Trypsin Synthesis Research Articles

Effect of an Early Mobilization Program on Functional Capacity after Coronary Artery Bypass Surgery a Systemic Review

Phenylalanine function stabilized by lysine which found on lysosomal surface, that Phe necessary to regulate the synthesis or secretion of α-amylase, trypsin and lipase, where the Severe acute pancreatitis in a child with phenylketonuria. The phenylketonuria due to decreased lysine followed by lysosomal dysfunction that will be result of deficiency in phenylalanine function that will not form pancreatic enzymes and stomach enzyme and will be accumulated. The Lysosomal dysfunction will be result of accumulation of undigested polymer including inflammation which play important roles in pathophysiology, that reflect reduction in lysine acylation followed by reduction in aminoacyl tRNAs which promote Phe and E coli functions for activating protein and mRNAs synthesis, that will be followed by increasing in TNf-a synthesis (and reduction in VEGF-As ) which activate NK (not beings related to human immune proper function ) which regulated by the undigested polymers in random pathways stimulated by TNf-a functional pathway (activated by NLRP3 inflammasome. Retinoic acid is necessary to promote lysine acylation which regulate aminoacyl-tRNAs production regulated by mitochondrial enzymes, which regulate mRNA production by E coli (regulated by phenylalanine) that necessary to run antioxidant functions and cellular biosynthesis. Lysine acetylation so important to regulates the activity of Escherichia coli Sadenosylmethionine synthase. RNA (produced by E coli) is 2′-O-methylated demonstrated higher stability that can improve immune by promoting anti-inflammatory processes and improve myocardial functions, and adopt heart constriction by activating NR4As pathway. The antihypertensive pathway mechanism is: the Lysine ¬¬>activate ATPase (stimulate retinoic acid “RA” ) ¬¬>Lysine acetylation ¬¬> promote Phe/ hydroxylase ¬¬> followed by ¬>activate Tyr/ hydroxylase ¬>activate dopamine ¬> activate NR4As pathway ¬>GC-beta ¬>activate both of Oxytocin and Nrf2 ¬> Ang2-AT2 and VEGF-A productive functions ¬>heme oxygenase ¬>anti-inflammatory growth and processes. Lysine Methylation has important role in adopt hypertension through activating pervious Antihypertensive pathway mediated by Phe hydroxylase synthesis and Tyr hydroxylase production followed by activating both of dopamine and NR4As pathway. The oxidation of phenylalanyl-tRNA synthetase positively regulates translational quality control, and regulates the initiation of digestive enzyme mRNA translation, where consecutive AAA-lysines is paused for encoding Phe followed by ribosome sliding on homopolymeric A sequence. That lysine activates mitochondria function followed by promoting Phenylalanine and phenylalanine-tRNAs productions by translations within E coli, followed by Phe/ hydroxylase activate Tyr hydroxylase production which activate dopamine synthesis. And positively, Lysine is the most effective amino acid for activating all of ATPase, GTPase, and lysosomes, and E coli functions, where ATPase necessary for activating lysine Phosphorylation for activating lysosome digestion, followed by activating E coli through activating Phe hydroxylase production, while GTPase necessary for activating mitochondrial repairs, which act on pro-inflammation and cholesterol for activating estrogen production and activating the NR4As pathway which activate all of Oxytocin, Nrf2 followed by Ang2-AT2 and VEGF-A production and then followed by activating heme oxygenase and anti-inflammatory growth and processes that prevent LV-hypertrophy, and prevent coronary calcification.

Impacts of pancreatic exocrine insufficiency on gut microbiota.

Pancreatic exocrine insufficiency (PEI) can be induced by various kinds of diseases, including chronic pancreatitis, acute pancreatitis, and post-pancreatectomy. The main pathogenetic mechanism of PEI involves the decline of trypsin synthesis, disorder of pancreatic fluid flow, and imbalance of secretion feedback. Animal studies have shown that PEI could induce gut bacterial overgrowth and dysbiosis, with the abundance of Lactobacillus and Bifidobacterium increasing the most, which could be partially reversed by pancreatic enzyme replacement therapy. Clinical studies have also confirmed the association between PEI and the dysbiosis of gut microbiota. Pancreatic exocrine secretions and changes in duodenal pH as well as bile salt malabsorption brought about by PEI may affect and shape the abundance and composition of gut microbiota. In turn, the gut microbiota may impact the pancreatic exocrine acinus through potential bidirectional crosstalk. Going forward, more and higher-quality studies are needed that focus on the mechanism underlying the impact of PEI on the gut microbiota.

