ABSTRACT
Studies have shown that mutant calreticulin (CALR) constitutively activates the thrombopoietin (TPO) receptor MPL and thus plays a causal role in the development of myeloproliferative neoplasms (MPNs). To further elucidate the molecular mechanism by which mutant CALR promotes MPN development, we studied the subcellular localization of mutant CALR and its importance for the oncogenic properties of mutant CALR. Here, mutant CALR accumulated in the Golgi apparatus, and its entrance into the secretion pathway and capacity to interact with N-glycan were required for its oncogenic capacity via the constitutive activation of MPL. Mutant CALR-dependent MPL activation was resistant to blockade of intracellular protein trafficking, suggesting that MPL is activated before reaching the cell surface. However, removal of MPL from the cell surface with trypsin shut down downstream activation, implying that the surface localization of MPL is required for mutant CALR-dependent activation. Furthermore, we found that mutant CALR and MPL interact on the cell surface. Based on these findings, we propose a model in which mutant CALR induces MPL activation on the cell surface to promote MPN development.
Subject(s)
Calreticulin/genetics , Mutation/genetics , Receptors, Thrombopoietin/genetics , Secretory Pathway/genetics , Cell Line , Cell Transformation, Neoplastic/genetics , Humans , Myeloproliferative Disorders/genetics , Signal Transduction/genetics , Trypsin/geneticsABSTRACT
Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes hold great potentials to predict pro-arrhythmic risks in preclinical cardiac safety screening, although the hiPSC cardiomyocytes exhibit rather immature functional and structural characteristics, including spontaneous activity. Our physiological characterization and mathematical simulation showed that low expression of the inward-rectifier potassium (IK1) channel is a determinant of spontaneous activity. To understand impact of the low IK1 expression on the pharmacological properties, we tested if transduction of hiPSC-derived cardiomyocytes with KCNJ2, which encodes the IK1 channel, alters pharmacological response to cardiac repolarization processes. The transduction of KCNJ2 resulted in quiescent hiPSC-derived cardiomyocytes, which need pacing to elicit action potentials. Significant prolongation of paced action potential duration in KCNJ2-transduced hiPSC-derived cardiomyocytes was stably measured at 0.1 µM E-4031, although the same concentration of E-4031 ablated firing of non-treated hiPSC-derived cardiomyocytes. These results in single cells were confirmed by mathematical simulations. Using the hiPSC-derived cardiac sheets with KCNJ2-transduction, we also investigated effects of a range of drugs on field potential duration recorded at 1 Hz. The KCNJ2 overexpression in hiPSC-derived cardiomyocytes may contribute to evaluate a part of QT-prolonging drugs at toxicological concentrations with high accuracy.
Subject(s)
Induced Pluripotent Stem Cells/metabolism , Myocytes, Cardiac/metabolism , Potassium Channel Blockers/adverse effects , Potassium Channels, Inwardly Rectifying/metabolism , Action Potentials/drug effects , Arrhythmias, Cardiac/chemically induced , Drug Evaluation, Preclinical/methods , HEK293 Cells , Humans , Induced Pluripotent Stem Cells/cytology , Models, Biological , Myocytes, Cardiac/cytology , Myocytes, Cardiac/drug effects , Patch-Clamp Techniques , Piperidines/adverse effects , Pyridines/adverse effectsABSTRACT
We report on the synthesis and the biological evaluation of a series of α-1-C-alkylated 1,4-dideoxy-1,4-imino-l-arabinitol (LAB) derivatives. The asymmetric synthesis of the derivatives was achieved by asymmetric allylic alkylation, ring-closing metathesis, and Negishi cross-coupling as key reactions. α-1-C-Butyl-LAB is a potent inhibitor of intestinal maltase, isomaltase, and sucrase, with IC50 values of 0.13, 4.7, and 0.032 µM, respectively. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis revealed that this compound differs from miglitol in that it does not influence oligosaccharide processing and the maturation of glycoproteins. A molecular docking study of maltase-glucoamylase suggested that the interaction modes and the orientations of α-1-C-butyl-LAB and miglitol are clearly different. Furthermore, α-1-C-butyl-LAB strongly suppressed postprandial hyperglycemia at an early phase, similar to miglitol in vivo. It is noteworthy that the effective dose was about 10-fold lower than that for miglitol. α-1-C-Butyl-LAB therefore represents a new class of promising compounds that can improve postprandial hyperglycemia.
Subject(s)
Enzyme Inhibitors/therapeutic use , Glycoside Hydrolase Inhibitors , Hyperglycemia/drug therapy , Hypoglycemic Agents/therapeutic use , Imino Sugars/therapeutic use , Administration, Oral , Animals , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/chemistry , Humans , Hypoglycemic Agents/administration & dosage , Hypoglycemic Agents/chemistry , Imino Sugars/administration & dosage , Imino Sugars/chemistry , Imino Sugars/pharmacology , Inhibitory Concentration 50 , Male , Mice , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
We examined 73 children with respiratory infections for Chlamydophila (Chlamydia) pneumoniae and Mycoplasma pneumoniae using real-time PCR assay and serological tests. C. pneumoniae and M. pneumoniae infections were found in 11 (15.1%) and 6 (8.2%) cases, respectively. The sensitivities and specificities of real-time PCR versus definite diagnosis of acute infection were 63.6% and 100% for C. pneumoniae, and 100% and 100% for M. pneumoniae, respectively. C. pneumoniae PCR-negative results appeared to be due to poor growth of the organism. The sensitivity and specificity of ImmunoCard tests were 33.3% and 82.1%, respectively, indicating that the efficacy of rapid diagnosis was disputable. The present results suggest that real-time PCR is suitable for rapid diagnosis as a first screening test to determine first-line antibacterial agents to be used against these infectious diseases.
