RESUMEN
In human pancreas two genes, reg I alpha and reg I beta, have been characterized but only the reg I alpha protein has been isolated from human pancreatic secretion. To examine their respective physiological roles in fetal and adult pancreas we have compared the patterns of gene expression using a specific RT-PCR method. No progressive evolution in the two mRNAs levels was observed during fetal development (16--41 weeks). A discoordinate expression of the two genes was found with a higher level of reg I alpha mRNA in fetus and a higher level of regI beta in adult. In addition, if reg I alpha mRNA level was correlated with the expression of genes encoding exocrine proteins in adults, reg I beta mRNA level presented no correlation with any ductular, endocrine, or exocrine gene expression. In human pancreatic cell lines we showed the only expression of reg I beta gene and protein. All these data suggest that the two reg genes and proteins could play different roles in the pancreas.
Asunto(s)
Proteínas de Unión al Calcio/biosíntesis , Proteínas del Tejido Nervioso , Páncreas/metabolismo , Anciano , Biomarcadores/análisis , Proteínas de Unión al Calcio/genética , Línea Celular , Regulación del Desarrollo de la Expresión Génica , Humanos , Hibridación in Situ , Litostatina , Persona de Mediana Edad , Páncreas/embriología , ARN Mensajero/biosíntesis , Transcripción GenéticaRESUMEN
We have previously shown a specific significant overexpression in the exocrine pancreatic tissue of two members of the regenerating gene multifamily: reg I and reg II in the non-obese diabetic (NOD) mouse during active diabetogenesis. To strengthen the hypothesis that the overexpression of these genes may represent a defence of the acinar cell against pancreatic endocrine agression, we studied the pancreatic expression and the localization of another member of this family: the pancreatitis-associated protein (PAP) in NOD mice under the same conditions. We found that NOD mice present significantly higher PAP mRNA levels than control IOPS-OF1 mice. There is no difference between female NOD mice which progressively develop type I diabetes between 100 and 200 days and male NOD mice which are protected. The only difference observed was in function of the age of onset of diabetes. Before 180 days, the PAP mRNA levels were similar to those found in NOD males and nondiabetic females, but above 180 days the levels of PAP mRNA increased significantly. More importantly immunohistological studies demonstrate a striking difference in the protein localization between normal or nondiabetic NOD mice and diabetic NOD mice. If the protein is mainly detected in the islet cells in the absence of diabetes, a specific and intense expression of PAP was observed in the acinar cells of diabetic NOD mice. In conclusion, our data demonstrates that the acinar cells may react to a long-lasting pancreatic endocrine aggression by an induction of PAP and underlines the existence of a symbiotic relationship between endocrine and exocrine tissue.
Asunto(s)
Antígenos de Neoplasias , Biomarcadores de Tumor , Proteínas de Unión al Calcio/análisis , Diabetes Mellitus Tipo 1/genética , Lectinas Tipo C , Lectinas/análisis , Páncreas/química , Pancreatitis/metabolismo , Proteínas , Animales , Northern Blotting , Western Blotting , Proteínas de Unión al Calcio/genética , ADN Complementario/análisis , Femenino , Inmunohistoquímica , Lectinas/genética , Masculino , Ratones , Ratones Endogámicos NOD , Pancreatitis/genética , Proteínas Asociadas a Pancreatitis , ARN Mensajero/análisis , Regulación hacia ArribaRESUMEN
A differential pancreatic behavior observed between male and female mice in diabetes and pancreatitis led us to study the gene and protein expressions of endocrine and exocrine pancreatic proteins in normal mice. We compared the levels of expression of six pancreatic genes and of four of the corresponding proteins in male and female mice OF1. Amylase gene expression was found to be significantly higher in females than in males, whereas trypsinogen and lipase gene expression were significantly lower. For chymotrypsinogen, reg, and insulin the differences were not significant. This sexual dimorphism did not exist in rat pancreas, where no gender difference was observed. After characterization of mice enzymes by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and antibodies directed to the closely related human pancreatic enzymes, we have compared the levels of these proteins in mice pancreatic homogenates. No significant difference was observed between males and females at the level of protein expression. These data suggest a hormonal sexual difference in the regulation of pancreatic protein synthesis at the pre- and posttranscriptional levels in normal mice, which may play a role in the development of mice pancreatic diseases.
