RESUMEN
Immunoreactive Trypsinogen (IRT) is a protein-based pancreatic proenzyme that has an important role in protein digestion in humans. In human body, once IRT present in the small intestine, the proteolytic cleavage activates trypsinogen into trypsin. When IRT is in the active form, it is capable to cleave antibodies, other proteins and even itself while it is desired to use in immunoassays. According to the literature, there are three important IRT isoforms called Immunoreactive Trypsinogen 1 (IRT1), Immunoreactive Trypsinogen 2 (IRT2), and Immunoreactive Trypsinogen 3 (IRT3). However, trypsinogen 1 (cationic trypsinogen, IRT1) and trypsinogen 2 (anionic trypsinogen, IRT2) are the major isoforms in human pancreatic juice and used in the diagnosis of cystic fibrosis (CF). In this study, it is aimed to restrain its proteolytic activity with K23D mutation, which changes lysine (K) residue at the 23rd position to aspartic acid (D). Because we wanted to produce a hassle-free human recombinant immune reactive trypsinogen proenzyme which has similar antigenic properties with the native form. It is also aimed that the mutant IRTs do not exhibit proteolytic activity for the development of durable detection kits with a longer shelf life for both two isoforms. The innovation was actualized in order to use IRTs as a standard antigen in Immunoassays such as ELISA kits. The gene was synthesized as mutated and expressed in P. pastoris X-33 strain. The loss of proteolytic activity has been proven with the BAEE test. Antigenic properties of K23D IRTs and the effect of proteolytic inactivation on their performance in immunoassays were assessed with ELISA and Western Blot. In ELISA results K23D mutated IRTs showed higher signals than Wild-Type forms.
Asunto(s)
Tripsina/biosíntesis , Tripsinógeno/biosíntesis , Antígenos/biosíntesis , Western Blotting/métodos , Clonación Molecular/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Inmunoensayo/métodos , Mutación/genética , Pichia/genética , Pichia/metabolismo , Isoformas de Proteínas/genética , Proteínas Recombinantes/inmunología , Tripsina/genética , Tripsina/inmunología , Tripsinógeno/genética , Tripsinógeno/inmunologíaRESUMEN
Recently, we engineered a Pichia pastoris Mut(+) strain to produce and secrete recombinant Litopenaeus vannamei trypsinogen. Despite the observed toxicity of the recombinant shrimp trypsinogen to the P. pastoris cell host, when high density cell cultures in shake flasks with alanine in the induction medium were used recombinant shrimp trypsinogen could be produced. To further improve the product yield, in this work, we evaluated L. vannamei trypsinogen production in P. pastoris using a bioreactor and two recombinant P. pastoris strains with different methanol utilization (Mut) phenotypes. The effect of pH and temperature during the induction step on the trypsinogen production was also evaluated. The results indicate that temperature, pH, and Mut phenotypes influence the production of the recombinant protein, with almost no observed effect on cell growth. All cultures with the Mut(+) strain had significant operational difficulties, such as in lowering the induction temperature, maintaining dissolved oxygen (DO) above 20%, and maintaining the methanol concentration at a constant value, and showed a decrease in metabolic activity due to trypsinogen toxicity to the cell host. In the culture with the Mut(s) strain, however, the temperature, methanol concentration, and DO could be more easily controlled, the temperature could be easily decreased, and the trypsinogen caused the lowest toxicity to the host cells. After 96 h of Mut(s) strain induction (pH 6 and 25°C), about 250 mg/L recombinant trypsinogen was detected in the culture medium.
