Bovine Pancreatic Trypsin Inhibitor-Trypsin Complex as a Detection System for Recombinant Proteins

NASA Astrophysics Data System (ADS)

Borjigin, Jimo; Nathans, Jeremy

1993-01-01

Bovine pancreatic trypsin inhibitor (BPTI) binds to trypsin and anhydrotrypsin (an enzymatically inactive derivative of trypsin) with affinities of 6 x 10-14 and 1.1 x 10-13 M, respectively. We have taken advantage of the high affinity and specificity of this binding reaction to develop a protein tagging system in which biotinylated trypsin or biotinylated anhydrotrypsin is used as the reagent to detect recombinant fusion proteins into which BPTI has been inserted. Two proteins, opsin and growth hormone, were used as targets for insertional mutagenesis with BPTI. In each case, both domains of the fusion protein appear to be correctly folded. The fusion proteins can be specifically and efficiently detected by biotinylated trypsin or biotinylated anhydrotrypsin, as demonstrated by staining of transfected cells, protein blotting, affinity purification, and a mobility shift assay in SDS/polyacrylamide gels.

Molecular cloning of trypsin cDNAs and trypsin gene expression in the salmon louse Lepeophtheirus salmonis (Copepoda: Caligidae).

PubMed

Johnson, S C; Ewart, K V; Osborne, J A; Delage, D; Ross, N W; Murray, H M

2002-09-01

The salmon louse, Lepeophtheirus salmonis, is a marine ectoparasitic copepod that infects salmonid fishes. We are studying the interactions between this parasite and its salmonid hosts, as it is a common cause of disease in both wild and farmed stocks of salmon. In this paper, we report on the cloning and sequencing of seven trypsin-like enzymes from a cDNA library prepared from whole body preadult female and male L. salmonis. The predicted trypsin activation peptides are 23 or 24 residues in length, considerably longer than previously reported activation peptides of other animals. Differences in the putative signal and activation peptide sequences of the trypsin isoforms suggest that these forms differ in their regulation and function. The calculated molecular weights of the trypsins range from 23.6 to 23.7 kDa. There are eight cysteine residues, which suggest the presence of four disulfide bridges. These trypsins are very similar (>or=46% aa identity) to other crustacean trypsins and insect hypodermins. Using in situ hybridization techniques trypsinogen expression could be identified in all three cell types of the midgut.

Plasma trypsin in chronic pancreatitis and pancreatic adenocarcinoma.

PubMed

Adrian, T E; Besterman, H S; Mallinson, C N; Pera, A; Redshaw, M R; Wood, T P; Bloom, S R

1979-10-01

We have used a simple and precise radioimmunoassay to measure trypsin in human plasma. Fasting plasma trypsin concentrations were extremely low in patients with chronic pancreatitis with steatorrhoea (5 +/- 2 ng/ml) when compared to healthy controls (86 +/- 7 ng/ml, p less than 0.001). In patients with chronic pancreatitis but no steatorrhoea basal plasma trypsin levels were similar to those of the normal controls (99 +/- 25 ng/ml). A small but significant postprandial rise in plasma trypsin concentrations was observed in normal subjects (mean increment 15 +/- 4%, p less than 0.005, paired t test) but was absent in patients with chronic pancreatitis with steatorrhoea. In contrast to exocrine deficient chronic pancreatitis, other malabsorptive conditions associated with steatorrhoea (active coeliac disease and acute tropical sprue) demonstrated mean fasting trypsin concentrations similar to controls. Patients with adenocarcinoma of the pancreas had basal trypsin concentrations similar to healthy subjects as did patients with adenocarcinoma of the stomach, colon, rectum, brochus, and breast. In some cases measurement of plasma trypsin may be of help in the differential diagnosis of steatorrhoea.

Chitosan nanoparticles-trypsin interactions: Bio-physicochemical and molecular dynamics simulation studies.

PubMed

Salar, Safoura; Mehrnejad, Faramarz; Sajedi, Reza H; Arough, Javad Mohammadnejad

2017-10-01

Herein, we investigated the effect of the chitosan nanoparticles (CsNP) on the structure, dynamics, and activity of trypsin. The enzyme activity in complex with the nanoparticles slightly increased, which represents the interactions between the nanoparticles and the enzyme. The kinetic parameters of the enzyme, K m and k cat , increased after adding the nanoparticles, resulting in a slight increase in the catalytic efficiency (k cat /K m ). However, the effect of the nanoparticles on the kinetic stability of trypsin has not exhibited significant variations. Fluorescence spectroscopy did not show remarkable changes in the trypsin conformation in the presence of the nanoparticles. The circular dichroism (CD) spectroscopy results also revealed the secondary structure of trypsin attached to the nanoparticles slightly changed. Furthermore, we used molecular dynamics (MD) simulation to find more information about the interaction mechanisms between the nanoparticles and trypsin. The root mean square deviation (RMSD) of Cα atoms results have shown that in the presence of the nanoparticles, trypsin was stable. The simulation and the calculation of the binding free energy demonstrate that the nonpolar interactions are the most important forces for the formation of stable nanoparticle-trypsin complex. This study has explicitly elucidated that the nanoparticles have not considerable effect on the trypsin. Copyright © 2017. Published by Elsevier B.V.

Comparison of the effects between animal-derived trypsin and recombinant trypsin on human skin cells proliferation, gene and protein expression.

PubMed

Manira, Maarof; Khairul Anuar, Khairoji; Seet, Wan Tai; Ahmad Irfan, Abd Wahab; Ng, Min Hwei; Chua, Kien Hui; Mohd Heikal, Mohd Yunus; Aminuddin, Bin Saim; Ruszymah, Bt Hj Idrus

2014-03-01

Animal-derivative free reagents are preferred in skin cell culture for clinical applications. The aim of this study was to compare the performance and effects between animal-derived trypsin and recombinant trypsin for skin cells culture and expansion. Full thickness human skin was digested in 0.6 % collagenase for 6 h to liberate the fibroblasts, followed by treatment with either animal-derived trypsin; Trypsin EDTA (TE) or recombinant trypsin; TrypLE Select (TS) to liberate the keratinocytes. Both keratinocytes and fibroblasts were then culture-expanded until passage 2. Trypsinization for both cell types during culture-expansion was performed using either TE or TS. Total cells yield was determined using a haemocytometer. Expression of collagen type I, collagen type III (Col-III), cytokeratin 10, and cytokeratin 14 genes were quantified via RT-PCR and further confirmed with immunocytochemical staining. The results of our study showed that the total cell yield for both keratinocytes and fibroblasts treated with TE or TS were comparable. RT-PCR showed that expression of skin-specific genes except Col-III was higher in the TS treated group compared to that in the TE group. Expression of proteins specific to the two cell types were confirmed by immunocytochemical staining in both TE and TS groups. In conclusion, the performance of the recombinant trypsin is comparable with the well-established animal-derived trypsin for human skin cell culture expansion in terms of cell yield and expression of specific cellular markers.

Trypsin from unicorn leatherjacket (Aluterus monoceros) pyloric caeca: purification and its use for preparation of fish protein hydrolysate with antioxidative activity.

PubMed

Zamani, Abbas; Benjakul, Soottawat

2016-02-01

Fish proteases, especially trypsin, could be used to prepare fish protein hydrolysates with antioxidative activities. In this study, trypsin from the pyloric caeca of unicorn leatherjacket was purified by ammonium sulfate precipitation and soybean trypsin inhibitor (SBTI)-Sepharose 4B affinity chromatography. Hydrolysate from Indian mackerel protein isolate with different degrees of hydrolysis (20, 30 and 40% DH) was prepared using the purified trypsin, and antioxidative activities (1,1-diphenyl-2-picrylhydrazyl and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical-scavenging activities, ferric-reducing antioxidant power and ferrous-chelating activity) of the hydrolysate were determined. Trypsin was purified 26.43-fold with a yield of 13.43%. The purified trypsin had a molecular weight (MW) of 23.5 kDa and optimal activity at pH 8.0 and 55 °C. It displayed high stability in the pH range of 6.0-11.0 and was thermally stable up to 50 °C. Both SBTI (0.05 mmol L(-1)) and N-p-tosyl-L-lysine-chloromethylketone (5 mmol L(-1)) completely inhibited trypsin activity. Antioxidative activities of the hydrolysate from Indian mackerel protein isolate increased with increasing DH up to 40% (P < 0.05). Based on sodium dodecyl sulfate polyacrylamide gel electrophoresis, the hydrolysate with 40% DH had a MW lower than 6.5 kDa. The purified protease from unicorn leatherjacket pyloric caeca was identified as trypsin based on its ability to hydrolyze a specific synthetic substrate and the response to specific trypsin inhibitors. The purified trypsin could hydrolyze Indian mackerel protein isolate, and the resulting hydrolysate exhibited antioxidative activity depending on its DH. © 2015 Society of Chemical Industry.

Using Trypsin & Soybean Trypsin Inhibitor to Teach Principles of Enzyme Kinetics

ERIC Educational Resources Information Center

Howard, David R.; Herr, Julie; Hollister, Rhiannon

2006-01-01

Trypsin and soybean trypsin inhibitor (Kunitz inhibitor) can be used in a relatively simple and inexpensive student exercise to demonstrate the usefulness of enzyme kinetics. The study of enzyme kinetics is essential to biology because enzymes play such a crucial role in the biochemical pathways of all living organisms. The data from enzyme…

In vitro assessment of phthalate acid esters-trypsin complex formation.

PubMed

Chi, Zhenxing; Zhao, Jing; Li, Weiguo; Araghi, Arash; Tan, Songwen

2017-10-01

In this work, interactions of three phthalate acid esters (PAEs), including dimethyl phthalate (DMP), diethyl phthalate (DEP) and dibutyl phthalate (DBP), with trypsin have been studied in vitro, under simulated physiological conditions using multi-spectroscopic techniques and molecular modeling. The results show that these PAEs can bind to the trypsin, forming trypsin-PAEs complexes, mainly via hydrophobic interactions, with the affinity order of DMP > DEP > DBP. Binding to the PAEs is found to result in molecular deformation of trypsin. The modeling results suggest that only DBP can bind with the amino acid residues of the catalytic triad and S1 binding pocket of trypsin, leading to potential competitive enzyme inhibition. Copyright © 2017 Elsevier Ltd. All rights reserved.

The serine proteinase inhibitory proteins of the chondrodystrophoid (beagle) and non-chondrodystrophoid (greyhound) canine intervertebral disc.

PubMed

Melrose, J; Taylor, T K; Ghosh, P

1997-06-01

Trypsin inhibitory proteins of low buoyant density (p < or = 1.35 g/mL) fractions were prepared by CsCl density gradient ultracentrifugation of 4 M guanidinium hydrochloride extracts of lumbar beagle and greyhound annulus fibrosus and nucleus pulposus from animals aged 1 to 6 years. Affinity blotting with biotinylated trypsin was used to identify active trypsin inhibitory protein species; these species were also identified immunologically by Western blotting using antibodies against bovine pancreatic trypsin inhibitor (BPTI), and human inter-alpha-trypsin inhibitor (ITI). None of the trypsin inhibitory species evident in Western blots were reactive with anti-human alpha1-proteinase inhibitor (alpha-1-PI), alpha2-macroglobulin or secretory leucocyte proteinase inhibitor. The greyhound intervertebral disc samples generally had higher levels of active trypsin inhibitor species per unit weight of tissue extracted, and a more extensive range of inhibitor species. Inhibitor species of 30, 32, 34 kDa were identified in both beagle and greyhound intervertebral disc samples; these species were generally most prominent in the annulus fibrosus samples. In contrast, the nucleus pulposus samples contained relatively large trypsin inhibitor species; the anti-BPTI detected an inhibitor species of approximately 85-90 kDa; anti-ITI detected species of 120-250 kDa; biotinylated trypsin detected species of 60-110 kDa. A small molecular mass trypsin inhibitor species of 6 kDa, which was of similar mobility to BPTI, was also detected in annulus fibrosus samples; however, this species did not react with anti-BPTI.

Practical and theoretical characterization of Inga laurina Kunitz inhibitor on the control of Homalinotus coriaceus.

PubMed

Macedo, Maria Lígia Rodrigues; Freire, Maria das Graças Machado; Franco, Octávio Luiz; Migliolo, Ludovico; de Oliveira, Caio Fernando Ramalho

2011-02-01

Digestive endoprotease activities of the coconut palm weevil, Homalinotus coriaceus (Coleoptera: Curculionidae), were characterized based on the ability of gut extracts to hydrolyze specific synthetic substrates, optimal pH, and hydrolysis sensitivity to protease inhibitors. Trypsin-like proteinases were major enzymes for H. coriaceus, with minor activity by chymotrypsin proteinases. More importantly, gut proteinases of H. coriaceus were inhibited by trypsin inhibitor from Inga laurina seeds. In addition, a serine proteinase inhibitor from I. laurina seeds demonstrated significant reduction of growth of H. coriaceus larvae after feeding on inhibitor incorporated artificial diets. Dietary utilization experiments show that 0.05% I. laurina trypsin inhibitor, incorporated into an artificial diet, decreases the consumption rate and fecal production of H. coriaceus larvae. Dietary utilization experiments show that 0.05% I. laurina trypsin inhibitor, incorporated into an artificial diet, decreases the consumption rate and fecal production of H. coriaceus larvae. We have constructed a three-dimensional model of the trypsin inhibitor complexed with trypsin. The model was built based on its comparative homology with soybean trypsin inhibitor. Trypsin inhibitor of I. laurina shows structural features characteristic of the Kunitz type trypsin inhibitor. In summary, these findings contribute to the development of biotechnological tools such as transgenic plants with enhanced resistance to insect pests. Copyright © 2010 Elsevier Inc. All rights reserved.

DNA-directed trypsin immobilization on a polyamidoamine dendrimer-modified capillary to form a renewable immobilized enzyme microreactor.

PubMed

Wu, Nan; Wang, Siming; Yang, Ye; Song, Jiayi; Su, Ping; Yang, Yi

2018-07-01

A novel type of trypsin capillary microreactor was developed based on a DNA-directed immobilization (DDI) technique applied to a fused-silica capillary modified with polyamidoamine (PAMAM) dendrimers. Trypsin binding to the inner wall of the capillary was confirmed by confocal laser scanning microscopy. The properties of the trypsin-DNA conjugated, PAMAM-modified capillary microreactor were investigated by monitoring hydrolysis of Nα-benzoyl- L -arginine ethyl ester. Through the hybridization and dehybridization of the DNA, the inner wall of the capillary functionalized with trypsin can be regenerated, thus indicating the renewability of this enzyme microreactor. In addition, these results demonstrated that introduction of PAMAM enabled higher amounts of trypsin to be immobilized, markedly improving the enzymolysis efficiency, compared with traditional modified capillaries. The digestion performance of the trypsin capillary microreactor was further evaluated by digesting cytochrome C, and a peptide numbers of 8, and a sequence coverage of 59% were obtained. This renewable and efficient immobilized trypsin capillary microreactor combines advantages of both DDI technology and PAMAM, and is potentially adaptable to high-throughput enzyme assays in biochemical and clinical research. Copyright © 2018. Published by Elsevier B.V.

Development of a rapid high-efficiency scalable process for acetylated Sus scrofa cationic trypsin production from Escherichia coli inclusion bodies.

PubMed

Zhao, Mingzhi; Wu, Feilin; Xu, Ping

2015-12-01

Trypsin is one of the most important enzymatic tools in proteomics and biopharmaceutical studies. Here, we describe the complete recombinant expression and purification from a trypsinogen expression vector construct. The Sus scrofa cationic trypsin gene with a propeptide sequence was optimized according to Escherichia coli codon-usage bias and chemically synthesized. The gene was inserted into pET-11c plasmid to yield an expression vector. Using high-density E. coli fed-batch fermentation, trypsinogen was expressed in inclusion bodies at 1.47 g/L. The inclusion body was refolded with a high yield of 36%. The purified trypsinogen was then activated to produce trypsin. To address stability problems, the trypsin thus produced was acetylated. The final product was generated upon gel filtration. The final yield of acetylated trypsin was 182 mg/L from a 5-L fermenter. Our acetylated trypsin product demonstrated higher BAEE activity (30,100 BAEE unit/mg) than a commercial product (9500 BAEE unit/mg, Promega). It also demonstrated resistance to autolysis. This is the first report of production of acetylated recombinant trypsin that is stable and suitable for scale-up. Copyright © 2015 Elsevier Inc. All rights reserved.

A trypsin inhibitor from Sapindus saponaria L. seeds: purification, characterization, and activity towards pest insect digestive enzyme.

PubMed

Macedo, Maria Lígia R; Diz Filho, Eduardo B S; Freire, Mariadas Graças M; Oliva, Maria Luiza V; Sumikawa, Joana T; Toyama, Marcos H; Marangoni, Sérgio

2011-01-01

The present paper describes the purification, characterization and determination of the partial primary structure of the first trypsin inhibitor isolated from the family Sapindaceae. A highly stable, potent trypsin inhibitor (SSTI) was purified to homogeneity. SDS-PAGE analysis revealed that the protein consists of a two-polypeptide chain with molecular masses of approximately 15 and 3 kDa. The purified inhibitor inhibited bovine trypsin at a 1:1 M ratio. Kinetic analysis revealed that the protein is a competitive inhibitor with an equilibrium dissociation constant of 10⁻⁹ M for trypsin. The partial NH₂- terminal sequence of 36 amino acids in SSTI indicates homology with other members of the trypsin-inhibitor family from different sources. This inhibitor is highly stable in the presence of denaturing agents. SSTI showed significant inhibitory activity against trypsin-like proteases present in the larval midgut on Anagasta kuehniella, Corcyra cephalonica, Diatreae saccharalis and Anticarsia gemmatalis.

The sequence and X-ray structure of the trypsin from Fusarium oxysporum.

PubMed

Rypniewski, W R; Hastrup, S; Betzel, C; Dauter, M; Dauter, Z; Papendorf, G; Branner, S; Wilson, K S

1993-06-01

The trypsin from Fusarium oxysporum is equally homologous to trypsins from Streptomyces griseus, Streptomyces erythraeus and to bovine trypsin. A DFP (diisopropylfluorophosphate) inhibited form of the enzyme has been crystallized from 1.4 M Na2SO4, buffered with citrate at pH 5.0-5.5. The crystals belong to space group P2(1) with cell parameters a = 33.43 A, b = 67.65 A, c = 39.85 A and beta = 107.6 degrees. There is one protein molecule in the asymmetric unit. X-ray diffraction data to a resolution of 1.8 A were collected on film using synchrotron radiation. The structure was solved by molecular replacement using models of bovine and S. griseus trypsins and refined to an R-factor of 0.141. The overall fold is similar to other trypsins, with some insertions and deletions. There is no evidence of the divalent cation binding sites seen in other trypsins. The covalently bound inhibitor molecule is clearly visible.

Mechanism of cinnamic acid-induced trypsin inhibition: A multi-technique approach

NASA Astrophysics Data System (ADS)

Zhang, Hongmei; Zhou, Qiuhua; Cao, Jian; Wang, Yanqing

2013-12-01

In order to investigate the association of the protease trypsin with cinnamic acid, the interaction was characterized by using fluorescence, UV-vis absorption spectroscopy, molecular modeling and an enzymatic inhibition assay. The binding process may be outlined as follows: cinnamic acid can interact with trypsin with one binding site to form cinnamic acid-trypsin complex, resulting in inhibition of trypsin activity; the spectroscopic data show that the interaction is a spontaneous process with the estimated enthalpy and entropy changes being -8.95 kJ mol-1 and 50.70 J mol-1 K-1, respectively. Noncovalent interactions make the main contribution to stabilize the trypsin-cinnamic acid complex; cinnamic acid can enter into the primary substrate-binding pocket and alter the environment around Trp and Tyr residues.

Purification and characterization of a trypsin inhibitor from the seeds of Artocarpus heterophyllus Lam.

PubMed

Lyu, Junchen; Liu, Yuan; An, Tianchen; Liu, Yujun; Wang, Manchuriga; Song, Yanting; Zheng, Feifei; Wu, Dan; Zhang, Yingxia; Deng, Shiming

2015-05-01

A proteinaceous inhibitor against trypsin was isolated from the seeds of Artocarpus heterophyllus Lam. by successive ammonium sulfate precipitation, ion-exchange, and gel-filtration chromatography. The trypsin inhibitor, named as AHLTI (A. heterophyllus Lam. trypsin inhibitor), consisted of a single polypeptide chain with a molecular weight of 28.5 kDa, which was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel-filtration chromatography. The N-terminal sequence of AHLTI was DEPPSELDAS, which showed no similarity to other known trypsin inhibitor sequence. AHLTI completely inhibited bovine trypsin at a molar ratio of 1:2 (AHLTI:trypsin) analyzed by native polyacrylamide gel electrophoresis, inhibition activity assay, and gel-filtration chromatography. Moreover, kinetic enzymatic studies were carried out to understand the inhibition mechanism of AHLTI against trypsin. Results showed that AHLTI was a competitive inhibitor with an equilibrium dissociation constant (Ki) of 3.7 × 10(-8) M. However, AHLTI showed weak inhibitory activity toward chymotrypsin and elastase. AHLTI was stable over a broad range of pH 4-8 and temperature 20-80°C. The reduction agent, dithiothreitol, had no obvious effect on AHLTI. The trypsin inhibition assays of AHLTI toward digestive enzymes from insect pest guts in vitro demonstrated that AHLTI was effective against enzymes from Locusta migratoria manilensis (Meyen). These results suggested that AHLTI might be a novel trypsin inhibitor from A. heterophyllus Lam. belonging to Kunitz family, and play an important role in protecting from insect pest. © The Author 2015. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

Mechanism of cinnamic acid-induced trypsin inhibition: a multi-technique approach.

PubMed

Zhang, Hongmei; Zhou, Qiuhua; Cao, Jian; Wang, Yanqing

2013-12-01

In order to investigate the association of the protease trypsin with cinnamic acid, the interaction was characterized by using fluorescence, UV-vis absorption spectroscopy, molecular modeling and an enzymatic inhibition assay. The binding process may be outlined as follows: cinnamic acid can interact with trypsin with one binding site to form cinnamic acid-trypsin complex, resulting in inhibition of trypsin activity; the spectroscopic data show that the interaction is a spontaneous process with the estimated enthalpy and entropy changes being -8.95 kJ mol(-1) and 50.70 J mol(-1) K(-1), respectively. Noncovalent interactions make the main contribution to stabilize the trypsin-cinnamic acid complex; cinnamic acid can enter into the primary substrate-binding pocket and alter the environment around Trp and Tyr residues. Copyright © 2013 Elsevier B.V. All rights reserved.

Modifications in trypsin digestion protocol for increasing the efficiency and coverage.

PubMed

Syal, Kirtimaan; Tadala, Raghu

2015-01-01

Standard trypsin digestion protocol of proteins followed by MALDI-MS analysis has been realized as an important tool for the identification and characterization of proteins. In this article, we proposed the elimination of the step of 'staining/de-staining of gel pieces' in in-gel digestion protocol in order to improve the efficiency of trypsin digestion. Coomassie dye is known to interfere with digestion of proteins by trypsin and the procedure of staining-de-staining could result in loss of photoaffinity probe, post translational modifications and catalytic activities of enzymes. Further, we studied parameters like hydrophobicity and isoelectric point, and attempted to quantitatively relate it to the efficiency of trypsin digestion. We suggest that properties of proteins should be considered and trypsin digestion protocol should be appropriately modified as per sequence and other information.

Effect of atropine and somatostatin on bombesin-stimulated plasma immunoreactive trypsin release in man.

PubMed

de Jong, A J; Klamer, M; Lamers, C B

1987-01-01

This study was undertaken to determine the effect of atropine and somatostatin, two inhibitors of intraduodenal pancreatic enzyme secretion, on bombesin-stimulated release of plasma immunoreactive trypsin in 6 healthy volunteers. Infusion of 5 ng/kg.min bombesin during 30 min induced significant increases in plasma trypsin from 206 +/- 20 to 334 +/- 44 ng/ml (p less than 0.01). Atropine (15 ng/kg as i.v. bolus followed by 5 ng/kg.h) had no influence on the bombesin-stimulated increase in plasma immunoreactive trypsin (207 +/- 20 to 326 +/- 54 ng/ml). Somatostatin (125 micrograms as i.v. bolus followed by 125 micrograms/h) also failed to inhibit the plasma trypsin response to bombesin (207 +/- 18 to 663 +/- 166 ng/ml). These results point to major differences in the regulation of plasma and intraduodenal trypsin secretion.

