RESUMEN
The effects of different environmental salinities (0, 12, 40, and 55 ppt) on pepsinogen 2 (pga2), trypsinogen 2 (try2), chymotrypsinogen (ctr), and pancreatic alpha-amylase (amy2a) gene expression, and on the total activities of their corresponding enzymes, were assessed in Chelon labrosus juveniles, after their corresponding full-complementary DNA sequences were cloned. Furthermore, the quantitative effect of different salinities on the hydrolysis of feed protein by fish digestive enzymes was evaluated using an in vitro system. Relative pga2 expression levels were significantly higher in animals maintained at 12 ppt, while a significantly higher gene expression level for ctr and try2 was observed at 40 ppt. amy2a gene expression showed its maximum level at 40 ppt and the lowest at 55 ppt. A significant reduction in the activity of amylase with the increase in salinity was observed, whereas the maximum activity for alkaline proteases was observed in individuals maintained at 40 ppt. A negative effect of high salinity on the action of proteases was confirmed by the in vitro assay, indicating a decreased efficiency in the digestive function in C. labrosus when maintained at high environmental salinities. Nevertheless, individuals can live under different environmental salinities, even though gene expression is different and the enzymatic activities are not maintained at the highest studied salinity. Therefore, compensatory mechanisms should be in place. Results are discussed on the light of the importance as a new species for aquaculture.
Asunto(s)
Digestión/fisiología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Salinidad , Smegmamorpha/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Quimotripsinógeno/genética , Quimotripsinógeno/metabolismo , ADN Complementario/genética , Mucosa Intestinal/metabolismo , alfa-Amilasas Pancreáticas/genética , alfa-Amilasas Pancreáticas/metabolismo , Pepsinógeno A/genética , Pepsinógeno A/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Cloruro de Sodio/farmacología , Tripsinógeno/metabolismoRESUMEN
OBJECTIVES: Genetic studies in adults/adolescent patients with chronic pancreatitis (CP) identified chymotrypsinogen C (CTRC) genetic variants but their association with CP risk has been difficult to replicate. To evaluate the risk of CP associated with CTRC variants in CP pediatric patients-control study. METHODS: The distribution of CTRC variants in CP pediatric cohort (nâ=â136, median age at CP onset 8 years) with no history of alcohol/smoking abuse was compared with controls (nâ=â401, median age 45). RESULTS: We showed that p.Arg254Trp (4.6%) and p.Lys247_Arg254del (5.3%) heterozygous mutations are frequent and significantly associated with CP risk in pediatric patients (odds ratio [OR]â=â19.1; 95% CI 2.8-160; Pâ=â0.001 and ORâ=â5.5; 95% CI 1.6-19.4; Pâ=â0.001, respectively). For the first time, we demonstrated that the c.180TT genotype of common p.Gly60Gly variant is strong, an independent CP risk factor (ORâ=â23; 95% CI 7.7-70; Pâ<â0.001) with effect size comparable to p.Arg254Trp mutation. The other novel observation is that common c.493+51C>A variant, both CA and AA genotype, is significantly underrepresented in CP compared with controls (15% vs 35%; ORâ=â0.33; 95% CI 0.19-0.59; Pâ<â0.001 and 2.8% vs 11%; ORâ=â0.24; 95% CI 0.06-0.85; Pâ=â0.027, respectively). CONCLUSIONS: Our study provides evidence that CTRC variants, including c.180TT (p.Gly60Gly) are strong CP risk factors. The c.493+51C>A variant may play a protective role against CP development.
