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PubMed2006

Expression of human cationic trypsinogen with an authentic N terminus using intein-mediated splicing in aminopeptidase P deficient Escherichia coli.

High-level expression of human trypsinogens as inclusion bodies in Escherichia coli requires deletion of the secretory signal sequence and placement of an initiator methionine at the N terminus. Trypsinogen preparations obtained this way contain a mixture of abnormal N termini, as a result of processing by cytoplasmic aminopeptidases. Here, we describe an expression system that produces recombinant human cationic trypsinogen with a native, intact N terminus, using intein-mediated protein splicing and an aminopeptidase P (pepP) deficient E. coli strain. As a first application of this system, the effect of the pancreatitis-associated mutation A16V on the autoactivation of human cationic trypsinogen was characterized. The use of the novel pepP knock-out E. coli strain should be generally applicable to the expression of recombinant proteins, which undergo unwanted N-terminal trimming by aminopeptidase P.

Aminopeptidases↗

PubMed1996

Urinary trypsinogen-2 test strip for acute pancreatitis.

BACKGROUND: A simple, rapid test is specific and sensitive enough to distinguish, in patients with clinically suspected acute pancreatitis, those whose abdominal pain is indeed of pancreatic origin has proved elusive. METHODS: In two consecutive series of surgical patients in a teaching hospital, whose acute abdominal pain turned out to be due to acute pancreatitis (n-57) or extrapancreatic in origin (n=40), we studied urinary trypsinogen-2 in two ways. A test strip, incorporating monoclonal antibodies to two epitopes on trypsinogen-2, recorded a blue line when concentrations exceeded 50 microgram/L; we also measured trypsinogen-2 concentrations in the laboratory. FINDINGS: In the patients with acute pancreatitis the test strip was positive in 52 and negative in five, whereas in the 40 extrapancreatic controls there were four false positives. In a further set of 57 orthopaedic controls, one urine was strip-test positive. Concentrations of urinary trypsinogen-2 and the test-strip results were in good agreement and in only three of the 154 patients were the two approaches discrepant, at the 50 microgram/L cut-off. INTERPRETATION: These findings, in patients whose acute abdominal pain was known to be pancreatic in origin or not, are encouraging but need to be confirmed in a consecutive series of patients in whom the diagnosis of pancreatitis is in doubt.

Abdominal Pain↗

PubMed2000

Cerebral trypsinogen expression in human and rat cerebrospinal fluid.

Trypsinogen was identified in cerebrospinal fluid (CSF), where it has not previously been reported and its activation state in experimental subarachnoid haemorrhage (SAH) in rats and in neurosurgical patients was determined. Trypsinogen activation peptide (TAP) release provided an equimolar marker for trypsinogen. Total TAP was significantly reduced to 26% of the baseline level (P<0.02) following experimental SAH in 15 rats but not in ten sham operated controls (P=0.3). TAP was also measured in patients with ruptured (n=11) and unruptured (n=9) aneurysms who underwent craniotomy to clip an aneurysm. Postoperatively there was a significant fall in TAP concentration (P<0.005) in both groups. Trypsinogen, as identified by CSF levels of TAP, is activated by SAH in rats and by craniotomy for aneurysmal clipping in patients.

Animals↗

PubMed2001

Activation peptide of carboxypeptidase B and anionic trypsinogen as early predictors of the severity of acute pancreatitis.

BACKGROUND: Early prediction of severity is important in the management of patients with acute pancreatitis. The presence of activation peptides and certain pancreatic proenzymes in plasma and urine has been shown to correlate with severity. This study was designed to assess the value of measuring levels of the activation peptide of carboxypeptidase B (CAPAP) and of anionic trypsinogen. METHODS: Concentrations of CAPAP and anionic trypsinogen were measured in the urine and serum in 60 patients with acute pancreatitis. Preset cut-off levels were used to analyse the accuracy of the tests. Severity was classified retrospectively according to the Atlanta classification. RESULTS: Concentrations of CAPAP in urine and serum and of anionic trypsinogen in urine correlated with the severity of the pancreatitis. CAPAP in urine showed the highest accuracy. The overall accuracy was 90 per cent, with a positive predictive value of 69 per cent and a negative predictive value of 98 per cent. CONCLUSION: In this study, measurement of CAPAP in urine was an accurate way to predict the severity of acute pancreatitis, and was superior to assay of anionic trypsinogen in urine and serum. Measurement of CAPAP in urine may be of value in the management of individual patients with pancreatitis and in the selection of patients for therapeutic trials.