Asunto(s)
Regulación de la Expresión Génica , Páncreas/metabolismo , Amilasas/genética , Animales , Quimotripsinógeno/genética , Femenino , Lipasa/genética , Masculino , Ratones , ARN Mensajero/análisis , Ratas , Caracteres Sexuales , Tripsinógeno/genéticaRESUMEN
We demonstrated pancreatic reg gene overexpression in non-obese diabetic (NOD) mice during active diabetogenesis. The aim of this study was to determine in which part of the pancreas (endocrine and/or exocrine) the gene(s) and the protein(s) were expressed and if their localization changed with progression of the disease. In situ hybridization analysis and immunocytochemical studies were carried out on pancreas of female and male NOD mice. Both develop insulitis but diabetes develops only in females and in males only when treated by cyclophosphamide. Our results show that whatever the age, sex, and presence of insulitis and/or diabetes, the expression of reg mRNAs and of the corresponding protein(s) was restricted to exocrine tissue. Moreover, reg remains localized in acinar cells in the two opposite situations of (a) cyclophosphamide-treated males in a prediabetic stage presenting a high level of both insulin and reg mRNAs, and (b) the overtly diabetic females with no insulin but a high level of reg mRNA. These findings suggest that overexpression of the reg gene(s) might represent a defense of the acinar cell against pancreatic aggression.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Proteínas del Tejido Nervioso , Páncreas/metabolismo , Animales , Northern Blotting , Femenino , Inmunohistoquímica , Hibridación in Situ , Insulina/metabolismo , Litostatina , Ratones , Ratones Endogámicos NOD , ARN/metabolismoRESUMEN
Gastrin (G) and cholecystokinin (CCK) are gastrointestinal neuropeptides that are released into circulation during a meal. G is also transiently expressed during embryogenic and early ontogenic development of the pancreas and is believed to act on islet-cell development. Both peptides act on pancreatic endocrine function; however, the effects are dependent on the species and on cellular and molecular underlying mechanisms that remain poorly characterized. Since CCK-B/G subtype receptor is predominant over the CCK-A subtype in the human pancreas, we hypothesized that it could be expressed by islet cells. Here we present reverse transcription-polymerase chain reaction and immunohistochemistry data demonstrating that the CCK-B/G receptor is expressed in islet cells and that islet glucagon-producing cells are the major site of CCK-B/G receptor expression in adult and fetal pancreas. Moreover, G immunoreactivity was detected in the fetal human pancreas at embryogenic week 22. G- and CCK-stimulated glucagon are released from purified human islets. Concentration of CCK and G eliciting a half-maximal level of glucagon secretion were 13 +/- 6 and 8 +/- 5 pmol/l, respectively. Maximal glucagon secretion was achieved in the presence of 30 pmol/l peptides and was similar to that obtained in the presence of 10 mmol/l L-arginine (1.6 pmol x ml(-1) x 90 min(-1)). The nonpeptide antagonist of the CCK-B/G receptor, RPR-101048, fully inhibited CCK- and G-stimulated glucagon secretion at 100 nmol/l concentration. These data are consistent with the view that the CCK-B/G receptor is involved in glucose homeostasis in adult humans and mediates the autocrine effects of G on islet differentiation and growth in the fetal pancreas.
Asunto(s)
Páncreas/fisiología , Receptores de Colecistoquinina/fisiología , Adulto , Células Cultivadas , Colecistoquinina/metabolismo , Clonación Molecular , Gastrinas/metabolismo , Regulación de la Expresión Génica , Glucagón/metabolismo , Humanos , Páncreas/embriología , ARN Mensajero/metabolismo , Receptor de Colecistoquinina B , Receptores de Colecistoquinina/genéticaRESUMEN
We localized REG protein in Paneth cells and nonmature columnar cells of the human small intestinal crypts and speculated that this protein was associated with growth and/or differentiation. The aim of this study was to determine whether REG protein is present in two human colon cancer cell lines that exhibit enterocytic differentiation after confluence and to investigate changes in the level of its expression during growth and differentiation. Results were compared to those obtained on cells that remain undifferentiated. Western immunoblotting and immunofluorescence demonstrated the presence of REG protein in the three cell lines. With the antisera against human REG protein, the staining was diffusely spread throughout the cytoplasm at Day 2, and after Days 3-4 it appeared to have migrated to cell boundaries. After confluence, we observed only a punctate staining array along cell boundaries, which disappeared at Day 15. REG mRNA expression was demonstrated by RTPCR and REG mRNA hybridization until Day 13, but not after, in the three cell types. REG protein may be involved in cellular junctions. Its presence appears to be associated with the cell growth period and the protein must be downregulated when growth is achieved and differentiation is induced.