Asunto(s)
Reactores Biológicos/microbiología , Técnicas de Cultivo de Célula , Penaeidae/metabolismo , Pichia/metabolismo , Tripsinógeno/biosíntesis , Animales , Células Cultivadas , Metanol/química , Penaeidae/genética , Pichia/citología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
BACKGROUND: The tumour-associated trypsin inhibitor TATI is expressed together with trypsin in many cancer forms, and an elevated serum level associates with poor prognosis. TATI can reduce tissue destruction by inhibiting trypsin and other proteinases, and in some cancer forms, its high tissue expression is associated with favourable prognosis. We analyzed the prognostic values of TATI, trypsinogen-1 and trypsinogen-2 immunoexpression from tissue array blocks constructed from surgical specimens of 592 colorectal cancer patients. RESULTS: TATI positivity correlated negatively with differentiation (p < 0.001) and positively with the histological type of adenocarcinoma (p < 0.001). Trypsinogen-1 and trypsinogen-2 positivity correlated with Dukes' stage (p = 0.045, p = 0.050); the percentage of trypsinogen-1- and trypsinogen-2-positive tumours was lower in metastasized (Dukes' stage C-D) than in local (Dukes' stage A-B) disease. In addition, trypsinogen-2 correlated inversely with differentiation (p = 0.012). In univariate analysis, the expression of TATI associated with more favourable cancer-specific survival (p = 0.010). In multivariate analysis, low TATI (p = 0.044), age (p < 0.001), Dukes' stage (p < 0.001), tumour differentiation (p = 0.020) and location in the rectum (p = 0.006) were independent prognostic factors for adverse outcome. Furthermore, TATI expression was an independent prognostic factor in a subgroup of trypsinogen-1- (p = 0.007) and trypsinogen-2-positive (p = 0.006) tumours. CONCLUSION: TATI tissue expression is an independent prognostic marker in colorectal cancer.
Asunto(s)
Biomarcadores de Tumor/biosíntesis , Neoplasias Colorrectales/metabolismo , Inhibidor de Tripsina Pancreática de Kazal/biosíntesis , Tripsina/biosíntesis , Tripsinógeno/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Pronóstico , Análisis de Supervivencia , Adulto JovenRESUMEN
Pure methanol, which is required as an inducer of the AOX1 promoter and a carbon/energy source in processes for recombinant protein production by Pichia pastoris, is impracticable and therefore generally undesirable. As an alternative, a procedure using double carbon substrate was examined (11.7g(carbon)l(-1), 60%/40% carbon from glucose/methanol). The effects on methanol metabolism, extracellular formation of porcine trypsinogen, biomass growth and cell viability were analyzed. In contrast to batch cultures, where the glucose and methanol were utilized sequentially, in carbon/energy-limited continuous cultures (operated between dilution rates 0.03 and 0.20h(-1)) the repressive effect of glucose on methanol utilization was eliminated up to 0.15h(-1) (ca. 130% of µ(max) with methanol). With the mixture, the yield of biomass (1.54±0.12) g(CDW)g(carbon)(-1) was found to be 1.4 times larger than the yield with methanol alone. Despite the current widespread view that glucose has a repressive effect on the AOX1 promoter, the product was synthesized over the entire range of dilution rates, with maximum productivities of (0.70±0.12)mgg(CDW)(-1) h(-1) at 0.07h(-1). Thus, glucose was shown to be a feasible partial substitute for methanol in recombinant protein production by P. pastoris Mut(+) strain while enhancing process productivity.
Asunto(s)
Medios de Cultivo , Glucosa/metabolismo , Metanol/metabolismo , Pichia/metabolismo , Proteínas Recombinantes/biosíntesis , Tripsinógeno/biosíntesis , Animales , Biomasa , Reactores Biológicos , Supervivencia Celular , Cinética , Pichia/genética , PorcinosRESUMEN
BACKGROUND AND AIMS: Premature intra-acinar activation of trypsinogen is widely considered key for both the initiation of acute pancreatitis and the development of chronic pancreatitis. However, the biological consequences of intracellular trypsinogen activation have not been directly examined. To do so, a new mouse model was developed. METHODS: Mice were engineered to conditionally express an endogenously activated trypsinogen within pancreatic acinar cells (PACE-tryp(on)). Hallmarks of pancreatitis were determined and findings were correlated to the level (zygosity) and extent (temporal and spatial) of conditional PACE-tryp(on) expression. Furthermore, the impact of acinar cell death in PACE-tryp(on) mice was assessed and compared with a model of selective diphtheria toxin (DT)-mediated induction of acinar apoptosis. RESULTS: Initiation of acute pancreatitis was observed with high (homozygous), but not low (heterozygous) levels of PACE-tryp(on) expression. Subtotal (maximal-rapid induction) but not limited (gradual-repetitive induction) conditional PACE-tryp(on) expression was associated with systemic complications and mortality. Rapid caspase-3 activation and apoptosis with delayed necrosis was observed, and loss of acinar cells led to replacement with fatty tissue. Chronic inflammation or fibrosis did not develop. Selective depletion of pancreatic acinar cells by apoptosis using DT evoked similar consequences. CONCLUSIONS: Intra-acinar activation of trypsinogen is sufficient to initiate acute pancreatitis. However, the primary response to intracellular trypsin activity is rapid induction of acinar cell death via apoptosis which facilitates resolution of the acute inflammation rather than causing chronic pancreatitis. This novel model provides a powerful tool to improve our understanding of basic mechanisms occurring during pancreatitis.
