SEARCH · Search PubMed

Results for “TRYPSINS”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 811 records · Page 45Linked to original sources

PubMed1997

Neonatal screening for cystic fibrosis in the Trent region (UK): two-stage immunoreactive trypsin screening compared with a three-stage protocol with DNA analysis as an intermediate step.

OBJECTIVES: To assess neonatal screening for cystic fibrosis using immunoreactive trypsin, either alone or in conjunction with DNA analysis for the delta F508 mutation. A novel three-stage screening protocol was compared with the previously introduced two-stage immunoreactive trypsin-DNA protocol. DESIGN: (a) Collection of data from a 4 1/2 year period (phase 1) of two-stage immunoreactive trypsin screening. The initial dried blood samples were obtained at 6 days of age and repeat samples at 27 days of age from babies with results above the 99.5th centile. Babies with persistent hypertrypsinaemia were referred for a diagnostic sweat test. (b) Retrospective DNA analysis: patients with cystic fibrosis diagnosed in phase 1 were genotyped and most samples from babies with increased initial immunoreactive trypsin but normal results in the second sample were analysed for the delta F508 mutation. (c) Phase 2, a prospective study of a three-stage neonatal screening protocol, in which only babies heterozygous for the delta F508 cystic fibrosis mutation progressed to the second immunoreactive trypsin test. SETTING: The Trent neonatal screening programme. SUBJECTS: 437 859 babies born between August 1989 and March 1996. MAIN OUTCOME MEASURES: Proportions of unaffected babies requiring a second blood sample or a sweat test. Overall sensitivity for the detection of cystic fibrosis. RESULTS: The two-stage screen failed to identify six out of 94 cases of cystic fibrosis (without meconium ileus). The introduction of the DNA analysis step would have resulted in one additional case being missed. With the three-stage screen there was a 92% reduction in babies requiring a second blood sample and an 80% reduction in negative sweat tests, results close to the predictions of the retrospective study. CONCLUSIONS: The three-stage screening protocol is a marked improvement on the two-stage immunoreactive trypsin strategy and on the two-stage immunoreactive trypsin-DNA strategy recently introduced in some other screening programmes.

Cystic Fibrosis↗

PubMed1980

Immunoreactive trypsins in sera from dogs before and after induction of experimental pancreatitis.

Radioimmunoassays for anionic and cationic dog trypsins are described. Characterization of the immunoreactivities in sera from fasting dogs demonstrated the presence of the two proenzymes only. Fasting sera from 10 dogs contained anionic and cationic trypsinogen in concentrations between 17-110 micrograms/l and 7-19 micrograms/l, respectively. Induction of experimental pancreatitis in dogs was accompanied by a large increase of immunoreactive anionic and cationic trypsins in the circulation. During the progress of the pancreatitis, immunoreactive trypsin with larger molecular weight than trypsinogen appeared. This high molecular weight immunoreactive trypsin was not seen in serum after intravenous injection of pancreatic juice in dogs. The high molecular weight immunoreactive trypsin probably consists of trypsin in complex with protease inhibitors. In vitro studies showed that the immunoreactivity of trypsin decreased considerably after binding to alpha 1-antitrypsin or alpha-macroglobulins.

Animals↗

PubMed1991

Activation of the complement cascade by trypsin.

In twenty-three patients with acute pancreatitis, the plasma levels of immunoreactive trypsin were determined with a RIA method. The patients were divided into groups according to the severity of the disease. Ranson's criteria and the development of multisystem organ failure were used for the classification. Elevated plasma levels of immunoreactive trypsin were found in all groups after admittance. Incubation of fresh human serum and plasma with bovine trypsin in concentrations between 10(-6) and 10(-4) M at 20 degrees C activated the complement cascade. The anaphylatoxins C3a and C5a were determined with a RIA and the terminal complement complex (TCC) with an ELISA method. C3a and C5a were released and TCC was formed. The effect of trypsin on leukocyte activation was determined with a chemiluminescence technique. Trypsin dissolved in saline did not activate the leukocytes. However, serum digested by trypsin-activated leukocytes in a dose-dependent manner. The present study supports the theory that trypsin can activate complement components and results in formation of split products which have potent vascular, and leukocyte activating effects.

