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The Markey Scholars Conference: Proceedings (2004)

Chapter: 11 Abstracts of Poster Sessions

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Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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Abstracts of Poster Sessions

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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CONFIGURATIONAL ENTROPY, BIOCHEMICAL COOPERATIVITY, AND SIGNAL TRANSDUCTION

Paul H. Axelsen, M.D.

Department of Pharmacology University of Pennsylvania

Cooperativity is a common biochemical phenomenon in which two or more otherwise independent processes are thermodynamically coupled. Because cooperative processes are usually attended by changes in molecular conformation, thermodynamic coupling is usually attributed to an enthalpy-driven mechanism. In the family of glycopeptide antibiotics that includes vancomycin, however, cooperative phenomena occur that cannot be explained by conformational change. We have demonstrated that cooperativity in these systems can arise solely from changes in vibrational activity using molecular dynamics simulation. This result has important implications for much larger systems because ligand-induced modulation of periodic motions in a macromolecular system is an eminently plausible means of communicating the presence of bound ligand over long distances. Indeed, the function of large membrane proteins such as those involved in transmembrane signal transduction may actually require a mechanism based on configurational entropy changes because the enthalpy changes generally involved in the binding of small ligands are small compared to the magnitude of potential energy fluctuations one would expect in systems of this size.

EMBRYONIC BEGINNINGS OF THE HEMATOPOIETIC AND VASCULAR SYSTEMS DURING MOUSE DEVELOPMENT

Margaret H. Baron, M.D., Ph.D.

Department of Medicine, Biochemistry and Molecular Biology Mount Sinai School of Medicine

Blood and vascular endothelial cells form in all vertebrates during gastrulation, a process in which the mesoderm of the embryo is induced and then patterned by molecules whose identity is still largely unknown. “Blood islands” of primitive hematopoietic cell clusters surrounded by a layer of endothelial cells form in the yolk sac, external to the developing embryo proper (epiblast). These lineages arise from a layer of extra embryonic mesoderm that is closely apposed with a layer of primitive (visceral) endoderm. Despite the identification of genes such as Flk1, SCL/tal-

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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1, Cbfa2/Runx1/AML1 and CD34 that are expressed during the induction of primitive hematopoiesis and vasculogenesis, the early molecular and cellular events involved in these processes are not well understood. We demonstrated previously that specification of these lineages requires a signal(s) secreted from the adjacent visceral endoderm (VE) and more recently that Indian hedgehog (Ihh) is a VE-secreted signal which alone is sufficient to induce formation of hematopoietic and endothelial cells. We have continued to investigate the mechanism by which Ihh activates these processes. As seen with VE, Ihh can also respecify prospective neural ectoderm (anterior epiblast) along hematopoietic and endothelial (posterior) lineages, as indicated by cell morphology, activation of specific transgenes (e.g., lacZ reporters controlled by embryonic globin, Flk1 and Cbfa2/Runx1/AML1 sequences), activation of endogenous markers of stem/progenitor cells (hemangioblasts) and more differentiated cells, and by immunostaining for PECAM1 and other proteins. Dispersed cells from recombinant human IHH-treated anterior epiblasts form primitive or definitive hematopoietic colonies in secondary cultures in the presence of appropriate cytokines, indicating that functional hematopoietic stem cells are produced, and endothelial cell sprouting is observed. As expected, downstream targets of the Hh signaling pathway (Ptch1, Smo, Gli1) are upregulated in anterior epiblasts cultured in the presence of Ihh protein. Blocking Ihh function in VE inhibits activation of hematopoiesis and vasculogenesis in the adjacent epiblast, suggesting that Ihh is an endogenous signal that plays a key role in the development of the earliest hemato-vascular system. The gene encoding Bone morphogenetic protein-4 (Bmp-4) is upregulated in the target epiblast in response to Ihh. Several more direct lines of evidence indicate that Ihh functions, at least in part, through activation of the Bmp signaling pathway. Hedgehog genes and protein are expressed by adult mouse and human bone marrow stromal cells and Ptch1 and Smo are expressed in hematopoietic stem/progenitor as well as endothelial cells. Therefore, these findings may have important implications for regulating hematopoiesis and vascular development for practical and therapeutic purposes.

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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FLUORESCENCE DETECTION TECHNOLOGIES AND BIOMEDICAL RESEARCH: PROTEOMICS, GENOMICS, IMAGING, ARRAYS, AND DISEASE

Joseph M. Beechem, Ph.D.

Molecular Probes, Inc.

In 1981, when I started graduate school at The Johns Hopkins University (Baltimore, MD), fluorescence spectroscopy was a technique mainly practiced by biophysicists. Everyone else in my graduate school class was “flocking-to” laboratories in really hot-areas of research, which at that time was molecular biology/genetics. During my Ph.D. training, instead of learning the latest cloning technique or expression system, items such as: fluorescence polarization/anisotropy, fluorescence resonance energy-transfer (FRET), excited-state reactions, solvent relaxation, protein folding, fast kinetics, etc., formed the heart of my research. The relevance of these “arcane” spectroscopic methodologies for biomedical research was tenuous (at best). Fortunately, as modern biomedical research methodologies evolved, these “arcane” fluorescence spectroscopic techniques became some of the key detection technologies associated with much of the modern biomedical research “revolution.” Fluorescence polarization /anisotropy spectroscopy has become an essential component of high throughput homogenous drug screening and protein-protein interaction assays. Fluorescence energy-transfer (FRET) became the key technology behind molecular “beacons,” and high-sensitivity DNA microarray techniques. Ultra-high sensitivity protein folding dyes are becoming the core technology associated with proteomics platform imaging of 2-D gels. Fluorophores covalently coupled to chelating reagents formed the key technology associated with discovering the important role of calcium as a universal “currency” of intracellular signaling. The list goes on-and-on and, most importantly, many key applications of fluorescence technology to biomedical research have yet to be developed. In this presentation, data will be described for the “next generation” of fluorescence reagents and methodologies, which are being developed at Molecular Probes. From high resolution deconvolved intracellular imaging of organelles, to mass-spectroscopy/fluorescence-combined approaches for whole cell post-translational modification mapping will be presented. These (and additional) fluorescence technologies will continue to play an active role in helping biomedical research solve the major health problems facing the modern world.

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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IDENTIFICATION OF THE PROTEIN 4.2 GENE AS A DIRECT TARGET OF THE TAL1/CL TRANSCRIPTION FACTOR IN DIFFERENTIATING MURINE ERYTHROID CELLS

Zhixiong Xu and Stephen J. Brandt, M.D.

Vanderbilt University and Veterans Affairs Medical Centers

The TAL1/SCL gene, originally identified through its involvement by a recurrent chromosomal translocation in leukemic T-cells, encodes a basic helix-loop-helix (bHLH) transcription factor essential for embryonic hematopoiesis and vascular remodeling. Although TAL1 is presumed to alter the transcription of a specific set of genes, no such targets have been definitively identified. Binding site selection assays using erythroid cell extracts have suggested that TAL1 contributes to a multi-protein DNA-binding complex that binds preferentially to a tandem E box (for bHLH proteins)-GATA motif and which also contains the zinc finger transcription factor GATA-1, the LIM domain protein LMO2, and the LIM domain binding protein Ldb1. We identified two such E box-GATA elements in the proximal promoter of the murine Protein 4.2 gene whose protein product plays an important role in maintaining the stability and flexibility of erythrocytes. To determine if transcription of this gene is regulated by such a complex, we analyzed the contributions of TAL1 and GATA-1 to Protein 4.2 DNA binding activity, promoter activity, and endogenous gene expression and in vivo occupancy of the Protein 4.2 promoter by TAL1. First, several TAL1-, GATA1-, LMO2-, and Ldb1-containing complexes were detected by gel mobility shift analysis of erythroid cell extracts using probes corresponding to either E box-GATA element in the Protein 4.2 promoter. An increase in these DNA-binding activities was observed with DMSO-induced differentiation of murine erythroleukemia (MEL) cells concomitant with expression of Protein 4.2 mRNA. Cold competitor studies and gel mobility shift assays with mutant probes indicated a requirement for both the E box and GATA sites in formation of these binding complexes and revealed an increased stability for the ternary complex relative to other E box- and GATA-binding complexes in MEL nuclear extracts. Using a novel modification of the gel mobility shift assay, it was shown that this TAL1- and GATA-containing complex could bridge in solution two double-stranded oligonucleotide probes corresponding to the two E box-GATA elements in the Protein 4.2 promoter. Reporter gene assays showed that DMSO-induced promoter activity was decreased by 75 percent and 90 percent, respectively, with mutation of either E box or GATA site, suggesting that both E box-GATA elements contribute to promoter activity and that both the E box and GATA sites within these elements are required for maximal induction of Protein 4.2

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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gene expression during MEL cell differentiation. In addition, a TAL1 expression vector increased Protein 4.2 promoter activity when cotransfected with vectors for its DNA-binding partner E47, GATA-1, LMO2, and Ldb1. Finally, an increase in endogenous Protein 4.2 gene expression and in E box-GATA DNA-binding activities was observed when TAL1 was overexpressed in MEL cells, a decrease in both was observed with enforced expression of a TAL1 mutant defective in DNA-binding or an Ldb1 mutant impaired in dimerization, and evidence for in vivo occupancy of the Protein 4.2 promoter by TAL1 was obtained through chromatin immunoprecipitation analysis. In sum, these data establish the Protein 4.2 gene as a direct target of a TAL1- and GATA-1-containing DNA-binding complex in differentiating erythroid cells.

CELL SIGNALING NETWORKS IN C. ELEGANS MORPHOGENESIS

Andrew Chisholm, Ph.D., Ian Chin-Sang, Mei Ding, Sean George, Bob Harrington, Tom Holcomb, Kris Larsen, and Sarah Moseley

Department of Biology

University of California

The epidermis of the nematode C. elegans is a simple model for analyzing mechanisms of epithelial morphogenesis. The worm epidermis undergoes several distinct morphogenetic movements, including epiboly, cell intercalation, directed dilation, and invagination. We have focused on the epiboly movements required for epidermal enclosure of the embryo. The epidermis develops from a sheet of cells lying on the dorsal part of the embryo. Changes in epidermal cell shape cause the epidermis to expand laterally and ventrally, moving over neuronal substrate cells. This results in epidermal enclosure in the embryo at the ventral midline. Using genetics we have found that signaling between underlying neuronal precursors is important for normal enclosure.

Mutations in the C. elegans Eph receptor VAB-1 and the ephrin ligand VAB-2/EFN-1 cause defects in neural and epidermal morphogenesis (George et al., 1998 Cell 92:633; Chin-Sang, et al., 1999 Cell 99:781). VAB-1 and VAB-2 are expressed in complementary sets of neurons during embryogenesis, and are required in neurons for epidermal morphogenesis. VAB-1 may have kinase-dependent and kinase-independent functions; vab-2 mutations synergise with vab-1 kinase domain mutations and not with a vab-1 extracellular domain mutation, suggesting that VAB-2 may mediate a kinase-independent function of VAB-1.

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×

The almost complete C. elegans genome encodes one Eph receptor (VAB-1) and four GPI-anchored ephrins (EFN-1/VAB-2, EFN-2, EFN-3, and EFN-4). Mutations in the gene mab-26 cause morphogenetic defects related to but distinct from those seen in vab-1 and vab-2 mutants. We have shown that mab-26 corresponds to the fourth worm ephrin (EFN-4). Strikingly, mab-26 mutations display synthetic lethality with vab-1 and vab-2 mutations, suggesting that MAB-26 may function in VAB-1-independent signaling.

Loss of function in the C. elegans LAR-like receptor tyrosine phosphatase PTP-1 causes morphogenetic defects. We have found that ptp-1 mutants display synthetic lethality with vab-1, vab-2, and mab-26 mutations. These results suggest that RPTP signaling may function in a parallel redundant pathway with Eph signaling. We are currently testing whether this interaction is specific to the Eph signaling mutants.

PAS PROTEINS AND THE REGULATION OF DEVELOPMENT AND PHYSIOLOGY

Stephen Crews, Ph.D.

Department of Biochemistry and Biophysics

University of North Carolina, Chapel Hill

The central nervous system consists of a large variety of neuronal and glial cell types. Neural precursor cells are first specified and those precursors then generate distinct CNS cell types. We have studied the formation of the cells that lie along the midline of the Drosophila CNS. This work has lead to an understanding of how distinct regions of the CNS are generated and the discovery of a class of regulatory proteins that control a wide variety of invertebrate and vertebrate developmental and physiological processes.

Single-minded and the control of CNS midline cell development. The CNS midline cells comprise a distinct set of functional neurons and glia, and also act as a signaling center that controls aspects of axon guidance, cell migration, and formation of epidermal, mesodermal, and neural cell types. We have broadly investigated CNS midline cell development using genetics and molecular techniques to identify and functionally analyze genes involved in establishing midline cell fate and function. The single-minded gene acts as a master regulator of CNS midline cell transcription and formation. Dorsal/ventral patterning proteins act in a concentration-dependent and cooperative mode in conjunction with the Notch signaling pathway to activate single-minded transcription in midline precursor cells.

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×

The single-minded gene encodes a basic-helix-loop-helix-PAS transcription factor that is required for all transcription and development of the midline cells. Single-minded protein dimerizes with the Tango bHLH-PAS protein. Together they enter the nucleus, bind to DNA control elements containing an ACGTG core sequence, and activate midline transcription in conjunction with additional unknown coregulators. Correct formation of the CNS midline cells involves activation of midline gene transcription and repression of lateral CNS transcription in the midline cells. Single-minded is required for both processes: it activates transcription of midline-expressed genes and the transcription of repressive factors. Single-minded continues to function throughout development. As midline precursor cells differentiate, the Single-minded: Tango protein complex interacts combinatorially with the Drifter and Fish-hook transcription factors to control midline glial transcription. Postembryonically, single-minded is expressed in the brain, including a group of neurons in the central complex that coordinate locomotion. Analysis of adult behavior using a single-minded temperature sensitive mutant shows defects in courtship and walking. Mutant flies only walk in circles—they can turn either left or right, but not both. Current work is focused on identifying genes that mediate CNS midline cell fate development, and understanding how Single-minded: Tango interacts with additional regulatory proteins to control their expression.