The effect of probiotics on digestive enzyme activity during larvae and juvenile stage of Yellow Fin Tuna (Thunnus albacares)

Yellow fin tuna fry production technology is not successful yet. This was assumed related to the biological and physiological characteristics of the fish. The physiological approach to regulating digestion through probiotics is needs to be studied. The aim of this study was to examine the effect of probiotics bacterial strains of the enzymatic activity in digestion system for the larvae to juvenile stages of yellow fin tuna, Thunnus albacares. The experiment was initiated by culturing 3 probiotic isolates (Bacillus subtilis strain TA-1, Bacillus amyloliquefaciens strain TN-2, Bacillus subtilis strain TO-4) and apply to the larval rearing of yellow fin tuna. In the experiment the treatments applied were supplemented probiotics and without probiotic, each treatment was held with 2 replications. Fish samples were collected every day and analyze the gene expression profile associated with digestion enzyme synthesis by total RNA isolation and cDNA synthesis followed analysis of its by using the RT –qPCR method. The results showed that the growth response of T. albacares tuna larvae reared with probiotics tended to be faster (P<0.05) than the control (without probiotics). The supplementation of probiotic could improved the expressed digestibility of the target genes associated with enzymatic synthesized. Amylase enzymatic synthesized increase 200-1200 times in larvae 10 –17 Day After Hatch (DAH), while in the lipase enzymatic activity started with 13 DAH ( 25 time) and 16 – 21 DAH by 150-300 times. In trypsin the highest enzymatic activity was at 16 DAH, which was 200 times. While in control, the expression of enzymatic synthesis of amylase, lipase, and trypsin was relatively low.

Leucine regulates α-amylase and trypsin synthesis in dairy calf pancreatic tissue in vitro via the mammalian target of rapamycin signalling pathway

Starch digestion in the small intestines of the dairy cow is low, to a large extent, due to a shortage of syntheses of α-amylase. One strategy to improve the situation is to enhance the synthesis of α-amylase. The mammalian target of rapamycin (mTOR) signalling pathway, which acts as a central regulator of protein synthesis, can be activated by leucine. Our objectives were to investigate the effects of leucine on the mTOR signalling pathway and to define the associations between these signalling activities and the synthesis of pancreatic enzymes using an in vitro model of cultured Holstein dairy calf pancreatic tissue. The pancreatic tissue was incubated in culture medium containing l-leucine for 3 h, and samples were collected hourly, with the control being included but not containing l-leucine. The leucine supplementation increased α-amylase and trypsin activities and the messenger RNA expression of their coding genes (P <0.05), and it enhanced the mTOR synthesis and the phosphorylation of mTOR, ribosomal protein S6 kinase 1 and eukaryotic initiation factor 4E-binding protein 1 (P <0.05). In addition, rapamycin inhibited the mTOR signal pathway factors during leucine treatment. In sum, the leucine regulates α-amylase and trypsin synthesis in dairy calves through the regulation of the mTOR signal pathways.

Phenylalanine regulates initiation of digestive enzyme mRNA translation in pancreatic acinar cells and tissue segments in dairy calves.

As new nutritional strategies for ruminant are designed to change production efficiency by improving the supply of rumen protect protein, lipid, and even starch, the digestive system must fit to utilize these increased nutrient supplies, especially the pancreas. The objective of this study was to investigate the effects of phenylalanine (Phe) on digestive enzymes synthesis or secretion and cellular signaling in pancreatic acinar (PA) cells of dairy calves. The PA cells isolated from fresh pancreas of dairy calves, and cultured in completed RIPA 1640 medium with no fetal serum but 0, 0.15 and 0.45 mM Phe at 37°C in CO2 incubator for 120 min. The pancreatic tissue segments (PTS) was cut approximately 2 × 2 mm from the fresh pancreas, and incubated in oxygenated Krebs-Ringer bicarbonate (KRB) buffer containing 0 or 0.35 mM Phe at 39°C for 180 min, and the samples were collected every 60 min after incubation. In PA cells, Phe increased (P < 0.05) the α-amylase secretion and mRNA expression, the phosphorylation of ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4EBP1). In PTS, the Phe increased (P < 0.05) α-amylase and trypsin synthesis, secretion and mRNA expression, as well as the phosphorylation of S6K1 and 4EBP1. Conclusively, these results suggested that Phe regulates the synthesis or secretion of α-amylase, trypsin and lipase through mRNA translation initiation factors – S6K1 and 4EBP1.

