• ParaDIME: Genome-wide differential DNA methylation analyses using next generation sequencing

      Pabla, Sarabjot; Institute of Molecular Medicine and Genetics (12/27/2016)
      Epigenetic modifications are key players in the regulation of a plethora of cellular and physiological processes. DNA methylation is one of the most widely studied epigenetic modification. Genomic abnormalities in DNA methylation have been implicated in various complex diseases including cancer and autoimmunity. With advent of next generation sequencing, investigating DNA methylation patterns at genome-wide scale has become increasingly feasible. However, the pace of developing appropriate statistical methods to analyze large scale DNA methylation data has been slower. This can be attributed to both statistical and computational challenges faced by current methods. In order to overcome these statistical and computational shortcomings, we developed ParaDIME, a web application for differential DNA methylation analysis. ParaDIME tests CpG dinucleotide sites or pre-defined regions of CpG sites for differential DNA methylation using Rao-Scott chi squared test. ParaDIME not only uses a nonparametric test that accounts for differential sequencing coverage but also uses permutation testing to compute exact p values. In order to overcome computation challenges of large amount of permutations, we use parallel computing to share the workload and decrease execution time significantly. To test ParaDIME in-silico, we initially simulated bisulfitesequencing data and tested it against two most widely used methods: MethylSig and MethylKit. It performed equal or better at accurately detecting differentially methylated regions than both the methods. Especially, at important, low differences of percent methylation, ParaDIME performed better than existing tools. In order to test ParaDIME’s ability to detect biologically relevant differentially methylation regions (DMRs), it was then tested on publically available methylation data from chronic lymphocytic leukemia patients. Our method was able to detect previously known and experimentally verified DMR in CLL, especially DMRs located in Nfatc1 and FOXA2 genes. Additionally, it was able to detect other DMRs in genes present in caner related pathways. Due to ParaDIME’s ability to detect biologically relevant DMRs, we employed it in an integrative analysis study to identify epigenetically regulated genes in Sjogren’s syndrome mouse model, B6.NOD aec1/aec2. We performed reduced representation bisulfite sequencing and RNA sequencing on salivary glands of four and eighteen weeks old B6.NOD aec1/aec2 compared to age and gender matched C57BL/6 mice. After removing age and mouse model effect, we discovered 89 differentially expressed as well as differentially methylated genes. Spearman rank order correlation analysis found a significant correlation between DNA methylation and gene expression. Autoimmunity related genes Klf9 and Nfkbid showed significant negative correlation whereas, other genes like Fgf12 and Coll11a2 genes showed significant positive correlation. Subnetwork enrichment using MATISSE showed three jointly active connected subnetworks that were highly enriched in Immune system related pathways, especially, T cell and B cell activation along with cytokine signaling and endocrine system development. Evidence presented in this report presents a novel and a robust differential DNA methylation analysis method with high accuracy to detect disease-relevant DMRs. ParaDIME is a user-friendly and scalable web application with appropriate test statistic to analyze large-scale DNA methylation studies.
    • Myr 8: The First Member of a Novel Myosin Class Is Expressed During Brain Development