Asunto(s)
Quimotripsinógeno/genética , Predisposición Genética a la Enfermedad , Pancreatitis Crónica/genética , Serina Endopeptidasas/genética , Adulto , Niño , Femenino , Variación Genética , Genotipo , Humanos , Masculino , Pancreatitis Crónica/diagnóstico , Factores de Riesgo , Inhibidor de Tripsina Pancreática de Kazal/genéticaAsunto(s)
Dolor Abdominal/etiología , Enfermedades Autoinmunes/complicaciones , Pancreatitis/complicaciones , Úlcera Gástrica/etiología , Enfermedades Autoinmunes/diagnóstico , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/cirugía , Biopsia , Quimotripsinógeno/genética , Análisis Mutacional de ADN , Duodenoscopía , Femenino , Gastrectomía , Gastroscopía , Predisposición Genética a la Enfermedad , Humanos , Mutación , Pancreaticoduodenectomía , Pancreatitis/diagnóstico , Pancreatitis/genética , Pancreatitis/cirugía , Factores de Riesgo , Serina Endopeptidasas/genética , Úlcera Gástrica/diagnóstico por imagen , Úlcera Gástrica/cirugía , Tomografía Computarizada por Rayos X , Adulto JovenRESUMEN
The study of digestive physiology is an important issue in species that have been introduced in aquaculture like the spotted rose snapper (Lutjanus guttatus). The aims of this study were to describe the expression of digestive enzymes (trypsinogen, chymotrypsinogen, α-amylase, lipoprotein lipase, phospholipase A and pepsinogen) and their relation with orexigenic (neuropeptide Y, NPY) and anorexigenic (cholecystokinin, CCK) factors during the larval development and to evaluate the effect of weaning in their expression. The results showed that the transcripts of all the assayed digestive enzymes, with the exception of pepsinogen, and NPY and CCK were already present in L. guttatus from the hatching stage. The expression of all the enzymes was low during the yolk-sac stage (0-2 days after hatching, DAH), whereas after the onset of exogenous feeding at 2 DAH, their expression increased and fluctuated throughout larval development, which followed a similar pattern as in other marine fish species and reflected changes in different types of food items and the progressive maturation of the digestive system. On the other hand, weaning of L. guttatus larvae from live prey onto a microdiet between 25 and 35 DAH significantly affected the relative expression of most pancreatic digestive enzymes during the first weaning days, whereas chymotrypsinogen 2 and lipoprotein lipase remained stable during this period. At the end of co-feeding, larvae showed similar levels of gene expression regardless of the diet (live prey vs. microdiet), which indicated that larvae of L. guttatus were able to adapt their digestive capacities to the microdiet. In contrast, feeding L. guttatus larvae with live feed or microdiet did not affect the expression of CCK and NPY. The relevance of these findings with regard to current larval rearing procedures of L. guttatus is discussed.
Asunto(s)
Digestión/genética , Perciformes/genética , Animales , Colecistoquinina/genética , Quimotripsinógeno/genética , Femenino , Expresión Génica , Larva/genética , Larva/crecimiento & desarrollo , Lipoproteína Lipasa/genética , Masculino , Neuropéptido Y/genética , Pepsinógeno A/genética , Perciformes/crecimiento & desarrollo , Perciformes/metabolismo , Fosfolipasas A2/genética , ARN Mensajero/metabolismo , Tripsinógeno/genética , alfa-Amilasas/genéticaRESUMEN
Even with significant advances in technology, few studies of structural variation have yet resolved to the level of the precise nucleotide junction. We examined the sequence of 408,532 gains, 383,804 losses, and 166 inversions from the first sequenced personal genome, to quantify the relative proportion of mutational mechanisms. Among small variants (<1 kb), we observed that 72.6% of them were associated with nonhomologous processes and 24.9% with microsatellites events. Medium-size variants (<10 kb) were commonly related to minisatellites (25.8%) and retrotransposons (24%), whereas 46.2% of large variants (>10 kb) were associated with nonallelic homologous recombination. We genotyped eight new breakpoint-resolved inversions at (3q26.1, Xp11.22, 7q11.22, 16q23.1, 4q22.1, 1q31.3, 6q27, and 16q24.1) in human populations to elucidate the structure of these presumed benign variants. Three of these inversions (3q26.1, 7q11.22, and 16q23.1) were accompanied by unexpected complex rearrangements. In particular, the 16q23.1 inversion and an accompanying deletion would create conjoined chymotrypsinogen genes (CTRB1 and CTRB2), disrupt their gene structure, and exhibit differentiated allelic frequencies among populations. Also, two loci (Xp11.3 and 6q27) of potential reference assembly orientation errors were found. This study provides a thorough account of formation mechanisms for structural variants, and reveals a glimpse of the dynamic structure of inversions.
