Biomedical subjects
Publications and source records attributed to J Kirsch.
We report a case of myocardial infarction due to occlusion of the left anterior descending artery seen on a routine chest CT performed in order to exclude aortic dissection. This underscores the need for the radiologist to look at the heart and coronary arteries on each thoracic CT-scan, even if acquired without EKG-gating.
The enteric nervous system is composed of neurones and glial cells. These enteric glia cells (EGC) appear to be essential for the maintenance of gut homeostasis and mucosal integrity. Neurotrophin nerve growth factor (NGF) also plays an important role for the gut integrity by regulating sensory and inflammatory processes in the intestines. Here, we demonstrate EGCs as one source of NGF and show increased levels of NGF mRNA/protein and tropomyosin receptor kinase A (TrkA) mRNA in cultured EGCs upon stimulation with proinflammatory cytokines and lipopolysaccharides. NGF is continuously secreted from cultured EGCs and proinflammatory cytokines and lipopolysaccharides stimulate the secretion of this neurotrophin in a time- and dose- dependent manner, whereas interleukin-4 had no effect on NGF expression. Furthermore, NGF secretion was sustained for more than 12 h after withdrawal of the proinflammatory cytokines, suggesting the involvement of transcriptional and/or translational processes. Thus, the release of proinflammatory cytokines can increase NGF secretion by EGCs and leads to a higher expression of TrkA in EGCs. NGF, in turn, can increase visceral sensitivity and, on the other hand, appears to improve gut inflammation. Therefore, NGF secreting EGCs may play a key role in modulating visceral sensitivity and might be involved in inflammatory processes of the gut.
BACKGROUND: Enteric glia protect the integrity of the gut, as loss of enteric glial fibrillary acidic protein (GFAP) positive (+) glia leads to a haemorrhagic jejunoileitis. Crohn's disease (CD) and necrotising enterocolitis (NEC) show pathological changes in enteric glia. Therefore, factors controlling GFAP+ enteric glia are of great interest. The aim of the present study was to characterise enteric glia and determine the effect of interleukin 1beta (IL-1beta), interleukin 4 (IL-4), tumour necrosis factor alpha (TNF-alpha), and lipopolysaccharides (LPS) on cultured enteric glia. METHODS: Dissected rat colon and cultured enteric glia cells were double labelled with anti-GFAP and anti-S-100 antibodies. For regulatory studies, enteric glia cells were treated with cytokines and LPS. Proliferation was assayed using bromodeoxyuridine (BrdU) and mitosis of enteric glia was blocked by demecolcine. RESULTS: We were able to distinguish GFAP negative (-) from GFAP+ glia subtypes in situ and in primary cultures. Incubation of cells with IL-1beta, TNF-alpha, and LPS led to a significant increase in GFAP+ enteric glia while IL-4 had no effect on GFAP expression. After incubation with IL-1beta, total intracellular GFAP of enteric glia cells was increased. Upregulation of GFAP+ enteric glia could also be observed after stimulation with IL-1beta on blocking mitosis. BrdU uptake in stimulated enteric glia showed no increased proliferation rate. CONCLUSIONS: Two different types of enteric glia based on GFAP expression exist in the gut. Proinflammatory cytokines and LPS cause a dramatic increase in GFAP+ enteric glia. This suggests that cytokines play an important role in controlling GFAP+ enteric glia which might in turn be involved in modulating the integrity of the bowel during inflammation.
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Gephyrin is essential for both the postsynaptic localization of inhibitory neurotransmitter receptors in the central nervous system and the biosynthesis of the molybdenum cofactor (Moco) in different peripheral organs. Several alternatively spliced gephyrin transcripts have been identified in rat brain that differ in their 5' coding regions. Here, we describe gephyrin splice variants that are differentially expressed in non-neuronal tissues and different regions of the adult mouse brain. Analysis of the murine gephyrin gene indicates a highly mosaic organization, with eight of its 29 exons corresponding to the alternatively spliced regions identified by cDNA sequencing. The N- and C-terminal domains of gephyrin encoded by exons 3-7 and 16-29, respectively, display sequence similarities to bacterial, invertebrate, and plant proteins involved in Moco biosynthesis, whereas the central exons 8, 13, and 14 encode motifs that may mediate oligomerization and tubulin binding. Our data are consistent with gephyrin having evolved from a Moco biosynthetic protein by insertion of protein interaction sequences.
This study considers the feasibility of defining a QALY from disease-specific data using the New York Heart Association (NYHA) classification of heart failure. The study derives health state values for the four different NYHA classifications of disease progression using the time trade-off (TTO) instrument associated with the five dimensional (EQ-5D) health state valuation method. Consistent mappings between the disease classification and the chosen QALY instrument are found. With this being the case, the assumption of constant proportionality, which is necessary to define the QALY as an acceptable measure of health related preferences, is considered. It is found that constant proportionality does not hold across the more severe health states, thus questioning the use of QALYs as representing cardinal preference structures.