Effect of Moringa oleifera flower extract on larval trypsin and acetylcholinesterase activities in Aedes aegypti.

PubMed

Pontual, Emmanuel Viana; Napoleão, Thiago Henrique; Dias de Assis, Caio Rodrigo; de Souza Bezerra, Ranilson; Xavier, Haroudo Satiro; Navarro, Daniela Maria do Amaral Ferraz; Coelho, Luana Cassandra Breitenbach Barroso; Paiva, Patrícia Maria Guedes

2012-03-01

Aedes aegypti control is crucial to reducing dengue fever. Aedes aegypti larvae have developed resistance to organophosporous insecticides and the use of natural larvicides may help manage larval resistance by increasing elements in insecticide rotation programs. Here, we report on larvicidal activity of Moringa oleifera flower extract against A. aegypti L(1), L(2), L(3), and L(4) as well as the effect of flower extract on gut trypsin and whole-larval acetylcholinesterase from L(4.) In addition, the heated flower extract was investigated for larvicidal activity against L(4) and effect on larval gut trypsin. Moringa oleifera flower extract contains a proteinaceous trypsin inhibitor (M. oleifera flower trypsin inhibitor, MoFTI), triterpene (β-amyrin), sterol (β-sitosterol) as well as flavonoids (kaempferol and quercetin). Larvicidal activity was detected against L(2), L(3), and L(4) (LC(50) of 1.72%, 1.67%, and 0.92%, respectively). Flower extract inhibited L(4) gut trypsin (MoFTI K(i) = 0.6 nM) and did not affect acetylcholinesterase activity. In vivo assay showed that gut trypsin activity from L(4) treated with M. oleifera flower extract decreased over time (0-1,440 min) and was strongly inhibited (98.6%) after 310 min incubation; acetylcholinesterase activity was not affected. Thermal treatment resulted in a loss of trypsin inhibitor and larvicidal activities, supporting the hypothesis that flower extract contains a proteinaceous trypsin inhibitor that may be responsible for the deleterious effects on larval mortality. © 2012 Wiley Periodicals, Inc.

Dissection of the binding of hydrogen peroxide to trypsin using spectroscopic methods and molecular modeling

NASA Astrophysics Data System (ADS)

Song, Wei; Yu, Zehua; Hu, Xinxin; Liu, Rutao

2015-02-01

Studies on the effects of environmental pollutants to protein in vitro has become a global attention. Hydrogen peroxide (H2O2) is used as an effective food preservative and bleacher in industrial production. The toxicity of H2O2 to trypsin was investigated by multiple spectroscopic techniques and the molecular docking method at the molecular level. The intrinsic fluorescence of trypsin was proved to be quenched in a static process based on the results of fluorescence lifetime experiment. Hydrogen bonds interaction and van der Waals forces were the main force to generate the trypsin-H2O2 complex on account of the negative ΔH0 and ΔS0. The binding of H2O2 changed the conformational structures and internal microenvironment of trypsin illustrated by UV-vis absorption, fluorescence, synchronous fluorescence, three-dimensional (3D) fluorescence and circular dichroism (CD) results. However, the binding site was far away from the active site of trypsin and the trypsin activity was only slightly affected by H2O2, which was further explained by molecular docking investigations.

Investigation on potential enzyme toxicity of clenbuterol to trypsin

NASA Astrophysics Data System (ADS)

Chai, Jun; Xu, Qifei; Dai, Jinping; Liu, Rutao

2013-03-01

Clenbuterol (CLB) is a kind of β2-adrenergic agonists which was illegally used as feed additives nowadays. The toxic interaction of CLB with trypsin, an important digestive enzyme, was studied in vitro using multi-spectroscopic methods and molecular modeling methods. CLB was proved to bind with trypsin in S1 pocket, forming a complex driven by the dominant force of H-bond. The binding constant was calculated to be 1.79887 × 105 L mol-1 at 289 K and 0.32584 × 105 L mol-1 at 310 K, respectively. The skeleton of trypsin became loosened and unfolded with the amino residues microenvironment changed. The secondary and tertiary structure of trypsin also varied. Molecular modeling studies illustrated specific display of the binding information and explained most of the experiment phenomena. The binding site of CLB induced the fluorescence quenching as well as inhibition of enzyme activity of trypsin. The study confirmed that CLB had potential toxicity on both the structure and function of trypsin and the effects enhanced with the increasing concentration of CLB.

Engineering Protein Allostery: 1.05 Å Resolution Structure and Enzymatic Properties of a Na[superscript +]-activated Trypsin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Page, Michael J.; Carrell, Christopher J.; Di Cera, Enrico

2008-05-28

Some trypsin-like proteases are endowed with Na{sup +}-dependent allosteric enhancement of catalytic activity, but this important mechanism has been difficult to engineer in other members of the family. Replacement of 19 amino acids in Streptomyces griseus trypsin targeting the active site and the Na{sup +}-binding site were found necessary to generate efficient Na{sup +} activation. Remarkably, this property was linked to the acquisition of a new substrate selectivity profile similar to that of factor Xa, a Na{sup -} activated protease involved in blood coagulation. The X-ray crystal structure of the mutant trypsin solved to 1.05 {angstrom} resolution defines the engineeredmore » Na{sup +} site and active site loops in unprecedented detail. The results demonstrate that trypsin can be engineered into an efficient allosteric protease, and that Na+ activation is interwoven with substrate selectivity in the trypsin scaffold.« less

Flow Cytometry Method Analysis of Apoptosis: No Significant Difference Between EDTA and EDTA-free Trypsin Treatment Procedure.

PubMed

Xu, Xiao-yan; Nie, Xiao-cui; Ma, Hai-ying; Song, Guo-qing; Zhang, Xiao-tong; Jin, Yu-nan; Yu, Yan-qiu

2015-04-01

Flow cytometry method (FCM) is a generally accepted tool to analyze apoptosis. Although apoptosis assay kit was applied by many companies, the manufacturers were not consistent with whether using Trypsin with EDTA to collect the adherent cells. In another words, the influence of EDTA on apoptotic ratio is not clear. In this work, we compared the proportion of apoptotic cells with EDTA or EDTA-free Trypsin treatment by FCM. We concluded that Trypsin with or without EDTA has little influence on the proportion of apoptotic cells. In addition, we found that the ratio of necrosis and apoptosis was different in cells collected by scraping. WAVE2 protein was analyzed as a typical example for movement related protein. WAVE2 expression is elevated in the EDTA Trypsin treated group, compared with EDTA-free Trypsin treatment and scrapping group. © The Author(s) 2014.

Immobilization of Trypsin in Lignocellulosic Waste Material to Produce Peptides with Bioactive Potential from Whey Protein

PubMed Central

Bassan, Juliana Cristina; de Souza Bezerra, Thaís Milena; Peixoto, Guilherme; da Cruz, Clariana Zanutto Paulino; Galán, Julián Paul Martínez; Vaz, Aline Buda dos Santos; Garrido, Saulo Santesso; Filice, Marco; Monti, Rubens

2016-01-01

In this study, trypsin (Enzyme Comission 3.4.21.4) was immobilized in a low cost, lignocellulosic support (corn cob powder—CCP) with the goal of obtaining peptides with bioactive potential from cheese whey. The pretreated support was activated with glyoxyl groups, glutaraldehyde and IDA-glyoxyl. The immobilization yields of the derivatives were higher than 83%, and the retention of catalytic activity was higher than 74%. The trypsin-glyoxyl-CCP derivative was thermally stable at 65 °C, a value that was 1090-fold higher than that obtained with the free enzyme. The trypsin-IDA-glyoxyl-CCP and trypsin-glutaraldehyde-CCP derivatives had thermal stabilities that were 883- and five-fold higher, respectively, then those obtained with the free enzyme. In the batch experiments, trypsin-IDA-glyoxyl-CCP retained 91% of its activity and had a degree of hydrolysis of 12.49%, while the values for trypsin-glyoxyl-CCP were 87% and 15.46%, respectively. The stabilized derivative trypsin-glyoxyl-CCP was also tested in an upflow packed-bed reactor. The hydrodynamic characterization of this reactor was a plug flow pattern, and the kinetics of this system provided a relative activity of 3.04 ± 0.01 U·g−1 and an average degree of hydrolysis of 23%, which were suitable for the production of potentially bioactive peptides. PMID:28773482

Bovine pancreatic trypsin inhibitor immobilized onto sepharose as a new strategy to purify a thermostable alkaline peptidase from cobia (Rachycentron canadum) processing waste.

PubMed

França, Renata Cristina da Penha; Assis, Caio Rodrigo Dias; Santos, Juliana Ferreira; Torquato, Ricardo José Soares; Tanaka, Aparecida Sadae; Hirata, Izaura Yoshico; Assis, Diego Magno; Juliano, Maria Aparecida; Cavalli, Ronaldo Olivera; Carvalho, Luiz Bezerra de; Bezerra, Ranilson Souza

2016-10-15

A thermostable alkaline peptidase was purified from the processing waste of cobia (Rachycentron canadum) using bovine pancreatic trypsin inhibitor (BPTI) immobilized onto Sepharose. The purified enzyme had an apparent molecular mass of 24kDa by both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. Its optimal temperature and pH were 50°C and 8.5, respectively. The enzyme was thermostable until 55°C and its activity was strongly inhibited by the classic trypsin inhibitors N-ρ-tosyl-l-lysine chloromethyl ketone (TLCK) and benzamidine. BPTI column allowed at least 15 assays without loss of efficacy. The purified enzyme was identified as a trypsin and the N-terminal amino acid sequence of this trypsin was IVGGYECTPHSQAHQVSLNSGYHFC, which was highly homologous to trypsin from cold water fish species. Using Nα-benzoyl-dl-arginine ρ-nitroanilide hydrochloride (BApNA) as substrate, the apparent km value of the purified trypsin was 0.38mM, kcat value was 3.14s(-1), and kcat/km was 8.26s(-1)mM(-1). The catalytic proficiency of the purified enzyme was 2.75×10(12)M(-1) showing higher affinity for the substrate at the transition state than other fish trypsin. The activation energy (AE) of the BApNA hydrolysis catalyzed by this enzyme was estimated to be 11.93kcalmol(-1) while the resulting rate enhancement of this reaction was found to be approximately in a range from 10(9) to 10(10)-fold evidencing its efficiency in comparison to other trypsin. This new purification strategy showed to be appropriate to obtain an alkaline peptidase from cobia processing waste with high purification degree. According with N-terminal homology and kinetic parameters, R. canadum trypsin may gathers desirable properties of psychrophilic and thermostable enzymes. Copyright © 2016 Elsevier B.V. All rights reserved.

Protease-Activated Receptor 2, Dipeptidyl Peptidase I, and Proteases Mediate Clostridium difficile Toxin A Enteritis

PubMed Central

COTTRELL, GRAEME S.; AMADESI, SILVIA; PIKIOS, STELLA; CAMERER, ERIC; WILLARDSEN, J. ADAM; MURPHY, BRETT R.; CAUGHEY, GEORGE H.; WOLTERS, PAUL J.; COUGHLIN, SHAUN R.; PETERSON, ANDERS; KNECHT, WOLFGANG; POTHOULAKIS, CHARALABOS; BUNNETT, NIGEL W.; GRADY, EILEEN F.

2008-01-01

Background & Aims We studied the role of protease-activated receptor 2 (PAR2) and its activating enzymes, trypsins and tryptase, in Clostridium difficile toxin A (TxA)-induced enteritis. Methods We injected TxA into ileal loops in PAR2 or dipeptidyl peptidase I (DPPI) knockout mice or in wild-type mice pretreated with tryptase inhibitors (FUT-175 or MPI-0442352) or soybean trypsin inhibitor. We examined the effect of TxA on expression and activity of PAR2 and trypsin IV messenger RNA in the ileum and cultured colonocytes. We injected activating peptide (AP), trypsins, tryptase, and p23 in wild-type mice, some pretreated with the neurokinin 1 receptor antagonist SR140333. Results TxA increased fluid secretion, myeloperoxidase activity in fluid and tissue, and histologic damage. PAR2 deletion decreased TxA-induced ileitis, reduced luminal fluid secretion by 20%, decreased tissue and fluid myeloperoxidase by 50%, and diminished epithelial damage, edema, and neutrophil infiltration. DPPI deletion reduced secretion by 20% and fluid myeloperoxidase by 55%. In wild-type mice, FUT-175 or MPI-0442352 inhibited secretion by 24%−28% and tissue and fluid myeloperoxidase by 31%−71%. Soybean trypsin inhibitor reduced secretion to background levels and tissue myeloperoxidase by up to 50%. TxA increased expression of PAR2 and trypsin IV in enterocytes and colonocytes and caused a 2-fold increase in Ca2+ responses to PAR2 AP. AP, tryptase, and trypsin isozymes (trypsin I/II, trypsin IV, p23) caused ileitis. SR140333 prevented AP-induced ileitis. Conclusions PAR2 and its activators are proinflammatory in TxA-induced enteritis. TxA stimulates existing PAR2 and up-regulates PAR2 and activating proteases, and PAR2 causes inflammation by neurogenic mechanisms. PMID:17570216

Flow Cytometry of Human Primary Epidermal and Follicular Keratinocytes

PubMed Central

Gragnani, Alfredo; Ipolito, Michelle Zampieri; Sobral, Christiane S; Brunialti, Milena Karina Coló; Salomão, Reinaldo; Ferreira, Lydia Masako

2008-01-01

Objective: The aim of this study was to characterize using flow cytometry cultured human primary keratinocytes isolated from the epidermis and hair follicles by different methods. Methods: Human keratinocytes derived from discarded fragments of total skin and scalp hair follicles from patients who underwent plastic surgery in the Plastic Surgery Division at UNIFESP were used. The epidermal keratinocytes were isolated by using 3 different methods: the standard method, upon exposure to trypsin for 30 minutes; the second, by treatment with dispase for 18 hours and with trypsin for 10 minutes; and the third, by treatment with dispase for 18 hours and with trypsin for 30 minutes. Follicular keratinocytes were isolated using the standard method. Results: On comparing the group treated with dispase for 18 hours and with trypsin for 10 minutes with the group treated with dispase for 18 hours and with trypsin for 30 minutes, it was observed that the first group presented the largest number of viable cells, the smallest number of cells in late apoptosis and necrosis with statistical significance, and no difference in apoptosis. When we compared the group treated with dispase for 18 hours and with trypsin for 10 minutes with the group treated with trypsin, the first group presented the largest number of viable cells, the smallest number of cells in apoptosis with statistical significance, and no difference in late apoptosis and necrosis. When we compared the results of the group treated with dispase for 18 hours and with trypsin for 10 minutes with the results for follical isolation, there was a statistical difference in apoptosis and viable cells. Conclusion: The isolation method of treatment with dispase for 18 hours and with trypsin for 10 minutes produced the largest number of viable cells and the smallest number of cells in apoptosis/necrosis. PMID:18350110

Flow cytometry of human primary epidermal and follicular keratinocytes.

PubMed

Gragnani, Alfredo; Ipolito, Michelle Zampieri; Sobral, Christiane S; Brunialti, Milena Karina Coló; Salomão, Reinaldo; Ferreira, Lydia Masako

2008-02-19

The aim of this study was to characterize using flow cytometry cultured human primary keratinocytes isolated from the epidermis and hair follicles by different methods. Human keratinocytes derived from discarded fragments of total skin and scalp hair follicles from patients who underwent plastic surgery in the Plastic Surgery Division at UNIFESP were used. The epidermal keratinocytes were isolated by using 3 different methods: the standard method, upon exposure to trypsin for 30 minutes; the second, by treatment with dispase for 18 hours and with trypsin for 10 minutes; and the third, by treatment with dispase for 18 hours and with trypsin for 30 minutes. Follicular keratinocytes were isolated using the standard method. On comparing the group treated with dispase for 18 hours and with trypsin for 10 minutes with the group treated with dispase for 18 hours and with trypsin for 30 minutes, it was observed that the first group presented the largest number of viable cells, the smallest number of cells in late apoptosis and necrosis with statistical significance, and no difference in apoptosis. When we compared the group treated with dispase for 18 hours and with trypsin for 10 minutes with the group treated with trypsin, the first group presented the largest number of viable cells, the smallest number of cells in apoptosis with statistical significance, and no difference in late apoptosis and necrosis. When we compared the results of the group treated with dispase for 18 hours and with trypsin for 10 minutes with the results for follical isolation, there was a statistical difference in apoptosis and viable cells. The isolation method of treatment with dispase for 18 hours and with trypsin for 10 minutes produced the largest number of viable cells and the smallest number of cells in apoptosis/necrosis.

Characterization of the Interaction Between Pancreatic Trypsin and an Enteric Copolymer as a Tool for Several Biotechnological Applications.

PubMed

Braia, Mauricio Javier; Loureiro, Dana Belén; Tubio, Gisela; Romanini, Diana

2015-12-01

Protein-polyelectrolyte complexes are very interesting systems since they can be applied in many long-established and emerging areas of biotechnology. From nanotechnology to industrial processing, these complexes are used for many purposes: to build multilayer particles for biosensors; to entrap and deliver proteins for pharmaceutical applications; to isolate and immobilize proteins. The enteric copolymer poly(methacrylic acid-co-methyl methacrylate) 1:2 (MMA) has been designed for drug delivery although its chemical properties allow to use it for other applications. Understanding the interaction between trypsin and this polymer is very important in order to optimize the mechanism of formation of this complex for different biotechnological applications.The formation of the trypsin-MMA complex was studied by spectroscopy and isothermal titration calorimetry. Structural analysis of trypsin was carried out by catalytic activity assays, circular dichroism and differential scanning calorimetry. Isothermal titration calorimetry experiments showed that the insoluble complex contains 12 trypsin molecules per MMA molecule at pH 5 and they interact with high affinity to form insoluble complexes. Both electrostatic and hydrophobic forces are involved in the formation of the complex. The structure of trypsin is not affected by the presence of MMA, although it interacts with some domains of trypsin affecting its thermal denaturation as seen in the differential scanning calorimetry experiments. Its catalytic activity is not altered. Dynamic light scattering demonstrated the presence of a soluble trypsin-copolymer complex at pH 5 and 8. Turbidimetric assays show that the insoluble complex can be dissolved by low ionic strength and/or pH in order to obtain free native trypsin. Copyright © 2015 Elsevier B.V. All rights reserved.

Interaction of sodium benzoate with trypsin by spectroscopic techniques

NASA Astrophysics Data System (ADS)

Mu, Yue; Lin, Jing; Liu, Rutao

2011-12-01

The toxicity of sodium benzoate to trypsin was investigated by fluorescence spectroscopy, synchronous fluorescence spectroscopy, UV-visible absorption spectroscopy and circular dichroism (CD) spectroscopy under mimic physiological conditions. Sodium benzoate could unfold trypsin by decreasing the β-sheet structure, which leads to more exposure of internal amino acid groups and the obvious intrinsic fluorescence quenching with the rising concentration of sodium benzoate. The results of spectroscopic measurements indicated that sodium benzoate changed the internal microenvironment of trypsin and induced the alteration of the whole molecule, which were performed toxic effects on the organism. Trypsin and sodium benzoate interacted with each other to produce a substance by van der Waals forces and hydrogen bond, the model of which was shown by AutoDock software.

Trypsin-like Proteins of the Fungi as Possible Markers of Phytopathogenicity

USDA-ARS?s Scientific Manuscript database

Sequences of peptidases with conserved motifs around the active site residues that are characteristic of trypsins (similar to trypsin peptidases, STP) were obtained from publicly available fungal genomes and related databases. Among the 74 fungal genomes, 30 species of parasitic Ascomycota contained...

21 CFR 866.5890 - Inter-alpha trypsin inhibitor immunological test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test... measure by immunochemical techniques the inter-alpha trypsin inhibitor (a protein) in serum and other body fluids. Measurement of inter-alpha trypsin inhibitor may aid in the diagnosis of acute bacterial...

21 CFR 866.5890 - Inter-alpha trypsin inhibitor immunological test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test... measure by immunochemical techniques the inter-alpha trypsin inhibitor (a protein) in serum and other body fluids. Measurement of inter-alpha trypsin inhibitor may aid in the diagnosis of acute bacterial...

21 CFR 866.5890 - Inter-alpha trypsin inhibitor immunological test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test... measure by immunochemical techniques the inter-alpha trypsin inhibitor (a protein) in serum and other body fluids. Measurement of inter-alpha trypsin inhibitor may aid in the diagnosis of acute bacterial...

21 CFR 866.5890 - Inter-alpha trypsin inhibitor immunological test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test... measure by immunochemical techniques the inter-alpha trypsin inhibitor (a protein) in serum and other body fluids. Measurement of inter-alpha trypsin inhibitor may aid in the diagnosis of acute bacterial...

Structural Insights into the Role of the Cyclic Backbone in a Squash Trypsin Inhibitor*

PubMed Central

Daly, Norelle L.; Thorstholm, Louise; Greenwood, Kathryn P.; King, Gordon J.; Rosengren, K. Johan; Heras, Begoña; Martin, Jennifer L.; Craik, David J.

2013-01-01

MCoTI-II is a head-to-tail cyclic peptide with potent trypsin inhibitory activity and, on the basis of its exceptional proteolytic stability, is a valuable template for the design of novel drug leads. Insights into inhibitor dynamics and interactions with biological targets are critical for drug design studies, particularly for protease targets. Here, we show that the cyclization and active site loops of MCoTI-II are flexible in solution, but when bound to trypsin, the active site loop converges to a single well defined conformation. This finding of reduced flexibility on binding is in contrast to a recent study on the homologous peptide MCoTI-I, which suggested that regions of the peptide are more flexible upon binding to trypsin. We provide a possible explanation for this discrepancy based on degradation of the complex over time. Our study also unexpectedly shows that the cyclization loop, not present in acyclic homologues, facilitates potent trypsin inhibitory activity by engaging in direct binding interactions with trypsin. PMID:24169696

A trypsin inhibitor from rambutan seeds with antitumor, anti-HIV-1 reverse transcriptase, and nitric oxide-inducing properties.

PubMed

Fang, Evandro Fei; Ng, Tzi Bun

2015-04-01

Nephelium lappaceum L., commonly known as "rambutan," is a typical tropical tree and is well known for its juicy and sweet fruit which has an exotic flavor. Chemical studies on rambutan have led to the identification of various components such as monoterpene lactones and volatile compounds. Here, a 22.5-kDa trypsin inhibitor (N . lappaceum trypsin inhibitor (NLTI)) was isolated from fresh rambutan seeds using liquid chromatographical techniques. NLTI reduced the proteolytic activities of both trypsin and α-chymotrypsin. Dithiothreitol reduced the trypsin inhibitory activity of NLTI at a concentration of 1 mM, indicating that an intact disulfide bond is essential to the activity. NLTI inhibited HIV-1 reverse transcriptase with an IC50 of 0.73 μM. In addition, NLTI manifested a time- and dose-dependent inhibitory effect on growth in many tumor cells. NLTI is one of the few trypsin inhibitors with nitric oxide-inducing activity and may find application in tumor therapy.

Action of trypsin on structural changes of collagen fibres from sea cucumber (Stichopus japonicus).

PubMed

Liu, Zi-Qiang; Tuo, Feng-Yan; Song, Liang; Liu, Yu-Xin; Dong, Xiu-Ping; Li, Dong-Mei; Zhou, Da-Yong; Shahidi, Fereidoon

2018-08-01

Trypsin, a representative serine proteinase, was used to hydrolyse the collagen fibres from sea cucumber (Stichopus japonicus) to highlight the role of serine proteinase in the autolysis of sea cucumber. Partial disaggregation of collagen fibres into collagen fibrils upon trypsin treatment occurred. The trypsin treatment also caused a time-dependent release of water-soluble glycosaminoglycans and proteins. Therefore, the degradation of the proteoglycan bridges between collagen fibrils might account for the disaggregation of collagen fibrils. For trypsin-treated collagen fibres (72 h), the collagen fibrils still kept their structural integrity and showed characteristic D-banding pattern, and the dissolution rate of hydroxyproline was just 0.21%. Meanwhile, Fourier transform infrared analysis showed the collagen within trypsin-treated collagen fibres (72 h) still retaining their triple-helical conformation. These results suggested that serine proteinase participated in the autolysis of S. japonicus body wall by damaging the proteoglycan bridges between collagen fibrils and disintegrating the latter. Copyright © 2018 Elsevier Ltd. All rights reserved.

