Pancreatic Proteases Research Articles

Purification and characterization of the pancreatic proteolytic enzymes trypsin and chymotrypsin from the Kamori goat of Sindh, Pakistan, using ion-exchange chromatography

ABSTRACT Trypsin and chymotrypsin were purified from the Kamori goat pancreas by affinity and ion exchange chromatography, and their activity was checked. The activity and stability of extracted enzymes under various conditions, including temperature, pH, and substrates, were studied. Extracted enzyme’s activity and stability studies demonstrated that both enzymes possessed excellent activity at pH 8 and reached maximum activity after 8 hours. Purity was increased to 22.1-and 12.8-fold, with 29.2% and 25.3% yields for trypsin and chymotrypsin, respectively. According to SDS-PAGE, their molecular weights were approximately 24 and 25.5 kDa, respectively. Trypsin and chymotrypsin displayed maximum activity at 50°C, pH 8, using BAPNA (Na-benzoyl-DL-arginine 4-nitroanilide hydrochloride) and BTEE (N benzoyl-L-tyrosine ethyl ester) as substrates. Both enzymes were stabilized in the presence of Ca++ ions. The enzymes activity declined continuously as the concentration of NaCl increased. Enzymes were effectively inhibited by SBTI and PMSF but not by pepstatin A. Kcat of trypsin and chymotrypsin was 16.8 and 36.7 S−1, and Km was 0.91 and 0.17 mm, respectively. Results suggest that Kamori goat pancreas is a promising source of trypsin and chymotrypsin due to their excellent activity and stability.

Identification of pancreatic cancer-specific protease substrates for protease-dependent targeted delivery

Pancreatic ductal adenocarcinoma (PDAC) presents significant challenges due to the inadequacy of existing chemotherapeutics, which often result in toxicity-dependent dose limitations and premature cessation of therapy. Targeted delivery of therapeutic molecules offers a promising solution. Given that PDAC is marked by a desmoplastic reaction with extensive aberrant protease activity, protease-dependent targeted delivery could minimize off-target toxicities and is of increasing interest. The efficacy of targeted delivery hinges on the specificity of the substrates used; insufficient specificity can lead to off-target effects, reducing the advantage over non-targeted methods. Here, we employ an unbiased library approach to screen over 7 million peptide substrates for proteolytic cleavage by PDAC cell lysates, identifying 37 substrates enriched by at least 500-fold after three rounds of selection. As systemically administered targeted delivery depends on the absence of substrate cleavage in circulation, the peptide library was also screened against whole blood lysates, and enriched substrates were removed from the PDAC-enriched dataset to obtain PDAC-specific substrates. In vitro validation using FRET-peptides showed that 13 of the selected 15 substrates are cleaved by a panel of PDAC cell line lysates. Moreover, evaluation against healthy murine organ and human blood lysates to assess off-target cleavage revealed that the identified substrates are indeed PDAC-specific and that several substrates may be superior with respect to PDAC specificity over the CAPN2-responsive substrate, which has recently shown preclinical potential in targeted therapy, but future animal models should address the potential superiority. Overall, we thus identified substrates with high selectivity and sensitivity for PDAC that could be employed in protease-dependent targeted therapies.

Modeling protease-sensitive human pancreatic lipase mutations in the mouse ortholog

Pancreatic lipase (PNLIP) is the major lipolytic enzyme secreted by the pancreas. A recent study identified human PNLIP variants P245A, I265R, F300L, S304F, and F314L in European chronic pancreatitis cohorts. Functional analyses indicated that the variants were normally secreted but exhibited reduced stability when exposed to pancreatic proteases. Proteolysis of the PNLIP variants yielded an intact C-terminal domain, while the N-terminal domain was degraded. The protease-sensitive PNLIP phenotype was strongly correlated with chronic pancreatitis, suggesting a novel pathological pathway underlying the disease. To facilitate preclinical mouse modeling, here we investigated how the human mutations affected the secretion and proteolytic stability of mouse PNLIP. We found that variants I265R, F300L, S304F, and F314L were secreted at high levels, while P245A had a secretion defect and accumulated inside the cells. Proteolysis experiments indicated that wild-type mouse PNLIP was resistant to cleavage, while variant I265R was readily degraded by mouse trypsin and chymotrypsin C. Variants F300L, S304F, and F314L were unaffected by trypsin but were slowly proteolyzed by chymotrypsin C. The proteases degraded the N-terminal domain of variant I265R, leaving the C-terminal domain intact. Structural analyses suggested that changes in stabilizing interactions around the I265R mutation site contribute to the increased proteolytic susceptibility of this variant. The results demonstrate that variant I265R is the best candidate for modeling the protease-sensitive PNLIP phenotype in mice.

