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Journal of Biochemistry Dec 1977Human pancreatic DNase I was purified extensively from duodenal juice of healthy subjects by a procedure including ammonium sulfate fractionation, ethanol fractionation,...
Human pancreatic DNase I was purified extensively from duodenal juice of healthy subjects by a procedure including ammonium sulfate fractionation, ethanol fractionation, phosphocellulose fractionation, isoelectric focusing, and gel filtration. The final preparation was free of DNase II, pancreatic RNase, alkaline phosphatase, and protease. The enzyme had a molecular weight of approximately 30,000, as determined by gel filtration on Sephadex G-100, and showed maximum activity at pH 7.2-7.6. It required divalent cations for activity, and caused single-strand breaks by endonucleolytic attack on double- as well as single-stranded DNA molecules. The enzyme was inhibited by actin and bovine pancreatic DNase I antibody.
Topics: Deoxyribonucleases; Humans; Immunodiffusion; Molecular Weight; Pancreas
PubMed: 413831
DOI: 10.1093/oxfordjournals.jbchem.a131875 -
Gastroenterology Oct 2018Intrapancreatic activation of the digestive proteases trypsin and chymotrypsin is an early event in the development of pancreatitis. Human genetic studies indicate that...
Intrapancreatic activation of the digestive proteases trypsin and chymotrypsin is an early event in the development of pancreatitis. Human genetic studies indicate that chymotrypsin controls trypsin activity via degradation, but there is no evidence of this from animal models. We used CRISPR-Cas9 to disrupt the chymotrypsinogen B1 gene (Ctrb1) in C57BL/6N mice and induced pancreatitis in CTRB1-deficient and C57BL/6N (control) mice by administration of cerulein. CTRB1-deficient mice given cerulein had significant increases in intrapancreatic trypsin activity and developed more severe pancreatitis compared with control mice. CTRB1 therefore protects against secretagogue-induced pancreatitis by reducing trypsin activity. Protease inhibitors developed for treatment of pancreatitis should be designed to target trypsin but not chymotrypsin.
Topics: Animals; Arginine; Ceruletide; Chymotrypsin; Disease Models, Animal; Enzyme Activation; Enzyme Stability; Female; Male; Mice, Inbred C57BL; Mice, Knockout; Pancreas; Pancreatitis; Proteolysis; Severity of Illness Index; Trypsin
PubMed: 30076839
DOI: 10.1053/j.gastro.2018.06.041 -
Journal of Visualized Experiments : JoVE Aug 2014Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion,...
Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.
Topics: Animals; Mice; Pancreas; RNA; Ribonuclease, Pancreatic
PubMed: 25145327
DOI: 10.3791/51779 -
Journal of Biochemistry Feb 1986A collagenolytic enzyme was purified to homogeneity from the activated pancreatic extract of the catfish Parasilurus asotus by chromatography on DEAE-cellulose,...
A collagenolytic enzyme was purified to homogeneity from the activated pancreatic extract of the catfish Parasilurus asotus by chromatography on DEAE-cellulose, hydroxylapatite, and Cellulofine columns. The molecular weight of the enzyme was estimated to be 29,500 by SDS-polyacrylamide gel electrophoresis. The enzyme is capable of degrading native, reconstituted calf skin collagen fibrils at pH 7.5 and 37 degrees C, and also of reducing the viscosity of native calf skin collagen at pH 7.5 and 20 degrees C. The SDS-polyacrylamide gel electrophoresis of thermally denatured enzyme-collagen reaction mixtures showed that the enzyme can cleave peptide bonds in the non-helical and triple-helical regions of the collagen. The enzyme was inhibited by DFP, PMSF, soybean trypsin inhibitor, and chicken ovoinhibitor, but not by metal-chelating reagents EDTA, EGTA, o-phenanthroline, or L-cysteine. These results indicate that the enzyme is a unique collagenolytic proteinase belonging to the group of serine proteinases and is a new member of the class of pancreatic enzymes.
