Separation of trypsin and peroxidase by ultrafiltration using crosslinked soybean trypsin inhibitor

The author describes the anatomical findings among the variants of brain arteries in 360 deceased where 160 of them had a hemorrhagic or ischemic stroke, while 200 had no brain diseases. Those cases where strokes were revealed the author found a separation in the posterior part of the circle of Willis--between the carotid and vertebro-basillar systems. This could happen due to an aplasia of one or both posterior connective arteries, or due to the absence of a connecting vessel between an atypically diverging posterior brain artery (posterior trifurcation) and the basillar artery. It is assumed that this circumstance has a definite significance in the pathogenesis of hemorrhages.     

Solution structure of bromelain inhibitor IV from pineapple stem: structural similarity with Bowman-Birk trypsin/chymotrypsin inhibitor from soybean

Bromelain inhibitor VI from pineapple stem (BI-VI) is a unique double-chain inhibitor with an 11-residue light chain and a 41-residue heavy chain by disulfide bonds and inhibits the cysteine proteinase bromelain competitively. The structure of BI-VI in aqueous solution was determined using nuclear magnetic resonance spectroscopy and simulated annealing-based calculations. Its three-dimensional structure was shown to be composed of two distinct domains, each of which is formed by a three-stranded antiparallel beta-sheet. Unexpectedly, BI-VI was found to share a similar folding and disulfide bond connectivities not with cystatin superfamily inhibitors which inhibit the same cysteine proteinases but with the Bowman-Birk trypsin/chymotrypsin inhibitor from soybean (BBI-I). BBI-I is a 71-residue inhibitor which has two independent inhibitory sites toward the serine proteinases trypsin and chymotrypsin. These structural similarities with BBI-I suggest that they have evolved from a common ancestor and differentiated in function during a course of molecular evolution.     

High performance in refolding of Streptomyces griseus trypsin by the aid of a mutant of Streptomyces subtilisin inhibitor designed as trypsin inhibitor

The platelet glycoprotein (GP) IIb/IIIa receptor antagonist appears to reduce the need for revascularization after coronary angioplasty. However, since the effect of GP IIb/IIIa receptor antagonist on the in-stent neointimal thickening has not been clarified, we examined it in the canine model. The beagle dogs were assigned to the control (n=7) or the GP IIb/IIIa receptor antagonist FK633 group (n=7). FK633 was administered by subcutaneous osmotic pumps (0.2 mg. kg-1. h-1) and an intravenous bolus injection (1 mg/kg) before stenting. A coil stent was implanted in the left circumflex coronary arteries. The platelet aggregation capability was significantly (<5%) and consistently reduced by FK633 except for the mild elevation (10% to 30%) on the next day of stenting. Hearts were excised 3 months after stent implantation. The area of intima and media and the area stenosis were obtained from the sections of the stented arteries. The area of intima and media and the area stenosis (1.3+/-0.2 mm2, 41.8+/-7.5% and 1.3+/-0.2 mm2, 33.9+/-6.7% in the FK633 and the control group, respectively) were not different between the groups. We conclude that, although GP IIb/IIIa antagonist FK633 prevented the platelet aggregation significantly and consistently, it could not prevent the neointimal thickening after stent implantation in canine coronary artery.     

The effects of dietary soybean versus skim milk protein on plasma and hepatic concentrations of zinc in veal calves

We assessed the zinc status of veal calves that were fed milk replacers containing either skim milk protein as the sole source of protein or a mixture of skim milk protein and soybean protein. After the milk replacers had been fed for 26 wk, mean body weight gain was 3 kg lower for calves fed the skim milk plus soybean proteins; this decrease was not significant. Inclusion of dietary protein from soybeans versus milk protein alone reduced plasma concentrations of zinc by 43% and reduced hepatic concentrations of zinc by 81%. The impairment of zinc status that was induced by the inclusion of soybean protein was probably caused by its phytate component. The effect of soybean protein on zinc status was rather specific because plasma and hepatic concentrations of copper were unaffected. Despite the high concentration of zinc (142 mg/kg of dry matter) in the milk replacer that contained milk plus soybean proteins, calves displayed a shortage of zinc because their plasma and hepatic concentrations of zinc were significantly reduced.     