Acute Disease↗

PubMed2005

Specificity of trypsin and chymotrypsin: loop-motion-controlled dynamic correlation as a determinant.

Trypsin and chymotrypsin are both serine proteases with high sequence and structural similarities, but with different substrate specificity. Previous experiments have demonstrated the critical role of the two loops outside the binding pocket in controlling the specificity of the two enzymes. To understand the mechanism of such a control of specificity by distant loops, we have used the Gaussian network model to study the dynamic properties of trypsin and chymotrypsin and the roles played by the two loops. A clustering method was introduced to analyze the correlated motions of residues. We have found that trypsin and chymotrypsin have distinct dynamic signatures in the two loop regions, which are in turn highly correlated with motions of certain residues in the binding pockets. Interestingly, replacing the two loops of trypsin with those of chymotrypsin changes the motion style of trypsin to chymotrypsin-like, whereas the same experimental replacement was shown necessary to make trypsin have chymotrypsin's enzyme specificity and activity. These results suggest that the cooperative motions of the two loops and the substrate-binding sites contribute to the activity and substrate specificity of trypsin and chymotrypsin.

Amino Acid Sequence↗

PubMed1976

Effects of trypsin on glucose-induced insulin secretion from the perfused rat pancreas.

The present study revealed that glucose-induced insulin secretion was significantly inhibited by perfusion with trypsin (10 mug/ml) in the isolated rat pancreas perfusion. However, when trypsin was interposed during perfusion of 16.6 mM glucose, glucose-induced insulin secretion was not suppressed during trypsin infusion. The insulin level was significantly lower in the trypsin treated animals than in the control after completion of trypsin infusion. The results suggest that trypsin does not attack the glucorecoptor bound with glucose but attacks the free glucoreceptor. Interestingly a biphasic pattern of glucose-induced insulin secretion disappeared by the pretreatment with trypsin. The possibility was suggested that tyrpsin prevented the initial binding of glucose to the B-cell.

Animals↗

PubMed1990

Trypsin stimulation of aldosterone and 18-hydroxycorticosterone production by rat adrenal zona glomerulosa tissue is mediated by activation of protein kinase C.

When rat adrenal whole capsules, containing the zona glomerulosa, were incubated, addition of the protein kinase C inhibitors TMB-8 (10 mumol/l), W7, H7, polymyxin-B and sphingosine (all 1 mumol/l) was found to inhibit the steroidogenic response to trypsin. Aldosterone and 18-hydroxycorticosterone were strongly, and corticosterone moderately, affected, while the production of 18-hydroxydeoxy-corticosterone was neither stimulated by trypsin nor inhibited by the protein kinase C inhibitors. Addition of neomycin, which prevents substrate interaction with phospholipase C, also inhibited the response to trypsin, while addition of phospholipase C itself stimulated aldosterone, 18-hydroxycorticosterone and corticosterone production with the same tissue sensitivity as trypsin. Addition of phospholipase A2 had no effect. Direct assay of protein kinase C activity showed that trypsin stimulation effected the translocation of Ca2+/phospholipid-activated protein kinase C from the cytosolic to the membrane fraction. When glomerulosa tissue was incubated with [32P]ATP, and cytosolic proteins were subjected to isoelectric focusing on polyacrylamide gels, autoradiography showed that incorporation of 32P into several protein components was increased by trypsin stimulation. It was concluded that trypsin exerts its stimulatory effects on steroidogenesis by activating protein kinase C; not, however, by generating the Ca2+/phospholipid-independent fragment, but possibly by enhancing the activity of phospholipase C.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1983

Regulatory effects on the pancreas of intraduodenal pancreatic juice and trypsin in the Syrian golden hamster.

