• Cellular and Molecular Mechanisms of Retinal Bipolar Regeneration in Zebrafish

      Ariga, Junko; Department of Pediatrics (2012-03)
      Human retinal degenerative diseases are characterized by slow progressive loss of retinal cells which induces reactive gliosis in Muller glia cells. In mammalian systems, this results in scar tissue formation which exacerbates loss of vision. Similar initial responses are observed following injury in highly regenerative species, such as zebrafish. However, Muller glia cells in these systems are capable of regenerating a functional retina. We are interested in determining how the regenerative potential of Muller glia cells is triggered and controlled. Thus, we are studying the cellular and molecular mechanisms governing how zebrafish regenerate specific retinal cell types. Ultimately, we seek to identify factors that could be harnessed to redirect mammalian Muller glia cells into regenerative pathways. Such insights could aid the development of regenerative therapies for degenerative diseases. Despite the relevance to disease, little is known about how the retina responds to loss of discrete cell types. Here, we focused on characterizing this paradigm to study four principle aspects of the regenerative process: 1) endogenous stem cell activation, 2) stem and progenitor cell proliferation, 3) progenitor cell differentiation, and 4) functional recovery. By studying all four of these aspects in relation to each other we were able to reveal fundamental insights into how retinal regeneration is governed. Specifically in Aim 1, we used transgenic and pharmacological techniques to induce ablation of /^-expressing retinal bipolar cell subtypes and asked whether the lost cells were subsequently regenerated in zebrafish larvae. We then sought to identify potential stem cell sources. In Aim 2, lineage tracing of retinal stem cell populations was used to ask whether the extent of bipolar cell loss altered the specificity of the regenerative response. In Aim 3, we manipulated the Wnt pathway to investigate the role of Wnt signaling in bipolar cell regeneration. Finally, in Aim 4 we used visual behavior assays to determine if functional deficits attend the loss of nyx-expressing bipolar cells and, if so, whether functional recovery was evident following their regeneration. In particular, our observations demonstrating opposing roles of the Wnt pathway in regeneration have implications regarding the development of age-appropriate and/or cell-specific regenerative therapies.
    • Cellular and Molecular Players in Neuromuscular Junction (NMJ) Formation and Function

      Barik, Arnab; Institute of Molecular Medicine and Genetics (2014-04)
      There are three distinct segments in this dissertation. First, I attempted to address the role of Schwann cells in mammalian neuromuscular junction (NMJ) development and function. Schwann cells at the NMJs do not form myelin sheaths and are known as terminal Schwann cells. Terminal Schwann cells are thought to be analogous to astrocytes in the central nervous system. Schwann cells (as described in details in the next section) provide trophic support to motor axons and modulate synaptic activity by sensing neurotransmitter release at the nerve terminal. However, the role of Schwann cells in synapse formation and maintenance remains unknown. Second, during NMJ formation, anterograde signals from nerve to muscle, and retrograde signals from muscle to nerve are critical for the establishment of a functional synapse. Research over the last three decades has contributed to our understanding of the role of the anterograde signaling at NMJ. However, identification of muscle-derived retrograde signals involved in motoneuron terminal differentiation remains scarce. Recent work from our laboratory suggests that genes that are transcriptionally regulated by p-catenin in muscles might play a crucial role in pre-synaptic differentiation at the NMJ.2 Third, Agrin-LRP4-MuSK signaling is critical for NMJ formation. At the NMJ, LRP4-mediated activation of MuSK by neural Agrin is required for post-synaptic differentiation. Mice that lack any one of the three genes fail to form NMJs and die at birth. Due to perinatal lethality of these null mice, less is known about how Agrin-LRP4-MuSK might regulate NMJ maintenance. Moreover, mutations in Agrin, LRP4, and MuSK have been reported in patients diagnosed with congenital myasthenic syndrome (CMS), and autoantibodies against MuSK and LRP4 have been detected in patients with myasthenia gravis (MG). However, the role of Agrin-LRP4-MuSK in the etiology of these neuromuscular disorders is not clear.
    • Central versus peripheral effects of scopolamine on performance in a delayed simple discrimination tas

