• The CB1 cannabinoid receptor : receptor states, activity and G protein sequestration

      Nie, Jingjiang; School of Graduate Studies (2001-01)
      The human CB I cannabinoid receptor is a member of the G prot~in coupled ' . •,' . ' receptor family. Th~ CB I cannabinoid ~eceptor couples to pertussis toxin sensitive Gi/o proteins and ·inhibits ri~uronal voltage-gated Ca2+· channels. The. hCB I r~ceptor has two . . - - ,·' i unusual properties: I) it is constitutively active in the absence ofago~~st and 2) it can prevent other G protein coupled receptors from signaling by sequestering: a common pool of Gi/o proteins. The mechanism of consJitutive activity and G protein ~equestration by the hCB I receptor is unknown. In this ·.stud~, two Cijrboxyl terminal truncation mutants (hCBI-417 and ~CBI-400) were used to test the hypothesis that the pnj,ximal carboxyl. ' ' ' terminal couples to G proteins while the distal ·carboxyl terminal modulates G protein j sequestration and constitutive activity. Additionally, mutation of a sing~e amino acid in the second transmembrane domain (rCB 1-D 164N) was used to test the: hypothesis that this amino acid plays a critical role in tlie structural basis of G protein coupling and constitutive activity: Receptor cDNA constructs were injected_ into the nucleus of s~perior cervical ganglion neurons. After an overnight incubation to allow for receptor expression, neurons were voltage clamped and Ca2+ current were recorded. Inhibition of the; Ca2+ current by the cannabinoid agonist WIN 55,212-2 was used as an index of C~l cannabinoid receptor G. protein coupling and activation. Ca2+ channels are inhibited by Gfly subunits_ -released from· activated Gi/o proteins. In contrast to the wild type C.B 1 cannabinoid receptor, the mutant receptor in which the entire carboxyl terminal (amino acids 401-472) was deleted (hCB 1-400) failed to inhibit the Ca2+ current. Deletion of only the distal carboxyl terminal ( amino acids 418-4 72; hCB 1-417) restored Ca2+ current inhibition. These results demonstrate the critical role of the proximal domain (amino acids 401-417) ' I of the carboxyl terminal of the hCB 1 receptor in coupling to G proteins. ; Truncation of the distal carboxyl terminal domain,· however, Ghanged the magnitude of Ca2+ current inhibition. The hCB 1-417 receptor produced significantly less inhibition of the Ca2+ current in the presence of WIN 55,212-2 c9~pared to the wild typ~- •. receptor (22.6±3.0% vs 43.7±6.5%, respectively). Thus, the distal c~rboxyl terminal domain is important in modulating the magnitude of Ca2+ current inhib~tion. In addition to the change in the magnitude of Ca2+ current inhibition, deletion of th¢ distal carboxyl terminal significantly slowed the kinetics of Ca2+ current inhibition byi WIN 55,212-2 (time to peak of effect: 146.0±8.7 second). The distal carboxyl terminal tail of the CBI canriabinoid receptor also played a· role in constitutive activity ~nd G protein : sequestration. The hCBI-417 receptor displayed. enhanced constitut~ve activity. In neurons injected with 50 ng/μl hCBI-417 cDNA the inverse agonist SR141716A increased the Ca2+ .current 101.1±21.3%. The inverse agonist acts ~to reverse the·. constitutive activity of the receptor. The effect of SR141716A on the hCBl-417 receptor was significantly_ greater than the 42.9±7.6% Ca2+ current increase in neu;rons expressing the wild type hCBI receptor .. · G protein sequestration was also enhanced in neurons expressing the truncated hCBI-417 receptor. Wild type h~Bl cannabinoid receptors when expres:sed by injecting · 100 ng/μl cDNA completely abolish signa~ing by other G protein coupled receptors including a2-adrenergic receptors. Normally activation of a 2-adrenergic r~ceptors inhibits the Ca2 + current 44.5±5.7%. In the presence of hCBI receptors activ~tion of the a 2.: adrenergic receptors by UK.14304 inhibited the Ca2+ current only 1.5±4.2%. Signaling by a2-adrenergic receptors can be partially restored by injecting a lower concentration of hCBI cDNA. In neurons injected with 50 ng/μl hCBl cDNA the a2-adrenergic agonist UK.14304 inhibited the Ca2+ current 20.0±3.7%. In neurons injected with: 50 ng/μl hCBI- 417 cDNA UK.14304 inhibited the Ca2+ current 7.0±1.2%. Thus, sign~lilJ,g by the a 2- _adrenergic receptor was abolished by the catboxyl terminal truncated hC~l-417 receptor. These results indica,te that deletion ~f the distal carboxyl terminal enhances the ability of the receptor to sequester· G proteins. ! The aspartic acid residue in the s~cond transmembr~ne_ dom~in of G protein ,.