      Patel, Krishna G.; Institute of Molecular Medicine and Genetics (2000-05)
      Neuronal cell migration and cellular differentiation, major phases in the assembly process o f the mammalian neocortex, involve considerable organelle and cellular motility. While the cytoskeletal organization of migrating neurons is well documented, and the involvement of the cytoskeleton in modulating intracellular membrane transport events during neuronal cell differentiation is well appreciated, identification of selective cytoskeletal components underlying these processes is only beginning to emerge. Observations over the past two decades reveal that myosin motors are involved intimately in multiple actin-dependent membrane movements, including vesicular trafficking, organelle localization and organization, endocytosis, exocytosis, phagocytosis, lamellopodial extension, and the more classically defined functions such as cytokinesis, contractility, and cell motility or migration. Accordingly, our studies have been directed toward the identification and characterization of unconventional myosins that may participate in neuronal cell migration and/or differentiation events within the developing mammalian brain. Our analyses identified two myosin isoforms that contribute to a novel unconventional myosin class. We have cloned, sequenced, and designated these myosin isoforms as myr 8a and 8b (8th unconventional myosin from rat). Structurally, the head domain o f myr 8 contains a large N-terminal extension composed of multiple ankyrin repeats similar to myosin phosphatase. The motor domain is followed by a single putative light chain binding domain. The tail domain of myr 8a is comparatively shortwith a net positive charge, whereas the elongated tail domain of myr 8b bears an overall neutral charge and reveals several streches o f poly-proline residues. Phylogenetic analysis indicates that myr 8 is sufficiently divergent from known myosins as to comprise a new class of myosins. Northern analyses demonstrate that the m yr 8 myosins are expressed predominantly in the nervous system, and are detected principally at developmental timeperiods. Indirect-im munofluorescent studies reveal a pattern of im m unoreactivity within forming neuronal and astroglial cell processes located throughout the developing brain. Taken together these data suggest that this novel myosin may play a crucial role in membrane biogenetic events during neuronal and astroglial cell differentiation. Given the increasing identification of neurological dysfunctions that arise as a consequence of defective myosins, as well as from other cytoskeletal components, it is essential to unravel the selective roles in which this novel unconventional myosin may participate during neocortical development.
    • Characterization of Cervical and Head and Neck Squamous Cell Carcinomas by Proteomic Analysis

      Merkley, Mark A.; Institute of Molecular Medicine and Genetics (2009-06)
      (First Paragraph) The oral cavity, oropharynx, larynx, esophagus, and ano-genital orifices are lined with stratified squamous nonkeratinized epithelium, which forms the barrier between the underlying tissue and the external environment. The proliferative nature of this epithelium, together with its potential exposure to environmental insults such as tobacco carcinogens, alcohol, or oncogenic viruses, makes it susceptible to carcinogenesis. Indeed, carcinomas of stratified squamous nonkeratinized epithelium are among the most common and deadly cancers worldwide. In particular head and neck squamous cell carcinoma and cervical squamous cell carcinoma together account for about 1 million new cases annually, worldwide. These cancers arise from similar tissues and share common risk factors, although they differ in that effective population-based screening exists only for cervical cancer.
    • Ceramide-mediated Regulation of Cell Polarity in Primitive Ectoderm Cells: A novel role for sphingolipids in morphogenesis

      Krishnamurthy, Kannan; Institute of Molecular Medicine and Genetics (2009-01)
      Ceramide is considered a key sphingolipid, regulating a variety of critical cellular processes. To facilitate the study of ceramide localization and its interaction with cellular proteins, we have developed a novel antibody against ceramide, raised in rabbit (rabbit IgG). The novel antibody specifically recognizes ceramide in lipid overlay assays and detects ceramide containing different fatty acid chain lengths (i.e. C2-, C16-, C18-, C20- and C24 ceramide). The new antibody was compared with the commercially available anti-ceramide mouse IgM antibody in immunocytochemistry experiments to study the localization of ceramide. Although both antibodies stain similar regions on the cell membrane, the rabbit IgG reveals the distribution of ceramide in intracellular compartments that are not well identified with the commercially available antibody. Pharmacological depletion or increase of ceramide levels results in a corresponding change in staining intensity, confirming the specificity of the antibody. These results indicate that the rabbit IgG is a suitable antibody to determine both the localization of ceramide, and its interaction with proteins by immunocytochemistry. To investigate the role of ceramide in early embryonic development, we used embryoid bodies (EBs) differentiated from mouse embryonic stem cells as a model. The primitive ectoderm cell layer of EBs represents the primitive ectoderm of the early embryo. In mammals, the primitive ectoderm is an epithelium of polarized cells that undergoes gastrulation and differentiates into all embryonic tissues. We find that in primitive ectoderm cells, ceramide was elevated and asymmetrically distributed to the apico-lateral cell membrane, where it was co-distributed with Cdc42 and F-actin. Pharmacological or siRNAmediated inhibition of ceramide biosynthesis impaired primitive ectoderm formation and concomitantly increased apoptosis in EBs. Primitive ectoderm formation was restored by incubation with ceramide or a ceramide analog, indicating that the observed defect was due to loss of ceramide. Ceramide depletion also prevented membrane translocation of atypical PKC (aPKC), interaction of aPKC with Cdc42, and phosphorylation of GSK-3|3. Recombinant aPKC, when bound to ceramide-containing lipid vesicles, formed a complex with the polarity protein Par6 and Cdc42. Taken together, our data suggest a novel mechanism by which a ceramide-induced, apico-lateral polarity complex with aPKC regulates primitive ectoderm cell polarity and morphogenesis.
    • Role of the NR2 Subunit and its Molecular Motifs on Memory and Cognition and the Physiological Outcomes after Experimental Intracerebral Hemorrhage