PAS Proteins. Single-minded, along with Arnt and Period, constitute the founding members of the PAS protein family. PAS proteins are found in all organisms, from bacteria to humans. Prokaryotic and plant PAS proteins are commonly environmental sensors that respond to and mediate the effects of changes in light, oxygen levels, redox, and metabolic state. Most animal PAS proteins belong to the bHLH-PAS class of DNA binding proteins. The Markey Trust has an impressive heritage in bHLH-PAS protein discovery and analysis, having sponsored research in our lab and those of Drs. Tessier-Lavigne, Montell, and Semenza. Drosophila and mammalian bHLH-PAS proteins control a variety of biological processes, including toxin metabolism, circadian rhythms, vasculogenesis, tissue-specific development, cell migration, and behavior. Clinically, they are important for understanding tumor growth, sleep disorders, birth defects, and obesity. Our lab has discovered or worked on a number of Drosophila bHLH-PAS proteins, including Single-minded, Trachealess (tracheal development), Spineless (appendage formation), Similar (response to hypoxia), Cranky, and Tango (dimerization partner for all of the above). This project has allowed us to generalize about the mechanisms of action of invertebrate and vertebrate developmental bHLH-PAS proteins. Given the fundamental role that bHLH-PAS proteins play in the formation of the nervous system and respiratory system in insects, we

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×

have begun to look for similar roles in other species. Drosophila trachealess is a master regulator of tracheal transcription and development. We have shown that the crustacean, Artemia, has a trachealess ortholog that is prominently expressed in the epipodal gills. Although the insect trachea and crustacean gills are divergent morphologically, these results suggest that their formation may be controlled by the same regulatory gene. Furthermore, evolutionary analysis of bHLH-PAS gene expression and function in organisms with distinct mechanisms of CNS and respiratory system development and anatomy may provide important insights into organismal evolution.

STRUCTURAL BASIS OF TRANSCRIPTION

Seth A. Darst, Ph.D.

Laboratories of Molecular Biophysics

The Rockefeller University

Transcription is the major control point of gene expression. RNA in all cells is synthesized by a complex molecular machine, the RNA polymerase (RNAP). In bacteria, RNAP comprises a ~400 kDa core (subunit composition alpha2/beta/beta’/omega), conserved from bacteria to man. Promoter-specific initiation requires additional proteins. In bacteria, a single polypeptide, the sigma factor, binds core RNAP to form holoenzyme.

Our goal is to understand the mechanism of transcription and its regulation. Our approach is to use a combination of structural and biophysical methods spanning resolution ranges (low-resolution: cryo-electron microscopy [EM]; medium: cross-linking analysis and X-ray crystallography; high: X-ray crystallography), complemented by the wealth of functional information already available, to determine the structure/function relationship of RNAP and its complexes with nucleic acids and regulatory factors. Our focus is on the bacterial RNAPs as a model for the cellular RNAPs in general.

Low-resolution EM structures (25 -12 Å) of RNAP revealed a molecule shaped like a crab-claw, with a groove or channel for accommodating double-helical DNA.1,2 In the first step towards high-resolution structural analysis of cellular RNAPs, we determined the 3.3 Å-resolution crystal structure of the 380 kDa core RNAP3,4 from T. aquaticus (Taq), providing a basis for further structural and functional studies. For example, the path of the transcript RNA and template DNA through RNAP was tracked using crosslink mapping, resulting in a detailed model of the elongation complex.5 A co-crystal structure of RNAP with rifampicin revealed the

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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mechanism of inhibition by this important antibiotic.4 The remarkable conformational flexibility of RNAP was analysed by comparing cryo-EM and X-ray results using newly developed computational tools.2

Recent progress is highlighted by crystal structures of the 430 kDa Taq holoenzyme (alpha2/beta/beta’/omega/sigma) at 4 Å,6 and a holoenzyme/promoter DNA complex at 6 Å resolution.7 These results depended on having high-resolution structures of core RNAP3,4 and sigma factor domains8 in-hand. These structures provide fundamental insight into sigma/core RNAP interactions and conformational changes to form holoenzyme, holoenzyme recognition of promoters, and sigma’s role in melting the DNA to form the transcription bubble. The structures also provide a basis for models of holoenzyme/promoter complexes along the pathway of open complex formation. The new structural information will guide future investigations at an unprecedented level of detail.

Notes

1  

Darst, S. A.; Kubalek, E. W.; Kornberg, R. D. (1989) Nature 340:730-732; Darst, S. A.; Edwards, A. M., Kubalek, E. W.; Kornberg, R. D. (1991) Cell 66:121-128; Polyakov, A.; Severinova, E.; Darst, S. A. (1995) Cell 83:365-373.

2  

Darst, S. A.; Opalka, N.; Chacon, P.; Polyakov, A.; Richter, C.; Zhang, G.; Wriggers, W. (2002) Proc. Natl. Acad. Sci., in press.

3  

Zhang, G.; Campbell, E.; Minakhin, L.; Richter, C.; Severinov, K.; Darst, S. A. (1999) Cell 98:811-824.

4  

Campbell, E. A.; Korzheva, N.; Mustaev, A.; Murakami, K.; Goldfarb, A.; Darst, S. A. (2000) Cell 104:901-912.

5  

Korzheva, N.; Mustaev, A.; Kozlov, M.; Malhotra, A.; Nikiforov, V.; Goldfarb, A.; Darst, S.A. (2000) Science 289:619-625.

6  

Murakami, K.; Masuda, S.; Darst, S.A. (2002) submitted.

7  

Murakami, K.; Masuda, S.; Campbell, E.A.; Muzzin, O.; Darst, S. A. (2002) submitted.

8  

Campbell, E. A.; Muzzin, O.; Chlenov, M.; Sun, J. L.; Olson, C. A.; Weinman, O.; Trester-Zedlitz, M. L.; Darst, S. A. (2002) submitted.

HIJACKING THE RIBOSOME: STRUCTURAL BASIS FOR TRANSLATION INITIATION IN HEPATITIS C VIRUS

Jennifer A. Doudna, Ph.D.

Molecular Biophysics and Biochemistry

Howard Hughes Medical Institute

Yale University

Initiation of protein synthesis is a key step in the control of gene expression in eukaryotes. In most cases, recruitment of the 40S ribosomal subunit to a messenger RNA (mRNA) involves recognition of a modified

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×

nucleotide cap on the mRNA 5’ end by several large translation initiation factors. However, a subset of mRNAs and viral RNAs lack the 5’ cap and instead contain a structured RNA element upstream of the coding region called an internal ribosome entry site (IRES) that recruits ribosomes without requiring some or most of the canonical translation initiation factors. In Hepatitis C virus (HCV), the IRES RNA binds directly to the 40S ribosomal subunit and requires just one initiation factor, eIF3, to form a complex primed to initiate translation upon assembly with initiator tRNA and the 60S ribosomal subunit. Images of HCV IRES-40S ribosomal subunit complexes, produced by cryo-electron microscopy in collaboration with J. Frank and C. Spahn, revealed a pronounced IRES-induced conformational change that clamps the 40S subunit on the viral message. This observation together with binding and translation assays suggests that the IRES RNA actively recruits and positions ribosomes during viral infection. Ongoing work focuses on cryo-EM structure determination of the complete HCV IRES preinitiation complex. We have also recently solved a high-resolution crystal structure of the IRES region containing the eIF3 binding site, providing a basis for understanding the role of eIF3 during initiation as well as a target for anti-viral drug design. Ultimately, we hope to elucidate a detailed mechanism for IRES-mediated translation initiation and to use structural data in the design of small molecule inhibitors of the HCV virus.

THE COMPLEX INTERPLAY OF MICROBIAL PATHOGENS WITH EUKARYOTIC CELLS

Joanne Engel, M.D., Ph.D.

Department of Medicine Division of Infectious Disease

University of California, San Francisco

My laboratory is interested in understanding and exploiting the complex interplay of microbial pathogens with eukaryotic cells. One project focuses on how Pseudomonas aeruginosa (PA), an important opportunistic pathogen of man, injures epithelial cells. This nosocomial gram negative bacterium is the leading cause of bacteremia and sepsis in patients receiving cytotoxic chemotherapeutic agents, the most common cause of nosocomial pneumonia, a killer of neutropenic and burn patients, the most common cause of corneal ulcers, and a contributing factor to severe pulmonary damage and consequent death in patients afflicted with cystic fibrosis. The virulence potential of PA is exacerbated by the rapid rate at which it develops drug resistance. Indeed, many clinical isolates are in-

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×

sensitive to most or all antibiotics, making the identification of new targets for diagnosis, therapy, and prevention essential for our ability to control this important nosocomial pathogen. Unraveling the complex interactions between PA and its host eukaryotic cells will lead to novel insights into how bacteria attack host cells and outmaneuver the immune system.

A critical determinant of PA infections is the requirement for pre-existing epithelial cell damage. Upon binding, this organism secretes a diverse array of virulence factors that lead to further tissue damage and dissemination. In a novel genetic screen designed to identify new virulence factors required for epithelial cell injury, we demonstrated a key role for the type III secretion system and its secreted effector molecules ExoU and ExoT. In a retrospective clinical study, we have shown that the presence of a functional type III secretion system correlates with outcome in ventilator-associated pneumonia. Such studies suggest that the type III secretion system may be a novel target for intervention in hospitalized patients with PA pneumonia. In addition, we have found that type IV pili are required for type III secretion and for virulence in vitro and in vivo. Using confluent MDCK cells as a model system for an epithelial monolayer, we have shown that the differentiation state of the monolayer affects the ability of PA to damage or enter epithelial cells.

ExoU is a novel cytotoxin that is directly translocated by the type III secretion system from the bacterium to the host cell where it induces necrosis of the host cell. It is required for full virulence in a mouse model of acute pneumonia. In addition to ExoU-mediated necrosis, PA can induce apoptosis in epithelial cells and macrophages. This process requires a functional type III secretion system and appears to work through the Fas pathway. Macrophages isolated from Fas receptor or Fas ligand-defective mice are resistant to PA-induced apoptosis. We are currently carrying out genetic screens to identify the bacterial molecule(s) required for induction of host cell apoptosis.

Using a combination of bacterial genetics and cell biology, we have shown that PA can modulate host cell signaling cascades to regulate its entry into host cells using pathways that are dependent upon the polarization state of the epithelium and that are upregulated in wounded epithelium. In injured or poorly polarized epithelial cells, binding of PA activates Rho GTPase, resulting in bacterial internalization. As epithelial cells regenerate an intact mucosal barrier, they become resistant to PA injury and internalization by downregulation of the entry pathway. These observations begin to explain why PA is such an effective pathogen in the setting of epithelial cell injury.

In addition, this organism can modulate its entry by directly translocating into the host cell cytoplasm the type III secreted effector ExoT,

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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which we have shown to act as a GTPase activating protein towards Rho, Rac, and CDC42. A second domain of ExoT has homology to ADP ribosyl transferases and is required for full activity of ExoT. In addition to its role as an anti-internalization factor, we have shown that ExoT possesses other important physiological activities: it prevents wound healing, disrupts the host actin cytoskeleton, and is required for full virulence in a mouse model of acute pneumonia. Identification of its eukaryotic host cell targets is under way.

Together these studies illustrate the power of combining bacterial genetics with host cell biology. We are currently focusing on understanding (i) the pathways by which ExoT alters the host cell cytoskeleton, (ii) the mechanism of Type III secretion dependent apoptosis, (iii) the role of type IV pili in type III secretion, and (iv) the signal transduction pathways that allow the bacterial pili to respond to environmental signals.

GENETICS OF MALARIAL INFECTION IN MICE

Simon Foote, Ph.D., Vikki Marshall, Rachel Burt, Tracey Baldwin, James Wagglen, and Enmoore Lin

The Walter and Eliza Hall Institute of Medical Research Melbourne, Australia

There are well-documented associations between host genetics and response to infection in humans. These are most evident in the case of malaria, where many genetic diseases involving the red blood cell are found in geographic coincidence with malarial endemnicity. While association with diseases is relatively straightforward, analysis of other genetic resistance factors which are not so obvious in homozygotes are difficult in human populations. We have elected to use mouse models to study the genetically determined response in mice in an attempt to identify pathways that may be involved in the regulation of the pathophysiological consequence of disease in humans.

We have mapped three loci in murine intercrosses which control various aspects of malarial infection. Two loci, Char1 and 2 control both outcome to infection and peak parasitaemia levels and Char3 controls clearance of parasites from the circulation. We have generated congenic mice for each locus and identified minimal congenic intervals still retaining a phenotype distinguishable from the backcross parental line.

A BAC map has been generated for Cha1. This covers some 3Mb DNA on murine chromosome 9. Approximately 50 percent of this region has been used to generate BAC congenic animals. One line has a malarial phenotype different from the non-transgenic littermates. This line con-

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
×

tains two overlapping BACs which have been sequenced and which contain 11 genes. Efforts to subdivide the region using further BAC transgenesis will be discussed.

MECHANISMS AND GENOMIC CONSEQUENCES OF RETROTRANSPOSON TY1 REPLICATION

Abram Gabriel, M.D.

Department of Molecular Biology and Biochemistry

Rutgers University

My laboratory is interested in two general areas associated with the chromosomal biology of the yeast Saccharomyces cerevisiae. The first is the replication fidelity of the endogenous retroelement, Ty1. The genome of S. cerevisiae contains ~35 copies of Ty1, which replicates in a manner strikingly similar to vertebrate retroviruses. We observed that Ty1 replication is highly error prone with changes occurring at specific terminal locations. Based on this we developed a model for a novel error generating mechanism, and are now using recombinant Ty1 reverse transcriptase to study the biochemical basis for this phenomenon. Additionally, we have generated active site mutations in the Ty1 RT in the three conserved aspartates. One of these mutants is still capable of efficient polymerization although it is incapable of transposition. Using second site suppressor analysis, in vivo examination of replication intermediates, and biochemical studies of the WT and mutant enzymes, we are examining the basis of RT catalysis, and the functional interactions of different regions of the enzyme.

Our second general area of interest is in the relationship of Ty1 to double-strand break (DSB) repair. We observed that under conditions of a potentially lethal HO endonuclease-induced DSB, chromosomal healing could occur by joining of a portion of Ty1 to the two broken ends. This process involves nonhomologous end joining and is distinct from transposition and homologous recombination with endogenous Ty1 elements. Interestingly, this insertional repair is in competition with other break repair processes such as gene conversion, deletion formation, and rear-rangements leading to inversions and translocations. We are studying the genetic determinants of this insertion process. We have also observed that other DNA fragments can insert at DSB sites, particularly mitochondrial DNA fragments. We are investigating the factors that influence the type of insertion event and the ratio of insertions to other repair mechanisms.

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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GENETIC BASIS OF SUDDEN CARDIAC DEATH AND OTHER CHANNELOPATHIES

Alfred L. George, Jr., M.D.

