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ItemControl of Skeletal Muscle Fiber Types by Calcium Signaling Pathways(2002-08-01) Wu, Hai; William, R. SandersDifferent patterns of motor nerve activity drive distinctive programs of gene expression in skeletal muscles, thereby establishing a high degree of metabolic and physiological specialization among myofiber subtypes. Previous studies have demonstrated that calcineurin activity is required to maintain slow myofiber identity. I am interested in determining the transcription factors downstream of calcineurin and other calcium-regulated signaling pathways in the control of myofiber specialization. By analyzing two fiber type-specific enhancers, I was able to demonstrate that there are functional NFAT (nuclear factor of activated T cells) and MEF2 (myocyte-specific enhancer factor 2) binding sites within the enhancer of troponin I slow, and both sites are required for slow fiber specific activity of this enhancer. Next, I identified MEF2 as a target of calcineurin in cultured myogenic cells. Calcineurin physically interacts with MEF2 and dephosphorylates MEF2. C-terminal transactivation domain, but not N-terminal DNA binding domain of MEF2, responds to calcineurin activation. The use of "MEF2 indicator" transgenic mice that harbor a MEF2-dependent lacZ transgene enabled us to monitor the endogenous activities of MEF2 transcription factors. MEF2 is selectively active in slow and oxidative myofibers. Calcineurin is both necessary and sufficient for MEF2 activation in skeletal muscles. I also found a dose-response relationship between calcineurin activity and expression level of slow, oxidative fiber-specific and MEF2 target genes. Furthermore, I observed that functional activity of MEF2 transcription factors was stimulated by sustained periods of endurance exercise or low-frequency motor nerve pacing in a calcineurin-dependent manner. In addition to calcineurin, CaMKs (calcium, calmodulin-dependent kinases) also transduce their signaling through MEF2. CaMKIV synergistically activates MEF2-dependent gene expression together with calcineurin. Transgenic mice expressing constitutively active CaMKIV in their skeletal muscles showed increased percentage of slow and oxidative myofibers, which was accompanied by increased mitochondrial biogenesis mediated through the upregulation of PGC-1 (PPARg co-activator). Taken together, these findings delineate a molecular pathway in which MEF2 and NFAT integrate signaling inputs from multiple calcium-regulated pathways in the control of skeletal muscle fiber types. ItemRole of I Kappa B Kinase Alpha and I Kappa B Kinase Beta in the Development and Function of B and T Lymphocytes(2002-12-19) Ren, Hong; Gaynor, Richard B.Transcription factor NF-κB plays a key role in regulating the expression of genes involved in the control of the inflammatory and immune response. NF-κB binds to a group of inhibitory proteins, IκBs, in the cytoplasm of non-stimulated cells. Activation of NF-κB is regulated by the IκB kinase complex that phosphorylates IκB proteins, leading to their polyubiquitination and degradation. The released NF-κB molecules translocate into the nucleus and activate gene transcription. IκB kinase complex contains two catalytic subunits, IκB kinase α (IKKα) and IκB kinase β (IKKβ), and a regulatory subunit, IκB kinase γ (IKKγ) or NEMO. To evaluate the functions of IKKα and IKKβ in the development and function of the immune system, transgenic mice expressing dominant negative forms of IKKα and IKKβ specifically in their B cells or T cells were generated. Phenotypic analysis of transgenic mice expressing dominant negative IKKβ in the B cells revealed that the proliferation of B cells from these mutant mice in response to B cell mitogens was reduced due to impaired cell cycle progression. Accordingly, in vitro secretion of immunoglobulins by the mutant B cells in response to these mitogens was also decreased. In addition, these mice displayed selective defects in the production of specific immunoglobulin subclasses in response to type 2 but not type 1 T cell independent antigens. Moreover, the levels of certain immunoglobulin subclasses were reduced in mutant mice challenged with a T cell dependent antigen. These results indicate that IKKβ is critical for the proliferation of B cells and the control of some aspects of the humoral response. Transgenic mice expressing one or both of the dominant negative IKK specifically in T cells exhibited distinct phenotypes in thymocyte proliferation, cytokine production, and cell survival. Proliferation of thymic T cells from IKKβ mutant mice and IKKα/β mutant mice was markedly reduced due to impaired cell cycle progression. In addition, inhibition of both IKKα and IKKβ appeared to suppress the expression of multiple cytokines by thymocytes. Furthermore, apoptosis of the double positive thymocytes induced by the administration of anti-CD3 antibody was significantly reduced in transgenic mice expressing dominant negative IKKβ, but increased in mice expressing only dominant negative IKKα. These results indicate that IKKα and IKKβ play different roles in regulating the activation and survival of T cells. ItemThe