Salmon trypsin stimulates the expression of interleukin-8 via protease-activated receptor-2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Larsen, Anett K.; Department of Pharmacology, Institute of Medical Biology, University of Tromso, Tromso; Seternes, Ole-Morten

2008-08-01

In this study, we focus on salmon trypsin as an activator of inflammatory responses in airway cells in vitro. The rationale behind the investigation is that salmon industry workers are exposed to aerosols containing enzymes, which are generated during industrial processing of the fish. Knowing that serine proteases such as trypsin are highly active mediators with diverse biological activities, the stimulation of nuclear factor-kappa B (NF-{kappa}B) and interleukin (IL)-8 and the role of protease-activated receptors (PAR) in inflammatory signal mediation were investigated. Protease-activated receptors are considered important under pathological situations in the human airways, and a thorough understanding of PAR-inducedmore » cellular events and their consequences in airway inflammation is necessary. Human airway epithelial cells (A549) were exposed to trypsin isolated from fish (Salmo salar), and we observed that purified salmon trypsin could generate secretion of IL-8 in a concentration-dependent manner. Furthermore, we demonstrate that PAR-2 activation by salmon trypsin is coupled to an induction of NF-{kappa}B-mediated transcription using a PAR-2 transfected HeLa cell model. Finally, we show that the release of IL-8 from A549 following stimulation with purified salmon trypsin is mediated through activation of PAR-2 using specific small interfering RNAs (siRNAs). The results presented suggest that salmon trypsin, via activation of PAR-2, might influence inflammation processes in the airways if inhaled in sufficient amounts.« less

Optimization of trypsins for influenza A/H1N1 virus replication in MDCK SI-6 cells, a novel MDCK cell line.

PubMed

Iskandar, Viska I; Sasaki, Yutaka; Yoshino, Naoto; Abubakar, Raden Z R; Sato, Shigehiro; Muraki, Yasushi

2018-02-01

A cell-based vaccine production method for influenza virus may be an effective and more rapid alternative to egg-based systems. For high-yield virus production, the effect of bovine, porcine, fungal, and recombinant trypsins on influenza A/H1N1 virus replication in MDCK SI-6 cells (SI-6 cells), a novel MDCK cell line developed by our research group, was examined. SI-6 cells infected with influenza A/H1N1 virus were incubated in the presence of four trypsin types at various concentrations, and virus yields in the culture medium were evaluated by a hemagglutination (HA) assay. Virus growth was most efficient in the presence of bovine and porcine trypsins. An analysis of the optimized concentration and definitive HA titer of each trypsin by Gaussian distribution revealed that comparable high virus yields (166.1 and 164.2 HAU/50μl) were obtained at the optimized concentrations of bovine (0.4μg/ml) and porcine (2.1μg/ml) trypsins, respectively, the yields of which were significantly higher than that of fungal and recombinant trypsins. We conclude that bovine and porcine trypsins are suitable for influenza A/H1N1 virus replication in SI-6 cells. This result complements our previous study and suggests the possible application of SI-6 cells to the development of cell-based influenza vaccines. Copyright © 2017 Elsevier B.V. All rights reserved.

Transient removal of proflavine inhibition of bovine beta-trypsin by the bovine basic pancreatic trypsin inhibitor (Kunitz). A case for "chronosteric effects".

PubMed

Antonini, E; Ascenzi, P; Bolognesi, M; Menegatti, E; Guarneri, M

1983-04-25

The formation of the bovine beta-trypsin-bovine basic pancreatic trypsin inhibitor (Kunitz) (BPTI) complex was monitored, making use of three different signals: proflavine displacement, optical density changes in the ultraviolet region, and the loss of the catalytic activity. The rates of the reactions indicated by the three different signals were similar at neutral pH, but diverged at low pH. At pH 3.50, proflavine displacement precedes the optical density changes in the ultraviolet and the loss of enzyme activity by several orders of magnitude in time (Antonini, E., Ascenzi, P., Menegatti, E., and Guarneri, M. (1983) Biopolymers 22, 363-375). These data indicated that the bovine beta-trypsin-BPTI complex formation is a multistage process and led to the prediction that, at pH 3.50, BPTI addition to the bovine beta-trypsin-proflavine complex would remove proflavine inhibition and the enzyme would recover transiently its catalytic activity before being irreversibly inhibited by completion of BPTI binding. The kinetic evidences, by completion of BPTI binding. The kinetic evidences, here shown, verified this prediction, indicating that during the bovine beta-trypsin-BPTI complex formation one transient intermediate occurs, which is not able to bind proflavine but may bind and hydrolyze the substrate. Thus, the observed peculiar catalytic behavior is in line with the proposed reaction mechanism for the bovine beta-trypsin-BPTI complex formation, which postulates a sequence of distinct polar and apolar interactions at the contact area.

Infectious rotavirus enters cells by direct cell membrane penetration, not by endocytosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaljot, K.T.; Shaw, R.D.; Greenberg, H.B.

1988-04-01

Rotaviruses are icosahedral viruses with a segmented, double-stranded RNA genome. They are the major cause of severe infantile infectious diarrhea. Rotavirus growth in tissue culture is markedly enhanced by pretreatment of virus with trypsin. Trypsin activation is associated with cleavage of the viral hemagglutinin (viral protein 3 (VP3); 88 kilodaltons) into two fragments (60 and 28 kilodaltons). The mechanism by which proteolytic cleavage leads to enhanced growth is unknown. To determine whether trypsin treatment affected rotavirus internalization, the authors studied the kinetics of entry of infectious rhesus rotavirus (RRV) into MA104 cells. Trypsin-activated RRV was internalized with a half-time ofmore » 3 to 5 min, while nonactivated virus disappeared from the cell surface with a half-time of 30 to 50 min. In contrast to trypsin-activated RRV, loss of nonactivated RRV from the cell surface did not result in the appearance of infection, as measured by plaque formation. Purified trypsin-activated RRV added to cell monolayers at pH 7.4 mediated {sup 51}Cr, ({sup 14}C)choline, and ({sup 3}H)inositol released from prelabeled MA104 cells. This release could be specifically blocked by neutralizing antibodies to VP3. These results suggest that MA104 cell infection follows the rapid entry of trypsin-activated RRV by direct cell membrane penetration. Cell membrane penetration of infectious RRV is initiated by trypsin cleavage of VP3. Neutralizing antibodies can inhibit this direct membrane penetration.« less

Maternally derived trypsin may have multiple functions in the early development of turbot (Scopthalmus maximus).

PubMed

Chi, Liang; Liu, Qinghua; Xu, Shihong; Xiao, Zhizhong; Ma, Daoyuan; Li, Jun

2015-10-01

Trypsin is an important serine protease that is considered to be involved in digestion of protein in teleost fish. Nevertheless, studies on trypsin/trypsinogen in fish embryos are very limited. In this study, the trypsinogen of turbot (Scophthalmus maximus) (tTG) was identified and the expression patterns and activity of trypsinogen/trypsin were investigated. The results showed that the tTG mRNA was evenly distributed in the oocytes and was also expressed along the yolk periphery in early embryos. At later embryo stages and 1 days after hatching (dph), the tTG mRNA concentrated at the alimentary tract and head. Quantitative expression analysis showed that the tTG transcripts decreased after fertilization until the gastrula stage, then increased with the embryo and larvae development. This result was also confirmed by the specific activity analysis of trypsin and in-situ-hybridization (ISH). All of the results indicated that tTG in early embryo stages was maternally derived and expressed by itself after gastrula stages. Additionally, location of tTG mRNA in embryos and larvae was investigated; we considered that trypsin may have multiple functions during the embryo development process. Based on our results regarding trypsinogen in embryos and early development, we concluded that the trypsin/trypsinogen in turbot embryos was inherited from a maternal source and we suggested that trypsin in early development has multiple functions in the process of development. Copyright © 2015 Elsevier Inc. All rights reserved.

21 CFR 866.5890 - Inter-alpha trypsin inhibitor immunological test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5890 Inter-alpha trypsin inhibitor immunological test system. (a) Identification. An inter... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Inter-alpha trypsin inhibitor immunological test...

THE INACTIVATION OF DILUTE SOLUTIONS OF CRYSTALLINE TRYPSIN BY X-RADIATION

PubMed Central

McDonald, Margaret R.

1954-01-01

The activity of dilute solutions of crystalline trypsin is destroyed by x-rays. The inactivation is an exponential function of the radiation dose. The reaction yield of inactivation is independent of the intensity at which the radiation is delivered or the quality of the x-rays. The reaction yield increases with increasing concentration of trypsin, varying from 0.06 to 0.7 micromoles per liter per 1000 r for trypsin solutions ranging from 1 x 10–7 to 2 x 10–4 M. PMID:13192318

Interaction of sodium benzoate with trypsin by spectroscopic techniques.

PubMed

Mu, Yue; Lin, Jing; Liu, Rutao

2011-12-01

The toxicity of sodium benzoate to trypsin was investigated by fluorescence spectroscopy, synchronous fluorescence spectroscopy, UV-visible absorption spectroscopy and circular dichroism (CD) spectroscopy under mimic physiological conditions. Sodium benzoate could unfold trypsin by decreasing the β-sheet structure, which leads to more exposure of internal amino acid groups and the obvious intrinsic fluorescence quenching with the rising concentration of sodium benzoate. The results of spectroscopic measurements indicated that sodium benzoate changed the internal microenvironment of trypsin and induced the alteration of the whole molecule, which were performed toxic effects on the organism. Trypsin and sodium benzoate interacted with each other to produce a substance by van der Waals forces and hydrogen bond, the model of which was shown by AutoDock software. Copyright © 2011 Elsevier B.V. All rights reserved.

Direct stimulation of immediate-early genes by intranuclear insulin in trypsin-treated H35 hepatoma cells.

PubMed Central

Lin, Y J; Harada, S; Loten, E G; Smith, R M; Jarett, L

1992-01-01

H35 hepatoma cells were treated with trypsin to abolish insulin binding and insulin-stimulated receptor kinase activity. Insulin was, however, internalized by fluid-phase endocytosis in trypsin-treated cells. Furthermore, nuclear accumulation of insulin was similar in control and trypsin-treated hepatoma cells. Northern blot analysis revealed insulin increased g33 and c-fos mRNA concentrations identically in control and trypsin-treated cells but had no effect on beta 2-microglobulin mRNA. Actinomycin D treatment prior to or after insulin addition demonstrated that insulin increased gene transcription and had no effect on mRNA degradation. These studies suggest that the accumulation of intact insulin in cell nuclei may be directly involved in the increased transcription of immediate-early genes. Images PMID:1409684

Vinyl functionalized silica hybrid monolith-based trypsin microreactor for on line digestion and separation via thiol-ene "click" strategy.

PubMed

Chen, Yingzhuang; Wu, Minghuo; Wang, Keyi; Chen, Bo; Yao, Shouzhuo; Zou, Hanfa; Nie, Lihua

2011-11-04

A novel thiol-ene "click" strategy for the preparation of monolithic trypsin microreactor was proposed. The hybrid organic-inorganic monolithic capillary column with ene-functionality was fabricated by sol-gel process using tetramethoxysilane (TMOS) and γ-methacryloxypropyltrimethoxysilane (γ-MAPS) as precursors. The disulfide bonds of trypsin were reduced to form free thiol groups. Then the trypsin containing free thiol groups was attached on the γ-MAPS hybrid monolithic column with ene-functionality via thiol-ene click chemistry to form a trypsin microreactor. The activity of the trypsin microreactor was characterized by detecting the substrate (Nα-p-tosyl-L-arginine methyl ester hydrochloride, TAME) and the product (Nα-p-tosyl-L-arginine, TA) with on-line capillary zone electrophoresis. After investigating various synthesizing conditions, it was found that the microreactor with poly(N,N'-methylenebisacrylamide) as spacer can deliver the highest activity, yielding a rapid reaction rate. After repeatedly sampling and analyzing for 100 times, the monolithic trypsin microreactor still remained 87.5% of its initial activity. It was demonstrated that thiol-ene "click" strategy for the construction of enzyme microreactor is a promising method for the highly selective immobilization of proteins under mild conditions, especially enzymes with free thiol radicals. Copyright © 2011 Elsevier B.V. All rights reserved.

In vitro excystation of Echinostoma paraensei (Digenea: Echinostomatidae) metacercariae assessed by light microscopy, morphometry and confocal laser scanning microscopy.

PubMed

Souza, Joyce; Garcia, Juberlan; Neves, Renata H; Machado-Silva, José Roberto; Maldonado, Arnaldo

2013-12-01

Trypsin and bile salts have been identified as important triggers for excystation of Echinostoma metacercariae. Although excystation in trematodes is a well-known phenomenon, some morphological developmental changes remain to be elucidated. In order to gain further insight into the in vitro development of metacercariae, we assayed different cultivating conditions: 0.5% trypsin and 0.5% bile salts; 1% trypsin and 1% bile salts; 1% trypsin and 0.5% bile salts; 0.5% bile salts; or 0.5% trypsin. By means of light microscopy and confocal microscopy, we characterized each encysted, activated, breached and excysted stage based on the morphological features. However, breached and excysted stages were not revealed in both bile salts and trypsin-free medium. Excretory concretions (25 ± 3.9) were visualized within excretory tubules, close to the ventral sucker and genital anlage. The oral sucker armed with spines and digestive system was similar to those of adult worms. The reproductive system is composed of a genital anlage and the cirrus sac primordium. In short, trypsin and bile salts associated were fundamental for the in vitro metacercariae excystation of Echinostoma paraensei. This article presents the first detailed information of all stages of metacercariae excystation obtained through light and confocal microscopy. Copyright © 2013. Published by Elsevier Inc.

Molecular glues for manipulating enzymes: trypsin inhibition by benzamidine-conjugated molecular glues† †Electronic supplementary information (ESI) available: Synthesis of TEG–BA, Gluen–BA, mGluen–BA and Gluen–Ph; 1H NMR, 13C NMR, MALDI-TOF MS, electronic absorption, and CD spectra; zeta potential distributions; SLS plots; DLS histograms; and related experimental procedures. See DOI: 10.1039/c5sc00524h Click here for additional data file.

PubMed Central

Mogaki, Rina

2015-01-01

Water-soluble bioadhesive polymers bearing multiple guanidinium ion (Gu+) pendants at their side-chain termini (Gluen–BA, n = 10 and 29) that were conjugated with benzamidine (BA) as a trypsin inhibitor were developed. The Gluen–BA molecules are supposed to adhere to oxyanionic regions of the trypsin surface, even in buffer, via a multivalent Gu+/oxyanion salt-bridge interaction, such that their BA group properly blocks the substrate-binding site. In fact, Glue10–BA and Glue29–BA exhibited 35- and 200-fold higher affinities for trypsin, respectively, than a BA derivative without the glue moiety (TEG–BA). Most importantly, Glue10–BA inhibited the protease activity of trypsin 13-fold more than TEG–BA. In sharp contrast, mGlue27–BA, which bears 27 Gu+ units along the main chain and has a 5-fold higher affinity than TEG–BA for trypsin, was inferior even to TEG–BA for trypsin inhibition. PMID:28706668

Engineering of Yersinia Phytases to Improve Pepsin and Trypsin Resistance and Thermostability and Application Potential in the Food and Feed Industry.

PubMed

Niu, Canfang; Yang, Peilong; Luo, Huiying; Huang, Huoqing; Wang, Yaru; Yao, Bin

2017-08-30

Susceptibility to proteases usually limits the application of phytase. We sought to improve the pepsin and trypsin resistance of YeAPPA from Yersinia enterocolitica and YkAPPA from Y. kristensenii by optimizing amino acid polarity and charge. The predicted pepsin/trypsin cleavage sites F89/K226 in pepsin/trypsin-sensitive YeAPPA and the corresponding sites (F89/E226) in pepsin-sensitive but trypsin-resistant YkAPPA were substituted with S and H, respectively. Six variants were produced in Pichia pastoris for catalytic and biochemical characterization. F89S, E226H, and F89S/E226H elevated pepsin resistance and thermostability and K226H and F89S/K226H improved pepsin and trypsin resistance and stability at 60 °C and low pH. All the variants increased the ability of the proteins to hydrolyze phytate in corn meal by 2.6-14.9-fold in the presence of pepsin at 37 °C and low pH. This study developed a genetic manipulation strategy specific for pepsin/trypsin-sensitive phytases that can improve enzyme tolerance against proteases and heat and benefit the food and feed industry in a cost-effective way.

Behaviour of the vitelline envelope in Bufo arenarum oocytes matured in vitro in blockade to polyspermy.

PubMed

Oterino, J; Sánchez Toranzo, G; Zelarayán, L; Ajmat, M T; Bonilla, F; Bühler, M I

2006-05-01

During activation of amphibian eggs, cortical granule exocytosis causes elaborate ultrastructural changes in the vitelline envelope. These changes involve modifications in the structure of the vitelline envelope and formation of a fertilization envelope (FE) that can no longer be penetrated by sperm. In Bufo arenarum, as the egg traverses the oviduct, the vitelline envelope is altered by a trypsin-like protease secreted by the oviduct, which induces an increased susceptibility of the vitelline envelope to sperm lysins. Full-grown oocytes of B. arenarum, matured in vitro by progesterone, are polyspermic, although cortical granule exocytosis seems to occur within a normal chronological sequence. These oocytes can be fertilized with or without trypsin treatment, suggesting that the vitelline envelope is totally sperm-permeable. Vitelline envelopes without trypsin treatment cannot retain either gp90 or gp96. This suggests that these glycoproteins are involved in the block to polyspermy and that trypsin treatment of matured in vitro oocytes before insemination is necessary to enable vitelline envelopes to block polyspermy. The loss of the binding capacity in vitelline envelopes isolated from B. arenarum oocytes matured in vitro with trypsin treatment and activated by electric shock suggests that previous trypsin treatment is a necessary step for sperm block to occur. When in vitro matured oocytes were incubated with the product of cortical granules obtained from in vitro matured oocytes (vCGP), vitelline envelopes with trypsin treatment were able to block sperm entry. These oocytes exhibited the characteristic signs of activation. These results support the idea that B. arenarum oocytes can be activated by external stimuli and suggest the presence of unknown oocyte surface receptors linked to the activation machinery in response to fertilization. Electrophoretic profiles obtained by SDS-PAGE of solubilized vitelline envelopes from oocytes matured in vitro revealed the conversion of gp40 (in vitro matured oocytes, without trypsin treatment) to gp38 (ascribable to trypsin activity or cortical granule product activity, CGP) and the conversion of gp70 to gp68 (ascribable to trypsin activity plus CGP activity). Taking into account that only the vitelline envelopes of in vitro matured oocytes with trypsin treatment and activated can block sperm entry, we may suggest that the conversion of gp70 to gp68 is related to the changes associated with sperm binding.

Trypsin treatment of reaction centers from Rhodobacter sphaeroides in the dark and under illumination: protein structural changes follow charge separation.

PubMed

Brzezinski, P; Andréasson, L E

1995-06-06

Reaction centers from Rhodobacter sphaeroides R-26 were treated with trypsin in the dark and during illumination (in the charge-separated state). Trypsination resulted in a time-dependent modification of the reaction centers, reflected in changes in the charge recombination rate, in the inhibition of QA- to QB electron transfer, and eventually to inhibition of charge separation. Comparisons of centers with ubiquinone or anthraquinone in the QA site, in which the charge recombination pathways are different, indicate that trypsination affects charges close to the QA(-)-binding site. Studies of light-induced voltage changes from moving charges in reaction centers incorporated in lipid layers on a Teflon film, a technique which allows the discrimination of effects on donor and acceptor sides, indicate that the acceptor side is preferentially degraded by trypsin in the dark. Tryptic digestion during illumination generally resulted in a marked strengthening and acceleration of the effects seen already during dark treatment, but new effects were also detected in gel electrophoretic peptide patterns, in optical spectra, and in the kinetic measurements. Optical kinetic measurements revealed that the donor side of the reaction centers became susceptible to modification by trypsin during illumination as seen in the value of the binding constant for soluble cytochrome c2 which increased by a factor of 2, whereas it was much less affected after trypsination of reaction centers in the dark. The influence of illumination on the rate and mode by which trypsin acts on reaction centers indicates that changes in the protein conformation follow charge separation.(ABSTRACT TRUNCATED AT 250 WORDS)

The on-bead digestion of protein corona on nanoparticles by trypsin immobilized on the magnetic nanoparticle.

PubMed

Hu, Zhengyan; Zhao, Liang; Zhang, Hongyan; Zhang, Yi; Wu, Ren'an; Zou, Hanfa

2014-03-21

Proteins interacting with nanoparticles would form the protein coronas on the surface of nanoparticles in biological systems, which would critically impact the biological identities of nanoparticles and/or result in the physiological and pathological consequences. The enzymatic digestion of protein corona was the primary step to achieve the identification of protein components of the protein corona for the bottom-up proteomic approaches. In this study, the investigation on the tryptic digestion of protein corona by the immobilized trypsin on a magnetic nanoparticle was carried out for the first time. As a comparison with the usual overnight long-time digestion and the severe self-digestion of free trypsin, the on-bead digestion of protein corona by the immobilized trypsin could be accomplished within 1h, along with the significantly reduced self-digestion of trypsin and the improved reproducibility on the identification of proteins by the mass spectrometry-based proteomic approach. It showed that the number of identified bovine serum (BS) proteins on the commercial Fe3O4 nanoparticles was increased by 13% for the immobilized trypsin with 1h digestion as compared to that of using free trypsin with even overnight digestion. In addition, the on-bead digestion of using the immobilized trypsin was further applied on the identification of human plasma protein corona on the commercial Fe3O4 nanoparticles, which leads the efficient digestion of the human plasma proteins and the identification of 149 human plasma proteins corresponding to putative critical pathways and biological processes. Copyright © 2014 Elsevier B.V. All rights reserved.

High-level production of recombinant trypsin in transgenic rice cell culture through utilization of an alternative carbon source and recycling system.

PubMed

Kim, Nan-Sun; Yu, Hwa-Young; Chung, Nguyen-Duc; Kwon, Tae-Ho; Yang, Moon-Sik

2014-09-01

Productivity of recombinant bovine trypsin using a rice amylase 3D promoter has been studied in transgenic rice suspension culture. Alternative carbon sources were added to rice cell suspension cultures in order to improve the production of recombinant bovine trypsin. It was demonstrated that addition of alternative carbon sources such as succinic acid, fumaric acid and malic acid in the culture medium could increase the productivity of recombinant bovine trypsin 3.8-4.3-fold compared to those in the control medium without carbon sources. The highest accumulated trypsin reached 68.2 mg/L on day 5 in the culture medium with 40 mM fumaric acid. The feasibility of repeated use of the cells for recombinant trypsin production was tested in transgenic rice cell suspension culture with the culture medium containing the combination of variable sucrose concentration and 40 mM fumaric acid. Among the used combinations, the combination of 1% sucrose and 40 mM fumaric acid resulted in a yield of up to 53 mg/L five days after incubation. It also increased 31% (W/W) of dry cell weight and improved 43% of cell viability compared to that in control medium without sucrose. Based on these data, recycling of the trypsin production process with repeated 1% sucrose and 40 mM fumaric acid supplying-harvesting cycles was developed in flask scale culture. Recombinant bovine trypsin could be stably produced with a yield of up to 53-39 mg/L per cycle during five recycling cycles. Copyright © 2014 Elsevier Inc. All rights reserved.

21 CFR 184.1914 - Trypsin.

Code of Federal Regulations, 2011 CFR

2011-04-01

... Substances Affirmed as GRAS § 184.1914 Trypsin. (a) Trypsin (CAS Reg. No. 9002-07-7) is an enzyme preparation... characterizing enzyme activity is that of a peptide hydrolase (EC 3.4.21.4). (b) The ingredient meets the general requirements and additional requirements for enzyme preparations in the Food Chemicals Codex, 3d ed. (1981), p...