      Jacobs, Stephanie A.; Institute of Molecular Medicine and Genetics (2013-10)
      The N-methyl-D-aspartate receptor is the main coincidence detector in the brain. It is known to be necessary for many forms of learning and memory. Interestingly, the NR2 subunit composition of the NMDA receptor is modulated endogenously according to the location in the brain and the age of the animal, with the NR1 subunit being ubiquitously expressed. In the forebrain regions, including the cortex, hippocampus, striatum, and amygdala, the NR2A and NR2B subunits are the primary subunits expressed. While it is known that a high NR2B:NR2A ratio enhances memory and cognition, it is not directly known, the effects of a low NR2B:NR2A ratio. In this project, the effects of modulating the NR2A:NR2B ratio on multiple forms of learning and memory are explored by the use of a NR2A transgenic mouse. Our transgenic mice overexpress the NR2A subunit in the forebrain regions, driving the NR2A:NR2B ratio toward the expression of the NR2A. As the NR2B subunit is known to favor learning and memory; we also further explore the role of the N-terminal and membrane domains and the Cterminal domain in the observed enhancements. Our data indicate that a high NR2A:NR2B ratio constrains multiple forms of long-term memory in our transgenic animals. Additionally, we have observed additional forms of enhanced learning and memory in the NR2B transgenic mice that were not tested previously. We were able to show that the NR2B C-terminal tail, and thus the intracellular signaling cascades, is responsible for the enhancements seen in the NR2B animals. Using the NR2A and NR2B transgenic mice, we also investigated the long-held hypothesis that a low NR2B:NR2A ratio would be beneficial to hemorrhagic stroke recovery. Until now this hypothesis has only been investigated by the use of pharmaceuticals, whereby the NR2B subunit is antagonized. We found that while several physiological factors, including neurological deficit and survival rate were unchanged, lesion size and percent edema were significantly less in the NR2A transgenic mice than the NR2B transgenic mice. This demonstrates that a low NR2B:NR2A ratio may be beneficial for some aspects of hemorrhagic stroke recovery.
    • Novel Role of Heat Shock Protein (HSP) 90 in Regulating ATR-CHK1 DNA Damage Response Pathway in Cancer Cells