IRIDESCENT System: An Automated Data-Mining Method to Identify, Evaluate, and Analyze Sets of Relationships Within Textual Databases(2003-02-01) Wren, Jonathan Daniel; Garner, Harold R.Individuals are limited in their ability to read, remember and compare relationships within the vast amount of scientific literature available. This is not only because the amount of literature is increasing exponentially, but the number of things being researched within is as well. Adding to the scale of analysis are new technologies that increase the rate by which data is being gathered from scientific experiments. For most areas of research interest, the scale of analysis exceeds an individual's ability to be aware of all the relationships contained within. Thus, an informatics approach is necessary to identify large-scale trends, shared relationships and novel relationships that are not contained within the literature, but are the logical consequence of the relationships that are. A system has been designed to establish a network of relationships between "objects" of research interest (e.g. genes, chemical compounds, drugs, diseases and clinical phenotypes) by extracting information from scientific text in an automated manner. This system, called IRIDESCENT (Implicit Relationship IDEntification by in-Silico Construction of an Entity-based Network from Text), enables the discovery of novel relationships by identifying and scoring objects sharing large sets of relationships with an object of interest. IRIDESCENT also allows sets of objects to be analyzed for shared relationships, such as responding genes from a microarray experiment. Herein is described the development and workings of IRIDESCENT as well as several well-developed applications of the system. ItemCharacterization of Class D VPS Proteins(2003-03-11) Friedberg, Andrew Seth; Sternweis, Paul C.The vacuole of the yeast Saccharomyces cerevisiae is functionally similar to the mammalian lysosome. The components of the VPS (vacuolar protein sorting) system are responsible for proper delivery of vacuolar biosythetic enzymes. Efforts to dissect the genetics of this system have revealed several classes of mutants, each defective in one transport step in the VPS pathway. The Class D VPS proteins are thought to control anterograde traffic between the late Golgi and late endosome. Although most of these proteins have homologues of known function in other systems, two exceptions are the Vps3p and Vps8p proteins. Analysis of Vps3p reveals that it is associated with a highdensity structure, possibly a coated vesicle or a large protein complex. The Vps8p protein contains a C-terminal H2 RING finger motif, a domain often associated with E3 ubiquitin ligase activity. In vitro analysis reveals that a Vps8p fragment containing this domain has this activity. Deletion of the RING finger reveals that the endocytic marker Ste3p accumulates in an abnormally large late-endosome-derived structure, but that sorting of the soluble vacuolar cargo CPY is relatively unaffected. These results suggest a division of function within the Vps8p molecule. ItemTelomere Position Effect in Human Cells(2003-04-01) Baur, Joseph Anthony; Shay, Jerry W.Telomeres are tracts of repetitive DNA that cap the ends of linear chromosomes. Each time the chromosome is duplicated, a small amount of telomeric DNA is lost from the end due to factors inherent in the mechanism of DNA replication. The result is a net shortening of telomeres with each cell division, unless new repeats are synthesized through the action of the enzyme telomerase. Most human somatic cells lack telomerase activity and so continued cell division leads to telomere shortening. After a limited number of divisions (the "Hayflick limit"), it is believed that a few critically shortened telomeres trigger a state of growth arrest termed replicative senescence. ItemMyogenic BHLH Transcription Factors: Their Overlapping Functions and Direct Regulation of MEF2C Provide a Regulatory Network for the Maintenance and Amplification of Vertebrate Myogenesis(2003-04-01) Valdez, Melissa Renee; Mangelsdorf, David J.The myogenic basic helix-loop-helix (bHLH) genes - Myf5, MyoD, myogenin and MRF4 - exhibit distinct, but overlapping expression patterns during vertebrate myogenesis. Loss-of-function mutations in these genes have defined an in vivo model for myogenesis in which MyoD and Myf5 have redundant functions in myoblast specification, whereas myogenin acts to control myoblast differentiation. A role for MRF4 in differentiation has been suggested by various studies, but not defined. Through the analysis of MyoD-/-MRF4-/- and myogenin-/-MRF4-/- mutants, we show that MRF4 plays a role in differentiation which it shares with MyoD, but not myogenin, thereby defining a novel myogenin-independent differentiation pathway. The functional redundancy of the myogenic bHLH factors demonstrated in these and other studies led us to investigate the ability of a single factor to direct the myogenic program in the absence of the other myogenic bHLH proteins. Analysis of myogenin-/-MyoD-/-MRF4-/- mutant animals showed that alone, Myf 5 was unable to bring about differentiation, although specification of