Hemorrhagic Cysts in the Pancreas: Risk Factors, Treatment, and Outcomes - Insights from a Single-Center Study.

BACKGROUND Hemorrhagic cysts are rarely discussed subtypes of pancreatic pseudocysts that occur in about 10% of these cases. They are caused by erosion of the walls of neighboring vessels by extravasated proteolytic pancreatic enzymes. A retrospective analysis was performed to clinically characterize risk factors, treatment, and outcome in patients with hemorrhagic cysts of the pancreas. MATERIAL AND METHODS The retrospective study included patients from the Department of Digestive Tract Surgery in Katowice, Poland, who were treated surgically for a pancreatic hemorrhagic cyst from January 2016 to November 2022. We gathered and assessed data on cyst etiology, symptoms, imaging examinations, risk factors, time, type, and complications of surgery. RESULTS The main symptom was abdominal pain, noted in 5 (62.5%) patients. The most common etiology of cyst was acute pancreatitis, which occurred in 5 patients (62.5%). The most common localization was the tail of pancreas, found in 3 patients (36.5%). The largest dimension of the cyst was 98±68 (30-200) mm. Every patient needed surgical intervention. Patients underwent distal pancreatectomy (n=3) or marsupialization (n=5). One (12.5%) postoperative complication was observed, while mortality was 0%. CONCLUSIONS Hemorrhagic cyst is a life-threatening complication of pancreatitis requiring immediate treatment. In most cases, open surgery is the treatment of choice. Despite the continuous development of minimally invasive techniques, surgical treatment remains the only effective treatment method. Depending on the cyst localization and technical possibilities, pancreatectomy or marsupialization can be applied, and both of them have low complication and mortality rates.

Chymotrypsin activity signals to intestinal epithelium by protease-activated receptor-dependent mechanisms.

Chymotrypsin is a pancreatic protease secreted into the lumen of the small intestine to digest food proteins. We hypothesized that chymotrypsin activity may be found close to epithelial cells and that chymotrypsin signals to them via protease-activated receptors (PARs). We deciphered molecular pharmacological mechanisms and gene expression regulation for chymotrypsin signalling in intestinal epithelial cells. The presence and activity of chymotrypsin were evaluated by Western blot and enzymatic activity tests in the luminal and mucosal compartments of murine and human gut samples. The ability of chymotrypsin to cleave the extracellular domain of PAR1 or PAR2 was assessed using cell lines expressing N-terminally tagged receptors. The cleavage site of chymotrypsin on PAR1 and PAR2 was determined by HPLC-MS analysis. The chymotrypsin signalling mechanism was investigated in CMT93 intestinal epithelial cells by calcium mobilization assays and Western blot analyses of (ERK1/2) phosphorylation. The transcriptional consequences of chymotrypsin signalling were analysed on colonic organoids. We found that chymotrypsin was present and active in the vicinity of the colonic epithelium. Molecular pharmacological studies have shown that chymotrypsin cleaves both PAR1 and PAR2 receptors. Chymotrypsin activated calcium and ERK1/2 signalling pathways through PAR2, and this pathway promoted interleukin-10 (IL-10) up-regulation in colonic organoids. In contrast, chymotrypsin disarmed PAR1, preventing further activation by its canonical agonist, thrombin. Our results highlight the ability of chymotrypsin to signal to intestinal epithelial cells via PARs, which may have important physiological consequences in gut homeostasis.