The formation of postsynaptic GABAA and glycine receptor clusters requires the receptor-associated peripheral membrane protein gephyrin. Here we describe two splice variants of a novel gephyrin-binding protein, termed collybistin I and II, which belong to the family of dbl-like GDP/GTP exchange factors (GEFs). Co-expression of collybistin II with gephyrin induced the formation of submembrane gephyrin aggregates that accumulate hetero-oligomeric glycine receptors. Our data suggest that collybistin II regulates the membrane deposition of gephyrin by activating a GTPase of the Rho/Rac family. Therefore, this protein may be an important determinant of inhibitory postsynaptic membrane formation and plasticity.
We analyzed the distribution of the receptor-anchoring protein gephyrin in the normal and deafferented rat dentate gyrus to investigate whether the expression of this postsynaptic protein is altered in response to the formation of new synaptic contacts. Confocal microscopy and digital image analysis revealed that in normal dentate gyrus immunolabeling was most prominent in the outer molecular layer and decreased successively in the direction of the granule cell layer. Simultaneous immunolabeling for gephyrin and cell-specific markers showed that granule cells and parvalbumin-positive interneurons express gephyrin. Large, intensely stained, gephyrin-positive clusters were distributed along distinct dendrites, and most of them were positive for parvalbumin. Calbindin-immunostained dendrites were associated with smaller, gephyrin-positive clusters. Lesion of the medial entorhinal cortex leads to deafferentiation of the middle molecular layer which resulted in an increased gephyrin immunoreactivity. These changes were due to a significantly increased concentration of the very small gephyrin-positive clusters. Parvalbumin-positive dendrites did not display any increase in co-localizing gephyrin-positive structures. The altered immunolabeling pattern persisted until 12 weeks after lesion, a time when the process of synaptic reorganization is complete. Our findings suggest that synaptogenesis following deafferentiation results in a cell-specific redistribution of gephyrin immunoreactivity at specific inhibitory synapses.
RAFT1 (rapamycin and FKBP12 target 1; also called FRAP or mTOR) is a member of the ATM (ataxia telangiectasia mutated)-related family of proteins and functions as the in vivo mediator of the effects of the immunosuppressant rapamycin and as an important regulator of messenger RNA translation. In mammalian cells RAFT1 interacted with gephyrin, a widely expressed protein necessary for the clustering of glycine receptors at the cell membrane of neurons. RAFT1 mutants that could not associate with gephyrin failed to signal to downstream molecules, including the p70 ribosomal S6 kinase and the eIF-4E binding protein, 4E-BP1. The interaction with gephyrin ascribes a function to the large amino-terminal region of an ATM-related protein and reveals a role in signal transduction for the clustering protein gephyrin.
The strychnine-sensitive glycine receptor (GlyR) is a pentameric chloride channel protein that exists in several developmentally and regionally regulated isoforms in the CNS. These result from the differential expression of four genes encoding different variants (alpha 1-alpha 4) of the ligand-binding subunit of the GlyR. Their assembly with the structural beta subunit is governed by "assembly cassettes" within the extracellular domains of these proteins and creates chloride channels of distinct conductance properties. GlyR gating is potentiated by Zn2+, a metal ion co-released with different neurotransmitters. Site-directed mutagenesis has unraveled major determinants of agonist binding and Zn2+ potentiation. During development, glycine receptors mediate excitation that results in Ca2+ influx and neurotransmitter release. Ca2+ influx triggered by the activation of embryonic GlyRs is required for the synaptic localization of the GlyR and its anchoring protein gepyhrin. In the adult, mutations in GlyR-subunit genes result in motor disorders. The spastic and spasmodic phenotypes in mouse as well as human hereditary startle disease will be discussed.
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The molybdenum cofactor (Moco), a highly conserved pterin compound complexing molybdenum, is required for the enzymatic activities of all molybdenum enzymes except nitrogenase. Moco is synthesized by a unique and evolutionarily old pathway that requires the activities of at least six gene products. Some of the proteins involved in bacterial, plant, and invertebrate Moco biosynthesis show striking homologies to the primary structure of gephyrin, a polypeptide required for the clustering of inhibitory glycine receptors in postsynaptic membranes in the rat central nervous system. Here, we show that gephyrin binds with high affinity to molybdopterin, the metabolic precursor of Moco. Furthermore, gephyrin expression can reconstitute Moco biosynthesis in Moco-deficient bacteria, a molybdenum-dependent mouse cell line, and a Moco-deficient plant mutant. Conversely, inhibition of gephyrin expression by antisense RNA expression in cultured murine cells reduces their Moco content significantly. These data indicate that in addition to clustering glycine receptors, gephyrin also is involved in Moco biosynthesis and illustrate the remarkable conservation of its function in Moco biosynthesis throughout phylogeny.
The postsynaptic membrane and the subsynaptic cell compartment are specialized for inter- and intracellular signaling. Recent work has focused on the role of synaptic activity in regulating the surface distribution of neurotransmitter receptors. In addition, several components of secondary signaling pathways involved in the long-term regulation of synaptic efficacy have been identified.