Acute Disease↗

PubMed2002

Thermal stress-induced HSP70 mediates protection against intrapancreatic trypsinogen activation and acute pancreatitis in rats.

BACKGROUND & AIMS: Prior thermal stress induces heat shock protein 70 (HSP70) expression in the pancreas and protects against secretagogue-induced pancreatitis, but it is not clear that this thermal stress-induced protection is actually mediated by HSP70 since thermal stress may have other, non-HSP related, effects. METHODS: In the present study, we have administered antisense (AS) oligonucleotides, which prevent pancreatic expression of HSP70 to rats, in vivo, to evaluate this issue. In a separate series of experiments, designed to examine the role of pancreatitis-induced HSP70 expression in modulating the severity of pancreatitis, rats not subjected to prior thermal stress were given AS-HSP70 before cerulein administration, and trypsinogen activation as well as the severity of pancreatitis were evaluated. RESULTS: Hyperthermia induced HSP70 expression, prevented intrapancreatic trypsinogen activation, and protected against cerulein-induced pancreatitis. Administration of AS-HSP70 but not sense-HSP70 reduced the thermal stress-induced HSP70 expression, restored the ability of supramaximal cerulein stimulation to cause intrapancreatic trypsinogen activation, and abolished the protective effect of prior thermal stress against pancreatitis. In non-thermally stressed animals, pretreatment with AS-HSP70 before the induction of pancreatitis exacerbated all the parameters associated with pancreatitis. CONCLUSIONS: These findings lead us to conclude that HSP70 induction, rather than some other thermal stress-related phenomenon, mediates the thermal stress-induced protection against pancreatitis and that it protects against pancreatitis by preventing intrapancreatic activation of trypsinogen. The worsening of pancreatitis, which occurs when non-thermally stressed animals are given AS-HSP70 before cerulein, suggests that cerulein-induced HSP70 expression in nontreated animals acts to limit the severity of pancreatitis.

Acute Disease↗

PubMed2001

The levels of trypsinogen isoenzymes in ovarian tumour cyst fluids are associated with promatrix metalloproteinase-9 but not promatrix metalloproteinase-2 activation.

Proteolysis mediated by matrix metalloproteinases (MMPs) and serine proteinases is associated with cancer invasion and metastasis. Activation of latent proMMPs, and especially the proforms of the type IV collagen degrading gelatinases A and B (proMMP-2 and proMMP-9), is thought to be a critical step in this process. We have recently found that human tumour-associated trypsin-2 is a potent activator of proMMP-9 and it also activates proMMP-2 in vitro. Trypsinogen, MMP-2, and MMP-9 are expressed in ovarian cancer. To elucidate the function of trypsin in vivo, we studied whether high concentrations of trypsinogen-1, trypsinogen-2, their alpha(1)-proteinase inhibitor (API) complexes, and tumour-associated trypsin inhibitor (TATI) are associated with proMMP-2 and proMMP-9 activation in ovarian tumour cyst fluids. Zymography and immunofluorometric analysis of 61 cyst fluids showed a significant association between high trypsin concentrations and the activation of MMP-9 (P = 0.003-0.05). In contrast, the trypsin concentrations were inversely associated with the activation of MMP-2 (P = 0.01-0.02). Immunohistochemical analysis of ovarian tumour tissue demonstrated expression of trypsinogen-2 and TATI in the secretory epithelium. MMP-2 was detected both in stromal and epithelial cells whereas MMP-9 was detected in neutrophils and macrophage-like cells in stromal and epithelial areas. These results suggest that trypsin may play a role in the regulation of the MMP-dependent proteolysis associated with invasion and metastasis of ovarian cancer.