· ' . ; coupled receptors is highly conserved. -Mutation of this aspartic acid (r~Bl-D164N) had profound effects on the constitutive activity of the CBI receptor as well :as.on the abilityof the receptor to sequester G proteins. Both the constitutive activity mid the ability to sequester G proteins were abolished by the rCBI-D164N· ieceptor. The inverse agonist increased the Ca2 + current only 11.6±6.9% in neurons expressing the mutant rCBl- : D164N receptors. The mutant rCB1-D164N receptors failed to block signaling by the a 2- adrengic receptor. UK.14304 inhibited the Ca2+ current 35.8±6.8% in neurons expressing the rCB l-Dl64N receptors and was not different from control neurons. Additionally, the D 164N mutation in the second transmembrane domain decreased the tim.e to peak of the WIN 55,212-2 inhibition of the Ca2+ current to 24±4 seconds. These results demonstrate that 1) the proximal carboxyl terminal domain of the hCB 1 receptor is critical for G protein coupling, 2) the distal carboxyl tbrminal domain regulates constitutive activity, G protein coupling kinetics and G protein sequestration and _3) the aspartic acid in the second tra~smembrane domain plays a critical role in G . ' 1 . protein sequestration, G protein coupling_ kinetics and constitutive activ~ty of the hCB 1 receptor. Taken together, the mutant receptors shift the CB 1 recept~r into different receptor states. The D164N-CB1 receptor exists primarily in an inactive'.state uncoupled from G proteins, the wild type CB 1 receptor e~sts in both active and inactive G protein coupled states and the carboxyl terminal truncated CB 1-417 receptor exists primarily in an active G protein coupled state.
    • The CB1 Cannabinoid Receptor: Receptor States, Activity and G Protein Sequestration

      Nie, Jingjiang; Department of Pharmacology and Toxicology (2001)
      The human CB1 cannabinoid receptor is a member o f the G protein coupled receptor family. The CB1 cannabinoid receptor couples to pertussis toxin sensitive Gi/o proteins and inhibits neuronal voltage-gated Ca2+ channels. The hCBl receptor has two unusual properties: 1) it is constitutively active in the absence o f agonist and 2) it can prevent other G protein coupled receptors from signaling by sequestering a common pool of Gi/o proteins. The mechanism o f constitutive activity and G protein sequestration by the hCB 1 receptor is unknown. In this study, two carboxyl terminal truncation mutants (hCB 1-417 and hCBl-400) were used to test the hypothesis that the proximal carboxyl terminal couples to G proteins while the distal carboxyl terminal modulates G protein sequestration and constitutive activity. Additionally, mutation o f a single amino acid in the second transmembrane domain (rCBl-D164N) was used to test the hypothesis that this amino acid plays a critical role in the structural basis o f G protein coupling and constitutive activity. Receptor cDNA constructs were injected into the nucleus of superior cervical ganglion neurons. After an overnight incubation to allow for receptor expression, neurons were voltage clamped and Ca2+ current were recorded. Inhibition of the Ca2r current by the cannabinoid agonist WIN 55,212-2 was used as an index o f CB1 cannabinoid receptor G protein coupling and activation. Ca2' channels are inhibited by G{3y subunits released from activated Gi/o proteins. In contrast to the wild type CB1 cannabinoid receptor, the mutant receptor in which the entire carboxyl terminal (amino acids 401-472) was deleted (hCB 1-400) failed to inhibit the Ca2+ current. Deletion o f only the distal carboxyl terminal (amino acids 418-472; hCB 1-417) restored Ca2+ current inhibition. These results demonstrate the critical role o