BG-4, a novel anticancer peptide from bitter gourd (Momordica charantia), promotes apoptosis in human colon cancer cells

USDA-ARS?s Scientific Manuscript database

Momordica charantia is a perennial plant with reported health benefits. BG-4, a novel peptide from Momordica charantia, was isolated, purified and characterized. The trypsin inhibitory activity of BG-4 is 8.6 times higher than purified soybean trypsin inhibitor. The high trypsin inhibitory activity ...

Effect of bombesin on serum immunoreactive trypsin in healthy subjects and in patients with chronic pancreatitis.

PubMed

Labò, G; Vezzadini, P; Gullo, L; Sternini, C; Bonora, G

1983-08-01

We studied the effect of bombesin (9 ng/kg X min for 30 min by intravenous infusion) on serum immunoreactive trypsin in healthy subjects and in chronic pancreatitis patients. Bombesin administration caused a marked and significant increase of serum immunoreactive trypsin concentration in healthy subjects. The increase occurred in the first 15 min after the beginning of bombesin infusion and persisted for the duration of the study (2 h). In patients with chronic pancreatitis, the increase was much less pronounced. In these patients, the integrated immunoreactive trypsin response to bombesin was significantly correlated with bicarbonate, lipase, and chymotrypsin outputs into the duodenum. The response of serum immunoreactive trypsin to bombesin stimulation seems to vary according to the degree of pancreatic exocrine dysfunction and to reflect the functional capacity of acinar cell mass.

Autocrine Extra-Pancreatic Trypsin 3 Secretion Promotes Cell Proliferation and Survival in Esophageal Adenocarcinoma

PubMed Central

Han, Song; Lee, Constance W.; Trevino, Jose G.; Hughes, Steven J.; Sarosi, George A.

2013-01-01

Trypsin or Tumor associated trypsin (TAT) activation of Protease-activated receptor 2 (PAR-2) promotes tumor cell proliferation in gastrointestinal cancers. The role of the trypsin/PAR-2 network in esophageal adenocarcinoma (EA) development has not yet been investigated. The aim of this study is to investigate the role of trypsin/PAR-2 activation in EA tumorogenesis and therapy. We found that esophageal adenocarcinoma cells (EACs) and Barrett’s Metaplasia (BART) expressed high levels of type 3 extra-pancreatic trypsinogen (PRSS3), a novel type of TAT. Activity of secreted trypsin was detected in cultured media from EA OE19 and OE33 cultures but not from BART culture. Surface PAR-2 expression in BART and EACs was confirmed by both flow cytometry and immunofluorescence. Trypsin induced cell proliferation (∼ 2 fold; P<0.01) in all tested cell lines at a concentration of 10 nM. Inhibition of PAR-2 activity in EACs via the PAR-2 antagonist ENMD (500 µM), anti-PAR2 antibody SAM-11 (2 µg/ml), or siRNA PAR-2 knockdown, reduced cell proliferation and increased apoptosis by up to 4 fold (P<0.01). Trypsin stimulation led to phosphorylation of ERK1/2, suggesting involvement of MAPK pathway in PAR-2 signal transduction. Inhibition of PAR-2 activation or siRNA PAR-2 knockdown in EACs prior to treatment with 5 FU reduced cell viability of EACs by an additional 30% (P<0.01) compared to chemotherapy alone. Our data suggest that extra-pancreatic trypsinogen 3 is produced by EACs and activates PAR-2 in an autocrine manner. PAR-2 activation increases cancer cell proliferation, and promotes cancer cell survival. Targeting the trypsin activated PAR-2 pathway in conjunction with current chemotherapeutic agents may be a viable therapeutic strategy in EA. PMID:24146905

Digestive peptidase evolution in holometabolous insects led to a divergent group of enzymes in Lepidoptera.

PubMed

Dias, Renata O; Via, Allegra; Brandão, Marcelo M; Tramontano, Anna; Silva-Filho, Marcio C

2015-03-01

Trypsins and chymotrypsins are well-studied serine peptidases that cleave peptide bonds at the carboxyl side of basic and hydrophobic L-amino acids, respectively. These enzymes are largely responsible for the digestion of proteins. Three primary processes regulate the activity of these peptidases: secretion, precursor (zymogen) activation and substrate-binding site recognition. Here, we present a detailed phylogenetic analysis of trypsins and chymotrypsins in three orders of holometabolous insects and reveal divergent characteristics of Lepidoptera enzymes in comparison with those of Coleoptera and Diptera. In particular, trypsin subsite S1 was more hydrophilic in Lepidoptera than in Coleoptera and Diptera, whereas subsites S2-S4 were more hydrophobic, suggesting different substrate preferences. Furthermore, Lepidoptera displayed a lineage-specific trypsin group belonging only to the Noctuidae family. Evidence for facilitated trypsin auto-activation events were also observed in all the insect orders studied, with the characteristic zymogen activation motif complementary to the trypsin active site. In contrast, insect chymotrypsins did not seem to have a peculiar evolutionary history with respect to their mammal counterparts. Overall, our findings suggest that the need for fast digestion allowed holometabolous insects to evolve divergent groups of peptidases with high auto-activation rates, and highlight that the evolution of trypsins led to a most diverse group of enzymes in Lepidoptera. Copyright © 2015 Elsevier Ltd. All rights reserved.

Atomic-scale investigation of the interactions between tetrabromobisphenol A, tetrabromobisphenol S and bovine trypsin by spectroscopies and molecular dynamics simulations.

PubMed

Ding, Keke; Zhang, Huanxin; Wang, Haifei; Lv, Xuan; Pan, Liumeng; Zhang, Wenjing; Zhuang, Shulin

2015-12-15

Tetrabromobisphenol A (TBBPA) and its replacement alternative tetrabromobisphenol S (TBBPS) are used widely as brominated flame retardants (BFRs). However, the potential risk of their effects on bovine trypsin remains largely unknown. We investigated the effects of TBBPA and TBBPS to bovine trypsin by the fluorescence spectroscopy, circular dichroism and molecular dynamics (MD) simulations. They statically quenched the intrinsic fluorescence of bovine trypsin in a concentration-dependent mode and caused slight red-shifted fluorescence. The short and long fluorescence lifetime decay components of bovine trypsin were both affected, partly due to the disturbed microenvironmental changes of Trp215. The β-sheet content of bovine trypsin was significantly reduced from 82.4% to 75.7% and 76.6% by TBBPA and TBBPS, respectively, possibly impairing the physiological function of bovine trypsin. TBBPA and TBBPS bind at the 8-anilinonaphthalene-1-sulfonate (ANS) binding site with an association constant of 1.09×10(4) M(-1) and 2.41×10(4) M(-1) at 298 K, respectively. MD simulations revealed that van der Waals interactions and hydrogen bond interactions are dominant for TBBPA, whereas electrostatic interactions are critical for TBBPS. Our in vitro and in silico studies are beneficial to the understanding of risk assessment and future design of environmental benign BFRs. Copyright © 2015 Elsevier B.V. All rights reserved.

Thermostable trypsin conjugates immobilized to biogenic magnetite show a high operational stability and remarkable reusability for protein digestion

NASA Astrophysics Data System (ADS)

Pečová, M.; Šebela, M.; Marková, Z.; Poláková, K.; Čuda, J.; Šafářová, K.; Zbořil, R.

2013-03-01

In this work, magnetosomes produced by microorganisms were chosen as a suitable magnetic carrier for covalent immobilization of thermostable trypsin conjugates with an expected applicability for efficient and rapid digestion of proteins at elevated temperatures. First, a biogenic magnetite was isolated from Magnetospirillum gryphiswaldense and its free surface was coated with the natural polysaccharide chitosan containing free amino and hydroxy groups. Prior to covalent immobilization, bovine trypsin was modified by conjugating with α-, β- and γ-cyclodextrin. Modified trypsin was bound to the magnetic carriers via amino groups using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide as coupling reagents. The magnetic biomaterial was characterized by magnetometric analysis and electron microscopy. With regard to their biochemical properties, the immobilized trypsin conjugates showed an increased resistance to elevated temperatures, eliminated autolysis, had an unchanged pH optimum and a significant storage stability and reusability. Considering these parameters, the presented enzymatic system exhibits properties that are superior to those of trypsin forms obtained by other frequently used approaches. The proteolytic performance was demonstrated during in-solution digestion of model proteins (horseradish peroxidase, bovine serum albumin and hen egg white lysozyme) followed by mass spectrometry. It is shown that both magnetic immobilization and chemical modification enhance the characteristics of trypsin making it a promising tool for protein digestion.

21 CFR 524.2620 - Liquid crystalline trypsin, Peru balsam, castor oil.

Code of Federal Regulations, 2013 CFR

2013-04-01

...) Conditions of use. The drug is used as an aid in the treatment of external wounds and assists healing by... delivered to the wound site contains 0.12 milligram of crystalline trypsin, 87.0 milligrams of Peru balsam... gram delivered to the wound site contains 0.1 milligram of crystalline trypsin, 72.5 milligrams of Peru...

21 CFR 524.2620 - Liquid crystalline trypsin, Peru balsam, castor oil.

Code of Federal Regulations, 2012 CFR

2012-04-01

...) Conditions of use. The drug is used as an aid in the treatment of external wounds and assists healing by... delivered to the wound site contains 0.12 milligram of crystalline trypsin, 87.0 milligrams of Peru balsam... gram delivered to the wound site contains 0.1 milligram of crystalline trypsin, 72.5 milligrams of Peru...

21 CFR 524.2620 - Liquid crystalline trypsin, Peru balsam, castor oil.

Code of Federal Regulations, 2011 CFR

2011-04-01

... delivered to the wound site contains 0.12 milligram of crystalline trypsin, 87.0 milligrams of Peru balsam... gram delivered to the wound site contains 0.1 milligram of crystalline trypsin, 72.5 milligrams of Peru...) Conditions of use. The drug is used as an aid in the treatment of external wounds and assists healing by...

21 CFR 524.2620 - Liquid crystalline trypsin, Peru balsam, castor oil.

Code of Federal Regulations, 2010 CFR

2010-04-01

...) Conditions of use. The drug is used as an aid in the treatment of external wounds and assists healing by... delivered to the wound site contains 0.12 milligram of crystalline trypsin, 87.0 milligrams of Peru balsam... gram delivered to the wound site contains 0.1 milligram of crystalline trypsin, 72.5 milligrams of Peru...

Trypsin activation pathway of rotavirus infectivity.

PubMed Central

Arias, C F; Romero, P; Alvarez, V; López, S

1996-01-01

The infectivity of rotaviruses is increased by and most probably is dependent on trypsin treatment of the virus. This proteolytic treatment specifically cleaves VP4, the protein that forms the spikes on the surface of the virions, to polypeptides VP5 and VP8. This cleavage has been reported to occur in rotavirus SA114fM at two conserved, closely spaced arginine residues located at VP4 amino acids 241 and 247. In this work, we have characterized the VP4 cleavage products of rotavirus SA114S generated by in vitro treatment of the virus with increasing concentrations of trypsin and with proteases AspN and alpha-chymotrypsin. The VP8 and VP5 polypeptides were analyzed by gel electrophoresis and by Western blotting (immunoblotting) with antibodies raised to synthetic peptides that mimic the terminal regions of VP4 generated by the trypsin cleavage. It was shown that in addition to arginine residues 241 and 247, VP4 is cleaved at arginine residue 231. These three sites were found to have different susceptibilities to trypsin, Arg-241 > Arg-231 > Arg-247, with the enhancement of infectivity correlating with cleavage at Arg-247 rather than at Arg-231 or Arg-241. Proteases AspN and alpha-chymotrypsin cleaved VP4 at Asp-242 and Tyr-246, respectively, with no significant enhancement of infectivity, although this enhancement could be achieved by further treatment of the virus with trypsin. The VP4 end products of trypsin treatment were a homogeneous VP8 polypeptide comprising VP4 amino acids 1 to 231 and a heterogeneous VP5, which is formed by two polypeptide species (present at a ratio of approximately 1:5) as a result of cleavage at either Arg-241 or Arg-247. A pathway for the trypsin activation of rotavirus infectivity is proposed. PMID:8709201

Extraction of gelatin from salmon (Salmo salar) fish skin using trypsin-aided process: optimization by Plackett-Burman and response surface methodological approaches.

PubMed

Fan, HuiYin; Dumont, Marie-Josée; Simpson, Benjamin K

2017-11-01

Gelatin from salmon ( Salmo salar ) skin with high molecular weight protein chains ( α -chains) was extracted using trypsin-aided process. Response surface methodology was used to optimise the extraction parameters. Yield, hydroxyproline content and protein electrophoretic profile via sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of gelatin were used as responses in the optimization study. The optimum conditions were determined as: trypsin concentration at 1.49 U/g; extraction temperature at 45 °C; and extraction time at 6 h 16 min. This response surface optimized model was significant and produced an experimental value (202.04 ± 8.64%) in good agreement with the predicted value (204.19%). Twofold higher yields of gelatin with high molecular weight protein chains were achieved in the optimized process with trypsin treatment when compared to the process without trypsin.

Porphyrin Induced Laser Deactivation of Trypsinogen-Trypsin Conversion

NASA Astrophysics Data System (ADS)

Perido, Joanna; Brancaleon, Lorenzo

2015-03-01

Pancreatitis is caused by the inflammation of the pancreas, where the digestive enzyme trypsin is activated from the precursor enzyme trypsinogen while still in the pancreas. The presence of trypsin in the pancreas causes auto-activation of trypsinogen, resulting in greater inflammation and auto-digestion of the pancreas. In severe cases, this cascade effect can lead to organ failure, diabetes, and pancreatic cancer. Our hypothesis is that if trypsinogen is prevented from auto-activating into trypsin, then this cascade can be stopped. We propose to do this by inducing conformational changes in trypsinogen when bound to a photoactive porphyrin dye. Porphyrins are comprised of four linked heterocyclic groups forming a flat ring, and bind well with proteins such as trypsinogen. In this study we used spectroscopic techniques to probe the binding of meso-tetrakis (4-sulfonatephenyl) porphyrin (TSPP) to trypsinogen in vitro, as a preliminary step to then prompt and characterize conformational changes of trypsinogen through irradiation. If conformational changes are detected the trypsinogen will be tested for trypsin inactivation. This investigation may provide promising initial results to the possible use of porphyrins as an inhibitor of the self-activation of trypsinogen into trypsin, and a potential inhibitor of pancreatitis. MARC*U-STAR.

Synthesis of Trypsin-Resistant Variants of the Listeria-Active Bacteriocin Salivaricin P▿

PubMed Central

O'Shea, Eileen F.; O'Connor, Paula M.; Cotter, Paul D.; Ross, R. Paul; Hill, Colin

2010-01-01

Two-component salivaricin P-like bacteriocins have demonstrated potential as antimicrobials capable of controlling infections in the gastrointestinal tract (GIT). The anti-Listeria activity of salivaricin P is optimal when the individual peptides Sln1 and Sln2 are added in succession at a 1:1 ratio. However, as degradation by digestive proteases may compromise the functionality of these peptides within the GIT, we investigated the potential to create salivaricin variants with enhanced resistance to the intestinal protease trypsin. A total of 11 variants of the salivaricin P components, in which conservative modifications at the trypsin-specific cleavage sites were explored in order to protect the peptides from trypsin degradation while maintaining their potent antimicrobial activity, were generated. Analysis of these variants revealed that eight were resistant to trypsin digestion while retaining antimicrobial activity. Combining the complementary trypsin-resistant variants Sln1-5 and Sln2-3 resulted in a MIC50 of 300 nM against Listeria monocytogenes, a 3.75-fold reduction in activity compared to the level for wild-type salivaricin P. This study demonstrates the potential of engineering bacteriocin variants which are resistant to specific protease action but which retain significant antimicrobial activity. PMID:20581174

Robust Trypsin Coating on Electrospun Polymer Nanofibers in Rigorous Conditions and Its Uses for Protein Digestion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ahn, Hye-Kyung; Kim, Byoung Chan; Jun, Seung-Hyun

2010-12-15

An efficient protein digestion in proteomic analysis requires the stabilization of proteases such as trypsin. In the present work, trypsin was stabilized in the form of enzyme coating on electrospun polymer nanofibers (EC-TR), which crosslinks additional trypsin molecules onto covalently-attached trypsin (CA-TR). EC-TR showed better stability than CA-TR in rigorous conditions, such as at high temperatures of 40 °C and 50 °C, in the presence of organic co-solvents, and at various pH's. For example, the half-lives of CA-TR and EC-TR were 0.24 and 163.20 hours at 40 ºC, respectively. The improved stability of EC-TR can be explained by covalent-linkages onmore » the surface of trypsin molecules, which effectively inhibits the denaturation, autolysis, and leaching of trypsin. The protein digestion was performed at 40 °C by using both CA-TR and EC-TR in digesting a model protein, enolase. EC-TR showed better performance and stability than CA-TR by maintaining good performance of enolase digestion under recycled uses for a period of one week. In the same condition, CA-TR showed poor performance from the beginning, and could not be used for digestion at all after a few usages. The enzyme coating approach is anticipated to be successfully employed not only for protein digestion in proteomic analysis, but also for various other fields where the poor enzyme stability presently hampers the practical applications of enzymes.« less

Mechanism of curcumin-induced trypsin inhibition: Computational and experimental studies

NASA Astrophysics Data System (ADS)

Wang, Yan-Qing; Zhang, Hong-Mei; Kang, Yi-Jun; Gu, Yun-Lan; Cao, Jian

2016-03-01

In the present study, the experimental and theoretical methods were used to analyze the binding interaction of food dye, curcumin with trypsin. The results of fluorescence spectroscopic measurements indicated that curcumin binding resulted in the obviously intrinsic fluorescence quenching with the increase concentration of curcumin. This binding interaction is a spontaneous process with the estimated enthalpy and entropy changes being -15.70 kJ mol-1 and 40.25 J mol-1 K-1, respectively. Hydrogen bonds and hydrophobic forces played an important role in the complex formation between curcumin and trypsin. Moreover, curcumin could enter into the primary substrate-binding pocket and makes the activity of trypsin decrease remarkably with the increasing concentration of curcumin.

The involvement of the cysteine proteases of Clonorchis sinensis metacercariae in excystment.

PubMed

Li, Shunyu; Chung, Young-Bae; Chung, Byung-Suk; Choi, Min-Ho; Yu, Jae-Ran; Hong, Sung-Tae

2004-05-01

The effects of trypsin, bile, trypsin-bile, pepsin, dithiothreitol (DTT) and metacercarial excretory-secretory product (ESP) on the in vitro excystment of Clonorchis sinensis metacercariae were investigated. The majority of metacercariae excysted immediately in trypsin-bile in PBS solution, a process which was complete after 30 min of incubation. When incubated in metacercarial ESP in PBS, excystment was potentiated in the presence of 5 mM DTT, but was inhibited dose-dependently by a cysteine protease inhibitor, iodoacetic acid. Two active protease bands of 28 and 40 kDa were identified in the ESP of metacercariae by gelatin substrate SDS-PAGE. Scanning electron microscopy demonstrated that the larvae in solutions of DTT and ESP migrated through a small hole on the metacercarial wall, whereas larvae were liberated by entire wall disruption in trypsin solution. These results suggest that trypsin is a major extrinsic factor of the rapid excystment of C. sinensis metacercariae, and that endogenous cysteine proteases are also involved in metacercarial excystment. Copyright 2004 Springer-Verlag

Amino acid sequence of a trypsin inhibitor from a Spirometra (Spirometra erinaceieuropaei).

PubMed

Sanda, A; Uchida, A; Itagaki, T; Kobayashi, H; Inokuchi, N; Koyama, T; Iwama, M; Ohgi, K; Irie, M

2001-12-01

A trypsin inhibitor that is highly homologous with bovine pancreatic trypsin inhibitor (BPTI) was co-purified along with RNase from Spirometra (Spirometra erinaceieuropaei). The amino acid sequence of this inhibitor (SETI) and the nucleotide sequence of the cDNA encoding this protein were determined by protein chemistry and gene technology. SETI contains 68 amino acid residues and has a molecular mass of 7,798 Da. SETI has 31 amino acid residues that are identical with BPTI's sequence, including 6 half-cystine and 5 aromatic amino acid residues. The active site Lys residue in BPTI is replaced by an Arg residue in SETI. SETI is an effective inhibitor of trypsin and moderately inhibits a-chymotrypsin, but less inhibits elastase or subtilisin. SETI was expressed by E. coli containing a PelB vector carrying the SETI encoding cDNA; an expression yield of 0.68 mg/l was obtained. The phylogenetic relationship of SETI and the other BPTI-like trypsin inhibitors was analyzed using most likelihood inference methods.

Gold nanoclusters-based chemiluminescence resonance energy transfer method for sensitive and label-free detection of trypsin.

PubMed

You, Xiaoying; Li, Yinhuan; Li, Baoping; Ma, Jie

2016-01-15

A chemiluminescence resonance energy transfer (CRET) platform was developed for sensitive and label-free detection of protease by using trypsin as a model analyte. In this CRET platform, bis(2,4,6-trichlorophenyl)oxalate-hydrogen peroxide chemiluminescence (CL) reaction was utilized as an energy donor and bovine serum albumin (BSA)-stabilized gold nanoclusters (Au NCs) as an energy acceptor. The BSA-stabilized Au NCs triggered the CRET phenomenon by accepting the energy from TCPO-H2O2 CL reaction, thus producing intense CL. In the presence of trypsin, the protein template of BSA-stabilized Au NCs was digested, which frustrated the energy transfer efficiency between the CL donor and the BSA-stabilized Au NCs, leading to a significant decrease in the CL signal. The decreased CL signal was proportional to the logarithm of trypsin concentration in the range of 0.01-50.0µg mL(-1). The detection limit for trypsin was 9ng mL(-)(1) and the relative standard deviations were lesser than 3% (n=11). This Au NCs-based CRET platform was successfully applied to the determination of trypsin in human urine samples, demonstrating its potential application in clinical diagnosis. Copyright © 2015 Elsevier B.V. All rights reserved.

Investigate the Binding of Catechins to Trypsin Using Docking and Molecular Dynamics Simulation

PubMed Central

Cui, Fengchao; Yang, Kecheng; Li, Yunqi

2015-01-01

To explore the inhibitory mechanism of catechins for digestive enzymes, we investigated the binding mode of catechins to a typical digestive enzyme-trypsin and analyzed the structure-activity relationship of catechins, using an integration of molecular docking, molecular dynamics simulation and binding free energy calculation. We found that catechins with different structures bound to a conservative pocket S1 of trypsin, which is comprised of residues 189–195, 214–220 and 225–228. In the trypsin-catechin complexes, Asp189 by forming strong hydrogen bonding, and Gln192, Trp215 and Gly216 through hydrophobic interactions, all significantly contribute to the binding of catechins. The number and the position of hydroxyl and aromatic groups, the structure of stereoisomers, and the orientation of catechins in the binding pocket S1 of trypsin all affect the binding affinity. The binding affinity is in the order of Epigallocatechin gallate (EGCG) > Epicatechin gallate (ECG) > Epicatechin (EC) > Epigallocatechin (EGC), and 2R-3R EGCG shows the strongest binding affinity out of other stereoisomers. Meanwhile, the synergic conformational changes of residues and catechins were also analyzed. These findings will be helpful in understanding the knowledge of interactions between catechins and trypsin and referable for the design of novel polyphenol based functional food and nutriceutical formulas. PMID:25938485

Specific lysine labeling by 18OH- during alkaline cleavage of the alpha-1-antitrypsin-trypsin complex.

PubMed Central

Cohen, A B; Gruenke, L D; Craig, J C; Geczy, D

1977-01-01

alpha-1-Antitrypsin is a serum protein that inhibits many proteolytic enzymes. Recently, it was suggested that the alpha-1-antitrypsin-trypsin complex is an acyl ester analogous to the acyl intermediate that forms between trypsin and its substrates. In previous work we showed that the alpha-1-antitrypsin-trypsin complex can be split at high pH, releasing a component of alpha-1-antitrypsin. This component had a new carboxyl-terminal lysine, and it had lost a peptide of about 4000 daltons. In order to determine whether the alpha-1-antitrypsin is bound to trypsin through the new carboxy-terminal lysine, as would be expected if the above hypothesis is correct, we split the complex in the presence of 18OH-. When the new carboxy-terminal lysine was cleaved with carboxypeptidase B, singly labeled, doubly labeled, and unlabeled lysine were recovered. These data support the hypothesis that the alpha-1-antitrypsin-trypsin complex is an acyl ester or a tetrahedral precursor that is transformed into the acyl ester form at high pH. If other enzymes are bound by a similar mechanism, the methods used may be useful in determining which amino acids on alpha-1-antitrypsin bind covalently to each enzyme. PMID:303770

Effect of bombesin and cholecystokinin on plasma immunoreactive trypsin in humans.

