176

Defining The Interaction Between The Nucleation Promoting Factor N-WASP And The Enterohemorrhagic E. Coli Translocated Protein Espfu

Brian Skehan, BS, University of Massachusetts Medical School, Worcester, MA, USA; Hui-Chun Cheng, PhD, University of Texas Southwestern Medical Center, Dallas, TX, USA; Michael Rosen, PhD, University of Texas Southwestern Medical Center, Dallas, TX, USA; John Leong, MD/PhD, University of Massachusetts Medical School, Worcester, MA, USA

During colonization of the mammalian intestine, enterohemorrhagic Escherichia coli (EHEC) generates filamentous actin “pedestals” on the surface of epithelial cells. To generate actin pedestals, EHEC stimulates the actin nucleation promoting factor N-WASP. The C-terminal domain of N-WASP, termed the VCA domain, is capable of binding and activating the actin nucleator Arp2/3, but when N-WASP is in a resting or inactive state, this domain is normally sequestered by interaction with a second N-WASP domain, the GTPase binding domain (GBD). Factors that stimulate N-WASP, such as the small GTPase Cdc42, release the VCA domain and permit its interaction with Arp2/3. To stimulate N-WASP, EHEC injects two bacterial actin assembly effectors, the translocated intimin receptor (Tir) and EspFu (also known as TccP), directly into the host cell. Tir integrates into the host plasma membrane and becomes clustered underneath the bacterium by binding the bacterial outer membrane protein intimin. Clustering results in the recruitment of EspFu, a 384 residue protein that consists of six tandem proline rich repeats. A single repeat unit is capable of binding the N-WASP GBD, thereby activating N-WASP and stimulating actin assembly. 

An amphipathic alpha helical region of the VCA binds to the GBD when N-WASP is in an autoinhibited conformation, and a portion of the EspFu proline rich repeat is predicted to also form an amphipathic alpha helix. Based on an alignment of the VCA and EspFu sequences, we predicted that a 30-residue segment of an EspFu proline rich repeat may bind the GBD. Yeast two hybrid studies confirmed that this fragment of EspFu bound the N-WASP GBD efficiently, whereas an 18 residue segment did not. In addition, the alignment suggested that EspFu residue leucine 91 may be essential for N-WASP binding, and we found that an alanine substitution at this position disrupted binding completely in yeast two-hybrid assay. When fragments of EspFu were artificially clustered at the plasma membrane of mammalian cells, a 30-residue construct induced pedestal formation as efficiently as a full 47 amino acid repeat. In contrast, an 18-residue fragment was devoid of activity. These finding delineate a small segment of EspFu that binds N-WASP, and the fact that this same segment retains the ability to trigger pedestal formation in cultured cells emphasizes that importance of N-WASP binding  by EspFu.

177

RNA-Dependent Eukaryal And Archaeal Selenocysteine Biosynthesis

Sotiria Palioura, B.Sc./M.Sc., Yale University School of Medicine, New Haven, CT, USA; Jing Yuan, PhD, Yale University, New Haven, CT, USA; Dan Su, PhD, Yale University, New Haven, CT, USA; Yuhei Araiso, B.Sc., Tokyo Institute of Technology, Kanagawa, NA, Japan; Osamu Nureki, PhD, Tokyo Institute of Technology, Kanagawa, NA, Japan; William Whitman, PhD, University of Georgia, Athens, GA, USA; Dieter Soll, PhD, Yale University, New Haven, CT, USA

The trace element selenium is found in proteins as selenocysteine (Sec). The human selenoproteome consists of 25 members implicated in health and disease. All three 5’-deiodinases that convert the thyroid hormone thyroxine (T4) into its active cellular form triiodothyronine (T3) contain an active site Sec residue. Several antioxidant enzymes such as glutathione peroxidases and thioredoxin reductases are selenoenzymes associated with carcinogenesis and male fertility. In organisms from all three domains of life this ‘21st amino acid’ is co-translationally inserted into proteins in response to an in-frame UGA codon. It is known that in bacteria Sec biosynthesis proceeds through a tRNA-dependent transformation of serine (Ser) to Sec. However, a homologous pathway has not been found in eukarya and archaea. We recently showed that in these two domains of life an additional phosphorylated intermediate is required for Sec formation. In this two-step pathway, O-phosphoseryl(Sep)-tRNA kinase converts Ser-tRNA to Sep-tRNA, which is the obligatory precursor for a Sep-tRNA:Sec-tRNA synthase (SepSecS). In contrast to the bacterial selenocysteine synthase SepSecS does not use Ser-tRNA as a substrate neither in vitro nor in vivo. We determined the crystal structure of Methanococcus maripaludis SepSecS at 2.5 Å resolution. It is a tetramer with a pyridoxal phosphate cofactor linked to the active site lysine. Mutation of this conserved residue abolishes activity of both the human and archaeal SepSecS enzymes. Further biochemical and genetic characterization of SepSecS has provided insight into the catalytic mechanism of RNA-dependent Sec formation.
References:
1. Yuan, J.*, Palioura, S.*, Salazar, J.C., Su, D., O’Donoghue, P., Hohn, M., Cardoso, A.M., Whitman, W.B., Söll, D. (2006). RNA-dependent conversion of phosphoserine forms selenocysteine in eukaryotes and archaea. Proc Natl Acad Sci USA 103, 18923-18927.
2. Ambrogelly, A., Palioura, S., Söll, D. (2007). Natural expansion of the genetic code. Nat Chem Biol 3, 29-35.
3. Araiso, A.*, Palioura, S.*, Ishitani, R., Sherrer, R.L., O’Donoghue, P., Yuan, J., Oshikane, O., Domae, N., DeFranco, J., Söll, D., Nureki, O. (2008). Structural insights into RNA-dependent eukaryal and archaeal selenocysteine formation. Nucleic Acids Res (in press).

178

Development of a Migraine Headache Model in Rodents: Behavioral Assessment of Photophobia Using the Light-Dark Box Assay

Max Opoku Agyemang, BS, University of New England College of Osteopathic Medicine, Biddeford, ME, USA; Alex Askorput, BS., University of New England College of Osteopathic Medicine, Biddeford, ME, USA; Ian Mang, PhD, University of New England College of Osteopathic Medicine, Biddeford, ME, USA; Ed. Bilsky, PhD, University of New England College of Osteopathic Medicine, Biddeford, ME, USA

Introduction: Migraine headache represents one of the most common chronic pain states, affecting approximately 22 million people in the United States. Current treatments are limited in terms of efficacy and/or side effects associated with pharmacotherapy. The lack of well-validated animal models of migraine has hampered the development of novel treatments. Given that greater than 90% of all migraine sufferers report photophobia, and that this manifestation is also considered one of the diagnostic criteria, we hypothesized that photophobia could be a useful measure of migraine-associated pain.

Methods: We have started the development and validation process using the classic light/dark box test of anxiety in rodents. The aims of the current study were to assess rat behaviors in the light-dark box test following either (a) pupil dilation with atropine or (b) corneal epithelial debridement, two conditions know to produce photophobia in humans. Adult, male Long Evans rats were used in all studies. Rats either received bilateral topical administration of 1 mg/ml atropine solution to the cornea or complete bilateral epithelial debridement. Controls for both groups received bilateral topical corneal administration of artificial tears. A third group received corneal epithelial debridement followed by immediate topical application of .5% tetracaine hydrochloride ophthalmic solution. Controls for this group received only tetracaine bilaterally. Rats were allowed to recover from the procedures for 30 minutes in a darkened holding area before being run in light-dark assay (15 minute session). Rats that received atropine showed a greater preference for the dark zone versus controls.

Results: Corneal lesioned rats showed a similar preference for the dark versus light zone compared to their respective controls. The apparent photophobic effect of corneal lesioning was prevented by the topical administration of tetracaine.

Conclusion: These results indicate that reliable changes in light-dark preference can be measured and altered by changes in pupillary size and damage to the corneal epithelium. Furthermore, the behaviors are indicative of an increase in photosensitivity, providing a useful endpoint for putative migraine models in rats.