Asunto(s)
Activación Enzimática/genética , Regulación de la Expresión Génica , Pancreatitis Aguda Necrotizante/genética , ARN Mensajero/genética , Tripsinógeno/genética , Animales , Líquido Intracelular/metabolismo , Ratones , Ratones Transgénicos , Pancreatitis Aguda Necrotizante/enzimología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vías Secretoras , Tripsinógeno/biosíntesisRESUMEN
A synthetic bovine trypsinogen (sbTrypsinogen) was synthesized on the basis of rice-optimized codon usage via an overlap PCR strategy, prior to being expressed under the control of the sucrose starvation-inducible rice α-amylase 3D (RAmy3D) promoter. Secretion of trypsin into the culture medium was achieved by using the existing signal peptide. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Dongjin), mediated by Agrobacterium tumefaciens. The integration of the sbTrypsinogen gene into the chromosome of the transgenic rice callus was verified via genomic DNA PCR amplification, and sbTrypsin expression in transgenic rice suspension cells was confirmed via Northern blot analysis. Western blot analysis detected glycosylated proteins in the culture medium, having masses from 24 to 26 kDa, following induction by sugar starvation. Proteolytic activity of the rice-derived trypsin was confirmed by gelatin zymogram, and was similar to that of the commercial bovine-produced trypsin. The yields of sbTrypsin that accumulated in the transgenic rice cell suspension medium were 15 mg/L at 5 days after sugar starvation.
Asunto(s)
Oryza , Plantas Modificadas Genéticamente , Proteínas Recombinantes/biosíntesis , Tripsina/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Células Cultivadas , Medios de Cultivo Condicionados , Vectores Genéticos , Datos de Secuencia Molecular , Tripsinógeno/biosíntesisRESUMEN
Heterologously expressed proteins in Escherichia coli may undergo unwanted N-terminal processing by methionine and proline aminopeptidases. To overcome this problem, we present a system where the gene of interest is cloned as a fusion to a self-splicing mini-intein. This fusion construct is expressed in an engineered E. coli strain from which the pepP gene coding for aminopeptidase P has been deleted. We describe a protocol using human cationic trypsinogen as an example to demonstrate that recombinant proteins produced in this expression system contain homogeneous, unprocessed N-termini.
Asunto(s)
Escherichia coli/metabolismo , Inteínas , Proteínas Recombinantes de Fusión/biosíntesis , Tripsinógeno/biosíntesis , Aminopeptidasas/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Eliminación de Gen , Humanos , Estructura Terciaria de Proteína , Tripsinógeno/genéticaRESUMEN
Shrimp (Litopenaeus vannamei) trypsinogen has never been isolated from its natural source. To assess the production of L. vannamei trypsinogen, we engineered Pichia pastoris strains and evaluated two culture approaches with three induction culture media, to produce recombinant shrimp trypsinogen for the first time. The trypsinogen II cDNA was fused to the signal sequence of the Saccharomyces cerevisiae alpha mating factor, placed under the control of the P. pastoris AOX1 promoter, and integrated into the genome of P. pastoris host strain GS115. Using standard culture conditions for heterologous gene induction of a GS115 strain in shake flasks, recombinant shrimp trypsinogen was not detected by SDS-PAGE and Western blot analysis. Growth kinetics revealed a toxicity of recombinant shrimp trypsinogen or its activated form over the cell host. Thus, a different culture approach was tested for the induction step, involving the use of high cell density cultures, a higher frequency of methanol feeding (every 12 h), and a buffered minimal methanol medium supplemented with sorbitol or alanine; alanine supplemented medium was found to be more efficient. After 96 h of induction with alanine supplemented medium, a 29-kDa band from the cell-free culture medium was clearly observed by SDS-PAGE, and confirmed by Western blot to be shrimp trypsinogen, at a concentration of 14 microg/mL. Our results demonstrate that high density cell cultures with alanine in the induction medium allow the production of recombinant shrimp trypsinogen using the P. pastoris expression system, because of improved cell viability and greater stability of the recombinant trypsinogen.
