Proceedings of the National Academy of Sciences of the United States of America
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Proc. Natl. Acad. Sci. U.S.A. · Jun 2000
Chimeric green fluorescent protein-aequorin as bioluminescent Ca2+ reporters at the single-cell level.
Monitoring calcium fluxes in real time could help to understand the development, the plasticity, and the functioning of the central nervous system. In jellyfish, the chemiluminescent calcium binding aequorin protein is associated with the green fluorescent protein and a green bioluminescent signal is emitted upon Ca(2+) stimulation. We decided to use this chemiluminescence resonance energy transfer between the two molecules. ⋯ Chemiluminescent and fluorescent activities of these fusion proteins have been assessed in mammalian cells. Cytosolic Ca(2+) increases were imaged at the single-cell level with a cooled intensified charge-coupled device camera. This bifunctional reporter gene should allow the investigation of calcium activities in neuronal networks and in specific subcellular compartments in transgenic animals.
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Proc. Natl. Acad. Sci. U.S.A. · May 2000
Mutations in the tuberous sclerosis complex gene TSC2 are a cause of sporadic pulmonary lymphangioleiomyomatosis.
Lymphangioleiomyomatosis (LAM) is a progressive and often fatal interstitial lung disease characterized by a diffuse proliferation of abnormal smooth muscle cells in the lungs. LAM is of unusual interest biologically because it affects almost exclusively young women. LAM can occur as an isolated disorder (sporadic LAM) or in association with tuberous sclerosis complex. ⋯ In all four patients from whom lung tissue was available, the same mutation found in the angiomyolipoma was present in the abnormal pulmonary smooth muscle cells. In no case was the mutation present in normal kidney, morphologically normal lung, or lymphoblastoid cells. Our data demonstrate that somatic mutations in the TSC2 gene occur in the angiomyolipomas and pulmonary LAM cells of women with sporadic LAM, strongly supporting a direct role of TSC2 in the pathogenesis of this disease.
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Proc. Natl. Acad. Sci. U.S.A. · May 2000
Preservation of myocardial beta-adrenergic receptor signaling delays the development of heart failure after myocardial infarction.
When the heart fails, there is often a constellation of biochemical alterations of the beta-adrenergic receptor (betaAR) signaling system, leading to the loss of cardiac inotropic reserve. betaAR down-regulation and functional uncoupling are mediated through enhanced activity of the betaAR kinase (betaARK1), the expression of which is increased in ischemic and failing myocardium. These changes are widely viewed as representing an adaptive mechanism, which protects the heart against chronic activation. ⋯ Rather than leading to deleterious effects, cardiac function is improved, and the development of heart failure is delayed. These results appear to challenge the notion that dampening of betaAR signaling in the failing heart is protective, and they may lead to novel therapeutic strategies to treat heart disease via inhibition of betaARK1 and preservation of myocardial betaAR function.
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Proc. Natl. Acad. Sci. U.S.A. · May 2000
Tissue factor- and factor X-dependent activation of protease-activated receptor 2 by factor VIIa.
Protease-activated receptor 2 (PAR2) is expressed by vascular endothelial cells and other cells in which its function and physiological activator(s) are unknown. Unlike PAR1, PAR3, and PAR4, PAR2 is not activatable by thrombin. Coagulation factors VIIa (FVIIa) and Xa (FXa) are proteases that act upstream of thrombin in the coagulation cascade and require cofactors to interact with their substrates. ⋯ Responses in keratinocytes and cytokine-treated endothelial cells suggested that PAR2 may be activated directly by TF/FVIIa and indirectly by TF/FVIIa-generated FXa at naturally occurring expression levels of TF and PAR2. These results suggest that PAR2, although not activatable by thrombin, may nonetheless function as a sensor for coagulation proteases and contribute to endothelial activation in the setting of injury and inflammation. More generally, these findings highlight the potential importance of cofactors in regulating PAR function and specificity.
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Proc. Natl. Acad. Sci. U.S.A. · Apr 2000
A synthetic agonist at the orphanin FQ/nociceptin receptor ORL1: anxiolytic profile in the rat.
The biochemical and behavioral effects of a nonpeptidic, selective, and brain-penetrant agonist at the ORL1 receptor are reported herein. This low molecular weight compound [(1S,3aS)-8- (2,3,3a,4,5, 6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza- spiro[4. 5]decan-4-one] has high affinity for recombinant human ORL1 receptors and has 100-fold selectivity for ORL1 over other members of the opioid receptor family. It is a full agonist at these receptors and elicits dose-dependent anxiolytic-like effects in a set of validated models of distinct types of anxiety states in the rat (i.e., elevated plus-maze, fear-potentiated startle, and operant conflict). ⋯ No significant change in intracranial self-stimulation performance and pain reactivity was observed in this dose range. Higher doses of this compound (>/=10 mg/kg) induced disruption in rat behavior. These data confirm the notable anxiolytic-like effects observed at low doses with the orphanin FQ/nociceptin neuropeptide given locally into the brain and support a role for orphanin FQ/nociceptin in adaptive behavioral fear responses to stress.