Asunto(s)
Variación Genética , Genoma Humano , Análisis de Secuencia de ADN/métodos , Deleción Cromosómica , Inversión Cromosómica , Cromosomas Humanos Par 16/genética , Quimotripsina/genética , Quimotripsina/metabolismo , Quimotripsinógeno/genética , Quimotripsinógeno/metabolismo , Frecuencia de los Genes , Haplotipos , Humanos , Repeticiones de Microsatélite , Repeticiones de Minisatélite , Retroelementos , Trisomía/genéticaRESUMEN
BACKGROUND/AIMS: Nutrition during fetal and early postnatal development can have permanent effects on physiology resulting in an increased risk for disease in later life. The aim of this study was to explore changes in gene expression related to maternal energy restriction during pregnancy in rat fetuses and in neonatal rat offspring. METHODS: From day 4 of gestation until parturition, energy-restricted dams received either 75 or 50% of ad libitum food intake. Microarray analyses were performed on whole 13- and 17-day fetuses and 1-day-old pups. Protein and fat contents of the dams' milk were analyzed in the different feeding groups. RESULTS: A surprisingly small number of genes were differentially expressed between the groups, probably due to the strict control of fetal development. Interestingly, the expressions of many pancreatic digestion enzymes were reduced in the 1-day-old pups of the energy-restricted dams. A statistically significant difference in milk protein content was observed on day 1 post-partum between the gestationally food-restricted groups. CONCLUSIONS: The expressions of several genes that may have an important role in the normal development of organs were affected by undernutrition during fetal development. In addition, undernutrition may have affected the function of the exocrine pancreas.
Asunto(s)
Dieta Reductora , Feto/fisiología , Perfilación de la Expresión Génica , Complicaciones del Embarazo/genética , Animales , Animales Recién Nacidos/genética , Calcineurina/genética , Quimotripsinógeno/genética , Cristalinas/genética , Sistema Enzimático del Citocromo P-450/genética , Femenino , Desarrollo Fetal/genética , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , Leche/química , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa/métodos , Embarazo , Complicaciones del Embarazo/etiología , Ratas , Ratas Sprague-Dawley , Serina Endopeptidasas/genéticaRESUMEN
BACKGROUND & AIMS: Acinar cells constitute 90% of the pancreas epithelium, are polarized, and secrete digestive enzymes. These cells play a crucial role in pancreatitis and pancreatic cancer. However, there are limited models to study normal acinar cell differentiation in vitro. The aim of this work was to generate and characterize purified populations of pancreatic acinar cells from embryonic stem (ES) cells. METHODS: Reporter ES cells (Ela-pur) were generated that stably expressed both beta-galactosidase and puromycin resistance genes under the control of the elastase I promoter. Directed differentiation was achieved by incubation with conditioned media of cultured fetal pancreatic rudiments and adenoviral-mediated co-expression of p48/Ptf1a and Mist1, 2 basic helix-loop-helix transcription factors crucial for normal pancreatic acinar development and differentiation. RESULTS: Selected cells expressed multiple markers of acinar cells, including digestive enzymes and proteins of the secretory pathway, indicating activation of a coordinated differentiation program. The genes coding for digestive enzymes were not regulated as a single module, thus recapitulating what occurs during in vivo pancreatic development. The generated cells displayed transient agonist-induced Ca(2+) mobilization and showed a typical response to physiologic concentrations of secretagogues, including enzyme synthesis and secretion. Importantly, these effects did not imply the acquisition of a mixed acinar-ductal phenotype. CONCLUSIONS: These studies allow the generation of almost pure acinar-like cells from ES cells, providing a normal cell-based model for the study of the acinar differentiation program in vitro.