The effect of intraduodenal trypsin activity on pancreatic exocrine secretion was studied in conscious Syrian golden hamsters provided with bile-pancreatic fistulae. The secretion (secretory volume, amylase and protein output) was stable during a collection period of 14 h without any duodenal infusions. Infusion into the duodenum of bicarbonate or bile did not affect the secretion. When, however, bile-pancreatic juice or trypsin was administered intraduodenally, a marked depression of amylase and protein output was found. After addition of trypsin inhibitor--in a dose sufficient to eliminate all trypsin activity--to either of the two infusates the secretion was restored to the initial values. In a long-term experiment (10 days) repeated subcutaneous injections of cholecystokinin caused a significant increase of pancreatic protein and amylase content in the hamster. Oral trypsin inhibitor administration for 10 days had similar, although not so pronounced effects. Subcutaneous secretin administration was without effect in this respect. The results show that pancreatic enzyme secretion in the Syrian golden hamster is controlled by a negative feedback regulation exerted by intraluminal trypsin. The findings also suggest that both cholecystokinin and orally administered trypsin inhibitor exert trophic effects on the pancreas.

Amylases↗

PubMed2006

Proteinase-activated receptor-2 expression in breast cancer and the role of trypsin on growth and metabolism of breast cancer cell line MDA MB-231.

Proteinase-activated receptor-2 (PAR-2) is a ubiquitous surface molecule participating in many biological processes. It belongs to the family of G protein-coupled receptors activated by the site-specific proteolysis of trypsin and similar proteases. Altered function of PAR-2 has been described in different malignant tumors. In the present study, we investigated the expression of PAR-2 in breast cancer surgical specimens and the role of trypsin in breast cancer cell line MDA MB-231 proliferation and metabolism. A total of 40 surgical samples of infiltrative ductal breast cancer and breast cancer cell line were included in this study. We analyzed PAR-2 expression by immunohistochemistry, RT-PCR and western blot. Activation of PAR-2 on cell line MDA MB-231 was measured using calcium mobilization assay determined by flow cytometry. MTT cell metabolism assay and cell count analysis were used to assess the trypsin influence on breast cancer cell line MDA MB-231 proliferation. Immunohistochemical examination showed the expression of PAR-2 in all samples of breast cancer surgical specimens and high levels of cell lines which was confirmed by RT-PCR and western blot. Calcium mobilization assay corroborated the activation of PAR-2 on cell line MDA MB-231 either by trypsin or by an agonistic peptide. Cell metabolism assay and cell count analysis showed significant differences of proliferative activity of breast cancer cells dependent on the presence or absence of trypsin and serum in the culture medium. PAR-2 is expressed by high levels in infiltrative ductal breast cancer tissue specimens. PAR-2 is also strongly expressed in studied breast cancer cell lines. PAR-2 is activated by trypsin and also by agonistic peptide in the model of breast cancer cell line MDA MB-231. Activation of PAR-2 in vitro influences proliferative and metabolic activity of breast cancer cell line MDA MB-231. The action of trypsin is modified by the presence of serum which is a potential source of protease inhibitors.

Blotting, Western↗

PubMed2000

Examination of CD11B/C, CD54 and CD62 expression on cells in trypsin-triggered emphysema model.

We studied the CD11b/c, CD54, CD62 expression on BAL (bronco-alveolar lavage) cells of rats by flow cytometry in a trypsin-triggered emphysema model. We made BAL sampling two and a half hour after trypsin infusion, in early inflammatory phase. Rats were divided into three groups: 1. negative controls, 2. saline-treated, 3. trypsin-infused rats. We found significantly (p < 0.05) increased number of neutrophil granulocytes in BAL of trypsin-treated group, comparing with controls. By flow cytometry in trypsin-treated group: 1). We found a significantly higher expression of CD54 on BAL macrophages (p < 0.05) 2). There was a lower, not significant CD11b/c expression on neutrophils and on macrophages in BAL, comparing with other groups. 3). A low, but not significant CD62 expression could be detected on neutrophils and on lymphocytes in BAL. We conclude: 1). Two and a half hour after trypsin infusion, macrophages are strongly activated, and play an important role in the neutrophil transendothelial migration in the early inflammatory phase of this model. 2). Neutrophils are high in number in BAL, but they are hardly activated in this early phase. 3). After trypsin infusion having a lower CD62 expression, lymphocytes seem to be involved as well.