      Ha, Kyungsoo; Institute of Molecular Medicine and Genetics (2011-10)
      DNA damage caused by environmental mutagens or reactive metabolic byproducts induces DNA damage response (DDR), which regulates cell cycle transit, DNA repair and apoptosis. DDR involves the phosphorylation and activation of Ataxia Telangiectasia Mutated (ATM) and ATM and RAD3-related (ATR) proteins. ATR regulates the firing of the replication forks during S phase, and the repair of damaged replication forks to prevent premature onset of mitosis. ATR phosphorylates and activates CHK1 which phosphorylates and inactivates CDC25, thereby inhibiting CDK1 activation and cell cycle progression. In the present studies, we determined that treatment with an hsp90 inhibitor AUY922, without affecting the mRNA levels, dose-dependently depletes the protein levels of p-ATR (Ser 428), ATR and CHK1 in human breast and cervical cancer cells. Additionally, treatment with the pan-histone deacetylase inhibitor panobinostat (PS), which is known to induce hyperacetylation and inhibition of hsp90 function, also depleted ATR and CHK1 levels in cancer cells. Co-treatment with the proteasome inhibitor bortezomib (BZ) partially reversed AUY922- or PS-mediated depletion of ATR and CHK1 expression, indicating proteasome-mediated degradation of ATR and CHK1. Treatment with either AUY922 or PS markedly inhibited the binding of ATR with hsp90, induced polyubiquitylation of ATR, and decreased the half-life of both ATR and CHK1 proteins. Treatment with AUY922 also abrogated ionizing radiation (IR)-induced cell cycle arrest and increased the amount of DNA damage in the cancer cells following IR. Treatment with AUY922 also inhibited the recruitment of p-ATR, ATR and 53BP1 to the site of DNA damage. In addition, HDAC3 binds to and deacetylates hsp90 in the nucleus. Depletion of HDAC3 by either short hairpin RNA or genetic knockout induced hyperacetylation of nuclear hsp90, resulting in the inhibition of chaperone association of ATR with hsp90 and depletion of ATR. These findings demonstrate that 1) ATR is chaperoned by hsp90, 2) Inhibition of chaperone function of hsp90 results in proteasomal degradation of ATR and inhibition of DDR, 3) pan-HDAC inhibitors abrogate ATRCHK1 cell cycle checkpoint pathway by modulating chaperone activity of hsp90 and 4) HD AC3 plays a critical role in the regulation of DNA damage response by stabilizing the chaperone activity of nuclear hsp90.
    • Mechanosensory Hair Cell Precursors in the Zebrafish Lateral Line

      Floyd, Tiffany L.; Institute of Molecular Medicine and Genetics (2009-07)
      The vertebrate inner ear mediates the senses of hearing and balance. Contained within both the auditory and vestibular compartments of the inner ear are specialized mechanosensory hair cells that function as receptors and transducers of environmental stimuli. In all vertebrates, these sensory hair cells are particularly susceptible to ototoxic insults resulting in cell death and, in mammals, the irreversible loss of hair cells underlies deafness and balance disorders. In contrast to mammals, several non-mammalian vertebrates (including zebrafish) possess the innate capacity to produce new hair cells throughout life as well as regenerate hair cells that have been lethally damaged. A long-term strategy of the hearing research field is to determine the molecular mechanisms of hair cell regeneration using regenerating model systems such as zebrafish, then to apply this information to mammalian models where sensory hair cell regeneration is limited or nonexistent. During embryogenesis, sensory hair cell fates are specified through a mechanism of Notch-Delta-mediated lateral inhibition. The gamma secretase complex is an upstream regulator of Notch signaling, responsible for proteolytic\ cleavage and activation of the Notch receptor. Recent evidence suggests that Notch signaling may also play a role during the process of hair cell regeneration in zebrafish (Ma et al., 2008). I used a chemical inhibitor of the gamma secretase complex to examine the role of Notch signaling in the regulation of hair cell number maintenance in larval zebrafish. Results presented in this thesis provide novel insight into the mechanisms regulating the maintenance of resident hair cell precursors within the sensory epithelium. Moreover, this new information is directly relevant to research efforts in mammalian models by providing the molecular framework for therapeutic strategies designed to replace or regenerate lethally damaged hair cells in the mammalian cochlea by reactivating resident precursors to differentiate into hair cells.
    • Design, Synthesis, and Initial Evaluation of D-Glyceraldehyde Crosslinked Gelatin-Hydroxyapatite as a Potential Bone Graft Substitute Material