179

Analysis of the Transcriptional Control of the Acinetobacter baumannii Biofilm-Associated Protein

Thomas Loehfelm, BA, University at Buffalo, Buffalo, NY, USA; Anthony Campagnari, PhD, University at Buffalo, Buffalo, NY, USA

Acinetobacter baumannii is a Gram negative bacterium and an important cause of medical device-related infections in hospitalized patients world-wide. Infection by A. baumannii is particularly troublesome because of its remarkable repertoire of antibiotic-resistance determinants.  Isolates are frequently multi-drug resistant, and pan-resistance, though fortunately less common, has been reported.  In situations with limited or no useful antibiotics, proper infection control measures are paramount.  Bacteria are able to resist these measures in part because they form biofilms that are resistant to physical and chemical disruption.  We have previously identified a conserved biofilm-associated protein (Bap) produced by A. baumannii strain 307-0294that contributes to wild-type biofilm development, and therefore may play a role in the persistence of this organism in the hospital environment.  Disruption of bap results in a mutant that forms thinner and less voluminous biofilms when compared to those formed by the wild-type.  In the current study, we show that Bap surface expression is limited to those bacteria within the biofilm itself, while free-floating cells in the same culture have much lower Bap surface-expression.  We demonstrate that bap transcription and surface expression increase during the late-log and stationary phases of the bacterial growth curve, and appear to be induced in response to quorum sensing signals.  Once induced, the maintenance of transcript levels is apparently regulated by physical cell-cell contact signaling.  Finally, confocal microscopy of biofilms growing in media containing fluorescently-labeled anti-Bap monoclonal antibody 6E3 indicate that the culture-density-dependent timing of Bap expression corresponds to the timing of the phenotypic divergence between wild-type and mutant, indicating that surface expression of Bap is directly correlated with the development of wild-type biofilm.

180

Integrity of Limbic Projections in Relation to Posterior Cingulate Metabolism in Mild Cognitive Impairment and Alzheimers Disease: Combined PET-DTI Study

Igor Korolev, BS, DO/PhD and Neuroscience Programs, Michigan State University, East Lansing, MI, USA; Andrea Bozoki, MD, Dept. of Neurology, Michigan State University, East Lansing, MI, USA; Nathan Davis, BS, Dept. of Neurology, Michigan State University, East Lansing, MI, USA; Lori Hoisington, MA, Dept. of Radiology, Michigan State University, East Lansing, MI, USA; Luke Fischer, BS, Dept. of Radiology, Michigan State University, East Lansing, MI, USA; Jie Huang, PhD, Dept. of Radiology, Michigan State University, East Lansing, MI, USA; Kevin Berger, MD, Dept. of Radiology, Michigan State University, East Lansing, MI, USA

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that causes memory impairment by damaging components of the limbic system. While hypometabolism in the posterior cingulate (PC) is recognized as the earliest manifestation of AD on positron emission tomography (PET) imaging, earliest histopathological changes occur in the medial temporal lobe. We hypothesized that changes in the integrity of limbic projections will reflect neuronal dysfunction occurring during incipient and early AD, and that these changes can be identified with diffusion tensor imaging (DTI). We further hypothesized that changes in projection integrity should correlate with a functional decline of metabolism in PC. We collected DTI data from 46 individuals in three groups: early AD (n=15), mild cognitive impairment (MCI; n=19), and normal controls (NC; n=12). DTI acquisition parameters were as follows: EPI, b=1000, 6 directions, 3 mm^2 in-plane resolution, interpolated to 1.5 mm^2, 3 mm slices, 4 NEX. We performed DTI tractography and designated several limbic structures as regions of interest: fornix body and superior, descending, and temporal portions of the cingulum, bilaterally. Fractional anisotropy (FA) and volume measurements were obtained for each region. Fluorodeoxyglucose (FDG)-PET data were also acquired from patients with AD and MCI following a 10 mCi injection of FDG. Pons-normalized z-scores of relative metabolism were obtained for the PC by comparison to an age-matched normal database. ANOVA revealed a significant effect of group on both FA and tract volume. Post-hoc comparisons indicated that NC had greater FA values and volume compared with MCI and AD groups; however, the latter two groups were indistinguishable from each other. When examining individual regions, the fornix, left superior, and bilateral temporal portions of the cingulum each exhibited declines in FA as a function of group. In contrast, the fornix, bilateral descending, and temporal portions of the cingulum each exhibited declines in volume as a function of group. The combined AD/MCI cohort showed an expected decline in PET z-scores (i.e. hypometabolism) in PC and also a difference between the two groups. In this cohort, multiple regression analysis revealed a significant relationship between bilateral PC z-scores and tract volume overall and volumes of the fornix and the left descending cingulum, specifically. There was also a significant relationship between volume of the fornix and bilateral PC metabolism in MCI but not AD. There were no reliable relationships between PC metabolism and FA values. SUMMARY: (1) The integrity of key limbic projections, assessed by DTI-based tract volume and FA, declines along a continuum of AD progression. (2) While the combined AD/MCI cohort exhibits a relationship between volume of limbic tracts (particularly the fornix body) and bilateral FDG hypometabolism in PC, this structure-function relationship is more robust in MCI than AD.

181

The Effects of Transforming Growth Factor Alpha on Articular Cartilage

Shirine Usmani, Bachelor of Science , The Schulich School of Medicine and Dentistry, London, ON, Canada; Christopher Appleton, PhD , The Schulich School of Medicine and Dentistry, London, ON, Canada; Suzanne Bernier, PhD, The CIHR Group in Skeletal Development and Remodeling, London, ON, Canada; Frank Beier, PhD, The CIHR Group in Skeletal Development and Remodeling, London, ON, Canada

Osteoarthritis (OA) involves the degradation of articular cartilage by catabolic factors.  Previously, we determined that transforming growth factor alpha (TGFα) gene expression was upregulated in articular chondrocytes in an experimental OA model. The purpose of the current study was to examine downstream effects of TGFα treatment in articular chondrocytes. We hypothesized that TGFα promotes catabolic factor expression in articular chondrocytes. 

Articular cartilage organ culture explants were treated with TGFα (10 ng/ml) for up to 7 days.  Immunohistochemisty showed that the expression of the catabolic enzyme matrix metalloproteinase 13 (MMP-13) increased with treatment when compared to controls. Monolayer cultures of articular chondorcytes were then treated with TGFα and the expression of genes implicated in OA was assessed by real-time PCR. Cxcr4, Tgfa and Il1b gene expression did not change after treatment, while the expression of Ednra (endothelin receptor A) and Tnfa (tumor necrosis factor alpha) increased. Further immunohistochemistry revealed that explants treated with TGFα expressed more endothelin receptor A (ETA) than did controls.

This study demonstrates that TGFα promotes the expression of MMP-13, ETA and other genes known to play a role OA. Thus, TGFα and its downstream signaling pathways may be potential therapeutic targets for OA treatment. Current studies involve examining the role of ETA in TGFα signaling. Articular chondrocyte cell cultures will be treated with Bosentan (an endothelin receptor inhibitor) as well as with co-treatments of TGFα and endothelin-1 (the ligand for ETA). Gene expression, chondrocyte morphology, and nitric oxide production will be assessed.  