myoblasts was not affected. These results suggest that these myogenic factors possess specialized functions. However, the remarkably low level of Myf5 available in triple mutant neonatal muscle leaves open the possibility that it is the total level of myogenic bHLH transcription factors that is critical to the completion of muscle differentiation. The auto- and cross-regulation that the myogenic bHLH factors provide for one another, combined with their functional redundancy, comprises a mechanism whereby myogenesis is induced and maintained. Members of the MEF2 family of transcription factors cooperate with the myogenic bHLH factors to control the expression of muscle specific genes, thereby contributing to the maintenance and amplification of muscle development. To determine the mechanisms that regulate the expression of MEF2C, the earliest of the MEF2 factors expressed in the myogenic lineage, the mouse MEF2C gene was analyzed for cis-regulatory elements that direct its expression in the skeletal muscle lineage in vivo. As described herein, such a control region was identified, characterized and shown to be a direct transcriptional target of myogenic bHLH and MEF2 proteins. These results further define the regulatory circuit that induces, amplifies and maintains myogenesis in vivo. ItemMuscle-Specific Regulation of Serum Response Factor by Differential DNA Binding Affinity and Cofactor Interactions(2003-04-01) Chang, Priscilla Shin-Ming; MacDonald, Raymond J.Serum response factor (SRF) is a MADS-box transcription factor that regulates muscle-specific and growth factor-inducible genes by binding the CArG box consensus sequence CC(A/T)6GG. Because SRF expression is not muscle-restricted, its expression alone cannot account for the muscle-specificity of some of its target genes. To further understand the role of SRF in muscle-specific transcription, two distinct approaches were taken. First, tandem multimers of different CArG boxes with flanking sequences were analyzed in transgenic mice. CArG elements from the SM22 and skeletal a-actin promoters directed highly restricted expression in developing smooth, cardiac, and skeletal muscle cells during early embryogenesis. In contrast, the CArG box and flanking sequences from the cfos promoter directed expression throughout the embryo, with no preference for muscle cells. Systematic swapping of the core and flanking sequences of the SM22 and c-fos CArG boxes revealed that cell type-specificity was dictated in large part by sequences immediately flanking the CArG box core. Sequences that directed widespread expression bound SRF more strongly than those that directed muscle-restricted expression. Therefore, sequence variations among CArG boxes influence cell type-specificity of expression and account, at least in part, for the ability of SRF to distinguish between growth factor-inducible and muscle-specific genes in vivo. Second, a novel transcriptional cofactor for SRF called Myocardin was characterized. Myocardin belongs to the SAP domain family of nuclear proteins, is expressed specifically in cardiac and smooth muscle cells, and is a potent activator of cardiac and smooth muscle genes, including SM22. Myocardin activates through CArG boxes, and its activation is dependent on its interaction with the MADS box domain of SRF. Myocardin is the founding member of a new class of muscle-specific transcription factors and provides another mechanism whereby SRF can convey myogenic activity to muscle-specific genes. These results describe two mechanisms for muscle-specific activation of target genes by SRF. Muscle-specific genes contain CArG boxes with relatively low affinities for SRF, and thus are only able to respond to the high levels of SRF found in muscle. Also, Myocardin, a muscle-specific transcription factor, is able to associate with SRF and cooperatively activate transcription of muscle genes. ItemCharacterization of the T122L Mutation in p53 and Its Protein Product in XPC Mutant Mice(2003-04-01) Nahari, Dorit; Friedberg, Errol C.Xeroderma Pigmentosum (XP) is a rare genetic disorder characterized by extreme sensitivity to sunlight, and a profound predisposition to skin cancer due to defects in nucleotide excision repair (NER) of DNA. XP patients can be divided into seven complementation groups (A-G) with corresponding genes Xpa thru Xpg. I have studied a novel UV-induced hot spot in codon 122 of the p53 gene in mice deficient in XPC protein and heterozygous for the p53 gene. The original Threonine residue is mutated to Leucine as a result of an AC->TT change. The main goal of this work was to characterize the T122L mutation and its protein product, and to elucidate the mechanism(s) that affect its appearance in XPC deficient skin. I have shown that the T122L mutation is rare in other NER deficient mouse models, suggesting that the XPC protein is required specifically to repair the unidentified damage in this codon. In addition, I have shown that the T122L mutant protein is not a loss of function mutant, but it retains some wild type protein functions including ransactivation of p53 regulated genes and promotion of cell cycle arrest in response to UV-induced DNA damage. The altered