Asunto(s)
Células Madre Embrionarias/citología , Células Madre Embrionarias/fisiología , Páncreas Exocrino/citología , Páncreas Exocrino/embriología , Amilasas/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Carbacol/farmacología , Carboxipeptidasas A/genética , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/fisiología , División Celular/fisiología , Células Cultivadas , Agonistas Colinérgicos/farmacología , Quimotripsinógeno/genética , Células Madre Embrionarias/ultraestructura , Exocitosis/efectos de los fármacos , Exocitosis/fisiología , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Genes Reporteros , Ratones , Microscopía Inmunoelectrónica , Elastasa Pancreática/genética , Factores de Transcripción/genética , TransfecciónRESUMEN
Extensive genetic studies of chronic pancreatitis over the past decade have highlighted the importance of a tightly regulated balance between activation and inactivation of trypsin within the pancreas to disease susceptibility and resistance. The recent identification of chymotrypsin C (CTRC) as enzyme Y, which was proposed to protect the pancreas by degrading prematurely activated trypsinogen within the pancreas 20 years ago, made CTRC an excellent candidate gene for disease-association studies. Here, we analyzed all eight exons of the CTRC gene for conventional genetic variants and copy number variations (CNVs) by direct sequencing and quantitative fluorescent multiplex PCR, respectively, in a total of 287 French white patients (idiopathic x 216; familial x 42; hereditary x 29). While no CNVs were found in any of the 287 subjects, 20 conventional variations including a nonsense mutation (p.W55X), a microdeletion mutation (p.K247_R254del) and nine missense mutations were found in the 216 patients with idiopathic chronic pancreatitis (ICP). Except for two common polymorphisms, all the remaining 18 mutational events represent rare variations, with a minor allele frequency of 0-0.3% in the control population. All these rare variants were always found more frequently in the ICP patients than in the controls, and their combined frequency in the ICP patients (26/216; 12.0%) is significantly different from that in the controls (4/350; 1.1%) (OR = 11.8 [3.9-40.6]), chi (2) = 31.58, P < 10(-6)). This genetic finding, when considered in the perceived role of CTRC in eliminating prematurely activated trypsin, indicated that CTRC is a new pancreatitis susceptibility gene.
Asunto(s)
Quimotripsinógeno/genética , Pancreatitis/genética , Serina Endopeptidasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Enfermedad Crónica , Quimotripsinógeno/química , Cartilla de ADN , Predisposición Genética a la Enfermedad , Humanos , Datos de Secuencia Molecular , Mutación Missense , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/químicaRESUMEN
Two apparently full-length cDNA clones encoding chymotrypsinogens I and II (CHTRI, 1022 bp; CHTRII, 909 bp) and one cDNA clone encoding trypsinogen II (TRPII, 848 bp) were isolated from a cDNA library prepared from gilthead sea bream (Sparus aurata) liver. The deduced amino acid sequences of the isolated cDNAs contain highly conserved residues essential for serine protease catalytic activity and conformational maintenance. The deduced amino acid sequences of CHTRI and CHTRII are 261 aa and 277 aa long, respectively, and share only 61% identity. Sea bream CHTRII appears to be the longest of all known teleostean chymotrypsinogen forms and contains a high number of methionine residues. Compared with CHTRI, CHTRII is more hydrophobic and has a lower isoelectric point. On the other hand, deduced amino acid sequence of TRPII is 241 aa long and has a signal peptide of thirteen amino acid residues and an activation peptide of seven amino acids long. In contrast to CHTRI and CHTRII, TRPII has a low isoelectric point (4.95), which makes it anionic at neutral pH. Northern blot analysis revealed that liver is the major transcription site for all zymogens. As expected, all zymogen transcripts were detected in parts of the digestive tract (stomach, pyloric caeca, anterior and posterior intestine) and pyloric caeca presented the most intense expression. In all tissues and amongst all zymogens, TRPII constitutive expression was the highest.