Concentrations of trypsin inhibitor and immunoglobulins in colostrum of Jersey cows

Colostrum samples from 49 Jersey cows were analyzed for concentrations of trypsin inhibitor, IgG, IgM, IgA, TS, fat, specific gravity, and N fractions. Colostrum (100 ml) was sampled from each cow as soon as possible after parturition. Mean concentrations of IgG, IgM, and IgA were 84.6, 3.4, and 4.5 g/L, respectively. Mean concentration of trypsin inhibitor was 56 mg of trypsin inhibited/dl of colostrum. Concentration of trypsin inhibitor was unaffected by lactation number and averaged 60, 53, and 54 mg of trypsin inhibited/dl of colostrum for cows in first, second, and third or later lactations, respectively. Colostral trypsin inhibitor and IgG were correlated (.54), although correlations between trypsin inhibitor and IgM and IgA were not significant. Trypsin inhibitor in colostrum was also positively correlated with fat, total N, protein N, noncasein N, and TS in colostrum. Variation in concentration of trypsin inhibitor from first-milking colostrum was closely related to colostral IgG concentration and may serve to protect IgG and other proteins from proteolytic degradation in the intestine of the neonatal calf.     

Protection against preterm delivery in mice by urinary trypsin inhibitor

OBJECTIVE: To investigate the precise mechanism by which urinary trypsin inhibitor suppresses cytokine production in the prevention of preterm delivery. METHODS: In vivo and in vitro studies were performed using ascites and peritoneal macrophages obtained on day 15 of pregnancy from female C3H/HeN mice that had been impregnated by B6D2F1 male mice. Lipopolysaccharide receptor, the intracellular signal transduction system, and nuclear factor-kappaB level were examined. RESULTS: In the in vivo study, we found that urinary trypsin inhibitor ameliorated the deterioration of intraperitoneal conditions induced by lipopolysaccharide (ie, increases in ascitic volume, peritoneal cell count, and tumor necrosis factor-alpha level) and caused a decrease in the binding of lipopolysaccharide to mouse macrophages. In the in vitro studies, urinary trypsin inhibitor decreased the binding capacity of lipopolysaccharide for its receptor, blocked the intracellular signal transduction induced by lipopolysaccharide, and decreased the nuclear factor-kappaB level. Increases were induced in the binding capacity of the macrophages for urinary trypsin inhibitor and its incorporation into them in the presence of lipopolysaccharide. CONCLUSION: We postulate that urinary trypsin inhibitor may suppress the production of inflammatory cytokines induced by lipopolysaccharide in mouse peritoneal macrophages through suppression of the lipopolysaccharide receptor, inhibition of the intracellular signal transduction system, and decrease in the nuclear factor-kappaB level.     

The nature of trypsin-pancreatic trypsin inhibitor binding: free energy calculation of Tyr39-->Phe39 mutation in trypsin

The main goal of this work is the detailed study of the binding interactions in the trypsin-pancreatic trypsin inhibitor (PTI) complex and, here, we present how meaningful the Tyr39-Ile19 interaction is to the stability of that particular complex using free energy methods. This knowledge should be very important in the design of new inhibitors for trypsin and enzymes homologous to it. In particular, it could help to decide whether it is possible to produce selective inhibitors for these enzymes by appropriate mutations of residues in the contact region of PTI.     

Failure of colostral immunoglobulin transfer in calves dying from infectious disease

Serum IgG1 concentrations of calves less than 3 weeks old and dying from infectious disease were significantly lower (P less than 0.01) than those of clinically normal calves. Fifty percent of the dead calves had serum IgG1 concentrations that were more than 2 standard deviations below the normal mean, and an additional 35% had IgG1 concentrations that were more than 1 standard deviation below the normal mean. Low IgG1 concentrations were attributed to failures in passive transfer of colostral immunoglobulin. The few calves dying of noninfectious causes generally had normal serum immunoglobulin concentrations. The results of this study emphasize the importance of adequate colostral intake and absorption to the neonatal calf. In view of the large numbers of calves that die from neonatal infection each year, it may be assumed that failure in passive transfer, as reflected by low serum immunoglobulin concentrations, is one of the most important factors influencing neonatal calf mortality.     

