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Thrombin and trypsin induce cell signaling through a subclass of G-protein-coupled receptors called the protease-activated receptors (PARs). In many cells, PAR signaling results in the activation of RhoA and other members of the Rho family of small GTPases which are involved in cytoskeletal reorganization. The expression of PARs and their role in the activation of Rho GTPases in prostate cancer cells are not clearly known. FACS analysis demonstrated that the androgen-dependent LNCaP cells express PAR1, PAR2, and PAR4 but not PAR3. Stimulation with thrombin and trypsin resulted in the rapid activation of RhoA in a dose-dependent manner with an EC(50) of 1.0 and 5 nM, respectively. Activation of RhoA was enhanced by, but not dependent on, the presence of 1 nM dihydrotestosterone. Inhibition of the proteolytic properties of thrombin by hirudin and trypsin by diisopropyl fluorophosphate abolished the observed RhoA activation. Stimulation with 150 microM PAR-activating peptides TFFLRN (PAR1), SLIGKV (PAR2), and AYPGKF (PAR4) demonstrated that PAR1 and PAR2 mediated protease-activated RhoA signaling. Fluorescent microscopy studies showed that LNCaP cells treated with either thrombin (10 nM) or trypsin (10 nM) developed an increased number of filopodia, stress fibers, and focal adhesions relative to untreated cells. These observations represent the first report of PAR signaling in prostate cancer cells as well as the ability of PAR2 to mediate RhoA activation. Since the activation of RhoA is important for cytoskeletal reorganization, we postulate that PAR-mediated RhoA activation may be a major signaling pathway in the biology of prostate cancer.

Gingipains are trypsin-like cysteine proteinases produced by Porphyromonas gingivalis, a major causative bacterium of adult periodontitis. HRgpA (95 kDa) and RgpB (50 kDa), products of 2 distinct but related genes, rgpA and rgpB, respectively, are specific for Arg-Xaa peptide bonds. Kgp, a product of the kgp gene, is specific for Lys-Xaa bonds. HRgpA and Kgp are non-covalent complexes containing separate catalytic and adhesion/ hemagglutinin domains, while RgpB has only a catalytic domain with a primary structure essentially identical to that of the catalytic subunit of HRgp. HRgpA and RgpB induce vascular permeability enhancement through activation of the kallikrein/kinin pathway and activate the blood coagulation system, which, respectively, are potentially associated with gingival crevicular fluid production and progression of inflammation leading to alveolar bone loss in the periodontitis site. Kgp is the most potent fibrinogen/fibrin degrading enzyme of the 3 gingipains in human plasma and is involved in the bleeding tendency at the diseased gingiva. HRgpA activates coagulation factors and degrades fibrinogen/fibrin more efficiently than RgpB due to the adhesion/hemagglutinin domains, which have affinity for phospholipids and fibrinogen. Gingipains degrade macrophage CD14, thus inhibiting activation of the leukocytes through the lipopolysaccharide (LPS) receptor, and thereby facilitating sustained colonization of P. gingivalis. Gingipains play a role in bacterial housekeeping and infection, including amino acid uptake from host proteins and fimbriae maturation. Based on the important activities of gingipains in the bacterial infection and the pathogenesis of periodontitis, the bacterial proteinases can be targets for periodontal disease therapy. Immunization with RgpB, HRgpA, or a portion of HRgpA catalytic domain attenuated P. gingivalis induced disorders in mice. In addition, a trypsin-like proteinase inhibitor retarded P. gingivalis growth specifically. Gingipains are potent virulence factors of P. gingivalis, and are likely to be associated with the development of periodontitis. It is, therefore, suggested that gingipain inhibition by vaccination and gingipain-specific inhibitors is a useful therapy for adult periodontitis caused by P. gingivalis infection.

The cytoplasmic trafficking of the prototype strain of minute virus of mice (MVMp) was investigated by analyzing and quantifying the effect of drugs that reduce or abolish specific cellular functions on the accumulation of viral macromolecules. With this strategy, it was found that a low endosomal pH is required for the infection, since bafilomycin A(1) and chloroquine, two pH-interfering drugs, were similarly active against MVMp. Disruption of the endosomal network by brefeldin A interfered with MVMp infection, indicating that viral particles are routed farther than the early endocytic compartment. Pulse experiments with endosome-interfering drugs showed that the bulk of MVMp particles remained in the endosomal compartment for several hours before its release to the cytosol. Drugs that block the activity of the proteasome by different mechanisms, such as MG132, lactacystin, and epoxomicin, all strongly blocked MVMp infection. Pulse experiments with the proteasome inhibitor MG132 indicated that MVMp interacts with cellular proteasomes after endosomal escape. The chymotrypsin-like but not the trypsin-like activity of the proteasome is required for the infection, since the chymotrypsin inhibitors N-tosyl-L-phenylalanine chloromethyl ketone and aclarubicin were both effective in blocking MVMp infection. However, the trypsin inhibitor Nalpha-p-tosyl-L-lysine chloromethyl ketone had no effect. These results suggest that the ubiquitin-proteasome pathway plays an essential role in the MVMp life cycle, probably assisting at the stages of capsid disassembly and/or nuclear translocation.

Midgut proteolytic enzymes contribute to the success or failure of Plasmodium infection of the mosquito. The present study investigated trypsin and aminopeptidase activities in the midgut of two strains of Anopheles dirus selected for susceptibility and refractoriness to Plasmodium yoelii nigeriensis. At intervals of 6 hours following a bloodmeal, the midguts of fully engorged female mosquitos were dissected, homogenized, and assayed for enzyme activity. No differences trypsin activity (nmole/min) were observed between the two strains throughout the course of blood digestion. By contrast, the aminopeptidase activity measured at 0 to 18 hours post-feeding was the same for the two strains, but at 24, 30 and 36 hours significantly less activity was observed in the refractory females. The results suggest neither trypsin nor aminopeptidase plays a role in the limitation of parasite development.

A clade of trypsins, known as group III, is identified by phylogenetic analysis. These trypsins occur in fish that spend all or part of their lives at temperatures near 0 degrees C and may represent extreme psychrophilic enzymes. A principal component analysis of amino acid compositions distinguishes group III from mesophilic trypsins, as do molecular trees and multidimensional scaling of molecular sequence distances. The primary sequences of group III trypsins, in conjunction with the known structures of mesophilic trypsins, permit insight into function and mechanisms of cold adaption. The techniques employed are broadly applicable to phylogenies characterized by a markedly different, or "fast-evolving," clade. An updated lactate dehydrogenase molecular tree illustrates an additional fast-evolving clade., (Copyright 2002 Wiley-Liss, Inc.)