Division of Genetic Medicine

Vanderbilt University

Ventricular arrhythmias are the single most important cause of sudden cardiac death (SCD) among adults living in industrialized nations. Evidence now exists indicating that genetic factors have a substantial influence in determining population-based risk for SCD and may also account for interindividual variability in susceptibility.

Specific genes underlying various Mendelian disorders associated with inherited arrhythmia susceptibility have been identified. The most well studied familial arrhythmia syndrome is the congenital long QT syndrome (LQTS), an inherited condition of abnormal cardiac excitability characterized by prolongation of the QT interval on surface electrocardiograms of affected individuals. A long QT interval correlates with prolongation of the ventricular action potential owing to delayed myocardial cell repolarization, an arrhythmia-prone cellular substrate. Many LQTS subjects harbor mutations in SCN5A encoding the cardiac voltage-dependent sodium channel. Another inherited cardiac arrhythmia, Brugada Syndrome, and certain forms of familial cardiac conduction system disease are also caused by mutations in SCN5A. These syndromes join a rapidly growing list of inherited disorders caused by mutations in genes encoding ion channels: the channelopathies.

We have been very successful in characterizing the molecular and biophysical defects caused by mutations in human voltage-gated sodium channels. Our studies utilize recombinant human sodium channels expressed heterologously in cultured cells and interrogated using electrophysiological tools such as the patch-clamp technique. In LQTS, mutations in SCN5A result in sodium channels that fail to “close” completely after activating. This functional defect leads to increased sodium influx into myocardial cells, delayed repolarization and increased susceptibility to triggered arrhythmias. By contrast to this “gain-of-function” disturbance in LQTS, sodium channel mutations in Brugada syndrome lead to loss-of-function and electrical imbalances (heterogeneity of repolarization) in the ventricular myocardium that predisposes to arrhythmias. Recently, we have characterized novel SCN5A mutations associated with familial cardiac conduction system disease that exhibit a curious combination of gain and loss of function characteristics that account for myocardial conduction slowing. Our studies have helped discern the molecular genotype-phenotype relationships among these three distinct cardiac dis-

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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orders and have contributed to improved diagnosis and therapy of cardiac arrhythmias.

Our work on the cardiac channelopathies is representative of other investigations into the molecular genetic basis of a variety of other familial disorders affecting muscle contraction (myotonia, periodic paralysis) and associated with epilepsy that have been the focus of my laboratories’ research effort.

REGULATION OF MACROPHAGE GENE EXPRESSION BY NUCLEAR HORMONE RECEPTORS

Christopher K. Glass, M.D., Ph.D.

Division of Cellular and Molecular Medicine

University of California, San Diego

My laboratory is interested in how members of the nuclear receptor gene family regulate macrophage development and function. Nuclear receptors are transcription factors that positively or negatively regulate gene expression in response to the binding of small molecular weight ligands. The human genome contains 48 members of this family that include receptors for steroid and thyroid hormones, vitamins, and metabolites of cholesterol and fatty acids. In addition to playing critical roles in the regulation of development and homeostasis, nuclear hormone receptors have been important targets for drug development. Examples include the development of synthetic glucocorticoids for treatment of inflammation and selective estrogen receptor modulators (SERMs) for treatment of breast cancer. Because the transcriptional functions of nuclear receptors can be switched on and off in vitro by addition of small molecules, these proteins have also provided some of the most powerful models for mechanistic studies of transcription. The nuclear receptor field thus spans an extraordinarily broad spectrum of investigation that ranges from basic lines of biochemical, cellular and molecular inquiry to clinical trials of new drugs in major human diseases such as diabetes and atherosclerosis. Owing to the potential medical importance of novel ligands for nuclear hormone receptors, they are the focus of intensive investigation by pharmaceutical companies, biotechnology companies, and academia.

Studies from my laboratory led to the discovery that many members of the nuclear receptor family bind to DNA recognition elements in target genes as heterodimers with retinoid X receptors (RXRs). More recently we have defined coactivators and corepressors that interact with nuclear receptors to mediate their transcriptional effects and have participated in

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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collaborative studies leading to crystal structures of nuclear receptor/ coactivator complexes. Currently, we are focusing on defining the biological roles of the peroxisome proliferator activated receptor γ. (PPAR γ) in macrophages. Several lines of evidence indicate that PPAR γ is required for fat cell development and that it regulates glucose homeostasis. PPAR γ has proven to be the molecular target of thiazolidinediones (TZDs), a class of insulin sensitizers that are in clinical use for the treatment of type 2 diabetes mellitus. Approximately three years ago, we reported that high levels of PPAR γ were also expressed in macrophages and that TZDs could inhibit the expression of pro-inflammatory genes. In a follow-up series of studies, we demonstrated that PPAR γ was also highly expressed in macrophage-derived foam cells of human atherosclerotic lesions and that TZDs dramatically reduced the development of atherosclerosis in hypercholesterolemic mice. However, several lines of evidence indicate that despite the net antiatherogenic effect, currently available TZDs also exert pro-atherogenic effects on a subset of macrophage target genes. These findings are of substantial clinical interest, because millions of patients with type 2 diabetes are at markedly increased risk of developing atherosclerosis and its clinical complications. These observations highlight the importance of defining the molecular mechanisms responsible for biological effects of synthetic nuclear receptor ligands and the potential to use this knowledge for the development of selective modulators of nuclear receptor function that are optimized for specific therapeutic outcomes.

SODIUM CHANNELS AND CNS DISEASE

Alan L. Goldin, M.D., Ph.D.

Department of Microbiology and Molecular Genetics

University of California, Irvine

We are studying the effects of mutations in the voltage-gated sodium channel to determine how specific alterations in channel function result in disease in the CNS, using three approaches. In the first part of our studies, we have examined the effects of a missense mutation in the mouse Scn8a gene encoding the Nav1.6 sodium channel, which is broadly distributed in brain and spinal cord. The mutation, termed jolting, causes cerebellar ataxia. It results in substitution of threonine for a conserved alanine in the cytoplasmic S4-S5 linker of domain III. Introduction of the mutation into the orthologous rat brain sodium channel shifted the voltage-dependence of activation by 10 mV in the depolarizing direction without any signifi-

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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cant changes in the kinetics of either inactivation or recovery from inactivation. The shift in the voltage-dependence of activation observed for the mutant channel would reduce the spontaneous activity of Purkinje cells and lead to a decrease in output from the cerebellum, which is consistent with the phenotype of cerebellar ataxia observed in jolting mice.

In the second part of our studies, we have constructed transgenic mice expressing a mutation of three residues (GAL 879-881 to QQQ) in the cytoplasmic S4-S5 linker of domain II in the rat Nav1.2 sodium channel. This mutation results in slowed inactivation and increased persistent current. The neuron-specific enolase promoter was used to direct in vivo expression of the mutant channel in transgenic mice. Three transgenic lines exhibited seizures, and one line was characterized in detail. The seizures in these mice began at two months of age and were accompanied by behavioral arrest and stereotyped repetitive behaviors. Continuous electroencephalogram monitoring detected focal seizure activity in the hippocampus, which in some instances generalized to involve the cortex. Hippocampal CA1 neurons isolated from presymptomatic mice exhibited increased persistent sodium current that may underlie hyperexcitability in the hippocampus. During the progression of the disorder there was extensive cell loss and gliosis within the hippocampus in areas CA1, CA2, CA3 and the hilus. The lifespan of the mice was shortened and only 25 percent survived beyond 6 months of age. Four independent transgenic lines expressing the wild-type sodium channel were examined and did not exhibit any abnormalities. The transgenic mice provide a genetic model that will be useful for testing the effect of pharmacological intervention on progression of seizures caused by sodium channel dysfunction.

In the final aspect of our studies, we have examined the effects of two mutations in the human SCN1A gene encoding the alpha subunit of the Nav1.1 sodium channel. These mutations cause generalized epilepsy with febrile seizures plus (GEFS+). Both mutations change conserved residues in putative voltage-sensing S4 segments, T875M in domain II and R1648H in domain IV. Each mutation was cloned into the orthologous rat channel, rNav1.1, and the properties of the mutant channels were determined in the absence and presence of the beta1 subunit in Xenopus oocytes. Neither mutation significantly altered the voltage-dependence of either activation or inactivation in the presence of the beta1 subunit. The most prominent effect of the T875M mutation was to enhance slow inactivation in the presence of beta1, with small effects on the kinetics of recovery from inactivation and use-dependent activity of the channel in both the presence and absence of the beta1 subunit. The most prominent effects of the R1648H mutation were to accelerate recovery from inactivation and decrease the use-dependence of channel activity with and without the beta1

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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subunit. The DIV mutation would cause a phenotype of sodium channel hyperexcitability while the DII mutation would cause a phenotype of sodium channel hypoexcitability, suggesting that either an increase or decrease in sodium channel activity can result in seizures.

A DECADE-LONG JOURNEY ON THE FRONT LINE OF THE HUMAN GENOME PROJECT

Eric D. Green, M.D., Ph.D.

National Human Genome Research Institute

National Institutes of Health

The Human Genome Project and the fruits of its effort are creating a new era in biomedical research. My laboratory has been firmly planted on the front line of this exciting endeavor for the past decade.

Initially, we focused on the systematic mapping and sequencing of human DNA. As a result of our detailed mapping of chromosome 7, this segment of the human genome was among the first to be sequenced in a large-scale fashion. The resulting mapping and sequencing infrastructure provided a powerful foundation for us to then pursue complementary studies in human genetics, in particular those aiming to identify genes implicated in human disease. Projects within my laboratory led to the identification of the genes responsible for Pendred syndrome (a deafness/goiter disorder), cerebral cavernous malformations (an inherited vascular disease), and an elusive tumor suppressor gene on chromosome 7q31. These studies vividly illustrated how the availability of genome mapping and sequencing data greatly accelerates the process of elucidating the genetic bases of human disease.

Most recently, my laboratory has developed a major program in comparative genomics. As a complement to whole-genome sequencing efforts, we are sequencing the same set of targeted genomic regions in >20 vertebrates. Specifically, large (~250-6,000 kb) chromosomal segments of particular biomedical interest are being isolated in overlapping bacterial artificial chromosome (BAC) clones from multiple species (to date including several non-human primates, ~8 other placental mammals, a marsupial, a monotreme, a bird, and several fish species). The mapped clones are then being sequenced, with the resulting data subjected to rigorous computational analyses. Together, our program is generating >125 Mb of comparative sequence data per year. Importantly, this unique sequence resource is facilitating the development of new computational tools for multi-species sequence comparisons, is revealing the benefits of sequenc-

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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ing species from a range of evolutionary distances, and is serving an important reconnaissance function that should help guide the selection of additional organisms for whole-genome sequencing.

In summary, our decade-long journal along the main trails of the Human Genome Project has allowed us to make important contributions towards the mapping and sequencing of the human genome, towards utilizing the resulting data for studies in human genetics, and, most recently, towards the exploration of myriad genomes in an evolutionarily-deep fashion for the purposes of unraveling the complexities and evolution of the vertebrate genetic blueprint.

NUCLEAR POSITIONING AND CONTROL OF DIVISION AXIS DURING MORPHOGENESIS IN C. ELEGENS

Dan Starr, Zhe Chen, and Min Han, Ph.D.

Howard Hughes Medical Institute

University of Colorado

Research in our laboratory aims at understanding the mechanisms of cell differentiation, cell migration, and tissue morphogenesis in C. elegans. Two representative projects involving nuclear movement/anchorage and morphogenesis are summarized below.

Nuclear position and migration within a cell are important for cell function in the growth and development of a wide variety of eukaryotes. Our genetic and molecular analyses of the unc-83, unc-84 and anc-1 genes have revealed protein functions at the nuclear membrane that are involved in nuclear migration and anchorage. UNC-84, a nuclear membrane protein, recruits UNC-83 and ANC-1 protein to the nuclear membrane where they function in nuclear migration and anchorage, respectively. Our results also indicate that ANC-1, a novel and giant coiled-coil protein, may directly mediate nuclear anchorage through a tethering mechanism. UNC-83, expressed at the nuclear envelope of specific cells with migrating nuclei, possibly facilitates migration partly by competing with ANC-1 for UNC-84 binding.

The last round of vulval cell division, particularly the characteristic change of division axis of specific cells is considered an early landmark of vulval morphogenesis. Analysis of nhr-25 and lin-40 mutants indicated that they control the asymmetry of vulval cells and proper execution of the division pattern. The nhr-25 gene encodes a homologue of Drosophila Ftz-F1 and mammalian SF-1 NHR proteins. An nhr-25 mutation that disrupts the DNA-binding activity of the protein specifically blocks the divi-

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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sion in cells where the division axes normally change to an orientation perpendicular to that in previous divisions and causes abnormal gene expression in these cells. In addition, the nhr-25 mutation leads to defects in cell migration and fusion that occur during vulval organogenesis. In contrast, mutations in lin-40, which encodes a homolog of MTA, do not block cell division but prevent the change of division axes for the same set of cells. We propose that lin-40 acts to impose a pause in cell cycle to allow the change of the division axis, while nhr-25 acts to initiate cell division with a new axis.

A ROLE FOR γδ T CELLS IN TISSUE REPAIR

Wendy L. Havran, Ph. D., Julie Jameson, Stephanie Rieder, and Richard Boismenu

Department of Immunology

The Scripps Research Institute

The focus of my laboratory is to determine the antigen specificity and function of epithelial resident γδ T cells. These cells appear to recognize and respond to self antigens expressed after malignancy, infection or trauma of neighboring epithelial cells. We have demonstrated that after antigen recognition, the epithelial γδ T cells uniquely produce tissue specific cytokines, secrete chemokines and lyse damaged epithelial cells. This data supports a unique role for epithelial γδ T cells in immune surveillance, wound repair, and protection from malignancy.

T cells bearing invariant γδ T cell antigen receptors (TCR) localize to distinct epithelial sites in the adult mouse. The Thy-1+ dendritic epidermal T cells (DETC) express a monoclonal Vγ3Vδ1 TCR that is not found elsewhere. We have demonstrated that the DETC recognize and respond to self antigens expressed by neighboring keratinocytes after damage or disease. This antigen recognition is mediated by the DETC γδ TCR. Interestingly, DETC do not express coreceptors CD4 or CD8 and do not express the costimulatory receptor CD28. This may mean that other unidentified molecules play similar roles in DETC activation. Studies in progress are directed towards identification of coreceptors and costimulatory molecules important for DETC function. Recent data from other groups have demonstrated that DETC can specifically kill skin tumor cells through recognition of particular costimulatory molecules. New molecules that are identified may be potential targets for immunotherapy.