182

In Vivo Comparisons Of Glutamate Neurotransmission In The Anesthetized and Freely-Moving Rat Hippocampus During Aging

Michelle Stephens, BA, University of Kentucky College of Medicine, Lexington , KY, USA; George Quintero, PhD, University of Kentucky College of Medicine, Lexington, KY, USA; Francois Pomerleau, MSc, University of Kentucky College of Medicine, Lexington, KY, USA; Peter Huettl, MS, University of Kentucky College of Medicine, Lexington, KY, USA; Greg Gerhardt, PhD, University of Kentucky College of Medicine, Lexington, KY, USA

L-glutamate (Glu) is the most prevalent modulator of excitatory neurotransmission in the mammalian CNS. When release and/or uptake are not appropriately regulated, elevated extracellular levels can initiate a cascade of biological events that ultimately lead to excitotoxicity. The aging hippocampus displays extensively investigated electrophysiological and morphological changes, but studies aimed at determining effects of age-associated alterations on dynamic neurotransmission in vivo have produced contradicting reports in the literature, most likely due to the techniques employed. Our laboratory has developed a mass fabricated enzyme-based microelectrode array (MEA) and recording system (FAST-16; Quanteon, L.L.C.) optimized for measuring real-time Glu fluctuations in the extracellular space. We hypothesize dysregulation of Glu neurotransmission may contribute to the positive correlation between increasing age and incidence of hippocampal-associated disease and dysfunction. Glu-selective MEAs were used to investigate second-by-second resting levels and evoked Glu release in the hippocampus of young (3-6 months), late-middle-aged (18 mo.) and aged (24 mo.) anesthetized and freely-moving Fischer 344 rats. Data were analyzed using a one way analysis of variance followed by Tukeys post hoc comparisons and reported as mean ± SEM. Under urethane (1.25 mg/kg), potassium stimulation (70mM, 50 nL, n=5 each group) evoked significantly less Glu release from synaptic input into the CA3 subregion of aged rats (0.12 ± 0.03 µM/nL KCl) as compared to young (0.29 ± 0.04, p<0.05) and late-middle aged (0.33 ± 0.05, p<0.01) rats. CA3 function is reported in the literature to be necessary for memory, which is often compromised during aging. In urethane-anesthetized rats, a comparison of resting Glu showed no difference across age groups (combined Glu mean = 2.9 ± 0.5 µM, n=15). However, in awake freely-moving animals implanted with MEAs for chronic recordings, Glu resting levels were elevated in the hippocampus of aged animals (Glu =19.9 ± 4.1 µM, n=16) as compared to young (Glu =7.7 ± 1.2 µM, n=11, p<0.05). Sustained elevations in extracellular Glu could initiate excitotoxic cascades contributing to the predisposition in aging populations for Alzheimer's disease and temporal lobe epilepsy. These data emphasize the attenuation of resting excitatory neurotransmission by urethane anesthesia, and highlight that alterations in Glu regulation may be masked during experimental procedures utilizing urethane. As we prepare for human CNS Glu recordings, an understanding of other anesthetic-effects is critical. The more relevant anesthetic isoflurane (2% with oxygen), did not alter hippocampal resting Glu levels when compared within animal to levels obtained when the rat was freely-moving (p<0.05, n=7), regardless of age or awake Glu concentration.

183

C4d Staining In Humoral Rejection Of Cardiac Allografts

Jeanann Suggs, BS, University of Mississippi Medical Center, Jackson, MS, USA; Julius Cruse, MD PhD, University of Mississippi Medical Center, Jackson, MS, USA; Robert Lewis, PhD, University of Mississippi Medical Center, Jackson, MS, USA; Joshua Goodin, BS, University of Mississippi Medical Center, Jackson, MS, USA; Bret Allen, MD, University of Mississippi Medical Center, Jackson, MS, USA; Steven Bigler, MD, University of Mississippi Medical Center, Jackson, MS, USA; Charles Moore, MD, University of Mississippi Medical Center, Jackson, MS, USA; Regina Thompson, MD, University of Mississippi Medical Center, Jackson, MS, USA; Holly McIntire, MD, University of Mississippi Medical Center, Jackson, MS, USA

C4d "staining" of interstitial capillaries of endomyocardial biopsies serves as an indicator of a humoral component of cardiac allograft rejection. In the present study, two cardiac allograft recipients were monitored serially for both cellular and humoral rejection using light microscopy and immunofluorescence, respectively. Cellular rejection, evaluated by light microscopy, and humoral rejection, as determined by C4d immunofluorescent "staining", were treated with appropriate immunosuppressive therapeutic protocols. Serial biopsies of the first patient, taken at weekly intervals for nine weeks post transplant, revealed moderate cellular rejection beginning with grade 1A, and increasing at times to 1B. Humoral rejection peaked at 3+ immunofluorescence "staining" for C4d one week post transplant, and then wavered between 2+ (moderate "staining") to weak (1+)"staining". Biopsies of the second patient, taken at weekly intervals for 15 weeks post transplant, revealed maximal (3+) humoral rejection one week after transplantation, and then diminished to 2+ thereafter. Cellular rejection was graded as 3A three weeks post transplant. Rejection levels declined steadily thereafter to negligible cellular rejection by week 15. Whereas, cellular rejection was most prominent 14-21 days post transplant, humoral rejection was greatest in the early post transplant period. Results of the present investigation revealed the significance not only of traditional light microscopy in evaluating the severity of cellular rejection in endomyocardial biopsies of cardiac allotransplants, but also the value of C4d immunofluorescent "staining" of interstitial capillaries as an indicator of humoral rejection episodes, which may require modified therapy.

184

New Pathogenic Role of the Human Polyomavirus JC in Neurons

Igor Koralnik, MD, Beth Israel Deaconess Medical Center, Boston, MA, USA; Xin Dang, PhD, Beth Israel Deaconess Medical Center, Boston, MA, USA; Christian Wuthrich, PhD, Beth Israel Deaconess Medical Center, Boston , MA, USA

Background: Most healthy adults are seropositive for the polyomavirus JC (JCV), which is not associated with any clinical manifestations in immunocompetent hosts. However, this virus can reactivate in immunosuppressed individuals and causes a lytic inection of oligodendrocytes, leading to a deadly demyelinating disease of the CNS called Progressive Multifocal Leukoencephalopathy (PML).We have demonstrated for the first time that JCV could also infect cerebellar granule cell neurons and cause a novel clinical entity which we named JCV granule cell neuronopathy (JCV GCN). We then characterized the full length sequence of a granule cell neuron-tropic JC virus (JCV) variant , JCVGCN1 associated with lytic infection of granule cell neurons and cerebellar atrophy in an HIV-infected patient with PML.This virus had a unique out of frame deletion causing a change in the c-terminus aa sequence of the VP1 capsid protein, which was absent in the JCVHWM strain found in hemispheric white matter PML lesions of this patient. We set up a in vitro study to verify and characterize the phenotype of the granule cell-tropic JCV variant. Methods: The full length clones of JCVGCN1 and JCVHWM were used to transfect the SVG human astroglial cell line, the human medulloblastoma cell line DAOY, derived from cerebellar granule cell layer, and the monkey kidney cell line COS-7 The prototype JCV Mad1, and the JCV Mad1D, containing a with JCVGCN1-type mutation located in the C termini of VP1 gene were used as controls. The four JCV strains were passaged 30 times in vitro. Quantitative PCR (Q-PCR) were performed to measure the JCV viral load in each cell line overtime, and the expression of viral proteins was tested by western blot. Results: The sequence of C termini of VP1 gene of all four JCV strains was stable after 30 passages. VP1 protein could be detected by Western Blot in all cell lysate samples. Q-PCR amplification of JCV from DNA extracted from supernatant and cell lysates showed that all four viral strains replicated equally at high levels in COS-7 monkey kidney cells while JCVGCN1 and JCV Mad1D DNA became decreased considerably and became undetectable at most time points after 7 passages in SVG cells. Conversely, all four viral strains showed low level of replication in DAOY medulloblastoma cell lines. Conclusions: These experiments confirm that the JCVGCN1deletion mutant isolated from the cerebellum of an HIV+ patient with cerebellar atrophy, is a replication competent virus in vitro. The GCN1 deletion constituted a disadvantage for replication in the human astroglial SVG cell line, consistent with the fact that only undeleted JCV was found in PML lesions of the same patient. However, GCN1 deletion mutants grew as well as undeleted strains in the human medulloblastoma cell line. The availability of a human cerebellar cell line able to sustain replication of JCVGCN1 will greatly facilitate the understanding of its pathogenesis in humans.