Asunto(s)
Proteínas de Unión al Calcio/biosíntesis , Intestino Delgado/citología , Intestino Delgado/metabolismo , Proteínas del Tejido Nervioso , Western Blotting , Células CACO-2 , Proteínas de Unión al Calcio/genética , Diferenciación Celular , División Celular , Técnica del Anticuerpo Fluorescente Indirecta , Células HT29 , Humanos , Litostatina , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: The biological function of the Reg protein, a non-enzymic protein produced in fairly large amounts by pancreatic acinar cells, remains elusive. Its susceptibility to proteolysis leading to precipitation of the proteolysis product at neutral pH suggests that it could contribute to the protein plugging observed in cystic fibrosis (CF). AIMS: To study its behaviour in the serum of CF patients with or without pancreatic insufficiency and to compare it with that of other pancreatic secretory proteins. PATIENTS: 170 patients (93 with CF, 55 controls, and 22 with chronic pancreatitis) were studied. METHODS: Reg protein was measured using a specific enzyme immunoassay and its molecular form in CF sera was characterised by gel filtration. Molecular gene expression was investigated by dot-blot hybridisation. RESULTS: Reg protein was present in all CF sera studied from patients with or without pancreatic insufficiency, and in all cases the level was significantly higher than in controls. Its chromatographic behaviour in CF sera was identical with that of the protein present in normal serum. No correlation was found between the levels of Reg protein and trypsin(ogen) (or lipase) in CF, nor in control sera or normal pancreatic juice. Molecular gene expression of the corresponding proteins investigated in pancreatic tissues showed an absence of correlation between the mRNA levels. CONCLUSIONS: Reg protein may not be a secretory exocrine protein like the digestive enzymes but rather a hormone-like secretory substance with an endocrine or paracrine function.
Asunto(s)
Proteínas de Unión al Calcio/sangre , Fibrosis Quística/sangre , Insuficiencia Pancreática Exocrina/sangre , Proteínas del Tejido Nervioso , Fosfoproteínas/sangre , Adolescente , Adulto , Proteínas de Unión al Calcio/química , Niño , Preescolar , Cromatografía en Gel , Quimotripsinógeno/sangre , Fibrosis Quística/complicaciones , Insuficiencia Pancreática Exocrina/etiología , Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Lactante , Recién Nacido , Lipasa/sangre , Litostatina , Jugo Pancreático/metabolismo , Fosfoproteínas/química , ARN Mensajero/genética , Tripsinógeno/sangre , Tripsinógeno/genéticaRESUMEN
A relationship between targeting of the protein CFTR (Cystic Fibrosis Transmembrane conductance Regulator) and cellular polarization has been observed in various types of epithelial cells. However, there are no reports on this in human exocrine pancreatic cells, which are functionally altered in patients with cystic fibrosis. The expression of CFTR and its targeting to apical plasma membranes was investigated during growth and polarization of human ductal pancreatic cancerous Capan-1 cells. Despite their neoplastic origin, the cancerous pancreatic duct cells of the Capan-1 line secrete Cl- and HCO3- ions. We showed by electron microscopy, impregnation of cells with tannin and freeze-fracture that these cells become polarized during growth in culture, and are joined by tight junctions. The expression of CFTR and the various stages in its anchorage to membranes was followed using a specific polyclonal antibody, ECL-885, directed against a synthetic peptide mimicking one of the extracellular loops of CFTR. Qualitative and quantitative confocal microscopic studies showed that: (i) the expression of CFTR was constant during growth, irrespective of cellular conformation, (ii) the number of cells presenting CFTR anchored to membranes increased with time in culture, (iii) the rise in membrane-bound CFTR-immunoreactivity accompanied the polarization of the cells, (iv) CFTR anchored to plasma membranes was distributed regularly over the surface of non-polarized cells, but was localized only at the apical membranes of the polarized cells. Moreover, patch-clamp studies indicated the presence of few Cl- cAMP-dependent conductance CFTR channels on unpolarized cells, and a larger number of CFTR channels on the apical plasma membranes of polarized cells. These results indicated that the anchorage of a functional CFTR to the plasma membrane is progressive and occurs in step with polarization of these human pancreatic duct cells in culture. We suggest that the targeting of CFTR to the apical membranes is directly linked to the process of cellular polarization.