Little is known about the specificity and function of T cells that reside in epithelial tissues. Our data supports the idea that these cells recognize

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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a tissue specific self-antigen expressed after epithelial distress. We have demonstrated a new functional role for epithelial-resident γδ T cells in epithelial homeostasis and tissue repair that is distinct from roles played by lymphoid αß and γδ T cells. We have performed wound healing studies and found defects in keratinocyte proliferation and tissue reepithelialization in the absence of DETC. The mechanism of the DETC effect is through local production of the potent epithelial mitogens KGF-1 and KGF-2. Production of KGFs is a specialized feature of activated epithelial γδ T cells and other T cells do not produce these factors. We also have data in a mouse model of ulcerative colitis demonstrating that intestinal intraepithelial γδ T cells play a role in disease severity and tissue repair that is mediated in part by KGF. Patient studies also suggest a role for resident γδ T cells in epithelial inflammatory disease and malignancy. Identification of molecules involved in DETC activation is an important and necessary step in fully understanding the physiological role played by these cells. The DETC are the prototypic epithelial γδ T cell. As such, information gained in understanding properties of antigen recognition of the DETC may be applicable to other populations of γδ T cells. Costimulatory molecules expressed by αß T cells have been identified as important targets for immunotherapy, raising the possibility of similar roles for molecules expressed by γδ T cells. Information obtained by these studies may also be useful in design of new treatment strategies for malignancy.

MOUSE MODELS OF X-LINKED DEVELOPMENTAL DISORDERS

Gail E. Herman, M.D., Ph.D.

Division of Molecular and Human Genetics

Children’s Research Institute and Department of Pediatrics

The Ohio State University

My laboratory uses genetic approaches to try to understand the basis for selected inherited developmental disorders. Because many of these human disorders are extremely rare and genes on the X chromosome are generally conserved among all mammals, we have focused on X-linked disorders where we study a human disease using a mouse model that often has the same phenotype. Currently, we are studying three groups of disorders in the laboratory, each of which is discussed briefly below.

X-linked dominant male lethal disorders: Recently, my laboratory identified genes responsible for 2 X-linked dominant male lethal disorders in mouse and human. The disorders produce skeletal, skin, and eye abnor-

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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malities and involve sequential steps in the cholesterol biosynthetic pathway. Cholesterol levels in affected male mouse embryos at the time of death are normal, suggesting that the pathogenesis of the male lethality must be caused by another mechanism. Placentas from affected male embryos are smaller than those of normal littermates (p < 0.001), and the labyrinthine layer of the placenta appears thinner, disorganized, and has fewer fetal vessels by PECAM staining. There are also statistically significant differences in placental thickness between affected male and affected female placentas. Since most cells in the female rodent placenta undergo preferential inactivation of the paternal X chromosome, we believe that cells derived from allantoic mesoderm that undergo random X-inactivation are responsible for or contribute to the male lethality. We are currently further investigating the pathogenesis of the defects in affected male and female mice using techniques such as in situ hybridization and microarray expression analysis. We are also pursuing analyses of sterol trafficking and regulation in cultured cells derived from affected male embryos.

X-linked myotubular myopathy (MTM1): This is a rare congenital myopathy that presents with hypotonia and respiratory insufficiency. Many affected patients die or are left ventilator dependent and wheel-chair bound for life. The MTM1 gene was isolated in 1996 by our collaborators and appears to be a lipid phosphatase. We have found mutations in the human gene in over 70 affected boys in the United States and have the largest set of clinical data on long-term survivors in the world. As a result of our studies, we now know that a significant number of boys with this disease develop medical complications involving other organ systems. We have employed the yeast two hybrid system to proteins that interact with the MTM protein and are developing a mouse model for the disorder using homologous recombination.

X-linked neural tube defects: Neural tube defects (NTDs) are the second most common human birth defect. The causes for the majority of NTDs are unknown but likely involve both genetic and environmental factors. We have determined that a transcription factor, Zic3, is deleted in an X-linked mouse mutant, Bent tail (Bn), that serves as a model for NTDs. In humans, mutations in ZIC3 are associated with laterality (situs) defects and complex congenital heart disease. We and others have identified similar defects in Bn mice. Depending on the exact genetic background, we can separate the NTD and situs phenotypes, and we are now mapping these modifiers in a series of genetic crosses. We are also trying to identify upstream regulators and downstream targets of Zic3 using promoter constructs in tissue culture and Xenopus model systems. The genes identified will be candidates for involvement in human NTDs and/or laterality disorders.

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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SUPPRESSORS OF CYTOKINE SIGNALING

Douglas J. Hilton, Ph.D.

TheWalter and Eliza Hall Institute of Medical Research

The Cooperative Research Centre for Cellular Growth Factors

PO Royal Melbourne Hospital

Suppressor of cytokine signaling-1 (SOCS-1) is an important negative regulator of IFNγ signal transduction. While SOCS1 deficient (SOCS1-/-) mice die before weaning of a severe inflammatory disease, mice lacking both SOCS1 and IFNγ survive normally to adulthood, but succumb to a range of diseases in their second year of life. In addition to an SH2 domain, which regulates interaction with tyrosine phosphorylated signaling proteins such as JAKs, SOCS1 contains a 40 amino acid motif, termed a SOCS box. Biochemical studies have demonstrated that the SOCS box interacts with elongin B and C, suggesting that SOCS proteins act as part of a ubiquitin ligase complex and that the termination of signal transduction by SOCS1 may occur in part by targeting signaling proteins for proteasomal degradation. To test this, we have generated mice (SOCS1Δ/Δ) in which the SOCS-box of SOCS1 has been specifically deleted. SOCS1Δ/Δ mice display a phenotype that is intermediate between SOCS1-/- and wild type mice. SOCS1Δ/Δ mice survive weaning but succumb to inflammatory disease at 2 to 6 months of age. In vivo and in vitro, cells from these mice respond to IFNγ for longer than cells from wild type mice, but not for as long as cells from SOCS1-/- mice. This suggests that while important, regulation of protein degradation by the SOCS box is not the only mechanism by which SOCS1 attenuates signaling.

IDENTIFICATION AND ANALYSIS OF A SMALL MOLECULE INHIBITOR OF THE BCL-XL ANTI-APOPTOTIC PROTEINS

David M. Hockenbery, M.D.

Divisions ofHuman Biology andClinical Research

Fred Hutchinson Cancer Research Center

The anti-apoptotic Bcl-2 family of proteins confers cellular resistance to a wide range of apoptotic triggers and multi-drug resistance for cancer cells. We developed a cell-based assay to screen for small molecule inhibitors of Bcl-xL, a closely related homolog of Bcl-2 with available NMR and crystallographically-determined solution structures. Cells with overexpressed Bcl-xL (5-6 fold) were highly resistant to diverse apoptotic

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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stimuli, including the anti-cancer agents doxorubicin and cisplatin, and tumor necrosis factor. Testing of a library of commercially available small molecules identified several compounds with selective toxicity for cells that highly expressed Bcl-xL compared to control cells. One of these was antimycin A, a known inhibitor of mitochondrial electron transfer complex III. Inhibitors of electron transport at other sites did not exhibit selective killing of Bcl-xL-expressing cells.

Using molecular docking analysis, antimycin was predicted to bind at a hydrophobic groove on the Bcl-xL surface. This groove forms an interaction surface for heterodimerization with Bcl-2 family proteins that possess opposite, pro-apoptotic activity. Physical interaction of antimycin and Bcl-xL was demonstrated by differential fluorescence spectroscopy and isothermal titration calorimetry, with a Kd ~1 mM. Binding was competitive with a hydrophobic groove-binding peptide from the BH3 domain of the pro-apoptotic Bak protein. Antimycin and a proapoptotic BH3 domain peptide both selectively triggered loss of mitochondrial DYm and swelling in Bcl-xL-expressing mitochondria. Bcl-2 family proteins form membrane pores in synthetic lipid bilayers, first recognized by the structural similarity of Bcl-xL to the translocation domain of diphtheria toxin. Pore activity of Bcl-xL, measured by fluorescent dye leakage from synthetic liposomes, was completely inhibited in the presence of antimycin.

These results demonstrated that small non-peptide ligands can directly influence the function of Bcl-2 proteins. Non-peptidyl compounds related to antimycin may be clinically useful to target drug-resistant tumor cells overexpressing Bcl-xL. However, the inhibitory effect of antimycin on mitochondrial oxidative phosphorylation would preclude further drug development. Chemical modification of antimycin to create the 2-methoxy derivative resulted in loss of respiratory inhibition with the selective toxicity for Bcl-xL-expressing cells retained. Furthermore, both antimycin and the 2-methoxy derivative inhibit the pore-forming activity and bind Bcl-xL with similar affinities. Administration of 2-methoxy antimycin A1 induced regression of human myeloma tumors grown in nod/ scid mice without apparent toxicity.

In order to characterize the molecular interaction between antimycin A and Bcl-xL in more detail, a series of Bcl-xL proteins with single amino acid substitutions were made to probe the binding pocket surface. We identified mutations that reduce or eliminate sensitivity to antimycin in cell-based assays without compromising the anti-apoptotic functions of Bcl-xL. Loss of antimycin sensitivity correlates with a reduced ability of antimycin to bind and inhibit pore activity of the recombinant mutant proteins. X-ray crystallographic structures have been obtained for several antimycin-resistant mutants. Of particular interest, the F146W Bcl-xL

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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mutant remains antimycin-sensitive, although without detectable antiapoptotic activity, therefore suggesting that allosteric regulation or additional functions of Bcl-xL mediate the effects of antimycin. These studies provide detailed structure-activity relationships that can be used for further refinement of Bcl-xL inhibitors.

CONTROL OF VERTEBRATE MUSCLE CHARACTER

Simon M. Hughes, Ph.D.

MRC Muscle and Cell Motility Unit and Developmental Biology

Research Centre

King’s College London

Skeletal muscle contractile properties are controlled by genetic and environmental factors. We are studying muscle development in the early vertebrate embryo using zebrafish as a model system. We have shown that hedgehog signals from the ventral midline are required for correct slow muscle formation. Hedgehogs act, at least in part, through maintaining expression of myogenic transcription factors of the MyoD family. We are investigating the role of other signals and factors in muscle patterning and growth by using the advantages of zebrafish genetics and the optical clarity of the embryo to permit analysis of the behaviour of living cells in response to manipulations. In later life, electrical activity is a major determinant of muscle fibre character. A second line of work examines how electrical activity regulates muscle fibre size and type in mice. We want to understand how electrical signals are integrated over time and turned into a binary decision controlling gene expression on long timescales. We are investigating the role of MyoD family proteins in adult muscle. Ultimately, we aim to unify our embryonic and adult work to understand how muscle character is controlled.

A PROTEOME-WIDE SCREEN FOR PROTEINS REQUIRED FOR CANCER CELL INVASIVENESS BY HIGH-THROUGHPUT CALI

Daniel Jay, Ph.D.

Department of Physiology

Tufts University School of Medicine

A critical aspect of cancer is that cancer cells invade healthy tissue and metastasize to form secondary tumors. Finding proteins required for cancer cell invasiveness would provide new targets for drugs that would

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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impede the spread of cancer and thus improve patient survival. The success of the Human Genome Project and the emergence of proteomics provide biologists with a large number of candidate proteins of potential disease importance. The major bottleneck is in efficient methods to test the functional relevance of these candidates. Previously, our laboratory developed Chromophore-Assisted Laser Inactivation (CALI) to use dye-labeled non-blocking antibodies and laser light to inactivate proteins in situ to validate their roles in cellular processes of clinical importance. We are now applying CALI to test which of the many proteins in the proteome is required for cancer cell invasiveness. We have coupled a multiplex version of CALI to a high-throughput cellular assay for cancer cell invasiveness and are screening antibody libraries directed against a significant fraction of the proteome. Antibodies that affect invasion upon light irradiation are subsequently used to immunoprecipitate the target antigen for identification by mass spectrometry. This screen is akin to mutagenesis but has the advantages of direct protein validation in cells of disease relevance such a human cancer cell lines. We believe that such a reverse proteomic approach is an efficient route to identifying and validating novel protein targets important for cancer cell invasion. Furthermore these studies establish the paradigm that CALI may be used to generate the “knockdown” of protein function to directly validate proteins for a wide array of cellular processes important in disease.

FUNCTIONAL ARCHITECTURE OF THE MAMMALIAN OLFACTORY SYSTEM

Lawrence C. Katz, Ph.D.

Department of Neurobiology

Howard Hughes Medical Institute

Duke University Medical Center

Insights into the functional representations of odors in mammals have been gleaned largely from 2-deoxglucose studies, which greatly limit the range of experiments. To overcome these limitations, we introduced the use of optical imaging of intrinsic signals to rapidly determine the representations of different odorants in the olfactory bulb. In both rats and mice we found that features predicted by molecular studies—bilateral symmetry, stereotypy between animals—had clear correlates in the functional representation of odorants. We uncovered a clear “odortopic” organization in which small changes in molecular structure resulted in spatial shifts in the ensemble of glomeruli representing an odorant. To examine

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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whether the distinctions observed in glomerular activation patterns could allow behavioral discrimination of closely related odorants, we have combined imaging of enantiomers—molecules which differ only in their ability to rotate light—with behavioral testing. Humans can only distinguish certain enantiomers, but rats reliably distinguished all enantiomers tested. The patterns of glomerular activation reflected this ability: some glomeruli were activated in common by both enantiomers, but others were activated exclusively by one or the other, implying the existence of enantiomer-selective odorant receptors.

The olfactory system has an especially intimate connection to the circuitry involved in learning and memory in the mouse brain. We are interested in the cellular and molecular mechanisms by which behaviorally relevant memories are acquired and stored. Using multiphoton imaging in transgenic mice, we’ve been able to visualize the structural stability of dendrites in the olfactory bulb of adult mice, and to probe whether learning is accompanied by morphological changes. In other experiments, we’ve employed a miniature, head-mounted microdrive to record from individual olfactory neurons in awake, behaving animals during and after the acquisition of olfactory memories.

METABOLIC “SWITCHES” IN THE DEVELOPING AND DISEASED HEART: FROM INBORN ERRORS TO THE PPAR GENE REGULATORY PATHWAY

Daniel P. Kelly, M.D.