transactivation properties of the mutant protein might support clonal expansion, giving cells that express it a growth advantage. In order to determine whether the p53 mutant protein function contributes significantly to the elevated mutation frequency observed in XPC deficient mice I have used a p53 knockout mouse model that retains the genomic region containing codon 122 without expressing p53 protein. I show that the mutation is observed in low frequencies in mice that do not express protein, suggesting that repair deficiency is the key factor for the appearance of the T122L mutation. However, once the mutation is formed its frequency is significantly increased as a result of the mutant protein function. The specific requirement for XPC protein and the location of the damage at a nondipyrimidine nucleotide site hints at an additional role of XPC in a repair pathway different from NER. ItemGenetic and Biochemical Analyses of the Necessity for Caspase Activation by the CED4-Domain Proteins, APAF-1 and DARK(2003-04-03) Oliver, George Reinhold; Abrams, John M.Activation of caspase proteases by Ced4 domain proteins is a critical step in the induction of programmed cell death, or apoptosis. Understanding of the genetic and biochemical regulation of the mammalian Ced-4 gene, Apaf-1, may be crucial to the understanding of autoimmune diseases, neurodegenerative disorders and cancer progression. Located in chromosomal band 12q22, Apaf-1 is in a locus frequently deleted in Male Germ Cell Tumors (GCT's). Though not homozygously inactivated in these tumors, Apaf-1 mediated caspase activation is impaired in GCT cell lines and may be a frequent event in other cancer types. Analysis of human genomic DNA facilitated the discovery of the homologous gene DARK, the Drosophila Apaf-1 Related Killer. Hypomorphic alleles of DARK cause developmental disruption, including wing defects, body wall defects, supernumerary bristles, male sterility and an enlarged nervous system. Mutation of DARK potently suppresses the apoptotic function of the genes reaper, grim, and hid. Recombinant Grim protein was shown to antagonize IAP-mediated caspase inhibition in vitro. Peptides corresponding to the conserved Amino-termini of the reaper, grim and hid genes could compete for a binding site also used by the mammalian anti-IAP protein SMAC to block IAP-caspase interaction. Despite these peptides' failure to allow for reconstitution of caspase activation in vitro, genetic inactivation of IAP's leads to significant activation of caspases in vivo. DARK plays a critical role in caspase activation in vivo, and mutations of DARK suppress several genetic measures of cell death due to IAP inactivation. These studies show DARK to be an important apoptosis gene in the fly and necessary for caspase amplification and apoptotic initiation in certain cell death pathways. Further understanding of the regulation of cell death in the genetically tractable Drosophila model may help shed light on the regulation of apoptosis in human cells and disease states as well. ItemRole of Mash1-E Protein Heterodimers in Mash1 Function in the Developing Neural Tube(2003-05-01) Collisson, Tandi Louise; Lu, Q. RichardNeural-specific Class II bHLH transcription factors heterodimerize with ubiquitous Class I bHLH E proteins to form complexes required for neural differentiation. There are four known E proteins, HEB, E12, E47 and E2.2, in the mammalian nervous system, which potentially form heterodimers with Mash1 in the neural tube. To test the relevance of particular Mash1-E protein heterodimer combinations in vivo, I constructed tethered Mash1-E protein heterodimers for over-expression in the chick neural tube. By comparing overexpression of Mash1 with over-expression of these Mash1-E protein heterodimers, their abilities to effect neural differentiation and cell-type specification were analyzed. Mash1-E protein heterodimers are interchangeable in the function of driving neurogenesis in the chick neural tube. The effects of Mash1-E protein heterodimers on cell-type specificity were different, suggesting non-redundant functions in effecting dorsal interneuron populations. Furthermore, additional Mash1 heterodimer partners may be required for the cell-type specification function of Mash1. ItemThe Development of an Informational Video Using Three-Dimentional [sic] Animation to Teach the Fundamentals of the Cellular Process of Apoptosis(2003-06-01) Litton, Rebecca; Calver, Lewis E.The goal of this thesis was to create an animated video, with narration, that explains the fundamentals of the process of apoptosis. The objectives were to produce a narrated 3D animation of apoptosis presented in an accurate efficient way, and format it for distribution on CD or DVD. Topics discussed in the video include: the difference between necrosis and apoptosis, the physical changes occurring in the cell during apoptosis, triggers of apoptosis and the effect apoptosis has on disease processes. The creation process began by determining subject, scope and audience. After these initial decisions were made a script was written and storyboards were produced. Narration was then recorded and combined with stills of the storyboards