Adolescent↗

PubMed1972

Inactivation of bovine trypsinogen and chymotrypsinogen by diisopropylphosphorofluoridate.

Diisopropylphosphorofluoridate reacts with trypsinogen and chymotrypsinogen and inhibits the potential activity of both zymogens. The reactions follow pseudo first-order kinetics and proceed approximately four orders of magnitude slower than diisopropylphosphorylation of the corresponding enzymes. Correlation of initial rates of inactivation with incorporation of the reagent indicates that zymogen inactivation results from incorporation of 1 mol of organic phosphate per mol of protein. Peptides isolated from the active-site region of trypsinogen account for more than 60% of the label originally present in the [(14)C]diisopropylphosphoryl zymogen. It is concluded that loss of activation of trypsinogen is due to alkylphosphorylation of Ser(183). It is proposed that reduced reactivity of the zymogen, as compared to the enzyme, primarily reflects inefficient binding of substrates and inhibitors, and that Ser(183) of the active site exists in trypsinogen in an activated state.

Animals↗

PubMed2006

Biochemistry and clinical role of trypsinogens and pancreatic secretory trypsin inhibitor.

Trypsinogens and PSTI/TATI/SPINK1 are expressed, usually together, at high levels by the pancreas but also by many other normal and malignant tissues. The present review describes studies on the expression and putative functions of trypsinogens and PSTI/TATI/SPINK1 in the human body. The clinical aspects are discussed, including the correlations between expression of trypsinogens and PSTI/TATI/SPINK1 in tissues, serum, and urine of patients with pancreatitis or cancer and clinicopathological characteristics, i.e., the roles of trypsinogens and PSTI/TATI/SPINK1 in spontaneous and hereditary pancreatitis, tumor progression, and prognosis.

Biomarkers, Tumor↗

PubMedDate not supplied

Gender difference in the pancreatic trypsinogen response to ethanol withdrawal in rat pups.

This study was designed to examine the effects of ethanol withdrawal on offspring rats that consumed ethanol during gestation and lactation, in order to examine whether there was an improvement in pancreatic trypsinogen and lipase activities at 2 months postpartum with respect to offspring that fed on ethanol until death. A second purpose for our study was to determine if a folic acid supplement during gestation and lactation was sufficient or insufficient to reverse the negative effects of ethanol consumption. Both genders were used with the aim of investigating any differential pancreatic behaviour. The animals were randomized into five groups: the control group (CG) received water and a basic rat diet during pregnancy, lactation and growth; the ethanol group (EG) was fed an ethanol diet during pregnancy, the suckling period and growth until death; the ethanol-water group's (E+WG) ethanol was eliminated after lactation; The ethanol-folic acid group (E+FG) received a folic acid supplemented diet during pregnancy and the suckling period and in the ethanol+folic acid group (E+FG+FG) this supplementation continued during growth. Our results showed that ethanol administration or ethanol withdrawal did not significantly alter lipase activity in the pancreas. Ethanol administration decreased trypsinogen levels in the pancreas of males and females. However, in males, as opposed to females, the withdrawal of ethanol did not recover the values of pancreatic trypsinogen content, nor did a folic acid supplementation significantly alter the parameters we studied. Our treatment produced no effect on lipase levels. There was a gender-related difference in pancreatic trypsinogen content, the implication being that in future all results on exocrine pancreas function in male and female animals should be analysed separately.

Alcohol Withdrawal Delirium↗

PubMed1985

The exocrine protein trypsinogen is targeted into the secretory granules of an endocrine cell line: studies by gene transfer.

The exocrine protein rat anionic trypsinogen has been expressed and is secreted from the murine anterior pituitary tumor cell line AtT-20. We examined which secretory pathway trypsinogen takes to the surface of this endocrine-derived cell line. The "constitutive" pathway externalizes proteins rapidly and in the absence of an external stimulus. In the alternate, "regulated" pathway, proteins are stored in secretory granules until the cells are stimulated to secrete with 8-Br-cAMP. On the basis of indirect immunofluorescence localization, stimulation of release, and subcellular fractionation, we find that trypsinogen is targeted into the regulated secretory pathway in AtT-20 cells. In contrast, laminin, an endogenous secretory glycoprotein, is shown to be secreted constitutively. Thus it appears that the transport apparatus for the regulated secretory pathway in endocrine cells can recognize not only endocrine prohormones, but also the exocrine protein trypsinogen, which suggests that a similar sorting mechanism is used by endocrine and exocrine cells.