Trypsin was isolated from ovine and porcine pancreas using affinity chromatography on immobilized p-aminobenzamidine. Molecular masses of the two proteins were 23900 and 23435 Da, determined by matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometry. The purified trypsins were compared using the kinetic properties K(m) and k(cat) which were determined at pH 8.0 and between 25 and 55 degrees C. Comparison of the Michaelis constants for ovine and porcine trypsins toward N-alpha-benzoyl-arginine-p-nitroanilide (BapNA) indicated that ovine trypsin had higher affinity for this substrate than the porcine enzyme. The rates of the reactions catalysed by the two enzymes correlated strongly over the range of temperatures and substrate concentrations tested, as did the k(cat) values. The specific activity of ovine trypsin for BapNA was, on average, approximately 10% higher than that of the porcine enzyme over the range of conditions tested. Porcine trypsin was less susceptible to denaturation at low pH or high temperature than was ovine trypsin. Porcine and ovine trypsin produced seven identically sized fragments from auto-catalytic hydrolysis. Proposed regions of identity between ovine and porcine trypsins were I(54)-K(77), L(98)-R(107), S(134)-K(178) and N(209)-K(116). Hydrolysis of beta-lactoglobulin, egg white lysozyme or casein by ovine or porcine trypsin yielded virtually identical patterns of fragments although the rate at which fragments were produced, in the case of beta-lactoglobulin, differed between the two enzymes. On balance the two enzymes appear to be functionally identical in their action.

Intestinal inflammatory disease or infection often results in the loss of the epithelial layer as a result mainly of the action of proteases, including the leucocyte serine proteinases (neutrophil elastase), lysosomal cathepsins and the matrix metalloproteinases from recruited inflammatory cells. Previous studies have shown that bronchial or intestinal epithelial cells (IEC) can respond to proteolytic attack by producing cytokines. In this study, we have determined the effect of protease treatment on interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) production by IEC lines. Both neutrophil elastase and trypsin treatment induced elevated levels of mRNA for IL-6 in rat IEC-6 cells. Non-proteolytic detachment of the IEC-6 cells also induced elevated levels of IL-6 mRNA, suggesting that the effect was not caused by a specific protease or degradation product, but probably by an effect on cell shape or cell detachment. Similar results were seen with the IEC-18 cell line. Trypsin treatment of the IEC-6 cells also enhanced unstimulated and IL-1 beta costimulated IL-6 secretion, but not MCP-1 secretion or mRNA levels. Finally, nuclear levels of the CCAAT/enhancer binding protein-beta (C/EBP-beta) were rapidly enhanced after proteolytic detachment of the IEC-6 cells, suggesting a mechanism for the enhancement of IL-6 mRNA responses. These data indicate that epithelial cells can respond to proteolytic attack or cell detachment by producing IL-6, a cytokine with several anti-inflammatory and antiprotease effects, which may be important in moderating the loss of the epithelial layer by its effects on nearby epithelial or inflammatory cells.

Background: Mechanisms regulating neuronal migration during development remain largely undefined. Extracellular matrix cues, target site released factors, and components of the migratory neurons themselves are likely all coordinated in time and space directing neurons to their appropriate locations. We have studied the effects of proteases and their inhibitors on the extracellular matrix and the consequences to the migration of gonadotropin releasing hormone (GnRH) neurons in the embryonic chick. Chick GnRH neurons differentiate in the olfactory epithelium, migrate along the olfactory nerve and enter the forebrain. The accessibility of this coherent cell group make it amenable for studying protease/inhibitor roles in migratory processes., Results: Affigel blue beads were used to deliver a serine protease inhibitor, protease nexin-1 (PN-1), and a target protease, trypsin, to the olfactory epithelium coincident with initiation of GnRH neuronal migration. PN-1 inhibited neuronal migration while trypsin accelerated their transit into the CNS. Prior to initiation of migration, neither PN-1 nor trypsin altered the timing of neuronal exit. Trypsin did, however, accelerate the timing of neuronal crossing into the nerve-forebrain junction., Conclusions: These data support the hypothesis that protease activity modulates neuronal movements across barriers. Moreover, the data suggest, for the first time, that aspects of GnRH neuronal migration may be cell autonomous but modulated by ECM alterations.

Background: Tissue injury mediated by matrix metalloproteinases (MMPs) is a hallmark of inflammatory lung diseases. Latent secreted proMMPs must be activated to be catalytically competent., Aim: Our aim was to analyse an involvement of the trypsin-2, trypsin-2-alpha1-proteinase inhibitor (PI) complex and tumour-associated trypsin inhibitor (TATI) in the in vivo activation of proMMP-8, -9 and -2., Methods: Concentrations of trypsin-2, trypsin-2-alpha1-PI complex and TATI in bronchoalveolar lavage fluid (BALF) were analysed by immunofluorometry. Molecular forms and expression of trypsin-2 and trypsin-2-alpha1-PI complex were identified by Western immunoblot and immunocytochemistry. Gelatinolytic and collagenolytic activities were measured by substrate-based activity assays., Results: BALFs from 16 of 43 patients and BALFs from five of 15 healthy controls contained trypsin-2-alpha1-PI complex. TATI was found in all healthy control BALFs (median 0.12 microg/L, range 0.02-0.66 microg/L) whereas 8 of 43 BALFs from patients (median 0, range 0-0.64 microg/L, P = 0.0001) contained TATI. Patient BALFs showed significantly increased activation of MMP-9 and MMP-8 compared with healthy controls. The concentrations of trypsin-2-alpha1-PI complex correlated with the in vivo activation of MMP-9 and -8 (r = 0.68, P = 0.002 and r = 0.61, P = 0.008) but not with the activation of MMP-2 in BALFs., Conclusion: Results show a key role of trypsin-2 in the in vivo activation of proMMP-8 and -9 in inflammatory lung diseases.

Transcription factor nuclear factor-kappaB (NF-kappaB) is activated in cerulein pancreatitis and mediates cytokine expression. The role of transcription factor activation in other models of pancreatitis has not been established. Here we report upregulation of NF-kappaB and inflammatory molecules, and their correlation with local pancreatic injury, in a model of severe pancreatitis. Rats received intraductal infusion of taurocholate or saline, and the pancreatic head and tail were analyzed separately. NF-kappaB and activator protein-1 (AP-1) activation were assessed by gel shift assay, and mRNA expression of interleukin-6, tumor necrosis factor-alpha, KC, monocyte chemoattractant protein-1, and inducible nitric oxide synthase was assessed by semiquantitative RT-PCR. Morphological damage and trypsin activation were much greater in the pancreatic head than tail, in parallel with a stronger activation of NF-kappaB and cytokine mRNA. Saline infusion mildly affected these parameters. AP-1 was strongly activated in both pancreatic segments after either taurocholate or saline infusion. NF-kappaB inhibition with N-acetylcysteine ameliorated the local inflammatory response. Correlation between localized NF-kappaB activation, cytokine upregulation, and tissue damage suggests a key role for NF-kappaB in the development of the inflammatory response of acute pancreatitis.