      Martin, Frances Fay Evans; Department of Pharmacology and Toxicology (1993-01)
      As an_ amnestic agent, scopolamine hydrobromide (SHB) has been used as· a classic model for amnesia. While it is generally accepted that SHB affects stimulus discrimination, attention, and acquisition, a conflict exists ~s to whether the time-dependent process of retention is affected by scopolamine. Wis tar rats were trained in·a simple discrimination task with tone and light stimuli and with delays of increasing length interposed between the stimulus and ,the response. When the rats were well-trained, 4 doses (5, 10, 25, and 50 μg/kg) of $HB and of scopolamine methylbromide (SMB), a quaternary analog that does not easily enter the CNS, were injected subcutaneously once or twice weekly before the daily session. With light as the stimulus, there were significant dose and delay effects .with both drugs and no difference in the effects of the two drugs except for a trend toward a greater effect of scopolamine hydrobromide on accuracy at the highest dose.
    • Ceramide Compartments and Protein Interaction: Structure Meets Function

      Kong, JiNa; Department of Neuroscience and Regenerative Medicine (12/27/2016)
      Ceramide is a key sphingolipid, regulating a variety of critical cellular processes. Although exosomes and cilia are derivatives of the membrane, little is known about the role of lipids in their formation. Here we examined the novel role of ceramide in two ceramide-enriched, subcellular compartments: 1) secreted, extracellular vesicles (EVs) termed exosomes, and 2) cell membrane protrusions termed cilia. Firstly, we attempted to address the role of ceramide in exosome secretion and breast cancer. Breast cancer cells acquire multidrug resistance (MDR) mediated by ABC transporters such as breast cancer resistance protein (BCRP). We show that incubation of human breast cancer MDA-MB-231 cells with the farnesoid X receptor antagonist guggulsterone (gug) and retinoid X receptor agonist bexarotene (bex) elevated ceramide, which is known to induce exosome secretion. Ceramide elevation by combined treatment with gug and bex induced BCRP secretion in exosomes and reduced cellular BCRP in cancer and cancer stem-like cells. Consistent with reduced BCRP, ABC transporter assays showed that gug+bex treatment increased doxorubicin retention and that the combination of gug+bex with doxorubicin enhanced cell death. Our results suggest a novel mechanism by which ceramide induces BCRP secretion and reduces MDR, which may be useful as adjuvant drug treatment for sensitizing breast cancer cells and cancer stem cells to chemotherapy. Secondly, to investigate the role of ceramide in ciliogenesis, in particular motile cilia, we used Chlamydomonas reinhardtii (Chlamydomonas) and murine ependymal cells as models. Motile cilia are specialized organelles formed by cell membrane protrusions to function in movement of body fluids. We show for the first time that Chlamydomonas expresses serine palmitoyl transferase (SPT), the first enzyme in the sphingolipid biosynthetic pathway. Ceramide depletion, by the SPT inhibitor myriocin and a neutral sphingomyelinase deficiency (fro/fro mouse), led to glycogen synthase kinase-3 (GSK3) dephosphorylation and defective flagella and cilia, respectively. A novel activation mechanism for GSK3 by the sphingolipids phytoceramide and ceramide is shown to be critical for ciliogenesis in Chlamydomonas and ependymal cells, respectively. We conclude that ceramide promotes exosome secretion to reduce MDR in MDA-MB-231 cells and regulates GSK3-mediated ciliogenesis in Chlamydomonas and murine ependymal cells.
    • 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.
    • Ceramide-mediated regulation of cell polarity in primitive ectoderm cells: a novel role for sphingolipids in morphogenesis

      Krishnamurthy, Kannan; School of Graduate Studies (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 lgG). 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 anti.body was compared with the commercially available anti-ceramide mouse lgM antibody in immunocytochemistry experiments to study the localization of ceramide. Although both antibodies stain similar regions on the cell membrane, the rabbit lgG 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 ofthe antibody. These results indicate that the rabbit lgG 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-3p. 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.
    • Changes in parental perceptions of stress in a pediatric intensive care units

      Sharp, Emily A.; School of Nursing (1985-05)
      . Pare~tal perceptions of stress associated wfth having a thild hospitalized in a Pediatric Intensive Care Unit {PICU) were studied. The Parental Stressor Scale: Pediatric Intensive Care Unit .{Miles & Carter, 1983) and a Demographic Data Sheet were used to assess nineteen parents• stress who had a child hospitalized in a PICU of a southwestern hospital {n = 19). Results of. the study indicated that parental perceptions of stress did not significaritly differ at three different times during their child's hospitalization in a PICU. The parental stress was assessed at the beginning, at the midpoint, and ·at the end of their child's hospitalization in the PICU. The parental stress was also analyzed for correlation with the child's age, admi~sion status, and parent's educational level. The parental stress was significantly greater if the. child's admission to-the PICU was unplanned., The study provides I some interesting data to be considered by profess-ionals caring for parents with a child hospitalized in a PICU.
    • Changes in the RANK/RANKL/OPG Signaling System as a Mechanism of Zoledronate-Induced Osteonecrosis of the Jaw

