RESUMEN
BACKGROUND: The understanding of genetic risk factors for chronic pancreatitis increased in the last decade with the discovery of mutations in the cationic trypsinogen gene (PRSS1). The first mutation was detected at the R122 autocleavage site of the protein (R122H) and subsequently two other mutations in this region, R122C and V123M, were described that resulted in a similar phenotype of hereditary pancreatitis. This study reports a novel A121T mutation within this region and characterises the resulting molecular properties at the autocleavage site. METHODS: Blood samples of a PRSS1 A121T carrier family were analysed for PRSS1 mutations using melting point curve analysis, restriction endonucleases and DNA sequencing. Conformation dependent properties of the mutated sequence were analysed by molecular modelling. The autodegradation kinetic of the mutated trypsin sequence was measured by a novel fluorescence resonance energy transfer (FRET) assay using designed 11 amino acid peptides from PRSS1 aa 118-aa 127 containing the trypsin cleavage site at aa 122 coupled to a Dabcyl/EDANS FRET system. The kinetic of tryptic peptide cleavage was measured in a fluorescence enzyme linked immunosorbent assay (ELISA) reader. RESULTS: DNA sequencing revealed a novel G to A transition at position 133279 of the published genomic sequence (#U66061 GenBank). The mutation results in an amino acid substitution of alanine by threonine at position 121 (A121T) of the cationic trypsinogen. Four additional mutation carriers could be identified among the relatives while only the first patient developed chronic pancreatitis. Molecular modelling of PRSS1 A121T revealed a change in the bond pattern between the R122 region and the calcium binding loop, whereas FRET assays showed an increased trypsin cleavage rate with a reaction kinetic elevated by more than 80%. CONCLUSION: The novel PRSS1 A121T mutation highlights the surface exposed region PRSS1 A121-R122-V123 as a hotspot for hereditary pancreatitis associated trypsinogen mutations. Molecular modelling and FRET assays provide evidence for an A121T mutation dependent increase in susceptibility to trypsin digestion at the R122 cleavage site suggesting an enhanced autodegradation and a loss-of-function at the autocleavage site.
Asunto(s)
Predisposición Genética a la Enfermedad , Pancreatitis Crónica/genética , Tripsinógeno/genética , Sustitución de Aminoácidos , Femenino , Transferencia Resonante de Energía de Fluorescencia , Humanos , Masculino , Persona de Mediana Edad , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Linaje , Penetrancia , Tripsinógeno/química , Tripsinógeno/metabolismoRESUMEN
Insulin-like growth factors (IGFs) stimulate both proliferation and differentiation of myogenic cell lines, and these actions are mostly mediated through the type I IGF receptor (type I IGF-R). To further investigate the role of this receptor in phenotypic characteristics of C2 murine myoblasts, we overexpressed the human type I IGF-R in the inducible clone of C2 cells, which requires IGFs in the differentiation medium to undergo terminal differentiation. Inducible myoblasts were transfected with either the eukaryotic expression vector pNTK or pNTK containing the human type I IGF-R complementary DNA, and we isolated two clones named Ind-Neo and Ind-R, respectively. Binding and autophosphorylation experiments indicate that Ind-R cells express about 10 times as much type I IGF-R compared with Ind-Neo control cells and that the transfected type I IGF-R is functional in Ind-R cells. We show that overexpression of the human type I IGF-R makes inducible myoblasts able to differentiate spontaneously, as assessed by expression of the myogenic transcription factors MyoD and myogenin, detection of the muscle-specific protein troponin T, and myotube formation. Moreover, when exposed to IGF-I, Ind-R cells lose contact inhibition, grow in the presence of a low level of growth factors and form colonies in soft agar, which is characteristic of a ligand-dependent transformed phenotype. It emerges from this study that 1) the type I IGF-R is strongly involved in the phenotypic differences between inducible and permissive cells with respect to the differentiation program; and 2) overexpression causes this receptor to act as a ligand-dependent transforming protein in muscle cells. We suggest that type I IGF-R abundance and level of activation may determine the efficiency of the autocrine mode of action of IGFs and discriminate their biological functions.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/metabolismo , Músculos/citología , Músculos/fisiología , Receptores de Somatomedina/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Ligandos , Ratones , Músculos/efectos de los fármacos , Fenotipo , Receptores de Somatomedina/inmunologíaRESUMEN
Cell-cell contacts are important regulatory elements in tissue development, organ morphogenesis and malignant tumor invasion. In recent in vivo studies we have identified the members of the cadherin/catenin family of cell adhesion proteins that are differentially expressed in the pancreas and have determined their cell biological dynamics during dissociation and repair of adherens junctions. To further characterize these events, epithelial cell culture systems were used and a number of type II protein tyrosine phosphatases (PTPs) were found to colocalize and interact with the cadherin/catenin complex. These observations suggest that tyrosine dephosphorylation in general and PTPs in particular are involved in cell contact formation. Our most recent experiments indicate 1) that inhibition of PTPs alone dissociates pancreatic adherens junctions, 2) that cytosolic and transmembrane PTPs are differentially expressed in acinar cells, and 3) that a subset of them can associate with proteins of the cadherin/catenin complex at pancreatic cell-cell adhesions.