Center for Cardiovascular Research

Departments of Medicine and Molecular Biology and Pharmacology

Washington University School of Medicine

The energy demands of the postnatal mammalian heart are met by high capacity mitochondrial pathways specialized to oxidize fatty acids to produce energy. In the early phases of this project, the identification and molecular genetic characterization of children with inborn errors in mitochondrial fatty acid oxidation (FAO) provided clues that single gene disturbances in cardiac energy metabolism can lead to heart failure and sudden death. Next, we found that the preference of the normal heart for fat versus carbohydrates as substrate for energy production is dynamically regulated during development and in diverse physiologic contexts. Importantly, in certain common acquired forms of heart disease, such as cardiac hypertrophy due to hypertension or coronary artery disease, the capacity of the myocardium to oxidize fats is dramatically reduced, a

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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metabolic phenotype remarkably similar to that of humans with genetic defects in the mitochondrial FAO pathway. Accordingly, we sought to delineate the molecular regulatory events involved in the physiologic control of mitochondrial energy production. We found that tight coordinate control of mitochondrial FAO enzyme gene expression is orchestrated, in part, by a lipid-activated nuclear receptor transcription factor, the peroxisome proliferator-activated receptor α (PPARα). Physiologic studies of genetically engineered “loss-of-function” and “gain-of-function” mice have demonstrated that PPARα maintains lipid and energy homeostasis in the mammalian heart in the context of diverse physiologic conditions, thus serving as a cellular energy metabolic “stress” factor. Mice lacking PPARα die suddenly following a physiologic stress such as fasting or ventricular pressure overload, a phenotype remarkably similar to that of children with genetic defects in the FAO pathway. The activity of PPARα is modulated by its interaction with the inducible transcriptional coactivator PGC-1. PGC-1 expression is induced in the heart following birth and in response to fasting or exercise, conditions known to trigger increased demands for mitochondrial ATP production. Gain-of-function studies in cardiac myocytes in culture and in transgenic mice have shown that PGC-1 not only activates PPARα but also promotes mitochondrial biogenesis through separate transcriptional regulatory pathways. Thus, the PPAR/PGC-1 complex serves as a master regulator of mitochondrial function in the heart. The expression and activity of the PGC-1/PPARα transcriptional regulatory complex is dysregulated in several common cardiovascular disease states. Pathologic cardiac hypertrophy or reduced oxygen availability, such as occurs with myocardial infarction or congenital heart disease, deactivates PPARα/PGC-1 at both transcriptional and post-transcriptional levels in rodents and humans. Conversely, the activity of PGC-1/PPAR, as a ligand-activated transcription factor, shows promise as a target for novel therapeutic strategies aimed at modulating myocardial metabolism in human disease states such as heart failure, diabetes, and myocardial infarction.

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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THE GENETIC BASIS OF VERTEBRATE EVOLUTION

David Kingsley, Ph.D.

Department of Developmental Biology

Howard Hughes Medical Institute

Stanford University School of Medicine

Despite great interest in the mechanisms that control the origin of new morphological and physiological traits in animals, we still know relatively little about the number of genetic changes required to evolve new traits, the types of genes involved, and whether evolution proceeds primarily by changes in coding or regulatory regions. To address these questions we have initiated a genome-wide linkage analysis of evolutionary change in fish. Three-spine sticklebacks are small teleost fish that have undergone one of the most recent and dramatic evolutionary radiations on earth. Following the widespread melting of glaciers 10,000 years ago, marine sticklebacks colonized thousands of newly created lakes and streams. Many of the freshwater populations subsequently diverged in response to local environmental conditions, generating a large number of isolated populations with dramatic changes in body size, skeletal armor, feeding modifications, and physiological traits at different locations around the world. Although many of these distinct stickleback populations meet a formal species definition, the reproductive barriers between contrasting fish types can be overcome using artificial fertilization in the laboratory, making it possible to carry out a formal genetic analysis of the mechanisms responsible for evolutionary change in vertebrates.

To take advantage of this system we have built the first genome-wide linkage map of three-spine sticklebacks. We have also collected and set up crosses between diverse types of sticklebacks around North America and Northern Europe, generating a large number of progeny segregating a wide range of interesting differences in body armor, feeding structures, anterior posterior patterning, and temperature and salinity preference. Initial mapping results suggest that many of these traits are controlled by a relatively small number of major chromosome regions. We are currently identifying the genes responsible for evolutionary change within these regions, using many of the same forward-genetic and positional cloning methods we have previously used successfully to identify genes responsible for classical morphological traits in mice. These studies should make it possible to determine the number and type of molecular alterations that underlie evolutionary changes in natural populations. In addition, the thousands of independent lakes represent a large number of independent evolutionary experiments. This will make it possible to test evolution

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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proceeds by similar or different mechanisms when similar fish evolve in independent locations around the world.

DID BLOOD VESSELS EVOLVE FROM BLOOD CELLS?

Mark Krasnow, M.D., Ph.D.

Department of Biochemistry

Howard Hughes Medical Institute

Stanford University School of Medicine

My lab is genetically dissecting the developmental programs that control formation of the tracheal (respiratory) system of Drosophila and the mammalian lung. Both of these organs are vast branching networks of epithelial tubes that serve as portals of oxygen entry and transport in the body. One major difference between them is that in Drosophila the tubes extend throughout the body whereas in mammals the tubes end in the lung and oxygen must be transferred to red blood cells and circulate through blood vessels to reach the rest of the body. Drosophila lack blood vessels, but they do have an open circulatory system in which the heart pumps blood through the body cavity and the blood and blood cells percolate around the tissues.

Genetic studies in a number of labs including my own have demonstrated remarkable parallels between Drosophila and mammals in the genetic programs that control development of the respiratory system, heart, and blood cells. The results imply that the Drosophila and mammalian organs are homologous structures, not just functional analogues as was believed for centuries. But Drosophila lack blood vessels, so how did blood vessels arise during evolution? If blood vessels arose late, how did they acquire their intimate relationship with the lung, and how did blood cells end up inside of the vessels? Recently, we discovered that the Vascular Endothelial Growth Factor (VEGF) pathway, a receptor tyrosine kinase signaling pathway that plays a central role in blood vessel development in mammals, controls blood cell development in Drosophila. I will describe how the Drosophila VEGF pathway controls developmental migrations of blood cells, and how these and other recent results point to an intimate evolutionary and developmental association between blood cells and blood vessels. This leads us to speculate that blood vessels arose from blood cells during evolution of the vascular system.

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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RNA-PROTEIN INTERACTIONS: ROLE IN THE REGULATION OF ONCOGENE EXPRESSION AND NUCLEAR RECEPTOR ACTION IN HORMONE-DEPENDENT CANCER

Peter J. Leedman, Ph.D.

Western Australian Institute for Medical Research

University of Western Australia

The proliferation of breast and prostate cancer cells is dependent upon a variety of growth factor and hormonal signals. Overexpression of members of the type I family of protein tyrosine kinase growth factor receptors (EGF-receptor and erbB-2) plays a critical role in the development of breast and prostate cancer. Estrogens and androgens, acting via nuclear hormone receptors (ER and AR, respectively), are also significant contributors to the proliferation of breast and prostate cancer cells. These growth factor receptors and their signaling pathways as well as the coregulators associated with these nuclear receptors are excellent targets for therapeutics. Interactions between RNA and proteins are recognised as making a major contribution to a variety of cellular processes. The field is expanding rapidly, with therapeutic targeting of RNA-protein interactions a growing industry. In the last few years, my laboratory has focused on the elucidation of novel RNA-protein interactions involving specific growth factor receptors, nuclear receptors and coregulators in breast and prostate cancer, with a view to developing specific modulators of tumor cell growth.

Oncogenes and Breast Cancer. Using yeast three hybrid screening we have cloned proteins from a human breast cancer library that target growth factor receptor RNA. One clone encodes a protein that binds growth factor receptor protein (via a SH2 domain) and also growth factor receptor mRNA (via a novel RNA-binding domain). Functional studies provide a role for these novel RNA-protein interactions in regulating receptor expression. Structural studies are in progress to design novel small organic molecule modulators of the interaction for therapeutics.

The Androgen Receptor and Prostate Cancer. AR mRNA is regulated in a cell-specific and divergent manner in prostate and breast cancer cells. We recently identified novel AR mRNA cis-trans interactions in these cells, and found that the AR is a target for a complex of RNA-binding proteins. One of these is HuR, a ubiquitously expressed member of the Elav/Hu family of RNA-binding proteins involved in the stabilization of several mRNAs. Poly(C) binding proteins, previously implicated in the control of mRNA turnover and translation, also bind AR mRNA and do so simultaneously with HuR. The functional role of these proteins in the regulation of AR expression in cancer cells is currently under investigation.

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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Novel RNA Coactivator Binding Proteins. The mechanisms underlying ER-mediated transactivation in human breast cancer cells involve a complex set of interactions between the ER and a variety of coregulators. SRA (steroid receptor RNA coactivator), the only described RNA coactivator, plays an important role in this process, and is aberrantly expressed in human breast cancer cells. SRA contains several stable stem-loops suggesting that it is the target for a variety of other coregulators. For example, SHARP is a potent transcriptional repressor that binds to SRA and represses SRA-potentiated ER transactivation. However, the identity and function of additional SRA-binding proteins, as well as the specific target SRA RNA-binding motifs remain to be clearly elucidated. We recently identified a novel family of SRA-binding proteins with a characteristic RNA-binding motif, which modulate SRA-mediated transactivation. Interestingly, these SRA-binding proteins are predominantly nuclear, consistent with their putative role as regulators of transcriptional coactivation. Determining their role in the regulation of estrogen action in breast cancer cells will be of great interest.

HAIRPIN OPENING AND OVERHANG PROCESSING BY AN ARTEMIS/DNA-DEPENDENT PROTEIN KINASE COMPLEX: ROLES IN V(D)J RECOMBINATION AND NONHOMOLOGOUS DNA END JOINING

Yunmei Ma, Ulrich Pannicke, Klaus Schwarz, and Michael R. Lieber, M.D., Ph.D.

Departments of Pathology and Biochemistry and Molecular Biology

University of Southern California School of Medicine

Mutations in the Artemis protein in humans result in hypersensitivity to DNA double-strand break-inducing agents and absence of B and T lymphocytes (radiosensitive severe combined immune deficiency [RS-SCID]). We have now shown that Artemis forms a complex with the 469 kDa DNA-dependent protein kinase (DNA-PKcs) in vitro and in vivo in the absence of DNA. The purified Artemis protein alone possesses singlestrand specific 5′ to 3′ exonuclease activity. Upon complex formation, DNA-PKcs phosphorylates Artemis, and Artemis acquires endonucleolytic activity on single- to double-strand DNA transitions (including 5′ and 3′ overhangs, as well as hairpins). Finally, the Artemis:DNA-PKcs complex can open hairpins generated by the RAG complex from a 12/23-substrate pair. Thus, DNA-PKcs regulates Artemis by both phosphorylation and complex formation to permit enzymatic activities that are critical

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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for the hairpin opening step of V(D)J recombination and for all of the 5′ and 3′ overhang processing in nonhomologous DNA end joining.

FLUCTUATIONS IN NEUROTRANSMITTER CONCENTRATIONS IN HUMAN BRAIN DIALYSATES DURING SLEEP OR WAKEFULNESS, DURING EPILEPTIC SEIZURES OR FOLLOWING COGNITIVE CHALLENGE

Nigel T. Maidment, Ph.D.,1 I. Fried,2 F. Lopez,1 J. M. Zeitzer,3 E. J. Behnke,3 L. C. Ackerson,1 J. Engel, Jr.,3 and C. L. Wilson3

1  

Department of Psychiatry and Biobehavioral Sciences Neuropsychiatric Institute, UCLA School of Medicine

2  

Division of Neurosurgery, UCLA School of Medicine

3  

Department of Neurology, UCLA School of Medicine

We have applied microdialysis to the measurement of extracellular concentrations of monoamine (dopamine, norepinephrine and serotinin) and amino acid (glutamate, aspartate, GABA, taurine) neurotransmitters in several regions of the human brain. Subjects were epilepsy patients who were candidates for neurosurgical resection of tissue encompassing the seizure focus. The data to be presented was collected during one to three weeks following an initial surgical procedure for implantation of deep EEG electrodes in order to determine the location of the seizure focus. Data were collected during quiet wakefulness and during several stages of sleep, during epileptic seizures, and during performance of cognitive tasks.

Materials and Methods. Each implanted assembly consisted of MRI-compatible, flexible, polyurethane probes with seven 1.5-mm-wide platinum contacts with intercontact separation of 1.5-4 mm. These contacts enabled recording of EEG at various sites along the electrode trajectory. Microdialysis probes and platinum/iridium microwires for single unit recording were inserted through the lumen of these probes. The microdialysis membrane (Cuprophan, 200±15-µm diameter, 10 mm length) protruded 10 mm beyond the EEG probe tip. Two fused silica tubes contained within the membrane were used for inflow and outflow of the dialysate. Electrode assemblies were stereotactically placed with MRI and angiographic guidance. Data described were collected in the amygdala, orbital frontal cortex, and anterior hippocampus.

Phosphate-buffered artificial cerebrospinal fluid (aCSF) (NaCl, 125 mM; KCl, 2.5 mM; NaH2PO4, 0.5 mM; Na2HPO4, 5 mM; CaCl2, 1.2 mM; MgCl2, 1 mM; ascorbic acid, 0.2 mM; pH 7.3 - 7.4) was perfused through

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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the probes at a flow rate of 1.2 µl/min. Sterile, disposable plastic 3-ml syringes were used for a CSF delivery, driven by a pair of CMA-102 minipumps. Flow was established before placement of the microdialysis probes in the brain, and maintained during application of the head dressing, in the recovery room and on the neurosurgery ward, where collection was started on a half-hour basis, using a CMA automated fraction collector. When a seizure occurred, the collection period was immediately reduced to 5 minutes. The “dead space” in the collection tubing allowed for collection of four 5-minute pre-seizure samples. In cases where there were multiple seizures, continuous 5-minute samples were taken. Similarly, the sampling interval was reduced to 5 or 10 minutes during cognitive tasks or sleep studies. Samples were stored at -80°C prior to analysis by HPLC with fluorometric or electrochemical detection.