Urinary trypsin inhibitor efficiently inhibits urokinase production in tumor necrosis factor-stimulated cells

We demonstrated that urinary trypsin inhibitor (UTI) efficiently inhibits soluble and tumor cell-associated plasmin activity and subsequently inhibits tumor cell invasion and metastasis. The effect of UTI on tumor necrosis factor-alpha (TNF)-induced stimulation of urokinase-type plasminogen activator (uPA) in cultured human umbilical vein endothelial cells (HUVEC) and in the promyeloid leukemia U937 cells was studied. uPA antigen was evaluated in the cell lysate and in the conditioned media by enzyme-linked immunosorbent assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and Western blot. TNF can promote the production of uPA in HUVEC and in U937 cells. The PKC inhibitors (H7, calphostin C, and staurosporine) inhibited TNF-induced uPA expression and secretion in a dose-dependent manner. Analysis of the expression of cell surface receptor-bound uPA by flow cytometry using uPA-specific MAb indicates that induction of uPA expression by TNF was inhibited when these cells were incubated with UTI. On the other hand, treatment of the cells with UTI alone failed to alter uPA production. UTI also reduced the secretion of uPA in TNF-treated cells. UTI was as effective as PKC inhibitors in inhibiting uPA expression by TNF. Incubation of the cells with UTI, however, had no effect on the ability of PMA to stimulate cell-associated uPA expression. These data suggest that UTI may influence the PKC-dependent protein kinase pathway in uPA expression. The study on intracellular pathways involved in UTI modulation of uPA will enhance our understanding of the role that UTI plays in uPA-mediated cellular invasion.     

Analysis of electrostatic interactions and their relationship to conformation and stability of bovine pancreatic trypsin inhibitor

The modified Tanford-Kirkwood electrostatic theory has been employed to evaluate pK values for all charge sites in the bovine pancreatic trypsin inhibitor (BPTI). 13C NMR titration data were obtained for all titrating groups except arginine residues in BPTI at nearly constant ionic strength in 0.1 M NaCl, at 41 degrees C. The chemical shifts of 46 resonances were found to be sensitive to pH. The pK values of these titrating resonances compared well with those computed by the modified Tanford-Kirkwood electrostatic theory. A conformational change involving the NH2- and COOH-terminal and nearby residues is shown to be partly electrostatically driven by the formation of a salt bridge between the alpha-amino and alpha-carboxyl groups at mid-pH values. The computed total electrostatic free energy of the molecule is found to be stabilizing at neutral pH despite the substantial net positive charge borne by the protein under such conditions.     

Dynamics of the conformational ensemble of partially folded bovine pancreatic trypsin inhibitor

A single-disulfide variant of bovine pancreatic trypsin inhibitor (BPTI), [14-38]Abu, is a partially folded ensemble which includes two, and in one case three, conformations that interconvert slowly enough to exhibit separate cross-peaks in the amide region of homonuclear and heteronuclear NMR spectra. Each conformation is itself composed of many subconformations in rapid equilibrium. Partially folded BPTI undergoes local motions that are slow, noncooperative, independent fluctuations of short segments within the chain. Cooperative global unfolding of the ensemble is also observed. Heteronuclear NMR has been used to measure interconversion rate constants of partially folded conformational substates; the rate constants differ for each residue and vary over an order of magnitude. For local fluctuation, the forward rate constants for amide protons of the antiparallel beta-sheet are significantly smaller than the rest of the molecule, consistent with other indications that this is the most stable part of the partially folded protein. The reverse rate constants also vary; they are the highest for Ala 27 in the turn between the strands in the sheet and for Phe 33 in the antiparallel beta-sheet. Global unfolding interconversion rate constants vary over a 3-fold range, consistent with previously observed deviations from two-state behavior. Fast backbone dynamics, from T1, T2, and NOE relaxation parameters, are obtained for the slowly interchanging conformations in the partially folded ensemble. Clear differences are observed between the two conformations; one is more flexible and less compact than the other. In the more flexible and disordered partially folded conformation, intermediate exchange is detected for some backbone amides, namely, those in the central beta-sheet and the turn. These same sheet and turn residues are more ordered in the globally denatured ensemble as well. Our results suggest that the turn initiates formation of a partially folded ensemble in which the slow-exchange core is the most stable region and in which segmental fluctuations reflect multiple nuclei for folding of the rest of the molecule.     