Supplementing lysolecithin in corn-oil based diet enhanced growth and improved body biochemical composition in juvenile stellate sturgeon (Acipenser stellatus)

A 56-days nutritional trial was conducted to examine the influence of including a corn-oil based diet with lysolecithin (LL) on stellate sturgeon (Acipenser stellatus) juveniles. Fish were fed four experimental diets (44% crude protein, 15% crude lipid) containing graded levels of LL [0 (control), 0.5, 1 and 2%)]. One hundred and eighty juvenile fish (45.3 ± 0.1 g, mean ± standard deviation) were stocked in twelve 300 L-polyethylene tanks containing ground water at 18.9 ± 0.5°C. Feeds were offered to fish at apparent satiation four times daily. Fish fed 1% LL-supplemented diet had higher growth and better feed conversion ratio than the control group (P < 0.05). The amounts of protein and lipid levels in the whole body increased in fish fed LL-supplemented diets compared to control, whereas the levels of n-6 PUFA, particularly linoleic acid, increased in fish fed 0.5 and 1% LL-supplemented diets. Total long chain polyunsaturated fatty acids increased in fish fed 1% LL-supplemented diet. Regarding digestive enzymes, trypsin (alkaline pancreatic proteases) and pepsin (acid gastric protease) activities decreased with increasing LL in the diet. Overall, these results indicated that 1% LL is recommended for better growth performance of stellate sturgeon juveniles.

Understanding the toxicity mechanism of gelsemine in zebrafish

Gelsemium elegans (GE), also known as Duanchangcao, is a plant associated with toxic symptoms related to the abdomen; however, the toxicity caused by GE remains unknown. Gelsemine (GEL) is an alkaloid extracted from GE and is one of the most toxic alkaloids. This study used zebrafish as an animal model and employed high-throughput gene sequencing to identify genes and signaling pathways related to GEL toxicity. Exposure to GEL negatively impacted heart rate, swim bladder development, and activity in zebrafish larvae. Transcriptomics data revealed the enrichment of inflammatory and phagocyte signaling pathways. RT-PCR analysis revealed a decrease in the expression of pancreas-related genes, including the pancreatic coagulation protease (Ctr) family, such as Ctrl, Ctrb 1, and Ctrc, due to GEL exposure. Furthermore, GEL exposure significantly reduced Ctrb1 protein expression while elevating trypsin and serum amylase activities in zebrafish larvae. GEL also resulted in a decrease in pancreas-associated fluorescence area and an increase in neutrophil-related fluorescence area in transgenic zebrafish. This study revealed that GEL toxicity in zebrafish larvae is related to acute pancreatic inflammation.

The primary prevention of pancreatic fistula using a vascularised rectus abdominis muscle flap – A porcine model

A pancreatic fistula is one of the most devastating complications following a Whipple's procedure. Fistula rates remain high despite various modifications to surgical techniques. We propose the use of a vascularised muscle flap in the primary prevention of pancreatic fistulas. A distal pancreatectomy was performed on 5 pigs in our porcine model. A pancreaticojejunal (PJ) anastomotic leak was simulated. The pigs were divided into treatment (4 pigs) and control groups (1 pig). A left pedicled rectus abdominis flap was wrapped around the PJ anastomosis for the treatment group and omitted for the control group. Serum and drain amylase levels were recorded. The PJ-rectus abdominis flap complex was evaluated histologically. There was no biochemical evidence of anastomotic leak in the treatment group. The drain-serum amylase ratio was less than 1.5 in the treatment group (p=0.006). Microscopically, the muscle adjacent to the anastomotic leak showed mild necrotic changes with an affected muscle depth of less than 10%. The vascularised rectus abdominis muscle is a durable flap to withstand proteolytic pancreatic enzymes. It is able to provide a water-tight seal around the PJ anastomosis and mitigate intraperitoneal haemorrhage and infection caused by erosion from the pancreatic fistula.

Thermal treatment of alkali lignin to eliminate its inhibition of pancreatic proteases in vitro

This study aims to investigate how alkali lignin inhibits protein digestion and explore thermal treatment as a potential solution. Solid alkali lignin species pre-heated at different temperatures (150, 200, and 250 °C) and soluble acid-differentiated fractions are subjected to in vitro protein digestion. A range of techniques, including Thermogravimetric Analysis (TGA), Size-Exclusion Chromatography (SEC), Zeta Potential Analyzer, 1H NMR, Isothermal Titration Calorimetry (ITC), and Molecular Docking, were used to investigate the inhibitory mechanism of alkali lignin on pancreatic proteases hydrolysis. Our results suggest that soluble alkali lignin inhibits pancreatic trypsin and chymotrypsin, with the acid-differentiated soluble fraction (LgpH<1) displaying the strongest inhibition and proteases’ binding affinity due to the abundance of polar groups (e.g., –OH, –CHO), which facilitate hydrogen-bond formation. Furthermore, pre-heating lignin (200 °C) was confirmed effective for removing LgpH<1 and its negative nutritional influence, providing a feasible strategy for overcoming the negative impact of alkali lignin on protein digestion.