PubMed

de Jong, A J; Klamer, M; Jansen, J B; Hopman, W P; Lamers, C B

1987-01-01

Since bombesin is a potent stimulus of the release of cholecystokinin (CCK), it has been suggested that the stimulatory effect of bombesin on pancreatic enzyme secretion is mediated by CCK. The present study was undertaken to determine the role of CCK in the bombesin-induced stimulation of plasma immunoreactive trypsin. Plasma CCK was measured by radioimmunoassay using the antibody T204, which binds to all biologically active sulfated COOH-terminal CCK-peptides. Plasma trypsin was also measured by radioimmunoassay. Infusion of 5 ng/kg/min bombesin in 6 healthy volunteers increased plasma CCK from 1.2 +/- 0.2-8.9 +/- 0.7 pM (p less than 0.0001). The peak increment in plasma CCK during bombesin (9.3 +/- 0.6 pM) was accompanied by a significant rise in plasma trypsin from 206 +/- 21-334 +/- 44 ng/ml (p less than 0.01). However, when similar increases in plasma CCK were achieved by infusion of 0.018 CU/kg/min CCK-33 (9.9 +/- 0.8 pM) or by intraduodenal instillation of 250 ml 20% Intralipid (9.7 +/- 1.9 pM), no significant changes in plasma trypsin were observed. It is therefore concluded that the stimulatory effect of bombesin on plasma immunoreactive trypsin is not mediated by CCK.

Structural stabilization of CNS synapses during postnatal development in rat cortex.

PubMed

Khaing, Zin Z; Fidler, Lazar; Nandy, Nina; Phillips, Greg R

2006-07-01

CNS synapses are produced rapidly upon pre- and post-synaptic recruitment. However, their composition is known to change during development and we reasoned that this may be reflected in the gross biochemical properties of synapses. We found synaptic structure in adult cortical synaptosomes to be resistant to digestion with trypsin in the presence and absence of calcium ions, contrasting with previous observations. We evaluated the divalent cation dependence and trypsin sensitivities of synapses using synaptosomes from different developmental stages. In contrast to adult synapses, at postnatal day (P) 10 EDTA treatment eliminated approximately 60% of the synapses, and trypsin and EDTA, together, eliminated all junctions. Trypsinization in the presence of calcium eliminated approximately 60% of the junctions at P10. By P35, all synapses were calcium independent, whereas full trypsin resistance was not attained until P49. To compare the calcium dependence and trypsin sensitivity of synapses in another region of the adult brain, we examined synapses from adult (P50) hippocampus. Adult hippocampus maintained a population of synapses that resembled that of P35 cortex. Our results show that synapses are modified over a long time period in the developing cortex. We propose a model in which the addition of synergistic calcium-dependent and -independent adhesive systems stabilize synapses.

Trypsin-catalyzed tandem reaction: one-pot synthesis of 3,4-dihydropyrimidin-2(1H)-ones by in situ formed acetaldehyde.

PubMed

Xie, Zong-Bo; Wang, Na; Wu, Wan-Xia; Le, Zhang-Gao; Yu, Xiao-Qi

2014-01-20

A simple, mild, one-pot tandem method catalyzed by trypsin was developed for the synthesis of 3,4-dihydropyrimidin-2(1H)-ones by the Biginelli reaction of urea, β-dicarbonyl compounds, and in situ-formed acetaldehyde. Trypsin was found to display dual promiscuous functions to catalyze transesterification and the Biginelli reaction in sequence. Copyright © 2013 Elsevier B.V. All rights reserved.

Targeting Trypsin-Inflammation Axis for Pancreatitis Therapy in a Humanized Pancreatitis Model

DTIC Science & Technology

2016-10-01

Award Number: W81XWH-15-1-0257 TITLE: Targeting Trypsin-Inflammation Axis for Pancreatitis Therapy in a Humanized Pancreatitis Model PRINCIPAL...AND SUBTITLE Targeting Trypsin-Inflammation Axis for Pancreatitis Therapy in a Humanized Pancreatitis Model 5a. CONTRACT NUMBER 5b. GRANT NUMBER...remains the same since it is covered under the institutional review. We set up monthly video conferences with our partnership PI to discuss any

Inhibition of trypsin by condensed tannins and wine.

PubMed

Gonçalves, Rui; Soares, Susana; Mateus, Nuno; de Freitas, Victor

2007-09-05

Phenolic compounds are abundant vegetable secondary metabolites in the human diet. The ability of procyanidin oligomers and wine polyphenols to inhibit trypsin activity was studied using a versatile and reliable in vitro method. The hydrolysis of the chromogenic substrate N-benzoyl-d,l-arginine-p-nitroanilide (BApNA) by trypsin was followed by spectrophotometry in the presence and absence of condensed tannins and wine. A clear relationship between the degree of polymerization of procyanidins and enzymatic inhibition was observed. Trypsin activity inhibition was also detected in several types of wine. In general, the inhibition increased with the concentration of phenolic compounds in wines. These results may be relevant when considering these compounds as antinutritional factors, thereby contributing to a reduced absorption of nutrients.

THE INACTIVATION OF DILUTE SOLUTIONS OF CRYSTALLINE TRYPSIN BY X-RADIATION

PubMed Central

McDonald, Margaret R.

1955-01-01

The proteolytic activity of dilute solutions of clystalline trypsin is destroyed by x-rays, the amount of inactivation being an exponential function of the radiation dose. The reaction yield increases steadily with increasing concentration of trypsin, varying, as the concentration of enzyme is increased from 1 to 300 µM, from 0.068 to 0.958 micromole of trypsin per liter inactivated per 1000 r with 0.005 N hydrochloric acid as the solvent, from 0.273 to 0.866 with 0.005 N sulfuric acid as the solvent, and from 0.343 to 0.844 with 0.005 N nitric acid as the solvent. When the reaction yields are plotted as a function of the initial concentration of trypsin, they fall on a curve given by the expression Y α XK, in which Y is the reaction yield, X is the concentration of trypsin, and K is a constant equal to 0.46, 0.20, and 0.16, respectively, with 0.005 N hydrochloric, sulfuric, and nitric acids as solvents. The differences between the reaction yields found with chloride and sulfate ions in I to 10 µM trypsin solutions are significant only in the pH range from 2 to 4. The amount of inactivation obtained with a given dose of x-rays depends on the pH of the solution being irradiated and the nature of the solvent. The reaction yield-pH curve is a symmetrical one, with minimum yields at about pH 7. Buffers such as acetate, citrate, borate and barbiturate, and other organic molecules such as ethanol and glucose, in concentrations as low as 20 µM, inhibit the inactivation of trypsin by x-radiation. Sigmoid inactivation-dose curves instead of exponential ones are obtained in the presence of ethanol. The reaction yields for the inactivation of trypsin solutions by x-rays are approximately 1.5 times greater when the irradiation is done at 26°C. than when it is done at 5°C., when 0.005 N hydrochloric acid is the solvent. The dependence on temperature is less when 0.005 N sulfuric acid is used, and is negligible with 0.005 N nitric acid. The difficulties involved in interpreting radiation effects in aqueous systems, and in comparing the results obtained under different experimental conditions, are discussed. PMID:14367774

Trypsin Reduces Pancreatic Ductal Bicarbonate Secretion by Inhibiting CFTR Cl- channel and Luminal Anion Exchangers

PubMed Central

Pallagi, Petra; Venglovecz, Viktória; Rakonczay, Zoltán; Borka, Katalin; Korompay, Anna; Ózsvári, Béla; Judák, Linda; Sahin-Tóth, Miklós; Geisz, Andrea; Schnúr, Andrea; Maléth, József; Takács, Tamás; Gray, Mike A.; Argent, Barry E.; Mayerle, Julia; Lerch, Markus M.; Wittmann, Tibor; Hegyi, Péter

2012-01-01

Background & Aims The effects of trypsin on pancreatic ductal epithelial cells (PDEC) vary among species and depend on localization of proteinase-activated receptor-2 (PAR-2). Bicarbonate secretion is similar in human and guinea pig PDEC; we compared its localization in these cell types and isolated guinea pig ducts to study the effects of trypsin and a PAR-2 agonist on this process. Methods PAR-2 localization was analyzed by immunohistochemistry in guinea pig and human pancreatic tissue samples (from 15 patients with chronic pancreatitis and 15 without pancreatic disease). Functions of guinea pig PDEC were studied by microperfusion of isolated ducts, measurements of intracellular pH (pHi) and Ca2+ concentration [Ca2+]i, and patch clamp analysis. The effect of pH on trypsinogen autoactivation was assessed using recombinant human cationic trypsinogen. Results PAR-2 localized to the apical membrane of human and guinea pig PDEC. Trypsin increased [Ca2+]i and pHi, and inhibited secretion of bicarbonate by the luminal anion exchanger and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Autoactivation of human cationic trypsinogen accelerated when the pH was reduced from 8.5 to 6.0. PAR-2 expression was strongly down-regulated, at transcriptional and protein levels, in the ducts of patients with chronic pancreatitis, consistent with increased activity of intraductal trypsin. Importantly, in PAR-2 knockout mice, the effects of trypsin were PAR-2 dependent. Conclusions Trypsin reduces pancreatic ductal bicarbonate secretion via PAR-2–dependent inhibition of the apical anion exchanger and the CFTR Cl- channel. This could contribute to the development of chronic pancreatitis, decreasing luminal pH and promoting premature activation of trypsinogen in the pancreatic ducts. PMID:21893120

Mesotrypsin has evolved four unique residues to cleave trypsin inhibitors as substrates [Mesotrypsin has evolved to cleave trypsin inhibitors as substrates using four unique residues

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alloy, Alexandre P.; Kayode, Olumide; Wang, Ruiying

Human mesotrypsin is highly homologous to other mammalian trypsins, and yet it is functionally unique in possessing resistance to inhibition by canonical serine protease inhibitors and in cleaving these inhibitors as preferred substrates. Arg-193 and Ser-39 have been identified as contributors to the inhibitor resistance and cleavage capability of mesotrypsin, but it is not known whether these residues fully account for the unusual properties of mesotrypsin. Here, we use human cationic trypsin as a template for engineering a gain of catalytic function, assessing mutants containing mesotrypsin-like mutations for resistance to inhibition by bovine pancreatic trypsin inhibitor (BPTI) and amyloid precursormore » protein Kunitz protease inhibitor (APPI), and for the ability to hydrolyze these inhibitors as substrates. We find that Arg-193 and Ser-39 are sufficient to confer mesotrypsin-like resistance to inhibition; however, compared with mesotrypsin, the trypsin-Y39S/G193R double mutant remains 10-fold slower at hydrolyzing BPTI and 2.5-fold slower at hydrolyzing APPI. We identify two additional residues in mesotrypsin, Lys-74 and Asp-97, which in concert with Arg-193 and Ser-39 confer the full catalytic capability of mesotrypsin for proteolysis of BPTI and APPI. Novel crystal structures of trypsin mutants in complex with BPTI suggest that these four residues function cooperatively to favor conformational dynamics that assist in dissociation of cleaved inhibitors. Our results reveal that efficient inhibitor cleavage is a complex capability to which at least four spatially separated residues of mesotrypsin contribute. As a result, these findings suggest that inhibitor cleavage represents a functional adaptation of mesotrypsin that may have evolved in response to positive selection pressure.« less

Efficacy of Trypsin in Treating Coral Snake Envenomation in the Porcine Model.

PubMed

Parker-Cote, Jennifer L; O'Rourke, Dorcas P; Brewer, Kori L; Lertpiriyapong, Kvin; Punja, Mohan; Bush, Sean P; Miller, Susan N; Meggs, William J

2015-12-01

Antivenom is the definitive treatment for venomous snakebites. Alternative treatments warrant investigation because antivenom is sometimes unavailable, expensive, and can have deleterious side effects. This study assesses the efficacy of trypsin to treat coral snake envenomation in an in vivo porcine model. A randomized, blinded study was conducted. Subjects were 13 pigs injected subcutaneously with 1 mL of eastern coral snake venom (10 mg/mL) in the right distal hind limb. After 1 min, subjects were randomized to have the envenomation site injected with either 1 mL of saline or 1 mL of trypsin (100 mg/mL) by a blinded investigator. Clinical endpoint was survival for 72 h or respiratory depression defined as respiratory rate <15 breaths per minute, falling pulse oximetry, or agonal respirations. Fisher's exact t test was used for between group comparisons. Average time to toxicity for the saline control was 263 min (191-305 min). The development of respiratory depression occurred more frequently in control pigs than treated pigs (p = 0.009). Four of the six pigs that received trypsin survived to the end of the 3-day study. No control pigs survived. Two of the trypsin treatment pigs died with times to toxicity of 718 and 971 min. Survival to 12 and 24 h was significantly greater in the trypsin treatment group (p = 0.002, p = 0.009, respectively). Local injection of trypsin, a proteolytic enzyme, at the site of envenomation decreased the toxicity of eastern coral snake venom and increased survival significantly. Further investigation is required before these results can be extended to human snakebites.

Trypsin induces biphasic muscle contraction and relaxation via transient receptor potential vanilloid 1 and neurokinin receptors 1/2 in porcine esophageal body.

PubMed

Xiaopeng, Bai; Tanaka, Yoshimasa; Ihara, Eikichi; Hirano, Katsuya; Nakano, Kayoko; Hirano, Mayumi; Oda, Yoshinao; Nakamura, Kazuhiko

2017-02-15

Duodenal reflux of fluids containing trypsin relates to refractory gastroesophageal reflux disease (GERD). Esophageal peristalsis and clearance are important factors in GERD pathogenesis. However, the function of trypsin in esophageal body contractility is not fully understood. In this study, effects of trypsin on circular smooth muscle (CSM) and longitudinal smooth muscle (LSM) of the porcine esophageal body were examined. Trypsin elicited a concentration dependent biphasic response, a major contraction and a subsequent relaxation only in CSM. In CSM, contraction occurred at trypsin concentrations of 100nM and relaxation at 1μM. A proteinase-activated receptor (PAR)2 activating peptide, SLIGKV-NH 2 (1mM), induced a monophasic contraction. Those responses were unaffected by tetrodotoxin though abolished by the gap junction uncouplers carbenoxolone and octanol. They were also partially inhibited by a transient receptor potential vanilloid type 1 (TRPV1) antagonist and abolished by combination of neurokinin receptor 1 (NK 1 ) and NK 2 antagonists, but not by an NK 3 antagonist, suggesting a PAR2-TRPV1-substance P pathway in sensory neurons. Substance P (100nM), an agonist for various NK receptors (NK 1 , NK 2 and NK 3 ) with differing affinities, induced significant contraction in CSM, but not in LSM. The contraction was also blocked by the combination of NK 1 and NK 2 antagonists, but not by the NK 3 antagonist. Moreover, substance P-induced contractions were unaffected by the TRPV1 antagonist, but inhibited by a gap junction uncoupler. In conclusion, trypsin induced a biphasic response only in CSM and this was mediated by PAR2, TRPV1 and NK 1/2 . Gap junctions were indispensable in this tachykinin-induced response. Copyright © 2017 Elsevier B.V. All rights reserved.

Mesotrypsin has evolved four unique residues to cleave trypsin inhibitors as substrates [Mesotrypsin has evolved to cleave trypsin inhibitors as substrates using four unique residues

DOE PAGES

Alloy, Alexandre P.; Kayode, Olumide; Wang, Ruiying; ...

2015-07-14

Human mesotrypsin is highly homologous to other mammalian trypsins, and yet it is functionally unique in possessing resistance to inhibition by canonical serine protease inhibitors and in cleaving these inhibitors as preferred substrates. Arg-193 and Ser-39 have been identified as contributors to the inhibitor resistance and cleavage capability of mesotrypsin, but it is not known whether these residues fully account for the unusual properties of mesotrypsin. Here, we use human cationic trypsin as a template for engineering a gain of catalytic function, assessing mutants containing mesotrypsin-like mutations for resistance to inhibition by bovine pancreatic trypsin inhibitor (BPTI) and amyloid precursormore » protein Kunitz protease inhibitor (APPI), and for the ability to hydrolyze these inhibitors as substrates. We find that Arg-193 and Ser-39 are sufficient to confer mesotrypsin-like resistance to inhibition; however, compared with mesotrypsin, the trypsin-Y39S/G193R double mutant remains 10-fold slower at hydrolyzing BPTI and 2.5-fold slower at hydrolyzing APPI. We identify two additional residues in mesotrypsin, Lys-74 and Asp-97, which in concert with Arg-193 and Ser-39 confer the full catalytic capability of mesotrypsin for proteolysis of BPTI and APPI. Novel crystal structures of trypsin mutants in complex with BPTI suggest that these four residues function cooperatively to favor conformational dynamics that assist in dissociation of cleaved inhibitors. Our results reveal that efficient inhibitor cleavage is a complex capability to which at least four spatially separated residues of mesotrypsin contribute. As a result, these findings suggest that inhibitor cleavage represents a functional adaptation of mesotrypsin that may have evolved in response to positive selection pressure.« less

The characterization of protein release from sericin film in the presence of an enzyme: towards fibroblast growth factor-2 delivery.

PubMed

Nishida, Ayumu; Naganuma, Tsuyoshi; Kanazawa, Takanori; Takashima, Yuuki; Yamada, Masaki; Okada, Hiroaki

2011-07-29

Aqueous preparations of silk protein (sericin) films were prepared to evaluate their biodegradation properties. In the absence of trypsin, sericin film swelled rapidly, kept its shape, and remained unaltered for 28 days or longer due to form β-sheet structures. In the presence of trypsin, sericin film gradually degraded; since the rate depended on the concentration of trypsin, the films likely underwent enzymatic hydrolysis. Sericin film incorporating the model protein drug fluorescein isothiocyanate-albumin (FA) also gradually degraded in the presence of trypsin and resulted in the sustained release of FA for 2 weeks or longer; in contrast, FA release was quite slow in the absence of trypsin. It is expected that sericin film has potential as a biodegradable and drug-releasing carrier. To evaluate the practical applicability of sericin film for the repair of defective tissues, fibroblast growth factor-2 (FGF-2) was incorporated into sericin films and the films were implanted on skull defects in rats. Whereas FGF-2 release was suppressed in the absence of trypsin in vitro, it appears that FGF-2, immobilized by ionic interactions between sericin and FGF-2, can be sustained-released in vivo from films incorporating 2500 or 250 ng of FGF-2 to support the growth of tissue around wounds. Copyright © 2011 Elsevier B.V. All rights reserved.

The incomplete anti-Rh antibody agglutination mechanism of trypsinized ORh+ red cells.

PubMed Central

Margni, R A; Leoni, J; Bazzurro, M

1977-01-01

The capacity for binding to trypsinized and non-trypsinized ORh+ red cells, of the IgG incomplete anti-Rh antibody and its F(ab')2 and Fc fragments has been investigated. An analysis has also been made of the capacity of non-specific human IgG, aggregated non-specific human IgG, human IgM (19S) and IgM (7S), and of fragments Fcgamma, Fcmu and Fc5mu to inhibit the agglutination of trypsinized ORh+ red cells by the IgG incomplete anti-Rh antibody. The results obtained indicate that these antibodies behave in a similar manner to that of nonprecipitating antibodies, and that the agglutination of trypsinized red cells seems to be a mixed reaction due to the interaction of an Fab fragment with its Rh antigenic determinant present in the surface of a red cell and the Fc of the same molecule with a receptor for Fc present in adjacent red cells. The trypsin treatment apparently results in the liberation of occult Fc receptors. It has also been demonstrated that in the agglutination of ORh+ red cells by IgG incomplete anti-Rh antibody in the presence of albumin, interaction must occur in some manner between the albumin and the Fc fragment since the F(ab')2 fragment does not give rise to agglutination under such conditions. Images Figure 1 PMID:415968

Trypsin-sensitive modulation of intestinal epithelial MD-2 as mechanism of lipopolysaccharide tolerance.

PubMed

Cario, Elke; Golenbock, Douglas T; Visintin, Alberto; Rünzi, Michael; Gerken, Guido; Podolsky, Daniel K

2006-04-01

Intestinal epithelial cells (IEC) are constantly exposed to both high concentrations of the bacterial ligand LPS and the serine protease trypsin. MD-2, which contains multiple trypsin cleavage sites, is an essential accessory glycoprotein required for LPS recognition and signaling through TLR4. The aim of this study was to characterize the expression and subcellular distribution of intestinal epithelial MD-2 and to delineate potential functional interactions with trypsin and then alteration in inflammatory bowel disease (IBD). Although MD-2 protein expression was minimal in primary IEC of normal colonic or ileal mucosa, expression was significantly increased in IEC from patients with active IBD colitis, but not in ileal areas from patients with severe Crohn's disease. Endogenous MD-2 was predominantly retained in the calnexin-calreticulin cycle of the endoplasmic reticulum; only a small fraction was exported to the Golgi. MD-2 expression correlated inversely with trypsin activity. Biochemical evidence and in vitro experiments demonstrated that trypsin exposure resulted in extensive proteolysis of endogenous and soluble MD-2 protein, but not of TLR4 in IEC, and was associated with desensitization of IEC to LPS. In conclusion, the present study suggests that endoplasmic reticulum-associated MD-2 expression in IBD may be altered by ileal protease in inflammation, leading to impaired LPS recognition and hyporesponsiveness through MD-2 proteolysis in IEC, thus implying a physiologic mechanism that helps maintain LPS tolerance in the intestine.

Trypsin inhibitors from Capsicum baccatum var. pendulum leaves involved in Pepper yellow mosaic virus resistance.

PubMed

Moulin, M M; Rodrigues, R; Ribeiro, S F F; Gonçalves, L S A; Bento, C S; Sudré, C P; Vasconcelos, I M; Gomes, V M

2014-11-07

Several plant organs contain proteinase inhibitors, which are produced during normal plant development or are induced upon pathogen attack to suppress the enzymatic activity of phytopathogenic microorganisms. In this study, we examined the presence of proteinase inhibitors, specifically trypsin inhibitors, in the leaf extract of Capsicum baccatum var. pendulum inoculated with PepYMV (Pepper yellow mosaic virus). Leaf extract from plants with the accession number UENF 1624, which is resistant to PepYMV, was collected at 7 different times (0, 24, 48, 72, 96, 120, and 144 h). Seedlings inoculated with PepYMV and control seedlings were grown in a growth chamber. Protein extract from leaf samples was partially purified by reversed-phase chromatography using a C2/C18 column. Residual trypsin activity was assayed to detect inhibitors followed by Tricine-SDS-PAGE analysis to determine the N-terminal peptide sequence. Based on trypsin inhibitor assays, trypsin inhibitors are likely constitutively synthesized in C. baccatum var. pendulum leaf tissue. These inhibitors are likely a defense mechanism for the C. baccatum var. pendulum- PepYMV pathosystem.

Quantitative proteomics reveals the kinetics of trypsin-catalyzed protein digestion.

PubMed

Pan, Yanbo; Cheng, Kai; Mao, Jiawei; Liu, Fangjie; Liu, Jing; Ye, Mingliang; Zou, Hanfa

2014-10-01

Trypsin is the popular protease to digest proteins into peptides in shotgun proteomics, but few studies have attempted to systematically investigate the kinetics of trypsin-catalyzed protein digestion in proteome samples. In this study, we applied quantitative proteomics via triplex stable isotope dimethyl labeling to investigate the kinetics of trypsin-catalyzed cleavage. It was found that trypsin cleaves the C-terminal to lysine (K) and arginine (R) residues with higher rates for R. And the cleavage sites surrounded by neutral residues could be quickly cut, while those with neighboring charged residues (D/E/K/R) or proline residue (P) could be slowly cut. In a proteome sample, a huge number of proteins with different physical chemical properties coexists. If any type of protein could be preferably digested, then limited digestion could be applied to reduce the sample complexity. However, we found that protein abundance and other physicochemical properties, such as molecular weight (Mw), grand average of hydropathicity (GRAVY), aliphatic index, and isoelectric point (pI) have no notable correlation with digestion priority of proteins.

Simple and label-free liquid crystal-based sensor for detecting trypsin coupled to the interaction between cationic surfactant and BSA.