Asunto(s)
Quimotripsinógeno/biosíntesis , Proteínas de Peces/biosíntesis , Regulación de la Expresión Génica/fisiología , Dorada/metabolismo , Tripsinógeno/biosíntesis , Secuencia de Aminoácidos , Animales , Quimotripsinógeno/química , Quimotripsinógeno/genética , Proteínas de Peces/química , Proteínas de Peces/genética , Datos de Secuencia Molecular , Especificidad de Órganos/fisiología , Filogenia , Dorada/genética , Análisis de Secuencia de ADN , Tripsinógeno/química , Tripsinógeno/genéticaRESUMEN
An ability to accurately simulate the dynamic behavior of concentrated macromolecular solutions would be of considerable utility in studies of a wide range of biological systems. With this goal in mind, a Brownian dynamics (BD) simulation method is reported here that allows systems to be modeled that comprise in excess of 1000 protein molecules, all of which are treated in atomic detail. Intermolecular forces are described in the method using an energy function that incorporates electrostatic and hydrophobic interactions and that is calibrated to reproduce experimental thermodynamic information with a single adjustable parameter. Using the method, BD simulations have been performed over a wide range of pH and ionic strengths for three proteins: hen egg white lysozyme (HEWL), chymotrypsinogen, and T4 lysozyme. The simulations reproduce experimental trends in second virial coefficients (B(22)) and translational diffusion coefficients, correctly capture changes in B(22) values due to single amino acid substitutions, and reveal a new explanation for the difficulties reported previously in the literature in reproducing B(22) values for protein solutions of very low ionic strength. In addition, a strong correlation is found between a residue's probability of being involved in a protein-protein contact in the simulations and its probability of being involved in an experimental crystal contact. Finally, exploratory simulations of HEWL indicate that the simulation model also gives a promising description of behavior at very high protein concentrations (approximately 250 g/L), suggesting that it may provide a suitable computational framework for modeling the complex behavior exhibited by macromolecules in cellular conditions.
Asunto(s)
Quimotripsinógeno/química , Muramidasa/química , Bacteriófago T4/enzimología , Quimotripsinógeno/genética , Simulación por Computador , Concentración de Iones de Hidrógeno , Cinética , Modelos Químicos , Modelos Moleculares , Muramidasa/genética , Mutación Puntual , Conformación Proteica , Sales (Química)/química , Soluciones , TermodinámicaRESUMEN
In some tissues, amino acids (AA) stimulate translation initiation via interactions between eukaryote initiation factor (eIF) 4E-binding protein 1 (4E-BP1), eIF4E and eIF4G. Dietary AA have been shown to induce pancreatic proteases independently of cholecystokinin in rats, the mechanism of which has not yet been clarified. In the present study, we examined the mechanism in rats for protease induction by dietary AA and determined the involvement of translation initiation. Male Wistar/ST rats were fed a 20 or 60% casein or AA mixture diet for 7 d and were intravenously injected with [35S] methionine (Met) 30 min before killing on d 7 (expt. 1). In expt. 2, rats were fed a 20 or 60% AA diet for 7 d and after food deprivation and refeeding with the respective diet on d 7 were killed at 0, 1 or 3 h. We measured mRNA and [35S] Met incorporation into chymotrypsinogen, phosphorylation status of 4E-BP1 and the association of eIF4E with 4E-BP1 or eIF4G. In expt. 1, chymotrypsin activity and synthesis were higher in both of the 60% diet groups than in the 20% diet groups, but the mRNA level and 4E-BP1 status did not differ. In expt. 2, chymotrypsin activity increased in the 60% AA diet group in a time-dependent manner. The translation initiation activity via the mTOR pathway indicated an increase similar to chymotrypsin activity. There were no differences in chymotrypsin mRNA level at any point. These results indicate that dietary AA induce chymotrypsin synthesis by promoting translation, and transient activation of translation initiation via mTOR may be associated with this induction.