Mechanisms of Digestive Enzyme Response to Acute Salinity Stress in Juvenile Yellowfin Tuna (Thunnus albacares).

This study investigates the effect of a sudden change in salinity for 48 h on the digestive enzyme activity of juvenile yellowfin tuna. The treatment included a control salinity of 32‱ in natural seawater and an experimental salinity of 29‱. Acute stress experiments were carried out on 72 juvenile yellowfin tuna (646.52 ± 66.32 g) for 48 h to determine changes in digestive enzyme activity in different intestinal sections over time (0 h, 12 h, 24 h, 48 h). The activities of pepsin, trypsin, α-amylase, lipase, and chymotrypsin in the digestive organs (stomach, foregut, and pyloric ceca) of juvenile yellowfin tuna were measured. Pepsin and pancreatic protease in the experimental group were significantly lower than in the control group (p < 0.05). α-amylase showed a fluctuating trend of decreasing and then increasing, and its activity trend was pyloric ceca > foregut > stomach. The lipase activity of gastric tissues decreased at the beginning and then increased, reaching a minimum at 24 h (2.74 ± 1.99 U·g protein-1). The change of lipase in the pyloric ceca and foregut was increasing and then decreasing. The lipase activity trend was pyloric ceca > foregut > stomach. The chymotrypsin showed a decreasing and increasing trend and then stabilized at 48 h with a pattern of pyloric ceca > foregut > stomach. Similarly, the gut villi morphology was not significantly altered in the acutely salinity-stressed compared to the non-salinity-stressed. This study suggests that salinity may change the digestive function of juvenile yellowfin tuna, thereby affecting fish feeding, growth, and development. On the contrary, yellowfin tuna is highly adapted to 29‱ salinity. However, excessive stress may negatively affect digestive enzyme activity and reduce fish digestibility. This study may provide a scientific basis for a coastal aquaculture water environment for yellowfin tuna farming, which may guide the development and cultivation of aquaculture.

Chitosan-based oral hydrogel formulations of β-galactosidase to improve enzyme supplementation therapy for lactose intolerance

β-Galactosidase supplementation plays an important role in the life of people with lactose intolerance. However, these formulations are rendered ineffective by the low pH and pepsin in the stomach and pancreatic proteases in the intestine. Therefore, it is necessary to develop oral transport systems for carrying this enzyme in the active form up to the intestine, where the lactose digestion occurs. In this research, a new hydrogel was developed that could potentially be used for enzyme supplement therapy. In this regard, the chitosan-based β-Gal formulations described in the manuscript are an alternative long-acting preparation to the so far available preparations that allow for enzyme protection and mucosal targeting. These hydrogels were prepared from chitosan and polyethylene glycol and contained a covalently immobilized β-galactosidase from Aspergillus oryzae. The β-galactosidase in the hydrogel was protected from degradation in a gastric medium at a pH of 2.5 and retained 75 % of its original activity under subsequent intestinal conditions. In the case of a simulated gastric fluid with a pH of 1.5, a copolymer containing methacrylic acid functional groups was sufficient to protect the hybrid hydrogel from the extremely acidic pH. In addition, the surface of the hydrogel was chemically modified with thiol and amidine groups, which increased the binding to intestinal mucin by 20 % compared with the unmodified hydrogel. These results represent a promising approach for oral transport as a reservoir for β-galactosidase in the small intestine to reduce the symptoms of hypolactasia.