185

The ApoA-I Mimetic Peptide 4F is Less Effective at Decreasing Plasma SAA Levels and Modifying Lipoprotein Distribution Compared to a Proline-Kinked Tandem Helix Peptide Despite Achieving Higher Plasma Concentrations

Geoffrey Wool, BA, The University of Chicago, Chicago, IL, USA; Catherine Reardon, PhD, The University of Chicago, Chicago, IL, USA; Godfrey Getz, MD/PhD, The University of Chicago, Chicago, IL, USA

We hypothesize that apoA-I mimetic peptides better mimicking the punctuated alpha-helical repeats of full-length apoA-I are more anti-inflammatory and anti-oxidative. In this study, we compared the lipoprotein-association, anti-inflammatory, and anti-oxidative properties of 4F (monomeric 18mer alpha-helix) to a proline-punctuated tandem helix peptide Pro (4F-proline-4F 37mer). Biotinylated 4F and Pro peptides were used to investigate peptide plasma clearance. After intraperitoneal injection of an equal mass of biotinylated peptides into apoE-/- mice, peak plasma levels occur in 1.5-3 hours for biotinPro and 1.5-5 hours for biotin4F. Peptide clearance t1/2 occurs for the biotinPro peptide within 12-22 hours and within 15-20 hours for biotin4F. Normalized AUC0-->t analysis demonstrated that biotin4F had a significantly higher (p<0.02) area under the curve than the biotinPro tandem peptide: 1 (0.633) vs. 0.263 (0.171), respectively. In order to determine the selective lipoprotein association behavior of the two peptides, biotinylated peptides were added to apoE-/- plasma ex vivo and separated by equilibrium density ultracentrifugation. Biotin4F associated with dense HDL as well as those fractions between HDL and free protein. The biotinPro peptide associated with all lipoproteins: chylomicron remnants/VLDL, LDL, and HDL. It also eluted in those fractions between HDL and free protein. Non-biotinylated peptides were injected intraperitoneally into mice for one week every other day starting at 8-10 weeks of age. Peptides were compared at equal molar concentrations of 4F or Pro, based on guidance from our clearance studies. This converts to ~1.25 mg/day/kg 4F or ~2.5mg/day/kg Pro. 4F treatment did not significantly decrease plasma levels of the acute phase protein SAA compared to control mice, while Pro-treated mice showed a significant 40% reduction in plasma SAA levels. There were no significant differences in plasma TBARS reactivity for 4F- or Pro-treated mice compared to controls. No change has been detectable in alpha versus pre-beta-migrating HDL after peptide treatment. 4F or Pro-treated mice did not show any significant difference in total serum PON arylesterase activity, as compared to PBS-treated controls. Also, 4F caused no shift in arylesterase activity between the HDL-bound or nonlipoprotein-associated fractions. Short term (one week) or long term (eight weeks) 4F-treated mice showed no change in plasma total cholesterol, lipoprotein distribution, or body weight compared to PBS controls. Long term Pro-treated mice showed no change in body weight or total cholesterol. However, LDL-C and HDL-C were significantly elevated in long term Pro-treated mice, compared to controls. The tandem apoA-I mimetic peptide Pro provides more anti-inflammatory ability and a unique HDL-boosting activity compared to the previously described 4F peptide. Atherosclerosis studies with these peptides are ongoing.

186

Regulation Of Microglia Migration And Inflammation By Glycogen Synthase Kinase-3

Christopher Yuskaitis, BA, University of Alabama at Birmingham, Birmingham, AL, USA; Richard Jope, PhD, University of Alabama at Birmingham, Birmingham, AL, USA

Glycogen synthase kinase-3 (GSK3) has been linked to modulation of inflammation as well as to a number of neurological disorders such as Alzheimer’s disease. Many neurological conditions, from traumatic brain injury to neurodegenerative diseases, have an inflammatory component which includes activation of microglia. In addition to activation, migration of microglia to sites of insults is an important function of these resident immune cells of the brain. Previous research has shown that GSK3 plays a role in migration of other cell types; however, the role of GSK3 in microglial migration has not been defined. Given that GSK3 has been shown to be involved in neurological disorders, inflammation, and cell migration, we hypothesized that GSK3 may regulate microglial migration. Using in vitro migration assays, transwell migration and scratch assay, we assessed whether several different GSK3 inhibitors can regulate the migration of immortalized BV-2 microglia. The results demonstrate that GSK3 promotes both random and directed migration of immortalized BV-2 microglia cells. We then tested if GSK3 inhibitors block migration of microglia in their native environment, using acute hippocampal preparations from GFP-labeled microglia. These results confirmed the in vitro findings that inhibition of GSK3 significantly inhibited microglial movement. Additionally, we tested if GSK3 plays a role in microglial production of pro-inflammatory molecules. BV-2 microglia cells were stimulated with lipopolysaccharide (LPS) and the activation of microglia was assessed by measuring cytokine production and nitric oxide (NO) release. The results show that inhibition of GSK3 suppresses pro-inflammatory IL-6 and NO production after stimulation with LPS. We then assessed microglial activation in situ using a mouse organotypic hippocampal slice culture model. GSK3 inhibition attenuated the LPS-induced pro-inflammatory molecule production in hippocampal slice cultures. We then tested if GSK3 inhibition protects from inflammation-induced neuronal cell death in hippocamal slice cultures. The results show that inhibition of GSK3 attenuates inflammation-induced neurotoxicity, supporting the hypothesis that a reduced inflammatory response is neuroprotective. Altogether, these results demonstrate that GSK3 contributes to microglial migration and pro-inflammatory cytokine production, supporting the concept that GSK3 promotes the neuroinflammatory functions of microglia and is a potential therapeutic target for neuroinflammatory conditions.

187

Stereological Immunohistochemical Investigation of Differential Susceptibility of Substantia Nigra Dopaminergic Neurons to MPTP Toxicity

Tyrell Simkins, BS, Michigan State University, East Lansing, MI, USA; John Goudreau, DO/PhD, Michigan State University, East Lansing, MI, USA; Keith Lookingland, PhD, Michigan State University, East Lansing, MI, USA

Parkinson’s disease is a common neurodegenerative disorder characterized by bradykinesia, resting tremor, rigidity and postural instability.  The discovery that the compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes a syndrome nearly identical to idiopathic Parkinson’s disease has led to its use as a tool in studying neurodegeneration of nigrostriatal dopamine (DA) neurons.  The chronic MPTP mouse model has been shown to closely mimic the progression of idiopathic Parkinson’s disease.  The purpose of this study is to investigate the occurrence of intranigral differences in the susceptibility of substantia nigra DA neurons to MPTP administration.  The midbrain containing the substantia nigra of MPTP treated mice were cut into 60 µm sections, immunostained for tyrosine hydroxylase (TH) and counterstained with Nissl. Estimations of TH positive cell populations we obtained using unbiased stereological methodology.  The substantia nigra was divided into rostrocaudal, mediolateral, and dorsoventral regions to determine if there was a differential susceptibility of subpopulations of substantia nigra dopaminergic neuron to MPTP.  Estimations of Nissl stained cells in the substantia nigra were also obtained.  The total number of TH positive cells bilaterally in the substantia nigra in controls was estimated to be 10644 ± 857.  Most of these cells were located in the rostral half of the substantia nigra.  In agreement with previous reports, progressive and selective loss of TH positive cells was observed following chronic MPTP administration.  These data also confirm previous reports that the lateral-ventral portion of the substantia nigra exhibits the greatest percent loss (42-78%) of all regions, although this area contained a minority (~6%) of the TH cells in the substantia nigra.  Significant differential loss of TH positive cells was not observed between the mediolateral or dorsoventral divisions. Additionally, no significant differential loss of TH positive cells was found along the rostrocaudal axis.  These data reveal that heterogeneously distributed populations of nigrostriatal DA neurons located in discrete subdivisions of the substantia nigra are not differentially susceptible to MPTP neurotoxicity.

