127

Tyrosine kinase inhibition in diffuse large B-cell lymphoma: Molecular basis for anti-tumor activity and drug resistance of dasatinib

128

Renal Subpopulations in Human Amniotic Fluid: A Novel Source of Structure Specific Progenitor Cells For Tissue Regeneration

129

Polyphenolic-Rich Fragaria Spp Modulates Inflammatory Markers And Insulin Response To High Fat Meal In Overweight Men And Women: Another Role For Phytonutrients In Medical Nutrition Therapy?

Collin Ellis, BS, UC Davis, Davis, CA, USA; Indika Edirisinghe, PhD, UC Davis, Davis, CA, USA; Amanda Linares, MS, UC Davis, Davis, CA, USA; Tissa Kappagoda, MD, PhD, UC Davis, Sacramento, CA, USA; Britt Burton-Freeman, PhD, Illinois Institute of Technology, Moffet Campus and UC Davis, Summit-Argo, IL, USA

BACKGROUND. Overweight patients are at risk for developing the metabolic syndrome, which is associated with elevated inflammatory markers and insulin resistance. Strawberries (Fragaria spp) have a varied polyphenolic composition with potential for modulating inflammatory status and insulin signaling. A high fat meal elevates inflammatory markers and promotes insulin resistance. It has also been shown that the beneficial effects of polyphenolic compounds on insulin signaling are associated with activation of the redox-sensitive phosphatidylinositol-3-kinase (PI3K)/protein kinase-B (Akt) pathway. HYPOTHESES. (i) Strawberry would reduce the postprandial inflammatory and insulin response to a high fat meal (HFM). (ii) An extract of strawberry activates the PI3K/Akt signaling pathway in human umbilical vein endothelial cells (HUVEC). METHODS. Hypothesis (i) was tested through a randomized, double-blind, placebo-controlled, 12-wk cross-over trial in overweight men and women (n=24). After a 10 day run-in, subjects consumed either a strawberry (Str) beverage (containing 10g freeze-dried fruit) or placebo (Pbo) beverage matched in energy and macronutrient composition for 6 wk. At defined intervals for 6 h, high sensitivity C-reactive protein (hsCRP), adipo-cytokines, insulin, and glucose were measured before and after HFM challenge with Str or Pbo during run-in (i.e. low Str intake) and again after 6 wks of daily Str or Pbo consumption. Hypothesis (ii) was tested on HUVEC by examining phosphorylation of Ser 473 in the absence and presence of the specific PI3-kinase inhibitor LY 294002 using immunoblotting. Strawberry was also analyzed for phytochemical/phytonutrient quantity and quality. RESULTS. In human subjects a single Str exposure significantly reduced hsCRP (Str, 2.8 mg/L +/- 0.1 and Pbo, 3.3 mg/L +/- 0.1, p<0.05) and insulin (p=0.002). After 6 wks Str or Pbo and HFM re-challenge, hsCRP was marginally lower after Str (Str, 2.9 mg/L +/- 0.2 and Pbo, 3.3 mg/L +/- 0.2, p=0.13) and insulin concentrations tended to be lower. Glucose concentrations did not differ by treatment. In HUVEC, the specific PI3-kinase inhibitor LY 294002 significantly attenuated the strawberry extract-induced phosphorylation of Akt (p<0.05). DISCUSSION/CONCLUSION. These data show that strawberry reduced the concentration of both hsCRP and insulin after a high fat meal in overweight subjects suggesting that it attenuated both the inflammatory response and insulin resistance. The finding that strawberry extract activated the PI3K/Akt pathway indicates that patients with the metabolic syndrome could benefit from consumption of strawberry. Our results support the importance of consuming strawberry and other foods rich in bioactive phytonutrients to promote health and reduce chronic disease risk. Funding and freeze-dried Str powder provided by the CA Strawberry Commission.

130

Structure and Function Studies of Xanthine Oxidoreductase

James Pauff, BS, The Ohio State University, Riverside, CA, USA; Charles Bell, PhD, The Ohio State University, Columbus, OH, USA; Russ Hille, PhD, University of California, Riverside, Riverside, CA, USA

Xanthine oxidoreductase (XOR) is a 290 kDa molybdenum-containing protein that catalyzes the final two steps of purine catabolism.  The enzyme exists as a homodimer in one of two forms; either as a dehydrogenase that reduces NAD+ to form NADH or as an oxidase that reduces O2 to form superoxide.   Both forms of the enzyme operate via the same redox mechanism while catalyzing the conversion of hypoxanthine to xanthine and then xanthine to uric acid at the two molybdenum-containing active sites, and it is only the preferred terminal electron acceptor that truly distinguishes XDH from XO.  Under physiological conditions, XOR exists predominately as the dehydrogenase XDH, but in certain pathological states, most notably ischemia/hypoxia, XDH is converted by oxidation and proteolysis to XO, which has the ability to exclusively produce reactive oxygen species such as superoxide and hydrogen peroxide.  It is the oxidase form of the enzyme that has been studied in human pathology for over 50 years.  Our work focuses on the structure and function of XOR, and in addition to studying the activity of both XDH and XO we have sought to elicit any differences between the two forms of the enzyme.  We have also investigated the in vitro inhibition of XOR, most often clinically accomplished by allopurinol.  Our results highlight the interface of in vitro biochemistry with in vivo studies and applications of biochemical research.