Adrenocorticotropic Hormone↗

PubMed1995

Calcium administration augments pancreatic injury and ectopic trypsinogen activation after temporary systemic hypotension in rats.

BACKGROUND: Calcium infusion and hypotension have been described as the most important risk factors for pancreatic injury after cardiopulmonary bypass. METHODS: Rats were randomly allocated to three experimental groups undergoing either sham operation and saline infusion (Control, n = 30), hemorrhagic reduction of mean arterial pressure to 30 mmHg for 30 min alone (hypotension, n = 51), or hypovolemic hypotension followed by bolus infusion of CaCl2 (200 mg.kg-1; hypercalcemia, n = 85). Serum ionized calcium, amylase activity, trypsinogen activation peptide in pancreatic tissue homogenates, pancreatic wet/dry weight ratio, histologic changes, and mortality were assessed for 24 h. RESULTS: Control rats showed no significant changes of any parameter throughout the experiments. In contrast, hypotension significantly increased serum amylase (P < 0.001), tissue trypsinogen activation peptide (P < 0.01), wet/dry weight ratio (P < 0.001), and histologic scores for edema (P < 0.001) and pancreatic necrosis (P < 0.05). Subsequent CaCl2 administration transiently increased [Ca2+] (P < 0.001) with the concentration rapidly returning to baseline within 3 h. That infusion of CaCl2 further increased amylase (P < 0.05), tissue trypsinogen activation peptide (P < 0.05), wet/dry weight ratio (P < 0.001), and histologic evidence of pancreatic edema (P < 0.05) and acinar necrosis (P < 0.05) when compared with hypotension alone. Whereas all Control animals survived the experiments, 22% (P < 0.05) and 47% (P < 0.05 vs. hypotension) of animals died in the hypotension and hypercalcemia groups, respectively. CONCLUSIONS: Temporary hypotension alone causes ectopic trypsinogen activation and lethal acute pancreatitis. Super-imposed hypercalcemia significantly aggravates hypotension-induced pancreatic injury and mortality in rats.

Animals↗

PubMed2000

Adaptive response of the rat pancreas to dietary substrates: parallel regulation of trypsinogen and pancreatic secretory trypsin inhibitor.

Chronic pancreatitis has been associated with malnutrition in alcoholic patients and malnourished juveniles. The composition of the diet, especially the protein content, regulates the synthesis of secretory proteins in the rat pancreas. Adaptive responses of the pancreas have shown that anionic proteases (e.g., trypsinogen) are upregulated during protein deprivation. We hypothesize that the (cationic) pancreatic secretory trypsin inhibitor (PSTI) is down-regulated after a protein-deficient diet. Low PSTI levels might cause a lack of protection from prematurely activated trypsin and therefore enhance the risk for pancreatic inflammation. Over a period of 1 month, rats were fed one of four isocaloric diets with a casein content varying from 0 to 82%. PSTI and trypsinogen mRNA remained fairly constant, irrespective of the diet composition. Trypsinogen and elastase secreted into pancreatic juice were upregulated after a protein-deficient diet relative to a control diet. Contrary to our hypothesis, PSTI was also upregulated. Parallel secretion of trypsinogen and PSTI appears to ensure protection against premature activation even under extreme dietary conditions.

Adaptation, Physiological↗

PubMed2001

Trypsinogen gene mutations in patients with chronic or recurrent acute pancreatitis.