Asunto(s)
Penaeidae/genética , Pichia/genética , Proteínas Recombinantes/biosíntesis , Tripsinógeno/biosíntesis , Análisis de Varianza , Animales , Clonación Molecular , Cinética , Metanol/metabolismo , Penaeidae/enzimología , Pichia/crecimiento & desarrollo , Pichia/ultraestructura , Proteínas Recombinantes/genética , Tripsinógeno/genéticaRESUMEN
It has been demonstrated that adipose-derived stem cells (ADSCs) secrete cytokines and exhibit diverse pharmacological actions. The present study examined the unknown pharmacological action of ADSCs regarding whitening effects. A conditioned medium of ADSCs (ADSC-CM) was harvested and the whitening effect of ADSC-CM was studied in melanoma B16 cells. ADSC-CM treatment inhibited the synthesis of melanin and the activity of tyrosinase in a dose dependent manner. To clarify the underlying mechanisms of the whitening action of ADSCs, protein levels of melanogenic proteins were measured by Western blot. Although expressions of microphthalmia-associated transcription factor and tyrosinase-related protein 2 (TRP2) remained unchanged, those of tyrosinase and TRP1 were down-regulated. Transforming growth factor-beta1 (TGF-beta 1), a potent regulator of melanogenic proteins, was neutralized by the addition of a blocking antibody to ADSC-CM, and down-regulated expression of tyrosinase and TRP1 was almost reversed. Collectively, these results indicate that secretary factors of ADSC inhibit melanin synthesis by down-regulating the expression of tyrosinase and TRP1, which are mainly mediated by TGF-beta1.
Asunto(s)
Melaninas/biosíntesis , Células Madre/fisiología , Factor de Crecimiento Transformador beta1/fisiología , Western Blotting , Supervivencia Celular , Células Cultivadas , Color , Humanos , Melanoma Experimental/metabolismo , Monofenol Monooxigenasa/biosíntesis , Monofenol Monooxigenasa/metabolismo , Pruebas de Neutralización , Proteómica , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tripsina , Tripsinógeno/biosíntesisRESUMEN
Although it is accepted that regulatory T cells (T regs) contribute to cancer progression, most studies in the field consider nonantigen-specific suppression. Here, we show the presence of tumor antigen-specific CD4(+) T regs in the blood of patients with metastatic melanoma. These CD4(+) T regs recognize a broad range of tumor antigens, including gp100 and TRP1 (melanoma tissue differentiation antigens), NY-ESO-1 (cancer/testis antigen) and survivin (inhibitor of apoptosis protein (IAP) family antigen). These tumor antigen-specific T regs proliferate in peripheral blood mononuclear cells (PBMC) cultures in response to specific 15-mer peptides, produce preferentially IL-10 and express high levels of FoxP3. They suppress autologous CD4(+)CD25(-) T cell responses in a cell contact-dependent manner and thus share properties of both naturally occurring regulatory T cells and type 1 regulatory T cells. Such tumor antigen-specific T regs were not detected in healthy individuals. These tumor antigen-specific T regs might thus represent another target for immunotherapy of metastatic melanoma.