      Florschutz, Anthony V; Institute of Molecular Medicine and Genetics (2012-07)
      Utilization of bone grafts for the treatment of skeletal pathology is a common practice in orthopaedic, craniomaxillofacial, dental, and plastic surgery. Autogenous bone graft is the established archetype but has disadvantages including donor site morbidity, limited supply, and prolonging operative time. In order to avoid these and other issues, bone graft substitute materials are becoming increasingly prevalent among surgeons for reconstructing skeletal defects and arthrodesis applications. Bone graft substitutes are biomaterials, biologies, and guided tissue/bone regenerative devices that can be used alone or in combinations as supplements or alternatives to autogenous bone graft. There is a growing interest and trend to specialize graft substitutes for specific indications and although there is good rationale for this indication-specific approach, the development and utility of a more universal bone graft substitute may provide a better answer for patients and surgeons. The aim of the present research focuses on the design, synthesis, and initial evaluation of D-glyceraldehyde crosslinked gelatin-hydroxyapatite composites for potential use as a bone graft substitutes. After initial establishment of rational material design, gelatinhydroxyapatite scaffolds were fabricated with different gelatin:hydroxyapatite ratios and crosslinking concentrations. The synthesized scaffolds were subsequently evaluated on the basis of their swelling behavior, porosity, density, percent composition, mechanical properties, and morphology and further assessed with respect to cell-biomaterial interaction and biomineralization in vitro. Although none of the materials achieved mechanical properties suitable for structural graft applications, a reproducible material design and synthesis was achieved with properties recognized to facilitate bone formation. Select scaffold formulations as well as a subset of scaffolds loaded with recombinant human bone morphogenetic protein-2 were implanted ectopically in a rodent animal model and histologically evaluated for biocompatibility, degradation, and bone formation in vivo. The gelatin-hydroxyapatite scaffolds retained dimensional structure over 28 days and did not elicit any undesirable systemic or local effects. Distinct areas of mineralization and osteoid/bone were noted in all the implanted scaffolds and quantitative differences were primarily dependent on the presence of hydroxyapatite.
    • The Clinical Phenotype of An Extended Pedigree with Late-onset Alzheimer's Disease

      Fennell, Eleanor M.; Institute of Molecular Medicine and Genetics (2009-03)
      Alzheimer's disease (AD) is a devastating neurodegenerative disease with a multifaceted etiology. This retrospective exploratory study used the family history method to develop a phenotype based on the personal history of cognitive decline, changes in behavioral characteristics, and the medical, social and environmental history of the five AD affected family members in one large extended family. Group family interviews provided a description of the phenotype and identified medical and environmental risk modifiers. Average age-of-onset for AD in 5 of 12 siblings, (all homozygous APOE4/4) was 69.2 years (range 66-72 years). Fisher's exact test identified neuropsychiatric behaviors as common phenotypic manifestations; delusions (p = 0.0455), hallucinations (p = 0.0101), irritability (p = 0.0455), personality change (p = 0.0013), pacing (p = 0.0013), aggressiveness (p = 0.0455), and poor judgment (p = 0.0013). Environmental variables that emerged as significant were a less than an eighth grade education (p — 0.0152), presence of stroke/TIA (p = 0.0455), presence of osteoarthritis (p = 0.0455), and vitamin B12 deficiency (p = 0.0013). Risk of stroke/TIA may be related to the increased risk from APOE4 while vitamin B12 deficiency may be associated with advanced age. There was no significant protective benefit from the management of hypertension (p = 0.5758), use of statins to control cholesterol (p = 0.9545), use of Vitamin C (p = 1.000), and/or Vitamin E (p = 0.9899) among family members. Potential modifiable health practices among the AD unaffected siblings demonstrated that 85% continued to engage in a sedentary lifestyle (p = 0.6818), 57% were overweight (p = 0.6894), 57% (p = 0.6894) consumed an unhealthy diet, and 56% smoked (p = 0.1591). Single nucleotide polymorphism (SNP) microarray Clinical Phenotype of Alzheimer's Disease analysis indicated that several SNPs in the gene, transient receptor potential cation channel, subfamily C, member 4 associated protein, (TRPC4AP), were significant. All five of the affected siblings and three unaffected siblings exhibited one haplotype; the unaffected siblings with the same haplotype as affected siblings were younger in age and did not have any cognitive problems at the time of the study (Poduslo, Huang, Huang, Smith, 2008).
    • Mechanisms of Neuroprotection by Estrogen and Selective Estrogen Receptor Modulators