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Conductos Pancreáticos/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Transporte Biológico , División Celular , Polaridad Celular , Células HT29 , Humanos , Ratones , Datos de Secuencia Molecular , Conductos Pancreáticos/citología , Células Tumorales CultivadasRESUMEN
A direct sandwich immunoassay was developed to quantify the human reg protein, a non enzymatic pancreatic acinar protein the biological function of which remains elusive. Polystyrene balls were coated with specific IgG fraction as the first antibody and horseradish peroxidase labelled IgG was used as a second antibody. The linearity of the assay was good over a concentration range of 1.25-100 ng/ml and the good parallelism obtained between the standard and the assay dilution curves in serum and pancreatic juice indicates the absence of non-specific interfering reactions. Gel filtration of serum showed that the reg protein was eluted in the fractions corresponding to the proteins of around 25 kDa and that the chromatographic behaviour of the serum protein was identical to that of the purified pancreatic protein when added to serum. This assay was simple, specific, sensitive and reproducible and may permit the determination of low levels of reg protein in different biological fluids.
Asunto(s)
Proteínas de Unión al Calcio/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , Proteínas del Tejido Nervioso , Fosfoproteínas/sangre , Proteínas de Unión al Calcio/análisis , Cromatografía en Gel , Humanos , Litostatina , Jugo Pancreático/química , Fosfoproteínas/análisis , Valores de Referencia , Sensibilidad y EspecificidadRESUMEN
The development of human endocrine pancreas has been the subject of many immunohistochemical studies but very little is known at the molecular level. We have determined the patterns of gene expression of glucagon, somatostatin and pancreatic polypeptide during fetal life (16-41 weeks of gestation) using the dot-blot hybridization method. In spite of some dispersion in the mRNA levels, no progressive increase or decrease during this period of fetal life could be found, as previously observed for insulin. In keeping with these molecular data, no increase in immunostaining of the four hormones was observed, but a dispersion of endocrine cells within the exocrine tissue was noticed at 20 weeks of gestation followed by a clear differentiation of the Langerhans islets at 31 weeks. Interestingly, the mRNA levels of the four hormones were always higher in the fetal pancreas than in the adult pancreas.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Islotes Pancreáticos/embriología , Northern Blotting , Sondas de ADN , Desarrollo Embrionario y Fetal , Femenino , Edad Gestacional , Glucagón/biosíntesis , Glucagón/genética , Humanos , Inmunohistoquímica , Insulina/biosíntesis , Insulina/genética , Islotes Pancreáticos/metabolismo , Polipéptido Pancreático/biosíntesis , Polipéptido Pancreático/genética , Embarazo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Somatostatina/biosíntesis , Somatostatina/genéticaRESUMEN
BACKGROUND: Very few studies have been reported on the expression of human pancreatic genes during fetal development. We have shown very low lipase immunoreactivity compared with elevated trypsinogen immunoreactivity in a previous immunohistological study of human fetal pancreas during development. METHODS: The expression of these two selectively expressed genes of the exocrine pancreas, trypsinogen and lipase were investigated. The developmental profiles of the corresponding mRNA's were determined from the 13th gestational week. RESULTS: For the two genes, fetal mRNA levels throughout gestation remained significantly lower than the corresponding adult levels. No correlation was found between trypsinogen and lipase gene expression in the fetal pancreas, whereas such a correlation was present in adult pancreas. This may be explained by differences in maturity of the pancreas.