Animals↗

PubMed1992

Urinary trypsin inhibitory activity for the diagnosis of bacterial infection: a prospective study in 690 patients.

OBJECTIVES: During the acute phase response, interleukin-1 induces production of inter-alpha-trypsin inhibitor. The measurement of urinary trypsin inhibitory activity which results from the effects of inter-alpha-trypsin inhibitor degradation products is easy, quick and inexpensive. We conducted a prospective study to investigate its value as a diagnostic tool in comparison with C-reactive protein. METHODS: Comparisons were made in 690 consecutive patients at admission to a department of internal medicine. RESULTS: The level of urinary trypsin inhibitory activity was significantly higher in patients with bacterial infection (mean = 123 IU/g creatinine) than in patients with either viral infection (34 IU), cancer (50 IU), elevated erythrocyte sedimentation rate without infection (45 IU), miscellaneous non-inflammatory diseases (27 IU) or in non-organic controls (19 IU) (Dunnet's test, p << 0.01). The receiver operating characteristic curve showed that sensitivity and specificity of urinary trypsin inhibitory activity were higher than those of C-Reactive protein for the diagnosis of bacterial infection. For levels > or = 60 IU, sensitivity was 75% and specificity 89%. Urinary trypsin inhibitory activity levels fell within 2 days in patients treated for acute bacterial infection. CONCLUSION: Urinary trypsin inhibitory activity could be a useful marker of bacterial infection particularly in patients with fever of unknown origin and/or elevated erythrocyte sedimentation rate.

Alpha-Globulins↗

PubMedDate not supplied

[Modification of the method of trypsin esterase activity determination].

Determination of esterase activity of trypsin in blood serum using the Erlanger's method leads to overestimation of enzyme activity due to the increased "dimness" of the reaction medium. V. A. Shaternikov suggested a correction, which is based on the use of the two-wavelength registration method. But this correction does not eliminate this secondary factor completely. So for the clearing of the solutions we offer to stop the reaction by putting tubes into ice followed by subsequent centrifugation the investigated samples at 0 degree C for 15 min, at 25,000 g. It gives the possibility to measure an actual increase of an optical density in accordance to the hydrolysis of the substrate. The results of the measuring of an esterase activity of trypsin are represented in a serum and in a peritoneal exudate of dogs with an acute experimental pancreatitis. The serum esterase activity of trypsin in a was not found in intact animals. Three hours after induction of pancreatitis trypsin activity was detected only in blood serum of two of seven animals [approximately 3.6 mmole/(min-l)]. Rats with acute pancreatitis also had very low esterase activity of trypsin in blood [0.38 +/- 0.16 mmole/(min-l)] that insignificantly deferred from the control. These data question applicability of this method for diagnostics of acute pancreatitis. The estarse activity of trypsin detected in blood and peritoneal exudate, does not indicate its proteolytic activity, because it was insensitive to soybean trypsin inhibitor.

Acute Disease↗

PubMed2006

Photoilluminated riboflavin/riboflavin-Cu(II) inactivates trypsin: Cu(II) tilts the balance.