Rational synthesis of MoS2-based immobilized trypsin for rapid and effective protein digestion

In this work, a novel MoS2-based immobilized trypsin reactor was designed and prepared. Pyrene-1-butyric acid was first assembled onto MoS2 nanosheets via the strong π-π stacking and then trypsin was covalently immobilized onto the nanocomposite supports through amidation reaction. Compared with traditional in-solution digestion, higher sequence coverage (84%) and shorter time (5min) could be achieved by the novel trypsin reactor during the digestion of BSA. The excellent performances of as-prepared trypsin reactor can be mainly attributed to the designed novel structure of the composites with high surface area resulting in high enzyme loading. In addition, strong reusability, good reproducibility and long storage of the trypsin reactor were also obtained. The novel immobilized trypsin reactor was further applied in large-scale proteomics research. The proteins extracted from HeLa cells and Amygdalus Pedunculata Pall. kernels were chosen to evaluate the digestion performance for the novel MoS2-based immobilized trypsin reactor, and the experimental results showed that the number of identified proteins from complex real bio-samples with 1h immobilized tryptic digestion was slightly more than that obtained by 12h in-solution digestion. The above results demonstrated that the protein digestion with our novel MoS2-based immobilized trypsin reactor is superior to the conventional protein digestion with free trypsin. Moreover, this simple, fast tryptic digestion method can effectively reduce the levels of artifacts in detection of oxidation and deamidation of peptides from proteins of Amygdalus Pedunculata Pall. kernels. Also, results of Gene Ontology analysis give an explanation for the good survival of Amygdalus Pedunculata Pall. in harsh desert environments from proteomics points of view. Therefore, the novel 2D-MoS2-based immobilized trypsin is potentially suitable for the high throughput proteome analysis and opening up a new avenue for Molybdenum disulfide in proteomics field.

Dietary and phylogenetic correlates of digestive trypsin activity in insect pests

AbstractBecause food proteins are crucial for insect survival, growth, and fecundity, enzymes involved in their digestion have attracted the attention of fundamental entomologists studying the mechanisms and patterns of dietary specialization, and applied entomologists searching for more efficient modes of pest control. Most insects digest proteins using trypsin, an endopeptidase that cleaves polypeptide chains on the carboxyl side of arginine and lysine, two basic amino acids. As the most ancestral proteinase, trypsin is wide‐spread in the digestive tract of insects from various orders and with various feeding habits. The present review focuses on biochemical and molecular characteristics, mechanisms of regulation, and adaptive/non‐adaptive changes of trypsin activity in response to heterogeneous food environments in a phylogenetic context. Within‐ and among‐species variations in trypsin structure and regulation that contribute to better matching with specific food types are emphasized. We also discuss the relevance of these data for choosing an appropriate strategy for control of insect pests. Pest control strategies to reduce trypsin activity by interfering with substrate binding or trypsin synthesis and secretion have been suggested. Successful application of these procedures requires a multidisciplinary approach and knowledge about digestive physiology of the pest, as well as the structural characteristics of trypsins that determine their interaction with proteinaceous inhibitors and other food compounds, about patterns of developmental changes in trypsin gene expression, adaptive responses of insects to food composition, and various environmental effects on trypsin activity.