Pancreatic proteases are mediators of pain in murine pancreatic cancer

Pancreatic proteases are mediators of pain in murine pancreatic cancer

The pancreatic proteases as new analgesic targets in acute and chronic pancreatitis

The pancreatic proteases as new analgesic targets in acute and chronic pancreatitis

Induction of Pancreatic Growth and Proteases by Feeding a High Amino Acid Diet Does Not Depend on Cholecystokinin in Rats

We examined differences in pancreatic growth, enzyme content and enzyme concentration between rats fed diets containing normal (2.49 g nitrogen/kg diet) or high (7.46 g nitrogen/kg diet) levels of an amino acid mixture and those in rats fed diets containing normal and high levels of casein for 11 d. Rats fed these diets were injected with a potent cholecystokinin antagonist, MK-329 (2.5 mg/kg body wt·d) or with vehicle only. Pancreatic contents (units in pancreas per 100 g body wt) of protein, trypsinogen and chymotrypsinogen were greater in rats fed a high amino acid diet compared with those in rats fed a normal amino acid diet. Proportionate increases in protein and the serine proteases in pancreata of the high amino acid group relative to those of the normal amino acid group were comparable to those of the high casein group relative to the normal casein group. The pancreatic protease concentrations (units/g pancreas) of rats fed the high casein diet and treated with MK-329 were lower than in rats fed high casein but not treated with MK-329. This difference was not observed in rats fed the high amino acid diet. These results demonstrate that pancreatic growth and proteases are induced by dietary amino acids in rats, and the stimulatory effects of amino acids on exocrine pancreas do not depend on cholecystokinin.

Diet Modifies Elastase I and II Activities and mRNA Levels during Postnatal Development and Weaning in Piglets ,

Little information is available on the expression of pancreatic elastase I and II, despite their role in protein milk digestion. We studied the developmental changes and the effects of diet composition on both elastase I and II expression in suckled and weaned piglets. We measured their activities and levels of their corresponding mRNA. Forty-two piglets were assigned to seven groups according to age and diet. Piglets were slaughtered at birth (Group 1), or suckled up to 13 d (Group 2) or 21 d (Group 3), fed a milk substitute from 14 to 21 d (Group 4) or to 56 d (Group 7), suckled up to 21 d and then fed a dry starter up to 56 d (Group 5), or fed a milk substitute from 14 to 21 d and then a dry starter up to 56 d (Group 6). At 21 d pancreatic function was not modified by the source and the form of milk consumed. The specific activity of elastase II was maximum at birth and declined sharply thereafter, whereas that of elastase I markedly increased after weaning. The presence of milk protein in the diet did not prevent the sharp decrease in elastase II activity observed with age. During the 13 d period of suckling sow's milk, the mRNA patterns indicated that the regulation was at the mRNA and post-transcriptional levels, whereas after weaning and depending on the source of dietary protein, it was essentially translational and/or post-translational. Taken together, our results provide evidence of the early expression of elastase I and II genes that could enhance protein digestion. It seems that elastase II might be a predominant pancreatic protease during the milk-feeding period, whereas elastase I might be related to weaning.

COLON TARGETED DELIVERY AND STABILIZATION OF MONOCLONAL ANTIBODIES FOR LOCAL TREATMENT OF INFLAMMATORY BOWEL DISEASES

Abstract In the era of biologics where IBD treatment has been transformed by potent antibody-based therapeutics, a key challenge still remains, to develop orally administered antibody medicines for chronic GI inflammation. This is primarily due to the challenges in accurate oral targeted delivery of drugs to the lower GI tract and the inherent enzymatic instability of antibodies in these harsh luminal environment. As a result, all antibody therapeutics in clinical development and on the market are available only as an injection leading to undesired adverse effects due to high systemic exposure and suboptimal therapeutic response due to insufficient antibody levels at the inflamed tissue. We have developed a suite of delivery technologies that are designed to bypass the harsher stomach and small intestinal conditions of the GI tract and achieve accurate targeted release and enzymatic stabilization of the antibody therapeutics in the colon. The delivery platform comprises of a film coating which is applied on the outside of the pill. The coating is a clinically validated polymer coating comprising of a combination of pH sensitive polymer and polysaccharide that is designed to start releasing drugs only when exposed to the correct pH and/or colonic bacteria, dramatically improving on the inconsistent colonic release profiles of solely pH-based coatings. The second component is in the core of the pill, a cocktail of amino acid-based excipients that are mixed together with the antibody and protects the drug from pancreatic and bacterial origin protease enzymes, allowing high luminal antibody concentration build-up that leads to inflamed tissue/systemic uptake through a combination of active and passive diffusion. We demonstrated in an acute DSS mouse model of colitis the effective colonic delivery and stabilization of an anti-IL6 antibody leading to superior uptake into the tissue compared IP injection. The superior tissue level demonstrated higher downregulation of target IL6. We are working to leverage the delivery technology for oral delivery of a variety of anti-inflammatory and epithelial wound healing protein modalities such as monoclonal antibodies, fusion proteins, cytokines, VHHs and peptides to allow for broad therapeutic interventions in indications such as Crohn’s disease, ulcerative colitis, pouchitis and celiac disease. Mechanism of oral colon targeted delivery of therapeutic antibodies in local treatment of inflammatory bowel disease. In-vivo PK of anti-IL6 antibody in acute DSS colitis mouse model. A. Impact of stabilization on antibody levels in the colon lumen compared to naked antibody and injected antibody. B. Impact of stabilization on colon tissue uptake. C. Effect of tissue conc. on colon tissue IL6 target downregulation.