Asunto(s)
Desarrollo Embrionario y Fetal , Expresión Génica , Lipasa/genética , Páncreas/embriología , Páncreas/enzimología , Tripsinógeno/genética , Adulto , Northern Blotting , Femenino , Edad Gestacional , Humanos , Embarazo , ARN Mensajero/metabolismoRESUMEN
Pancreatic dysfunction in cystic fibrosis (CF) begins in utero and, at birth, in most cases, cystic fibrosis is characterized by an elevated level of serum immunoreactive trypsin (IRT). If most patients with CF typically present insufficient pancreatic exocrine function, 10-15% of CF patients have pancreatic sufficiency and this status is genetically determined by one or two 'mild' mutations in CF transmembrane conductance regulator (CFTR). However, with age, these patients can develop pancreatic insufficiency.
Asunto(s)
Fibrosis Quística/fisiopatología , Páncreas/fisiopatología , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Insuficiencia Pancreática Exocrina/etiología , Insuficiencia Pancreática Exocrina/fisiopatología , Feto/fisiopatología , Expresión Génica , Humanos , Pancreatitis/fisiopatologíaRESUMEN
Beta-cell regeneration in adult pancreas is usually considered to be limited. However, various animal models suggest that this tissue is still capable of regeneration under certain conditions. Reg protein could be responsible for this replicative process. The reg gene codes for a 166 amino-acid protein usually synthesized and secreted by pancreatic acinar cells but expressed in islet beta cells during experimental regenerative processes in animals (90% pancreatectomy + nicotinamide, or insulinoma tumor removal in rats, or the "wrapping pancreas model" in the hamster). In addition, recombinant rat reg protein can stimulate beta-cell replication in vivo and in vitro. In animal models of Type 1 diabetes mellitus, reg gene overexpression occurs during active phases of diabetogenesis and could be a defence mechanism. During human pancreatic development, reg gene is expressed at an early stage but is not associated with the expression of other pancreatic genes. Conversely, gene expression for reg and insulin are correlated in adult pancreas. Accordingly, reg protein could be a beta-cell-specific growth factor implicated in the maintenance of beta-cell mass, especially in adult pancreas.
Asunto(s)
Proteínas de Unión al Calcio/fisiología , Sustancias de Crecimiento/fisiología , Islotes Pancreáticos/fisiología , Proteínas del Tejido Nervioso , Regeneración/fisiología , Animales , Proteínas de Unión al Calcio/genética , Diabetes Mellitus Tipo 1/fisiopatología , Desarrollo Embrionario y Fetal/fisiología , Genoma Humano , Sustancias de Crecimiento/genética , Humanos , Litostatina , Páncreas/embriología , Páncreas/crecimiento & desarrolloRESUMEN
The X-ray structure of human trypsin 1 has been determined in the presence of diisopropyl-phosphofluoridate by the molecular replacement method and refined at a resolution of 2.2 A to an R-factor of 18%. Crystals belong to the space group P4, with two independent molecules in the asymmetric unit packing as crystallographic tetramers. This study was performed in order to seek possible structural peculiarities of human trypsin 1, suggested by some striking differences in its biochemical behavior as compared to other trypsins of mammalian species. Its fold is, in fact, very similar to those of the bovine, rat and porcine trypsins, with root-mean-square differences in the 0.4 to 0.6 A range for all 223 C alpha positions. The most unexpected feature of the human trypsin 1 structure is in the phosphorylated state of tyrosine residue 151 in the present X-ray study. This feature was confirmed by mass spectrometry on the same inhibited sample and also on the native enzyme. This phosphorylation strengthens the outstanding clustering of highly negative or highly positive electrostatic surface potentials. The peculiar inhibitory behaviour of pancreatic secretory trypsin inhibitors of the Kazal type on this enzyme is discussed as a possible consequence of these properties. A charged surface loop has also been interpreted as an epitope site recognised by a monoclonal antibody specific to human trypsin 1.