188

IL-17-Independent Actions of IL-23 in Pseudomonas Aeruginosa Pulmonary Infection

Patricia Dubin, MD, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA, USA; Jay Kolls, MD, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA, USA

Background:Chronic P. aeruginosa (PA) pulmonary infection is the most significant cause of morbidity and mortality in individuals with cystic fibrosis (CF).Because PA is a highly adaptable pathogen, the CF host immune response is unable to eradicate it and causes significant collateral airway damage through recruitment and activation of neutrophils.IL-23 and IL-17 constitute a pro-inflammatory axis that is critical to neutrophil recruitment in PA infection.In IL-23/IL-17 proinflammatory axis models, neutrophil recruitment is most commonly attributed to the actions of IL-17; however, the IL-17-independent contributions of IL-23 have not been studied.Objectives:Define the IL-17-independent role of IL-23 in the acute host inflammatory response to PA pulmonary infection.Determine if IL-23 acts independently or additively/synergistically with IL-1ß in effecting this response.Design/Methods:In vivo studies to determine if IL-23 is critical to the PA-induced inflammatory response were conducted. WT and IL-23-deficient mice were infected with PA and sacrificed at 3 hours, a time point prior to the production of IL-17. BAL inflammatory cell counts and cytokines/chemokines were measured.Bacterial load was measured in lung homogenate. Additional in vivo studies were conducted to determine the relationship of IL-23 and IL-1ß in the inflammatory response.Recombinant murine IL-23, IL-1ß and IL-23 + IL-1ß were instilled into WT and IL-23-deficient mice. BAL inflammatory cell counts and cytokines/chemokines were measured at 3 hours. Results: In PA infection, IL-23 deficient mice had significantly lower percent neutrophils (p<0.02) and lower MIP1α, KC, and IL-6 (p<0.001) compared to WT mice. At this timepoint, IL-17 was undetectable in both groups and bacterial load was the same; thus it did not explain the differences in neutrophil recruitment and cytokine/chemokine levels.IT instillation of recombinant mouse IL-23 resulted in no discernible inflammatory response compared to control, suggesting that though IL-23 is necessary, it is not sufficient to initiate the immune response. The IT administration of IL-23 + IL-1ß elicited neutrophil recruitment and cytokine/chemokine induction in excess of that of IL-23 alone and IL-1ß alone (p<0.01). These results suggest that IL-23 and IL-1ß act synergistically. Statistical analysis for all studies was completed with t test or ANOVA.Conclusions: In addition to its role in the IL-23/IL-17 proinflammatory axis, IL-23 is critically important to neutrophil recruitment in PA infection through its synergistic actions with IL-1ß, acting in an IL-17-independent manner. These findings have not been published before and highlight the critical role that IL-23 plays as a master regulator in both the innate and adaptive immune response to PA pulmonary infection.

189

Gab2 is essential for Bcr-Abl-Induced Leukemogenesis

Wayne Chan, BSE, Tufts-New England Medical Center, Boston, MA, USA; Golam Mohi, PhD, SUNY Upstate Medical University, Syracuse, NY, USA; Benjamin Neel, PhD, MD, Ontario Cancer Institute, Toronto, ON, Canada; Richard Van Etten, MD, PhD, Tufts-New England Medical Center, Boston, MA, USA

The BCR-ABL oncogene encodes an activated tyrosine kinase fusion protein that causes chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL) in humans. The autophosphorylation site Tyr 177 of Bcr-Abl is known to recruit Grb2, and is required for efficient induction of CML-like myeloproliferative disorder. We showed previously that the scaffolding adapter protein Gab2 is recruited to Tyr 177 of BCR-ABL via a Grb2/Gab2 complex. Using Gab2-/- mice, we also showed that Gab2 is critical for myeloid transformation by Bcr-Abl, whereas lymphoid transformation is markedly reduced. Biochemical analysis showed that the Erk and Akt pathways are defective in Gab2-/- myeloid and lymphoid cells expressing BCR-ABL. Using a bone marrow transduction and transplantation assay, we now demonstrate an essential requirement of Gab2 in myeloid leukemogenesis by BCR-ABL. Whereas recipients of Gab2+/+ BM all died within 3 weeks due to CML-like myeloproliferative disease, Gab2-/- BM recipients failed to develop CML. Eventually, these Gab2-/- BM recipients developed T-cell acute lymphoblastic leukemia at a much later stage and died, similar to the phenotype observed with the Y177F Bcr-Abl mutant. Because Gab2 deficiency phenocopies the effect of BCR-ABL Y177F mutation, these results suggest that the key transforming signal from Tyr177 of BCR-ABL is mediated through Gab2. Co-expression of Gab2 with BCR-ABL by retroviral transduction into Gab2-/- BM rescued the defects in myeloid and lymphoid transformation by BCR-ABL. However, expression of Gab2 mutants defective in binding of either Shp2 or p85PI3K failed to restore efficient myeloid and lymphoid transformation. These results argue that Gab2 is essential for BCR-ABL-evoked leukemogenesis, and suggest the key downstream signals for transformation/leukemogenesis from Gab2 are mediated through recruitment of both Shp2 and p85-PI-3K.

190

Evaluation Of A Novel Connexin-Based Peptide For The Treatment Of Diabetic Wounds

GAUTAM GHATNEKAR, PhD, FirstSting Research, Inc., North Charleston, SC, USA; GAUTAM GHATNEKAR, PhD, FirstSting Research, Inc., North Charleston, SC, USA; ABHIJIT GURJARPADHYE, MS, Medical University of South Carolina, Charleston, SC, USA; SPENCER ROBERT, JD, MBA, FirstSting Research, Inc., North Charleston, SC, USA; JOSEPH PALATINUS, BS, Medical University of South Carolina, Charleston, SC, USA; ROBERT GOURDIE, PhD, Medical University of South Carolina, Charleston, SC, USA

Wound healing is a complex process and induces a cascade of events including inflammation, proliferation, and scar production/tissue remodeling. Diabetic patients commonly demonstrate impaired wound healing. According to the American Diabetes Association, there are more than 16 million people in the United States with known diabetes. A well-established reason as to why diabetic wounds are tough to heal is that they do not progress through the normal healing phases. Instead, it is thought that diabetic wounds are caught and remain trapped in the initial inflammatory phase of wound healing. It is well established that intercellular communication via gap junction channels is a key aspect of wound repair; and that gap junctions, and their connexin subunits, have functions in coordinating the cellular activities necessary for inflammation, wound repair and scar tissue formation following injury. Previously, we reported the generation and characterization of a novel bioengineered peptide (dubbed ACT1) based on the extreme carboxy terminus of Cx43 that had been rationally designed to inhibit binding of Cx43-interacting proteins and its use in acute wound healing. Recently, we undertook the study to determine the effects of the ACT1 peptide in diabetic wounds using adult diabetic C57BL/KsJ-m+/+Leptdb (db+/db+) mice. The db+/db+ mouse is a well-accepted animal model that is known to exhibit slow and pathologic healing of skin wounds. Animals were divided into 2 groups (n = 20/group) and received one 5mm in diameter full-thickness excisional wounds between the shoulder blades on the dorsal mid-line. Wounds were treated with either ACT1 peptide or vehicle control. Peptide was formulated in a 20% Pluronic F-127 gel and applied on the wounds immediately after injury and again 24 hr post-wounding. No subsequent applications were made. Wounds were photographed on day 0, 1, 4, 7, 10, 12, 15 and 21 post-wounding. Wounds treated with ACT1 peptide demonstrated significant improvement in wound closure rate (p<0.05) as compared to controls. Subjective parameters such as redness and overall appearance were substantially improved in ACT1 treated wounds. In addition, the peptide treated wounds demonstrated pronounced fur growth at the wound edges around day 15, which was not seen in controls. These results suggest that Cx43 function in wound healing is mediated via signaling by its carboxy-terminal-most sequences and also implicate Cx43-interacting proteins in the process. Approaches based on the peptide may provide novel therapies in cutaneous wound healing. In particular, our focus is on development of our peptide into a pharmaceutically acceptable topical agent for application to diabetic foot ulcers. (Supported by NIH-NIDDK R43DK080567-01)

191

Upregulation of Arachidonic Acid 5-Lipoxygenase in Papillary Thyroid Carcinoma Correlates With Increased Tumor Invasion In-Vivo and Increases Extracellular Matrix Degradation and Invasion In-Vitro.