131

Racial Differences Between Depression and Post-Myocardial Infarction Outcomes

Susmita Parashar, MD, Emory University, Atlanta, GA, USA; Kimberly Reid, MS, Mid America Heart Institute , Kansas City, MO, USA; John Spertus, MD, PhD, Mid America Heart Institute , Kansas City, MO, USA; Viola Vaccarino, MD, PhD, Emory University, Atlanta, GA, USA; John Rumsfeld, MD, PhD, University of Colorado Health Sciences Center, Denver, CO, USA

Background: Depression affects approximately 20% of patients during myocardial infarction (MI) hospitalization. Depressive symptoms, even in the absence of a formal diagnosis of major depression, predict patient health status, costs of care, morbidity and mortality post-MI. To date, however, little is known about the prevalence of depression and the association between depression and outcomes among racial minorities. Methods: In a prospective multi-center registry of acute MI (PREMIER), depressive symptoms were measured in 2,226 patients with the Patient Health Questionnaire (PHQ) during hospitalization. Depression was defined as a PHQ score ≥10 (at least moderate depressive symptoms). Outcomes included: 3-yr mortality, 1-yr rehospitalization, and 1-yr angina using the Seattle Angina Questionnaire.We compared the prevalence of depression and the relationship between depression and outcomes after MI between African Americans and Caucasians, adjusting for medical history, disease severity, and quality of care. Results: Depression was more prevalent among African Americans compared with Caucasians (30% vs 20%, P<0.001). In unadjusted analysis, depressed African Americans were more likely to have 3-yr mortality rates (for depressed African Americans , non-depressed African Americans, depressed Caucasians and non-depressed Caucasians: 36, 22, 16 and 11 percent, respectively; P

132

Contribution of Helix VII Microdomain to the Sweet Receptor Function.

Elvera Baron, BS, Mount Sinai School of Medicine, New York, NY, USA; Yoel Rodriguez, PhD, Mount Sinai School of Medicine, New York, NY, USA; Meng Cui, PhD, Mount Sinai School of Medicine, New York, NY, USA; Marinna Max, PhD, Mount Sinai School of Medicine, New York, NY, USA; Roman Osman, PhD, Mount Sinai School of Medicine, New York, NY, USA

The main goal of our laboratory is to understand the molecular events underlying Class C G-protein coupled sweet receptor (T1R2+T1R3) function.  Sweet receptor responses to sugars and sweeteners require both T1R2 and T1R3, yet each monomer likely serves different functions.  We are particularly interested in how the transmembrane domains (TMDs) of each T1R monomer contribute to binding, activation, and signal transduction.  Specifically, human T1R3 (hT1R3) exhibits sequence-conserved microdomains shown to be functionally important in rhodopsin-like Class A receptors.  The focus of these molecular and computational studies is on the contribution of one such motif, xPR(K)xY in Helix VII, to the sweet receptor function.  Based on rhodopsin TMD structure, we created hT1R3 comparative homology model.  Interaction energy analysis was conducted, aiding in choosing candidate residues for site-directed mutagenesis studies.  Briefly, we calculated a pair-wise residue interaction energy matrix by averaging the corresponding non-bonded energy components over a MD trajectory starting from the initial homology structure.  This matrix was then decomposed into eigenvalues and associated eigenvectors, allowing detection of functionally significant residues regulating receptor activity.  As a result, R813 of xPR(K)xY motif in hT1R3 was pinpointed as a ‘hot residue’.  Further local energy calculations identified L806, A807, and F809 backbone interactions with the side-chain of R813 as the strongest in this region.  The R813N mutant showed much weaker interactions with L806, A807, and F809 computationally than the wild-type R813.  In vitro R813N mutant correlated with its energy calculations, showing a significant decrease in receptor function in response to a panel of sweeteners.  Similar computational analysis for R813K mutant demonstrated an intermediate strength of interactions of this mutant compared to wild-type and R813N.  This predicted graded response is being characterized through ongoing functional studies.  These studies reveal that the R in the xPRxY motif of hT1R3 has a Pro-kink stabilizing function.  Since the positively charged residue is conserved in Class C members, we suggest that this mechanism may be shared by other Class C receptors.  Based on the integration between in vitro experiments and molecular modeling, these studies will enhance our understanding at the molecular level of sweet taste receptor function with the hope of future means to control our sweet cravings and aid in the fight against obesity and diabetes.

133

Effective Connectivity In Acute Cocaine Withdrawal

Sanjida Saklayen, BA, The Ohio State University, Columbia, MO, USA; Ananth Narayanan, BS, The Ohio State University, Columbus, OH, USA; Catherine White, MA, The Ohio State University, Columbus, OH, USA; Amir Abduljalil, PhD, The Ohio State University, Columbus, OH, USA; Petra Schmalbrock, PhD, The Ohio State University, Columbus, OH, USA; Thomas Pepper, MD, The Ohio State University, Columbus , OH, USA; Bradley Lander, MD, The Ohio State University, Columbus, OH, USA; David Beversdorf, MD, University of Missouri-Columbia, Columbia, MO, USA

Subjects withdrawing from cocaine have increased noradrenergic activity and are plagued by a high rate of relapse. Individuals acutely withdrawing from cocaine are impaired in tasks that require a high degree of cognitive flexibility, particularly tasks that require flexibility of access to the semantic network. Previous research suggests noradrenergic antagonists improve performance on semantic network flexibility tasks for subjects acutely withdrawing from cocaine. Recent data in autism (a population with impaired flexibility of semantic networks of a different cause) showed decreased effective connectivity in the task-relevant regions. Therefore, we wished to examine effective connectivity in acute cocaine withdrawal in language tasks. Individuals acutely withdrawing from cocaine and matched controls were scanned on a Philips 3T scanner while performing phonological and semantic tasks. Data were analyzed using FEAT, part of FSL, and effective connectivity was compared between groups by comparing the correlations of extracted time series between different regions of interest (ROI). Several ROI pairs showed a decrease in effective connectivity for cocaine subjects as compared to matched controls in several ROI pairs, as had been reported with autism. Future research should further explore the role of this decreased effective connectivity in acute cocaine withdrawal and the possible effects of pharmacological treatment.