and preliminary animation to create an animatic. All animation was created in 3D Studio Max. Editing was accomplished using Adobe Premiere. The final product was then copied to CD and to DVD. This document discusses the process of creating this video from formation of the idea to DVD creation. Results of an informal test of the video are also iscussed as well as ideas for further research. ItemThe Development of an Instructional Module on the Basics of Web Design for Scientists Interested in Creating a Web Page(2003-06-01) Wilkins, Marla Rochelle; Calver, Lewis E.The purpose of this thesis is to document and explain the development of an instructional module on the basics of web design for scientists interested in developing a web page. This instructional module was created as an aid for scientific researchers who are interested in either creating a web page or having a web page created on their behalf, but who have limited Internet experience and are unfamiliar with the basic terminology and design processes involved in developing a web page. The module was posted online by the author for evaluation by selected scientists and researchers at UT Southwestern Medical Center at Dallas. It contains information on initial web page design (purpose, audience, organization and navigation), graphic design issues (color, typography, consistency and redundancy), the technical aspects of web design (computer specifications, "internetiquette", print production versus web production, editing and submission), and a glossary of important terms. The thesis focuses on the development of a web site for Dr. Chandra Mohan's Lupus Research Laboratory at UT Southwestern, the creation of an interactive web design guide using Mohan Lab's web site as an example, and the creation of a printable pdf "Shortcuts Guide" to accompany the module. ItemBody Fat Assessment and Adipocytokine Levels in the Lipodystrophy Syndrome in HIV-infected Patients(2003-06-30) Dinges, Warren L.; Garg, AbhimanyuThe lipodystrophy syndrome in HIV-infected patients (LDHIV) is characterized by body morphologic and metabolic changes. This case-control study compared 16 male, HIV-infected, severely lipodystrophic patients on protease inhibitor (PI) therapy to 14 HIV-infected, non-lipodystrophic men not on PI-inclusive HAART. Body fat distribution was assessed using anthropometry, whole-body MRI, and DEXA analysis. Adipocytokines were compared using fasting plasma adiponectin, leptin, and insulin levels. LDHIV patients compared to control subjects had lower peripheral fat with gluteal, suprailiac and triceps skinfolds (all p<0.01, with MRI thigh subcutaneous fat (ThSCF)(mean%±SD, LDHIV 12±5 vs. Controls 22±12 p=0.01) and with DEXA leg fat (%, 12±5 vs. 22±9, p=0.0006). Dorsocervical fat (DCF) accumulation was significantly greater in LDHIV subjects by MRI depth (mm, 47±24 vs. 19±7, p=0.0009) and DEXA head fat (%, 18±3 vs. 16±1, p=0.01), but not significantly by subscapular skinfold p=0.30. In LDHIV patients, abdominal fat was greater by waist circumference (cm, 98±5 vs. 86±9 p=0.0008), nearly significantly greater by MRI Intra-abdominal fat (cm2, 218±90 vs.157±70 p=0.057), and greater by DEXA Trunk percent total fat (%TF, 65±7 vs. 53±8 p=0.0005). In the correlation analysis of LDHIV patients, MRI IAF/ThSCF showed a strong negative relationship to DCF in all three methods r<-0.80, p<0.001, implying a novel split phenotype of either DCF or IAF accumulation. Five combined lipodystrophy parameters were derived, that well delineated the two groups (all p<0.00003). LDHIV patients compared to control subjects also had lower adiponectin levels (3.2±3.1 vs. 5.9±2.9 µg/mL, p=0.01), similar leptin levels (3.5±1.5 vs. 3.0±1.6 ng/mL, p=0.49), and higher insulin levels (20±11/19 vs. 12±6/11 µU/mL, p=0.04). There was a strong negative relationship between adiponectin and insulin levels in the LDHIV group (r=-0.73, p=0.01), but not in control subjects (p=0.33). Conversely, leptin and insulin levels in the control subjects were highly correlated (r=0.86, p<0.0001), but not in LDHIV patients (p=0.82). In conclusion, plasma adiponectin levels are low in patients with LDHIV and may be a better marker than leptin for the hyperinsulinemia of LDHIV. Also, this study has defined consistent methods to quantitatively assess the regional body fat changes and lipodystrophy severity in LDHIV using Anthropometry, DEXA, and MRI. ItemSignal Specific Ubiquitination and Degradation of IkBa(2003-10-08) Hakala, Kevin William; Kodadek, Thomas J.The transcription factor Nuclear Factor kB (NF-kB) is retained in the cytoplasm by the action of its inhibitor IkB. Upon phosphorylation by the IKK complex, IkB is rapidly ubiquitinated and targeted for 26S proteasome mediated degradation, thus liberating NF-kB for transport to its nuclear destination. The current project was initiated to reconstitute this pathway in vitro by using the purified ubiquitination and degradation machinery to degrade IkBa, and activate NF-kB. While signal dependant IkBa ubiquitination was achieved early in the project, this substrate was not degraded by a number of different 26S protein preparations. Instead, an integral or associated isopeptidase activity