Results and Discussion. Transient increases in glutamate, aspartate, GABA and taurine were observed in several brain regions sampled during seizure activity, similar to previous reports in the literature. However, this was not a consistent finding. In many cases seizures produced little or no perturbation of extracellular amino acid levels. Dialysate concentrations of dopamine and serotonin in the amygdala varied with state. Dopamine levels were high during waking and low during all stages of sleep but showed no difference between stages of sleep. On the other hand, serotonin was significantly lower during slow wave sleep than during wakefulness, and lower still in REM sleep. Significant increases in dopamine (but not serotonin) were observed in the amygdala during the transition between quiet waking and performance of a simple cognitive task such as reading and during a task requiring working memory. Furthermore, the time-course of the increase in dopamine during an extended learning task correlated with the learning curve during the task.

These data demonstrate that robust and sustained changes in the concentrations of extracellular monoamine neuromodulators occur in the human brain during different behavioral states that are readily measured by microdialysis. The application of recent improvements in the speed and sensitivity of analytical procedures for measurement of these, and other, neuromodulators/neurotransmitters promises to provide further insights into their role in mediating changes in human behavior.

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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IDENTIFICATION OF NOVEL PROTEIN-PROTEIN INTERACTION DOMAINS INVOLVED IN CELL SIGNALING AND PROTEIN TARGETING

Benjamin L. Margolis, M.D.

Department of Internal Medicine and Biological Chemistry

Howard Hughes Medical Institute

University of Michigan Medical School

With the sequencing of the human genome complete it becomes increasingly important to understand the role of newly identified but uncharacterized proteins. One mechanism to understand the biological role of proteins is to identify functional domains within the sequence of the proteins. One type of motif found in many proteins is the protein-protein interaction domain. Our laboratory works at defining these domains and trying to understand their biological function in mammalian cells. Presently we are focused on understanding the role of protein-protein interaction domains in signal transduction and protein targeting. Our group identified and has extensively studied a protein-protein interaction domain called the Phosphotyrosine Binding (PTB) domain.

Our initial work showed that proteins such as Shc contain a PTB domain and play an important role in signaling by growth factor receptors. The PTB domain in Shc allows it to bind to tyrosine phoshorylated growth factor receptors and connect certain growth factor receptors to the Ras signaling pathway. More recent work has shown that the PTB domains do not always bind to tyrosine phosphorylated proteins and can interact with proteins in a nonphosphotyrosine dependent fashion. This has led to discoveries by our group and others into the important role of PTB domains in cell signaling, neurological development, and cholesterol homeostasis.

Our most recent work has focused on the PTB domain protein, X11. This protein is the mammalian homologue of the C. elegans Lin-10 protein. Work of another Markey Scholar, Stuart Kim, has demonstrated that Lin-10 is complexed with two other proteins Lin-2 and Lin-7 and these proteins are crucial for the targeting of the worm EGF-Receptor to the basolateral surface. Our work has demonstrated a similar complex in mammalian brain and a complex of mLin-2 and mLin-7 in mammalian epithelia. In mammalian epithelia we have found that mLin-2 is not the only partner of mLin-7 but that at least five different proteins can bind mLin-7. Two new proteins we identified that associate with mLin-7 are called Pals for Proteins Associated with Lin-7. By identifying these additional mLin-7 binding partners we have been able to define a new domain in the Pals and mLin-2 proteins that allow them to interact with mLin-7.

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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We have also demonstrated that this domain is present in the aminoterminus of Lin-7 itself. We call this new domain the L27 domain because we first identified it in Lin-7 and Lin-2 proteins. Proteins like Lin-2 and Pals1 contain two L27 domains, the carboxy-terminal L27 (L27C) domain binds to the L27 domain of mLin-7 whereas the partners of the amino terminal L27 (L27N) domain were unknown. We have been examining the role of the L27N domains in the targeting of the Pals1 protein. We have found that Pals1 is targeted to the tight junction of epithelia cells by the L27N domain and that this domain binds a novel PDZ domain protein called Pals1 Associated Tight Junction Protein (PATJ). Our data finds that Pals1 and PATJ form a tight junction complex in mammalian epithelia that is conserved in Drosophila proteins known to be crucial for epithelial polarity.

GLOBAL GENE EXPRESSION IN SALMONELLA TYPHIMURIUM, AND GENE CONTENT SHARED WITH OTHER SALMONELLA, DETERMINED BY MICROARRAYS

Michael McClelland, Ph.D.,1 Jonathan Frye,1 Rick Wilson,2 Sandy Clifton,2 John Spieth,2 Ken Sanderson,3 Steffen Porwollik1

1  

Sidney Kimmel Cancer Center

2  

Genome Sequencing Center, Washington University School of Medicine

3  

University of Calgary

Following our sequencing of the Typhimurium genome we have arrayed the complete open reading frame set by spotting PCR amplified ORFs on glass slides. We have begun a survey of expression changes in Typhimurium in response to stresses that simulate aspects of the host response to infection, and expression changes induced by mutations of the major known regulators of pathogenesis. In addition, genomes from other Salmonella were hybridized to the array. Despite being closely related, the Salomonella differ widely in host range and pathogenic routes. We present data on the presence and absence of homologues of S. typhimurium LT2 genes obtained from members of all seven Salmonella subspecies, and from Salmonella bongori. Genes that were acquired during key stages of Salmonella evolution were determined including those gained at the branching point of subspecies I, which contains all the major Salmonella pathogens of warm-blooded animals.

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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THE MAP OF CHEMICAL SPACE ON THE OLFACTORY BULB

Markus Meister, Ph.D.

Molecular and CellularBiology

Harvard University

Our nose senses odors using several hundred types of olfactory neurons. Each type expresses a distinct receptor protein to bind ligands in the air stream. Neurons of the same type project their axons to the same location in the olfactory bulb, a small ball of fibers ~100 µm across, called a glomerulus. It is now possible to monitor the neural activity in ~200 of these glomeruli simultaneously, through methods of optical recording from the brain surface of anesthetized animals. We have combined this recording method with a stimulating machine that can rapidly deliver ~1000 odors to the animal. In this way, one can measure the sensitivity of 200 olfactory receptor proteins to hundreds of different ligands. This is providing new insight into the affinity spectra of these receptors, the nature of the neural code for smells, and the structure and development of the olfactory bulb.

PROTEOME STARGAZING

Jonathan Minden, Ph.D.

Department of Biological Sciences

Carnegie Mellon University

Proteomics is a new and burgeoning field that has emerged as a major focus since the sequencing of the human genome and the genomes of many other model organisms. The proteome is defined as the collection of proteins found in cells, tissues, and organisms. Individual cells may express 5,000-10,000 different proteins, while a whole organism can express >1,000,000 protein isoforms. One of the main goals of proteomics is to identify protein changes that occur normally during development and tissue differentiation and as a result of disease, drug treatment, and environmental change. There are two main components to proteomics: separation of cellular proteins into individual components and identification of the genes that encode individual proteins. Protein separation is usually conducted by two-dimensional gel electrophoresis, which separates proteins based on size and charge. Identification of the gene encoding isolated proteins is done by mass spectrometry where one compares the

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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mass of specifically-generated protein fragments to a database of predicted protein fragments.

The scale of the genome sequencing projects pales in comparison to the goal of proteome projects. There are plans to search for protein changes in dozens of different tissues at dozens of different stages of development and dozens of different diseases from hundreds of samples and in the presence of thousands of different drugs and conditions. The number of possible experiments is truly staggering. There is great need to increase the rate of proteome analysis 10-100 fold, or more. We have developed a method, called difference gel electrophoresis (DIGE), to rapidly identify protein changes between two or three samples on the same two-dimensional electrophoresis gel. This method relies on fluorescently tagging all proteins in each sample with one of a set of matched fluorescent dyes that do not affect the relative mobility of proteins during electrophoresis. This method greatly reduces the complexity of the search for protein differences from several thousand proteins observed in the protein extract to several dozen candidate proteins that are significantly different between the samples. DIGE is more sensitive than silver staining and can detect changes as little as 0.01 percent of total protein. The imaging system can detect a 10,000-fold range of protein concentrations; silver staining can only detect a 30-fold concentration range. This level of sensitivity and discrimination challenges the performance of mass spectrometers to identify the genes that encode the proteins difference that we can detect using DIGE. This presentation will describe the uses of DIGE and future challenges for proteome analysis.

FUNCTIONAL GENOMIC ANALYSES OF CELLULAR MORPHOLOGY USING HIGH-THROUGHPUT RNAI SCREENS

A. Kiger,* 1 B. Baum,* 1,4 S. Armknecht,1 M. Chang,1 S. Jones,2 B. Sönnichsen,2 C. Echeverri,2 M. Jones,3 A. Coulson,3 Norbert Perrimon, Ph.D.1

1  

Department of Genetics, HHMI/Harvard Medical School

2  

Cenix BioScience GmbH, Dresden

3  

Sanger Centre, Cambridge

4  

Ludwig Institute, University College of London

*  

These authors contributed equally.

The vast potential of the genome sequence relies on new technologies capable of functional and systematic analyses of the ~14,000 predicted Drosophila genes. The use of RNA-interference (RNAi) in Drosophila cell

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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cultures by the simple addition of dsRNA to the culture medium provides an effcient method to reduce or eliminate the expression of specific genes, yielding phenocopies of loss-of-function mutations. The availability of predicted gene-specific sequences in combination with RNAi application in cell cultures enables the design of a large-scale approach to study of cell-biological functions. I will describe our efforts at developing high-throughput screens based on RNAi methodology using Drosophila cell cultures in 384-well plates and automated microscopic imaging.

In a pilot screen, we assembled a set of 1000 dsRNAs representing genes predicted to encode for regulators central to many fundamental cellular processes, including all the small GTPases and GTPase regulators, kinases, and phosphatases. We screened this collection for genes that alter cell morphology and cytoskeletal organization in different Drosophila cell lines. With analysis of actin filaments, microtubules, and DNA, we were able to visibly distinguish phenotypic classes that revealed specific functions associated with specifc members within each gene family. The identification of all the dsRNAs that shared similar phenotypes revealed genes that delineate specific pathways that affect cell form and function. To identify additional components in the pathway, we carried-out RNAi modifier screens. By using RNAi as a screening tool we have been able to identify functions for specific GTPases and associated pathways.

CATHEPSIN B: A NOVEL DRUG TARGET FOR TOXOPLASMOSIS

Sharon Reed, M.D.

Department of Pathology and Medicine

University of California, San Diego

Toxoplasmosis is one of the most common parasitic infections of man, with infection rates approaching 90 percent in some countries. Although the majority of patients have asymptomatic, dormant infection for life, serious complications result from congenital infection or reactivation disease in immunocompromised patients, particularly with AIDS. Toxoplasma gondii encephalitis is the most common cause of central nervous system infection in patients with AIDS and is uniformly fatal unless diagnosed and treated early. Current treatment regimens are often limited by toxic side effects of the drugs. Thus, further understanding of the pathogenesis of infection by T. gondii and the identification of potential drug targets is critical.

Cysteine proteinases play a key role in a number of host-parasite

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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interactions and can be targeted by specific inhibitors. We have cloned a cathepsin B gene, TgCP1, from T. gondii. The enzyme contains a classic pre-pro sequence, an active site triad, and dipeptidyl carboxypeptidase activity characteristic of cathepsin B’s of higher eukaryotes. When models of the T. gondii enzyme were compared to human and rat cathepsin B, a high degree of similarity between the active site regions was found. The structure of the enzyme predicts substrate specificity for positively charged amino acids, which was confirmed with both purified native and active recombinant enzyme.

Cathepsins in higher eukaryotes are located in acidic lysosomes, where they play an important role in protein processing and breakdown. Classic lysosomes are not detected in toxoplasma. Instead, TgCP1 localizes to the rhoptry, a unique club-shaped organelle at the apical end of the parasite. Rhoptries are critical for host cell invasion and are packaged with specialized hydrolases, cholesterol, and membranes for secretion, characteristic of lysosomal-like organelles. All rhoptry proteins are synthesized as prepro-proteins, which must be processed to their active form. We found that specific cathepsin B inhibitors blocked the processing of ROP2, suggesting that it was involved in rhoptry protein processing. When specific inhibitors were added to infecting parasites, invasion of host cells was blocked. If cell-permeable inhibitors were added after infection was initiated, significantly fewer host cells were infected. These findings suggest that specific inhibitors may block both invasion and intracellular survival of the parasite.

In future studies, we plan to evaluate the role of TgCP1 in parasite survival using specific inhibitors and anti-sense RNA, crystallize the enzyme for drug modeling to identify novel new drugs, and test these compounds in animal models of infection. These studies should not only provide important information about the pathogenesis of one of the most serious opportunistic infections of AIDS patients, but could also establish a role for cysteine proteinase inhibitors as novel new therapeutic agents for toxoplasmosis.

DEFINING THE FUNCTIONS OF HNRNP PROTEINS USING GENOMICS AND DROSOPHILA GENETICS

Marco Blanchette, Emmanuel Labourier, and Donald Rio, Ph.D.

Department of Molecular and Cell Biology

University of California, Berkeley

Alternative splicing is a strategy that eukaryotic cells have evolved to generate multiple protein isoforms from a single gene. Computer analysis

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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of the human genome predicts that 40 to 60 percent of all genes undergo alternative splicing. Despite the fact that the biochemistry of the spliceosome is well understood, little is known about the mechanism governing alternative splice site selection. The Drosophila hrp48 and PSI genes encode RNA binding proteins structurally related to the mammalian hnRNP (heterogeneous nuclear ribonucleoprotein) A/B and hnRNP K proteins, respectively. HnRNP proteins are a large family of nucleic acid binding proteins involved in many essential cellular processes, such as alternative pre-mRNA splicing regulation, RNA stability, RNA transport, translational regulation, as well as telomere maintenance and transcriptional regulation. Previous studies have shown that hrp48 and PSI are involved in the soma-specific inhibition of the P-element transposase third intron pre-mRNA splicing. In addition to their roles in the regulation of P-element transposase expression, both hrp48 and PSI play other essential functions in Drosophila since mutant hrp48 and PSI strains exhibit larval lethality. Identification of cellular RNAs bound by both proteins will help to clarify their cellular functions. Using high density cDNA microarrays, a genome-wide search for PSI and hrp48 RNA target has been performed. Briefly, hrp48- or PSI-containing ribonucleoprotein particles were purified from embryonic nuclear extracts using gradient sedimentation and immunoaffinity chromatography. The protein-associated RNAs were then extracted, reverse-transcribed, labeled and hybridized to Drosophila cDNA microarrays representing more than 5000 different genes. Using this approach, several putative targets have been identified for both hrp48 and PSI. Using Drosophila strains expressing a mutant form of the PSI protein, we have found that alternative splicing of one such target, the sqd/hrp40 gene was affected in the PSI mutant. We are currently in the process of developing in vitro assays to address how PSI controls alternative splicing of the sqd pre-mRNA. We are also investigating if expression or processing of some of the hrp48 target RNAs are affected in an hrp48 mutant genetic background. The splicing pattern of intron-containing targets will be assayed by RT-PCR experiments while the level of mRNA expression is being tested using cDNA microarrays. Finally, we will look at the cellular localization of the putative hrp48-regulated target RNAs in the RNA interference hrp48-disrupted cells using whole mount embryo in situ hybridization. Together, these molecular genetic analyses and the identification of putative hrp48- and PSI-regulated cellular target RNAs will help to understand the roles these RNA binding protein in play for Drosophila mRNAs. More generally, these studies will give us tools to explore how these classes of hnRNP proteins function in RNA processing, transport and/or stability.