English

This work describes the larvicidal activity of the ethanolic crude extract (EEB) as well as the hexanic (FH), chloroform (FCL), ethyl acetate (FAE) and remaining (FR) fractions obtained from Byrsonima intermedia leaves and bark against Aedes aegypti. Hexanic and remaining fractions from the leaves demonstrated larvicide activity against A. aegypti larvae on the 3rd stage, with LC50 values of 250 and 125 &mu;g/ml-1, respectively, with less mortality as compared to the other fractions. Hexanic and remaining fraction from the leaves demonstrated larvicide acitivity against A. aegypti larvae on the 3rd stage, with LC50 values of 250 and 125 &mu;g/ml-1, respectively, with less mortality as compared to the other fractions. Trypsin activities from gut of larvae treated with the extract or the fractions were significant (p &lt; 0.05), and the major effects occurred with hexanic fraction (0.06 &mu;mol min-1) and remaining fraction (0.09 &mu;mol min-1) in the 1000 &mu;g/ml-1 concentration, respectively. The same fractions inhibited chymotrypsin activity in 0.06 &mu;mol/ml-1 (hexanic fraction) and 0.13 &mu;mol/ml-1 (remaining fraction) at 1000 &mu;g/ml-1. The inhibition was dose-dependent. The results show the inhibitory effects of the hexanic and remaining fractions from the leaves of B. intermedia on trypsin and chymotrypsin activity, which may be responsible for the deleterious effects on larvae mortality. The crude ethanolic extract and fractions of the bark does not inhibit the synthesis of trypsin compared to control. The results show the inhibitory effects of the hexanic and remaining fraction from the leaves of B. intermedia on trypsin and chymotrypsin activity, which may be responsible for the deleterious effects on larvae mortality. &nbsp; Key words: Digestive system, dengue fever, larvicidal activity, natural products.

Editorial introductions

Current Opinion in Gastroenterology was launched in 1985. It is one of a successful series of review journals whose unique format is designed to provide a systematic and critical assessment of the literature as presented in the many primary journals. The field of gastroenterology is divided into 12 sections that are reviewed once a year. Each section is assigned a Section Editor, a leading authority in the area, who identifies the most important topics at that time. Here we are pleased to introduce one of the Section Editors for this issue. SECTION EDITORS Fergus ShanahanFergus ShanahanFergus Shanahan is Professor and Chairman of the Department of Medicine at University College Cork (UCC), National University of Ireland. Born and educated in Dublin, he attended medical school at University College Dublin, Ireland, where he graduated with honours in 1977. After internship and residency in internal medicine in Dublin, he did a two-year fellowship in clinical immunology at McMaster University in Ontario, Canada, followed by a two-year fellowship in gastroenterology at the University of California, Los Angeles (UCLA), USA. Following completion of his fellowship at UCLA, he was appointed to the faculty there, rising to the rank of Associate Professor before making the decision, in 1993, to return to his native Ireland. Together with colleagues from several departments and different faculties within University College Cork and Teagasc (a research agency of the Irish Ministry of Food and Agriculture), Dr Shanahan led a team of clinicians, clinician-scientists, and basic scientists to successfully compete for seed funding from Science Foundation Ireland to create a multi-disciplinary research center, the Alimentary Pharmabiotic Centre, which investigates host-microbe interactions in the gut in health and disease. Under Dr Shanahan's directorship, the center now has a membership of 168 staff, scientists, and students and has expanded its funding and research base by securing research alliances with indigenous and multinational companies within the food and pharmaceutical sectors. Dr Shanahan has published more than 450 scientific papers and several on the medical humanities and has co-edited several books. He is a Fellow of the Royal College of Physicians in Ireland, Canada, and the United Kingdom as well as of the American College of Physicians. He served as President of the Irish Society of Gastroenterology from 2007–2009. He was recently named to the “Irish Life Science 50” a list of the top 50 Irish and Irish Americans in the life science industry. His interests are in mucosal immunology, gut microbiota, inflammatory bowel disease, and most things that affect the human experience. Stephen J.D. O’KeefeStephen J.D. O’KeefeDr O’Keefe's research is in the field of nutritional gastroenterology. Most of his investigations are translational in nature, evaluating the physiological and pathophysiological responses to dietary intake and interventional feeding. He has had NIH funding for his studies on the effects of enteral and parenteral feeding on the synthesis and secretion of trypsin in humans which have formed the basis for his latest NIH funded multicenter study on distal jejunal feeding in acute pancreatitis. He has applied stable isotope labeling techniques to the study of in vivo amino acid turnover and protein synthesis, with particular attention to the measurement of mucosal protein turnover and the synthesis of trypsin in patients with acute severe pancreatitis. His second area of current interest is the management of short bowel by enteral and IV feeding, and by small bowel transplantation. Third, he is investigating the influence of diet on colonic bacterial metabolism and colon cancer risk.