134

DC-STAMP As A Potential Marker Of Erosive Versus Non-Erosive Arthritis

Kofi Mensah, BS, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA; Yahui Chiu, PhD, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA; Colleen Hock, MS, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA; Christopher Ritchlin, MD, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA; Edward Schwarz, PhD, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA

Introduction: Inflammatory arthropathies include a broad spectrum of joint disorders that range from non-erosive arthritis in patients with SLE to the extensive erosions observed in rheumatoid arthritis. Given that osteoclasts (OC) are the pivotal bone resorbing cells, understanding factors that control osteoclastogenesis and dictate the erosive phenotype of inflammatory arthritis is centrally important to diagnosis and treatment. To explain the observation of erosions in some inflammatory arthropathies and not in others, we proposed a myelopoiesis model of autoimmune disease in which an individual’s “erosive potential” is determined by systemic factors that stimulate differentiation of peripheral blood mononuclear cells (PBMC) towards or away from OC in a mutually exclusive fashion. Additionally, we propose that there is a surface marker on myeloid PBMC that faithfully predicts a cell’s osteoclastogenic ability and can serve as a biomarker of erosive potential. One candidate biomarker is DC-STAMP, which was first discovered in myeloid dendritic cells (mDC) but is also essential for the fusion of mononuclear OC precursors (OCP) to form mature multinucleated OC. Interestingly, DC-STAMP was found to colocalize with ER proteins in mDC, while plasma membrane DC-STAMP expression was described in OC. Based on this differential expression during myelopoiesis we hypothesize that DC-STAMP can serve as a biomarker to distinguish OC and mDC precursors.

Methods: The RAW 264.7 monocyte cell line and primary macrophages were used to evaluate the localization of DC-STAMP after culture with RANKL to generate OC in-vitro. Gene and protein expression for DC-STAMP were measured using qRT-PCR or flow cytometry/immunofluorescence microscopy respectively.

Results: We found that RANKL transiently increases dc-stamp mRNA levels while dc-stamp mRNA levels continually rise in mDC formation under IL-4 + GM-CSF treatment. DC-STAMP protein expression analysis demonstrated that RANKL stimulation generates two OCP populations, DC-STAMPhi and DC-STAMPlo. In contrast, little change in DC-STAMP surface expression was detected during mDC formation. Sorting the DC-STAMPhi and DC-STAMPlo populations demonstrated that cells retaining high surface DC-STAMP after RANKL stimulation are not capable of forming OC, while co-culture of these cells with OCP that have down-regulated surface DC-STAMP resulted in larger, more nucleated OC.

Conclusions: Based on the model of mutually exclusive OC vs. mDC differentiation in response to systemic factors, we postulated that DC-STAMP could be a biomarker to assess the potential of erosive vs. non-erosive arthritis based on its differential expression and unique function in each cell type. Our finding that fusogenic OCP down-regulate surface DC-STAMP suggests that this event defines cells that generate processes that attach and fuse with mononuclear cells.

135

Medical Doctors Of The People’s Republic Of China: The Profession, Professionalization, And Professionalism

Belinda Chow, MPhil, The Hong Kong Polytechnic University, Hong Kong, NA, Hong Kong; Carlos Lo, Professor, The Hong Kong Polytechnic University, Hong Kong, NA, Hong Kong; Edward Snape, Professor, The Hong Kong Polytechnic University, Hong Kong, NA, Hong Kong; Xu Huang, Associate Professor, The Hong Kong Polytechnic University, Hong Kong, NA, Hong Kong

Professionals play a critical role in accelerating the welfare of our society through their wealth of specialized knowledge while professionalization is the key indicator on their professional development through institutionalization. In the case of China, how far the medical profession has been professionalized in the Peoples Republic of China? To what extent the institutional constrains encumbered their process of professionalization? Under the rapid institutional change in the Peoples Republic of China in recent decades, state intervention is believed to play a critical role in the process of professionalization. Based on 30 in-depth interviews with doctors in three different age groups in Guangzhou, with constitutions, national statistics and newspaper cuttings to serve as analysis enhancement, the findings would contribute to our understandings of the role of institutional constrains on professionalization. In terms of theoretical contribution, the current research would add a new dimension in institutional theory in understanding human behaviors through exploration of contextual factors. Professionalization of Chinese doctors in the past three decades can be conceptualized into three distinct stages. After the resumption of medical postgraduate education in the early 1980s till the establishment of China Health Law Society in 1993 can be classified as institution-building stage by fulfilling standardization process under the dominance of Ministry of Health, which provided the think tank of legal foundation for later promulgation of Law on Practical Doctors of Peoples Republic of China at the turn of twentieth century. From 2002 to the present, ever since the establishment of the Chinese Medical Doctors Association, the process of doctors professionalization has entered the initial de-statization stage but still under strong supervision of Ministry of Health. In some respects, the professionalization of doctors in China has elements in common with the West. They have standardized medical education, state licensure and code of ethics. However, they have changed to be a unit-based employee of their working unit and serve the interest of the hospital they belong to but somehow still have close connection with the State and bear the status of a healthcare policy front-line executors in recent decades. Compare with the West, the Chinese doctors only get limited access to autonomy under the State corporatism. Their professional identity is vague as their professional status is not widely recognized. The situation has been further jeopardized under the fix price policy and introduction of the MSA which indirectly intensified the doctor-patient relationship and destructed the general morale of the Chinese doctors. Hence, the role of China government is still salient in fueling the professionalization of the medical profession especially in bridging and alleviating the relationship between the doctors and their patients.

