chimpanzee troops against population reduction attributable to HBV infection.

Michelle Cook, Combined-Degree Student - Dr. Narayan Avadhani, Sponsor
PROTEIN KINASE A-MEDIATED PHOSPHORYLATION TARGETS HUMAN CYTOCHROME P450 2D6 TO THE MITOCHONDRIAL COMPARTMENT:  IDENTIFICATION OF GENETIC VARIANTS WITH IMPAIRED MITOCHONDRIAL TARGETING

Cytochrome P450 2D6 (CYP2D6) is responsible for the metabolism of approximately 25% of drugs in common clinical use. It is widely accepted that CYP2D6 is localized in the endoplasmic reticulum of cells; however, in this study we show that it is present in the mitochondria of human liver samples, and demonstrate that there is extensive inter-individual variability in the mitochondrial levels.  Our results show that CYP2D6 contains an N-terminal chimeric signal that mediates its bimodal targeting to the ER and mitochondria.  The mitochondrial targeting signal and protein kinase A mediated phosphorylation are both required for mitochondrial import in isolated mitochondria as well as in intact COS cells. Screening of the liver samples by RT-PCR has enabled us to identify several variant forms with mutations involving the ER targeting signal, the proline rich domain, as well as the PKA-specific phosphorylation site.  These mutations affect the efficiency of mitochondrial targeting thus providing a valuable system in which to study the role of mitochondrial CYP2D6 in modulating both pharmacological efficacies and adverse toxic effects.

Ann P. Klocke, V'07 - Dr. Regina Turner, Sponsor; Karen Schlingmann
MOLECULAR AND CELLULAR REGULATION OF SPERM MOTILITY IN MAMMALIAN SPECIES, USING AN EQUINE MODEL

Sperm motility is of central importance to male fertility. Thus, significant research has been devoted to understanding the basic mechanisms that regulate flagellar function. Among the most important sperm motility regulatory mechanisms are calcium signaling and the cAMP/Protein Kinase-A (PK-A) pathways (1-7). In sperm, the majority of cAMP is produced by an atypical, “soluble” adenylyl cyclase (sAC) that is genetically and biochemically distinct from the classical transmembrane cyclase(8-15). sAC, and the cAMP pathway, are required for normal sperm motility and male fertility (16). One goal of this laboratory is to improve fertility and reproductive efficiency of male domestic animals, in particular equines. However, there are few studies designed to elucidate the mechanism(s) for regulation and modulation of equine sperm motility.

Most studies on the molecular biology of sperm motility have been performed either in the mouse model system or in the human. Fortunately, flagellar anatomy and function appear to be highly conserved among mammals and it is likely that much of the existing data also will apply to domestic species including equines. Since sperm motility is a central, necessary component of the natural fertilization process, a better understanding of the signaling pathways responsible for flagellar motility will benefit the ability to manipulate and potentially enhance equine sperm motility and therefore fertility.
This study is composed of three components. The first portion involves adding N-(6-Aminohexyl)-5-chloro-1-napthalenesulfonamide hydrochloride (W-7), a calmodulin inhibitor, in varying concentrations to equivalent samples of stallion semen in order to support the hypothesis that calcium regulation through calmodulin’s activity is a necessary component to maintaining sperm motility. Once this is accomplished, part two will consist of using consistent concentrations of W-7 to inhibit sperm motility without affecting cell viability. Then attempts will be made to restore motion through the addition of the following components: Thimerasol (a compound that causes release of calcium from intracellular stores), dibutyrl cyclic adenosine monophosphate (db cAMP - a suspected component of the signaling cascade that leads to sperm motility), as well as methylxanthine drugs (caffeine and 3-isobutryl-1-methylxanthine, or IBMX). Finally, the last portion of the study will involve the addition of these same compounds to samples of extended (using Kenney extender), cooled, stored semen once motility has dropped below a preferred value (60%). It is hypothesized that the addition of these compounds to viable, yet nonmotile cells will cause an increase in the values of total motility and possibly progressive motility in each sample.

Thus far, results have shown that W-7 inhibits sperm motility in a concentration dependent manner, indicating that effective activation of signaling pathway involving calmodulin is essential essential to sperm motility. Results for the remaining two portions of the study are pending.

Mary Robinson, Combined-Degree Student - Dr. Cynthia Otto, Sponsor
PHYSIOLOGIC OXYGEN TENSIONS REGULATE RAPID CHANGES IN ENDOGENOUS NO PRODUCTION BY CYTOKINE-STIMULATED MACROPHAGES

Nitric oxide (NO) is synthesized by inducible nitric oxide synthase (iNOS) from molecular oxygen and L-arginine during inflammation.  Hypoxia clearly decreases NO production.  However, the duration and degree of hypoxia, and the mechanisms mediating this decrease, have not been clearly defined due to the technical limitations of standard cell culture. To overcome those limitations, a novel forced convection cell culture system was used to deliver various oxygen tensions directly to the cell monolayer while measuring real-time NO production.  Decreased oxygen tension immediately inhibited NO production by cytokine-stimulated macrophages with an apparent Km for oxygen of 28 Torr.  NO was not consumed by the cells, and iNOS protein concentration was independent of oxygen tension, eliminating these possible mechanisms as influences on the measured Km for the model.  We conclude that changes in oxygen tension within a physiologic and patho-physiologic range rapidly regulate NO production by unknown mechanism(s). Identification of these mechanisms will facilitate our understanding of the affects of tissue hypoxia during inflammation.