Asunto(s)
Isoenzimas/química , Isoenzimas/metabolismo , Tripsina/química , Tripsina/metabolismo , Tirosina/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , Humanos , Isoenzimas/aislamiento & purificación , Datos de Secuencia Molecular , Jugo Pancreático/enzimología , Fosforilación , Homología de Secuencia de Aminoácido , Temperatura , Tripsina/aislamiento & purificaciónRESUMEN
The reg gene characterized in the exocrine pancreas has been found to be expressed in regenerating islets of 90% depancreatized rats and not in normal islets. In humans, it was identified only in the exocrine pancreas. Because the reg protein has been found to be related to islet cell replication and/or beta cell regeneration, we compared the expression of the reg gene with that of chymotrypsinogen of exocrine origin and insulin of endocrine origin. We investigated the expression of the three pancreatic genes in the fetal pancreas during human development using dot-blot analysis. The levels of expression of the corresponding mRNAs did not appear to undergo great changes between the 17th and the 29th wk of gestation. Nevertheless, the fetal mRNA levels for reg and chymotrypsinogen were below that of the adult, with very low levels of reg gene expression in more than half of the studied pancreases. In contrast, the insulin mRNA levels were significantly higher in fetal than in adult pancreases, suggesting that insulin may function as a growth factor during fetal development. Our results indicate that no correlation between reg and insulin gene expression exists in the fetal pancreas during the developmental period studied but, on the contrary, such a correlation was present in the adult pancreas.
Asunto(s)
Proteínas de Unión al Calcio/genética , Expresión Génica/genética , Insulina/genética , Proteínas del Tejido Nervioso , Páncreas/metabolismo , Adulto , Secuencia de Bases , Northern Blotting , Quimotripsinógeno/genética , Cartilla de ADN , Feto , Humanos , Litostatina , Datos de Secuencia Molecular , Páncreas/embriología , ARN Mensajero/genéticaRESUMEN
A direct sandwich enzyme immunoassay using two monoclonal antibodies was developed in order to quantify mucin M1 antigens produced by two pancreatic adenocarcinoma cell lines: CAPAN-1 and CFPAC-1. As a solid phase, the wells of a microtiter plate were coated with a first monoclonal antibody, 1-13 M1 and the biotinylated monoclonal conjugate 9-13 M1 was used as the second antibody. The assay was optimized with streptavidin-peroxidase. The detection limit of the assay is 1.6 ng/ml. This ELISA is highly specific, sensitive, reproducible and quickly performed. It will permit the comparison of mucin exocytosis by the two cell lines in response to secretagogue agents and may help in the study of the pathogenesis of mucus hypersecretion such as cystic fibrosis.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Epítopos/inmunología , Mucinas/química , Moco/metabolismo , Páncreas/metabolismo , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Proteínas Bacterianas , Biotina , Línea Celular , Fibrosis Quística/metabolismo , Humanos , Técnicas para Inmunoenzimas , Mucinas/inmunología , Moco/química , Moco/inmunología , Neoplasias Pancreáticas/metabolismo , Proteínas/análisis , Estreptavidina , Células Tumorales CultivadasRESUMEN
A complex of two calcium binding proteins, MRP8 [also called cystic fibrosis (CF) antigen] and MRP14, proteins known to be expressed in cells of myeloid origin, has been shown to be present in higher amounts in the serum of CF patients and heterozygotes compared with normal subjects. We demonstrated here for the first time, by dot-blot analysis and immunocytochemistry, the expression and the presence of these S100 calcium binding proteins in the pancreatic cell lines CAPAN-1 and CFPAC-1, the latter provided from a patient with CF. Moreover, using immunocytochemical methods, we showed that the localization of MRP8 and MRP14 on the plasma membrane seems to be restricted to the cells expressing a cystic fibrosis transmembrane conductance regulator (CFTR) wild-type protein such as CAPAN-1 cells and CFPAC-1 cells transfected with a plasmid containing the nonmutated CFTR gene (CFPAC-PLJ-CFTR-6 cells). In CFPAC-1 cells, immunoreactivity remains in the cytoplasm throughout the stationary phase. We also showed an increased level of the mRNAs of the two proteins in the CFPAC-1 cells compared with those transfected with the nonmutated CFTR. The demonstration of a difference in the cellular localization of these two proteins and in their mRNA levels in the cell line of CF origin leads us to assume the existence of a possible correlation in the expression of the MRPs with that of the CFTR protein.