The deleterious effects of fungicides and herbicides on Xenopus laevis embryos

Groups of 30 Xenopus laevis embryos, at "tail-bud" stage (Nieukoop-Faber stages 22-24) were exposed to 0.1-2 ppm concentrations of various pesticides for 1 to 10 days. The pesticides used were chloranil and dichlone (both are fungicidal and herbicidal); diquat (herbicide); and nabam (fungicide). The parameters examined were mortality, gross morphology, histology, and behavior. Chloranil (1.25 to 1.75 ppm) treated embryos showed abnormalities of the otolith, optic cup, and general pigmentation. Their movement was sporadically convulsive and they were unable to maintain proper balance. Dichlone (0.1 to 0.15 ppm) disrupted the development of the cephalic end of the embryo. Many of these embryos developed a slightly retarded trunk and tail only. These headless embryos lived for a time and were relatively lethargic. Diquat (0.75 to 2.0 ppm) administration reduced body size and pigmentation, and altered body shape. When embryos were treated with both 1.0 ppm of diquat and 2.0 ppm of nabam the integrity of myomeres and myocommata of the musculature was disrupted. The histological bases of these morphological and behavioral changes are discussed.     

Primary structure and possible functions of a trypsin inhibitor of Bombyx mori

A protein with a low molecular mass of 6027 was purified from cocoon shell of silkworm, Bombyx mori. Two-dimensional polyacrylamide gel electrophoresis (2D/PAGE) resolved this protein into a single spot with pI 4.3 and Mr 6000. Amino acid sequence analysis revealed that this protein consists of 55 amino acids, six of these being cysteine residues and is highly homologous to bovine pancreatic trypsin inhibitor-type inhibitors. The 6-kDa protein is heat stable and acid stable and inhibits bovine trypsin by forming a low-dissociation complex with trypsin in a 1 : 1 molar ratio (Ki = 2.8 x 10-10), but does not alpha-chymotrypsin. This cocoon shell-associated trypsin inhibitor (CSTI) was thus concluded to belong to the bovine pancreatic trypsin inhibitor class. CSTI was developmentally regulated in the silk gland at the final stage of larval growth, and its specific distribution in the middle silk gland, an organ in which silk proteins are stored during the final larval instar, occurred before the onset of spinning. This inhibitor protects the tryptic degradation of fibroin light (L) chain in vitro. These results suggest that this trypsin inhibitor may play an important part on regulating proteolytic activity in the silk gland or protecting silk proteins from degradation during histolysis.     

Identification and characterization of the cell-associated binding protein for urinary trypsin inhibitor

Urinary trypsin inhibitor (UTI) inhibits not only tumor cell invasion but also production of experimental and spontaneous metastasis. Cell-binding experiments indicated that human choriocarcinoma SMT-cc1 cells have specific binding sites for UTI on their cell surface. [Kobayashi et al., J. Biol. Chem. 269, 1994, 20,642-20,647]. UTI binding protein (UTIBP) was purified to homogeneity by a combination of UTI-coupled affinity beads, preparative polyacrylamide gel electrophoresis and reverse phase HPLC. This protein is very similar to a truncated form of human cartilage link protein (LP). LP was identified structurally by its apparent molecular mass with and without deglycosylation treatment: Immunologically by the reactivity with anti-UTIBP antibody, and functionally by its ability to bind the NH2-terminal domain of UTI. UTI and UTIBP are distributed uniformly in the cytoplasm and/or over the cell surface of tumor cells and fibroblasts. The level of staining for hyaluronic acid, UTIBP and UTI is much lower in sections digested with hyaluronidase. These results suggest that the cell membrane-derived UTI-associated binding protein is the LP of proteoglycan-hyaluronic acid aggregates, which interacts with hyaluronic acid. Cell-associated LP may play a role in modulating protease activity to the environment close to tumor and fibroblast cell surface.     