Among periodontal anaerobic pathogens, the oral spirochetes, and especially Treponema denticola, have been associated with periodontal diseases such as early-onset periodontitis, necrotizing ulcerative gingivitis, and acute pericoronitis. Basic research as well as clinical evidence suggest that the prevalence of T denticola, together with other proteolytic gram-negative bacteria in high numbers in periodontal pockets, may play an important role in the progression of periodontal disease. The accumulation of these bacteria and their products in the pocket may render the surface lining periodontal cells highly susceptible to lysis and damage. T. denticola has been shown to adhere to fibroblasts and epithelial cells, as well as to extracellular matrix components present in periodontal tissues, and to produce several deleterious factors that may contribute to the virulence of the bacteria. These bacterial components include outer-sheath-associated peptidases, chymotrypsin-like and trypsin-like proteinases, hemolytic and hemagglutinating activities, adhesins that bind to matrix proteins and cells, and an outer-sheath protein with pore-forming properties. The effects of T. denticola whole cells and their products on a variety of host mucosal and immunological cells has been studied extensively (Fig. 1). The clinical data regarding the presence of T. denticola in periodontal health and disease, together with the basic research results involving the role of T. denticola factors and products in relation to periodontal diseases, are reviewed and discussed in this article.

Acute pancreatitis represents a local inflammatory disorder with severe systemic consequences. Significant progress in understanding the pathophysiology of acute pancreatitis has been achieved in recent years. However, there is no clear concept about initialization and propagation of the disease both in experimental models and in humans. Furthermore, reliable strategies to evaluate prognosis and perform therapy are still missing. The review focuses on mechanisms originating from acinar cells leading to a systemic inflammatory response in experimental pancreatitis.

The triggering events by which mononuclear cells throughout the body are induced to produce large amounts of cytokines during acute pancreatitis are unclear. However, recent work in our laboratory demonstrated that three specific pancreatic enzymes (elastase, carboxypeptidase A, and lipase) induced dramatic tumor necrosis factor-alpha (TNF-alpha) protein production from macrophages, whereas all others could not. This series of experiments was designed to examine the second messenger system by which this occurs. The rat macrophage cell line NR8383 was incubated for 3 hours with elastase, carboxypeptidase A, lipase, trypsin, or lipopolysaccharide (positive control). Activation of nuclear factor kappa B (NF-kappa B) was demonstrated by electrophoretic mobility shift assay, presence of inhibitory kappa B alpha and beta (I kappa B-alpha and I kappa B-beta) by Western blot analysis, and TNF-alpha protein production by enzyme-linked immunosorbent assay. Elastase, carboxypeptidase A, and lipase induced degradation of I kappa B-beta (but not I kappa B-alpha), activation of NF-kappa B, and production of TNF-alpha protein, whereas inhibition of I kappa B with pyrrolidine dithiocarbamate attenuated this response. Trypsin was unable to elicit any of these responses. Macrophages can be induced by specific activated pancreatic enzymes-elastase, carboxypeptidase A, and lipase-to produce TNF-alpha. This process is dependent on I kappa B-beta degradation and NF- kappa B activation, suggesting that these enzymes trigger this second messenger system through specific membrane-bound receptors.

In a number of chronic inflammatory conditions resulting in fibrosis, perivascular mononuclear cell infiltration including mast cells (MC) has been shown before the onset of vascular injury and interstitial fibrosis. These observations suggest a role for MC in inducing endothelial cell (EC) injury and fibroblast (FB) proliferation and collagen synthesis. In view of these observations, the interactions of MC with EC and FB were studied. MC adhesion to EC and FB showed time-dependent increase reaching a plateau at 1 and 3 hrs, respectively. With added MC, the proliferation of EC showed a dose-dependent decrease, but that of FB, a dose-dependent increase. MC, MC surpernatant and sonicated MC induced dose-dependent cytotoxic activity to EC, whose cytotoxicy was inhibited by trypsin inhibitor. FB cocultured with MC showed 9.95 times collagen synthesis and 11.0 times protein synthesis compared with FB without MC. These results showed that 1) MC attached to EC inhibited the proliferation by cytotoxic activity to EC, which was due to a kind of proteolytic enzyme involving trypsin, 2) MC had proliferative and collagen synthetic activity to FB. These results suggest the possibility that MC have a role in a number of chronic inflammatory diseases resulting in vascular injury and interstitial fibrosis.

Pancreaticobiliary diversion (PBD) and biliodigestive shunt (BDS) cause long-standing hypercholecystokininemia followed by pancreatic hyperplasia. These changes have been suggested to be due to the lack of intraluminal trypsin and bile, respectively, in the upper small intestine. The aim of these experiments was to study the effect of restoration of intraluminal trypsin and bile on plasma levels of cholecystokinin (CCK) and the changes found in exocrine and endocrine pancreas after PBD and BDS. Male Sprague-Dawley rats were used. PBD was done in 16 rats, eight of which had trypsin dissolved in 50 mM sodium bicarbonate (SB), and eight had SB only by gastric intubation twice daily. BDS was done in another 16 rats, eight of which had bile dissolved in SB, and eight had SB in a similar manner. Sham-operated rats had SB and served as controls. After 4 weeks, the rats were killed, and the concentrations of circulating CCK, gastrin, glucose, glucagon, and insulin were determined. The pancreas was removed, weighed, and analyzed for contents of water, protein, and DNA. In another study, PBD-operated rats got trypsin in varying dosages or trypsin and taurocholate in combination for 2 weeks before death. The concentrations of plasma CCK and glucagon were elevated after both PBD and BDS. PBD decreased the concentration of gastrin in plasma. PBD caused an increase of pancreatic weight and the contents of protein and DNA. Trypsin substitution to PBD-operated rats did not affect plasma CCK or glucagon levels, but the PBD-induced increases in weight and DNA content were counteracted by trypsin. Higher dosages of trypsin did not further influence the effects seen after PBD. Pancreatic weight and DNA content were increased after BDS. Bile administration completely abolished the increase in plasma CCK and glucagon, as well as the gain in pancreatic weight, and reduced the increase in pancreatic DNA. Substitution with bile to BDS-operated rats abolished the increase in the plasma levels of CCK and glucagon, as well as the trophic effects on the pancreas. Trypsin substitution to PBD-operated rats partly reversed the trophic effects on the pancreas but not the hormonal changes in plasma. Thus the trophic effects on the pancreas exerted by BDS seem to be dependent on the lack of bile in the upper small intestine, whereas the effects of PBD only partly are a consequence of the absence of intraluminal trypsin.

The basal exocrine secretion of the pancreas is maintained at a constant level in conscious rats. We examined the changes in basal secretion with respect to the effect of various time periods of pancreatic duct occlusion (PDL). Male Wistar rats were prepared with cannulae that separately drained bile and pancreatic juice as well as with a duodenal cannula. Rats were placed in restraint cages, and experiments were conducted without anesthesia 4 days after the operation. Cholecystokinin (CCK) release was artificially prevented by the continuous infusion of bile with trypsin into the duodenal lumen throughout the experimental period to avoid the modification of pancreatic response by CCK. After 2-h basal collection, a pancreatic secretion was interrupted for 0.5-4 h, and then the collection of pancreatic juice was initiated again for an additional 2-4.5 h. The pancreatic secretion after the reopening of the 0.5-to 3-h PDL was comparable to basal secretion levels. However, protein secretion was significantly inhibited after the removal of 4-h PDL. Both vagotomy and capsaicin treatment abolished this inhibition, and the protein secretion after 1-h PDL in vagotomized rats increased 1.5-fold high compared with the basal value. These observations indicate that protein secretion was ceased during PDL via vagal nerve, and this may be a self-protective mechanism.