136

Mechanisms of IL-17 Induced Gene Expression

Justin Hartupee, BA, Case Western, Cleveland, OH, USA; Thomas Hamilton, PhD, Cleveland Clinic, Cleveland, OH, USA

The T cell derived cytokine IL-17 plays an important role in driving acute inflammation by inducing cells resident within tissues to produce a variety of pro-inflammatory genes, including CXC chemokines, resulting in the influx of neutrophils.  While IL-17 is recognized to be a critical player driving inflammation in both host defense and autoimmunity, the molecular mechanisms by which it regulates target gene expression are poorly understood.  Expression of many pro-inflammatory genes, including those induced by IL-17, is regulated at multiple levels including modulation of transcription and post-transcriptional events such as mRNA stability. We have used the mouse CXC chemokine KC as a model to study IL-17 induced gene expression.  KC expression requires both NFκB driven transcription and stabilization of the constitutively unstable mRNA.  IL-1 and toll-like receptor ligands can induce both transcription and stabilization, while TNFα only drives transcription.  We have found that IL-17 alone exerts little effect on KC expression, but induces a cooperative response when used in combination with TNFα, a pattern of expression that has been observed for other IL-17 target genes.  IL-17 had little effect on KC transcription or activation of the transcription factor NFκB.  However, multiple experimental systems indicate that IL-17 serves as a strong stimulus to stabilize KC mRNA.   This pattern of regulation extended beyond KC to other IL-17 target genes.  Thus TNFα and IL-17 work cooperatively because they each provide one of the requirements for expression.  TNFα induces transcription while IL-17 prolongs the half-life of the mRNA.  IL-17 is the first stimulus we are aware of that acts predominately post-transcriptionally to regulate pro-inflammatory gene expression without also effecting transcription.  Although the signaling molecules Act1 and TRAF6 have both been linked to IL-17, we find that only Act1 is required for IL-17 induced mRNA stabilization.  Consistent with stabilization being the major mechanism by which IL-17 regulates expression, we find that IL-17 induced gene expression is lost in Act1 deficient cells but remains intact in TRAF6 knockouts. 

137

Methyl Binding Domain Protien 2 (MBD2) Mediates Epigenetic Silencing of Maspin in MCF7 Cells and Contributes to Elevated Tumor Grade in 3D Culture.

Omar Mian, MS, Virginia Commonwealth University, Richmond, VA, USA; Gordon Ginder, MD, Massey Cancer Center, Richmond, VA, USA

Tumor-suppressor associated CpG island hyper-methylation is a frequent epigenetic lesion in neoplastic disease. Methyl-CpG binding proteins (MCBPs), which recruit histone-modifying complexes and lead to transcriptional silencing, are therefore promising targets for anti-cancer therapy. To date, however, functional studies that support targeting MCBPs as a therapeutic strategy are limited. Our findings suggest MBD2 promotes abnormal growth of MCF7 cells in 3D culture. Stable shRNA knockdown of MBD2 in MCF7 cells results in increased contact inhibition as well as an increased proportion of differentiated epithelial structures (e.g. aciniar and ductal forms) when compared to transfection with scrambled shRNA controls. In these cells, knockdown of MBD2 also resulted in restoration of Maspin expression. Maspin is a multi-functional tumor suppressor known to be epigenetically silenced in breast cancer. In addition to MBD2 occupancy of the promoter CpG-rich region, we found MBD2 bound to downstream CpG rich sites. However, MBD2 occupancy at all sites we examined did not change following treatment with the DNMT inhibitor, 5-aza-2-dCytidine (5-aza). Moreover, the effects of MBD2 knockdown and 5-aza are more than additive with respect to Maspin expression, suggesting a role for MBD2 or recruited co-repressor proteins in opposing de-methylation in the presence of 5-aza. Our data are particularly interesting in light of reports showing 5-aza decreases repressive H3K9 di-methylation without changes in DNA methylation. In this context, MBD2 residency intimates a tumor-specific mechanism for recruiting the relevant histone methyl-transferase to this gene. We conclude that MBD2 contributes to the undifferentiated phenotype of breast cancer cells and that transcriptional repression of the Maspin gene occurs in part through an MBD2 dependent silencing mechanism.

138

Transforming Growth Factor-Beta Induces Fibroblast Proliferation Via Activation Of Mtor Signaling

Rod Rahimi, BA, Mayo Clinic College of Medicine, Rochester, MN, USA; Mahefatiana Andrianifahanana, PhD, Mayo Clinic College of Medicine, Rochester, MN, USA; Edward Leof, PhD, Mayo Clinic College of Medicine, Rochester, MN, USA

Transforming growth factor-beta (TGF-ß) regulates a wide variety of biological processes by signaling through receptor serine/threonine kinases to activate the transcription factor Smad proteins. Interestingly, TGF-ß is capable of inducing variable cellular responses dependent on cell type despite activing the same Smad proteins. These cell type specific responses include the ability of TGF-ß to promote growth arrest of epithelial cells while causing fibroblasts to proliferate despite activating Smad2 and Smad3 in both cell types. Recent evidence suggests that TGF-ß non-Smad effectors are critical in mediating these cell type specific responses. Here we investigate the role of mammalian target of rapamycin (mTOR), a known regulator of cell growth, in the fibroblast response to TGF-ß. We show that TGF-ß activates mTORC1 in fibroblasts but not epithelial cells via a PI3K-Akt-TSC2 dependent pathway. Inhibition of mTORC1 with rapamycin inhibits TGF-ß mediated anchorage-independent growth of fibroblasts without affecting Smad or non-Smad transcriptional responses. Given the recent evidence that rapamycin can inhibit both mTORC1 and mTORC2, we investigate the role of mTORC2 in TGF-ß signaling. Consistent with its role in receptor tyrosine kinase signaling, mTORC2 is required for TGF-ß induced Akt Ser473 phosphorylation but not mTORC1 signaling. Interestingly, both mTORC1 and mTORC2 are required for TGF-ß mediated anchorage-independent growth of fibroblasts. While mTORC1 is not required for either Smad or non-Smad transcriptional responses, mTORC2 is required for efficient transcription of extracellular matrix components fibronectin and Type I collagen. These results suggest that inhibitors of mTOR signaling may be useful in treating TGF-ß mediated fibroblast pathologies in vivo.