Asunto(s)
Antígenos de Neoplasias/sangre , Melanoma/sangre , Melanoma/patología , Linfocitos T Reguladores/metabolismo , Adulto , Anciano , Antígenos de Neoplasias/biosíntesis , Linfocitos T CD4-Positivos/metabolismo , Femenino , Factores de Transcripción Forkhead/metabolismo , Humanos , Proteínas Inhibidoras de la Apoptosis , Interleucina-10/metabolismo , Subunidad alfa del Receptor de Interleucina-2/biosíntesis , Leucocitos Mononucleares/metabolismo , Masculino , Glicoproteínas de Membrana/biosíntesis , Proteínas de la Membrana/biosíntesis , Proteínas Asociadas a Microtúbulos/metabolismo , Persona de Mediana Edad , Metástasis de la Neoplasia , Proteínas de Neoplasias/metabolismo , Survivin , Tripsina , Tripsinógeno/biosíntesis , Tripsinógeno/metabolismo , Antígeno gp100 del MelanomaRESUMEN
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
Tumor-associated trypsin inhibitor (TATI) is a marker of mucinous ovarian carcinoma, but it is also widely expressed in other malignant tumors and normal human tissues. Elevated serum concentrations of TATI are of prognostic value in ovarian, kidney, and bladder cancer. Tumor-associated trypsin is co-expressed with TATI in many malignancies and is thought to be involved in tumor invasion. TATI mRNA has been shown to be overexpressed in bladder cancer. We therefore studied whether trypsinogen expression also can be detected in bladder cancer and how this and TATI expression are associated with the clinicopathological characteristics of the tumors. We used RT-PCR, in situ hybridization and immunohistochemistry to detect trypsinogen- and TATI mRNA and protein in tissue samples from 28 bladder cancer patients and ten benign urothelia. TATI expression was detected in all benign tissues and non-invasive tumors. However, the expression was lower in the muscle-invasive tumors (pT2; n=5), whereas trypsinogen expression was seen in all but one non-invasive tumor. We conclude that trypsinogen is expressed in both malignant and benign bladder epithelium, whereas TATI expression decreases with increasing stage and grade of the tumor. This may suggest that a balanced expression of TATI and trypsinogen is required in normal tissue and that this balance is disrupted during tumor progression.
Asunto(s)
Inhibidor de Tripsina Pancreática de Kazal/biosíntesis , Tripsinógeno/biosíntesis , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patología , Adulto , Anciano , Anciano de 80 o más Años , Transformación Celular Neoplásica , Progresión de la Enfermedad , Epitelio , Femenino , Humanos , Inmunohistoquímica , Hibridación in Situ , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We have found that dietary protein markedly induced pancreatic serine protease activity via a mechanism independent of luminal trypsin activity in pancreaticobiliary-diverted (PBD) rats. The aim of this study was to examine the effects of dietary protein on the synthesis of trypsinogen isoforms by comparing in vivo incorporation of [35S] L-methionine into isoform proteins in PBD and sham-operated rats. A small duodenal segment including the ampulla of Vater was sectioned and transposed to the upper ileum with end-to-side anastomosis (PBD) or duodenal transection was followed by reanastomosis (sham) in male Sprague-Dawley rats. After recovery, PBD and sham rats were fed a 25% or 60% casein-sucrose-based diet (NC or HC) for 14 days. Rats were then intravenously injected with [35S] L-methionine (15 MBq/kg body weight) 30 mins before being sacrificed for analysis of pancreatic enzymes by two-dimensional SDS-polyacrylamide gel electrophoresis. By using electrophoresis with narrow range of isoelectric focusing (pI 4.5-5.5), five trypsinogen 2 (2-x) isoform spots were identified using both [35S] incorporation and Coomassie brilliant blue (CBB) staining in PBD rats, but not in sham rats. N-terminal sequences of these trypsinogen 2-x spots were identical to known rat trypsinogen 2 with the exception that the third valine was changed to isoleucine in one isoform. In PBD rats, feeding of HC specifically increased the [35S] and CBB intensities of these trypsinogen 2-x isoforms and trypsinogen 3. The degree of induction of the five trypsinogen 2-x molecules by HC varied greatly. Trypsinogen 1 and 4, which are the major trypsinogens in normal rats, showed no changes. We conclude that increases in synthesis of a few newly identified trypsinogen 2-x isoforms mainly contribute to the induction of trypsin activity in the pancreas by HC in PBD rats.