      Lane, Jonathan; Department of Oral Biology (3/22/2016)
      Bisphosphonates (BPs) are widely used for the treatment of osteoporosis, hypercalcemia of malignancy, skeletal-related events associated with bone metastases, and for managing lytic lesions of multiple myeloma. A serious risk associated with the use of BPs is the development of Bisphosphonate Related Osteonecrosis of the Jaw (BRONJ), a painful and inflamed area of exposed bone in the oral cavity that fails to heal after 6-8 weeks. The cause of BRONJ is unknown, but it is believed to be due primarily to a longterm suppression of bone remodeling, caused by BP’s potent inhibition of osteoclastic activity. At the cellular level, it is generally accepted that bisphosphonates are taken in by osteoclasts at sites of relatively greater bone remodeling, owing to the strong affinity of bisphosphonates for the mineralized matrix and the increased activity of osteoclasts at active sites of resorption. The accumulation of intracellular bisphosphonates ultimately leads to osteoclast dysfunction or apoptosis through the formation of nonhydrolyzable ATP-analogues, or due to inhibition of the mevalonate pathway responsible for synthesis of sterols and lipids necessary for proper cellular membrane structure. However, the refined details of the pathophysiology of BRONJ remain elusive. The RANK/RANKL/OPG system is a well-known signaling pathway for the recruitment and differentiation of osteoclasts. RANK is a surface-bound receptor on osteoclasts, and requires binding of its ligand, RANKL, for cell activation and ultimately resorption of bone. On the other hand, OPG is a soluble decoy receptor for RANKL. Therefore, osteoclastic activity is effectively regulated by the ratio of RANKL to OPG. For years, it has been generally accepted that osteoblasts are the primary source of both RANKL and OPG. However, it is now recognized that the master orchestrator of bone activity, the osteocyte, contributes to the pathway. Furthermore, it has been shown that in localized tissue damage or hypoxia, such as in a dental extraction, immediately adjacent surviving nonapoptotic osteocytes upregulate RANKL and downregulate OPG. It is unknown to what extent BPs may alter the normal osteocyte response to injury and hypoxia or, ultimately, the dynamics of the RANK/RANKL/OPG system. Furthermore, the extent to which this could contribute to the development of BRONJ is unexplored.There is a paucity of studies concerning how the fundamental system responsible for bone remodeling, RANK/RANKL/OPG, is effected by BPs. It may be that changes in this system, especially in signals derived from the osteocyte, contribute to the pathophysiology of BRONJ.
    • Characterization o f the DNA Ligase IV and XRCC4 complex in the DNA double-strand break repair

      Lee, Kyung-Jong; Institute of Molecular Medicine and Genetics (2002-11)
      DNA double-strand breaks (DSBs) are among the most lethal forms of DNA damage. The nonhomologous end-joining (NHEJ) pathway is the principal mechanism for repairing DSBs in mammalian cells. It is also required for V(D)J recombination. There are at least four essential proteins in this pathway. These include Ku protein, DNA PKcs, and the DNA Ligase IV/XRCC4 (DNL IV/XRCC4) complex. This dissertation reports the determination of the quaternary structure of the DNL IV/XRCC4 complex, the mapping of a major human autoimmune epitope in XRCC4, the identification of DNAPKcs phosphorylation sites in XRCC4, and an investigation of the biochemical significance of XRCC4 phosphorylation. Biochemical characterization shows that DNA Ligase IV and XRCC4 form a stable mixed heterotetramer. This is the active form of the enzyme and is essential for in vitro DNA end joining in the presence of additional factors derived from cell extracts. Data shown here also demonstrate that the DNL IV/XRCC4 complex is a human autoantigen. The major autoimmune epitope maps to amino acids 251-266. This epitope coincides with several sites where XRCC4 is potentially modified in response to radiation or inflammation, including a DNA-PKcs phosphorylation site at serine 260. Results raise the possibility that radiation-induced post-translational modifications contribute to development of an autoimmune response in susceptible individuals. Previous work has shown that DNA-PKcs kinase activity is required for NHEJ, but the critical physiological target of this enzyme is not yet known. Current work shows that DNA-PKcs phosphorylates serine 318 of XRCC4, in addition to the serine 260 site described above. The presence of serine 260 increases phosphorylation at serine 318, suggesting that phosphorylation can occur sequentially. Mutation o f serine 260 reduced DNA end-joining activity and sensitivity to the PI3 kinase inhibitor (LY294002). These data provide preliminary evidence that phosphorylation of XRCC4 by DNA-PKcs contributes to regulation of DNA repair.
    • Characterization o f Zebrafish Mutant m erlot as a Non-Mammalian Vertebrate Model for Congenital Anemia Due to Protein 4.1 Deficiency