was observed with each 26S preparation. The development of new 26S protein purification methods has enabled the isolation of highly purified 26S proteins that exhibits low degradative activity towards the ubiquitinated IkBa substrate without excess isopeptidase activity. In an effort to increase substrate degradation, the IkBa ubiquitination reaction was carefully scrutinized. The current literature reports that Ubch5 is the relevant E2 that works in conjunction with the IkBa SCFᔲCP E3 complex, however, Cdc34/Ubc3 can also ubiquitinate IkBa, and may also be a relevant E2. While both E2s carry out in vitro signal dependant ubiquitination of IkBa, the ubiquitin conjugates made by Ubc3 are specific for Lysine-48 linked isopeptide bonds, whereas Ubch5 is able to utilize a variety of ubiquitin surface Lysine residues in isopeptide bond formation. Because K-48 linked ubiquitin conjugates are believed to target substrates for 26S mediated degradation, it was not surprising to find that my 26S proteasome preparations exhibited higher levels of IkBa degradation when ubiquitin conjugation reactions were carried out with Ubc3 instead of Ubch5. Using small interfering RNA to knock down the protein levels of each E2 in vivo, we have found that Ubc3 has no effect on IkBa degradation, whereas the Ubc5/7 double knockdown exhibits partial inhibition of IkBa degradation which is comparable to knocking down the levels of the IkBa E3 specificity factor ᔲCP. The completion of this project has established an in vitro ubiquitination and degradation system that will be instrumental for future studies aimed at determining how the 26S proteasome unfolds and degrades its protein substrates. ItemIdentification of Acyloxyacyl Hydrolase, a Lipopolysaccharide Detoxifying Enzyme, in the Murine Urinary Tract(2003-10-8) Feulner, J. Amelia; Munford, Robert S.Acyloxyacyl hydrolase (AOAH) is a lipase that removes the secondary fatty acyl chains that are substituted to the hydroxyl groups of glucosamine-linked 3-hydroxyacyl residues in lipid A, the bioactive center of Gram-negative bacterial lipopolysaccharides (LPS). Such limited deacylation has been shown to attenuate cytokine and chemokine responses to LPS, suggesting a role for AOAH in modulating (downregulating) inflammatory responses to invading Gramnegative bacteria. Prior to the experiments described in this report, AOAH had only been found in myeloid lineage cells (monocyte-macrophages, neutrophils and dendritic cells). In the work presented here, AOAH was found in murine renal proximal tubule cells and in human renal v cortex. Proximal tubule cells are known targets for invading Gram-negative uropathogens and we hypothesize that possessing AOAH may help them degrade the LPS contained within these bacteria. I further found that AOAH is secreted from proximal tubules in vitro and that it can be detected in murine urine, where it is able to deacylate purified LPS. AOAH may also associate with downstream bladder epithelial cells (which do not express AOAH) and be processed by them to its more enzymatically active, mature form. Bladder cells that have taken up AOAH in vitro are able to deacylate LPS. To determine the in vivo role of AOAH, I induced ascending urinary tract infections (UTIs) in wild type and AOAH null mice. To my surprise, AOAH null mice were able to clear bacteria from their urine faster than did wild type mice. An analysis of the immune response by histological analysis of bladder tissue and enumeration of neutrophils in the urine did not show a significant difference between wild type and AOAH null mice at any of the time points examined. Although I do not yet understand the mechanism for such increased clearance in AOAH null animals, we hypothesize that, due to their inability to deacylate LPS, they might have a more effective immune response to invading Gram-negative bacteria. A more detailed analysis of such responses to invading Gram-negative uropathogens will be important for understanding the in vivo role of AOAH in the urinary tract. ItemAcanthamoeba spp. Secrete a Mannose-Induced Protein that Correlates with Ability to Cause Acanthamoeba Keratitis(2004-01-14) Hurt, Michael Allen; Niederkorn, Jerry Y.Acanthamoeba spp. are ubiquitously distributed in the environment. The trophozoite form can infect the cornea and cause sight-threatening corneal inflammation known as Acanthamoeba keratitis. The pathogenic cascade of Acanthamoeba keratitis begins when Acanthamoebae bind to mannose expressed on traumatized corneas. Published reports indicate that mannose is upregulated on the corneal surface during wound healing. Experiments in laboratory animals have shown that corneal abrasion prior to infection is essential for generating Acanthamoeba keratitis. Furthermore, supernatants from Acanthamoeba ItemAcyloxyacyl Hydrolase: Studies on Its Regulation and Function in Mus Musculus(2004-01-14) Lu, Mingfang; Munford, Robert S.Acyloxyacyl hydrolase (AOAH) is an enzyme that detoxifies Gram-negative bacterial lipopolysaccharides (LPS) by selectively removing secondary acyl chains from the lipid A moiety. Originally found in neutrophils, it is also produced by monocyte-macrophages and renal proximal tubule