f the proximal domain (amino acids 401-417) o f the carboxyl terminal of the hCB 1 receptor in coupling to G proteins. Truncation o f the distal carboxyl terminal domain, however, changed the magnitude o f Ca2+ current inhibition. The hCBl-417 receptor produced significantly less inhibition of the Ca2+ current in the presence o f WIN 55,212-2 compared to the wild type receptor (22.6±3.0% vs 43.7±6.5%, respectively). Thus, the distal carboxyl terminal domain is important in modulating the magnitude of Ca2* current inhibition. In addition to the change in the magnitude o f Ca2+ current inhibition, deletion o f the distal carboxyl terminal significantly slowed the kinetics o f Ca2l~ current inhibition by WIN 55,212-2 (time to peak o f effect: 146.0+8.7 second). The distal carboxyl terminal tail of the CB1 cannabinoid receptor also played a role in constitutive activity and G protein sequestration. The hCB 1-417 receptor displayed enhanced constitutive activity. In neurons injected with 50 ng/pl hCBl-417 cDNA the inverse agonist SR141716A increased the Ca2+ current 101.1±21.3%. The inverse agonist acts to reverse the constitutive activity o f the receptor. The effect o f SRI 41716 A on the hCBl-417 receptor was significantly greater than the 42.9±7.6% Ca2+ current increase in neurons expressing the wild type hCB 1 receptor. G protein sequestration was also enhanced in neurons expressing the truncated hCBl-417 receptor. Wild type hCBl cannabinoid receptors when expressed by injecting 100 ng/p.1 cDNA completely abolish signaling by other G protein coupled receptors including a 2 -adrenergic receptors. Normally activation o f a 2 -adrenergic receptors inhibits the Ca2+ current 44.5±5.7%. In the presence o f hCBl receptors activation of the a 2- adrenergic receptors by UK14304 inhibited the Ca2+ current only 1.5±4.2%. Signaling by a 2-adrenergic receptors can be partially restored by injecting a lower concentration o f hCBl cDNA. In neurons injected with 50 ng/pl hCBl cD N A the ct2 -adrenergic agonist UK14304 inhibited the Ca2+ current 20.0±3.7%. In neurons injected with 50 ng/pl hCBl- 417 cDNA UK14304 inhibited the Ca2+ current 7.0±1.2%. Thus, signaling by the a 2- adrenergic receptor was abolished by the carboxyl terminal truncated hCBl-417 receptor. These results indicate that deletion of the distal carboxyl terminal enhances the ability o f the receptor to sequester G proteins. The aspartic acid residue in the second transmembrane domain of G protein coupled receptors is highly conserved. Mutation o f this aspartic acid (rCBl-D164N) had profound effects on the constitutive activity o f the C B 1 receptor as well as on the ability of the receptor to sequester G proteins. Both the constitutive activity and the ability to sequester G proteins were abolished by the rCBl-D164N receptor. The inverse agonist increased the Ca2+ current only 11.6+6.9% in neurons expressing the mutant rC Bl- D164N receptors. The mutant rCBl-D164N receptors failed to block signaling by the a 2- adrengic receptor. UK14304 inhibited the Ca2+ current 35.8+6.8% in neurons expressing the rCBl-D164N receptors and was not different from control neurons. Additionally, the D164N mutation in the second transmembrane domain decreased the time to peak o f the WIN 55,212-2 inhibition o f the Ca2+ current to 24+4 seconds. These results demonstrate that 1) the proximal carboxyl terminal domain o f the hCB 1 receptor is critical for G protein coupling, 2) the distal carboxyl terminal domain regulates constitutive activity, G protein coupling kinetics and G protein sequestration and 3) the aspartic acid in the second transmembrane domain plays a critical role in G protein sequestration, G protein coupling kinetics and constitutive activity o f the hCBl receptor. Taken together, the mutant receptors shift the CB1 receptor into different receptor states. The D164N-CB1 receptor exists primarily in an inactive state uncoupled from G proteins, the wild type CB1 receptor exists in both active and inactive G protein coupled states and the carboxyl terminal truncated CB 1-417 receptor exists primarily in an active G protein coupled state.
