Tumor-associated trypsin inhibitor (TATI) and tumor-associated trypsin-2 (TAT-2) predict outcomes in gastric cancer
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Introduction: Tumor-associated trypsin inhibitor (TATI) limits serine proteases, promotes carcinogenesis in several cancers and functions as an acute-phase reactant. Tumor-associated trypsin-2 (TAT-2), a proteolytic target enzyme for TATI, can enhance invasion by promoting extracellular matrix degradation. Here, we aimed to study serum TATI and TAT-2 levels, including the TAT-2/TATI ratio, as prognostic and diagnostic biomarkers in gastric cancer. We compared the results with the plasma level of C-reactive protein (CRP). Material and Methods: We selected 240 individuals operated on for gastric adenocarcinoma at the Helsinki University Hospital, Finland, between 2000 and 2009. We determined the preoperative serum TAT-2, TATI and plasma CRP levels using time-resolved immunofluorometric assays using monoclonal antibodies. Results: The medium serum TAT-2 level was higher among gastric cancer patients [8.68 ng/ml; interquartile range (IQR) 5.93–13.2] than among benign controls (median 5.41 ng/ml; IQR 4.12–11.8; p = .005). Five-year survival among patients with a high serum TAT-2 was 22.9% [95% confidence interval (CI) 11.7–34.1], compared to 52.2% (95% CI 44.6–59.8; p p p = .037). Conclusions: This study shows for the first time that a high serum TAT-2 may function as a prognostic biomarker in gastric cancer and that TAT-2 levels may be elevated compared to controls. Additionally, we show that the prognosis is worse among gastric cancer patients with a high serum TATI. These biomarkers serve as prognostic factors particularly among patients with a metastatic or a locally advanced disease.Keywords:
Trypsin inhibitor
In a previous report(l) we showed that trypsin (1-300) fed at 0.15% of the diet neutralized most of the effect of the growth inhibitor present when one-fourth of the protein in the diet of the chick was provided as raw soybean meal. This relative amount of raw meal caused practically as much growth inhibition as did higher proportions. It has been indicated that the substance in crude trypsin, which is able to reverse the action of the soybean growth inhibitor in rat diets, is not trypsin but some associated impurity(2). In a critical test of this question we have fed crystalline lyophilized trypsin in chick diets containing raw soybean protein. Methods. The procedure was the same as previously described(l). Chicks were carefully selected for uniform size and vitality and then divided equally into pens of 9 each. Results. The results of 2 experiments are given in Table I. These show a distinct increase in weight gains when the crystalline trypsin was added. Discussion. It is not certain that the amount of trypsin added was optimal, but this amount was clearly sufficient to demonstrate the anti-inhibitor activity of highly purified trypsin. We have shown that a low level of trypsin may exert no discernible effect, presumably because a low level may be completely inactivated in the presence of a surplus of the antitrypsin known to be present in the raw meal(1). Enough trypsin must be added to neutralize the antitrypsin to the extent that proteolytic enzymes secreted by the animal may be allowed a moderate to complete freedom of action.
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Abstract Cultivar differences in the heat deactivation temperature of trypsin inhibitors in an extract of chopped tissue were examined. In addition, the distribution of trypsin inhibitor activity in the root and existence of different trypsin inhibitor proteins within and among 4 cultivars were investigated. There was a significant cultivar-by-temperature interaction during the heat deactivation test. Activity in ‘Jewel’ was decreased by 50% at a significantly lower temperature (65°C) than the other cultivars (75°) indicating that ‘Jewel’ contains more heat labile trypsin inhibitors. The longitudinal distribution of trypsin inhibitor activity was dependent on the cultivar. All cultivars had increased levels of trypsin inhibitor activity in the proximal or stem end. Sectioning of roots, in order to eliminate high trypsin inhibitor activity, would not be reliable since there is a cultivar-by-section interaction, and the gradients are not large enough to eliminate the majority of trypsin inhibitor activity by removal of one section. The cross-sectional gradient of trypsin inhibitor activity in the roots also depended on the cultivar. There was a high level of trypsin inhibitor activity in the cortical region of all cultivars. The concentration (over 50%) of trypsin inhibitor activity in the cortical area of the root in ‘Jewel’ and ‘Caromex’ could be important when heat deactivating the whole root. Disc-gel electrophoresis revealed that the trypsin inhibitor fraction isolated via affinity chromatography was heterogenous. There were 7 different trypsin inhibitor bands after electrophoresis at pH 8.9 in 7.5% acrylamide gels. The quantitative distribution of these seven trypsin inhibitor proteins differed among cultivars. These different distributions may be responsible for the cultivar differences previously observed.