      Shafizadeh, Ebrahim; Department of Biochemistry and Molecular Biology (2002-08)
      The zebrafish mutant merlot (mot) is characterized by onset o f a severe anemia at 96 hours post fertilization. We performed whole mount RNA in situ hybridization and showed that the process o f primitive erythropoiesis is not interrupted in the mot embryos. Blood analysis demonstrated that mot suffers from a severe congenital hemolytic anemia. Using the TU N E L assay, we detected apoptotic erythroid progenitors in the kidneys. We performed electron microscopic analysis and detected membrane abnormalities and a loss o f the cortical membrane organization in the mot cells. We used positional cloning techniques w ith a candidate gene approach to demonstrate that mot encodes the erythroid specific isoform o f protein 4.1R, a critical component o f the red blood cell membrane skeleton. Sequence analysis o f 4.IR cD N A detected nonsense point mutations in both alleles o f mot resulting in premature stop codons. We performed linkage analysis and transgenic rescue experiments to provide further confirmation that the molecular defect in the protein 4 .1R is the underlying cause o f anemic phenotype in mot fish. This study presents the zebrafish mutant merlot as the first characterized non-mammalian vertebrate model o f congenital anemia due to a defect in protein 4.1R integrity.
    • Characterization of a high-affinity, highly selective tryptophan transport system in the human macrophage and the effects of overexpression of tryptophanyl t-RNA synthetase on Jurkat proliferation

      Seymour, Robert L.; Department of Pediatrics (2004-04)
      Suppression of T cell activation by macrophages/dendritic cells via tryptophan degradation has been shown to play an important role in immunotolerance. Tryptophan degradation is carried out by the enzyme indolamine-2,3-dioxegenase (IDO). This model raises many questions. This study addresses two of these questions. First, how does the macrophage gain access to and degrade tryptophan to a level below 50 nM in culture medium? This is achieved despite the fact that the known high affinity tryptophan transport systems accept other amino acids and have Km values for tryptophan ranging fi-om 10-100 pM. In this study we show that the macrophage possesses a high-affinity, highly selective, and Na-independent tryptophan transport system with a Km for tryptophan of about 300 nM. This would allow the macrophage to have effective access to tryptophan at concentrations in the nanomolar range. We also show T cells do not possess this transport system. Second, how does the T cell sense the level of intracellular free tryptophan? It has been shown in the past that if T cells are stimulated in medium containing less than SOOnM tryptophan that they attempt to activate but arrest in mid-Gl of the cell cycle. The enzyme tryptophanyl t-RNA S5mthetase (WRS) charges tRNA*'^ with tryptophan. This enzjmie has two protein isoforms, with one having a non-canonical N-terminal kinase domain. WRS is also upregulated by interferon gamma (IFNy). These characteristics put WRS in a position to be an intracellular free tryptophan sensor. Here we show that transient transfection of the T cell line, Jurkat, with cDNA encoding the kinase-containing isoform of WRS inhibits proliferation. In addition, to the above we have ereated a subline of Jurkat, which stably arrests in the absence of tryptophan. We also show that this new subline is resistant to the drug G418 but is sensitive to hygromycin. When treated with rapamycin the Jurkat sub line will stably arrest in the presence or absence of tryptophan. Rapamycin is a known immunosuppressive agent, which inhibits T cell proliferation. This leads to the speculation of a possible link between the signaling pathways involved in tryptophan sensing and those involved in the effects of rapamycin.
    • Characterization of a microsomal androgen receptor in the rat ventral prostate

      Steinsapir, Jaime; Department of Physiology and Endcrinology (1988)
    • Characterization of Cardiac L-Type and T-Type Calcium Channels During Normal and Defective Chick Heart Development