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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HYPOXIA-INDUCIBLE FACTOR 1: MASTER REGULATOR OF OXYGEN HOMEOSTASIS IN HEALTH AND DISEASE

Gregg L. Semenza, M.D., Ph.D.

McKusick-Nathans Institute of Genetic Medicine

The Johns Hopkins University School of Medicine

Hypoxia-inducible factor 1 (HIF-1) is a global regulator of O2 homeostasis that activates the transcription of >50 known target genes encoding proteins which mediate cellular adaptation to O2 deprivation (via induction of glycolytic metabolism and growth factor signaling pathways) or increased O2 delivery (via regulation of the erythroid, cardiac, vascular, and respiratory systems). Progress in elucidating the mechanisms and consequences of HIF-1 activity since our isolation of the factor in 1995 is summarized below.

Mechanism of O2-regulated HIF-1α activity. HIF-1α is a heterodimer consisting of a constitutively-expressed HIF-1ß subunit and a HIF-1α subunit, the expression of which is determined by the cellular O2 concentration. HIF-1α is modified by prolyl hydroxylases (PHs) which require O2 at concentrations that are rate-limiting under physiologic conditions. The von Hippel-Lindau tumor suppressor protein (VHL) binds to HIF-1α only when the protein has been prolyl-hydroxylated and targets it for ubiquitination and proteasomal degradation. In addition to the regulation of HIF-1α protein expression, the transcriptional activity of HIF-1α is also negatively regulated under normoxic conditions by interactions with VHL and the corepressor FIH-1α, which both recruit histone deacetylases to HIF-1, thus providing a molecular basis for the regulation of gene expression by HIF-1 in response to changes in cellular oxygenation.

Consequences of HIF-1 deficiency in knockout mice. Analysis of knockout mice has revealed that HIF-1α expression is required for embryogenesis. HIF-1α-null mice die at midgestation with neural tube defects, cardiac and vascular defects, and massive mesenchymal cell death. Mice that are heterozygous for the null allele develop normally and are indistinguishable from their wild-type littermates under normoxic conditions. When wild-type mice are subjected to chronic hypoxia, they develop pulmonary vascular remodeling and right ventricular hypertrophy. These responses are markedly impaired in the heterozygotes, implicating HIF-1α in the pathogenesis of pulmonary hypertension, a lethal complication of chronic lung disease. The heterozygous mice also have ventilatory abnormalities due to a failure of the carotid body chemoreceptors to sense and/or respond to hypoxia.

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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Role of HIF-1 in angiogenesis. Among the targets regulated by HIF-1 is the gene encoding vascular endothelial growth factor (VEGF) which plays a critical role in angiogenesis. Pre-clinical trials of therapeutic angiogenesis utilizing VEGF gene therapy for ischemic disorders indicate that VEGF stimulates neovascularization but that the resulting vessels have excessive permeability. In contrast, increased HIF-1α expression results in increased vessels with normal permeability. These results suggest that HIF-1α controls the expression of angiogenic factors in addition to VEGF, resulting in a more physiologic outcome. Clinical trials of HIF-1 gene therapy in patients with limb or myocardial ischemia are currently in progress.

Role in HIF-1α in protecting ischemic cells from infarction. In models of myocardial and cerebral ischemia, subjecting animals to brief episodes of ischemia protects against prolonged ischemia 24 hours later that would otherwise result in infarction (delayed preconditioning). Recent studies indicate that HIF-1α activity is required for delayed preconditioning in the heart.

Involvement of HIF-1α in tumor progression. In animal models, genetic manipulations that increase or decrease HIF-1 expression are associated with increased or decreased tumor growth and angiogenesis, respectively. Immunohistochemical analysis of human tumor biopsies has revealed that HIF-1α is overexpressed in common human cancers and their metastases. In brain and breast tumors, HIF-1 expression is correlated with tumor grade and angiogenesis. Studies of oropharyngeal squamous cell cancers, early-stage breast and cervical cancer, and ovarian cancer have demonstrated that increased HIF-1α expression predicts radiation resistance and patient mortality. These studies indicate, first, that the determination of HIF-1α levels at the time of diagnosis may identify patients who require more aggressive therapy in order to survive their disease and, second, that pharmacologic inhibition of HIF-1α activity may be of therapeutic utility. The Developmental Therapeutics Program at the NCI is presently screening for small molecule inhibitors of HIF-1α that can be utilized for proof-of-principle experiments in animal models and as lead compounds for the development of novel chemotherapeutic agents.

Conclusion. Heart disease, cancer, stroke, and chronic lung disease account for two thirds of all deaths in the U.S. Alterations in O2 homeostasis and HIF-1α expression play major roles in the pathophysiology of these disorders. Genetic or pharmacologic manipulation of HIF-1α activity represents a novel therapeutic approach to these common causes of mortality.

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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IN VIVO ANALYSES OF T CELL COSTIMULATORY PATHWAYS

Arlene H. Sharpe, M.D., Ph.D.

Department of Pathology and Brigham and Women’s Hospital

Harvard Medical School

My research focuses on the in vivo biology of costimulation and its rolein regulating immune responses. Costimulation appears pivotal in determining whether T cell antigen recognition leads to T cell activation or anergy. For antigen-specific T cell activation to occur, two signals are needed. The interaction between the T cell receptor (TCR) complex and antigen-MHC is necessary, but not sufficient for T cell activation. Costimulatory signals, provided cell surface molecules are expressed on antigen-presenting cells, determine the outcome of TCR engagement, since they augment T cell proliferation and effector functions, such as lymphokine production. The interaction between CD3/TCR and antigen-MHC in the absence of costimulation not only results in a failure to induce an immune response, but often also results in functional inactivation of mature T cells, leading to a state of T cell unresponsiveness or death. Thus, T cell costimulatory signals play a critical role in determining the fate of a T cell. Several receptor-ligand pairs, which are primarily either members of the immunoglobulin supergene family or tumor necrosis factor receptor family, have costimulatory function because they induce activated T cells to proliferate after TCR signaling. The B7-CD28/CTLA4 costimulatory pathway appears to be particularly important, because of its unique capacity to prevent the induction of anergy.

The main approach that my laboratory has been taking to analyze the in vivo function of costimulatory pathways is to generate and analyze the immune capabilities of mice lacking costimulatory ligands and receptors using gene targeting approaches. We have focused primarily upon the B7:CD28 superfamily. Our mouse strains have provided a genetic means for dissecting the hierarchy of costimulatory pathways in the development of an immune response. Our studies have revealed striking and unexpected functions of costimulatory pathways. Our studies of B7-1 deficient mice provided the first evidence for the existence of additional functional CD28/CTLA-4 counter-receptors in vivo. As a result of these findings, a second CD28/CTLA-4 counter-receptor, B7-2, was cloned. Our studies revealed that B7-2 is the major early activating costimulator in this pathway. Mice lacking both B7-1 and B7-2 exhibit profound immunologic deficits and have demonstrated a critical role for this pathway in IgG class switching and germinal center formation. Our CTLA-4 deficient mouse revealed a critical role for CTLA-4 in turning off activated T cells and a previously unsuspected means by which costimulation can regulate re-

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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sponses to self antigens and potentially offers new approaches for regulating Tcell activation and tolerance.

There is currently great interest in manipulating costimulatory signals for therapeutic purposes. Specifically, learning how to inhibit costimulatory pathways may enable new methods for achieving tolerance for tissue transplantation and for controlling autoimmune diseases and allergies, whereas learning how to use costimulatory pathways to augment immune responses may lead to new immunization strategies for infectious agents and tumor immunity. Clinical trials using costimulatory blockade to block transplant rejection and ameliorate autoimmune diseases are in progress.

LESSONS FROM ABL: IMPLICATIONS FOR THE FUTURE OF TARGETED CANCER THERAPEUTICS

Richard A. Van Etten, M.D., Ph.D.

Center for Blood Research and Department ofGenetics

Harvard Medical School

A new era of targeted cancer therapy was inaugurated in May 2001 with the FDA approval of STI-571 (Gleevec®/imatinib mesylate) for the treatment of chronic myeloid leukemia (CML). STI-571 is a phenylamino-pyrimidine compound that is a potent and selective inhibitor of the Abl, PDGFbR, and Kit tyrosine kinases. An ATP mimetic, STI-571 binds to the ATP-binding site of the Abl catalytic domain and effectively inhibits Abl kinase activity in vitro and in vivo at concentrations of 0.1-1.0 mM. In phase I trials, STI-571 was remarkably effective as a single agent in interferon-resistant CML chronic phase patients, inducing durable hematologic remissions in 90 percent and major cytogenetic responses in 55 percent of patients. This agent has already radically altered the way patients with CML are managed, and is being heralded as the paradigm for the development of anti-cancer drugs in the future. From my perspective as a hematologist studying the molecular pathophysiology of leukemia, the STI-571 story raises several important issues: (1) The importance of precise knowledge of molecular drug targets in cancer. Work from our laboratory and many others over the previous decade demonstrated that the Bcr/Abl fusion tyrosine kinase, the product of the Philadelphia chromosome, is the direct cause of CML. Definition of the fundamental genetic abnormalities in cancer cells is a prerequisite to developing targeted therapies. Our work strongly argues that accurate animal models of human cancer are critical to this effort, as results obtained in cultured cells have

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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often been misleading. (2) The remarkable activity of STI-571 in CML as a single agent, and whether this can be generalized to other cancers. Our work suggests that solitary expression of Bcr/Abl in a hematopoietic stem cell is sufficient for induction of CML, and this may account for the extreme sensitivity of this leukemia to STI-571. However, solid tumors may have additional abnormalities in addition to activated tyrosine kinases that contribute to the oncogenic phenotype. Thus, while a subset of gastrointestinal stromal cell tumors that express activated c-Kit tyrosine kinase receptors also respond to STI-571, no complete remissions are observed. Trials of EGFR inhibitors in head and neck and glial tumors are just beginning. (3) Acquired drug resistance is a central problem in cancer therapy, and targeted therapeutics will be no different. Although STI-571 is effective in CML chronic phase, in patients with advanced disease, including accelerated phase, myeloid and B-lymphoid blast crisis, and those with de novo Ph-positive B-lymphoblastic leukemia, STI-571 is less effective. Although 50-70 percent of such patients initially respond to the drug, 60 percent of myeloid blast crisis patients and all B-lymphoid leukemia patients relapse within 3 to 6 months of starting therapy. Our laboratory and others have shown that a major mechanism of resistance is point mutations in the Abl catalytic domain that directly confer drug resistance. (4) Combinations of targeted therapies may be a strategy for improving responses to STI-571 and preventing the development of resistance. By analogy to HIV infection (like CML, another acquired dominant genetic disease of the blood) where treatment targeting both viral reverse transcriptase and protease is more efficacious than monotherapy, it is plausible that targeting critical signaling pathways downstream of Bcr/Abl will synergize with kinase inhibitor therapy. Again, animal models are the best way to validate such pathways. (5) Can the success with STI-571 be repeated? This is a complex question with both scientific and economic/social aspects. The development of STI-571 is often cited as a successful example of high-throughput drug screening and/or rational drug design. Both are incorrect, as the screen that identified the basic pharmacophore of STI-571 used protein kinase C, and the structural basis for the selectivity of action of the compound against Abl is still not understood. In addition, economic forces will play a major role in dictating which drugs will be developed for which diseases. The STI-571 program was nearly cancelled by Novartis less than 18 months before FDA approval due to the small perceived market opportunity, and resurrected only after direct pressure from leukemia patients. There are other leukemias with excellent tyrosine kinase targets for which drug development may never proceed due to the comparatively small number of patients with the disease.

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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TWO FAMILIES OF BIR-CONTAINING PROTEINS: INHIBITORS OF APOPTOSIS OR REQUIRED FOR MITOSIS

David L. Vaux, Ph.D.

The Walter and Eliza Hall Institute ofMedical Research

Melbourne, Australia

Inhibitor of apoptosis (IAP) proteins all bear one or more copies of a motif termed a baculoviral IAP repeat (BIR), a novel zinc finger fold. Certain BIR bearing proteins from baculoviruses, Drosophila and mammals inhibit cell death by binding to processed caspases. The Drosophila IAPs are antagonized by small pro-apoptotic molecules Grim, HID and Reaper. Mammalian IAPs are antagonized by mitochondrial proteins Diablo/Smac and Htra2, which interact via their processed amino-termini. Survivin is a protein that bears a single, structurally distinct BIR, and is expressed in all cancer cells, but is usually undetectable in cells from normal adult tissues. We have identified and deleted surviving homologues in the yeasts S. pombe and S. cerevisiae, in C. elegans and in the mouse. The phenotypes of these mutant organisms, and the pattern of Survivin expression revealed by antibodies, indicates that Survivin, inner centromere protein (INCENP) and aurora kinase 1 and their respective homologues function in concert to coordinate chromosome segregation and cytokinesis. The requirement for Survivin for cell division explains why it is expressed in cancer cells, which are dividing, but not in most normal adult cells, which are not.

A COMMON THEME IN THE PATHOGENESIS OF BACTERIAL INFECTION OF THE HUMAN RESPIRATORY TRACT

Jeffrey N. Weiser, M.D.

Departments of Microbiology and Pediatrics

University of Pennsylvania

Choline, a major component of eukaryotic membrane lipids, has been considered to be a highly unusual feature in prokaryotes. This laboratory has recently shown that choline in the form of phosphorylcholine (ChoP) decorates the oligosaccharide portion of the LPS of Haemophilus influenzae. Choline is obtained directly from the airway surface fluid or stripped off of host membrane lipids by a cell-surface glycerophosphodiesterase causing a cytotoxic effect on the ciliated epithelium. A MAb that recognizes ChoP shows that the ChoP epitope is a common feature of other major

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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pathogens that reside primarily on the mucosal surface of the human respiratory tract. In addition to Streptococcus pneumoniae, where ChoP has long been recognized as a constituent of its teichoic acids, the ChoP epitope is found on pili of Neisseria meningitidis and gonorrheae, a temperature regulated cell-envelope protein in Pseudomonas aeruginosa, the LPS of commensal Neisseria species and Actinobacillus actinomycetem-comitans, and a polar membrane lipid in various mycoplasma species.