HSP27 and kidney tubulointerstitial fibrosis

Arjang Djamali, MD, UW Madison, Madison, WI, USA; Aparna Vidyasagar, BS, UW Madison, Madison, WI, USA; Shannon Reese, BS, UW Madison, Madison, WI, USA

140

Two Distinct Cell Populations are Capable of Repopulating Mammary Gland

Stina Mui, MD/PhD, University of California, San Diego, La Jolla, CA, USA; Andreas Beyer, PhD, Technical University Dresden, Dresden, NA, Germany; Trey Ideker, PhD, University of California, San Diego, San Diego, CA, USA

Characterization of cancer stem cells in the breast has been challenging largely due to our inability to identify and characterize normal mammary stem cells. Although markers such as CD24 (heat-stable antigen), CD29 (beta1-integrin), and CD49f (alpha6-integrin) have been used to isolate stem-cell enriched populations in the normal mouse mammary gland, the exact identity of the mammary stem cell remains elusive. Our goal is to examine global gene expression in stem and progenitor cell enriched mammary populations and to identify potential functional markers and regulatory pathways necessary to maintain stem and progenitor cells in the mouse mammary gland. We used the existing known markers, CD24, CD29 and CD49f on lineage negative mouse mammary cells to select for a stem-cell enriched population that is devoid of hematopoietic lineage cells (Lin-) and is either CD24+CD49f++ or CD24+CD29++. To determine if these represent the same underlying population of cells, we adopted all the positive selection markers (CD24, CD29, CD49f) for fluorescence activated cell sorting (FACS). Surprisingly, we find two distinct populations using Lin-CD24+CD49f++ and Lin- CD24+CD29++. We demonstrate in vivo that these two populations not only have enhanced ability to repopulate cleared mammary fat pads compared to lineage-depleted cells but they also have similar repopulating frequencies when transplanted into cleared mammary fat pads. To find regulatory networks and candidate functional markers distinguishing these two populations, we use expression microarrays for these two stem- and progenitor- enriched populations. We find 684 genes that are significantly differentially expressed, with multiple genes highlighting pathways that are implicated in mammary stem cell function or carcinogenesis, including CXCR4, CXCL12, Wnt4, Wisp2, Wif1, Dkk, Notch1, Notch 3, Jag1, Dll1, PDGFR-alpha, and kit. We subsequently validated the expression of all of these genes by real-time PCR. Furthermore, by using existing protein-protein interaction networks and pathway analysis, we find that Wnt and Notch signaling genes may be important for maintaining the identities of these two stem- and progenitor- cell enriched populations in the mammary gland. In addition, immunocytochemistry studies of kit, CXCR4, and Jag1 suggest that they may be novel functional cell-surface markers for stem and progenitor enriched populations in the mammary gland. Characterization of these markers in both cancer and normal mammary gland may shed light on mechanisms of carcinogenesis in the breast.

141

Controlled Experimental Study Depicting Moving Objects in View-Shared Time-Resolved 3D MRA

Petrice Mostardi, BS, Mayo Clinic, Rochester, MN, USA; Clifton Haider, BS, Mayo Clinic, Rochester, MN, USA; Phillip Rossman, BS, Mayo Clinic, Rochester, MN, USA; Stephen Riederer, PhD, Mayo Clinic, Rochester , MN, USA

Introduction: A variety of methods have been used for time-resolved contrast enhanced MR Angiography (CE-MRA), many involving view sharing. When central k-space is updated more frequently than non-central k-space, the net image frame rate is higher than the intrinsic image acquisition time. However, it is not always clear to what extent the resultant image time series truly represents the dynamic behavior of the object of interest. Although simulations can be used to estimate various aspects of performance, an experimental study can allow more realistic characterization. The purpose of this work was to develop and use a computer-controlled motion phantom for study of the fidelity with which 3D time-resolved sequences can portray dynamic phenomena. This allowed determination not only of such properties as ghosting, blurring, and undesired vascular enhancement, but also the fidelity of portraying a rapid influx of contrast into a vessel as well as the smooth passage of contrast across an extended field of view. Methods: Studies were performed using diluted gadolinium-filled vials that were moved along tabletop tracks through the bore of the MRI scanner by a computer-controlled motor. Three pulse sequences (S1, S2, S3), which vary in their degree of centricity about the k-space origin (S1>S2>S3), were implemented. Each sequence was used to acquire continuous images as the vials were moved through one field of view. To test the accuracy in portrayal of continuous linear motion, the image sequences were analyzed to find the position of bolus leading edge at each frame and the sharpness of the leading edge. To explore the effects of increased temporal resolution with parallel imaging we compared S1 without SENSE and with 2D SENSE. Results: In leading edge analysis, S1 and S2 accurately portrayed linear motion, while S3 did not; indicating that linear depiction of motion is dependent on the consistency of the sequence in sampling the center of k-space for all timeframes. S1 showed the least amount of leading edge artifact due to its inherent centricity. The sharpness of the bolus leading edge declined from S1 to S3, corresponding to the longer time period over which high frequency data are acquired as the bolus continues to advance. There was decreased blurring and a sharper leading edge when SENSE was applied due to reduction in image update time and footprint while maintaining k-space coverage. Conclusions: A computer-controlled phantom can be used to carefully characterize the experimental performance of pulse sequences used for time-resolved 3D CE-MRA. Consistency of view ordering within each reconstruction frame, centricity in both kY and kZ, and 2D SENSE acceleration provide improved performance. These studies helped to identify desirable characteristics of a pulse sequence used for time-resolved studies to see contrast in vessels with high fidelity and low artifact in order to more accurately diagnose disease of the vasculature.