Asunto(s)
Aminoácidos/administración & dosificación , Proteínas en la Dieta/administración & dosificación , Endopeptidasas/biosíntesis , Páncreas/enzimología , Biosíntesis de Proteínas/efectos de los fármacos , Amilasas/biosíntesis , Amilasas/genética , Amilasas/metabolismo , Animales , Carboxipeptidasas/biosíntesis , Carboxipeptidasas/metabolismo , Proteínas Portadoras/metabolismo , Caseínas/administración & dosificación , Quimotripsina/biosíntesis , Quimotripsina/genética , Quimotripsina/metabolismo , Quimotripsinógeno/biosíntesis , Quimotripsinógeno/genética , Quimotripsinógeno/metabolismo , Ingestión de Alimentos , Precursores Enzimáticos/biosíntesis , Precursores Enzimáticos/metabolismo , Factor 4E Eucariótico de Iniciación/metabolismo , Factor 4G Eucariótico de Iniciación/metabolismo , Alimentos , Privación de Alimentos , Péptidos y Proteínas de Señalización Intracelular , Lipasa/biosíntesis , Lipasa/metabolismo , Masculino , Elastasa Pancreática/biosíntesis , Elastasa Pancreática/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , ARN Mensajero/análisis , Ratas , Ratas Wistar , Aumento de PesoRESUMEN
The purification of human chymotrypsinogen B (hCTRB) after expression and secretion by the yeast Pichia pastoris is described based on two different approaches using integrated initial recovery. Extraction employing aqueous two-phase systems (ATPS) from poly(ethylene glycol) and sodium sulfate allows direct processing of cell containing yeast suspensions of 50% wet weight. The target protein is obtained partially purified in the top phase while cells and cell debris are partitioned to the bottom phase of the system. hCTRB is further purified by adsorption from the top phase to the cation exchanger SP Sepharose Big Beads and elution in a salt step. The single step isolation of hCTRB is possible by expanded bed adsorption (EBA) using a fluidized cation exchanger (Streamline SP XL). A design strategy is shown taking both target protein binding and stable fluidization of the stationary phase in cell containing suspensions into consideration. For the example of hCTRB isolation from cell containing P. pastoris suspensions, a successful use of this strategy is demonstrated. Both initial recovery strategies deliver a product that can be further purified and formulated by ultrafiltration/diafiltration followed by lyophilization, resulting in a homogeneous product. Scale-up to 30-90 L of culture suspension was shown for both methods, resulting in a product of similar quality. Comparing both strategies reveals that the two-step ATPS route is better suited for high cell density cultures, while the single step EBA method is preferred for cultures of moderate cell density. This is due to the fact that application of EBA is restricted to suspensions of 10-12.5% wet weight cell concentration, thus necessitating dilution of the original broth prior to sample application. The data presented show that integrated recovery operations are a valuable alternative to traditional processing for systems that are problematic during initial solid-liquid separation.
Asunto(s)
Quimotripsinógeno/aislamiento & purificación , Quimotripsinógeno/metabolismo , Microbiología Industrial/métodos , Pichia/metabolismo , Quimotripsinógeno/genética , Fermentación , Humanos , Microbiología Industrial/instrumentación , Pichia/genética , Proyectos Piloto , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Based on an integrated approach of genetic engineering, fermentation process development, and downstream processing, a fermentative chymotrypsinogen B production process using recombinant Pichia pastoris is presented. Making use of the P. pastoris AOX1-promotor, the demand for methanol as the single carbon source as well as an inducer of protein secretion enforced the use of an optimized feeding strategy by help of on-line analysis and an advanced controller algorithm. By using an experimental system of six parallel sparged column bioreactors, proteolytic product degradation could be minimized while also optimizing starting conditions for the following downstream processing. This optimization of process conditions resulted in the production of authentic chymotrypsinogen at a final concentration level of 480 mg.L(-)(1) in the whole broth and a biomass concentration of 150 g.L(-)(1) cell dry weight, thus comprising a space-time yield of 5.2 mg.L(-)(1).h(-)(1). Alternatively to the high cell density fermentation approach, a continuous fermentation process was developed to study the effects of reduced cell density toward oxygen demand, cooling energy, and biomass separation. This development led to a process with a highly increased space-time yield of 25 mg.L(-)(1).h(-)(1) while reducing the cell dry weight concentration from 150 g.L(-)(1) in fed-batch to 65 g.L(-)(1) in continuous cultivation.