The peripheral membrane protein gephyrin is essential for the postsynaptic localization of inhibitory glycine receptors (GlyRs). Binding of gephyrin to the GlyR beta subunit is mediated by a sequence motif located in the intracellular loop region connecting transmembrane segments 3 and 4. Here, insertion of this binding motif is shown to alter the subcellular distribution of an excitatory neurotransmitter receptor in transfected mammalian cells. Upon coexpression with gephyrin, a mutant N-methyl-D-aspartate (NMDA) receptor containing NMDA receptor 1 (NR1) subunits which harboured a gephyrin-binding motif within its cytoplasmic tail region, was targeted to intracellular gephyrin-rich domains, as previously observed for the GlyR beta subunit. Our data indicate that a gephyrin-binding motif located in a cytoplasmic domain of an integral membrane protein suffices for routing to gephyrin-rich domains.
Glycine receptors (GlyRs) are ligand-gated chloride channel proteins composed of alpha- and beta-subunits. GlyRs are located to and anchored at postsynaptic sites by the receptor-associated protein gephyrin. Previous work from our laboratory has identified a core motif for gephyrin binding in the cytoplasmic loop of the GlyR beta-subunit. Here, we localized amino acid residues implicated in gephyrin binding by site-directed mutagenesis. In a novel transfection assay, a green fluorescent protein-gephyrin binding motif fusion protein was used to monitor the consequences of amino acid substitutions for beta-subunit interaction with gephyrin. Only multiple, but not single, replacements of hydrophobic side chains abolished the interaction between the two proteins. Our data are consistent with gephyrin binding being mediated by the hydrophobic side of an imperfect amphipathic helix.
M-cadherin, a calcium-dependent intercellular adhesion molecule, is expressed in skeletal muscle cells. Its pattern of expression, both in vivo and in cell culture as well as functional studies, have implied that M-cadherin is important for skeletal muscle development, in particular the fusion of myoblasts into myotubes. M-cadherin formed complexes with the catenins in skeletal muscle cells similar to E-cadherin in epithelial cells. This suggested that the muscle-specific function of the M-cadherin catenin complex might be mediated by additional interactions with yet unidentified cellular components, especially cytoskeletal elements. These include the microtubules which also have been implicated in the fusion process of myoblasts. Here we present evidence that the M-cadherin catenin complex interacts with microtubules in myogenic cells by using three independent experimental approaches. (1) Analysis by laser scan microscopy revealed that the destruction of microtubules by nocodazole leads to an altered cell surface distribution of M-cadherin in differentiating myogenic cells. In contrast, disruption of actin filaments had little effect on the surface distribution of M-cadherin. (2) M-cadherin antibodies coimmunoprecipitated tubulin from extracts of nocodazole-treated myogenic cells but not of nocodazole-treated epithelial cells ectopically expressing M-cadherin. Vice versa, tubulin antibodies coimmunoprecipitated M-cadherin from extracts of nocodazole-treated myogenic cells but not of nocodazole-treated M-cadherin-expressing epithelial cells. (3) M-cadherin and the catenins, but not a panel of control proteins, were copolymerized with tubulin from myogenic cell extracts even after repeated cycles of assembly and disassemly of tubulin. Moreover, neither M-cadherin nor E-cadherin could be found in a complex with microtubules in epithelial cells ectopically expressing M-cadherin. Our data are consistent with the idea that the interaction of M-cadherin with microtubules might be essential to keep the myoblasts aligned during fusion, a process in which both M-cadherin and microtubules have been implicated.
Glycine receptors are anchored at inhibitory chemical synapses by a cytoplasmic protein, gephyrin. Molecular cloning revealed the similarity of gephyrin to prokaryotic and invertebrate proteins essential for synthesizing a cofactor required for activity of molybdoenzymes. Gene targeting in mice showed that gephyrin is required both for synaptic clustering of glycine receptors in spinal cord and for molybdoenzyme activity in nonneural tissues. The mutant phenotype resembled that of humans with hereditary molybdenum cofactor deficiency and hyperekplexia (a failure of inhibitory neurotransmission), suggesting that gephyrin function may be impaired in both diseases.
In this report we demonstrate that in HEK293 cells stably expressing the human V2 vasopressin receptor, ligand-induced internalization of the hormone receptor occurs via the clathrin-dependent pathway. Studies of receptor trafficking either by direct visualization of the V2 receptor by confocal microscopy or binding experiments show a rapid internalization (half-time 6-7 min). Blocking of the clathrin-dependent pathway by hypertonic sucrose increased vasopressin-induced cellular cAMP production and decreased the desensitization of the V2 receptor-adenylyl cyclase system. Thus, internalization appears to be a major regulatory mechanism terminating vasopressin action in HEK293 cells. Two antagonists of the vasopressin V2 receptor exerted different effects on receptor internalization, as determined by confocal fluorescence microscopy. The nonpeptidic antagonist OPC31260 did not induce any visible receptor internalization, whereas the peptidic antagonist d(CH2)5[D-Tyr(Et)2,Val4,Lys8,Tyr-NH29]VP induced a slow but substantial receptor internalization. These results suggest that long-term treatment with peptidic V2 receptor antagonists might lead to desensitization.