Three-point mutations (R117H, N211, A16V) within the cationic trypsinogen gene have been identified in patients with hereditary pancreatitis (HP). A genetic background has also been discussed for idiopathic juvenile chronic pancreatitis (IJCP), which closely mimicks the clinical pattern of HP, and alcoholic chronic pancreatitis because only a small number of heavy drinkers develop pancreatitis. This prompted us to screen 104 patients in our well-defined pancreatitis cohort for the currently known cationic trypsinogen gene mutations. The R117H mutation was detected in seven patients (six patients of two clinically classified HP families, one patient with clinically classified IJCP) and the A16V mutation in one IJCP patient. No cationic trypsinogen gene mutations were found in the remaining 96 patients with chronic and recurrent acute pancreatitis of various etiologies. Our results demonstrate the need for genetic testing to exclude HP, particularly in the presence of an atypical or unknown family history. In addition, cationic trypsinogen gene mutations are no predisposing factor in patients with chronic and recurrent acute pancreatitis of different etiologies.

Acute Disease↗

PubMed1990

Serum immunoreactive anodal trypsinogen and urinary amylase as biochemical markers for rejection of clinical whole-organ pancreas allografts having exocrine drainage into the urinary bladder.

Rejection of pancreas allografts is best measured today by co-monitoring the creatinine of a simultaneously transplanted kidney allograft from the same donor. This methodology discourages pancreas transplantation for patients who have previously received a kidney allograft and preuremic patients. Thus, an early, graft-specific marker of rejection is desirable. In this study we compared 2 putative biochemical markers for rejection of pancreas allografts, serum immunoreactive anodal trypsinogen and urinary amylase, with serum creatinine in 15 simultaneously transplanted type I diabetics. Serial values during hospitalizations were determined. Follow-up ranged from 18 to 134 postoperative days. Rejection was diagnosed clinically and considered real if the patient received a course of anti-rejection medication. Ten of these 15 patients experienced a total of 21 rejection episodes. For all episodes of rejection, serum trypsinogen rose from a baseline of 398.1 +/- 25 ng/ml to 1686.2 +/- 317.9 ng/ml (P less than 0.001) on the day of rejection. Urinary amylase fell from 88,310 +/- 7877 U/24 hr to 37,508 +/- 7142 U/24 hr (P less than 0.001). For 10 patients in whom rejection was diagnosed on the initial hospitalization so that serial prediagnosis sera and urines were available, anodal trypsinogen rose from a baseline of 756 +/- 263 ng/ml to 1936 +/- 582 ng/ml (P less than 0.001). Urinary amylase values for these same 10 patients did not change significantly (baseline = 55,788 +/- 18,404 U/24 hr, rejection = 47,133 +/- 14,737 U/24 hr, (P = 0.7). We conclude that serum anodal trypsinogen behaves as a graft-specific biochemical marker for rejection of vascularized pancreas allografts.

Amylases↗

PubMed2003

The endothelin-1 receptor antagonists ameliorate histology and ultrastructural alterations in the pancreas and decrease trypsinogen activation in severe taurocholate pancreatitis in rats.

The role of potent vasoconstrictor endothelin-1 (ET-1) in acute pancreatitis (AP) remains controversial. The aim was to compare the effect of nonselective ET RA/B (LU-302872) and selective ET RA (LU-302146) antagonists on pancreatic histology, ultrastructure and trypsinogen activation in severe taurocholate AP in rats. Male Wistar rats with AP were treated with an intraperitoneous injection of 1, 5 and 10 mg/kg of body weight of each antagonist at 0, 6, 12 and 18 h after taurocholate administration. After 24 h, the samples of pancreases were taken for histological and ultrastructural examinations and for assessment of trypsinogen activation. Both antagonists, at all investigated doses, decreased the damage to the acinar cells detected in the light microscope and ultrastructurally. Trypsinogen activation increased to 29.7 +/- 3.9% in the AP untreated in comparison to the control group [12.7 +/- 1.4% (P<0.001)]. This increase was attenuated to 13.8 +/- 2.2% in AP treated with a high dose of the nonselective antagonist and to 8.4 +/- 1.7% with low dose of selective antagonist. The obtained results indicate that ET-1 could participate in the damage to the pancreas during AP. Both antagonists of ET-1 receptors exerted a similar beneficial effect on the morphological changes of the pancreas in AP. One of the probable mechanism could be the attenuation of trypsinogen activation.

Acute Disease↗

PubMed1990

Isolation and nucleotide sequence of cDNA clone for bovine pancreatic anionic trypsinogen. Structural identity within the trypsin family.