cells. In the studies described here, I found that both immature dendritic cells (DCs) of the XS52 cell line and bone marrow-derived DCs produce AOAH. AOAH expression decreased when DCs were incubated with IL-4, IL-1ᬠTNFa and an agonistic CD40 antibody (maturation cocktail), and increased following treatment with microbial agonists that engage 3 distinct Toll-like receptors (LPS, TLR4; CpG oligodeoxynucleotides, TLR9; and a Gram-positive bacterium (Micrococcus luteus), TLR2). Maturation cocktail treatment also diminished, while LPS treatment enhanced or maintained, the cells' ability to kill E. coli, deacylate LPS, and degrade bacterial proteins. Enzymatic deacylation of LPS is thus an intrinsic, regulated mechanism by which DCs may modulate host responses to this potent bacterial agonist. To study the biological functions of AOAH, AOAH-deficient mice were generated by targeted gene disruption. AOAH did not protect mice from lethal doses of LPS or Gram-negative bacterial challenge. In response to subcutaneous injections of LPS, however, AOAH-deficient mice produced significantly higher levels of non-specific (polyclonal) IgM and IgG3 than did wild type mice. Anti-double-stranded DNA and anti-nucleosome IgM and IgG antibody levels were also higher in LPS-immunized AOAH-deficient mice than in wild type control mice. In addition, the partially-deacylated LPS product (dLPS) induced lower polyclonal antibody responses in vivo than did mock-treated LPS, yet the anti-LPS specific responses to dLPS and LPS were equivalent. These results suggest that AOAH may diminish potentially harmful polyclonal antibody responses to Gram-negative infection but maintain the protective anti-LPS specific response. Since B cells do not produce the enzyme, my results also point to an important role for macrophages and DCs in modulating B-cell responses to LPS antigens. In addition, the absence of AOAH did not alter the ability of LPS to function as an adjuvant, indicating that this activity is mechanistically distinct from stimulation of polyclonal antibody production. Finally, the ability of a bacterial lipopeptide to stimulate polyclonal antibody production only in AOAH -/- mice suggests that the enzyme may also regulate immune responses to non-LPS bacterial agonists. ItemTranscriptional Regulation of Cardio-Pulmonary Development(2004-01-14) Aiyer, Aparna R.; Srivastava, DeepakOrganogenesis is a complex process, disruption of which results in developmental anomalies. In recent years, genetic dissection of the pathways involved in cardiogenesis, have shown a striking similarity in molecular mechanisms across species. One conserved protein is dHAND, a basic helix-loop-helix (bHLH) transcription factor that is required for normal development of the right ventricle, the pharyngeal arches and limb buds. Loss of dHAND leads to apoptosis in the aforementioned tissues and to embryonic lethality at E10.0. A differential display analysis was performed to identify genes dysregulated in dHAND-/- hearts. Characterization of such genes could potentially shed light on the molecular mechanisms involved in the defects seen in dHAND mutants, while also identifying genes required for normal embryonic development. This thesis represents work on two molecules that were identified in this screen. Bnip3, a hypoxia inducible, pro-apoptotic molecule that can induce mitochondrial damage, was upregulated in the dHAND-/- pharyngeal arches and heart, suggesting a role for mitochondrial damage in the observed apoptosis. I have shown that while Apaf-1, a downstream mediator of mitochondrial-induced apoptosis, is required for the apoptosis observed in dHAND-null pharyngeal and aortic arch mesenchyme, cardiomyocyte apoptosis in dHAND mutants is Apaf-1 independent. Rescue of pharyngeal arches revealed that premature closure of the pharyngeal arch arteries likely contributes to the early lethality observed in dHAND-/- embryos. The mouse ortholog of Bcl-2 associated transcription factor (Btf), which was similar to thyroid hormone receptor associated protein 150 (TRAP150), was down regulated in dHAND mutants. TRAPs are a family of transcriptional co-activators that are required for normal cardiac and embryonic development. Mice lacking Btf showed normal cardiac development, however, the animals had hypercellular lungs and died within 24 hours after birth. Analysis of lung ultrastructure and cell specific markers showed presence of immature secretory cells in the proximal airways of the lung and aberrant proximal-distal patterning. The ectopic presence of stem cell-like proximal epithelial cells (Clara cells) in the distal epithelium may explain the hypercellularity observed in btf-null lungs. These results show that Btf is required for normal maturation and patterning of the pulmonary epithelium and survival of the animal. ItemCharacterization of the FXYD Protein Family in the Regulation of Insulin Exocytosis(2004-05-04) Hays, Lori Beth; Rhodes, Christopher J.; Roth, Michael G.; Cobb, Melanie H.; Uyeda, KosakuInsulin exocytosis is a complex, regulated process involving numerous exocytotic proteins to coordinate the release of insulin. Syncollin has been implicated in zymogen granule exocytosis