Asunto(s)
Proteínas en la Dieta/farmacología , Páncreas/enzimología , Tripsinógeno/biosíntesis , Anastomosis Quirúrgica , Animales , Bilis/enzimología , Caseínas , Duodeno/cirugía , Electroforesis en Gel Bidimensional , Íleon/cirugía , Isoenzimas/biosíntesis , Isoenzimas/aislamiento & purificación , Masculino , Jugo Pancreático/enzimología , Ratas , Ratas Sprague-Dawley , Tripsina , Tripsinógeno/aislamiento & purificaciónRESUMEN
Distant metastasis is the predominant cause of death in early-stage non-small cell lung cancer (NSCLC). Currently, it is impossible to predict the occurrence of metastasis at early stages and thereby separate patients who could be cured by surgical resection alone from patients who would benefit from additional chemotherapy. In this study, we applied a comparative microarray approach to identify gene expression differences between early-stage NSCLC patients whose cancer ultimately did or did not metastasize during the course of their disease. Transcriptional profiling of 82 microarrays from two patient groups revealed differential expression of several gene families including known predictors of metastasis (e.g., matrix metalloproteinases). In addition, we found S100P, S100A2, trypsinogen C (TRY6), and trypsinogen IVb (PRSS3) to be overexpressed in tumors that metastasized during the course of the disease. In a third group of 42 patients, we confirmed the induction of S100 proteins and trypsinogens in metastasizing tumors and its significant correlation with survival by real-time quantitative reverse transcription-PCR. Overexpression of S100A2, S100P, or PRSS3 in NSCLC cell cultures led to increased transendothelial migration, corroborating the role of S100A2, S100P, and PRSS3 in the metastatic process. Taken together, we provide evidence that expression of S100 proteins and trypsinogens is associated with metastasis and predicts survival in early stages of NSCLC. For the first time, this implicates a role of S100 proteins and trypsinogens in the metastatic process of early-stage NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Proteínas S100/genética , Tripsinógeno/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/metabolismo , Metástasis de la Neoplasia , Análisis de Secuencia por Matrices de Oligonucleótidos , Valor Predictivo de las Pruebas , Proteínas S100/biosíntesis , Proteínas S100/farmacología , Tripsinógeno/biosíntesis , Tripsinógeno/farmacologíaRESUMEN
PURPOSE: The purpose is to study the prognostic significance of tissue expression of trypsinogen-1, trypsinogen-2, and tumor-associated trypsin inhibitor (TATI) and serum concentration of trypsinogen-2, trypsin-2-API (complex of trypsin-2 with alpha-1-proteinase inhibitor), and TATI in epithelial ovarian cancer. EXPERIMENTAL DESIGN: Expression of trypsinogen-1, trypsinogen-2, and TATI was determined by immunohistochemistry with monoclonal antibodies in tissue sections of tumors from 119 patients with untreated primary epithelial ovarian cancer. Preoperative serum concentrations of trypsinogen-2, trypsin-2-API and TATI were analyzed using specific immunoassays. RESULTS: Fifty-four percent of the tumors expressed trypsinogen-1, 45% expressed trypsinogen-2, and 30% expressed TATI. In patients with stage III and IV disease, TATI tissue expression (P = 0.002) and elevated TATI concentration in serum (P = 0.048) were associated with adverse cancer-specific and progression-free survival in univariate analysis. In multivariate analysis, TATI tissue expression (P = 0.005), tumor grade (P = 0.0001), histological type (P = 0.02), and stage (P = 0.0005) were independent prognostic factors for adverse cancer-specific survival and TATI tissue expression (P = 0.006) and grade (P = 0.0003) for progression-free survival. In multivariate analysis of all patients and those with advanced disease, serum trypsin-2-API concentration was an adverse prognostic factor for cancer-specific and progression-free survival, and it was independent of stage and histological type of the tumor (P Asunto(s)
Neoplasias Ováricas/patología
, Inhibidores de Tripsina/biosíntesis
, Tripsina/biosíntesis
, Tripsinógeno/biosíntesis
, Adulto
, Anciano
, Anciano de 80 o más Años
, Femenino
, Humanos
, Inmunoensayo
, Inmunohistoquímica
, Persona de Mediana Edad
, Análisis Multivariante
, Estadificación de Neoplasias
, Neoplasias Ováricas/genética
, Neoplasias Ováricas/metabolismo
, Pronóstico
, ARN Neoplásico/genética
, ARN Neoplásico/metabolismo
, Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
, Análisis de Supervivencia
, Tripsina/sangre
, Tripsina/genética
, Inhibidores de Tripsina/sangre
, Inhibidores de Tripsina/genética
, Tripsinógeno/sangre
, Tripsinógeno/genética
RESUMEN