The attachment of two strains of Mycoplasma bovoculi to erythrocytes was measured using 35S-methionine-labelled organisms. Receptor sites of M. bovoculi involved in this attachment are trypsin-sensitive, since mild trypsin treatment of the intact organisms abolished this process completely. Pretreatment of erythrocytes with trypsin or increasing concentrations of neuraminidase resulted in no measurable effect. Monoclonal antibody MA25.5 directed against a M. bovoculi surface antigen of 94 kDa termed p94 blocked 40% of the attachment, while MA18.13 directed against a 57 kDa protein band of M. bovoculi had no effect on the attachment process. Other properties of M. bovoculi were tested using six strains of the mycoplasma and erythrocytes from several animal species. None of the strains showed haemagglutinating or haemadsorbing activities.

The contractile actions of the proteinase-activated receptor-2-activating peptides (PAR2APs), SLIGRL-NH2 (SL-NH2), SLIGKV-NH2 (KV-NH2), trans-cinnamoyl-LIGRLO-NH2 (tc-NH2), and the PAR1-AP. TFLLR-NH2 (TF-NH2) as well as trypsin and thrombin were studied in endothelium-denuded and intact human umbilical vein (HUV) ring preparations. In HUV rings with, but not without an intact endothelium, PAR2APs caused a concentration-dependent contractile response, whereas LSIGRL-NH2 trypsin and PAR1APs were inactive. The contractile response was not affected by the endothelin ETA receptor antagonist, BQ123, the cyclooxygenase inhibitor, indomethacin, the leukotriene synthesis inhibitor, MK886, or the epoxygenase/P450 inhibitor, SKF-525A. Other pharmacological antagonists (prazosin, Losartan") were similarly inactive. The order of potencies of the PAR2APs to cause a contraction in the endothelium-intact preparation was: SL-NH2 > > KV-NH2 > or = tc-NH2. Using an endothelium-free rat aorta ring as a reporter tissue, surrounded with endothelium-intact HUV as a donor tissue in a 'sandwich assay,' we also monitored the ability of SL-NH2, TF-NH2, trypsin and thrombin to release either contractile (EDCF) or relaxant (EDRF) factors. In the 'sandwich assay' done in the presence of L-NAME (0.1 mM), the endothelium-intact HUV tissue (but not endothelium-denuded HUV) released a contractile factor (EDCF) in response to SL-NH2 (50 microM) but not to trypsin or LSIGRL-NH2. The SL-NH2-mediated release/action of the EDCF was not affected by BQ123, indomethacin, MK886 or SKF-525A. In the 'sandwich assay', trypsin (4-10 nM), SL-NH2, KV-NH2 and tc-NH2 caused the release of a relaxant activity (EDRF) from the endothelium-intact (but not the denuded) HUV preparation. The release of EDRF was blocked by 0.1 mM (omega)nitro-L-arginine-methylester (L-NAME). Neither thrombin (10 u ml(-1), 100 nM) nor TF-NH2 (50 microM) were active in this EDRF-release assay. The relative potencies of the PAR2 agonists for causing the release of EDRF in the HUV sandwich assay were: trypsin> >SL-NH2> >tc-NH2>KV-NH2. This order of potencies differed from the one observed for the same agonists in the HUV contraction assay (above) and in an intracellular calcium signalling assay, conducted with cloned human PAR2 that was expressed in cultured rat kidney KNRK cells: trypsin > > SL-NH2 = tc-NH2 > KV-NH2. We conclude that PAR2APs (but not PAR1APs) via a receptor distinct from PAR2, can cause a contractile response in endothelium-intact HUV tissue via the release of a diffusable EDCF, that is different from previously recognized smooth muscle agonists (e.g. prostanoid metabolites, endothelin, noradrenaline, angiotensin-II, acetylcholine).

It has previously been reported that the trypsinogen gene is expressed in various human cancers. To investigate the possible role of trypsin in tumor malignancy, trypsinogen-1 cDNA was introduced into the human gastric carcinoma cell line MKN-1. The overexpression of trypsinogen-1 in MKN-1 cells stimulated cellular growth and adhesion to fibronectin and vitronectin when the trypsinogen activator enterokinase was added into the culture. Enterokinase treatment of the conditioned medium of the MKN-1 transfectants partially converted the proforms of gelatinases B and A to their apparent active forms. When the MKN-1 transfectants expressing trypsinogen-1 were intraperitoneally transplanted into nude mice, the mice frequently produced tumors in the colon, spleen and liver. However, the mice implanted with control MKN-1 cells produced no tumors. These results strongly suggest that tumor-derived trypsin contributes to the disseminated growth of some types of cancer cells including gastric cancer.

For over a century it has been assumed that acute pancreatitis represents an autodigestion of the pancreas by its own, physiologically inactive proteases. Whether, how and where digestive proteases are being activated in the pancreas has remained the topic of much controversy and speculation. We review a number of recent studies that have been undertaken to elucidate the mechanisms and identify the initial subcellular localization of this process. These studies suggest that a premature and intrapancreatic protease activation does, indeed, occur early in pancreatitis and can be experimentally induced in vivo and in vitro. Activation begins within minutes of the induction of pancreatitis and is initially confined to cytoplasmic vacuoles at the apical pole of acinar cells. From here trypsin activity as well as its activation peptide are transferred to the cytosol of acinar cells where autodigestion may begin.

For an effective CD8+ cytotoxic T cell response to occur during infection, MHC class I molecules must be loaded with antigenic peptides in the endoplasmic reticulum. The cytosolic factor responsible for peptide generation is believed to be the proteasome, with the TAP heterodimer mediating peptide transport into the endoplasmic reticulum. However, the rate-determining step(s) in this intracellular pathway of Ag presentation is currently unresolved. The availability of a specific and irreversible proteasome inhibitor called lactacystin has enabled us to determine the amount of proteasomes required for the peptide loading of MHC class I molecules in four cell types. In the absence of the IFN-gamma-inducible proteasome subunits LMP2 and LMP7, the trypsin-like (but not the chymotrypsin-like) activity of the proteasome is directly related to MHC class I peptide loading. However, IFN-gamma stimulation or assimilation of catalytic LMP2 and LMP7 subunits into proteasomes causes both chymotrypsin- and trypsin-like activities of the proteasome to become limiting for the loading of class I molecules. Our data suggest that upon full IFN-gamma stimulation, peptide supply by the proteasome is the limiting step in the assembly of MHC class I polypeptides. This mechanism may enable the cell to prevent competition between novel Ags and the pool of endogenous proteins for binding to MHC class I molecules.