PubMed

Wang, Yi; Zhou, Lele; Kang, Qi; Yu, Li

2018-06-01

Trypsin plays a central role in catalyzing the hydrolysis of peptide bonds, so a technique with simple operation is needed to monitor the activity of trypsin. Here a simple and label-free senor based on liquid crystals (LCs) was developed by employing bovine serum albumin (BSA) as the enzyme substrate and dodecyl trimethyl ammonium bromide (DTAB) as the controller for the alignment of LC. It was found that DTAB could form a self-assembled monolayer at the aqueous/LC interface to produce the dark optical images of LCs. And the addition of BSA could disturb the monolayer, so that the optical signal of LCs turned bright from dark. But the hydrolysis of BSA by trypsin resulted in the dark appearance. The sensing platform allows detection as low as 1 U/mL under the polarized light microscope based on at least three measurements. Moreover, this method was successfully applied in the detection of trypsin in human urines, suggesting its potential applications in clinic diagnosis. Copyright © 2018 Elsevier B.V. All rights reserved.

Trypsin cleavage of the baculovirus occlusion-derived virus attachment protein P74 is prerequisite in per os infection.

PubMed

Slack, Jeffrey M; Lawrence, Susan D; Krell, Peter J; Arif, Basil M

2008-10-01

Baculovirus occlusion-derived virions (ODVs) contain a number of infectivity factors essential for the initiation of infection in larval midgut cells. Deletion of any of these factors neutralizes infectivity by the per os route. We have observed that P74 of the group I alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is N-terminally cleaved when a soluble form of the protein was incubated with insect midgut tissues under alkaline conditions and that cleavage was prevented by soybean trypsin inhibitor (SBTI). Presently, biological assays were carried out that suggest SBTI inhibits and trypsin enhances baculovirus per os infectivity. We developed a method to rescue per os infectivity of a P74 null virus involving co-transfection of viral DNA with a plasmid that transiently expresses p74. We used this plasmid rescue method to functionally characterize P74. A series of site-directed mutants were generated at the N terminus to evaluate if trypsin cleavage sites were necessary for function. Mutagenesis of R195, R196 and R199 compromised per os infectivity and rendered P74 resistant to midgut trypsin.

Isolation, expression and characterization of a novel dual serine protease inhibitor, OH-TCI, from king cobra venom.

PubMed

He, Ying-Ying; Liu, Shu-Bai; Lee, Wen-Hui; Qian, Jin-Qiao; Zhang, Yun

2008-10-01

Snake venom Kunitz/BPTI members are good tools for understanding of structure-functional relationship between serine proteases and their inhibitors. A novel dual Kunitz/BPTI serine proteinase inhibitor named OH-TCI (trypsin- and chymotrypsin-dual inhibitor from Ophiophagus hannah) was isolated from king cobra venom by three chromatographic steps of gel filtration, trypsin affinity and reverse phase HPLC. OH-TCI is composed of 58 amino acid residues with a molecular mass of 6339Da. Successful expression of OH-TCI was performed as the maltose-binding fusion protein in E. coli DH5alpha. Much different from Oh11-1, the purified native and recombinant OH-TCI both had strong inhibitory activities against trypsin and chymotrypsin although the sequence identity (74.1%) between them is very high. The inhibitor constants (K(i)) of recombinant OH-TCI were 3.91 x 10(-7) and 8.46 x10(-8)M for trypsin and chymotrypsin, respectively. To our knowledge, it was the first report of Kunitz/BPTI serine proteinase inhibitor from snake venom that had equivalent trypsin and chymotrypsin inhibitory activities.

Isolation and characterization of the chicken trypsinogen gene family.

PubMed Central

Wang, K; Gan, L; Lee, I; Hood, L

1995-01-01

Based on genomic Southern hybridizations and cDNA sequence analyses, the chicken trypsinogen gene family can be divided into two multi-member subfamilies, a six-member trypsinogen I subfamily which encodes the cationic trypsin isoenzymes and a three-member trypsinogen II subfamily which encodes the anionic trypsin isoenzymes. The chicken cDNA and genomic clones containing these two subfamilies were isolated and characterized by DNA sequence analysis. The results indicated that the chicken trypsinogen genes encoded a signal peptide of 15 to 16 amino acid residues, an activation peptide of 9 to 10 residues and a trypsin of 223 amino acid residues. The chicken trypsinogens contain all the common catalytic and structural features for trypsins, including the catalytic triad His, Asp and Ser and the six disulphide bonds. The trypsinogen I and II subfamilies share approximately 70% sequence identity at the nucleotide and amino acid level. The sequence comparison among chicken trypsinogen subfamily members and trypsin sequences from other species suggested that the chicken trypsinogen genes may have evolved in coincidental or concerted fashion. Images Figure 6 Figure 7 PMID:7733885

Evaluation of Mannosidase and Trypsin Enzymes Effects on Biofilm Production of Pseudomonas aeruginosa Isolated from Burn Wound Infections.

PubMed

Banar, Maryam; Emaneini, Mohammad; Satarzadeh, Mhboubeh; Abdellahi, Nafiseh; Beigverdi, Reza; Leeuwen, Willem B van; Jabalameli, Fereshteh

2016-01-01

Biofilm is an important virulence factor in Pseudomonas aeruginosa and has a substantial role in antibiotic resistance and chronic burn wound infections. New therapeutic agents against P. aeruginosa, degrading biofilms in burn wounds and improving the efficacy of current antimicrobial agents, are required. In this study, the effects of α-mannosidase, β-mannosidase and trypsin enzymes on the degradation of P. aeruginosa biofilms and on the reduction of ceftazidime minimum biofilm eliminating concentrations (MBEC) were evaluated. All tested enzymes, destroyed the biofilms and reduced the ceftazidime MBECs. However, only trypsin had no cytotoxic effect on A-431 human epidermoid carcinoma cell lines. In conclusion, since trypsin had better features than mannosidase enzymes, it can be a promising agent in combatting P. aeruginosa burn wound infections.

STRANDEDNESS OF VICIA FABA CHROMOSOMES AS REVEALED BY ENZYME DIGESTION STUDIES

PubMed Central

Trosko, James E.; Wolff, Sheldon

1965-01-01

Chromosomes and nuclei isolated from neutral formalin-fixed Vicia faba lateral roots were treated with trypsin, pepsin, RNase, or DNase. Only trypsin affected the morphology of the chromosomes and nuclei. The appearance of the chromosomes after trypsin digestion indicated that each chromatid contained four strands that could be seen with an ordinary light microscope. The experiments are interpreted as indicating that mitotic chromosomes of Vicia faba are multistranded and that the linear continuity of the chromosome is dependent on protein. PMID:5323605

A bioactive film based on cashew gum polysaccharide for wound dressing applications.

PubMed

Moreira, Bruna R; Batista, Karla A; Castro, Elisandra G; Lima, Eliana M; Fernandes, Kátia F

2015-05-20

This work presents the development of a new bioactive material for wound therapeutics which may play a dual role of modulate metallo proteinases activity while prevents infection blocking out pathogenic microorganisms and foreign materials. A CGP/PVA film was activated by covalent immobilization of trypsin. Results from biocompatibility test revealed that PDL fibroblasts grown on the surface of CGP/PVA and the high amount of viable cells proved absence of cytotoxicity. Trypsin immobilized onto CGP/PVA film remained 100% active after 28 days stored dried at room temperature. In addition, CGP/PVA-trypsin film could be used for 9 cycles of storage/use without loss of activity. After immobilization, trypsin retained its collagenolytic activity, indicating this material as a promising material for wound dressing applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

Studies on Cation-induced Thylakoid Membrane Stacking, Fluorescence Yield, and Photochemical Efficiency 1

PubMed Central

Jennings, Robert Charles; Forti, Giorgio; Gerola, Paolo Domenico; Garlaschi, Flavio Massimo

1978-01-01

Trypsin digestion of photosynthetic membranes isolated from spinach (Spinacia oleracea L.) leaves eliminates the cation stimulation of chlorophyll fluorescence. High concentrations of cations protect the fluorescence yield against trypsin digestion, and the cation specificity for this protection closely resembles that required for the stimulation of fluorescence by cations. Trypsin digestion reverses cation-induced thylakoid stacking, and the time course of this effect seems to parallel that of the reversal of cation fluorescence. High concentrations of cations protect thylakoid stacking and cation-stimulated fluorescence alike. The cation stimulation of photosytem II photochemistry remains intact after trypsinization has reversed both cation-induced thylakoid stacking and fluorescence yield. It is concluded that cation-stimulated fluorescence yield, and not the cation stimulation of photosystem II photochemistry, is associated with thylakoid membrane stacking. ImagesFig. 2Fig. 3 PMID:16660630

Effect of conformational mobility and hydrogen-bonding interactions on the selectivity of some guanidinoaryl-substituted mechanism-based inhibitors of trypsin-like serine proteases.

PubMed

Rai, R; Katzenellenbogen, J A

1992-11-13

Previously, we have reported that some guanidino-substituted alpha- and beta-aryl enol lactones I and II behaved as selective, mechanism-based inhibitors of some trypsin-like proteases (Rai, R.; Katzenellenbogen, J.A. J. Med. Chem., submitted). In this study, we describe the synthesis and kinetic evaluation of some related, guanidino-substituted enol lactones having greater conformational mobility and affording additional hydrogen-bonding sites at the active site. The alpha-aryl-substituted lactones 1 and 2, which have greater conformational mobility in the guanidinoaryl linkage than I, selectively inhibited the trypsin-like enzymes, and they were relatively poor inactivators of alpha-chymotrypsin and human neutrophil elastase (HNE). The iodo enol lactone 2 permanently inactivated trypsin, urokinase, tissue plasminogen activator, and plasmin, showing exceptionally high specificity in its interaction with trypsin and urokinase. The selectivity pattern exhibited by the closely related, conformationally less mobile alpha-aryl-substituted iodo lactone Ib, which was previously shown to be a selective suicide substrate of urokinase and plasmin, provides an interesting comparison. The alpha-benzamido-substituted lactones 3 and 4, which afford an additional site for active-site hydrogen bonding, were found to be very potent alternate substrate inhibitors of trypsin and urokinase. In addition, the iodo lactone 4 permanently inactivated alpha-chymotrypsin. The importance of secondary interactions in increasing the specificities in the case of alpha-chymotrypsin is discussed.

Bio-potency of a 21 kDa Kunitz-type trypsin inhibitor from Tamarindus indica seeds on the developmental physiology of H. armigera.

PubMed

Pandey, Prabhash K; Jamal, Farrukh

2014-11-01

A trypsin inhibitor purified from the seeds of Tamarindus indica by Sephadex G-75, DEAE-Sepharose and Trypsin-Sepharose CL-4B columns was studied for its antifeedant, larvicidal, pupicidal and growth inhibitory activities against Helicoverpa armigera larvae. Tamarindus trypsin inhibitor (TTI) exhibited inhibitory activity towards total gut proteolytic enzymes of H. armigera (~87%) and bovine trypsin (~84%). Lethal doses which caused mortality and weight reduction by 50% were 1% w/w and 0.50% w/w, respectively. IC50 of TTI against Helicoverpa midgut proteases and bovine trypsin were ~2.10 µg/ml and 1.68 µg/ml respectively. In larval feeding studies the 21 kDa Kunitz-type protein was found to retard growth and development, prolonged the larval-pupal development durations along with adversely affecting the fertility and fecundity of H. armigera. In artificial diet at 0.5% w/w TTI, the efficiency of conversion of ingested food as well as of digested food, relative growth rate, growth index declined whereas approximate digestibility, metabolic cost, relative consumption rate, consumption index and total developmental period enhanced for H. armigera larvae. These results suggest that TTI has toxic and adverse effect on the developmental physiology of H. armigera and could be useful in controlling the pest H. armigera. Copyright © 2014 Elsevier Inc. All rights reserved.

Expanding proteome coverage with orthogonal-specificity α-Lytic proteases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meyer, Jesse G.; Kim, Sangtae; Maltby, David A.

2014-03-01

Bottom-up proteomics studies traditionally involve proteome digestion with a single protease, trypsin. However, trypsin alone does not generate peptides that encompass the entire proteome. Alternative proteases have been explored, but most have specificity for charged amino acid side chains. Therefore, additional proteases that improve proteome coverage by cleavage at sequences complimentary to trypsin may increase proteome coverage. We demonstrate the novel application of two proteases for bottom-up proteomics: wild type alpha-lytic protease (WaLP), and an active site mutant of WaLP, M190A alpha-lytic protease (MaLP). We assess several relevant factors including MS/MS fragmentation, peptide length, peptide yield, and protease specificity. Bymore » combining data from separate digestions with trypsin, LysC, WaLP, and MaLP, proteome coverage was increased 101% compared to trypsin digestion alone. To demonstrate how the gained sequence coverage can access additional PTM information, we show identification of a number of novel phosphorylation sites in the S. pombe proteome and include an illustrative example from the protein MPD2, wherein two novel sites are identified, one in a tryptic peptide too short to identify and the other in a sequence devoid of tryptic sites. The specificity of WaLP and MaLP for aliphatic amino acid side chains was particularly valuable for coverage of membrane protein sequences, which increased 350% when the data from trypsin, LysC, WaLP, and MaLP were combined.« less

Systematic Design of Trypsin Cleavage Site Mutated Exendin4-Cysteine 1, an Orally Bioavailable Glucagon-Like Peptide-1 Receptor Agonist.

PubMed

Sai, Wenbo; Tian, Hong; Yang, Kangmin; Tang, Daoqi; Bao, Jinxiao; Ge, Yang; Song, Xiaoda; Zhang, Yu; Luo, Cheng; Gao, Xiangdong; Yao, Wenbing

2017-03-08

Exendin-4 is a strong therapeutic candidate for the treatment of metabolic syndrome. Related receptor agonist drugs have been on the market since 2005. However, technical limitations and the pain caused by subcutaneous injection have severely limited patient compliance. The goal of the study is to investigate a biologically active exendin-4 analog could be administered orally. Using intraperitoneal glucose tolerance tests, we discovered that exendin4-cysteine administered by oral gavage had a distinct hypoglycemic effect in C57BL/6J mice. Using Rosetta Design and Amber, we designed and screened a series of exendin4-cysteine analogs to identify those that retained biological activity while resisting trypsin digestion. Trypsin Cleavage Site Mutated Exendin4-cysteine 1 (TSME-1), an analog whose bioactivity was similar to exendin-4 and was almost completely resistant to trypsin, was screened out. In addition, TSME-1 significantly normalized the blood glucose levels and the availability of TSME-1 was significantly higher than that of exendin-4 and exendin4-cysteine. Collectively orally administered TSME-1, a trypsin-resistant exendin-4 analog obtained by the system, is a strong candidate for future treatments of type 2 diabetes.

Effect of sorbitol and glycerol on the stability of trypsin and difference between their stabilization effects in the various solvents.

PubMed

Pazhang, Mohammad; Mehrnejad, Faramarz; Pazhang, Yaghub; Falahati, Hanieh; Chaparzadeh, Nader

2016-01-01

The effect of glycerol and sorbitol on the stability of porcine pancreas trypsin was investigated in this work. Molecular dynamics simulation and thermostability results showed that trypsin has two flexible regions, and polyols (sorbitol and glycerol) stabilize the enzyme by decreasing the flexibility of these regions. Radial distribution function results exhibited that sorbitol and glycerol were excluded from the first water layer of the enzyme, therefore decrease the flexibility of the regions by preferential exclusion. Also, results showed that the stabilization effect of sorbitol is more than glycerol. This observation could be because of the larger decrease in the fluctuations of trypsin in the presence of sorbitol. We also examined the role of solvent's hydrophobicity in enzyme stabilization by sorbitol and glycerol. To do so, the thermostability of trypsin was evaluated in the presence of solvents with different hydrophobicity (methanol, ethanol, isopropanol and n-propanol) in addition to the polyols. Our results depicted that glycerol is a better stabilizer than sorbitol in the presence of hydrophobic solvents (n-propanol), whereas sorbitol is a better stabilizer than glycerol in the presence of hydrophilic solvents (methanol). © 2015 International Union of Biochemistry and Molecular Biology, Inc.

Characterization and Pharmacological Properties of a Novel Multifunctional Kunitz Inhibitor from Erythrina velutina Seeds

PubMed Central

Machado, Richele J. A.; Monteiro, Norberto K. V.; Migliolo, Ludovico; Silva, Osmar N.; Pinto, Michele F. S.; Oliveira, Adeliana S.; Franco, Octávio L.; Kiyota, Sumika; Bemquerer, Marcelo P.; Uchoa, Adriana F.; Morais, Ana H. A.; Santos, Elizeu A.

2013-01-01

Inhibitors of peptidases isolated from leguminous seeds have been studied for their pharmacological properties. The present study focused on purification, biochemical characterization and anti-inflammatory and anticoagulant evaluation of a novel Kunitz trypsin inhibitor from Erythrina velutina seeds (EvTI). Trypsin inhibitors were purified by ammonium sulfate (30–60%), fractionation followed by Trypsin-Sepharose affinity chromatography and reversed-phase high performance liquid chromatography. The purified inhibitor showed molecular mass of 19,210.48 Da. Furthermore, a second isoform with 19,228.16 Da was also observed. The inhibitor that showed highest trypsin specificity and enhanced recovery yield was named EvTI (P2) and was selected for further analysis. The EvTI peptide fragments, generated by trypsin and pepsin digestion, were further analyzed by MALDI-ToF-ToF mass spectrometry, allowing a partial primary structure elucidation. EvTI exhibited inhibitory activity against trypsin with IC50 of 2.2×10−8 mol.L−1 and constant inhibition (Ki) of 1.0×10−8 mol.L−1, by a non-competitive mechanism. In addition to inhibit the activity of trypsin, EvTI also inhibited factor Xa and neutrophil elastase, but do not inhibit thrombin, chymotrypsin or peptidase 3. EvTI was investigated for its anti-inflammatory and anti-coagulant properties. Firstly, EvTI showed no cytotoxic effect on human peripheral blood cells. Nevertheless, the inhibitor was able to prolong the clotting time in a dose-dependent manner by using in vitro and in vivo models. Due to anti-inflammatory and anticoagulant EvTI properties, two sepsis models were here challenged. EvTI inhibited leukocyte migration and specifically acted by inhibiting TNF-α release and stimulating IFN-α and IL-12 synthesis. The data presented clearly contribute to a better understanding of the use of Kunitz inhibitors in sepsis as a bioactive agent capable of interfering in blood coagulation and inflammation. PMID:23737945

Identification by GeLC-MS/MS of trypsin inhibitor in sarcoplasmic proteins of three tropical fish and characterization of their inhibitory properties.

PubMed

Siriangkanakun, Siriphon; Li-Chan, Eunice C Y; Yongsawadigul, Jirawat

2014-07-01

Sarcoplasmic proteins from 3 fish species were fractionated by 50% to 70% ammonium sulfate precipitation. Lyophilized fractionated sarcoplasmic proteins of threadfin bream (TB-SP), bigeye snapper (BS-SP), and yellow croaker (YC-SP) showed 80% to 92% trypsin inhibitory activity. Trypsin inhibitory activity staining gel electrophoresis revealed bands at 32, 33, 37, 45, 48, and 50 kDa for the 3 species, and a band at 95 kDa was observed for TB-SP and YC-SP. Alpha-1-antitrypsin with molecular mass of 45 to 50 kDa was identified in YC-SP by gel-based liquid chromatography-tandem mass spectrometry (GeLC-MS/MS). Other major protein bands appeared on trypsin activity staining included phosphorylase, glyceraldehyde-3-phosphate dehydrogenase, and creatine kinase with molecular mass of 95 and 35 to 40 kDa, respectively. But, these 3 proteins did not show true trypsin inhibitory activity. Trypsin inhibitory activity of fractionated sarcoplasmic proteins showed good stability, with >80% activity retained at 60 °C and up to 0.6 M NaCl. TB-SP showed the highest inhibitory activity against autolysis of washed threadfin bream mince at 65 °C. Addition of 0.5% or 1% TB-SP improved textural properties of threadfin bream surimi gels preincubated at 37 or 65 °C followed by heating at 90 °C. Therefore, TB-SP could be a promising protein ingredient for enhancing surimi gel texture. Threadfin bream, bigeye snapper, and yellow croaker are the main species used as raw material for tropical surimi production. Sarcoplasmic proteins from 3 species contain trypsin inhibitor(s) that can minimize proteolytic activity and improve gel texture of proteinase-laden fish muscle. Therefore, sarcoplasmic proteins that are byproducts from surimi processing of these species could be recovered, fractionated, and utilized as a functional protein ingredient. © 2014 Institute of Food Technologists®

A novel poly(deep eutectic solvent)-based magnetic silica composite for solid-phase extraction of trypsin.

PubMed

Xu, Kaijia; Wang, Yuzhi; Li, Yixue; Lin, Yunxuan; Zhang, Haibao; Zhou, Yigang

2016-11-23

Novel poly(deep eutectic solvent) grafted silica-coated magnetic microspheres (Fe 3 O 4 @SiO 2 -MPS@PDES) were prepared by polymerization of choline chloride-itaconic acid (ChCl-IA) and γ-MPS-modified magnetic silica composites, and were characterized by vibrating sample magnetometer (VSM), Fourier transform infrared spectrometry (FT-IR), X-ray photoelectron spectra (XPS), thermal gravimetric analysis (TGA) and transmission electron microscope (TEM). Then the synthetic Fe 3 O 4 @SiO 2 -MPS@PDES microspheres were applied for the magnetic solid-phase extraction (MSPE) of trypsin for the first time. After extraction, the concentration of trypsin in the supernatant was determined by a UV-vis spectrophotometer. Single factor experiments were carried out to investigate the effects of the extraction process, including the concentration of trypsin, the ionic strength, the pH value, the extraction time and the temperature. Experimental results showed the extraction capacity could reach up to 287.5 mg/g under optimized conditions. In comparison with Fe 3 O 4 @SiO 2 -MPS, Fe 3 O 4 @SiO 2 -MPS@PDES displayed higher extraction capacity and selectivity for trypsin. According to the regeneration studies, Fe 3 O 4 @SiO 2 -MPS@PDES microspheres can be recycled six times without significant loss of its extraction capacity, and retained a high extraction capacity of 233 mg/g after eight cycles. Besides, the activity studies also demonstrated that the activity of the extracted trypsin was well retained. Furthermore, the analysis of real sample revealed that the prepared magnetic microspheres can be used to purify trypsin in crude bovine pancreas extract. These results highlight the potential of the proposed Fe 3 O 4 @SiO 2 -MPS@PDES-MSPE method in separation of biomolecules. Copyright © 2016 Elsevier B.V. All rights reserved.

Improvement of porcine islet isolation by inhibition of trypsin activity during pancreas preservation and digestion using α1-antitrypsin.

PubMed

Shimoda, Masayuki; Noguchi, Hirofumi; Fujita, Yasutaka; Takita, Morihito; Ikemoto, Tetsuya; Chujo, Daisuke; Naziruddin, Bashoo; Levy, Marlon F; Kobayashi, Naoya; Grayburn, Paul A; Matsumoto, Shinichi

2012-01-01

Porcine islets are considered to be a promising resource for xenotransplantation. However, it is difficult to isolate porcine islets because of the marked fragility and rapid dissociation. Endogenous trypsin is one of the main factors to damage islets during the isolation procedure. Recent studies have suggested that trypsin inhibitors during the preservation of pancreas or the collagenase digestion can improve the result of islet isolation. In this study, we examined whether α1-antitrypsin (Aralast™), which inhibits several endogenous proteases and has immunomodulatory properties, can protect islets from the proteases and improve the results of porcine islet isolation. Twelve porcine pancreata were divided into three groups: without Aralast group (standard, n = 5), preserved with Aralast using the ductal injection (DI) method (DI, n = 3), and with Aralast using the DI method and in the collagenase solution (DI+C, n = 4). Efficacy of islet isolation was assessed by islet yields, purity, and viability. The trypsin activity of the preservation and the digestion solution during the isolation procedure was measured. During islet isolation, the trypsin activity in DI+C group was significantly inhibited compared to the standard group, whereas DI group showed less effect than DI+C group. The average of postpurification islet equivalents (IEQ) per pancreas weight in the DI+C group was significantly higher than the standard group (standard: 3516 ± 497 IEQ/g, DI: 4607 ± 1090 IEQ/g, DI+C: 7097 ± 995 IEQ/g; p = 0.017 between standard and DI+C). In the DI+C group, stimulation index was higher than in other groups, although there was no significant difference. The presence of Aralast in both DI solution and collagenase solution markedly inhibited trypsin activity during pancreas digestion procedure and improved the porcine islet isolation. Inhibition of trypsin activity by Aralast could improve porcine islet isolation.