Riboflavin (RF) upon irradiation with fluorescent light generates reactive oxygen species like superoxide anion, singlet and triplet oxygen, flavin radicals and substantial amounts of hydrogen peroxide (H2O2). H2O2 can freely penetrate cell membrane and react with a transition metal ion like Cu(ll), generating hydroxyl radical via the modified metal-catalyzed Haber-Weiss reaction. Earlier, it was reported that trypsin-chymotrypsin mixture served as an indirect antioxidant and decreased free radical generation. Thus, in the present study, we used photoilluminated RF as a source of ROS to investigate the effect of free radicals on the activity of trypsin. We also compared the damaging effect of photoilluminated RF and RF-Cu(ll) system using trypsin as a target molecule. RF caused fragmentation of trypsin and the effect was further enhanced, when Cu(II) was added to the reaction. Results obtained with various ROS scavengers suggested that superoxide radical, singlet and triplet oxygen were predominantly responsible for trypsin damage caused by photoilluminated RF. On the other hand, when Cu(ll) was added to the reaction, hydroxyl radical was mainly responsible for trypsin damage. A mechanism of generation of various ROS in the reaction is also proposed. Trypsin did not show any antioxidant effect with RF alone or with RF-Cu(II) combination.

Copper↗

PubMed1990

A plausible identification of the secondary binding site in trypsin and trypsinogen.

1. The determination of the binding of 4,4'-diazoamino-bis-benzamidine (DABB) to alpha-trypsin by equilibrium measurements in columns indicated a stoichiometry of 2 mol ligand/mol enzyme. One molecule of ligand is bound to the active center, as shown by competitive experiments and Ki. The second molecule binds to the secondary binding site, with Ki2 = 0.63 mM at pH 8.0, 25 degrees C. 2. Bovine pancreatic trypsin inhibitor (BPTI) prevented binding of DABB to both sites, indicating that they are topographically close and within the interface of the trypsin-BPTI complex. 3. On the basis of data from the literature regarding the tertiary structure of the trypsin-BPTI complex, we concluded that the secondary binding site of trypsin is plausibly identified as the same site in trypsin that binds the Arg-17 residue of BPTI, i.e., Tyr-39 and Tyr-151 in bovine trypsin. This site would then correspond to subsite S'2 on the enzyme surface.

Benzamidines↗

PubMedDate not supplied

Problems related to the use of serum and trypsin in the growth of monkey kidney cells.

A function of serum in the growth medium for primary monkey kidney cells has been shown to be inhibition of proteolytic enzymes. Serum inactivates the residual trypsin remaining from enzymatic digestion of the kidneys and the proteolytic enzymes subsequently synthesized by the cells. Freshly trypsinized cells could be grown to monolayers in the absence of serum provided that they were repeatedly washed to remove residual trypsin. In the absence of serum, cell growth ceased on the 4-5th day after initiation of the culture, at which time the culture fluids became active proteolytically. When the 5th day fluids were replaced with fresh serum-free medium, cell growth was accelerated and a monolayer was attained by the 7th day. If cells were grown in the absence of whole serum but in the presence of medium containing alpha globulins or fetuin which inhibit both trypsin and cell proteases, such cultures grew as well as cultures containing serum. The sterilization of trypsin for use in digestion of tissues and cell cultures poses a serious problem. After filtration through 0.22 micron filters, trypsin preparations may still contain adventitious viruses, mycoplasma and minute forms of pseudomonas and other bacteria or bacteria-produced toxins, which pass the membrane pores. A process of purifying and sterilizing trypsin without deleteriously affecting its proteolytic activity is described.

Animals↗

PubMed1985

Characterization of trypsin stimulation of cardiac adenylate cyclase.

The activity of adenylate cyclase present in a purified dog heart sarcolemmal preparation in the presence of magnesium as cosubstrate is biphasically influenced by increasing concentrations of trypsin: Stimulation at low concentrations (0.5 to 1 microgram/mL) is followed by inhibition at higher concentrations. In the presence of manganese in place of magnesium, the stimulation phase is abolished but the inhibition is still observed at the same trypsin concentrations. The trypsin stimulatory effect does not occur when trypsin is preincubated with cardiac membranes prior to the addition of ATP. When trypsin is added with ATP, the stimulation is expressed by an increase in the Maximal Velocity (Vmax) rather than a decrease in the Michaelis constant (Km). The stimulatory effect of trypsin on AC activity is rapid, linear and irreversible. GTP, Gpp(NH)p and adrenaline stimulatory curves are shifted to the left in the presence of trypsin. These results suggest that protease stimulation of cardiac AC involves the GTP-binding protein (N) activity, but the exact mechanism remains to be determined.