We expand the already large number of known trypsinogen nucleotide and amino acid sequences by presenting additional trypsinogen sequences from the tunicate (Boltenia villosa), the lamprey (Petromyzon marinus), the pufferfish (Fugu rubripes), and the frog (Xenopus laevis). The current array of known trypsinogen sequences now spans the entire vertebrate phylogeny. Phylogenetic analysis is made difficult by the presence of multiple isozymes within species and rates of evolution that vary highly between both species and isozymes. We nevertheless present a Fitch-Margoliash phylogeny constructed from pairwise distances. We employ this phylogeny as a vehicle for speculation on the evolution of the trypsinogen gene family as well as the general modes of evolution of multigene families. Unique attributes of the lamprey and tunicate trypsinogens are noted.

The effect of age and weaning on the activities of digestive enzymes with emphasis on the lipolytic enzymes before and after weaning was investigated. The activities of amylase, chymotrypsin, trypsin, carboxyl ester hydrolase, pancreatic lipase, and colipase in pancreatic tissue and the activity of gastric lipase in the cardiac mucosa of the stomach in 45 pigs were response variables. The activity of trypsin was not affected by weaning and the rate of increase was similar during the whole experiment. The activities of chymotrypsin and amylase decreased at weaning (P < .05). After weaning the activity of chymotrypsin increased more slowly than before weaning (P < .001), whereas the rate of increase of amylase activity remained unchanged. Lipase, colipase, and carboxyl ester hydrolase activities decreased at weaning (P < .001), whereas gastric lipase activity increased at weaning (P < .01). The development of lipase, colipase, and carboxyl ester hydrolase activity decreased postweaning (P < .01), whereas gastric lipase activity increased before weaning and remained constant after weaning. Pancreatic lipase had a considerably higher capacity for hydrolyzing tributyrin, and the total activity of pancreatic lipase was up to 600 times higher than that of gastric lipase. The lipolytic enzymes displayed a non-parallel pattern of development, and we suggest that this reflects the importance of these enzymes during the suckling and postweaning phases, respectively. However, the significance of gastric lipase for the digestion of fat in pigs remains to be elucidated.

The extracellular proteolytic activity of Streptomyces albogriseolus 444 was investigated. S. albogriseolus 444 was shown to synthesize an extracellular proteolytic complex with metal-protease and trypsin-like activity defining differentiation of the substrate mycelium to the aerial one as well as the spore formation. The synthesis of the complex proceeded in the absence of the inductor in the medium i.e. constitutively. The complex heterogeneity was confirmed chromatographically. Low concentration of calcium ions (1.5 mM) stimulated the total caseinolytic activity and suppressed the activity of the trypsin-like proteases.

Effects of proteases and protease inhibitors on generation of long-term potentiation (LTP) were investigated in the CA1 and dentate regions of rat hippocampus. Plasmin, a serine protease, and its precursor plasminogen significantly enhanced short-term potentiation (STP) induced by a weak tetanic stimulation, without affecting basal responses. The STP-enhancing effect of plasmin disappeared by concomitant perfusion of alpha 2-antiplasmin, an endogenous plasmin inhibitor. Other proteases, such as thrombin, trypsin and cathepsin B, did not affect STP. On the other hand, alpha 2-antiplasmin and leupeptin significantly attenuated LTP induced by a strong tetanus though plasminogen or plasmin itself did not influence LTP. Furthermore, plasminogen and plasmin did not affect NMDA receptor-mediated synaptic responses in the absence of extracellular Mg2+. These results suggest that endogenous plasmin is involved in the mechanism of LTP in CA1 and dentate regions of rat hippocampus and that the STP-enhancing effect of plasmin is independent of NMDA receptors.

The peritrophic matrix (PM) that forms around a blood meal is a potential barrier of Plasmodium development in mosquitoes. Previously, we have shown that to traverse the PM, Plasmodium ookinetes secrete a prochitinase and that an inhibitor of chitinase blocks further parasite development. Here we report that it is the mosquito trypsin that activates the Plasmodium prochitinase. Trypsin was identified as the chitinase-activating enzyme by two criteria: (i) trypsin activity and activating activity comigrated on one-dimensional gels, and (ii) activating activity and penetration of the PM by Plasmodium parasites were both hindered by trypsin-specific inhibitors. Subsequently, we examined the effect of antitrypsin antibodies on the parasite life cycle. Antibodies prepared against a recombinant blackfly trypsin effectively and specifically inhibited mosquito trypsin activity. Moreover, when incorporated into an infective blood meal, the antitrypsin antibodies blocked infectivity of Aedes aegypti mosquitoes by Plasmodium gallinaceum. This block of infectivity could be reversed by exogenously provided chitinase, strongly suggesting that the antibodies act by inhibiting prochitinase activation and not on the parasite itself. This work led to the identification of a mosquito antigen, i.e., midgut trypsin, as a novel target for blocking malaria transmission.

The objective of this research was to formulate a mixture of commercial proteases that would mimic the rate and extent of protein degradation obtained using strained ruminal fluid. The proteolytic activity of strained ruminal fluid and several commercial proteases was characterized using 13 L-amino acid p-nitroanilides as artificial substrates. A mixture of Streptomyces griseus protease, chymotrypsin, and proteinase K at .042, 2.5, and .5 enzyme units/mL, respectively, was similar to the activity of strained ruminal fluid against the same artificial substrates. However, degradative activities were different in incubations with feed proteins as substrates. The rates of degradation of expeller soybean meal, solvent soybean meal, and casein were .08, .05, and .08/h, respectively, using the enzyme mixture and .03, .15, and .24/h using strained ruminal fluid. A second experiment compared degradative activity of S. griseus protease at .066 enzyme units/mL, ficin at .5 enzyme units/mL, and a mixture of trypsin, carboxypeptidase B, chymotrypsin, and carboxypeptidase A at 116.6, .5, 2.5, and .5 enzyme units/mL, respectively. Protein degradation rates obtained with strained ruminal fluid were two to six times faster than those obtained with the enzyme mixtures. A third experiment compared the degradability of 15 feed proteins with the mixture of trypsin, carboxypeptidase B, chymotrypsin and carboxypeptidase A to that with strained ruminal fluid. Degradation rates obtained using strained ruminal fluid ranged from .007 to .217/h; degradation rates using the enzyme mixture ranged from .010 to .079/h and were lower (P = .004) than with strained ruminal fluid. Overall, the experiments indicated that the commercial enzymes tested did not mimic the protein degradative activity of strained ruminal fluid.