Nicolas Kummer, BS., NYMC, Valhalla, NY, USA; Cordon Iacob, MD, New York Eye and Ear Infirmary, New York, NY, USA; Stimson Schantz , MD, New York Eye and Ear Infirmary, Otolaryngology Head and Neck Surgery, New York, NY, USA; Jan Geliebter, PhD, New York Medical College, Valhalla, NY, USA

Upregulation of arachidonic acid 5-lipoxygenase (ALOX5) expression has been observed in a variety of carcinomas including bladder, breast and prostate; it is reported to inhibit apoptosis in the respective in-vitro tissue culture systems. Here we report that increased ALOX5 expression, in clinical samples of papillary thyroid carcinoma (PTC), correlates with tumor invasion. Additionally, over-expressing ALOX5 in PTC cells increased invasive phenotype, but did not affect cell viability. Thyroid carcinoma is the most prevalent endocrine malignancy; its incidence and mortality have risen ~84% and ~33% respectively during the past decade. PTC accounts for ~85% of thyroid carcinomas and is a major source of morbidity, thus improvements in both diagnosis and treatment are necessary. Microarray analysis of 7 pairs of matched PTC-normal patient samples revealed a 5 fold increase (P. = 0.004) in ALOX5 mRNA expression in tumors. Real time RT-PCR analysis of an expanded set of 17 matched PTC-normal patient samples showed an average 23 fold increase (P. = 0.0011) in ALOX5 mRNA in tumors and showed a linear trend correlating ALOX5 mRNA expression with Tumor Invasive Score (TIS), as determined from clinical pathology reports. ALOX5 expression in the patient samples was also validated by immunohistochemistry. It has been reported in the literature that inhibition of ALOX5 in cancer cells by pan-lipoxygenase inhibitors induces apoptosis in-vitro. To investigate the therapeutic potential of targeting ALOX5 expression in PTC, we tested the effect of Zileuton (0.01uM to 50 uM) on the PTC cell lines, BCPAP, BCPAP transfected with ALOX5 (ALOX5-BCPAP) and BCPAP transfected with Green Fluorescence Protein (GFP) fused ALOX5 (ALOX5-GFP-BCPAP). XTT [2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanilide] analysis after 48hr of treatment revealed no change in cell viability. Furthermore, the same cell lines treated with arachidonic acid (AA) or 5-hydroxyeicosatetraenoic acid (5-HETE) (0.01 uM to 1.00 uM) also showed no change in cell viability at 48hrs. However, ALOX5-BCPAP showed a 3.54 fold increase (P. = 0.0003) in percent invasion through 8uM membrane pores overlaid with Matrigel Matrix compared to BCPAP transfected with vector alone; 50 uM Zileuton treatment of ALOX-BCPAP reduced invasion by 56% (P. = 005). ALOX5-BCPAP was also shown to degrade type I collagen, further supporting the invasion assay results. Additionally, ALOX5-BCPAP exhibited a 2.17 (P = 0.006) fold increase in anchorage independent growth in 0.33% agarose. To our knowledge, this is the first report demonstrating ALOX5 expression correlating to tumor invasion both in-vivo and demonstrating invasion in-vitro. Thus, our data indicate that ALOX5 may contribute to tumor invasion and may be useful as a potential biomarker and therapeutic target for PTC.

192

Temporal Regulation Of Kinetochore-Microtubule Dynamics Is Essential For Error-Free Mitosis

Samuel Bakhoum, BS, Dartmouth Medical School, Hanover, NH, USA; Sarah Thompson, BA, Dartmouth Medical School, Hanover, NH, USA; Duane Compton, PhD, Dartmouth Medical School, Hanover, NH, USA

Chromosome mis-segregation during mitosis causes aneuploidy, a hallmark of cancer cells.  To properly segregate, sister chromatids use kinetochores to attach to microtubules emanating from opposite spindle poles and maloriented microtubule attachments must be released.  Thus, the dynamics of kinetochore-microtubule attachments must be optimized to provide both stable and error-free attachment, but how that balance is achieved is unknown.  Here we examine the contributions of the kinesin-13 proteins, Kif2b and MCAK, to mitotic fidelity and the precise temporal regulation of kinetochore-microtubule dynamics.  Kif2b is recruited in an Aurora kinase-dependent manner to tensionless kinetochores where it promotes microtubule turnover during prometaphase to correct improper kinetochore-microtubule attachments that lead to chromosome mis-segregation.  Conversely, MCAK promotes kinetochore microtubule turnover in metaphase to prevent the additional formation of improper attachments.  Finally, overexpression of Kif2b or MCAK suppresses chromosome mis-segregation in chromosomally unstable cancer cell lines.  Thus, global control of kinetochore microtubule dynamics at different stages of mitosis provides a basis for ensuring faithful chromosome segregation.

193

Lipopolysaccharide Signaling Disruption Fails to Ameliorate High Fat Diet-Induced Glucose Intolerance and Insulin Resistance

James Young, AB, University of Massachusetts Medical School, Worcester, MA, USA; Alfonso Mora, DVM, University of Massachusetts Medical School, Worcester, MA, USA; Michael Czech, PhD, University of Massachusetts Medical School, Worcester, MA, USA; Douglas Golenbock, MD, University of Massachusetts Medical School, Worcester, MA, USA; Evelyn Kurt-Jones, PhD, University of Massachusetts Medical School, Worcester, MA, USA; Robert Finberg, MD, University of Massachusetts Medical School, Worcester, MA, USA; Silvia Corvera, MD, University of Massachusetts Medical School, Worcester, MA, USA

Glucose intolerance and insulin resistance are hallmarks of the type 2 diabetes population, which is estimated to afflict 180 million people globally and anticipated to double by the year 2030. Despite a predominance of obesity in the type 2 diabetic patient population, it is estimated that the majority of obese individuals are not overtly diabetic, suggesting that nutrient excess alone is a strong but not singular risk factor for glucometabolic dysfunction. It has been proposed that obesity is associated with a chronic low-grade inflammatory state that may potentiate glucose intolerance and insulin resistance.

As a function of its potency and wide distribution, bacterial endotoxin or lipopolysaccharide (LPS) may fuel chronic inflammation associated with diabetes. To test this hypothesis, we employed mice deficient in components of the LPS signaling complex, namely CD14, TLR4 and MyD88, to investigate glucose homeostasis and insulin sensitivity in the context of a high fat diet challenge.

Surprisingly, whole body disruption of LPS signaling led to impaired glucose tolerance and insulin resistance in cd14-/- and tlr4-/- mice with no alterations in adipose or total body mass from wild type controls regardless of dietary regimen. In contrast, myd88-/- mice gained ~20% more weight primarily due to adipose expansion; however, developed milder glucose intolerance and insulin resistance only when challenged with a high fat diet. In cd14-/- and tlr4-/-, but not myd88-/- mice, a marked rebound phase in the insulin tolerance test was associated with enhanced catecholamine response to hypoglycemia. Furthermore, adrenal hypertrophy was observed, suggesting structural compensation that may support prolonged hyperactive sympathoadrenal drive.

The novel employment of three genetic knockouts, namely cd14-/- , tlr4-/-, and myd88-/-, localizes metabolic observations to these genes shared role in LPS signaling in lieu of alternate biological functions. The overlay of these mouse models suggests an intimate association of CD14 and TLR4 in the process of glucose intolerance and insulin resistance demonstrated here in part due to an enhanced sympathoadrenal tone and exaggerated counter-regulatory response to hypoglycemia.

194

Pancreatic Injury in Response to Dietary Challenge and Hyperlipidemia-A New Path to Inflammation?

Jon Mallen-St. Clair, MS, NYU School of Medicine, NY, NY, USA; Victor Joaquin, MS, NYU School of Medicine, NY, NY, USA; Edward Fisher, MD PhD, NYU School of Medicine, NY, NY, USA; Dafna Bar-Sagi, PhD, NYU School of Medicine, NY, NY, USA

    Pancreatic cancer is the fourth leading cause of cancer related death in the United States and is almost uniformly fatal. Epidemiological studies have shown that high fat diet (HFD), obesity, and chronic pancreatic inflammation are associated with an increased risk of pancreatic cancer. Given the high incidence of obesity and its association with pancreatic cancer, it is imperative to determine the mechanistic link between these disease entities.
    To model the effects of obesity and consequent hyperlipidemia on pancreatic disease, we set out to establish a mouse model of hyperlipidemia driven pancreatic injury using Apolipoprotein E deficient mice (APOE-/-).    APOE-/- mice have a profound defect in intermediate density lipoprotein and chylomicron clearance that leads to hypercholesterolemia and hypertryglyceridemia when placed on a high fat diet (HFD).  These mice provide a more accurate model of the elevated lipid levels frequently found in obese humans.  In order to determine the consequences of prolonged hyperlipidemia on the pancreas, we examined the effect of HFD on the pancreata of APOE-/- mice.  Our studies demonstrate that HFD causes a mild inflammation in the pancreas of wild-type mice.  In the setting of APOE deficiency the inflammation was exacerbated and associated with fat deposition and acinar to ductal metaplasia.  Using immunohistochemical analysis, we found that APOE -/- mice had an increased rate of ductal and acinar proliferation, an expansion of Pdx-1 expressing pancreatic progenitor cells within the acinar and ductal compartments, and islet neogenesis within the ductal epithelium. Furthermore, in a model of acute pancreatitis, pancreatic edema and inflammation was specifically exacerbated in APOE-/- HFD mice.
    These findings demonstrate that hyperlipidemia contributes to pancreatic injury and causes a compensatory inflammatory and proliferative response.  Furthermore, this pancreatic injury is associated with an expansion of progenitor cells and cellular plasticity.  Future experiments will focus on the mechanistic basis of hyperlipidemic pancreatic inflammation and determine the consequences of hyperlipidemia in the context of genetically engineered mice that harbor mutations found in pancreatic cancer.