Adenylyl Cyclases↗

PubMed1987

[Trypsin inhibitor activity in the colostrum of cows].

The activity of the colostral trypsin inhibitor (CTI) was studied in seven milkings of colostrum in the course of the first five days after calving in 15 cows. The activity of the total trypsin inhibitor in the colostrum was ascertained in the course of the first two days after calving in ten cows in their second to fourth lactations and also in the colostrum of the first milking after calving in the case of 11 cows. The greatest CTI activity was found in the colostrum of the first milking after calving (301 micrograms X ml-1). Between the CTI activity and the concentration of total protein (CB) in the whey of the colostrum from the first and second day after calving there was found to be a statistically significant correlation (r = +0.929). The activity of total trypsin inhibitor was also highest in the colostrum from the first milking (499 micrograms X ml-1). After calving the CTI and the total trypsin inhibitor activities in the colostrum gradually dropped. Between the activity of the total trypsin inhibitor and the CB concentration in whey from the first, third, fifth and seventh milkings after birth a statistically significant correlation was found (r = +0.972). Statistically significant correlations were also found between the activity of the total trypsin inhibitor, the specific gravity of colostrum, the concentration of total protein and gamma-globulins in the whey of the colostrum from the first milking after calving. In metabolic alkalosis the concentration of total protein in the whey and the activity of trypsin inhibitor were found to be considerably lower than the average values.

Animals↗

PubMed1988

Different sensitivity to trypsin of the human platelet plasma and intracellular membrane Ca2+ pumps.

The Ca2+ pumps associated with human platelet plasma and intracellular membranes have been further characterized by their sensitivity to trypsin. (a) Tryptic degradation of the Ca2+-ATPases has been followed by immunoblotting. It resulted in fragmentation into peptides of 80, 55, 35, and 24 kDa for both enzymes. Subcomplete hydrolysis obtained with a ratio of trypsin/membrane protein of 0.05-0.1 for the two Ca2+ pumps resulted in the total disappearance of the 100-, 80-, and 35-kDa fragments. However, maximum degradation was reached within 1 min for the intracellular enzyme but needed 5 min of incubation for the plasma membrane enzyme. (b) This effect of trypsin has been correlated with its effect on both the Ca2+-ATPase activities. The plasma membrane enzyme showed a maximum inhibition of 50-60% which was obtained using a trypsin/protein ratio of 0.1 and 5 min of incubation. A much higher trypsin sensitivity was observed for the intracellular enzyme because the maximum inhibition reached 80% after only 1 min of incubation. (c) Finally, the two Ca2+ transport systems studied showed different trypsin reactivities; the Ca2+ uptake by the plasma membrane vesicles was inhibited by 20-25%, and this maximum inhibition was observed after 5 min of incubation with trypsin. In contrast, the Ca2+ transport associated with the intracellular membrane vesicles was difficult to detect after trypsin treatment. Taken together, the results show that the two Ca2+ pumps can be distinguished by their trypsin sensitivity.

Animals↗

PubMed1982

In vitro generation of splenic suppressor cells by trypsin.

Rat spleen cells were mitogenically stimulated with concanavalin A (Con A) or sodium periodate and cultured for 14-20 hr with trypsin. When these trypsin-activated cells were co-cultured with fresh mitogenically stimulated cells, [3H]-thymidine incorporation into the fresh cells was suppressed. Artifactual and trivial effects of trypsin (e.g. increased release of cold thymidine, decreased cell viability, and a change in thymidine transport) could not account for the suppressor effect. Suppressor activity was not affected by removing B cells or macrophages before trypsin activation nor by treating the trypsinized cells with mitomycin C. Suppressor activity could only be generated when the spleen cells were stimulated with Con A or periodate during culture with trypsin, and supernates from the trypsin-activated cells did not have suppressor activity. The physiological significance of the results and possible mechanisms of action of the suppressor cells are discussed.

Animals↗