Melissa Sanchez, Combined Degree Student - Dr. Robert Doms, Sponsor; Theodore C. Pierson, Marciela M. DeGrace, Fabio Del Piero, Sheri L. Hanna
CHARACTERIZATION OF NEUTRALIZING ANTIBODIES TO WEST NILE VIRUS

West Nile virus (WNV), the etiologic agent of West Nile virus fever and encephalitis, is a significant human and veterinary pathogen. The humoral immune response plays an important role in prevention and clearance of WNV infection. We have produced a panel of monoclonal antibodies (MAbs) against the WNV strain present in the United States (Lineage I). All MAbs recognized the envelope (E) protein of WNV. Virus neutralization by the MAbs was assessed by plaque reduction neutralization assays. All neutralizing MAbs mapped to a group of residues on the lateral surface of Domain III of the E protein, involving residues 306, 307, 330 and 332. The remaining MAbs mapped to other residues in Domains I or III, and did not neutralize virus infection. Therefore, the epitope created by these four amino acid residues on the lateral surface of Domain III is an important target of neutralizing antibodies in mice. Further work aims to characterize neutralizing antibodies against WNV present in a naturally infected host. Serum samples from normal, vaccinated and WNV-infected horses are currently being analyzed for the presence and types of WNV-specific neutralizing antibodies. Neutralizing activity of sera was measured by a novel reporter virus particle (RVP) assay developed in our laboratory. WNV RVPs produce luciferase or other reporter genes within 24-48 hours after infection, making it possible to rapidly and quantitatively measure WNV neutralization by MAbs or sera. Infected horses produce antibodies against the described neutralizing epitope in Domain III, although antibodies against other epitopes are also present. In addition, for a subset of the horses, a large number of neutralizing antibodies target this dominant epitope. Therefore, the neutralizing epitope on the lateral surface of Domain III appears to be an important target of neutralizing antibodies during natural WNV infection in horses.

LeAndra Thompson, V'08 - Dr. Erika L. F. Holzbaur, Sponsor; Karen E. Wallace
THE EFFECTS OF CYTOPLASMIC DYNEIN DISRUPTION ON THE DE-STABILIZATION OF THE MAMMALIAN NEUROMUSCULAR JUNCTION

A transgenic mouse line, referred to as M21, , M21, has been generated as a model for the late-onset, human neurodegenerative disease, amyotrophic lateral sclerosis (ALS), more commonly known as Lou Gehrig’s disease. Tissue pathology of ALS patients is characterized by axonal aggregates of neurofilaments (NFs), de-stabilized neuromuscular junctions (NMJs), and muscle atrophy. Evidence suggests that deficient axonal transport may lead to the axonal accumulation of NFs, resulting in subsequent NMJ de-stabilization and degeneration of muscle function.

The protein complex dynactin has been implicated in the stabilization of NMJs in developing Drosophila. Dynactin mediates cytoplasmic dynein, the primary motor for retrograde axonal transport of cellular components in neurons. A subunit of dynactin, dynamitin (p50), is crucial to dynactin’s function within the cell. Overexpression of dynamitin leads to disruption of the dynactin-dynein complex, inhibiting retrograde transport. The transgenic mouse line, M21, has M21 mice have an overexpression of dynamitin in its their motor neurons and exhibit s some similar signs of muscle weakness to ALS patients.
We hypothesized that disruption of dynein-dynactin interaction in M21 mice would lead to de-stabilization of neuromuscular junctions (NMJs). We tested this hypothesis by using immunohistochemistry to compare NMJ morphology in the soleus muscle of age-matched (6 or 16 months) wild-type and transgenic M21 mice. NMJs were analyzed for acetylcholine receptor shape, degree of nerve terminal-receptor overlap at the end plate, and number of axonal aggregates.

Our data indicates that disruption of dynein-dynactin interaction is not critical to the de-stabilization of the mammalian NMJ. It may, however, facilitate axonal NF aggregation in older, transgenic M21 mice.

Tracy Zeldis, V'07 - Dr. Michael May, Sponsor; Laura Solt
THE EFFECTS OF NEMO ON NF-kB SIGNALING IN T CELLS

NF- kB is an inducible transcription factor that is required for immune inflammatory responses and pro-survivial signals. Induction of NF- kB is usually rapid and transient and dysregulation of NF- kB has been implicated in various cancers and chronic inflammatory conditions. Consequently, understanding the signals leading to normal NF- kB induction is critical for defining the defects in aberrant NF- kB activity. NF- kB activation requires the I kB kinase complex (IKK), the activity of which must be highly regulated to maintain the proper homeostasis in an organism.

The IKK complex consists of a core of three proteins. The two catalytic subunits, IKK a and IKK b, are responsible for the highly specific phosphorylation events required for NF- kB to translocate to the nucleus and activate gene transcription. NEMO, ( NF- kB essential modulator) is the regulatory subunit of the complex and possesses no inherent kinase activity. To date, two distinct NF- kB pathways have been described that involve the IKK complex. The best studied mechanism, named the classical pathway, has been shown to be absolutely dependent upon the interaction of IKK b with NEMO. The second and more recently identified mechanism is named the alternative pathway and is dependent solely on the catalytic activity of IKK a. This pathway appears to function independently of NEMO and IKK b.

We have identified a 6 amino acid sequence that is necessary for the interaction of IKK b with NEMO that we have named the NEMO binding domain (NBD). Paradoxically, this sequence also exists in IKK a and has been shown to facilitate the interaction of NEMO with this kinase. Current understanding of the alternative pathway would dictate that the NEMO:IKK a interaction is unnecessary for p100 processing to occur. However, in vitro experimentation shows that there is an increased amount of basal p100 processing in NEMO deficient T lymphocytes comparable to wild-type cells, indicating one possible function for this conserved interaction. Our findings strongly suggest that the interaction of NEMO with IKK a indeed plays an as-yet unknown role in regulating both the classical and non-canonical pathways. Our ongoing study will further elucidate this interaction and the role that it plays in NF- kB activation.