195

Regulation Of Huntingtin Palmitoylation And Its Role In Huntington Disease

Fiona Young, BSc.(Hon), University of British Columbia, Vancouver, BC, Canada; Michael Hayden, MB, ChB, PhD, University of British Columbia, Vancouver, BC, Canada

Huntington Disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor, cognitive, and psychiatric deficits and selective neuronal cell death. The causative mutation in HD is an expansion of the N-terminal polyglutamine tract in huntingtin (htt), which results in altered trafficking of mutant htt and enhanced toxicity to striatal neurons.

Post-translational modification by the lipid palmitate has been shown to play a critical role in the trafficking and function of many proteins, including htt. It has been previously demonstrated that huntingtin-interacting protein 14 (HIP14) is a palmitoyl transferase that palmitoylates htt. Previous characterization of HIP14 demonstrated a reduced interaction with mutant htt resulting in reduced palmitoylation, suggesting that palmitoylation may play a role in the pathogenesis of HD. Most recently, we have identified cysteine 214 as a major site of htt palmitoylation in the N-terminus of htt, close to the site of polyglutamine expansion. It was demonstrated that mutation of this site, rendering htt palmitoylation-resistant, results in increased neuronal toxicity, enhanced inclusion formation, and in altered trafficking of htt. Remarkably, mutation of the palmitoylation site in wild type htt also resulted in enhanced toxicity similar to that seen in mutant htt. Together, these previous studies suggest a critical role of palmitoylation in htt trafficking and function.

Based on this preliminary work, we are characterizing the enzymatic regulation of htt palmitoylation in a number of existing and new mouse models. These studies will impart key insights into how this process is regulated in vivo, as well as setting the precedent to understand the general role of palmitoylation in a wide range of other human diseases. Ultimately, this may lead to identification of new therapeutic targets and treatments for patients.

F.B.J.Y. is supported by a Canadian Institutes of Health Research Walter and Jessie Boyd & Charles Scriver and Child & Family Research Institute –UBC MD/PhD Studentship Award. She also receives funding from the Michael Smith Foundation for Health Research as a Junior Trainee.

196

Antioxidant Treatment Lowers Blood Pressure And Improves Sympathetic Nerve Function In A Salt-Sensitive Model Of Hypertension

Stacie Demel, BS, Michigan State University, East Lansing, MI, USA; James Galligan, PhD, Michigan State University, East Lansing, MI, USA; Hua Dong, BS, Michigan State University, East Lansing, MI, USA

Hypertension (HTN) afflicts 50 million people in the US alone, and despite treatment rates of 75% or greater, the HTN of only 30 to 50% of patients have their blood pressure controlled. Common to both human hypertension and animal models of hypertension is an increase in the formation of reactive oxygen species (ROS) resulting in oxidative stress. ROS damages proteins and tissues, perpetuating the hypertension and causing end-organ damage. Hypertension is also associated with an increase in norepinephrine (NE) release from sympathetic nerves as a result of alterations in both central and peripheral nervous systems. ATP is a cotransmitter with NE from sympathetic nerves, but hypertension associated changes in purinergic neurotransmission have not been well characterized. New techniques allow for ATP and NE to be studied independently and with greater temporal and spatial resolution. ATP binds to ligand-gated P2X receptors on arterial smooth muscle cells (SMCs) resulting in a transient depolarization of the membrane potential which can be measured via intracellular recordings from blood vessels maintained in vitro. Microlectrodes coupled to electroanalytical techniques can be used to measure NE. NE adheres to the surface of the microlectrode where it is oxidized and the resulting oxidation current at the surface of a carbon fiber microelectrode represents the number of molecules of NE released. In these studies, the deoxycorticosterone acetate (DOCA)-salt model of hypertension was used to study the role of ROS on alterations in neurotransmission from periarterial sympathetic nerve endings in rats. We hypothesized that increased ROS in DOCA-salt hypertension targets, and damages, sympathetic nerve terminals resulting in altered neurotransmission. To assess this, DOCA-salt rats were given antioxidants in their drinking water (1% saline). Control rats received 1% saline only. Rats were implanted with telemetric pressure transducers fixed to femoral artery catheters for hemodynamic measurements. After four weeks, animals were sacrificed and neurotransmission between the three treatment groups was assessed using the techniques described above. It was found that antioxidant treatment significantly lowered blood pressure compared to control. In addition alterations to NE and ATP release seen in hypertension were attenuated, suggesting that antioxidants can be neuroprotective at sympathetic nerve endings. These data suggests that sympathetic nerve endings are significant regulators of blood pressure, and reveals their importance as a potential therapeutic target for drugs that can be used to treat HTN.

197

Pyruvate-Enhanced Cardioplegia Preserves Myocardial Hsp 70 And Endothelial NOS In A Porcine Model Of Cardiopulmonary Bypass

Devin Flaherty, BA, University of North Texas Health Science Center, Fort Worth, TX, USA; Myoung-Gwi Ryou, MS, University of North Texas Health Science Center, Fort Worth, TX, USA; Besim Hoxha, MD, University of North Texas Health Science Center, Fort Worth, TX, USA; Jie Sun, BS, University of North Texas Health Science Center, Fort Worth, TX, USA; Heather Ferlitch, High School Diploma, Juniata College, Huntingdon, PA, USA; Albert Olivencia-Yurvati, DO, University of North Texas Health Science Center, Fort Worth, TX, USA; Robert Mallet, PhD, University of North Texas Health Science Center, Fort Worth, TX, USA

Background: The use of pyruvate-fortified cardioplegia to arrest the heart during cardiopulmonary bypass (CPB) enhances post-surgical recovery of cardiac function, compared with conventional cardioplegia solutions. Pyruvate’s energy-yielding and antioxidant effects wane as pyruvate is cleared from the circulation, yet its functional benefits persist, suggesting a protein-mediated cardioprotective mechanism. Hypothesis: Pyruvate cardioplegia during CPB prevents subsequent depletion of the cytoprotective proteins eNOS and Hsp-70. Methods: After sternotomy, swine were subjected to 60 min cardioplegic arrest and 30 min myocardial reperfusion on CPB, then were weaned from bypass. The crystalloid portion of the 4:1 blood:crystalloid cardioplegia contained 24 mM lactate or pyruvate. Left ventricular myocardium was sampled at 4 h post-CPB for extraction and immunoblot detection of Hsp70 and eNOS; actin served as loading control. Sham pigs underwent the same surgical and analytical procedures, but without CPB. Results: Actin-normalized myocardial eNOS and Hsp-70 contents fell by 32 and 68%, respectively, and NOS activity fell from 132 ± 10 to 91 ± 6 nmol/min/g protein in the lactate cardioplegia group, vs. respective sham values (P < 0.05). Pyruvate cardioplegia prevented loss of eNOS and Hsp70, and maintained NOS activity at 134 ± 8 nmol/min/g protein (P < 0.05 vs. lactate group). Conclusions: Cardioplegic arrest on CPB can deplete myocardium of the cytoprotective proteins eNOS and Hsp70. Protection of these proteins by pyruvate-enriched cardioplegia may support improved post-CPB cardiac performance. Support: Osteopathic Heritage Foundation 02-18-522

198

ATP Binding Site Analysis and Inhibitor Design for Hepatitis C Virus Helicase

Craig Belon, BS, New York Medical College, Valhalla, NY, USA; David Frick, PhD, New York Medical College, Valhalla, NY, USA