Asunto(s)
Quimotripsinógeno/metabolismo , Microbiología Industrial/métodos , Pichia/metabolismo , Quimotripsinógeno/genética , Fermentación , Humanos , Metanol/metabolismo , Pichia/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
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
The cDNA encoding Atlantic cod (Gadus morhua) chymotrypsinogen B has been isolated and sequenced. Its deduced amino acid sequence consists of a 16-residue signal sequence and a mature polypeptide of 247 residues, being two residues longer than its vertebrate analogs. This mature polypeptide corresponds to a calculated molecular mass of 26.5 kDa and shares 70% sequence identity with cod chymotrypsinogen A. However, the identity between cod chymotrypsinogen B and its other vertebrate analogues is 63-66%. In common with most fish serine proteases, cod chymotrypsinogen B contains a high number of methionine residues. The presence of a threonine instead of a highly conserved serine residue at position 189 is a novel characteristic of this enzyme. Cod chymotrypsin B, as its type B vertebrate analogs, has an alanine at position 226, whereas a glycine is most commonly found at this position in the type A chymotrypsins.
Asunto(s)
Quimotripsinógeno/genética , Peces/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Biblioteca de Genes , Datos de Secuencia Molecular , Filogenia , Señales de Clasificación de Proteína/genética , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Advances in molecular genetics have provided the powerful tools necessary to identify the key molecules and mechanisms that underly the disease process. Continued work in this area promises to reveal new insights as new disease genes are discovered. This article focuses on the insights into the cause of acute and chronic pancreatitis gained by investigation of the HP genes, the diagnosis of the known mutations, the fascinating observation of nonpenetrance, and a look at future directions.
Asunto(s)
Predisposición Genética a la Enfermedad/genética , Pancreatitis/genética , Enfermedad Aguda , Secuencia de Aminoácidos , Enfermedad Crónica , Quimotripsinógeno/genética , Análisis Mutacional de ADN , Pruebas Genéticas , Humanos , Datos de Secuencia Molecular , Penetrancia , Tripsinógeno/genéticaRESUMEN
A cDNA encoding a chymotrypsinogen-like protein in midguts of the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae) was cloned and sequenced. The 901 bp cDNA contains an 816-nucleotide open reading frame encoding 272-amino acids. The predicted molecular mass and pI of the mature enzyme are 23.7 kDa and 4.64, respectively. The encoded protein includes amino acid sequence motifs that are conserved with 5 homologous chymotrypsinogen proteins from other insects. Features of the putative chymotrypsin-like protein from R. dominica include the serine proteinase active site (His(90), Asp(133), Ser(226)), conserved cysteine residues for disulfide bridges, the residues (Gly(220), Gly(243), Asp(252)) that determine chymotrypsin specificity, and both zymogen activation and signal peptides. A TPCK-sensitive caseinolytic protein (P6) with an estimated molecular mass of 24 kDa is present in midgut extracts of R. dominica and can be resolved by electrophoresis on 4-16% polyacrylamide gels. The molecular mass of this caseinolytic enzyme is similar to that of the chymotrypsin deduced from cDNA. Midgut extracts of R. dominica readily hydrolyzed azocasein and N-succinyl-alanine-alanine-proline-phenylalanine-p- nitroanilide (SAAPFpNA), a chymotrypsin-specific substrate. Properties of the enzymes responsible for these activities were partially characterized with respect to distribution in the gut, optimum pH, and sensitivity toward selected proteinase inhibitors. Optimal activity against both azocasein and SAAPFpNA occurs in a broad pH range from about 7 to 10. Both azocasein and SAAPFpNA activities, located primarily in the anterior midgut region, are inhibited by aprotinin, phenylmethyl sulphonylfluoride (PMSF), and soybean trypsin inhibitor (STI). TPCK (N-alpha-tosyl-L-phenylalanine chloromethyl ketone) and chymostatin inhibited more than 60% of SAAPFpNA but only about 10-20% of azocasein activity. These results provide additional evidence for the presence of serine proteinases, including chymotrypsin, in midguts of R. dominica. Arch. Insect Biochem. Physiol. 43:173-184, 2000.Published 2000 Wiley-Liss, Inc.