Topics: Amino Acids; Animals; Collagen; Endopeptidases; Enzyme Precursors; Fishes; Hydrogen-Ion Concentration; Molecular Weight; Pancreas; Protease Inhibitors; Serine Endopeptidases
PubMed: 3516983
DOI: 10.1093/oxfordjournals.jbchem.a135500 -
World Journal of Gastroenterology Feb 2007To study the effect of early intrajejunal nutrition on enzyme-protein synthesis and secretion during acute pancreatitis.
AIM
To study the effect of early intrajejunal nutrition on enzyme-protein synthesis and secretion during acute pancreatitis.
METHODS
Fifteen dogs were randomly divided into parenteral nutrition (n = 7) and early intrajejunal nutrition groups (n = 8). An acute pancreatitis model was induced by injecting 5% sodium taurocholate and trypsin into the pancreas via the pancreatic duct. Intrajejunal nutrition was delivered with a catheter via a jejunostomy tube after the model was established for 24 h. On d 1 and 7 and at the beginning of nutritional support, radioactive tracing and electron microscopes were used to evaluate the enzyme-protein synthesis in acinar cells, the subcellular fractionation and the change in zymogen granules after 1.85 x 10(6) Bq L-(3)H phenylalanine was infused at 30, 60, 120, and 180 min.
RESULTS
The 3H radioactivity in pancreatic acinar cells reached its peak level at 60 min, and the contents in the early intrajejunal nutrition group were higher than those in the parenteral nutrition group, which were then decreased. The mean number and area of zymogen granules did not show any significant statistical difference in both groups on d 1 or on d 7 (P > 0.05).
CONCLUSION
Early intrajejunal nutrition might be effective in dogs with acute pancreatitis.
Topics: Acute Disease; Amylases; Animals; Cathepsin B; Detergents; Disease Models, Animal; Dogs; Electron Transport Complex IV; Enteral Nutrition; Lysosomes; Pancreas; Pancreatitis; Parenteral Nutrition; Secretory Vesicles; Taurocholic Acid
PubMed: 17373751
DOI: 10.3748/wjg.v13.i7.1123 -
The Journal of Cell Biology Apr 1980Rat pancreases were minced and treated with collagenase or collagenase supplemented with chymotrypsin to yield a mixture of ducts, islets, acinar cell clusters, blood...
Rat pancreases were minced and treated with collagenase or collagenase supplemented with chymotrypsin to yield a mixture of ducts, islets, acinar cell clusters, blood vessels, and nerves. Histologically and ultrastructurally, the isolated tissues resembled their in situ counterparts in most respects, the major difference being the destruction of the basement membranes (basal laminae). Ducts ranging in size from the common bile/main pancreatic duct to the intercalated ducts were identified in the digest, although interlobular ducts were most frequently observed. Acinar tissue fragments were separated from nonacinar structures either by flotation through discontinuous gradients of Ficoll or by sieving, the latter technique being the more efficient. Common bile/main ducts, interlobular ducts, and blood vessels were selected manually from the nonacinar fractions. Biochemical analyses showed that the entire nonacinar fraction, as well as isolated ducts and blood vessels, contained larger alkaline phosphatase, carbonic anhydrase, and Mg-ATPase specific activities than acinar tissue, whereas acinar tissue contained larger gamma-glutamyltranspeptidase and amylase activities. However, greater than 63% of the total recovered activity of each enzyme was associated with the acinar tissue. Both the association of the majority of each of these enzyme activities with the acinar tissue and the similarity in specific activities associated with ducts and blood vessels indicate that none of the enzymes tested is a unique marker for interlobular and larger ducts of the pancreas of the rat.