Porphyromonas gingivalis possesses a large number of enzymatic activities which might be important in the virulence of this putative periodontopathogen. The purpose of this study was to examine these enzymatic activities in vivo in a murine model to assess their role in soft tissue destruction. Whole cells of P. gingivalis strains whether grown on blood agar plates or in broth exhibited high levels of alkaline phosphatase (ALPase), a trypsin-like protease (TLPase), acid phosphatase (ACPase), N-acetyl beta-glucosaminidase (Na beta-Gase) enzymes and collagenolytic activities. P. gingivalis W50 treated with 2 mM Na-P-tosyl-L-lysine chloromethyl ketone (TLCK)/phenylmethylsulfonyl fluoride (PMSF) prior to subcutaneous infection of mice failed to induce a phlegmonous abscess and lethality characteristic of animals challenged with untreated P. gingivalis. Comparison of wild type P. gingivalis strain 3079.03 with its protease-deficient (TLPase-negative) mutant NG4B19 revealed the mutant to be avirulent (no lesion and no death) in this model. P. gingivalis BEI and SW5 mutants (parent W50), which partially lacked TLPase enzyme activity produced only localized lesions, and no death. Thus, the TLPase enzyme appears to be correlated with the lesion type (spreading or localized), lesion size, and death in this mouse abscess model. Therefore, the enzymatic activities of P. gingivalis and specifically the TLPase enzyme could play an important role in periodontal disease by enhancing bacterial spread and degrading gingival tissues.

Day-old male, meat-type chicks raised in brooder batteries were infected by orally administering an inoculum prepared from intestines of broiler chicks infected with stunting syndrome (SS). Naive controls were kept in a parallel room. The chicks were fed a commercial starter diet supplemented with two levels of enzyme preparations to 14 d of age. The experiment was continued to the age of 6 wk in order to estimate compensatory feed intake and growth. In a parallel study, digestibility of the feed was determined from 1 to 3 wk of age with control or inoculated chicks. The enzymes amylase and proteases were produced by Bacillus subtilis and Penicillium emersonii. Enzyme supplementation had no effect on feed intake, growth, or feed utilization, or on digestibility of fat, starch, protein, or energy. Because enzyme supplementation did not consistently affect performance of chicks and no interactions were observed between enzyme supplementation and infection status, data are presented for effects of infection only. Inoculation of SS-infective material reduced performance to 4 wk. Compensatory growth and feed intake were observed from the age of 4 wk onward. At the age of 6 wk the slight retardation of the inoculated chicks was not significant. On Week 1, retention of fat, starch, protein, and energy was significantly depressed in the inoculated chicks. At the age of 2 wk, retention of starch was not depressed, and at the age of 3 wk, the only consistent depression was that observed for fat. The proventriculus weight and content were consistently higher in inoculated chicks, as were the small intestine and intestinal content. The pH of the gizzard content was higher, and that of the small intestine content was lower, in the inoculated birds than in their control counterparts. Stunting syndrome infection was accompanied by a significant depression of trypsin activity in the pancreas at the age of 1 and 2 wk. At these periods, amylase and chymotrypsin were not affected. At 6 wk of age, the activities of amylase, trypsin, and chymotrypsin in the pancreas were higher in the inoculated than in the control birds. In the intestinal chime, amylase, trypsin, and chymotrypsin activities were lower in the inoculated birds on Week 1 and 2 (NS for amylase on Week 1). On Week 6, the activity of all enzymes assayed was higher in the inoculated birds (NS for amylase). It is suggested that the main factors depressing feed intake and growth in SS-infected birds are most probably beyond those of digestion.

Using specific substrates, benzyloxycarbonyl-Gly-Gly-Leu-p-nitroanilide, benzyloxycarbonyl-Gly-Gly-Arg-2-naphthylamide and benzyloxycarbonyl-Leu-Leu-Glu-2-naphthylamide, cytosolic chymotrypsin-like, trypsin-like and cucumsin-like activities were determined, respectively, in rat epithelial tissues and differentiated human Caco-2 cells. The cytosolic fractions of rat colonic, rectal, nasal, and alveolar epithelial cells and differentiated human Caco-2 cells contained these three distinct enzyme activities. However, effects of enzyme inhibitors revealed that these three distinctive activities were not extensively involved in cytosolic or homogenate degradation of insulin and insulin-like growth factor I (IGF-I). It is concluded that proteasome-like activities may not significantly limit nonparenteral absorption of peptide and protein drugs such as insulin and IGF-I.

Streptomyces exfoliatus SMF13 produced leupeptin, chymotrypsin-like protease (CTP), metalloprotease, and trypsin-like protease (TLP) extracellularly. The activity of TLP was specifically inhibited by leupeptin. Production of leupeptin was closely associated with growth but leupeptin was inactivated by leupeptin-inactivating protein (LIP) when growth reached the stationary phase in submerged cultures, or when aerial mycelia started to form on surface cultures. Autolysis of mycelia after the stationary phase in submerged cultures was apparently retarded by the addition of leupeptin; on surface cultures, aerial mycelium formation was clearly retarded by the addition of leupeptin. We propose that CTP participates primarily in utilization of a proteinaceous nitrogen source, that TLP functions as an essential enzyme involved in the metabolism of mycelial protein, that leupeptin inhibits the activity of TLP and that LIP inactivates leupeptin. The cascade of regulatory actions of the compounds, which are produced sequentially during mycelium development, may provide selective advantages in adverse culture conditions.

The influences of (a) intraluminal bile deficiency due to common bile duct obstruction and (b) intraduodenal administration of pooled own bile and bovine trypsin on the plasma cholecystokinin (CCK) response to oral fat (Lipomul) ingestion were investigated in seven patients with periampullary tumors and 10 healthy volunteers. Basal and fat-stimulated plasma CCK levels in the patients were significantly higher than in the normal controls. Intraduodenal administration of pooled own bile at a rate of 100 ml/h significantly suppressed both basal and fat-stimulated CCK secretion. Simultaneous administration of pooled own bile (100 ml/hr) and bovine trypsin (600 mg/hr) caused further significant suppression of fat-stimulated CCK secretion compared with that under bile infusion alone. These results indicate that both intraluminal bile and trypsin exert a negative feedback effect on the release of CCK in humans.

To examine the reasons for the high frequency of biliary tract carcinogenesis in individuals with anomalous arrangement of the pancreaticobiliary duct (AAPBD), we investigated the effects on cellular functions of bile acid and trypsin, which are possible risk factors for carcinogenesis found in stagnant bile juice, using a chick embryo fibroblast culture system. Bile acid was found to increase PGE2 synthesis which has been shown to be increased in premalignant lesions, but to suppress the incorporation of [3H]-labelled TdR into DNA. On the other hand, trypsin increased the incorporation of [3H]TdR into DNA, but did not increase PGE2 synthesis. These results suggest that both the bile acid and trypsin present in the stagnant bile juice in AAPBD act to stimulate cell proliferation, but that their mechanisms of action on cell growth differ. Therefore, the combination of these effects of different types of tumor promoters in the stagnant bile juice in AAPBD may account for the high incidence of carcinogenesis.