      Dhandapani, Krishnan M.; Institute of Molecular Medicine and Genetics (2003-03)
      Specific Aim #1: To determine whether 17b-E2 and SERMs directly influence neuronal survival. Specific Aim #2: To determine whether astrocyte-derived TGF-b protects neurons from cell death. Specific Aim #3: To determine whether 17b-E2 or SERMs regulate the release of TGF-b from astrocytes. Specific Aim #4:To elucidate the mechanism of TGF-b mediated neuroprotection in GT1-7 Neurons. Specific Aim #5: To identify genes potentially mediating the neuroprotective effects of 17b-E2 and/or tamoxifen through the use of high density gene chip arrays.
    • Signaling in the Late Phase of T Cell Activation

      Chang, Jing-Wen; Institute of Molecular Medicine and Genetics (2004-12)
      Engagement of the T cell antigen receptor (TCR) induces multiple signaling pathways, including the activation of extracellular signal-regulated kinase (ERK), a mitogen-activated protein kinase (MAPK). We previously reported the importance of sustained ERK activation for interleukin-2 (IL-2) production. Inhibition o f ERK activation from 2 to 6 hours after TCR stimulation significantly impaired IL-2 production and activation of the nuclear factor-kappaB (NF-kB) family transcription factor, c-Rel, whereas inhibition during the first 4 hours had no effect. Loss o f the adaptor protein, She, results in impaired ERK activation during the late phase of TCR stimulation, and leads to severely reduced IL-2 production and c-Rel activation. These data suggest a novel activation process following TCR stimulation that involves She and late ERK activation-dependent regulation of c-Rel activation and IL-2 production. To further understand the mechanisms underlying this pathway, we employed a two-dimensional differential in-gel electrophoresis/ mass spectrometry (2D-DIGE/MS)-based proteomics approach. This approach to identify members of a Shc-containing signaling complex revealed alpha tubulin and beta actin as She associated proteins. Furthermore, we identified proteins whose expression and modification are triggered by TCR stimulation and are under control of the ERK signaling pathway, by comparing TCR-stimulated samples with or without MEK (MAPK kinase) inhibitor treatment. Here we report heterogeneous nuclear ribonucleoprotein K (hnRNP-K) as a novel downstream target of ERK in TCR signaling. Functional studies using small RNA interference showed that hnRNPK regulated IL-2 production at the transcriptional level. We also showed that knockdown o f hnRNP-K expression specifically impaired NF-kB activity, but caused a relatively minor effect on activating protein-1 (AP-1) activity and expression of CD69 or CD25. Biochemical analysis showed that knockdown of hnRNP-K caused enhanced proteolysis of the protooncogene Vav. MEK inhibitor treatment during the late phase of stimulation also enhanced proteolysis of Vav. Moreover, knockdown of hnRNP-K impaired Vav-mediated transcriptional activation of IL-2 gene. Taken together; these results indicate that ERK signaling modulates IL-2 production by regulating Vav activity through the function of hnRNP-K. We also examined changes in phosphoprotein profiles upon TCR stimulation and MEK inhibitor treatment. Results obtained from these three different approaches provide a further understanding of the mechanisms that regulate late phase T cell activation as well as the components required for full activation o f T cells.
    • Evidence Supporting Glial Derived TGF-B1 as a Modulator of Luteinizing Hormone-Releasing Hormone