A cDNA clone encoding an anionic form of bovine trypsinogen was isolated from a pancreatic cDNA library. The corresponding 855-nucleotide mRNA contains a short 5' noncoding region of 8 nucleotides and a long 3' noncoding region of 56 nucleotides in addition to a poly(A) tail of at least 50 nucleotides. The deduced amino acid sequence for the anionic pretrypsinogen (247 residues) includes the N-terminal 15-amino-acid signal peptide followed by an 8-amino-acid activation peptide. The zymogen (232 residues) contains an additional C-terminal serine, compared with the amino acid sequence of bovine cationic trypsinogen. The identity between the anionic and cationic forms of bovine trypsinogen (65%) is lower than that existing between the anionic protein and other mammalian anionic trypsinogens (73-85%), suggesting that trypsin gene duplication in mammals occurred prior to the evolutionary events responsible for the species divergence. Bovine pancreatic anionic trypsin possesses all the key amino acids characteristic of the serine protease family.

Amino Acid Sequence↗

PubMed1996

Unfolding kinetics of bovine trypsinogen.

The unfolding kinetics of bovine trypsinogen were studied by a fluorescence-detected stopped-flow technique at pH 5.8. Trypsinogen unfolding appeared to be a rather complex reaction. Two phases, fast (with a time constant in the millisecond range) and slow, were detected in the range 2-7 M guanidium chloride (GdmCl). The natural logarithm of the rate constant of the slow phase exhibited strong dependence on [GdmCl], changing from hundreds of seconds at low denaturant concentration to about 20 ms at 7 M GdmCl. The curvature of this dependence further suggests a complex mechanism of unfolding. Generally, similar kinetics were observed for the trypsinogen.Ca complex. Small differences could be noticed, however, for the fast phase. In agreement, Ca2+ influenced only this stage of the reaction. Analysis of the dependence of the time constant of the fast phase on [CaCl2] indicates that at 4 M GdmCl, trypsinogen.Ca unfolds about sixfold slower than free zymogen, and that native trypsinogen at 4 M GdmCl still exhibits high affinity for Ca2+. Limited data on trypsin unfolding show virtually an identical dependence of the slow phase on [GdmCl]; the fast phase, however was not observed. Moreover, in the 3-4.5 M GdmCl range, a separate phase was detected. It is postulated that this phase is a manifestation of the activation-domain unfolding. The Eyring plots for the fast phase of . trypsinogen and trypsinogen.Ca unfolding are linear, indicating little change in heat capacity for this stage of reaction. The slow step of unfolding, however, shows significant curvature which indicates a substantial increase in heat capacity.

Animals↗

PubMed1999

Mutations in exons 2 and 3 of the cationic trypsinogen gene in Japanese families with hereditary pancreatitis.

BACKGROUND/AIMS: Single-point mutations in the cationic trypsinogen gene have been reported in hereditary pancreatitis kindreds in the white population. The aim of the present study was to investigate whether similar gene mutations are present in Japanese hereditary pancreatitis kindreds. METHODS: All five exons of the cationic trypsinogen gene were amplified by polymerase chain reaction and sequenced in six Japanese families with hereditary pancreatitis. RESULTS: Two types of single-point mutation in the cationic trypsinogen gene, which were identical with those reported in white families with hereditary pancreatitis, were observed in separate Japanese families with hereditary pancreatitis: 21Asn (AAC) to Ile (ATC) (N21I) in exon 2 and 117Arg (CGC) to His (CAC) (R117H) in exon 3. Pancreatitis occurred at more advanced ages in patients with the N21I mutation than in those with the R117H mutation. Besides normal polymorphisms in exons 4 and 5, no mutation was found in patients in the remaining four families with hereditary pancreatitis, 21 patients with sporadic chronic pancreatitis, or five normal subjects. CONCLUSIONS: These results show heterogeneity, but no racial specificity, in the cationic trypsinogen gene mutations in hereditary pancreatitis kindreds. A distinctive clinical feature for each of the mutation types is suggested: adult onset for the N21I mutation and childhood onset for the R117H mutation.

Adolescent↗