Understanding the interaction of the atherogenic lipoprotein, lipoprotein(a) [Lp(a)], with macrophages may provide important insight into the physiology and pathophysiology of this lipoprotein. We have recently shown that cholesterol loading of macrophages, such as occurs in atheroma foam cells, leads to marked upregulation of a novel receptor activity for native Lp(a) and its plasminogen-like protein component, apoprotein(a) [apo(a)]. We show here that the Lp(a)/apo(a) receptor activity on cholesterol-loaded macrophages is trypsin sensitive, indicating that a cell-surface protein is involved and that the upregulation by cholesterol loading requires new protein synthesis. Ligand studies revealed that the foam cell receptor activity recognizes Lp(a) containing both small and large isoforms of apo(a) as well as rhesus monkey Lp(a), which contains an inactive kringle-4(37) (K4(37) lysine-binding domain. Elastase degradation products of plasminogen did not compete for 125I-labeled recombinant apo(a) [125I-r-apo(a)] internalization and degradation by foam cells, indicating that the K4(37) sequence, as well as the K5 and "protease" domains of apo(a), are not sufficient for receptor interaction. Consistent with these data, the degradation of 125I-r-apo(a) was completely blocked by an anti-Lp(a) polyclonal antibody that does not cross-react with plasminogen. Furthermore, the multiple sialic residues of apo(a) are also not involved in receptor interaction, since desialylated r-apo(a) interacted with foam cells as well as native r-apo(a). In contrast, reduced and denatured r-apo(a) was degraded by foam cells only slightly better than by control cells [28% increased degradation by foam cells versus 450% for native r-apo(a)], suggesting that the upregulated receptor activity recognizes certain secondary and tertiary structural features of apo(a).(ABSTRACT TRUNCATED AT 250 WORDS)

In conscious rats, bile inhibits pancreatic secretion. The role of luminal taurocholate (TC), a major component of rat bile, in the regulation of pancreatic secretion was studied in conscious rats with external bile and pancreatic fistulae. On the fourth postoperative day, after the basal collection of bile and pancreatic juice (PJ) returned to the duodenum, graded doses of TC (0, 0.4, 4, 40 mM) containing 10 mM CaCl2 were infused into the duodenum instead of bile and PJ for 2 hr (1 ml/hr), with or without 1 mg/ml of porcine trypsin. Luminal trypsin activities were not affected by any dose of TC. The increases in pancreatic secretion in response to diversion of bile and PJ were progressively inhibited with increasing doses of infused TC from 0 mM to 4 mM both with and without trypsin infusion. The effects with 4 and 40 mM TC were not significantly different. Changes in plasma cholecystokinin concentrations roughly correlated with changes in protein output in rats without trypsin infusion. We concluded that TC directly inhibited pancreatic secretion independent of the luminal trypsin activity and that its inhibitory action was concentration dependent.

We recorded esophageal alkaline exposure time (AET) in 52 patients with gastroesophageal reflux and in 20 control subjects to determine whether esophageal pH monitoring can measure reflux of bile acids and trypsin from the duodenum. Patients underwent a further 16-h study (divided into 2-h periods) in which AET was correlated with bile acid and trypsin concentrations in esophageal aspirates. Patients had greater nocturnal AET than controls (22.7 versus 0.9%, p = 0.005). Patients with a stricture had a greater AET than patients with erosive esophagitis (25.2 versus 13%, p less than 0.05). There was no relationship between esophageal bile acid concentrations and AET, and total bile acid concentrations were similar regardless of whether a 2-h period contained alkaline episodes. Esophageal bile acid concentrations were no different, in patients with a normal esophagus, esophagitis, stricture, or Barrett's esophagus. Trypsin was found in only 5% of aspirates, and could not be predicted by AET. We conclude that measurement of AET is not useful in the clinical evaluation of duodeno-esophageal bile reflux, and bile acids and trypsin are not important in the pathogenesis of reflux esophagitis.

Sucrase-alpha-dextrinase, a hybrid digestive carbohydrase of the intestinal brush border, is initially synthesized and transported to the surface membrane as a single-chain glycoprotein, P, which is then cleaved to alpha- and beta-subunits, presumably by one or more pancreatic proteases. However, efforts to convert P under controlled conditions to authentic alpha and beta have been unsuccessful. Sucrase-dextrinase immunoprecipitates from rats intraintestinally labeled with [3H]leucine or [35S]methionine without presence of biliary-pancreatic secretions revealed only the 230-kDa P precursor. Restoration of intestinal flow converted the brush border P to the alpha- (140 kDa) and beta- (125 kDa) subunits. Biliary plus pancreatic secretions facilitated this postinsertional cleavage, but bile alone played no role in conversion. When isolated brush borders, prelabeled in vivo, were exposed to a mixture of pancreatic proteases at physiological concentrations, P was converted to authentic alpha and beta, but only trypsin was responsible for the conversion. Kinetic analysis in prelabeled isolated brush-border vesicles revealed the appearance of several intermediate species (205-145 kDa) produced either by endogenous membrane proteases or by trypsin itself. Reconstituted duodenal luminal contents yielded a fragmentation pattern identical to that produced by trypsin alone. Trypsin was necessary and sufficient for processing of the intermediate precursors to the final authentic alpha- and beta-subunits. Based on the alpha- to beta radioactivity ratio and the known amino acid composition of the subunits, differential cleavage occurred with relatively greater production of the beta-subunit (alpha-to-beta molar ratio = 0.77). The conversion of P to the alpha- and beta-units, rather than occurring in a single step after membrane insertion, is differentially catalyzed by trypsin trimming to unequal amounts of the subunits involving a complex series of cleavage steps.

The cellular mechanisms for aldosterone biosynthesis are incompletely understood. Although the enzymes involved are now well characterized, the dynamics of aldosterone secretion in a variety of rat adrenal preparations are not consistent with the concept that freshly synthesized corticosterone is an important intermediate. In whole glomerulosa tissue preparations, aldosterone is more readily formed from endogenous precursors than from an added radioactive precursor, such as [3H]pregnenolone, and in the in situ perfused gland preparation, aldosterone responses to stimulation, for example by ACTH, are significantly more rapid than those of corticosterone, suggesting a tissue source of steroid substrate for aldosterone production other than corticosterone. The only steroid which is stored in rat adrenal glomerulosa tissue to any extent is 18-hydroxydeoxycorticosterone (18-OH-DOC), and this pool has been located in plasma membrane fractions. It is lost on preparation of collagenase dispersed glomerulosa cells. Since dispersed glomerulosa cell preparations produce significantly less aldosterone, relative to corticosterone, than incubated intact whole glomerulosa, it is plausible that this tissue pool (which is not found in the inner zones) is the immediate precursor for aldosterone formation. Further evidence shows that trypsin, which stimulates aldosterone (and 18-hydroxycorticosterone) production in rat intact glomerulosa tissue, but not in dispersed cells, stimulates translocation of protein kinase C to the plasma membrane. It is plausible that one function of protein kinase C in the rat adrenal zona glomerulosa is to mobilize membrane sequestered 18-OH-DOC for conversion to aldosterone.