      Nichols, Carol A.; Department of Cellular Biology and Anatomy (2000-03)
      (First Paragraph) The human heart is vital for survival from early in embryonic development throughout life. It begins developing around the third week of gestation from a pair of endocardial tubes that fuse to form a single primitive heart tube. The single-lumen heart tube develops a series of expanded areas and infoldings that divide it into four presumptive chambers. As the embryo grows, the heart begins looping. This looping process serves to bring the four presumptive chambers into the appropriate orientation for septation. The developing heart remodels itself into four separated chambers (two atria or holding chambers, two ventricles or pumping chambers) which provide for separate systemic and pulmonary circulation at birth. In most mammals, oxygenated blood enters the left atrium through four pulmonary veins. The blood is forced into the left ventricle when the left atrium contracts. When the left ventricle contracts, blood is pumped through the aorta and carried throughout the body. Deoxygenated blood returns to the right atrium via the superior and inferior vena cavae. Blood is forced into the right ventricle by contraction of the right atrium. Blood is then pumped through the pulmonary trunk and arteries to the lungs to be re-oxygenated. The four- chambered heart is formed by the eighth week o f gestation. (Larsen, 1997; de la Cruz & Markwald, 1998).
    • 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.
    • Characterization of gastric tubulovesicles: implications for vesicle fusion and recycling

      Calhoun, Benjamin Carlisle; School of Graduate Studies (1997-06)
      Gastric parietal cells undergo a massive morphological transformation in response t. to· secretagogues such as histamine and acetylcholine. The surface area of the apical secretory canaliculus increases, and the volume occupied by the intracellular tubulovesicles, which sequester the H/K-ATPase in resting parietal cells, decreases. These morphological changes are thought to correspond to the delivery of the H/K-ATPase from the intracellular tubulovesicles to the cell surface in a regulated vesicle fusion event. The scope of this reversible membrane addition makes the parietal cell an ideal model for the study of the molecular regulation of regulated apical vesicle fusion and recycling. The aim of this study was to determine if candidate regulators of vesicle fusion in other well-:-characterized systems also participate in the fusion of the tubulovesicles with the secretory canaliculus of the parietal cell. Highly purified ·tubulovesicles were prepar(?d by gradient fractionation and immunoisolation on magnetic beads coated with monoclonal antibodies against the ex.subunit_ ofH/K-ATPase. The association of Rab proteins, vesicle-associated membrane protein (V AMP-2), syntaxins, and secretory carrier membrane proteins (SCAMPs) with inimunoisolated tubulovesicles was determined by western blot analysis. Rab 11 and Rab25 are two ras-related small OTP-binding proteins enriched in gastric parietal cells. The vesicle prote!n V AMP-:-2 forms a putative vesicle docking-and fusion complex with the target membrane proteins syntaxin and synaptosome-associated protein (SNAP-25). The SCAMPs are markers for vesicles that recycle from the cell surface. The gastric H/KA TPase, Rabl 1, Rab25, V AMP-2, syntaxin 3 and SCAMPs were recovered in tubulovesicles immunoisolated with the anti-H/K-ATPase antibody. The association of Rabl 1, V AMP-2 and SCAMPs with H/K-ATPase-containing tubulovesicles was confirmed in immunoisolation experiments using monoclonal antibodies specific for V AMP-2 and SCAMPs. In contrast, immunoreactivity for syntaxin lA/B, syntaxin 4 and SNAP-25 was detected in gradient isolated vesicles but not in the immunoisolated tubulovesicles. 'The association of V AMP-2 and two Rab proteins with immunoisolated tubulovesicles sugges~s that tubulovesicles undergo regulated· ve_sicle fusion with the secretory canaliculus. · The observation that syntaxin 3, a putative target membrq11e docking protein, is a component of immunoisolated tubulovesicles suggests that tubulovesicles undergo vesicle-vesicle fusion during the delivery of the H/K.-ATPase to the secretory canaliculus. In addition~ the association of SCAMPs, along ~ith Rabll and Rab25, with immunoisolated tubulovesicles suggests that tubulovesicles likely comprise a compartment I •of apical recycling vesicles. These data are all consistent with the role of tubulovesicles as an expanded apical recycling system that delivers the H/K.-ATPase to the surface of the gastric parietal cell in a regulated vesicle fusion event.
    • Characterization of Neurotransmitter Transporter Gene Family in C. Elegans