Synthesis, Characterization and Bio-Labeling Studies of Trypsin Stabilized Silver Quantum Clusters

Quantum clusters are very small sized nano materials with significant photo luminescence. Noble metal quantum clusters can be synthesized using protein as an etching substance. Here silver quantum clusters (Ag-QCs) are synthesized by etching with intestinal protease enzyme Trypsin. It has photoluminescence at bluish green region with a significant quantum yield. The synthesized clusters were characterized using tools like UV-Vis Spectroscopy, Fluorescence Spectroscopy, Zeta Potential, DLS, FTIR and TEM. Cyclic RGD peptides are conjugated to the quantum clusters by EDAC coupling method. These conjugated clusters were used for rabbit adipose derived stem cells imaging and they were found out to be suitable for bio-labeling application. MTT assay and AFM studies were performed to check the bio compatibility of the silver quantum clusters. They were found to be non cytotoxic.

Synthesis of the Backbone Cyclic Peptide Sunflower Trypsin Inhibitor‐1 Promoted by the Induced Peptidyl‐tRNA Drop‐off

Shifting around: A rapid one-pot preparation of a backbone cyclic peptide from peptidyl-tRNA, which has been ejected from the ribosome is described. A series of intramolecular rearrangements release tRNA and afford 1. A cyclization gives 2 and on enzymatic removal of the formylated methionine (fM) the backbone cyclic peptide is generated.

Synthesis of the Backbone Cyclic Peptide Sunflower Trypsin Inhibitor‐1 Promoted by the Induced Peptidyl‐tRNA Drop‐off

Peptide aus „Abfall“: Ein Eintopfverfahren ermöglicht die schnelle Synthese von Peptiden mit cyclischem Rückgrat ausgehend von abgestoßener Peptidyl-tRNA aus dem Ribosom. Eine Reihe intramolekularer Umlagerungen setzt die tRNA frei und liefert 1. Eine Cyclisierung ergibt 2, und durch enzymatische Abspaltung von formyliertem Methionin (fM) wird das cyclische Peptid erzeugt. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

A viral chitinase enhances oral activity of TMOF

In this study we investigate the combined effect on Heliothis virescens (Lepidoptera, Noctuidae) larvae of Aedes aegypti-Trypsin Modulating Oostatic Factor ( Aea-TMOF), a peptide that inhibits trypsin synthesis by the gut, impairing insect digestive function, and Autographa californica nucleopolyhedrovirus Chitinase A (AcMNPV ChiA), an enzyme that is able to alter the permeability of the peritrophic membrane (PM). Aea-TMOF and AcMNPV ChiA were provided to the larvae by administering transgenic tobacco plants, co-expressing both molecules. Experimental larvae feeding on these plants, compared to those alimented on plants expressing only one of the two molecules considered, showed significantly stronger negative effects on growth rate, developmental time and mortality. The impact of AcMNPV ChiA on the PM of H. virescens larvae, measured as increased permeability to molecules, was evident after five days of feeding on transgenic plants expressing ChiA. This result was confirmed by in vitro treatment of PM with recombinant ChiA, extracted from the transgenic plants used for the feeding experiments. Collectively, these data indicate the occurrence of a positive interaction between the two transgenes concurrently expressed in the same plant. The hydrolytic activity of ChiA on the PM of tobacco budworm larvae enhances the permeation of TMOF molecules to the ectoperitrophic space, and its subsequent absorption. The permeation through the paracellular route of Aea-TMOF resulted in a spotted accumulation on the basolateral domain of enterocytes, which suggests the occurrence of a receptor on the gut side facing the haemocoel. The binding of the peptide, permeating at increased rates due to the ChiA activity, is considered responsible for the enhanced insecticide activity of the transgenic plants expressing both molecules. These data corroborate the idea that ChiA can be effectively used as gut permeation enhancer in oral delivery strategies of bioinsecticides targeting haemocoelic receptors.

Nutrient control of release of pancreatic enzymes in yellowtail ( Seriola quinqueradiata): Involvement of CCK and PY in the regulatory loop

Cholecystokinin (CCK) and neuropeptide Y (NPY)-related peptides are key regulators of pancreatic enzyme secretion in vertebrates. CCK stimulates enzyme secretion whereas peptide Y (PY), a NPY-related peptide, plays an antagonistic role to that of CCK. In fish, very little is known about how different nutrients affect the synthesis of CCK and PY in the digestive tract, and the mechanism by which CCK and PY actually regulate digestive enzyme secretion is not well understood. In order to determine how different nutrients stimulate the synthesis of CCK and PY in yellowtail ( Seriola quinqueradiata), CCK and PY mRNA levels in the digestive tract were measured after oral administration of a single bolus of either phosphate-buffered saline (PBS: control), starch (carbohydrate), casein (protein), oleic acid (fatty acid) or tri-olein (triglyceride). In addition, in order to confirm the synthesis and secretion of digestive enzymes, the mRNA levels and enzymatic activities of three digestive enzymes (lipase, trypsin and amylase) were also analyzed. Casein, oleic acid and tri-olein increased the synthesis of lipase, trypsin and amylase, while starch and PBS did not affect the activity of any of these enzymes. CCK mRNA levels rose, while PY mRNA levels were reduced in fish administered casein, oleic acid and tri-olein. These results suggest that in yellowtail, CCK and PY maintain antagonistic control of pancreatic enzyme secretion after intake of protein and/or fat.