Prorenin can be converted to renin by limited proteolysis with trypsin. In the current study we compared conditions for activation of human renal and ovarian prorenin and cat renal prorenin with either liquid-phase trypsin or trypsin bound to sepharose (solid phase). Higher concentrations of trypsin were required to activate cat prorenin than human prorenin. Human prorenin was destroyed by high concentrations of trypsin, while cat prorenin was not destroyed by up to 2 mg/mL solid-phase trypsin. For both human and cat prorenin, addition of the competitive serine protease inhibitor benzamidine--HCl increased the concentration of trypsin needed to activate prorenin, resulting in slightly higher levels of human prorenin but lower levels of cat prorenin. For human samples, activation with solid-phase trypsin resulted in slightly higher estimates of prorenin than liquid-phase trypsin. These results demonstrate species differences in the susceptibility of prorenin to trypsin cleavage. Cat prorenin requires more trypsin to be activated and is less susceptible to destruction than human prorenin.

The intestinal sucrase-isomaltase precursor is cleaved at the brush border membrane by luminal proteases. Whether the lactase precursor also is cleaved by luminal proteases is uncertain. Lactase synthesis and processing was studied in 0- and 15-day-old rats after IP administration of [35S]methionine, and changes in precociously cortisone-induced sucrase-isomaltase were used as an internal control. Mucosal lactase and sucrase-isomaltase were separately immunoprecipitated and analyzed by autoradiography after electrophoresis. In both 0- and 15-day-old rats, mucosal lactase appeared as a 200K lactase precursor band at 30 minutes and as 200K and 225K lactase precursor bands at 60 minutes and was cleaved to form a 130K lactase band 120-240 minutes after labeling; sucrase-isomaltase similarly appeared as 210K and 220K bands at 30-60 minutes and was cleaved to form 140K I and 120K S subunits by 240 minutes in day 15 rats. To determine the role of luminal proteases, intestinal segments were isolated in situ and the luminal contents were flushed 30 minutes after labeling. Unflushed segments were used as controls. Only lactase precursor and sucrase-isomaltase precursor were present 240 minutes after labeling in flushed intestinal segments, but lactase precursor and sucrase-isomaltase precursor were cleaved in unflushed segments. Addition of trypsin or elastase into the lumen of flushed segments resulted in partial cleavage of lactase precursor but not of sucrase-isomaltase precursor. Luminal contents collected from the small intestine of day 15 rats 120 and 240 minutes after labeling showed 35S-labeled 130K and 80K polypeptides in lactase immunoprecipitates. It is concluded that intestinal lactase is synthesized as lactase precursor and transported to brush border membrane and cleaved by luminal proteases, and the amino end polypeptide cleaved from lactase precursor is released into the lumen.

We have recently demonstrated that many cancer cell lines produce a novel trypsinogen isoenzyme called tumor-associated trypsinogen 2 (TAT-2). It was found during a search of the target protease for tumor-associated trypsin inhibitor (TATI). We now show that degradation of subendothelial cell extracellular matrix (ECM) by four different cell lines (COLO 205 colon carcinoma, K-562 erythroleukemia, CAPAN-1 pancreatic carcinoma, and HT 1080 fibrosarcoma) can be partially inhibited by TATI or neutralizing trypsin antibodies. When cells were cultured in serum-free medium on ECM, TATI and trypsin antibodies inhibited the release of immunoreactive fibronectin fragments from ECM by 47-54 and 40%, respectively. Degradation of isotopically labeled ([3H]serine, [3H]proline, and [35S]sulfate) ECM was also significantly prevented by TATI. At its maximum, it exerted a 57% inhibition on the degradation of [3H]serine-labeled ECM. Plasminogen added exogenously to the culture medium further potentiated the proteolysis of ECM. Interestingly, addition of enteropeptidase, an activator of TAT-2, also enhanced cell-mediated proteolysis as assessed by degradation of purified fibronectin coated onto the surface of wells. Immunoblot analysis showed that enteropeptidase-mediated proteolysis generated a pattern of fibronectin fragments similar to that obtained by digestion of purified fibronectin by TAT-2. These results demonstrate the existence of a proteolytic system in tumor cells which is dependent on the activation of TAT-2. We suggest that TAT-2 is involved in a protease cascade-stimulating tumor cell invasion and degradation of extracellular matrix.

Recent studies have supported the existence of a feedback regulation of pancreatic enzyme secretion in man. The aim of present study was to evaluate whether a feedback regulation of pancreatic enzyme secretion exists in cholecystectomized patients. The study was carried out in 12 healthy volunteers and in 6 cholecystectomized patients, 7-14 years (mean 9 years) after the operation. Pancreatic secretion was stimulated by intraduodenal infusion of L-phenylalanine (100 mmo 1.1-1). The calculated outputs of pancreatic alpha-amylase and lipase were estimated. The feedback phenomenon was studied using consecutive intraduodenal infusion of trypsin (300 mg/h) and aprotinin (1.5 x 10(6) KIU/30 min). In 6 healthy persons, the infusion of trypsin caused a significant decrease (35-45%) in phenylalaninestimulated alpha-amylase and lipase outputs (p less than 0.05). This effect was completely reversed by intraduodenal aprotinin infusion. In contrast, the increase or the inhibition of tryptic activity in the duodenum was without any effect on pancreatic secretion in cholecystectomized patients. A feedback control of pancreatic secretion could not be demonstrated in patients 6-14 years after cholecystectomy.

The duodenogastric reflux (DGR) is a suspected cause in some esogastric pathologies in adults: esophagitis, peptic gastric ulcers, stress ulcers, ulcers secondary to drugs, gastric cancer, and gastritis. The toxic substances of the reflux are essentially bile acids, lysolecithin, and trypsin. A number of diagnostic methods have been proposed in the adult. This study suggests a diagnosis technique for DGR in the child. Fasting gastric juice was collected by gastric intubation during 1 h and three substances were measured: phospholipids as markers of biliary reflux, trypsin as a marker of pancreatic reflux, and sialic acid as a marker of the degradation of gastric mucus. The sialic acid enabled us to evaluate some of the toxicity of DGR on the stomach. The study of 49 child subjects permitted us to show the existence, in the normal child, of biliopancreatic markers in the stomach under fasting conditions through a physiological DGR; to define the norms in the child, varying according to three age groups: 0-2 months, 2-12 months, and 1-4 years (the maximum values for an age above 4 years seemed to correspond to those in the adult); and to suggest the existence of a pathological DGR in children with antral gastritis or ulcers.

1. The effect of duodenal infusion of pancreatic juice on basal pancreatic and gastric secretion was studied in five conscious dogs provided with pancreatic fistulae, gastric fistulae and Heidenhain fundic pouches. 2. Pancreatic juice and trypsin stimulated a pancreatic secretion rich in protein. 3. Autodigested juice without proteolytic activities also stimulated the secretion. Boiling the juice or addition of trypsin inhibitor to the juice diminished the augmented secretion. 4. It seems, therefore, that trypsin is necessary even in proteolytically inactive autodigested juice for pancreatic stimulation. 5. In dogs, unlike rats and pigs, basal pancreatic secretion is not under negative feed-back control by duodenal tryptic activity. 6. Basal gastric secretion was not significantly changed by duodenal infusion of pancreatic juice.