      Jiang, Gouliang; Department of Biochemistry and Molecular Biology (2005-10)
      GABA functions as an inhibitory neurotransmitter in body muscles and as an excitatory neurotransmitter in enteric muscles in C. elegans. No transporter specific for this neurotransmitter has been identified to date in this organism. Here we report on the cloning and functional characterization of a GABA transporter from C. elegans (ceGAT- 1) and on the functional relevance of the transporter to the biology of body muscles and enteric muscles. ceGAT-1 is coded by snf-11 gene, a member of the sodium-dependent neurotransmitter symporter gene family in C. elegans. The cloned ceGAT-1 functions as a Na+/C f -coupled high-affinity transporter selective for GABA with aK t of~15 uM. The Na+:C1':GABA stoichiometry for ceGAT-1-mediated transport process is 2:1:1. The process is electrogenic as evidenced from GABA-induced inward currents in X laevis oocytes that express ceGAT-1 heterologously. The transporter is expressed exclusively in GABAergic neurons and in two other additional neurons. We also investigated the functional relevance of ceGAT-1 to the biology of body muscles and enteric muscles by ceGAT-1-specific RNAi in rrf-3 mutant, a strain of C. elegans in which neurons are not refractory to RNAi as in wild type strain. Downregulation of ceGAT-1 by RNAi leads to an interesting phenotype associated with altered function of body muscles and enteric muscles and also with altered sensitivity to aldicarb-induced paralysis. These findings provide unequivocal evidence for a modulatory role of GABA and ceGAT-1 in the biology of cholinergic neurons and in the function of body muscles and enteric muscles in this organism. We also cloned and functionally characterized for the first time a sodium-coupled transporter for amino acids in C. elegans. This transporter, designated ceNAT-1 (sodiumcoupled amino acid transporter-1), is identified in Worm database as snf-5, also a member of the sn f gene family (sodium/neurotransmitter symporter gene family). When expressed heterologously in mammalian cells, ceNAT-1 mediates the uptake of a broad spectrum of neutral amino acids in a Na+ dependent manner. The transport process exhibits a Na+: amino acid stoichiometry of 1:1. There is no involvement of C f in the transport process. When expressed heterologously in A! laevis oocytes, ceNAT-1 induces inward currents in response to neutral amino acids under voltage-clamp conditions, indicating that the transport process is electrogenic. Based on functional features, NAT-1 seems to be the C. elegans counterpart of the amino acid transporter B°AT in mammals. Mutations in the gene coding for B°AT cause Hartnup disease in humans. The clinical phenotype of Hartnup disease varies markedly depending on the environmental conditions. The present study shows that RNAi-mediated knockdown of NAT-1 or genetic deletion of NAT-1 in C. elegans is not associated with any detectable phenotype. This may be similar to the situation in humans where environmental conditions influence the clinical outcome of Hartnup disease. Further studies with altered experimental conditions are needed to determine if C. elegans with deletion of NAT-1 is a useful model system for investigations of Hartnup disease. Recently, a second isoform of B°AT has been identified in mammals. This transporter is expressed predominantly in the brain. Therefore, it is not clear at present whether the ceNAT-1 represents the worm counterpart of the Hartnup gene or the recently identified second isoform. We also report here on the cloning and functional characterization of a C. elegas betaine transporter which is encoded by snf-3, another member of the C. elegans sn f gene family. We named this transporter ceBGT-1. ceBGT-1 exhibits high specificity for betaine when expressed heterologouly in mammalian expression system and the uptake process mediated by ceBGT-1 is dependent on both sodium and chloride. The Na+: Cl': betaine stoichiometry for ceBGT-1-mediated transport process is 2:1:las confirmed y two-microelectrode voltage-clamp study. The Kt of ceBGT-1 for betaine is about 0.32 mM. Consistent with its role in osmoregulation, in vivo expression study using transgenic GFP fusion technique shows ceBGT-1 is expressed in the canal cells of C. elegans which represent the excretory represent the excretory organ in this organism. Investigation of the effects of hypertonicity on the expression of ceBGT-1 shows that hypertonicity increases its expression in C. elegans cultured with medium containing 350 mM NaCl compared to C. elegans cultured under normal conditions (50 mM NaCl).
    • Characterization of neurotransmitter transporter gene family in c. elegans

      Jiang, Guoliang; School of Graduate Studies (2005-10)