142

Determinants of Sensitivity to Halothane-Induced Hepatotoxicity in Mice

Christine Dugan, MS, Physician Scientist Program at Michigan State University, East Lansing, MI, USA; Robert Roth, PhD, DABT, Pharmacology and Toxicology Dept, East Lansing, MI, USA; Patricia Ganey, PhD, Pharmacology and Toxicology Dept, East Lansing, MI, USA

    The once widely used anesthetic halothane (2-bromo-2-chloro-1, 1, 1-trifluoro-ethane) induces a mild hepatitis in 20% of patients and idiosyncratic fulminate liver failure in approximately 1 in 30,000.   The mechanism of the severe form of halothane hepatic injury is not entirely understood.  We examined several factors for their ability to influence sensitivity of mice to halothane-induced liver injury.  To study the influence of gender, male and female Balb/C mice were treated with halothane (15- 45 mmol/kg), and liver injury was assessed 24 hrs later by the activity of serum alanine aminotransferase (ALT) and liver histopathology.  In both males and females, the injury was dose-dependent.  The female maximal ALT response (10,000 U/L) was greater than that in males and corresponds to severe hepatocellular injury.   Female mice also developed liver injury at smaller halothane doses than males.  Livers from both male and female Balb/C mice had centrilobular necrosis and accumulation of neutrophils consistent with the appearance of postmortem histological changes in patients who died from halothane-induced liver failure.  Fasting female Balb/C mice for 15 hr increased the sensitivity to halothane-induced hepatic injury.  To test the possibility that a concurrent inflammatory episode could sensitize a mouse to halothane-induce hepatocellular injury, male Balb/C mice were given a mildly hepatotoxic dose of the halothane (15mmol/kg) followed by a nonhepatotoxic dose of bacterial lipopolysaccharide (LPS; 5x106 EU/kg), and serum ALT activity was assessed 24hrs later.   The cotreated animals had a 10-fold increase in serum ALT activity relative to mice treated with either agent alone.  These results suggest that multiple intersecting determinants of sensitivity including gender, fasting, and inflammatory stress can result in severe liver injury from halothane exposure.  (Supported by NIH grant GM075865.)

143

All Four Interferon Antagonist Proteins Of Nipah Virus Are Expressed In Infected Cells

Sachin Kulkarni, MS, Mount Sinai School of Medicine, New York, NY, USA; Valentina Volchkova, PhD, University Claude Bernard Lyon-1, Lyon, NA, France; Viktor Volchkov, PhD, University Claude Bernard Lyon-1, Lyon, NA, France; Peter Palese, PhD, Mount Sinai School of Medicine, New York, NY, USA; Megan Shaw, PhD, Mount Sinai School of Medicine, New York, NY, USA

Nipah virus (NiV) is a paramyxovirus which is highly pathogenic in several species, including humans.  The P, W, V, and C proteins of NiV have all been shown to antagonize the innate immune response.  The P, V and W proteins inhibit interferon (IFN) signaling and the V and W proteins also hinder IFN synthesis.  The P gene of NiV is predicted to encode all four proteins. The C protein is derived from an alternate open-reading frame in the P gene, while the V and W proteins are encoded by edited P mRNA transcripts that result from the insertion of 1 or 2 G residues, respectively at an editing region in the P gene.  The ratio of P:V:W transcripts that are produced in an infected cell as well as the levels of protein expressed from these transcripts have not been previously shown. Using antibodies raised against the P, V, W and C proteins, we evaluated the expression of these proteins over time in extracts from NiV-infected cells.   We confirm that all four proteins are in fact expressed in infected cells.  Furthermore, our data suggest that the C protein may be expressed late in infection.  RT-PCR was performed using primers designed to amplify a 340 base-pair fragment spanning the editing region of the mRNA purified from the infected cells, and the resulting product was cloned and sequenced.  Initially the majority of transcripts code for the P protein but as the infection progresses there is a marked increase in the number of V and W transcripts.  At times, the percentage of edited transcripts equals or exceeds the percentage of unedited transcripts.  In addition, we find that the number of G residue insertions is not limited to 1 or 2, but can range from 1 to 10.  Having determined the relative transcript numbers and expression levels of the NiV interferon antagonist proteins in the context of an infected cell, we may be able to better appreciate the contribution of each of these proteins to the overall pathogenicity of the virus.  

144

Beta-cell ABCA1 Influences Insulin Secretion, Glucose Homeostasis and Response to Thiazolidinedione Treatment

Liam Brunham, BSc, University of British Columbia, Vancouver, BC, Canada; Janine Kruit, PhD, University of British Columbia, Vancouver, BC, Canada; Bruce Verchere, PhD, University of British Columbia, Vancouver, BC, Canada; Michael Hayden, MD PhD, University of British Columbia, Vancouver, BC, Canada

Type 2 diabetes is a significant risk factor for cardiovascular disease and occurs when beta-cells are unable to secrete sufficient insulin to meet the increasing metabolic requirements associated with insulin resistance and obesity. The reasons for beta-cells failure in type 2 diabetes are incompletely understood, but may involve the build-up of toxic lipids in islets. We examined the role of ABCA1, a cellular cholesterol transporter, in cholesterol homeostasis and insulin secretion in beta-cells. Mice with specific inactivation of ABCA1 in beta-cells have markedly impaired glucose tolerance but normal insulin sensitivity. Deletion of beta-cell ABCA1 results in impaired insulin secretion in vivo, and islets isolated from these mice show altered cholesterol homeostasis and impaired insulin secretion in vitro. We show that rosiglitazone, an activator of the peroxisome proliferator-activated receptor delta, which upregulates ABCA1 in beta-cells, requires beta-cell ABCA1 for its beneficial effects on glucose tolerance. These studies establish a novel role for ABCA1 in beta-cell cholesterol homeostasis and insulin secretion, and suggest that cholesterol accumulation may be a pathogenic mechanism involved in beta cell dysfunction in type 2 diabetes.