    • Cell drinking: a closer look at how macropinocytosis drives cholesterol uptake in atherosclerotic vessels

      Lin, Huiping; Vascular Biology Center (Augusta University, 2020-05)
      Atherosclerotic vascular disease is the underlying cause of myocardial infarction, stable and unstable angina, stroke, peripheral artery disease and sudden cardiac death. Collectively, these cardiovascular diseases are responsible for the majority of deaths worldwide. Internalization of modified apolipoprotein B–containing lipoproteins by macrophages through scavenger receptor (SR)-mediated pathways is generally viewed as an essential step for the initiation and progression of atherosclerosis. Our studies were designed to investigate the contribution of receptor-independent LDL macropinocytosis to arterial lipid accumulation and atherosclerosis. We developed novel genetic and pharmacological approaches, utilized high resolution imaging techniques and employed unique in vivo lipid quantification assays to investigate the role of macrophage macropinocytosis in the pathogenesis of atherosclerosis. My results demonstrate that the macropinocytosis inhibitor EIPA and selective deletion of a key pathway regulating macropinocytosis in myeloid cells substantially decreased lesion size in both hypercholesterolemic wild type (WT) and SR knockout (CD36-/-/SR-A-/-) mice. Stimulation of macropinocytosis using genetic and physiologically relevant approaches promotes lipoprotein internalization by WT and CD36-/-/SR-A-/- macrophages, leading to foam cell formation. Serial section high-resolution imaging of murine and human atherosclerotic arteries identified for the first time subendothelial macrophages for the first time that demonstrate plasma membrane ruffling, cupping and macropinosome internalization. Immunoelectron microscopy, 3D reconstruction of macrophage foam cells and in vivo LDL tracking demonstrate macrophage internalization of LDL in human and murine atherosclerotic arteries via macropinocytosis. We next performed a large, unbiased-screen of an FDA-approved drug library to identify clinically relevant therapeutic agents that can be repurposed as pharmacological inhibitors of macropinocytosis. Our studies identified a low MW compound (imipramine) that inhibits macrophage macropinocytosis in vitro and in vivo. Imaging, toxicity and selectivity studies demonstrated that imipramine is a potent (IC50 = 130.9 nM), non-toxic (selectivity index CC50/IC50 > 300) and selective inhibitor of macropinocytosis. Repurposing of imipramine to inhibit macropinocytosis in hypercholesterolemic mice substantially decreased plaque development compared with control treatment. Taken together, our findings challenge the SR paradigm of atherosclerosis and identify inhibition of receptor-independent macrophage macropinocytosis as a new therapeutic strategy that may be beneficial in the treatment of atherosclerosis and its cardiovascular consequences.
    • Cellular and Immunocytochemical Response to Mandibular Distraction Using an Implanted Lengthening Device

      Elbokle, Nadar N; Department of Oral Biology (2004)
      Distraction osteogenesis (DO) is a biologic process that generates new bone between surfaces of bone segments, which are gradually separated by traction forces. It is a uniquely effective method with multiple applications in the craniofacial region. This concept has been the basis of all bone-lengthening operations; it involved an osteotomy of the shortened bone and an external/internal fixator device, which slowly elongates the bone to its new dimension while a bony callus is being formed at the side to distraction. The biology of DO is similar to callus fracture healing. The bony regenerate passes through the same phases: formation of a collagen fibril template, mineralization, bony union and finally remodeling. The mechanisms by which the mechanical stresses applied to the bone tissue cause the cells to proliferate and form new bone are not well understood. More studies are needed to understand the cellular events underlying DO and the effects of the strains applied during DO on cellular proliferation and mineral apposition.
    • 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; 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.
    • 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.
    • 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).