Invertebrate trypsins: a review

Food protein hydrolysis, a crucial step in digestion, is catalyzed by trypsin enzymes from the digestive apparatus of invertebrates. Trypsin appeared early in evolution and occurs in all phyla and, in the digestive systems of invertebrates, it became the most abundant proteinase. As in vertebrates, invertebrate trypsin is also present in several forms (isoenzymes). Its physiological importance in food protein digestion in several invertebrate species has emerged with compelling evidence; and several other physiological functions, such as regulation of digestive functions, are now settled. Recent advances in the knowledge of invertebrate trypsin synthesis, regulation, genetics, catalytic characteristics; structure, evolution, as well as inhibition, especially in non-Drosophilidae insects and in some crustaceans are reviewed. Most of the existing information is largely based on the use of several tools, including molecular techniques, to answer many still open questions and solve medical, agricultural, and food quality problems.

Physiological Evaluation of the Severity of Pancreatic Exocrine Dysfunction During Endoscopy

Despite the advances in pancreatic imaging, there continues to be a need to measure exocrine function to determine which patient requires enzyme supplementation. To evaluate the potential use of a rapid endoscopic test that can be conducted by nonacademic centers, we investigated whether concentration of trypsin in food-stimulated secretion is related to trypsin synthesis and secretion. Subjects include 22 chronic pancreatitis patients (10 mild, 5 moderate, and 7 severe radiological disease) and 11 healthy controls. During upper gastrointestinal endoscopy, pancreatic secretion was stimulated by a single 30-mL duodenal injection of an enteral diet, followed 5 minutes later by periampullary juice aspiration (endoscopic pancreatic function test [ePFT]). This was followed by a conventional 2-hour marker-perfusion diet-stimulated pancreatic trypsin secretion and synthesis study (2-hour PFT [2hPFT]). Severity of radiological disease was associated with a progressive loss of enzyme secretion measured by the 2hPFT. The endoscopic PFT correlated positively with 2hPFT (r2 = 0.48; P < 0.0001) and an activity of less than 5% of the average normal had a 96% specificity and 75% sensitivity for the detection of pancreatic insufficiency as defined by a loss of greater than 90% of pancreatic secretion. The diagnostic power of endoscopy may be enhanced by the collection of a pancreatic juice sample after enteral feed stimulation because measurement of the trypsin content will identify chronic pancreatitis patients who will be benefited by enzyme supplementation.

Reevaluation of the role of early trypsin activity in the transcriptional activation of the late trypsin gene in the mosquito Aedes aegypti

In the female mosquito Aedes aegypti, trypsin expression is largely biphasic. Early trypsin synthesis, which is regulated at the translational level relative to feeding, peaks in the first few hours post-blood meal. Late trypsin expression is regulated at the transcriptional level, and peaks 18–24 h post-blood meal. It was proposed that early trypsin activity released unknown factors during digestion of a meal that caused activation of transcription of the late trypsin gene. This connection between early trypsin activity and late trypsin expression was dependent on the fact that feeding a single trypsin inhibitor, soybean trypsin inhibitor (STI), which blocked early trypsin activity, also blocked late trypsin expression. We show in this study that feeding different trypsin inhibitors which effectively blocked early trypsin activity did not result in reduced late trypsin expression. We also found that a different lot of STI failed to cause inhibition of late trypsin transcription, although it was effective in inhibiting early trypsin activity. In addition, using RNAi methodology to reduce the level of early trypsin expression had no effect on the level of late trypsin expression. We conclude that early trypsin activity is not necessary for the transcriptional activation of late trypsin and that the previous results were due to the effect of a cytotoxic agent present in some, but not all preparations of STI.