Topics: Adenosine Triphosphatases; Alkaline Phosphatase; Amylases; Animals; Carbonic Anhydrases; DNA; Histological Techniques; Hydrolases; Pancreatic Ducts; Proteins; Rats; gamma-Glutamyltransferase
PubMed: 6154056
DOI: 10.1083/jcb.85.1.122 -
Journal of Physiology and Pharmacology... Jun 2016Apelin is considered as important gut regulatory peptide ligand of APJ receptor with a potential physiological role in gastrointestinal cytoprotection, regulation of...
Apelin is considered as important gut regulatory peptide ligand of APJ receptor with a potential physiological role in gastrointestinal cytoprotection, regulation of food intake and drinking behavior. Circulating apelin inhibits secretion of pancreatic juice through vagal- cholecystokinin-dependent mechanism and reduces local blood flow. Our study was aimed to determine the effect of fundectomy and intraperitoneal or intragastric administration of apelin-13 on pancreatic and gastric enzymes activities in adult rats. Fundectomy is a surgical removal of stomach fundus - maine site apelin synthesis. Three independent experiments were carried out on Wistar rats. In the first and second experiment apelin-13 was given by intragastric or intraperitoneal way twice a day for 10 days (100 nmol/kg b.w.). Control groups received the physiological saline respectively. In the third experiment the group of rats after fundectomy were used. Fundectomized rats did not receive apelin and the rats from control group were 'sham operated'. At the end of experiment rats were sacrificed and blood from rats was withdrawn for apelin and CCK (cholecystokinin) radioimmunoassay analysis and pancreas and stomach tissues were collected for enzyme activity analyses. Intragastric and intraperitoneal administrations of apelin-13 increased basal plasma CCK level and stimulated gastric and pancreatic enzymes activity in rats. In animals after fundectomy decreased activity of studied enzymes was observed, as well as basal plasma apelin and CCK levels. In conclusion, apelin can effects on CCK release and stimulates some gastric and pancreatic enzymes activity in adult rats while fudectomy suppresses those processes. Changes in the level of pancreatic lipase activity point out that apelin may occurs as a regulator of lipase secretion.
Topics: Amylases; Animals; Apelin; Cholecystokinin; Chymosin; Intercellular Signaling Peptides and Proteins; Lipase; Male; Pancreas; Rats, Wistar; Stomach; Trypsin
PubMed: 27512001
DOI: No ID Found -
The Journal of Biological Chemistry Oct 1978When pancreatic DNase I is used as a specific biochemical reagent in the preparation of nuclear ribonucleic acids or nuclear proteins, freedom from contaminating...
When pancreatic DNase I is used as a specific biochemical reagent in the preparation of nuclear ribonucleic acids or nuclear proteins, freedom from contaminating ribonucleases or proteases is an important property of the enzyme preparation. A simple one-step procedure has been developed to effect complete removal of trypsin, chymotrypsin, and chymotrypsinogen by a combination of affinity chromatography and salting-out adsorption on lima bean protease inhibitor coupled to Sepharose (a column (0.9 X 60 cm) operated in series with a regeneratable 1-ml bed). Commercial preparations of DNase (about 10 mg) give a quantitative yield of the enzyme that is protease-free as evidenced by full stability for more than 10 days at pH 8 and 37 degrees C even in the absence of the protecting action of Ca2+. Removal of the last traces of RNase has been accomplished by affinity chromatography on a column (0.4 X 72 cm) of 5'-(4-aminophenyl-phosphoryl)-uridine 2'(3')-phosphate-Sepharose; the product is a highly active DNase that gives no detectable hydrolysis of RNA by assay on radioactive substrates.
Topics: Chromatography, Affinity; Deoxyribonucleases; Pancreas; Peptide Hydrolases; Ribonucleases
PubMed: 701244
DOI: No ID Found -
Gut Jun 1996It is hypothesised that nutrients increase pancreatic enzyme secretion by converting cyclical interdigestive secretion to a non-cyclical pattern. This study tested the... (Clinical Trial)
Clinical Trial
BACKGROUND AND AIMS
It is hypothesised that nutrients increase pancreatic enzyme secretion by converting cyclical interdigestive secretion to a non-cyclical pattern. This study tested the hypotheses that nutrients do not interrupt cycles and determined the relation of nutrients, calories, and osmotic load to the rate of pancreatic secretion.