in acinar cells. It was hypothesized that either syncollin or a ‘syncollin-like’ protein may be expressed in β-cells and influence insulin exocytosis. Adenoviral mediated expression of either long or short forms of syncollin in isolated islets and INS-1 cells showed both forms underwent N-terminal signal peptide cleavage to yield the same 14kD mature protein. Immunofluorescence revealed that adenovirally-expressed syncollin was specifically targeted to the ß-granule lumen. In perifused islets, syncollin expression significantly inhibited first-phase glucose-induced insulin secretion compared to AdV-GFP infected islets. GLP-1 and glyburide potentiation of insulin secretion was inhibited; whereas constitutive secretion and insulin content were normal in syncollin-infected islets indicating syncollin-mediated inhibition of insulin secretion was not due to inadequate insulin production or secondary stimulus-coupling signals. Thus, syncollin likely inhibited the distal stages of insulin exocytosis providing the first evidence that an intragranular protein is capable of influencing regulated insulin secretion. Syncollin fluorescent fusion proteins were localized to ß-granules, but did not influence insulin secretion implicating these chimeras as ß-granule specific markers for emerging imaging technology. Real-time confocal microscopy demonstrated syncollin-GFP could be used to examine spatiotemporal dynamics of exocytosis. Furthermore, consecutive infection of syncollin-GFP and syncollin-dsRFP labeled distinct pools of β-granules. Expression of syncollin was not identified in β-cells; however, a 10Kd ‘syncollin-like’ protein was expressed, which when sequenced corresponded to FXYD6. Comparison of syncollin and FXYD6 protein structure revealed several conserved domains, indicating syncollin is likely a pseudo-FXYD family member. FXYD6 was the only FXYD protein endogenously expressed in β-cells, which localized to distinct regions of the plasma membrane. Overexpression of FXYD6-Myc enhanced β-granule transport to distinct regions of the plasma membrane that also expressed FXYD6; however, there was no significant effect on glucose-stimulated insulin secretion in isolated islets. SiRNA-mediated reduction of FXYD6 resulted in no obvious changes in β-granule distribution; however, β-granule movement during glucose stimulation was erratic and misdirected. These data implicate FXYD6 as a molecular beacon on the plasma membrane guiding β-granules to the active site of exocytosis. ItemUncovering the Complexity of RAS Signaling Networks(2004-05-04) Matheny, Sharon A.; Anderson, Richard G. W.The Raf/MEK/MAP Kinase signal transduction cascade is a key Ras effector pathway that mediates diverse cellular responses to environmental cues, and makes a major contribution to Ras-dependent oncogenic transformation. Here we describe two important aspects to understanding the signaling complexity of any multiphenotypic arrangement, namely, the identification and examination of new effectors and the direct authentication of protein-protein interactions. We have identified a new Ras effector, IMP (Impedes Mitogenic signal Propagation) that negatively regulates MAP kinase activation by limiting formation of Raf/MEK complexes. The mechanism of inhibition appears to be through inactivation of the KSR1 adaptor/scaffold protein, demonstrating that in addition to promoting signal transmission, scaffold proteins can function to restrict signal propagation. Ras can inactivate IMP through induction of IMP auto-ubiquitination, facilitating KSR-dependent engagement of MEK by activated Raf. These observations reveal dual Ras effector inputs to the MAP kinase cascade: induction of Raf kinase activity concomitant with derepression of KSR-dependent Raf/MEK complex formation. This relationship provides a mechanism to limit engagement of the MAP kinase cascade in the absence of Ras activation. MAP kinase activation contributes to multiple diverse cellular responses to environment. The capacity to control the amplitude of this response via molecules like IMP likely contributes to flexible and adaptive cellular behavior in the context of complex regulatory signals. Analysis of the primary amino acid sequence of IMP indicated that it may regulated on multiple levels in different ways. We show that IMP can be phosphorylated by ERK and that this correlates with IMP translocation to the nucleus. We also demonstrate that SUMO-1 exerts negative effects on IMP inhibitory activity. Additionally, a predicted UBP-ZnF was examined for regulation of autoubiquitination. Together, these data point to the existence of multiple regulatory inputs that control IMP activity. Finally, we discuss the importance of establishing true binding relationships between associated proteins for the characterization of signaling pathways. Using Ras and its putative effector RalGDS as an example, we describe a simple method to this end. By identifying loss-of-function mutations in both proteins that reciprocally compensate to restore a biological outcome, we confirm that RalGDS is a bona fide effector of Ras, thereby proving the validity of this technique for authenticating other protein-protein interactions.