Characterization of the interaction between bovine pancreatic trypsin inhibitor and thiocyanate by NMR

The interaction between Bovine Pancreatic Trypsin Inhibitor and thiocyanate was studied using NMR spectroscopy following several experimental approaches. The chemical shift variations of the BPTI protons in the absence and in the presence of increasing thiocyanate concentrations (up to 0.2 M) were significant (> 0.05 ppm) for 30 protein protons belonging to 20 residues. The largest deviation, 0.2 ppm, was observed for the amide backbone proton of Arg42 in the absence of thiocyanate and in the presence of 40 molar equivalents of thiocyanate. The influence of the presence of thiocyanate on the electrostatic potential surrounding the protein was demonstrated by NOESY spectra selective at the water frequency: the presence of SCN- favours acid catalysed exchange and disfavours base catalysis. However, a specific effect of thiocyanate was pointed out since the comparison of the chemical shifts in the presence of 40 molar equivalents of KSCN and KCl, respectively, showed much more as well as larger deviations compared to measurements in the absence of salt. A dissociation constant, KD, for a 1/1 complex between BPTI and thiocyanate was calculated from chemical shifts measurements: KD = 89 +/- 8 mM. A second value, KD = 99 +/- 10 mM, was extracted from SC15N relaxation time measurements.     

Monitoring serum tryptic activity and effect of trypsin inhibitor on rat acute pancreatitis

The effect of a novel synthetic trypsin inhibitor, 4-(2-succinimidoethylthio)-phenyl 4-guanidinobenzoate methanesulfonate (E3123), on severe acute pancreatitis was studied in trypsin-taurocholate-induced acute experimental pancreatitis in rats. Rats were divided into four groups according to difference of subdivided doses of E3123 with fixing the total dose at 3 mg/kg body weight. Group A: 1.5 mg/kg of E3123 subcutaneously (SC) each at 1 h before and after induction of pancreatitis. Group B: 1 mg/kg SC each at 1 h before, 1 and 3 h after induction. Group C: 1.5 mg/kg SC each at 1 and 3 h after induction. Group D: 1.5 mg/kg SC each at 3 and 5 h after induction of pancreatitis. The survival rate at 24 h was significantly improved in group B (77% in group B, vs. 36% in paired control; p < 0.01) and in group C (70 vs. 38%; p < 0.05), but not in group A or D. Residual tryptic activity of serum alpha 2-macroglobulin trypsin complex (alpha 2M-TRY) was reduced after the injection of E3123 though immunoreactive trypsin remained unchanged in the early phase of pancreatitis. The reduction of alpha 2M-TRY reflected the inhibitory capacity of E3123 in plasma. E3123 showed favorable effects on the initial stage of severe acute pancreatitis and the effects were probably based on the inhibition of alpha 2M-TRY activity in serum.     

Isolation and characteristics of a new trypsin and chymotrypsin inhibitor from potato tubers

A novel trypsin and chymotrypsin inhibitor has been isolated from potato (Solanum tuberosum L.) tubers. The isolation procedure included ammonium sulfate precipitation, gel-chromatography on Sephadex G-75 and ion-exchange chromatography on DEAE-cellulose. The inhibitor interacts with trypsin and chymotrypsin at a molar ratio of 1:1. The substrate-dependent dissociation of the enzyme-inhibitor complexes is observed. The inhibitor displays no activity towards subtilisin and pancreatic elastase. The ability of the inhibitor to form a ternary complex containing simultaneously both trypsin and chymotrypsin molecules testifies to the presence of two independent reactive sites for these enzymes.