Trypsin inhibitor
Plant Physiology
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Abstract For expressing trypsin inhibitor activity (TIA), trypsin units inhibited (TUI), trypsin inhibited, and trypsin inhibitors have been used. Although the last two units are preferred, their calculations in current practices require refinement. With the proposed AOCS method Ba 12a‐2020, four experiments were conducted, using four trypsin preparations having specific activity of 11,625, 12,602, 13,728, and 14,926 Nα ‐benzoyl‐L‐arginine ethyl ester (BAEE) units/mg protein, respectively. Experiment 1 determined the relationship between absorbance at 410 nm (A410) and trypsin concentration. Experiment 2 involved assaying raw and heated soybeans, expressing TIA as TUI/mg sample and μg trypsin inhibited/mg sample, and determining conversion factors between the two units. Experiment 3 resembled Experiment 2 except for using purified soybean Kunitz inhibitor (KTI) and Bowman‐Birk inhibitor (BBI). Conversion factors determined correlated highly with trypsin‐specific activity (R 2 = 0.9789). After standardizing against a reference trypsin having 15,000 BAEE units/mg protein, a standardized conversion factor of 0.03 A410 (1.5 TUI) = 1 μg trypsin inhibited was determined. It remained consistent regardless of trypsin specific activity, with or without inhibitors, and type of inhibitor samples. By using purified inhibitors (Experiment 3), conversion values between TUI and μg trypsin inhibitor and between μg trypsin inhibited and μg trypsin inhibitor could also be calculated, enabling expression of TIA in amounts of pure KTI, BBI or their equivalents. Furthermore, when the AOCS method was modified with half substrate concentration, half trypsin concentration or half both (Experiment 4), TIA values in TUI could change with modifications but values in mg trypsin inhibited (standardized) or trypsin inhibitor remained consistent.
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Trypsin inhibitor was isolated from the vegetative portion of alfalfa and purified 270-fold by affinity chromatography on Trypsin-Sepharose. The inhibitor was eluted by gel-filtration as a single peak with molecular weight of 6900. Disc-electrophoresis of the purified inhibitor revealed the presence of only one protein band. Trypsin inhibition is a mixed process. The trypsin inhibitor from alfalfa does not prevent the activity of cathepsin D from bovine brain. Trypsin inhibitor was immobilized on BrCN-activated Sepharose 4B. The binding of trypsin to the immobilized trypsin inhibitor was studied: 5 mg of the immobilized trypsin inhibitor were found to bind 1 mg of trypsin.
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Trypsin inhibitor
Denaturation (fissile materials)
Electroblotting
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Trypsin inhibitor
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Trypsin inhibitor
Aleurone
Cotyledon
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A rice trypsin inhibitor with molecular of around 18 KDa has been obtained through cationic and gel filtration columns from coleoptiles grown under submerge condition. This trypsin inhibitor was further characterized toward proteases of chymotrpsin and trypsin. It was found that the inhibition mode when competing with substrate L-N-α-benzoyl-arginine-para- nitroanilide (L-BAPNA) toward chymotrypsin was not a typical competitive mode. However, the inhibition mode when competing with L-BAPAN toward trypsin was found a typical competitive mode as that of soybean trypsin inhibitor. The EI complex dissociation constant, Kd, for rice trypsin inhibitor, toward trypsin was 4.0 x 10-7M, while it was 7.4 x 10-7M for soybean trypsin inhibitor. When the molar ratio of Rice trypsin inhibitor to trypsin was about 0.3, 50% of trypsin activity had been inhibited; while it was about 0.5 for soybean trypsin inhibitor. This study shows that rice trypsin inhibitor has better inhibition activity than soybean trypsin inhibitor does toward trypsin. Thus, it would be interesting and important to investigate further in the application of this inhibitor in medicinal and food chemistry.
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