Severe impairment of exocrine pancreatic secretion has recently been demonstrated in a clinical study in sepsis and septic shock patients. The purpose of this study was to further evaluate involvement of the pancreas in the acute phase reaction in sepsis. Using a normotensive rat model of Pseudomonas pneumonia-induced sepsis, we assessed the expression of PAP-I, amylase and trypsinogen mRNA, PAPI protein levels, and cytokine expression in the pancreas by Northern and Western blot analysis and RT-M PCR, respectively. Presence of several well-established features of pancreatitis in sepsis-induced animals were examined by biochemical and histopathological methods as well as by a determination of both water and myeloperoxidase content. Sepsis resulted in an up-regulation of PAP-I gene expression and increase in its protein level in pancreas while the mRNA levels of amylase and trypsinogen were down-regulated. Differences in the pancreatic cytokine expression, serum amylase and serum lipase levels, the occurrence of pancreatic edema as well as the severity of inflammatory infiltration and necrosis were not significantly different between sham and pneumonia groups. Acinar cells showed increased vacuolization in pneumonia animals 24 hours after the treatment. These findings demonstrate that the pancreas is actively involved in the acute phase reaction in sepsis of remote origin. This involvement occurs without concomitant biochemical and histopathologic alterations observed in pancreatitis. Taken all together, these features are indicative of a sepsis-specific dysfunction of the pancreas.
Asunto(s)
Reacción de Fase Aguda , Antígenos de Neoplasias/biosíntesis , Biomarcadores de Tumor/biosíntesis , Regulación de la Expresión Génica , Lectinas Tipo C/biosíntesis , Páncreas/metabolismo , Pancreatitis/etiología , Neumonía Bacteriana/complicaciones , Infecciones por Pseudomonas/metabolismo , Sepsis/metabolismo , Reacción de Fase Aguda/genética , Amilasas/biosíntesis , Amilasas/sangre , Amilasas/genética , Animales , Antígenos de Neoplasias/genética , Biomarcadores de Tumor/genética , Citocinas/biosíntesis , Citocinas/genética , Lectinas Tipo C/genética , Recuento de Leucocitos , Lipasa/sangre , Masculino , Necrosis , Páncreas/patología , Proteínas Asociadas a Pancreatitis , Peroxidasa/análisis , Infecciones por Pseudomonas/complicaciones , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Sepsis/etiología , Factores de Tiempo , Tripsinógeno/biosíntesis , Tripsinógeno/genética , Vacuolas/ultraestructuraRESUMEN
The expression of recombinant trypsinogens from different mammalian origins in Escherichia coli typically leads to the formation of insoluble aggregates. This work describes the high level expression of human trypsinogen 1 in E. coli using the T7 expression system. Direct expression of trypsinogen was not possible, but the N-terminal fusion of the first 11 amino acids of the T7 protein 10 resulted in an expression level of 200 mg g(-1) bacterial dry mass. A refolding procedure was optimized, and a method using continuous feed of denatured product was developed. Thus the working concentration of trypsinogen could be raised four-fold, while the yield of active protein could be maintained at 20-35%. The refolded trypsinogen was converted to trypsin by autocatalytic activation, and the utility for the detachment of mammalian cells in culture was proven.
Asunto(s)
Escherichia coli/genética , Proteínas Recombinantes de Fusión/biosíntesis , Tripsinógeno/biosíntesis , Animales , Secuencia de Bases , Células CHO , Proteínas de la Cápside/genética , Clonación Molecular , Cricetinae , Cricetulus , Escherichia coli/metabolismo , Vectores Genéticos/genética , Cuerpos de Inclusión/metabolismo , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Desnaturalización Proteica , Pliegue de Proteína , Renaturación de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Tripsina/biosíntesis , Tripsina/genética , Tripsina/aislamiento & purificación , Tripsinógeno/química , Tripsinógeno/genéticaRESUMEN
A double mutant of rat trypsinogen (Asp189Ser, DeltaAsp223) was constructed by site-directed mutagenesis. The recombinant protein was produced in Escherichia coli under the control of a periplasmic expression vector. The purified and enterokinase-activated enzyme was characterized by synthetic fluorogenic tetrapeptide and natural polypeptide substrates and by a recently developed method. In case of this latter method the specificity profile of the enzyme was examined by simultaneous digestion of a mixture of oligopeptide substrates each differing only at the P(1) site residue, and the results were analyzed by high-performance liquid chromatography. All these assays unanimously demonstrated that the recombinant proteinase lacks trypsin-like activity but acquired a rather unique selectivity: it preferentially hydrolyses peptide bonds C-terminal to tyrosyl residues. This narrow specificity should be useful in peptide-analytical applications such as sequence-specific fragmentation of large proteins prior to sequencing.