      Buchanan, Clint D.; Institute of Molecular Medicine and Genetics (2000-03)
      The overall objective of this research is to elucidate mechanisms involved in glial cell regulation of reproductive function. Regulation of LHRH secretion is a complex process that involves a multiplicity of inputs of both excitatory and inhibitory nature, and recent evidence has demonstrated the significance of glial cell-neuron interactions in modifying the activity of LHRH producing neurons. Evidence exists indicating that glial derived growth factors may play a role in the functional control o f the LHRH neuronal network as conditioned medium from astrocytes has been shown to stimulate LHRH secretion from immortalized LHRH neurons (52-55,57,58). However, there is a controversy concerning the identity of the active factor from astrocytes that is responsible for the LHRH releasing activity of conditioned medium. Melcangi and colleagues have provided evidence that TGF-Pi may be responsible for astrocyte-conditioned medium induced LHRH release in the GTl-l cell line (55). However, many of these studies supporting a role for TGF-pi were performed using cortical astrocytes, and additionally, no attempt was made to measure TGF-Pi levels in astrocyte-conditioned medium and correlate it to conditioned medium ability to induce LHRH release. Furthermore, these studies did not discuss potential regulators of TGF-pi secretion and also failed to investigate whether TGF-p receptors, which are necessary for TGF-pi action, are expressed in the GT1 cell line or hypothalamic tissue of the female rat (55,57,58). A second group suggests that TGF-a rather than TGF-Pi may be the active astrocyte factor that regulates LHRH release (53). Although TGF-a mRNA expression and precursor peptide immunoreactivity have been reported in the female rat hypothalamus, these studies failed to demonstrate the ability of hypothalamic astrocyte cultures to produce Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 15 TGF-a and relied upon addition of exogenous TGF-a to astrocyte cell cultures (50ng/ml 16 hours) in formation of astrocyte TGF-a-conditioned medium (25,46,52,53,76,77).
    • Genomic and Functional Analysis of Vesicular Inhibitory Amino Acid Transporter During Mouse Embryogenesis

      Oh, Won-Jong; Institute of Molecular Medicine and Genetics (2006-01)
      The specification of particular neuronal phenotypes during embryonic development requires the appropriate activation and regulation of genes encoding the proteins required for neurotransmitter synthesis, vesicular packaging and re-uptake from the synaptic cleft. Each neurotransmitter is packaged into synaptic vesicles by its own distinct vesicular transporter. In addition, neurotransmitter packaging is well controlled by other co-factors (reviewed in Ahnert-Hilger et al., 2003). Components of GABAergic neurons GABAergic neurons are the principal inhibitory neurons in the mammalian central nervous system (CNS), where GABA is synthesized from glutamate by two glutamate decarboxylases (GAD), namely GAD65 (Gad2) and GAD67 (Gad1) (Erlander et al., 1991). GABA is then loaded into synaptic vesicles by the vesicular inhibitory amino acid transporter (VIAAT, also known as VGAT). Four GABA transporters (GAT 1-4) are responsible for the re-uptake of GABA from the synaptic cleft through the plasma membrane. Inhibitory GABAergic transmission is mediated by binding of GABA to its ionotropic receptors, GABAA and GABAC, which are ligand-gated chloride channels, and its metabotropic receptor, GABAB (Fig. 1).
    • Influence of DNA Ends on Structure and Function of the DNA-dependent Protein Kinase

      Jovanovic, Marko; Institute of Molecular Medicine and Genetics (2006-12)
      Non-homologous end joining is a major DNA double-strand break repair pathway in mammalian cells. The DNA-dependent protein kinase (DNA-PK), consisting of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and Ku heterodimer, is hypothesized to be a key regulator of the pathway. Available data suggest DNA-PKcs may exert this regulatory function by controlling access to the DNA termini and by phosphorylation of itself and other proteins. I further characterized DNA-PK-DNA interaction by studying binding of DNA-PKcs and Ku to oligonucleotides with chemically defined end structures under conditions that preclude synapsis between opposing DNA ends. Binding of DNA-PKcs to DNA varied with the end structure in a manner that suggests that partial melting of DNA ends is necessary for the formation of a stable, enzymatically active complex. Unexpectedly, these studies also revealed that ATP, as well as its nonhydrolyzable analog AMP-PNP, have an allosteric effect on the interaction of DNA-PKcs with DNA.