Asunto(s)
Adhesión Celular , Páncreas/citología , Proteínas Tirosina Fosfatasas/metabolismo , Tirosina/metabolismo , Células Epiteliales , Páncreas/enzimología , FosforilaciónRESUMEN
Two simple and sensitive calorimetric procedures for the determination of p-aminophenol with 3-cyano-N-methoxypyridinium perchlorate are presented. One is based on reaction in methoxyethanol in presence of sodium acetate, with direct measurement at 410 nm. The reaction product obtained by this procedure has been separated and identified. The other is based on reaction in methoxyethanol in presence of chloramine-T and direct measurement at 448 nm. The method has been applied to the determination of p-aminophenol in pure form and as an impurity in paracetamol and paracetamol- containing tablets, with a coefficient of variation less than 2%.
Asunto(s)
Pancreatitis/genética , Inhibidor de Tripsina Pancreática de Kazal/genética , Tripsinógeno/metabolismo , ADN/química , ADN/genética , Análisis Mutacional de ADN , Salud de la Familia , Femenino , Humanos , Masculino , Mutación , Mutación Missense , Pancreatitis/metabolismo , Pancreatitis/patología , Linaje , Fenotipo , Polimorfismo Genético , Índice de Severidad de la Enfermedad , Inhibidor de Tripsina Pancreática de Kazal/metabolismoRESUMEN
The lysosomal protease cathepsin B is thought to play a crucial role in the intracellular activation cascade of digestive proteases and in the initiation of acute pancreatitis. Although cathepsin B has been shown to be physiologically present in the secretory pathway of pancreatic acinar cells it has been suggested that premature activation of zymogens requires an additional redistribution of cathepsin B into the secretory compartment. Here, we studied the role of cathepsin B targeting during caerulein-induced pancreatitis in mouse mutants lacking the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (CI-MPR) which normally mediates the trafficking of cathepsin B to lysosomes. Absence of the CI-MPR led to redistribution of cathepsin B to the zymogen granule enriched subcellular fraction and to a substantial formation of large cytoplasmic vacuoles that contained both, trypsinogen and cathepsin B. However, this did not cause premature intracellular trypsin activation in saline-treated control animals lacking the CI-MPR. During caerulein-induced pancreatitis, trypsinogen activation in the pancreas of CI-MPR-deficient animals was about 40% higher than in wild-type animals but serum amylase levels were reduced and lung damage was unchanged. These data suggest that subcellular redistribution of cathepsin B, in itself, induces neither spontaneous trypsinogen activation nor pancreatitis. Furthermore, we clearly show that a marked increase in intracellular trypsinogen activation is not necessarily associated with greater disease severity.
Asunto(s)
Catepsina B/metabolismo , Páncreas/metabolismo , Pancreatitis/metabolismo , Tripsina/metabolismo , Amilasas/sangre , Amilasas/metabolismo , Animales , Ceruletida/metabolismo , Progresión de la Enfermedad , Factor II del Crecimiento Similar a la Insulina/metabolismo , Lisosomas/metabolismo , Ratones , Ratones Noqueados , Páncreas/ultraestructura , Pancreatitis/inducido químicamente , Pancreatitis/patología , Péptido Hidrolasas/metabolismo , Receptor IGF Tipo 2/metabolismo , Vesículas Secretoras/metabolismo , Tripsinógeno/metabolismo , Vacuolas/metabolismoRESUMEN
BACKGROUND: We have previously shown that cell contacts between pancreatic acinar cells dissociate early in pancreatitis and that this is a prerequisite for the development of pancreatic oedema. Here we studied the underlying mechanism. METHODS: Employing experimental caerulein induced pancreatitis in vivo and isolated pancreatic acini ex vivo, in conjunction with protein chemistry, morphology, and electron microscopy, we determined whether cell contact regulation in the pancreas requires or involves: (1) changes in cadherin-catenin protein expression, (2) tyrosine phosphorylation of adhesion proteins, or (3) alterations in the actin cytoskeleton. RESULTS: During initial cell-cell contact dissociation at adherens junctions, expression of adhesion proteins remained stable. At time points of dissociated adherens junctions, the cadherin-catenin complex was found to be tyrosine phosphorylated and internalised. The receptor type protein tyrosine phosphatase (PTP)kappa was constitutively associated with the cadherin-catenin complex at intact cell contacts whereas following the dissociation of adherens junctions, the internalised components of the cadherin-catenin complex were tyrosine phosphorylated and associated with the cytosolic PTP SHP-1. In isolated acini, inhibition of endogenous protein tyrosine phosphatases alone was sufficient to induce dissociation of adherens junctions analogous to that found with supramaximal caerulein stimulation. Dissociation of actin microfilaments had no effect on adherens junction integrity. CONCLUSIONS: These data identify tyrosine phosphorylation as the key regulator for cell contacts at adherens junctions and suggest a definitive role for the protein tyrosine phosphatases PTPkappa and SHP-1 in the regulation, maintenance, and restitution of cell adhesions in a complex epithelial organ such as the pancreas.