Asunto(s)
Antígenos de Diferenciación/metabolismo , Proteínas de Unión al Calcio/metabolismo , Fibrosis Quística/metabolismo , Páncreas/metabolismo , Antígenos de Diferenciación/genética , Proteínas de Unión al Calcio/genética , Calgranulina A , Calgranulina B , Línea Celular , Membrana Celular/metabolismo , Fibrosis Quística/patología , Humanos , Immunoblotting , Inmunohistoquímica , Microscopía Electrónica , Páncreas/patología , ARN Mensajero/metabolismo , Distribución TisularRESUMEN
An immunoradiometric assay using two monoclonal antibodies directed to human trypsin 1 was developed for measuring trypsin(ogen) in biological fluids. The assay is different from other assays in that it is specific for cationic trypsinogen and does not recognize the alpha-1-proteinase inhibitor-trypsin complex. It can be used as a complement to classical immunoassays to characterize trypsinogen activation in pathological cases. The evaluation and the specificity of the assay are presented.
Asunto(s)
alfa-Globulinas/análisis , Ensayo Inmunorradiométrico/métodos , Inhibidores de Tripsina/análisis , Tripsina/análisis , Tripsinógeno/análisis , Líquido Amniótico/enzimología , Animales , Humanos , Jugo Pancreático/enzimología , Radioinmunoensayo , Ratas , Porcinos , Tripsina/sangre , Inhibidores de Tripsina/sangre , Tripsinógeno/sangreRESUMEN
Using immunoenzymometric assays, the production of elastase, alkaline protease and exotoxin A was determined in culture supernatants of 35 strains of Pseudomonas aeruginosa isolated from patients suffering from cystic fibrosis. The assays were simple, specific, sensitive and reproducible, and permitted the determination of low levels of exoproteins. A large strain variability of exoprotein production was found. Most of the strains secreted all three exoproteins, but six out of the 35 strains (17%) did not secrete at least one of the three (< 0.3 microgram/l). A significant correlation was observed between elastase and exotoxin A productions (r = 0.697, p < 0.001).
Asunto(s)
ADP Ribosa Transferasas , Proteínas Bacterianas/análisis , Toxinas Bacterianas , Fibrosis Quística/microbiología , Exotoxinas/análisis , Pseudomonas aeruginosa/química , Serina Endopeptidasas/análisis , Factores de Virulencia , Animales , Especificidad de Anticuerpos , Electroforesis en Gel de Poliacrilamida , Humanos , Immunoblotting , Técnicas para Inmunoenzimas , Elastasa Pancreática , Pseudomonas aeruginosa/enzimología , Conejos , Sensibilidad y Especificidad , Dodecil Sulfato de Sodio , Esputo/microbiología , Exotoxina A de Pseudomonas aeruginosaRESUMEN
We previously demonstrated that two human pancreatic adenocarcinoma cell lines, CFPAC-1 (established from a patient with cystic fibrosis) and CAPAN-1, were able to secrete trypsinogens 1 and 2 specifically. In order to analyze the relation of trypsin secretion to differentiation and cell growth, we undertook a comparative study of immunoreactive trypsin 1 (IRT) secretion by the two cell lines during cell growth in the presence and in the absence of various differentiating agents: sodium butyrate (NaBut), dimethylsulfoxide (DMSO), and dexamethasone (DX). In the presence of NaBut, IRT levels in the supernatants of both cell lines were slightly increased, whereas the cellular growth of both cell lines decreased significantly. In the presence of DX, IRT levels in cell culture conditioned media immediately and dramatically decreased, but the cell growth of neither cell line was affected by DX. An important increase in IRT levels was observed when CFPAC-1 cells and CAPAN-1 cells were grown in the presence of DMSO, but for both cell lines the cellular growth decreased in the presence of DMSO. Our data show that neither the IRT secretion level nor the differentiation state of these cell lines correlates with cellular growth, and suggests that the expression of pancreatic proteases by these two tumor cell lines could be either related to a common stem cell with this potential or to a possible acinar origin of pancreatic cancer, as recently proposed by others.