Hepatitis C virus (HCV) is a positive-sense, single stranded RNA virus infecting an estimated 1-2% of the worlds population. Of those infected, a large proportion go on to develop serious complications including cirrhosis, hepatocellular carcinoma and, eventually, death. Current therapy for HCV infection, a combination of the broad-coverage antiviral ribavirin and peg-interferon, is nonspecific and in many cases ineffective. When drug therapy fails, the only option is liver transplant. Indeed, in the United States, cirrhosis caused by HCV is the leading cause of orthotopic liver transplant. One key HCV protein, nonstructural protein 3 (NS3), is a multifunctional enzyme possessing ATPase, helicase, and protease activities and is necessary for viral replication in vivo. Specific drugs targeted against the protease functionality of this enzyme have been attempted and have entered clinical trials. Because the helicase portion of the enzyme is fueled by ATP, the NS3 helicase functionality also makes an attractive target for small molecule inhibitors. Previous studies on HCV have demonstrated the geometry and relevant residues involved at the catalytic site of the NS3 protein in exquisite detail, elucidating the specific amino acids necessary for metal binding, stabilization of ATPs gamma-phosphate, and mechanism of cleavage to ADP and inorganic phosphate. While contributing much valuable information regarding the catalytic activity and mechanism of the enzyme at the phosphate groups, they have largely neglected the influence of the rest of the ATP molecule- namely the ribose sugar and adenine base moieties- on binding and subsequent usage by the enzyme to fuel unwinding. Data suggest the unwinding rate varies considerably when fueled by the 8 canonical nucleoside triphosphates (NTPs). This indicates that the identity of the NTP directly influences the rate determining step of the unwinding reaction. Not only is the specific structure of the ribose or base moiety of the NTP important, it is an absolute requirement for defining the complete NTP binding site. Reported X-ray crystal structures of NS3 helicase have not been determined with a bound, non-hydrolysable ATP analog. Consequently, specific interactions between the non-phosphate portions of ATP and the helicase are largely unknown. Here, a newly developed unwinding assay is used to determine structure-activity relationships using the 8 canonical NTPs as well as various non-naturally occurring NTP analogs. This information is used to generate a working model of the NTP binding site for NS3 helicase. This information is a valuable first step for the rational design of specific, tight-binding inhibitors for treatment of HCV infection.

199

A New Locus On Chromosome 1 Increases Susceptibility To Asthma In Children

Eric Schauberger, BS, Michigan State University, East Lansing, MI, USA; Susan Ewart, DVM, PhD, Michigan State University, East Lansing, MI, USA; Karen Friderici, PhD, Michigan State University, East Lansing, MI, USA; S Arshad, MD, University of Southampton/Asthma and Allergy Research Centre, Southampton, NA, United Kingdom; Marianne Huebner, PhD, Michigan State University, East Lansing, MI, USA; Wilfried Karmaus, MD, Dr.med, MPH , University of South Carolina, Columbia, SC, USA; Stephen Holgate, MD, DSc, University of Southampton, Southampton, NA, United Kingdom; Marsha Wills-Karp, PhD, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA; John Holloway, PhD, University of Southampton, Southampton, NA, United Kingdom; Qutayba Hamid, MD, PhD, Meakins-Christie Labs, McGill University, Montreal, QB, Canada; John Harley, MD, PhD, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA

RATIONALE: The results of a genome-wide association study implicated SNPs in neighboring genes ATPAF1 and KIAA0494 as associated with asthma. Here, we report the results of haplotype group analysis, a replication study, and preliminary functional studies, which support a new paradigm in asthma pathogenesis.

METHODS: The initial study was conducted on a subset of children (n=277) from the 1989-90 birth cohort of children from the Isle of Wight, UK. Cohort children were assessed for asthma (physician diagnosed at age 10) and atopy status (positive skin prick test). A genome-wide association study of pooled DNA showed strong association with the region containing the ATPAF1 and KIAA0494 genes. Fourteen tagging SNPs within and surrounding ATPAF1 and KIAA0494 genes were selected based on Hapmap CEU data and genotyped in individual children. A replication study was performed on asthmatic sib-pair families (341 families, n=1,481 total) from the Southampton, UK area. Both populations were predominantly Caucasian. Odds ratios and P values were calculated for individual SNPs and haplotypes. ATPAF1 and S9 mRNA expression was measured by the StepOnePlus PCR system in bronchial biopsies from normal (N=4), mild asthmatic (N=6) and severe asthmatic (N=9) Caucasian adults from Montreal, Canada. Sputum samples (N=25) from Isle of Wight children were analyzed using ATPlite assays.

RESULTS: Eleven-SNP haplotype groups across ATPAF1 and KIAA0494 genes were constructed and analysis revealed three haplotype groups with a frequency of ≥10%. In the Isle of Wight population, 10 SNPs (P<0.05) and two haplotype groups (P<0.05) were significant for asthma with one haplotype (haplotype I) with an OR of 2.2 when compared to the referent. The haplotype results were replicated in the Southampton population (with haplotype I conferring risk), however individuals SNPs were not significant for asthma in families. Analysis of sputum collected from a subset (N=25) of Isle of Wight cohort children under resting condition indicated a trend toward higher mean ATP levels in children with haplotype I . Preliminary functional studies show that ATPAF1 to have a 50 fold increase in expression in biopsied bronchial tissue from asthmatics when compared to control.

CONCLUSIONS: The results from two different populations (a case-control and a family based study) support haplotype groups across the ATPAF1 and KIAA0494 region being associated with asthma. This study provides new support for the involvement of ATP in the pathogenesis of asthma. Future studies will focus on the relative importance of these two adjacent genes.

200

Treatment of Pancreatic Cancer in Mice with a Monoclonal Antibody Against Anaplastic Lymphoma Kinase

Joseph LaConti, BS, Georgetown University School of Medicine, Washington, DC, USA; Sungeun Kim, MD, Lombardi Cancer Center, Georgetown University, Washington, DC, USA; Anton Wellstein, MD PhD, Lombardi Cancer Center, Georgetown University, washington, DC, USA

Anaplastic lymphoma kinase (ALK) is a transmembrane receptor that has an increased expression in various tumors and areas of active angiogenesis. Our laboratory determined that pleiotrophin (PTN), a secreted heparin binding protein, is a ligand of ALK. We have developed an antagonistic mouse monoclonal IgG antibody to the ALK receptor (anti-ALK IgG). In vitro, this antibody can recognize the ligand binding domain of ALK in ELISA and prevent the attachment of ALK dependent MDA MB 231 cells on plastic. To test the antibodys effect during angiogenesis, mice were implanted subcutaneously with FGF imbedded matrigel plugs. This stimulates angiogenesis, and we observed an increase in ALK mRNA expression in newly formed vessels invading the plug as opposed to pre-existing vessels surrounding the plug. Mice treated with anti-ALK IgG show a decrease in endothelial cell invasion, a surrogate marker of active angiogenesis. Our laboratory utilizes a unique transgenic mouse model which can recapitulate the progression of pancreatic cancer. This strain has been developed with a floxed stop sequence upstream of a mutant KRAS sequence containing a G to A transition in codon twelve, resulting in a glycine to aspartic acid substitution (KRAS G12D). These mice were bred to a strain with cre recombinase downstream of a pancreatic acinar promoter, p48. Double transgenic mice have the stop sequence before KRAS G12D removed, and the mutant allele produces a product which results in the full progression of pancreatic cancer in the mouse, beginning with typical pancreatic intraepithelial neoplastic (PanIN) lesions and progressing to pancreatic adenocarcinoma. In agreement with data from other groups using these mice, PanIN 1 develops by four months, PanIN 3 by 10 months, and adenocarcinoma by 15 months of age. We have initiated a study aimed at identifying the effect of anti-ALK IgG treatment on the progression of PanIN lesions to adenocarcinoma. Mice were treated with the antibody inta-peritonealy for six weeks. Treatment was begun at four months and 15 months in two different cohorts to determine the antibodys ability to abrogate the progression of the pancreas to PanIN 1 or PanIN 3 and adenocarcinoma, respectively. Most notably, anti-ALK IgG treated mice developed less adenocarcinoma, metastasis, and PanIN 3 lesions in the 15 month cohort. Our future experiments involve examining the various signaling and biomarker changes at the tissue level.