Increased (/sup 125/I)trypsin-binding in serum from cystic fibrosis patients

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cox, K.L.; Frates, R.C. Jr.; Sheikholislam, B.M.

1982-01-01

The capacities of normal and cystic fibrosis (CF) sera to bind to exogenous human (/sup 125/I)trypsin were compared. Sera from eight older CF patients bound significantly more exogenous human (/sup 125/I)trypsin than did sera from eight normal subjects (p less than 0.001). Disregarding the increased trypsin-binding (TB) of CF sera, serum immunoreactive trypsinogen (SIRT) levels were not detectable in these eight older CF patients. However, when SIRT levels were corrected for TB, four CF patients had normal SIRT concentrations and four had low but detectable SIRT levels. As compared to five normal newborns' sera, serum from a newborn with CFmore » had normal TB and the SIRT levels were very high. In conclusion, increased TB in CF serum lowers results of SIRT assays. Therefore, unless SIRT levels are corrected for TB, results obtained from currently available SIRT kits may be invalid.« less

Effects of Mucuna pruriens protease inhibitors on Echis carinatus venom.

PubMed

Hope-Onyekwere, Nnadozie Stanley; Ogueli, Godwin Ifeanyi; Cortelazzo, Alessio; Cerutti, Helena; Cito, Annarita; Aguiyi, John C; Guerranti, Roberto

2012-12-01

The medicinal plant Mucuna pruriens, with reputed anti-snake venom properties has been reported to contain a kunitz-type trypsin inhibitor. This study was undertaken to further evaluate the protease inhibitory potential of gpMuc, a multiform glycoprotein, and other protein fractions from M. pruriens seeds against trypsin, chymotrypsin, Echis carinatus snake venom, ecarin and thrombin. The results showed that gpMuc inhibited both trypsin and chymotrypsin activities and was thermally stable, maintaining its trypsin inhibitory activity at temperatures of up to 50°C. Its structural conformation was also maintained at pH ranges of 4-7. Immunoreactivity study confirms that it contains protease-recognizing epitope on one of its isoforms. The whole protein extract of M. pruriens seeds inhibited prothrombin activation by ecarin and whole E. carinatus venom, and also thrombin-like activity using chromogenic assay. Copyright © 2012 John Wiley & Sons, Ltd.

Partial trypsin digestion as an indicator of mis-folding of mutant alanine:glyoxylate aminotransferase and chaperone effects of specific ligands. Study of a spectrum of missense mutants.

PubMed

Coulter-Mackie, M B; Lian, Q

2008-07-01

Alanine:glyoxylate aminotransferase (AGT) is a liver peroxisomal enzyme whose deficiency results in primary hyperoxaluria type 1 (PH1). More than 75 PH1 mutations are now documented in the AGT gene (AGXT), of which about 50% are missense. We have previously demonstrated that many such mutants expressed by transcription/translation are subject to generalized degradation by the proteasome and a specific limited trimming by an endogenous ATP-independent protease activity. Here, we report the results of partial digestion using trypsin as a mimic for the endogenous non-proteasomal protease and the use of N-terminal protein sequencing to determine the sensitive site. Partial trypsin digestion also provided an indicator of proper folding of the mutant enzyme. For selected mutations the sensitivity to trypsin could be ameliorated by addition of pyridoxal phosphate or aminooxy acetic acid as specific pharmacological chaperones.

Protease-dependent hemagglutinin cleavage contributes to alteration in chicken hemagglutination by the H3N2 influenza A virus.

PubMed

Yamaoka, Masaoki; Makino, Akiko; Sasahara, Kenji; Nastri, Aldise Mareta; Krisna, Luh Ade Wilan; Purhito, Edith Frederika; Poetranto, Emmanuel Djoko; Wulandari, Laksmi; Yudhawati, Resti; Setiawati, Landia; Setyoningrum, Retno Asih; Shinya, Kyoko

2013-01-01

The human influenza A virus (H3N2) has been the predominant influenza strain since 1992, and one property of this virus is non-agglutination of chicken erythrocytes [Ch(-) virus]. The Ch(-) virus in our study was able to acquire chicken hemagglutination [Ch(+)] by trypsin passage but not by chymotrypsin passage. Moreover, the trypsin-passaged Ch(+) viruses reacquired the Ch(-) property after a further chymotrypsin passage. In particular, genetic analysis showed no evidence of mutations in the hemagglutinin (HA) gene during either trypsin or chymotrypsin passages: the only differences found were in the HA cleavage sites between the trypsin-passaged virus and the chymotrypsin-passaged virus as determined by the N-terminal amino acid sequence. These results suggested that protease-dependent differences at the viral HA cleavage site, rather than genetic mutations, are likely to have a significant effect on the viral ability to produce chicken hemagglutination.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jung, D.W.; Laties, G.G.

Potato mitochondria (Solanum tuberosum var. Russet Burbank), which readily phosphorylate ADP in oxidative phosphorylation, show low levels of ATPase activity which is stimulated neither by Mg/sup 2 +/, 2,4-dinitrophenol, incubation with respiratory substrates, nor disruption by sonication or treatment with Triton X-100, individually or in concert. Treatment of disrupted potato mitochondria with trypsin stimulates Mg/sup 2 +/-dependent, oligomycin-sensitive ATPase activity 10- to 15-fold, suggesting the presence of an ATPase inhibitor protein. Trypsin-induced ATPase activity was unaffected by uncoupler. Oligomycin-sensitive ATPase activity decreases as exposure to trypsin is increased. Incubation at alkaline pH or heating at 60/sup 0/C for 2 minutesmore » also activates ATPase of sonicated potato mitochondria. Disruption of cauliflower (Brassica oleracea), red sweet potato (Ipomoea batatas), and carrot (Daucus carota) mitochondria increases ATPase activity, which is further enhanced by treatment with trypsin. The significance of the tight association of the inhibitor protein and ATPase in potato mitochondria is not clear.« less

Framework for Interpretation of Trypsin–antitrypsin Imbalance and Genetic Heterogeneity in Pancreatitis

PubMed Central

Lin, Kun; Gao, Feng; Chen, Qingquan; Liu, Qicai; Chen, Shu

2015-01-01

Early intracellular premature trypsinogen activation was interpreted as the key initiator of pancreatitis. When the balance in the homeostasis of trypsin and antitrypsin system is disequilibrated, elevated aggressive enzymes directly attack the pancreatic tissue, which leads to pancreatic destruction and inflammation. However, trypsin alone is not enough to cause complications in pancreatitis, which may play a crucial role in modulating signaling events in the initial phase of the disease. NFκB activation is the major inflammatory pathway involved in the occurrence and development of pancreatitis and it can be induced by intrapancreatic activation of trypsinogen. Synthesis of trypsinogen occurs in endoplasmic reticulum (ER), and ER stress is an important early acinar cell event. Components of ER stress response are known to be able to trigger cell death as well as NFκB signaling cascade. The strongest evidence supporting the trypsin-centered theory is that gene mutations, which lead to the generation of more trypsin, or reduce the activity of trypsin inhibitors or trypsin degradation, are associated with pancreatitis. Thus, trypsin–antitrypsin imbalance may be the first step leading to pancreatic autodigestion and inducing other pathways. Continued experimental studies are necessary to determine the specific relationships between trypsin–antitrypsin imbalance and genetic heterogeneity in pancreatitis. In this article, we review the latest advances that contributed to the understanding of the basic mechanisms behind the occurrence and development of pancreatitis with a focus on the interpretation of trypsin–antitrypsin imbalance and their relationships with other inflammation pathways. We additionally highlight genetic predispositions to pancreatitis and possible mechanisms associated with them. PMID:26228362

Carrier free immobilization and characterization of trypsin.

PubMed

Menfaatli, Esra; Zihnioglu, Figen

2015-04-01

Pancreatic trypsin was immobilized by cross-linked enzyme aggregates (CLEA) which is a carrier free immobilization method. Ammonium sulfate was chosen for enzyme precipitation which was followed by cross linking of formed aggregates via glutaraldehyde. Concentrations of precipitant and cross linker were respectively optimized as 60% ammonium sulfate and 1% glutaraldehyde. Optimum pH and temperature for CLEA was increased compared to free enzyme. Furthermore, pH, thermal and storage stability were improved. Presence of additives had no effects on enzyme activity. Prepared cross-linked trypsin aggregates are convenient for in situ protein fragmentation and can be used for protein identification.

High-Yield Method for Dispersing Simian Kidneys for Cell Cultures

PubMed Central

de Oca, H. Montes; Probst, P.; Grubbs, R.

1971-01-01

A technique for dispersion of animal tissue cells is described. The proposed technique is based on the concomitant use of trypsin and disodium ethylenediamine tetraacetate (EDTA). The use of the two dispersing agents (trypsin and disodium EDTA) markedly enhances cell yield as compared with the standard cell dispersion methods. Moreover, significant reduction in the amount of time required for complete tissue dispersal, presence of a very low number of nonviable cells, less cell clumping, and more uniform monolayer formation upon cultivation compare favorably with the results usually obtained with the standard trypsinization technique. Images PMID:4993235

Biochemical Characterization of An Arginine-Specific Alkaline Trypsin from Bacillus licheniformis.

PubMed

Gong, Jin-Song; Li, Wei; Zhang, Dan-Dan; Xie, Min-Feng; Yang, Biao; Zhang, Rong-Xian; Li, Heng; Lu, Zhen-Ming; Xu, Zheng-Hong; Shi, Jin-Song

2015-12-17

In the present study, we isolated a trypsin-producing strain DMN6 from the leather waste and identified it as Bacillus licheniformis through a two-step screening strategy. The trypsin activity was increased up to 140 from 20 U/mL through culture optimization. The enzyme was purified to electrophoretic homogeneity with a molecular mass of 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the specific activity of purified enzyme is 350 U/mg with Nα-Benzoyl-L-arginine ethylester as the substrate. The optimum temperature and pH for the trypsin are 65 °C and pH 9.0, respectively. Also, the enzyme can be significantly activated by Ba(2+). This enzyme is relatively stable in alkaline environment and displays excellent activity at low temperatures. It could retain over 95% of enzyme activity after 180 min of incubation at 45 °C. The distinguished activity under low temperature and prominent stability enhance its catalytic potential. In the current work, the open reading frame was obtained with a length of 1371 nucleotides that encoded a protein of 456 amino acids. These data would warrant the B. licheniformis trypsin as a promising candidate for catalytic application in collagen preparation and leather bating through further protein engineering.

Effects of trypsinization and of a combined trypsin, collagenase, and DNase digestion on liberation and in vitro function of satellite cells isolated from juvenile porcine muscles.

PubMed

Miersch, Claudia; Stange, Katja; Röntgen, Monika

2018-06-01

Muscle stem cells, termed satellite cells (SC), and SC-derived myogenic progenitor cells (MPC) are involved in postnatal muscle growth, regeneration, and muscle adaptability. They can be released from their natural environment by mechanical disruption and tissue digestion. The literature contains several isolation protocols for porcine SC/MPC including various digestion procedures, but comparative studies are missing. In this report, classic trypsinization and a more complex trypsin, collagenase, and DNase (TCD) digestion were performed with skeletal muscle tissue from 4- to 5-d-old piglets. The two digestion procedures were compared regarding cell yield, viability, myogenic purity, and in vitro cell function. The TCD digestion tended to result in higher cell yields than digestion with solely trypsin (statistical trend p = 0.096), whereas cell size and viability did not differ. Isolated myogenic cells from both digestion procedures showed comparable proliferation rates, expressed the myogenic marker Desmin, and initiated myogenic differentiation in vitro at similar levels. Thus, TCD digestion tended to liberate slightly more cells without changes in the tested in vitro properties of the isolated cells. Both procedures are adequate for the isolation of SC/MPC from juvenile porcine muscles but the developmental state of the animal should always be considered.

TsAg5, a Taenia solium cysticercus protein with a marginal trypsin-like activity in the diagnosis of human neurocysticercosis.

PubMed

Rueda, Analiz; Sifuentes, Cecilia; Gilman, Robert H; Gutiérrez, Andrés H; Piña, Ruby; Chile, Nancy; Carrasco, Sebastián; Larson, Sandra; Mayta, Holger; Verástegui, Manuela; Rodriguez, Silvia; Gutiérrez-Correa, Marcel; García, Héctor H; Sheen, Patricia; Zimic, Mirko

2011-12-01

Neurocysticercosis is an endemic parasitic disease caused by Taenia solium larva. Although the mechanism of infection is not completely understood, it is likely driven by proteolytic activity that degrades the intestinal wall to facilitate oncosphere penetration and further infection. We analyzed the publicly available T. solium EST/DNA library and identified two contigs comprising a full-length cDNA fragment very similar to Echinococcus granulosus Ag5 protein. The T. solium cDNA sequence included a proteolytic trypsin-like-domain in the C-terminal region, and a thrombospondin type-1 adherence-domain in the N-terminal region. Both the trypsin-like and adherence domains were expressed independently as recombinant proteins in bacterial systems. TsAg5 showed marginal trypsin-like activity and high sequence similarity to Ag5. The purified antigens were tested in a Western immunoblot assay to diagnose human neurocysticercosis. The sensitivity of the trypsin-like-domain was 96.36% in patients infected with extraparenchymal cysts, 75.44% in patients infected with multiple cysts, and 39.62% in patients with a single cyst. Specificity was 76.70%. The thrombospondin type-1 adherence-domain was not specific for neurocysticercosis. Copyright © 2011 Elsevier B.V. All rights reserved.

Conformational flexibility in the catalytic triad revealed by the high-resolution crystal structure of Streptomyces erythraeus trypsin in an unliganded state

PubMed Central

Blankenship, Elise; Vukoti, Krishna; Miyagi, Masaru; Lodowski, David T.

2014-01-01

With more than 500 crystal structures determined, serine proteases make up greater than one-third of all proteases structurally examined to date, making them among the best biochemically and structurally characterized enzymes. Despite the numerous crystallographic and biochemical studies of trypsin and related serine proteases, there are still considerable shortcomings in the understanding of their catalytic mechanism. Streptomyces erythraeus trypsin (SET) does not exhibit autolysis and crystallizes readily at physiological pH; hence, it is well suited for structural studies aimed at extending the understanding of the catalytic mechanism of serine proteases. While X-ray crystallographic structures of this enzyme have been reported, no coordinates have ever been made available in the Protein Data Bank. Based on this, and observations on the extreme stability and unique properties of this particular trypsin, it was decided to crystallize it and determine its structure. Here, the first sub-angstrom resolution structure of an unmodified, unliganded trypsin crystallized at physiological pH is reported. Detailed structural analysis reveals the geometry and structural rigidity of the catalytic triad in the unoccupied active site and comparison to related serine proteases provides a context for interpretation of biochemical studies of catalytic mechanism and activity. PMID:24598752

A crystalline protein-proteinase inhibitor from pinto bean seeds.

PubMed

Wang, D

1975-06-26

A crystalline protein-proteinase inhibitor has been isolated from seeds of Pinto bean (Phaseolus vulgaris cultvar. Pinto). It has an average molecular weight of 19 000 as estimated by gel filtration. This crystalline inhibitor is highly active against both bovine pancreatic trypsin and alpha-chymotrypsin. Complexes of both trypsin-inhibitor and alpha-chymotrypsin-inhibitor have been isolated. The inhibitor which was derived from the dissociated trypsin-inhibitor complex was only 62% as effective as the original compound against either enzyme. In contrast, the inhibitor obtained from alpha-chymotrypsin-inhibitor complex retained its full original inhibitory activity for trypsin, but only 25% of its original activity against alpha-chymotrypsin. The dissociated inhibitor from alpha-chymotrypsin-inhibitor compex, despite its full inhibitory activity, had been modified to such an extent that it could no longer form any precipitable complex with trypsin. The crystalline protein-proteinase inhibitor is not homogeneous and has been resolved into two distinct inhibitors in terms of their physical and chemical properties. These two inhibitors are designated as Pinto bean proteinase inhibitor I and II and their respective minimum molecular weights are 9100 and 10 000. They differ most strikingly in their amino acid composition in that inhibitor II is void of both valine and methionine.

Trypsin inhibitory activity and gel-enhancing effect of sarcoplasmic proteins from common carp.

PubMed

Siriangkanakun, Siriphon; Yongsawatdigul, Jirawat

2012-10-01

Proteinase inhibitory activity of sarcoplasmic protein (SP) extracted from common carp (Cyprinus carpio) muscle and its gel-improving ability were investigated. SPs displayed 89% and 54% inhibitory activity toward trypsin at 40 and 65 °C, respectively. Protein bands with molecular mass of 69, 50, 44, 41, and 35 kDa appeared on trypsin inhibitory activity staining under nonreducing condition when incubated at 40 °C, while 2 protein bands at 54 and 35 kDa were observed at 65 °C. Addition of SP at 0.18 g protein/100 g increased textural properties of threadfin bream surimi gel. However, when SP was added in combination with various CaCl(2) concentrations (0.1% to 0.5%) it did not further improve textural properties as compared to the addition of SP alone. Retention of myosin heavy chain of threadfin bream surimi was greater with the addition of SP. These results indicated that the gel-enhancing effect of common carp SP was due to the inhibitory activity toward endogenous trypsin-like proteinases in threadfin bream surimi. Sarcoplasmic protein from common carp muscle could be used as a functional protein ingredient that minimizes muscle proteolysis and improves textural properties of surimi containing trypsin-like endogenous proteinases. © 2012 Institute of Food Technologists®

Conformational flexibility in the catalytic triad revealed by the high-resolution crystal structure of Streptomyces erythraeus trypsin in an unliganded state.

PubMed

Blankenship, Elise; Vukoti, Krishna; Miyagi, Masaru; Lodowski, David T

2014-03-01

With more than 500 crystal structures determined, serine proteases make up greater than one-third of all proteases structurally examined to date, making them among the best biochemically and structurally characterized enzymes. Despite the numerous crystallographic and biochemical studies of trypsin and related serine proteases, there are still considerable shortcomings in the understanding of their catalytic mechanism. Streptomyces erythraeus trypsin (SET) does not exhibit autolysis and crystallizes readily at physiological pH; hence, it is well suited for structural studies aimed at extending the understanding of the catalytic mechanism of serine proteases. While X-ray crystallographic structures of this enzyme have been reported, no coordinates have ever been made available in the Protein Data Bank. Based on this, and observations on the extreme stability and unique properties of this particular trypsin, it was decided to crystallize it and determine its structure. Here, the first sub-angstrom resolution structure of an unmodified, unliganded trypsin crystallized at physiological pH is reported. Detailed structural analysis reveals the geometry and structural rigidity of the catalytic triad in the unoccupied active site and comparison to related serine proteases provides a context for interpretation of biochemical studies of catalytic mechanism and activity.

The Activity of Trypsin

ERIC Educational Resources Information Center

Russo, Salvatore F.; Holzman, Tom

1977-01-01

Describes an experiment that illustrates the following points concerning the experimental determination of trypsin activity: (1) there is a difference in basing enzyme concentration on weight, absorbance, or active sites; and (2) the method of expressing enzyme concentration determines the value of specific, molecular, and catalytic center…

Mesotrypsin Has Evolved Four Unique Residues to Cleave Trypsin Inhibitors as Substrates.

PubMed

Alloy, Alexandre P; Kayode, Olumide; Wang, Ruiying; Hockla, Alexandra; Soares, Alexei S; Radisky, Evette S

2015-08-28

Human mesotrypsin is highly homologous to other mammalian trypsins, and yet it is functionally unique in possessing resistance to inhibition by canonical serine protease inhibitors and in cleaving these inhibitors as preferred substrates. Arg-193 and Ser-39 have been identified as contributors to the inhibitor resistance and cleavage capability of mesotrypsin, but it is not known whether these residues fully account for the unusual properties of mesotrypsin. Here, we use human cationic trypsin as a template for engineering a gain of catalytic function, assessing mutants containing mesotrypsin-like mutations for resistance to inhibition by bovine pancreatic trypsin inhibitor (BPTI) and amyloid precursor protein Kunitz protease inhibitor (APPI), and for the ability to hydrolyze these inhibitors as substrates. We find that Arg-193 and Ser-39 are sufficient to confer mesotrypsin-like resistance to inhibition; however, compared with mesotrypsin, the trypsin-Y39S/G193R double mutant remains 10-fold slower at hydrolyzing BPTI and 2.5-fold slower at hydrolyzing APPI. We identify two additional residues in mesotrypsin, Lys-74 and Asp-97, which in concert with Arg-193 and Ser-39 confer the full catalytic capability of mesotrypsin for proteolysis of BPTI and APPI. Novel crystal structures of trypsin mutants in complex with BPTI suggest that these four residues function cooperatively to favor conformational dynamics that assist in dissociation of cleaved inhibitors. Our results reveal that efficient inhibitor cleavage is a complex capability to which at least four spatially separated residues of mesotrypsin contribute. These findings suggest that inhibitor cleavage represents a functional adaptation of mesotrypsin that may have evolved in response to positive selection pressure. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

T-cell epitope-containing hypoallergenic β-lactoglobulin for oral immunotherapy in milk allergy.

PubMed

Ueno, Hiroshi M; Kato, Teruhiko; Ohnishi, Hidenori; Kawamoto, Norio; Kato, Zenichiro; Kaneko, Hideo; Kondo, Naomi; Nakano, Taku

2016-12-01

Optimally hydrolyzed β-Lactoglobulin (βLg) is a promising milk oral immunotherapy (OIT) candidate with respect to showing reduced B-cell reactivity but retaining the T-cell epitope. To demonstrate that an edible hypoallergenic βLg hydrolysate containing the T-cell epitope is suitable for OIT. We tested how chymotrypsin affected the retention of the T-cell epitope of βLg when preparing βLg hydrolysates using food-grade trypsin. We investigated the effect of chymotrypsin activity on the formation of the T-cell epitope-containing peptide of βLg (βLg 102-124 ) and prepared an edible βLg hydrolysate containing βLg 102-124 using screened food-grade trypsins. B-cell reactivity was determined using immunoassays in which ELISA was performed with anti-βLg rabbit IgG and Western blotting was performed with a milk-specific IgE antiserum. In βLg hydrolysis performed by varying the activity of trypsin and chymotrypsin, chymotrypsin activity inhibited the formation of βLg 102-124 with an increase in hydrolysis time in a dose-dependent manner. βLg 102-124 was generated by two of five food-grade trypsins used at a ratio of 1:50 (w/w, enzyme/substrate) for 20 h at 40°C. The edible βLg hydrolysate retained βLg 102-124 and showed a reduction in molecular weight distribution and antigenicity against IgG and IgE. Chymotrypsin activity inhibited the formation of βLg 102-124 in the trypsin hydrolysate of βLg. This βLg trypsin hydrolysate is a novel candidate for peptide-based OIT in cow's milk allergy for safely inducing desensitization. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Imaging trypsin activity through changes in the orientation of liquid crystals coupled to the interactions between a polyelectrolyte and a phospholipid layer.

PubMed

Hu, Qiong-Zheng; Jang, Chang-Hyun

2012-03-01

In this study, we developed a new type of liquid crystal (LC)-based sensor for the real-time and label-free monitoring of enzymatic activity through changes in the orientation of LCs coupled to the interactions between polyelectrolyte and phospholipid. The LCs changed from dark to bright after an aqueous solution of poly-l-lysine (PLL) was transferred onto a self-assembled monolayer of the phospholipid, dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), at the aqueous/LC interface. Interactions between the positively charged PLL and the negatively charged DOPG drove the reorganization of the phospholipid membrane, which induced an orientational transition in the LCs from a homeotropic to planar state. Since the serine endopeptidase trypsin can enzymatically catalyze the hydrolysis of PLL, the dark-to-bright shift in the optical response was not observed after transferring a mixed solution of PLL and trypsin onto the DOPG-decorated LC interface, indicating that no orientational transitions in the LCs occurred. However, the optical response from dark to bright was observed when the mixture in the optical cell was replaced by an aqueous solution of PLL. Control experiments with trypsin or an aqueous mixture of PLL and deactivated trypsin further confirmed the feasibility of this approach. The detection limit of trypsin was determined to be ~1 μg/mL. This approach holds great promise for use in the development of LC-based sensors for the detection of enzymatic reactions in cases where the biological polyelectrolyte substrates of enzymes could disrupt the organization of the membrane and induce orientational transitions of LCs at the aqueous/LC interface. © 2012 American Chemical Society

A novel helper phage enabling construction of genome-scale ORF-enriched phage display libraries.