Asunto(s)
Quimotripsina/genética , Escarabajos/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Quimotripsina/antagonistas & inhibidores , Quimotripsina/metabolismo , Quimotripsinógeno/genética , Clonación Molecular , Escarabajos/genética , ADN Complementario , Sistema Digestivo/enzimología , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Inhibidores de Proteasas/farmacología , Homología de Secuencia de AminoácidoRESUMEN
A reliable protocol was designed for fast expression and purification of recombinant chymotrypsin(ogen). The zymogen was overexpressed in soluble form as a (His)6-fusion construct in the cytoplasm of the thioredoxin reductase deficient Escherichia coli strain AD494(DE3). This allowed purification of chymotrypsinogen in a highly selective affinity chromatography capture step using a Ni-NTA column. After activation with enterokinase, the enzymatically active chymotrypsin was purified in a polishing step using a modified soybean trypsin inhibitor agarose column. This expression system and the use of affinity chromatography for capture and polishing, offers an easier and faster route to recombinant chymotrypsin(ogen) than the previously described use of Saccharomyces cerevisiae.
Asunto(s)
Quimotripsina/genética , Quimotripsinógeno/genética , Escherichia coli/genética , Reductasa de Tiorredoxina-Disulfuro/genética , Secuencia de Bases , Quimotripsina/aislamiento & purificación , Quimotripsina/metabolismo , Quimotripsinógeno/aislamiento & purificación , Quimotripsinógeno/metabolismo , ADN , Electroforesis en Gel de Poliacrilamida , Vectores Genéticos , Histidina/genética , Cinética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
In order to determine which physiological functions can be regulated by the pancreatic CCKB/gastrin receptor, studies were carried out on pancreatic acini from mice expressing transgenic CCKB/gastrin receptors in the exocrine pancreas (ElasCCKB mice). Acini were stimulated by sulfated gastrin in the presence of SR 27897 (1.8 microM), blocking endogenous CCKA receptors. After 30 min incubation with gastrin, the secretion of chymotrypsinogen and amylase showed superimposable monophasic dose-response curves. Enzyme secretion was detectable and maximal at 100 pM and 1 nM of gastrin, respectively. No increase in chymotrypsinogen and amylase mRNAs was detected for doses of gastrin which specifically occupy the CCKB/gastrin receptor. In contrast, gastrin stimulated total protein synthesis in isolated acini from ElasCCKB mice. [35S]Methionine incorporation into total proteins was increased dose-dependently to a maximum for 30 pM gastrin and inhibited with higher doses (> 300 pM). Gastrin stimulated p70 S6 kinase activity for concentrations ranging from 10 pM to 1 nM. Gastrin-stimulated p70 S6 kinase activity and protein synthesis were blocked by rapamycin and wortmannin. Therefore, in ElasCCKB mice acinar cells, the CCKB/gastrin receptor mediates enzyme release and protein synthesis. However, a more efficient coupling of the CCKB/gastrin receptor to protein synthesis than to enzyme secretion was demonstrated. CCKB/gastrin receptor-stimulated protein synthesis likely results from an enhancement of mRNA translation and involves phosphatidyl inositol 3-kinase and p70 S6 kinase.
Asunto(s)
Páncreas/metabolismo , Biosíntesis de Proteínas , Receptores de Colecistoquinina/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Secuencia de Aminoácidos , Amilasas/genética , Amilasas/metabolismo , Androstadienos/farmacología , Animales , Quimotripsinógeno/genética , Quimotripsinógeno/metabolismo , Gastrinas/farmacología , Humanos , Técnicas In Vitro , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Páncreas/citología , Páncreas/efectos de los fármacos , Péptidos/genética , Fosfatidilinositol 3-Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Receptor de Colecistoquinina B , Receptores de Colecistoquinina/efectos de los fármacos , Receptores de Colecistoquinina/genética , Sirolimus/farmacología , WortmaninaRESUMEN
Hereditary pancreatitis is an unusual form of acute and chronic pancreatitis that is usually associated with two specific mutations in the cationic trypsinogen gene. The extensive information available on the biochemistry, cell biology, and molecular biology of cationic trypsinogen and its gene provides the groundwork for development of a variety of therapeutic strategies, including gene therapy. Several features of this disease, however, make gene therapy unlikely in the near future. Further research using new models, including transgenic animals, is required before breakthroughs in therapy can be expected.