The cell-surface expression of ChoP is subject to phase variation at a frequency of 10-3/generation due to slip-stranded mispairing of multiple tandem 5′-CAAT-3′ repeats within the open reading frame of the gene expressing choline kinase, licA. This suggests that it is not always advantageous for the organism to express ChoP. Our ability to define the genetic basis of choline incorporation in H. influenzae has allowed us to compare constitutive ChoP+ and ChoP- phenotypes to demonstrate that on the mucosal surface in both animal models and in human carriage there is strong selective pressure for bacteria expressing ChoP(ChoP+). In contrast, during invasive disease there is a selection for bacteria with an out-of-frame number of 5′-CAAT-3′ repeats in licA(ChoP- phenotype).

ChoP appears to have multiple effects on the ability of respiratory tract pathogens to interact with their host. ChoP may function in binding and invasion of host cells through mimicry of the natural ligand for the receptor for platelet activating factor (rPAF) expressed on the apical surface of the respiratory epithelium. In addition, ChoP renders the organism more resistant to the bactericidal effects of the antimicrobial peptide LL-37, which is present in airway surface fluid and targets structural differences between host and microbial membranes. In contrast, for some strains ChoP confers sensitivity to killing mediated by the binding of the serum acute phase reactant, C-reactive protein (CRP). Binding of CRP to ChoP causes the activation of the classical pathway of complement by ligation of C1q. In this regard, CRP seems to be a component of innate host defense that specifically targets microbes bearing cell-surface ChoP. Humans may depend on innate immunity based on CRP because the pathogens involved can quickly overwhelm their host and unlike other host species their antibody against ChoP does not appear to be protective. CRP, recently noted to be present in airway surface fluid, also inhibits binding of ChoP-expressing bacteria to rPAF. The inhibitory effect of CRP on adherence of both H. influenzae and S. pneumoniae is blocked by human surfactant, an abundant component of the lower airway that is composed largely of choline phosphate and binds to CRP. The effect of surfactant could contribute to the susceptibility of the lower airway to organisms that asymptomatically colonize the upper airway where there is no sur-

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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factant. It appears, therefore, that ChoP on the cell-surface of a variety of otherwise dissimilar pathogens may be 1) important for successful colonization of the mucosal surface, and 2) evasion of innate host defenses mechanisms.

THE ROLE OF TGFSS SIGNALS DURING EMBRYOGENESIS

Malcolm Whitman, Ph.D.

Department of Cell Biology

Harvard Medical School

Our laboratory studies the mechanisms by which intercellular signals regulate the differentiation and patterning of the early vertebrate embryo. Our work is currently focused on the role of TGFβ signals during embryogenesis. TGFβ superfamily ligands have long been known to play essential roles in patterning at multiple steps in vertebrate embryogenesis, but, until recently, the signal transduction pathways by which TGFβ factors act have been obscure. Several years ago, we discovered a novel transcription factor, FAST-1, that interacts in a ligand regulated manner with the TGFβ signal transducers Smad2 and Smad4, and demonstrated that this interaction targets the Smads for specific, developmentally regulated promoters. The Smads are regulated directly by TGFβ receptors, and therefore the identification of the Smad/FAST DNA binding complex provided the first example of a direct pathway by which TGFβs regulate a specific set of transcriptional responses. We have subsequently shown that FAST-1 is an essential component of the TGFβ signaling pathway that establishes the early embryonic body plan.

More recently, we have used antibodies specific for the phosphorylated, activated forms of the Smads to examine how TGFβ signaling is regulated endogenously during embryogenesis. We have found that the ability of embryonic cells to respond to specific TGFβ ligands is regulated as development progresses, and that a small extracellular protein, cripto, acts as a specific co-receptor for one subset of TGFβ ligands, the nodals, during early development. We are currently studying how cripto is regulated in the early embryo. We have also shown that nodals act via a novel mechanism, heterodimerization, to antagonize other TGFβ superfamily ligands during early embryogenesis. These studies suggest that direct interactions among distantly related TGFβ ligands may be important determinants of their activity in the embryo.

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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RO SMALL RNPS FUNCTION IN THE RECOVERY OF CELLS FROM RADIATION DAMAGE

X. Chen, H. Shi, J. Smith, D. Yang,1 L. Evangelisti,1 R. Flavell1 and Sandra Wolin, M.D., Ph.D.

Department of Cell Biology

Yale University

1  

Section of Immunobiology, HHMI, Yale University School of Medicine

The Ro 60 kDa autoantigen is an RNA-binding protein that is normally bound to small cytoplasmic RNAs known as Y RNAs. Although these RNPs are components of most vertebrate cells, their function has long been mysterious. In Xenopus oocyte nuclei, the Ro protein is also complexed with a large class of variant 5S rRNA precursors. Because these variant RNAs are inefficiently processed to mature 5S rRNA and eventually degraded, the Ro protein may recognize improperly folded 5S rRNA precursors as part of a quality control pathway (O’Brien and Wolin, Genes & Dev. 8:2891-2903).

Although Ro RNPs have not been detected in either S. cerevisiae or S. pombe, the genome of the radiation-resistant eubacterium Deinococcus radiodurans contains an orthologue of the Ro protein. The Ro protein orthologue, Rsr (Ro Sixty Related) contributes to the resistance of D. radiodurans to ultraviolet irradiation. D. radiodurans cells lacking rsr are more sensitive to UV irradiation than wild-type cells. During recovery from irradiation, the levels of Rsr increase approximately fourfold. Rsr binds several small RNAs, encoded upstream of rsr, that also accumulate during recovery from UV irradiation. Remarkably, one of these RNAs resembles the Y RNAs bound by the Ro autoantigen in higher eukaryotes (Chen et al., Genes & Dev. 14:777-82).

We have been examining the role of Ro RNPs in the recovery of higher cells following UV irradiation. Using gene knockout technology, we generated mouse embryonic stem cells that lack the Ro protein. Mouse cells lacking Ro have drastically reduced levels of Y RNAs, suggesting that Ro protein binding stabilizes these RNAs from degradation. Most interestingly, cells lacking the Ro protein are more sensitive to ultraviolet light than wild-type cells. Thus, in both mouse and bacterial cells, Ro RNPs contribute to survival following radiation damage. Although the mechanism is under investigation, one possibility is that the Ro protein binds to misfolded, mutant RNAs that are transcribed from DNA molecules containing radiation-induced mutations.

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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ENVIRONMENTAL RESPONSIVENESS OF THE DIMORPHIC FUNGAL PATHOGEN HISTOPLASMA CAPSULATUM

Jon P. Woods, M.D., Ph.D.

Department of Medical Microbiology and Immunology

University of Wisconsin, Madison

Histoplasma capsulatum (Hc) is a thermally dimorphic fungus that is a significant cause of respiratory and systemic disease in humans and other mammals. Its clinical importance has increased along with the growing immunodeficiency of the human population associated with HIV/AIDS, cancer and its treatments, immunosuppressive therapy for transplants and inflammatory syndromes, aging, and hospitalization. Hc lives saprobically in the soil as a mold, which is a successful member of a competitive polymicrobial ecosystem. The host-adapted parasitic morphotype is a budding yeast which is a facultative intracellular pathogen of macrophages. This microbe faces a variety of different environments and must survive under harsh conditions or modulate its microenvironment to achieve success as a pathogen in a professionally antimicrobial host cell. We have used several molecular techniques to identify fungal genes that are differentially expressed during infection of host macrophages and/or mice, when Hc is subjected to a complex range of environmental conditions. These methods have included in vivo expression technology (IVET), differential display, and cDNA representational difference analysis (RDA). Such approaches do not provide exhaustive genomic surveys in this eukaryotic microorganism, but we have identified several interesting genes. One differentially expressed gene encodes a small transcript in antisense orientation to a homolog of a negative regulatory protein kinase gene from another fungus, which is important in mating and starvation responses. We are examining both upstream and downstream aspects of this potential regulatory system in Hc, such as the specific environmental stimuli influencing expression of the antisense transcript, whether expression of the antisense transcript affects sense transcript expression, whether the sense transcript encodes a protein kinase functional in Hc, what the downstream targets of the putative kinase are, and the role of this locus in Hc biology and pathogenesis. A second target gene is expressed specifically in the yeast morphotype and not in mold, and the predicted encoded protein displays significant sequence homology with epidermal growth factor (EGF) domains found in a variety of proteins from other organisms, that typically function in attachment or intercellular signaling. Finally, we have preliminarily identified homologs of genes in other organisms that are involved in iron uptake. This finding interfaces with our separate interest in iron acquistion and fungal responses to

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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the specific environmental stress of iron limitation. The essential nutrient iron lies at the competitive interface between the mammalian host and nearly all microbial pathogens, including Hc. The host displays both constitutive and inducible iron sequestration mechanisms. Iron limitation acts as an important host defense mechanism against Hc in human and mouse macrophage cell culture infection models. As a successful pathogen, Hc must express iron acquisition mechanisms to obtain this nutrient in the competitive host environment in which it resides during infection. Hc previously has been shown to produce hydroxamate siderophores, which typically act as iron-scavenging compounds. We have demonstrated ferric reduction by Hc via at least three moieties—an extracellular ferric reductase enzyme, extracellular ferric reductant(s), and cell-surface ferric reducing agent(s). Reduction of ferric to ferrous iron causes removal from both host (e.g., transferrin) and fungal (siderophore) iron-binding compounds. Siderophore-mediated and reductive processes may provide important alternate, complementary, or interactive mechanisms for acquiring iron in the soil and/or the host.

MERGING BIOLOGY WITH DRUG DISCOVERY IN OBESITY, INFLAMMATION, ANGIOGENESIS, MUSCLE DISEASE AND OTHER SETTINGS

George D. Yancopoulos M.D., Ph.D.

President, Regeneron Laboratories

Chief Scientific Officer, Regeneron Pharmaceuticals, Inc.

Growth factor and cytokines, released by one cell and acting via cell surface receptors on a second cell, mediate intercellular communications required for the initiation and/or regulation of all biologic processes. A major focus for us at Regeneron has been to identify new growth factors and/or their receptor systems, with the notion that identification of such critical master regulatory systems would present new therapeutic opportunities. We have particularly focused on growth factor/receptor systems that specifically act on a single or limited number of cell types, so that manipulation of these systems could be attempted so as to benefit diseases involving those cell types, without having widespread side effects. Over the last decade, our efforts have led to the discovery and characterization of multiple growth factor/receptor systems (e.g., neurotrophins and their Trk receptors; CNTF/IL6 family and their gp130-related receptors; agrin and its MuSK receptor; collagens and their DDR receptors; ephrins and their Eph receptors; angiopoietins and their Tie receptors;

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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cartilage-specific ROR receptors), as well as to novel approaches for blocking these and other growth factor systems using engineered versions of soluble receptors we term Traps.

In many cases, the growth factors or their blockers are in, or are approaching, clinical testing. I will discuss the progress of a second-generation version of CNTF, termed Axokine, which is in Phase III clinical testing for obesity, as well as Traps that we are using to block IL-1 in rheumatoid arthritis, VEGF in cancer, and IL-4 and IL-13 in asthma and allergy. I will also discuss our new high-throughput knockout and transgenic technology, termed Velocigene, which we use to rapidly assign function to genes, with examples involving genes we have realized play key roles in the biology and pathology of muscle, cartilage, blood vessels, and lymphatic vessels.

STUDIES OF 53BP1 MAY REVEAL AN UNEXPECTED LINK BETWEEN DNA DAMAGE AND MITOSIS

S.T. Liu, Y. Adachi,1 and Tim J. Yen, Ph.D.

Fox Chase Cancer Center

1  

University of Edinburgh, Edinburgh, U.K.

53BP1 was identified as a yeast two-hybrid interactor of the p53 tumor suppressor but the functional significance of this interaction remains unclear. 53BP1 contains two copies of a BRCT motif that is found in a large number of proteins that are involved with various aspects of DNA replication, repair, and recombination. Furthermore, 53BP1 has been shown to accumulate at dozens of foci within nuclei that contain damaged DNA. Although the functionality of DNA damage induced foci remain to be clarified, it is generally believed to represent a macromolecular assembly of multiple proteins at or near the site of broken DNA. These findings strongly suggest that 53BP1 is a component of the DNA damage response pathway.

We have recently discovered that 53BP1 may have a different function besides DNA damage. 53BP1 accumulates in several large aggregates in nuclei of normal cycling cells. In mitotic cells, 53BP1 is dispersed from these aggregates and becomes concentrated at kinetochores, a structure that links chromosomes to the mitotic spindle. We determined that the earliest time that 53BP1 can be detected at kinetochores is shortly after nuclear envelope breakdown. 53BP1 assembles onto kinetochores after several other proteins that assemble onto kinetochore just prior to nuclear envelope breakdown. The temporal pattern of kinetochore binding exhib-

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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ited by 53BP1 suggests that it is likely to provide functions important for the final steps of kinetochore assembly. To begin to understand the importance of 53BP1 to kinetochore function, we have localized the kinetochore targeting domain to lie within the region that contains the BRCT repeats.

Given that 53BP1 form foci of similar size at sites of DNA damage and kinetochores, we speculate that 53BP1 provides functions that are shared by these two different cellular functions. Thus, our studies of the mitotic functions of 53BP1 may provide novel insights into the mechanism by which cells monitor and respond to double stranded DNA breaks.

Suggested Citation:"11 Abstracts of Poster Sessions." National Research Council. 2004. The Markey Scholars Conference: Proceedings. Washington, DC: The National Academies Press. doi: 10.17226/11001.
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This is the second of five reports to emerge from the evaluation of the Markey Trust. As part of this assessment, the NRC hosted a scientific conference for Markey Scholars and Visiting Fellows in Rio Grande, Puerto Rico on June 28-30, 2002. The purpose of the conference was to enable the Scholars and Fellows to share their research experiences, just as they did at the annual Scholars Conferences previously conducted by the Markey Trust. All of the attending Scholars and Fellows submitted abstracts of their poster sessions. Six scholars, along with other experts in the biomedical sciences, made formal presentations. These proceedings consist of shortened versions of the individual presentations and the poster session abstracts.

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