LRB is supported by a Canadian Institutes for Health Research MD/PhD Studentship.

145

Identification Of The HIV-1 Rev Binding (Hrb) Protein As An Important Downstream Target Of Oncogenic Notch Signaling

Shariq Khwaja, BS, Mayo Clinic College of Medicine, Rochester, MN, USA; Jan van Deursen, PhD, Mayo Clinic College of Medicine, Rochester, MN, USA; Richard Bram, MD, PhD, Mayo Clinic College of Medicine, Rochester, MN, USA

A pathophysiologic increase in Notch signaling has been implicated as a causal or contributing factor in the development of T-cell leukemias/lymphomas. The first demonstration of Notch’s role was the identification of a chromosomal translocation [t(7;9)] found in a subset of these human cancers. Further studies revealed Notch1 activating mutations in more than 50% of human T-ALL (T-cell acute lymphoblastic leukemias) samples. Notch is activated as a result of three proteolytic cleavage events, culminating in the release of the intracellular domain (ICN) of the Notch receptor. ICN translocates to the nucleus where it binds to the CSL family of transcription factors, converting a repressive transcriptional program into an activated one, ultimately resulting in the transcriptional upregulation of downstream Notch target genes.

Several studies reveal that overactive Notch dramatically alters the cell’s transcriptional program. Despite these observations, signaling pathways downstream of Notch are poorly defined. There are published reports showing that the HIV-1 Rev Binding (Hrb) protein is one of several proteins regulated by Notch. We are interested in determining whether Hrb has any role in the Notch signaling pathway. To date, studies of Hrb have focused on its role in nucleocytoplasmic transport and vesicle fusion/docking events during spermatogenesis. Hrb has been implicated as an essential cofactor for the nucleocytoplasmic export of HIV spliced and unspliced RNAs. Hrb seems to participate in a conserved nuclear export pathway involving Ran G-proteins and the major cellular exportin, Crm1. In addition to nucleocytoplasmic transport, Hrb has shown to function in the fusion of post-Golgi derived vesicles to form the membranous organelle called the acrosome during spermatogenesis. Here, we provide evidence that the HIV-1 Rev Binding (Hrb) protein is a downstream target of Notch signaling in thymocytes.

Our research in focused on understanding the role of Hrb in both physiologic and pathologic Notch signaling. We have examined thymic development, a Notch-dependent process, from Hrb knockout mice and found a normal developmental program. We have also utilized an in vitro thymocyte development system to examine the effect of transducing an activated form of Notch (ICN-X) into both Hrb knockout and wildtype thymocytes. Hrb wildtype thymocytes transduced with ICN-X have a dramatic upregulation of Hrb at the protein level compared to controls. Moreover, we are in the process of determining the role of Hrb in the development of leukemias/lymphomas induced by overactive Notch in mice. To this end, we have evidence that loss of Hrb reduces the incidence of fatal Notch-mediated T-cell lymphomas caused by conditional deletion of the CREB Binding Protein (CBP). Thus, we propose that pathological Notch signaling may require Hrb expression to exert its cellular effects.

147

Cyclic Transition States in the Catalysis of Proton Transfer in Glutamate Racemase: Implications for Antibacterial Drug Design.

Dylan Dodd, M.S., Microbiology, University of Illinois, Urbana, IL, USA; Jerome Baudry, PhD, School of Chemical Sciences, Urbana, IL, USA; Katherine Pankow, BS, Biochemistry, Urbana, IL, USA; Joseph Reese, BS, Biochemistry, Urbana, IL, USA; Steven Blanke, PhD, Microbiology, Urbana, IL, USA; M. Ashley Spies, PhD, Biochemistry, Urbana, IL, USA

Glutamate racemases (EC 5.1.1.3) catalyze the co-factor independent stereoinversion of D- and L-glutamate, and are important for viability in Gram-negative and -positive bacteria. As the sole enzyme capable of converting L- to D-glutamate for peptidoglycan biosynthesis, glutamate racemase is an attractive target for the design of antibacterial agents and has received a great deal of attention in this regard. However, the development of tight binding reversible inhibitors has been so far largely unsuccessful. Detailed knowledge of the mechanism employed by glutamate racemases during catalysis may provide significant insight into the design of effective inhibitors. Despite the recent release of a number of crystal structures of cofactor independent epimerases and racemases, the source of these enzymes' catalytic power and their ability to acidify the C-alpha of amino acids remains unknown. Here we report results from a comprehensive computational and experimental study of the reaction catalyzed by the glutamate racemase from B. subtilis. Our results suggest that the reactive form of glutamate proton abstraction from C-alpha is "cyclic", forming an intramolecular hydrogen bond between the gamma-carboxylate oxygen and the ammonium nitrogen. Analysis of the transition states for Ca proton abstraction by QM/MM, in both the D→L and L→D directions, revealed a migration of the His187 imidazolium ring towards the gamma-carboxylate oxygen of glutamate, resulting in enhanced electrostatic interactions. Furthermore, the large number of hydrogen bond donors to the alpha-carboxylate in the active site are sufficient to achieve C-alpha proton abstraction in both the L→D and D→L directions, without the need for alpha-carboxylate protonation. Results from experimental inhibition studies of B. subtilis glutamate racemase with cyclic, carboxylate-containing compounds combined with in silico docking studies provide insight into a new class of small molecule inhibitors targeting this important enzyme.