Thrombosis of Extrasplanchnic and Splanchnic Venous System in Acute Pancreatitis- A Case with Rare Combination of Vascular Complication

Vascular complications in acute pancreatitis are common and seen in 25% of cases. While it is common to have venous thrombosis in the Superior Mesenteric Vein (SMV), portal vein, and splenic vein, thrombosis of extra-splanchnic vessels such as Inferior Vena Cava (IVC) and left renal vein due to acute pancreatitis is a rare entity, with more adverse outcomes. A 48-year-old male presented with severe epigastric pain, vomiting, and constipation for seven days. Outside Ultrasonography (USG) report was suggestive of acute pancreatitis. His serum amylase, serum lipase, and D-dimer levels were raised. Computed Tomography (CT) of abdomen and pelvis revealed acute necrotising pancreatitis with peripancreatic fluid collection and thrombosis of splenic vein, left renal vein, and IVC. Thrombosis in pancreatitis can occur due to pancreatic proteolytic enzymes which can cause intimal injury. An enlarged pancreas, walled-off necrosis, and pancreatic pseudocyst can compress veins, resulting in venous stasis. Pancreatitis has a systemic hypercoagulable or prothrombotic state. The patient was given symptomatic treatment along with an injection (inj.) of clexaine and monocef. He showed improvement in 10 days and was symptomatically well on follow-up. Early detection of these findings and targeted treatment for the same is crucial to prevent morbidity and mortality of such patients.

Патогенетические аспекты острого панкреатита на субклеточном уровне

The pathogenesis of acute pancreatitis (AP) is based on a cascade of reactions of immunologicalinflammation due to dysfunction of important structures at the subcellular level. At an early stage of AP,excessive activation of leukocytes and the subsequent release of an inflammatory mediator are crucial forthe development of early organ failure. Regardless of the cause, trigger events lead to premature activationof pancreatic proteases simultaneously with the activation of lysosomal enzymes. The initiating events ofpancreatic necrosis, including zymogen activation and cell death, occur in the pancreatic acinar cells (PAC),where the earliest immune response is also noted in the form of activation of circulating immune cells,which triggers a systemic inflammatory response. Further in-depth study of pathophysiological disordersat the subcellular level is necessary for targeted and effective therapy of AP.

PRIMARY HYPERPARATHYROIDISM REVEALED BY ACUTE ALITHIASIC PANCREATITIS: A RARE CASE

Acute pancreatitis is a rare presentation of Primary hyperparathyroidism (PHPT), its prevalence during PHPT is about 1%. The physiopathology of the implication of PHPTs hypercalcemia is not completely elucidated. Several hypotheses plead for the direct or indirect role of hypercalcemia via the activation of pancreatic proteases. Acute alithiasic pancreatitis can be a revealing feature of PHPT wich is a curable disease with the mastery of parathyroid surgery and hypercalcemia treatment. Herein, the authors report a rare case of a patient with PHPT revealed by acute alithiasic pancreatitis with a dreaded complication.

The pancreatic proteases as new analgesic targets in acute and chronic pancreatitis

The pancreatic proteases as new analgesic targets in acute and chronic pancreatitis