Trypsin restoration time in the pyloric ceca of yellowtail Seriola quinqueradiata

The time required for trypsin restoration in the pyloric ceca-pancreatic tissue of yellowtail Seriola quinqueradiata (average body weight 942 g) was estimated. Fish were collected after starvation for 24, 48 or 72 h, and 6 h after being fed after each of the three tested starvation periods. Pyloric ceca isolated to gether with the pancreas were used for measurements of trypsin activity, quantity and expression rate. In the starved group, trypsin activities after 72 and 48 h starvation were significantly higher than after 24 h. Trypsin quantity, which was estimated by a Western blot test, showed similar results. The expression rate, estimated by reverse transcription polymerase chain reaction, was high after 24 h, but decreased after 48 h starvation. Thus, it was concluded that trypsin reaches the maximum storage level after 48 h starvation. In the fed group, the reduction in trypsin activity was greater when fish were fed after 48 and 72 h starvation periods that after 24 h. Trypsin expression rates 6 h after feeding were higher than before feeding. This indicates that trypsin synthesis is initiated after feeding or trypsin secretion and continues until 48 h of food deprivation. These results indicate that yellowtail of this size require feeding intervals of about 48 h to maximize trypsin storage.

Trypsin and splanchnic protein turnover during feeding and fasting in human subjects

Knowledge of the stimulatory effects of enteral and parenteral (intravenous) feeding on the synthesis and turnover of trypsin would help in the management of acute pancreatitis, because the disease is caused by the premature activation of trypsin. To investigate this, we labeled intravenous infusions with [1-(13)C]leucine and enterals with [(2)H]leucine and measured isotope enrichment of plasma, secreted trypsin, and duodenal mucosal proteins over 6 h by duodenal perfusion/aspiration and endoscopic biopsy. Thirty healthy volunteers were studied during fasting (n = 7), intravenous feeding (n = 6), or postpyloric enteral feeding [duodenal polymeric (n = 6), elemental duodenal (n = 6), and jejunal elemental (n = 5)]. All diets provided 1.5 g x kg(-1) x day(-1) protein and 40 kcal x kg(-1) x day(-1) energy. Results demonstrated that compared with fasting, enteral feeding increased the rate of appearance (71 +/- 4 vs. 91 +/- 5 min, P = 0.01) and secretion (546 +/- 80 vs. 219 +/- 37 U/h, P = 0.01) of newly labeled trypsin and expanded zymogen stores (1,660 +/- 237 vs. 749 +/- 133 units, P = 0.03). These differences persisted whether the feedings were polymeric or elemental, duodenal, or jejunal. In contrast, intravenous feeding had no effect on basal rates. Differential labeling of the plasma amino acid pool by enteral and intravenous isotope infusions suggested that 35% of absorbed amino acids were retained within the splanchnic bed during enteral feeding and that mucosal protein turnover increased from a fasting rate of 34 +/- 6 to 108 +/- 8%/day (P < 0.05) compared with no change after intravenous feeding. In conclusion, all common forms of enteral feeding stimulate the synthesis and secretion of pancreatic trypsin, and only parenteral nutrition avoids it.

Development of the digestive tract, trypsin activity and gene expression in eggs and larvae of the bullseye puffer fish Sphoeroides annulatus

Hatchery-produced Sphoeroides annulatus were studied from fertilized egg until day 32 post-hatch to examine the digestive tract development and to evaluate its digestive capacity during the larval period. Fish larvae were progressively fed microalgae, rotifers, Artemia nauplii and a formulated microdiet. Digestive tract development, trypsin activity, and trypsinogen gene expression in fish samples were analyzed by histology, histochemistry and reverse-transcription coupled to the polymerase chain reaction (RT–PCR) respectively. The intestine and liver started to develop on day 1 after hatch, followed by the pancreas. The mouth opened at day 4 after hatch, which was the start of rotifer feeding. Trypsinogen gene expression was detected very early in development, starting in the fertilized egg, showing a sharp increase in eggs at 75 h after fertilization, and then a gradual increase after hatching as the larvae developed. Trypsin activity by histochemistry was first detected at day 2 post-hatch, and maximum expression and activity were observed at days 16 to 24 after hatch, which corresponded to the period of Artemia nauplii feeding. No gastric glands were observed during the whole period of study. From day 28 onwards the fish were fed with a formulated microdiet, at this time both trypsin synthesis and activity decreased, suggesting a more important role for other enzymes in the digestion process.