148

Inhaled Carbon Monoxide as a Potential Modulator of Inflammation and Oxidative Stress in Sickle Cell Disease

Joan Beckman, B.S., University of Minnesota, Minneapolis , MN, USA; Julie Vineyard, B.S., University of Minnesota, Minneapolis, MN, USA; John Belcher, Ph.D., University of Minnesota, Minneapolis, MN, USA; Gregory Vercellotti, M.D., University of Minnesota, Minneapolis, MN, USA

Sickle cell disease (SCD) is a hemolytic condition in which inflammation plays a prominent role. The heme oxygenase-1 (HO-1) enzyme plays a critical role in metabolizing the excess heme generated during hemolysis, and modulates vaso-occlusion in murine models of SCD. The products of HO-1 activity, carbon monoxide (CO), Fe2+, and biliverdin/bilirubin have demonstrable anti-oxidant and anti-inflammatory effects. We have observed that brief inhalation of CO in SCD mice modulates vaso-occlusion. Therefore, we hypothesize that prolonged treatment with inhaled CO will significantly decrease the inflammatory phenotype of SCD mice, C57BL-6J, heterozygous BERK (mβ/hßs), and the more severe sickle phenotype, S+S Antilles mice, were treated with inhaled CO at 0, 25, or 250 ppm (1 h/d x 3 d/wk x 10 wks). Upon completion of the ten week treatment period animals were euthanized, blood was removed by cardiac puncture, and organs were harvested for analysis. Untreated S+S Antilles and heterozygous BERK mice had a higher white blood cell count (WBC) than C57BL-6J mice (p<0.005). Treatment for 10 weeks with either 25 or 250 ppm CO significantly decreased total WBC in both SCD models (p<0.05), but had no effect on C57BL-6J WBC. The decrement in total WBC count was primarily due to a significant decrease in neutrophils (p<0.05) and leukocytes (p<0.001). 25 ppm inhaled CO significantly (p<0.05) increased the hematocrit S+SAntilles mice, whereas it significantly reduced (p<0.05) reticulocyte count. There was no significant difference in either the mean hematocrits or reticulocyte counts in the other CO-treated mice. In untreated heterozygous BERK mice, redox-active iron was two-fold greater than C57BL-6J mice (p<0.05) and treatment with 25 or 250 ppm CO significantly (p<0.01, p<0.02 respectively) decreased the redox-active iron Fe 2+ to levels comparable to untreated C57BL-6J. In addition, the heme-content of untreated heterozygous BERK mice was three-fold (p<0.05) higher than untreated C57BL-6J. Treatment with 25 or 250 ppm CO significantly (p<0.05) decreased the heme-content of the livers to levels comparable to untreated C57BL-6J.The cytoprotective proteins HO-1 and ferritin heavy-chain were significantly increased in the livers of untreated heterozygous BERK mice compared to C57BL-6J (p<0.02). However, CO treatments had no significant effects on expression of these proteins. In summary, we conclude that inhaled CO treatments decrease total WBC as well as liver redox-active iron and heme content in SCD mice. In addition, in the more severe sickle model, CO has a beneficial effect on both hematocrit and reticulocyte count. We speculate that inhaled CO treatments may be a potential therapy for patients with SCD by acting as a modulator of oxidative stress and inflammation.

149

Calcium Dependent Nox 5 NADPH Oxidase Contributes To Vascular Oxidative Stress In Human Coronary Artery Disease.

M Gongora, MD, Emory University, Atlanta, GA, USA; Tomasz Guzik, MD, Emory University, Atlanta, GA, USA; Wei Chen, Md PhD, Emory University, Atlanta, GA, USA; Tomasz Guzik, MD, Emory University, Atlanta, GA, USA; Wei Chen, PhD, Emory University, Atlanta, GA, USA; Henrich Lob, PhD, Emory University, Atlanta, GA, USA; Nyssa Hoch, PhD, Emory University, Atlanta, GA, USA; Deepa Mangalat, MD, Emory University, Atlanta, GA, USA; Sergey Dikalov, PhD, Emory University, Atlanta, GA, USA; David Harrison, MD, Emory University, Atlanta, GA, USA

The Nox based NADPH oxidases are major sources of ROS in human vasculature. Several Nox homologs have been identified but their relative contribution to vascular ROS production in coronary artery disease (CAD) is unclear. Nox5, a calcium dependent homolog, has been previously identified in immature lymphatic tissues, cancer cells and in endothelial cells but has not yet been studied in human vasculature. Moreover, the role of calcium-dependent superoxide production in human coronary artery disease remains unknown. We hypothesized that Nox5 is present in human coronary arteries and that its expression and activity is increased by CAD. Western blotting using a novel anti-Nox5 antibody yielded a single 65 kDa band, consistent with known Nox5 molecular weight, in both human aortic endothelial cells (HAEC) and human umbilical vein endothelial cells (HUVEC). Small interfering RNA against Nox5 reduced this band and inhibited Ca++-dependent NADPH oxidase activity as measured by electron paramagnetic spin resonance. Studies of human coronary arteries indicated calcium-dependent NADPH oxidase activity , Nox 5 mRNA of and Nox5 protein are dramatically increased in human coronary arteries obtained from patients with CAD. This increase is observed predominantly in the endothelium at early stages of atherosclerotic lesions and in the intima of the atherosclerotic coronary arteries. Moreover levels of Nox5 mRNA were significantly correlated to the Ca++ dependent NADPH oxidase activity measured from artery homogenates. These studies identify Nox5 as a novel, calcium dependent source of reactive oxygen species atherosclerosis. It also raises the possibility that calcium channel antagonists could reduce ROS production in CAD.