Asunto(s)
Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Tripsina/química , Tripsina/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Escherichia coli/genética , Escherichia coli/metabolismo , Vectores Genéticos/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/biosíntesis , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Tripsina/metabolismo , Tripsinógeno/biosíntesis , Tripsinógeno/química , Tripsinógeno/genéticaRESUMEN
The human pancreas secretes two major trypsinogen isoforms, cationic and anionic trypsinogen. To date, 19 genetic variants have been identified in the cationic trypsinogen gene (PRSS1) of patients with hereditary, familial, or sporadic chronic pancreatitis. A common feature of cationic trypsinogen mutants studied so far is an increased propensity for autocatalytic activation (autoactivation). This is thought to lead to premature intrapancreatic digestive protease activation. In contrast, no pancreatitis-associated mutations have been found in the anionic trypsinogen gene (PRSS2), suggesting that this isoform might play a relatively unimportant role in pancreatitis. To challenge this notion, here we describe the unique properties of the E79K cationic trypsinogen mutation (c.235G>A), which was identified in three European families affected by sporadic or familial pancreatitis cases. In vitro analysis of recombinant wild-type and mutant enzymes revealed that catalytic activity of E79K trypsin was normal, and its inhibition by pancreatic secretory trypsin inhibitor was unaffected. Although the E79K mutation introduces a potential new tryptic cleavage site, autocatalytic degradation (autolysis) of E79K-trypsin was also unchanged. Furthermore, in contrast to previously characterized disease-causing mutations, E79K markedly inhibited autoactivation of cationic trypsinogen. Remarkably, however, E79K trypsin activated anionic trypsinogen two-fold better than wild-type cationic trypsin did, while the common pancreatitis-associated mutants R122H or N29I had no such effect. The observations not only suggest a novel mechanism of action for pancreatitis-associated trypsinogen mutations, but also highlight the importance of interactions between the two major trypsinogen isoforms in the development of genetically determined chronic pancreatitis.
Asunto(s)
Mutación , Pancreatitis/genética , Tripsinógeno/genética , Adulto , Anciano , Proteínas Portadoras , Catálisis , Catepsinas/metabolismo , Enfermedad Crónica , Análisis Mutacional de ADN , Activación Enzimática , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Isoenzimas/biosíntesis , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Masculino , Persona de Mediana Edad , Pancreatitis/enzimología , Linaje , ARN Mensajero/biosíntesis , Tripsina/metabolismo , Inhibidor de Tripsina Pancreática de Kazal , Tripsinógeno/biosíntesis , Tripsinógeno/metabolismoRESUMEN
Trypsin is a serine protease family member with a potential role in cancer invasion. We investigated trypsinogen expression at the RNA level in 49 esophageal squamous cell carcinomas (ESCCs) and 72 gastric adenocarcinomas. Almost all primary ESCC tissues (95%) showed reduced expression, and 9 of 13 ESCC cell lines were silenced for trypsinogen expression. Absent expression correlated with promoter hypermethylation of trypsinogen-4 by bisulfite DNA sequence. Moreover, we detected promoter hypermethylation in 50% of primary ESCCs by methylation-specific PCR. A subset of gastric adenocarcinomas (71%) also showed reduced trypsinogen accompanied by reduction in PAR2, a G protein activated by trypsin, and a propensity to penetrate beyond the gastric wall (P = 0.001). Our results support the notion that trypsin plays a tumor-suppressive role in human carcinoma.