METHODS
Twenty six healthy persons were intubated with oroduodenal and orogastric tubes. Each had one of four different solutions containing 12 to 36% of calories as protein, 24 to 48% as fat, and 40 to 64% as carbohydrate infused into the duodenum at 40, 90, or 160 kcal/h for 300 minutes. Nine g/l sodium chloride (290 mOsm) was added to 16 infusates; osmolality of the other 10 infusates was 24 to 98 mOsm. Pancreatic enzyme outputs were measured every 15 minutes and peaks of enzyme secretion were identified.
RESULTS
The number of enzyme peaks was similar for the different infusates and the proportion of nutrients in the infusates did not affect secretion of individual enzymes. The nadir, but not the peak of the cycles of enzyme outputs correlated with increasing the caloric load (r = 0.55, p < 0.003 for nadir:peak ratio). Increasing osmolality did not affect cycling but reduced (p < 0.001) enzyme output.
CONCLUSION
Nutrients entering the duodenum do not abolish cycles of enzyme secretion; instead they modulate cycles by increasing the nadir. Forty and 90 kcal infusions submaximally stimulate pancreatic secretion and might be used in patients with pancreatitis without producing pain; adding sodium chloride to solutions should increase this effect.
Topics: Amylases; Chymotrypsin; Cyclization; Humans; Hydrolases; Pancreas; Parenteral Nutrition; Random Allocation; Trypsin
PubMed: 8984034
DOI: 10.1136/gut.38.6.920 -
Cell Transplantation Jul 2018Despite huge advances in the field of islet transplantation over the last two decades, current islet isolation methods remain suboptimal, with transplantable yields...
Despite huge advances in the field of islet transplantation over the last two decades, current islet isolation methods remain suboptimal, with transplantable yields obtained in less than half of all pancreases processed worldwide. Successful islet isolation is dependent on the ability of collagenase-based enzyme blends to digest extracellular matrix components at the islet-exocrine interface. The limited availability of donor pancreases hinders the use of full-scale islet isolations to characterize pancreas digestion by different enzyme components or blends, or allow the influence of inter-pancreatic variability between donors to be explored. We have developed a method that allows multiple enzyme components to be tested on any one pancreas. Biopsies of 0.5 cm were taken from seven standard (age ≥45) and eight young (age ≤35) pancreases. Serial cryosections were treated with Serva collagenase, neutral protease (NP), or the two enzymes together at clinically relevant concentrations. Following digestion, insulin and either collagen IV or laminin-α5 were detected by immunofluorescent labeling. Protein loss at the islet-exocrine interface was semi-quantified morphometrically, with reference to a control section. Differential digestion of the two proteins based on the enzyme components used was seen, with protein digestion significantly influenced by donor age. Treatment with collagenase and NP alone was significantly more effective at digesting collagen IV in the standard donor group, as was the NP mediated digestion of laminin-α5. Collagenase alone was not capable of significantly digesting laminin-α5 in either donor group. Combining the two enzymes ameliorated the age-related differences in the digestion of both proteins. No significant differences in protein loss were detected by the method when analyzed by two independent operators, demonstrating the reproducibility of the assay. The development of this simple yet reproducible assay has implications for both enzyme batch testing and identifying inter-donor digestion variability, while utilizing small amounts of both enzyme and human tissue.
Topics: Adult; Cell Separation; Collagenases; Extracellular Matrix; Extracellular Matrix Proteins; Female; Humans; Islets of Langerhans; Islets of Langerhans Transplantation; Male; Middle Aged; Pancreas; Proteolysis
PubMed: 29954221
DOI: 10.1177/0963689718779778