Asunto(s)
Uniones Adherentes/metabolismo , Comunicación Celular , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Páncreas Exocrino/metabolismo , Pancreatitis/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Receptores de Superficie Celular/metabolismo , Actinas/metabolismo , Animales , Cadherinas/metabolismo , Adhesión Celular/fisiología , Moléculas de Adhesión Celular/metabolismo , Comunicación Celular/fisiología , Ceruletida/administración & dosificación , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Masculino , Páncreas/metabolismo , Pancreatitis/inducido químicamente , Fosforilación , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Quinasas/metabolismo , Ratas , Ratas Wistar , Tirosina/metabolismoRESUMEN
The preparation of ditertiary aliphatic diamines 3 designed as drugs protecting CNS acetylcholinesterase against organophosphate inhibition, is described. Owing to the radicals at the basic nitrogen atoms, these compounds should exist in appreciable amounts both as base and as diammonium ion at biological pH's.
Asunto(s)
Reactivadores de la Colinesterasa/síntesis química , Diaminas/síntesis química , Antídotos/síntesis química , Concentración de Iones de Hidrógeno , Insecticidas/envenenamiento , Métodos , Intoxicación por OrganofosfatosRESUMEN
With an automated method, four antacid gel formulations were tested for antacid in vitro activity. In addition, some elements involved in the buffer effect were assayed in the solution parallel to the in vitro test. Of the four formulations, one showed only insufficient buffer effect. Compared with the common proton/metal ion exchange of antacid buffering, experimental data of another formulation suggest a partially different proton binding mechanism.
Asunto(s)
Antiácidos/farmacología , Aluminio/farmacología , Tampones (Química) , Concentración de Iones de Hidrógeno , Magnesio/metabolismo , Dióxido de Silicio/farmacología , Sodio/farmacología , Factores de TiempoRESUMEN
A method for the colorimetric assay of praziquantel has been developed. For the colorimetric assay, it was necessary to hydrolyse praziquantel with 3 mol dm-3 NaOH, 6 mol dm-3 HCl and 85% phosphoric acid separately. 4-Chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) reacts with the basic hydrolysis product in methanolic aqueous phosphate buffer (pH 7.4), resulting in the formation of an orange product with a characteristic absorption maximum at 478 nm. The red-orange product of the interaction between the hydrochloric acid hydrolysis product and NBD-Cl showed an absorption maximum at 486 nm. The colours obtained were stable for 24 h. The colour system obeyed Beer's law in the concentration range 2-15 and 2-18 micrograms ml-1 for the basic hydrolysis product and the acid hydrolysis product, respectively. The results obtained showed good recoveries with relative standard deviations of 0.378 and 0.47% for the basic and the acid hydrolysis product, respectively. The determination limit was found to be 0.124 and 0.150 micrograms ml-1 for the praziquantel basic hydrolysis product and the acid hydrolysis product, respectively. The coloured reaction products obtained with the proposed method were synthesized. The structures of these products were studied and the compounds identified.
Asunto(s)
Praziquantel/análisis , 4-Cloro-7-nitrobenzofurazano , Colorimetría/métodos , Indicadores y Reactivos , ComprimidosRESUMEN
Insulin signal transduction involves the multisite docking protein insulin receptor substrate-1 (IRS-1) and a number of Src homology-2 (SH2) domain factors, including p85/p110 phosphatidylinositol 3-kinase, p110 GTPase-activating protein, and the phosphotyrosine-specific phosphatase PTP1D. In transfected baby hamster kidney cells, Rat1 fibroblasts, and normal IM9 lymphoblasts, PTP1D directly binds activated insulin receptor. This interaction is mediated by catalytic domain-proximal SH2 determinants of the phosphatase and phosphotyrosine 1146 of the activated insulin receptor. While the receptor and the phosphatase do not serve as substrates for each other, their interaction promotes IRS-1 binding to the receptor, indicating that PTP1D functions as an adapter for insulin receptor and IRS-1. The formation of a multiprotein signaling complex involving the insulin receptor, PTP1D, and IRS-1 enhances cellular glucose uptake, a critical process in the physiological action of insulin.