PubMed

Gupta, Amita; Shrivastava, Nimisha; Grover, Payal; Singh, Ajay; Mathur, Kapil; Verma, Vaishali; Kaur, Charanpreet; Chaudhary, Vijay K

2013-01-01

Phagemid-based expression of cloned genes fused to the gIIIP coding sequence and rescue using helper phages, such as VCSM13, has been used extensively for constructing large antibody phage display libraries. However, for randomly primed cDNA and gene fragment libraries, this system encounters reading frame problems wherein only one of 18 phages display the translated foreign peptide/protein fused to phagemid-encoded gIIIP. The elimination of phages carrying out-of-frame inserts is vital in order to improve the quality of phage display libraries. In this study, we designed a novel helper phage, AGM13, which carries trypsin-sensitive sites within the linker regions of gIIIP. This renders the phage highly sensitive to trypsin digestion, which abolishes its infectivity. For open reading frame (ORF) selection, the phagemid-borne phages are rescued using AGM13, so that clones with in-frame inserts express fusion proteins with phagemid-encoded trypsin-resistant gIIIP, which becomes incorporated into the phages along with a few copies of AGM13-encoded trypsin-sensitive gIIIP. In contrast, clones with out-of-frame inserts produce phages carrying only AGM13-encoded trypsin-sensitive gIIIP. Trypsin treatment of the phage population renders the phages with out-of-frame inserts non-infectious, whereas phages carrying in-frame inserts remain fully infectious and can hence be enriched by infection. This strategy was applied efficiently at a genome scale to generate an ORF-enriched whole genome fragment library from Mycobacterium tuberculosis, in which nearly 100% of the clones carried in-frame inserts after selection. The ORF-enriched libraries were successfully used for identification of linear and conformational epitopes for monoclonal antibodies specific to mycobacterial proteins.

Trypsin from the digestive system of carp Cirrhinus mrigala: purification, characterization and its potential application.

PubMed

Khangembam, Bronson Kumar; Chakrabarti, Rina

2015-05-15

Trypsin was purified 35.64-fold with 4.97% recovery from the viscera of carp Cirrhinus mrigala (mrigal) by ammonium sulfate precipitation, ion exchange and affinity chromatography. The purified enzyme was active at a wide range of pH (7.0-9.2) and temperature (10-50°C). The purified enzyme exhibited high thermal stability up to 50°C for 1h. The enzyme activity was stabilized by Ca(+2) (2mM) up to 7h at 40°C. The Km and kcat values of purified enzyme were 0.0672 mM and 92.09/s/mM, respectively. Soybean trypsin inhibitor and phenylmethylsulphonylflouride completely inhibited the enzyme activity. The specific inhibitor of trypsin, N-α-p-tosyl-L-lysine chloromethyl ketone inhibited 99.67% activity. Na(+), K(+) and Li(+) inhibited 20.99 ± 5.25%, 16.53 ± 4.80% and 18.99 ± 1.42% of enzyme activity, respectively. Divalent ions Mg(+2), Zn(+2), Co(+2), Hg(+2) and Cd(+2) inhibited 21.61 ± 2.22%, 31.62 ± 1.78%, 31.62 ± 1.96%, 85.68 ± 1.51% and 47.95 ± 2.13% enzyme activity, respectively. SDS-PAGE showed that the molecular mass of purified enzyme was 21.7 kDa. MALDI-TOF study showed a peptide sequence of AFCGGSLVNENKMHSAGHCYKSRIQV at the N-Terminal. This sequence recorded 76-84% identity with trypsin from Thunnus thynnus and other fish species. This confirmed that the purified protein was trypsin. The purified enzyme has potential applications in detergent and food industry because of its thermal stability and alkaline nature. Copyright © 2014 Elsevier Ltd. All rights reserved.

A novel dianionic amino acid ionic liquid-coated PEG 4000 modified Fe3O4 nanocomposite for the magnetic solid-phase extraction of trypsin.

PubMed

Yang, Qin; Wang, Yuzhi; Zhang, Hongmei; Xu, Kaijia; Wei, Xiaoxiao; Xu, Panli; Zhou, Yigang

2017-11-01

A novel magnetic extractant, PEG 4000 modified Fe 3 O 4 nanomaterial that coated with dianionic amino acid ionic liquid (Fe 3 O 4 @PEG@DAAAIL), was successfully synthesized and characterized. X-ray diffraction (XRD), transmission electron microscope (TEM), vibrating sample magnetometer (VSM), fourier transform infrared spectrometry (FT-IR), thermal gravimetric analysis (TGA) and zeta potentials were used to confirm that the novel nanocomposite was successfully synthesized. Subsequently, the prepared Fe 3 O 4 @PEG@DAAAIL nanocomposite was used as the extractant for trypsin coupled with magnetic solid-phase extraction (MSPE). The concentrations of trypsin in the supernatant were detected by UV-vis spectrophotometer at 278nm. The extraction ability turned out to be better than the other four kinds of extractants prepared in this work. Furthermore, the influence of a series of factors, such as extraction time and temperature, initial trypsin concentration, the value of pH and ionic strength, was systematically investigated. Under the optimal extraction condition, the extraction capacity for trypsin could reach up to 718.73mg/g, absolutely higher than that of other adsorbents reported. This satisfactory extraction capacity could be maintained unchangeable after at least eight days, and kept over 90% of initial extraction capacity after eight recycles. What's more, the activity of trypsin after extraction retained 92.29% of initial activity, verifying the biocompatibility of the prepared extractant. Finally, the developed Fe 3 O 4 @PEG@DAAAIL-MSPE method was successfully applied to the real sample analysis with satisfactory results. All of above proves the potential value of Fe 3 O 4 @PEG@DAAAIL-MSPE in the analysis of biomass. Copyright © 2017 Elsevier B.V. All rights reserved.

Purification and Partial Characterization of Trypsin-Specific Proteinase Inhibitors from Pigeonpea Wild Relative Cajanus platycarpus L. (Fabaceae) Active against Gut Proteases of Lepidopteran Pest Helicoverpa armigera

PubMed Central

Swathi, Marri; Mishra, Prashant K.; Lokya, Vadthya; Swaroop, Vanka; Mallikarjuna, Nalini; Dutta-Gupta, Aparna; Padmasree, Kollipara

2016-01-01

Proteinase inhibitors (PIs) are natural defense proteins of plants found to be active against gut proteases of various insects. A pigeonpea wild relative Cajanus platycarpus was identified as a source of resistance against Helicoverpa armigera, a most devastating pest of several crops including pigeonpea. In the light of earlier studies, trypsin-specific PIs (CpPI 63) were purified from mature dry seeds of C. platycarpus (ICPW-63) and characterized their biochemical properties in contributing to H. armigera resistance. CpPI 63 possessed significant H. armigera gut trypsin-like proteinase inhibitor (HGPI) activity than trypsin inhibitor (TI) activity. Analysis of CpPI 63 using two-dimensional (2-D) electrophoresis and matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry revealed that it contained several isoinhibitors and small oligomers with masses ranging between 6 and 58 kDa. The gelatin activity staining studies suggest that these isoinhibitors and oligomers possessed strong inhibitory activity against H. armigera gut trypsin-like proteases (HGPs). The N-terminal sequence of the isoinhibitors (pI 6.6 and pI 5.6) of CpPI 63 exhibited 80% homology with several Kunitz trypsin inhibitors (KTIs) as well as miraculin-like proteins (MLPs). Further, modification of lysine residue(s) lead to 80% loss in both TI and HGPI activities of CpPI 63. In contrast, the TI and HGPI activities of CpPI 63 were stable over a wide range of temperature and pH conditions. The reported results provide a biochemical basis for pod borer resistance in C. platycarpus. PMID:27656149

Methylselenium and Prostate Cancer Apoptosis

DTIC Science & Technology

2007-02-01

adherent cells were collected by gentle trypsinization and were combined with the floaters for pelleting by centrifugation. After gentle lysis of the...Cancer Ther 2006;5(7). July 2006 trypsinization and were combined with the floaters for pelleting by centrifugation. After gentle lysis of the cells with

Modification of host erythrocyte membranes by trypsin and chymotrypsin treatments and effects on the in vitro growth of bovine and equine Babesia parasites.

PubMed

Okamura, Masashi; Yokoyama, Naoaki; Takabatake, Noriyuki; Okubo, Kazuhiro; Ikehara, Yuzuru; Igarashi, Ikuo

2007-02-01

In the present study, we investigated the effects of protease pretreatments of host erythrocytes (RBC) on the in vitro growth of bovine Babesia parasites (Babesia bovis and B. bigemina) and equine Babesia parasites (B. equi and B. caballi). The selected proteases, trypsin and chymotrypsin, clearly modified several membrane proteins of both bovine and equine RBC, as demonstrated by SDS-PAGE analysis; however, the protease treatments also modified the sialic acid content exclusively in bovine RBC, as demonstrated by lectin blot analysis. An in vitro growth assay using the protease-treated RBC showed that the trypsin-treated bovine RBC, but not the chymotrypsin-treated ones, significantly reduced the growth of B. bovis and B. bigemina as compared to the control. In contrast, the growth of B. equi and B. caballi was not affected by any of these proteases. Thus, the bovine, but not the equine, Babesia parasites require the trypsin-sensitive membrane (sialoglyco) proteins to infect the RBC.

Biopolymers conjugated with magnetite as support materials for trypsin immobilization and protein digestion.

PubMed

Zdarta, Jakub; Antecka, Katarzyna; Jędrzak, Artur; Synoradzki, Karol; Łuczak, Magdalena; Jesionowski, Teofil

2018-05-08

In the presented study synthesized magnetic nanoparticles were used as an inorganic precursor for the preparation of novel magnetite-lignin and magnetite-chitin hybrid supports for enzyme immobilization. Effective synthesis of the hybrids was confirmed by Fourier transform infrared spectroscopy and powder X-ray diffraction analysis. The materials exhibited good thermal stability and surface areas of 4.3 and 5.6 m 2 /g respectively. The magnetite-lignin + trypsin and magnetite-chitin + trypsin systems were found to have good storage stability and reusability. After 20 days they retained over 75% and 90% respectively of their initial activity, and after 10 consecutive biocatalytic cycles retained over 60% and 80% respectively of their initial activity. The kinetic parameters of the free and immobilized enzyme were also comprehensively examined and compared. The results of peptide digestion tests confirmed the high proteolytic activity of the produced trypsin-based magnetic biocatalytic systems. Copyright © 2018 Elsevier B.V. All rights reserved.

Pretreatment of flaxseed protein isolate by high hydrostatic pressure: Impacts on protein structure, enzymatic hydrolysis and final hydrolysate antioxidant capacities.

PubMed

Perreault, Véronique; Hénaux, Loïc; Bazinet, Laurent; Doyen, Alain

2017-04-15

The effect of high hydrostatic pressure (HHP) on flaxseed protein structure and peptide profiles, obtained after protein hydrolysis, was investigated. Isolated flaxseed protein (1%, m/v) was subjected to HHP (600MPa, 5min or 20min at 20°C) prior to hydrolysis with trypsin only and trypsin-pronase. The results demonstrated that HHP treatment induced dissociation of flaxseed proteins and generated higher molecular weight aggregates as a function of processing duration. Fluorescence spectroscopy showed that HHP treatment, as well as processing duration, had an impact on flaxseed protein structure since exposition of hydrophobic amino acid tyrosine was modified. Except for some specific peptides, the concentrations of which were modified, similar peptide profiles were obtained after hydrolysis of pressure-treated proteins using trypsin. Finally, hydrolysates obtained using trypsin-pronase had a greater antioxidant capacity (ORAC) than control samples; these results confirmed that HHP enhanced the generation of antioxidant peptides. Copyright © 2016 Elsevier Ltd. All rights reserved.

Fractionation and identification of novel antioxidant peptides from buffalo and bovine casein hydrolysates.

PubMed

Shazly, Ahmed Behdal; He, Zhiyong; El-Aziz, Mahmoud Abd; Zeng, Maomao; Zhang, Shuang; Qin, Fang; Chen, Jie

2017-10-01

Buffalo and bovine caseins were hydrolysed by alcalase and trypsin to produce novel antioxidant peptides. The casein hydrolysates were purified using ultrafiltration (UF) and further characterized by RP-HPLC. The fractions produced higher antioxidant activities were identified for their peptides using LC MS/MS. All UF-VI (MW<1kDa) fractions showed higher antioxidant activity. Hydrolysate produced by alcalase for buffalo casein (UF-VI with 54.84-fold purification) showed higher antioxidant activity than that obtained by trypsin. Trypsin hydrolysate contained high amount of hydrophobic amino acids while alcalase hydrolysate consisted mainly of Ser, Arg, Ala and Leu. The antioxidant peptides identified by LC MS/MS were RELEE, MEDNKQ and TVA, EQL in buffalo casein hydrolysates produced by trypsin and alcalase, respectively. Mechanism and reaction pathways of selected antioxidant peptides with ABTS were proposed. Conclusively, buffalo casein provided antioxidant peptides similar to bovine, suggesting that buffalo casein is a novel source of antioxidant. Copyright © 2017 Elsevier Ltd. All rights reserved.

Trypsin-dependent production of an antibacterial substance by a human Peptostreptococcus strain in gnotobiotic rats and in vitro.

PubMed

Ramare, F; Nicoli, J; Dabard, J; Corring, T; Ladire, M; Gueugneau, A M; Raibaud, P

1993-09-01

An antibacterial substance appeared within 1 day in feces of gnotobiotic rats harboring a human intestinal Peptostreptococcus strain. It disappeared when the rat bile-pancreatic duct was ligatured or when the rats ingested a trypsin inhibitor. Anaerobic cultures of the Peptostreptococcus strain in a medium supplemented with trypsin also exhibited an antibacterial activity, which was also inhibited by the trypsin inhibitor. In vitro the antibacterial substance from both feces and culture medium was active against several gram-positive bacteria, including other Peptostreptococcus spp., potentially pathogenic Clostridium spp. such as C. perfringens, C. difficile, C. butyricum, C. septicum, and C. sordellii, Eubacterium spp., Bifidobacterium spp., and Bacillus spp. Whatever the order of inoculation of the strains, a sensitive strain of C. perfringens was eliminated within 1 day from the intestine of rats monoassociated with the Peptostreptococcus strain. These findings demonstrate for the first time that very potent antibacterial substances can be produced through a mechanism involving intestinal bacteria and exocrine pancreatic secretions.

Comparison of polymer induced and solvent induced trypsin denaturation: the role of hydrophobicity.

PubMed

Jasti, Lakshmi S; Fadnavis, Nitin W; Addepally, Uma; Daniels, Siona; Deokar, Sarika; Ponrathnam, Surendra

2014-04-01

Trypsin adsorption from aqueous buffer by various copolymers of allyl glycidyl ether-ethylene glycol dimethacrylate (AGE-EGDM) copolymer with varying crosslink density increases with increasing crosslink density and the effect slowly wears off after reaching a plateau at 50% crosslink density. The copolymer with 25% crosslink density was reacted with different amines with alkyl/aryl side chains to obtain a series of copolymers with 1,2-amino alcohol functional groups and varying hydrophobicity. Trypsin binding capacity again increases with hydrophobicity of the reacting amine and a good correlation between logPoctanol of the amine and protein binding is observed. The bound trypsin is denatured to the extent of 90% in spite of the presence of hydrophilic hydroxyl and amino groups. The behavior was comparable to that in mixtures of aqueous buffer and water-miscible organic co-solvents where the solvent concentration required to deactivate 50% of the enzyme (C50) is dependent on logPoctanol of the co-solvent. Copyright © 2014 Elsevier B.V. All rights reserved.

Fresh and aged human lymphocyte metaphase slides are equally usable for GTG banding.

PubMed

Sajjad, Naheed; Haque, Sayedul; SBurney, Syed Intesar; Shahid, Syed Muhammad; Zehra, Sitwat; Azhar, Abid

2014-09-01

The identification of chromosomes for routine cytogenetic analysis is based on quality of metaphases and good banding pattern. Fresh slides of human lymphocytes have been shown to produce good bands for the identification of chromosomes morphology. G-bands by Trypsin using Giemsa (GTG) banding of aged slides is generally considered hard to get desired band pattern of chromosomes persistently. The current study is focused on GTG banding of aged slides. A total of 340 subjects including 290 primary infertile and 50 fertile were selected. The blood samples were drawn aseptically for cytogenetic analysis. Lymphocytes were cultured and GTG banding was done on 1440 glass slides. Giemsa trypsin banding of aged slides were done by adjusting average trypsin time for each month according to the slide age and metaphase concentration. Correlation analyses showed a significant and positive correlation between slide ageing and trypsin pre-treatment time. The results of this study suggest that, the fresh and aged human lymphocyte metaphases are equally usable for GTG banding.

Medullary thyroid carcinoma: ectopic production of peptides with ACTH-like, corticotrophin releasing factor-like and prolactin production-stimulating activities.

PubMed

Birkenhäger, J C; Upton, G V; Seldenrath, H J; Krieger, D T; Tashjian, A H

1976-10-01

A 45-year-old women had medullary tyroid carcinoma associated with Cushing's syndrome and galactorrhoea. Elevated plasma immunoreactive ACTH and cortisol were partially suppressed by intravenous dexamethasone, appreciably raised by lysine vasopressin, and urinary excretion of 17-oxogenic steroids slightly elevated by metyrapone. A large arterio-venous increase in plasma corticotrophin releasing factor-like activity across the thyroid gland was observed and tumour tissue contained corticotrophin releasing factor-like activity. Biologically active ACTH was not detected in tumour extracts before incubation with trypsin, but after trypsinization a value of 3.2 mU per gram was obtained. Arterial plasma contained biologically active ACTH (1.5 mU/100 ml) prior to trypsinization. Venous effluent from the thyroid gland contained biologically active (9.6 mU/100 ml) and immunoreactive ACTH (970 pg/ml) before trypsinization. Tumour extracts also contained prolactin production-stimulating activity. These findings can explain the Cushing's syndrome and the galactorrhoea both of which disappeared completely after thyroidectomy.

Dual core quantum dots for highly quantitative ratiometric detection of trypsin activity in cystic fibrosis patients

NASA Astrophysics Data System (ADS)

Castelló Serrano, Iván; Stoica, Georgiana; Matas Adams, Alba; Palomares, Emilio

2014-10-01

We present herein two colour encoded silica nanospheres (2nanoSi) for the fluorescence quantitative ratiometric determination of trypsin in humans. Current detection methods for cystic fibrosis diagnosis are slow, costly and suffer from false positives. The 2nanoSi proved to be a highly sensitive, fast (minutes), and single-step approach nanosensor for the screening and diagnosis of cystic fibrosis, allowing the quantification of trypsin concentrations in a wide range relevant for clinical applications (25-350 μg L-1). Furthermore, as trypsin is directly related to the development of cystic fibrosis (CF), different human genotypes, i.e. CF homozygotic, CF heterozygotic, and unaffected, respectively, can be determined using our 2nanoSi nanospheres. We anticipate the 2nanoSi system to be a starting point for non-invasive, easy-to-use and cost effective ratiometric fluorescent biomarkers for recessive genetic diseases like human cystic fibrosis. In a screening program in which the goal is to detect disease and also the carrier status, early diagnosis could be of great help.We present herein two colour encoded silica nanospheres (2nanoSi) for the fluorescence quantitative ratiometric determination of trypsin in humans. Current detection methods for cystic fibrosis diagnosis are slow, costly and suffer from false positives. The 2nanoSi proved to be a highly sensitive, fast (minutes), and single-step approach nanosensor for the screening and diagnosis of cystic fibrosis, allowing the quantification of trypsin concentrations in a wide range relevant for clinical applications (25-350 μg L-1). Furthermore, as trypsin is directly related to the development of cystic fibrosis (CF), different human genotypes, i.e. CF homozygotic, CF heterozygotic, and unaffected, respectively, can be determined using our 2nanoSi nanospheres. We anticipate the 2nanoSi system to be a starting point for non-invasive, easy-to-use and cost effective ratiometric fluorescent biomarkers for recessive genetic diseases like human cystic fibrosis. In a screening program in which the goal is to detect disease and also the carrier status, early diagnosis could be of great help. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr03952a

Hypotrypsinaemia in diabetes mellitus.

PubMed

Adrian, T E; Barnes, A J; Bloom, S R

1979-10-01

Plasma trypsin concentrations were measured in 403 fasting diabetics and 106 healthy controls. Basal trypsin concentrations in the normal subjects were 88 +/- 6 ng/ml (mean +/- S.E.M.). Mean plasma trypsin concentrations in diabetics treated with diet alone (n = 74) were 45 +/- 2 ng/ml, while in a group of young (less than 35 years, n = 88) insulin-dependent diabetics, they were very low at 29 +/- 2 ng/ml and these levels were inversely related to insulin dosage. The findings may help in the understanding of the pathophysiological changes in the exocrine pancreas in the diabetic state and may also shed some light on the physiological interrelationship between the endocrine and exocrine pancreas.

Trypsin level in gallbladder bile and ductitis and width of the cystic duct.

PubMed

Vracko, J; Wiechel, K L

2000-01-01

The change from laparotomy to laparoscopy for cholecystectomy has raised the question of how to manage concomitant bile duct stones. The present-day interest--and controversy--has focused on a transcystic approach reported to be feasible in 66-96% of cases, but without explaining the necessary prerequisite: the widening of the cystic duct. The cystic duct, wide mainly in patients with bile duct stones, has been reported to be highly variable: from strictured to very wide. The present study aims at comparing the trypsin level in the gallbladder bile and the cystic duct morphology and width in patients with and without bile duct stones. A prospective series of 63 gallstone patients, 30 with and 33 without bile duct stones (controls), underwent cholecystectomy and bile duct clearance. The study includes the trypsin level in the gallbladder bile, the width and morphology of the cystic duct, and the size of the gallstones. The patients with bile duct stones had, in contrast to the controls, higher trypsin levels in the gallbladder bile (P < 0.001) and wider cystic ducts (P < 0.001) with more pronounced signs of chronic ductitis. The obtained results strongly suggest that the increased trypsin level, a sign of reflux of pancreatic juice, caused changes in the cystic duct that facilitate gallstone migration, which also ought to render a transcystic stone extraction feasible.

Optimising the decellularization of human elastic cartilage with trypsin for future use in ear reconstruction.

PubMed

Rahman, Shafiq; Griffin, Michelle; Naik, Anish; Szarko, Matthew; Butler, Peter E M

2018-02-15

Decellularized scaffolds can induce chondrogenic differentiation of stem cells. This study compares different methods to optimise the decellularization of auricular cartilage. The process consisted of an initial 12 hour dry freeze thaw which froze the cartilage specimens in an empty tube at -20 °C. Samples were allowed to thaw at room temperature followed by submersion in phosphate buffer solution in which they were frozen at -20 °C for a 12 hour period. They were then allowed to thaw at room temperature as before. Protocol A subsequently involved subjecting specimens to both deoxyribonuclease and sodium deoxycholate. Protocol B and C were adaptations of this using 0.25% trypsin (7 cycles) and a 0.5 molar solution of ethylenediaminetetraacetic acid (3 hours for each cycle) respectively as additional steps. Trypsin accelerated the decellularization process with a reduction in DNA content from 55.4 ng/μL (native) to 17.3 ng/μL (P-value < 0.05) after 14 days. Protocol B showed a faster reduction in DNA content when compared with protocol A. In comparison to protocol C after 14 days, trypsin also showed greater decellularization with a mean difference of 11.7 ng/μL (P-value < 0.05). Histological analysis with H&E and DAPI confirmed depletion of cells at 14 days with trypsin.

Proteases induce secretion of collagenase and plasminogen activator by fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Werb, Z.; Aggeler, J.

1978-04-01

We have observed that treatment of rabbit synovial fibroblasts with proteolytic enzymes can induce secretion of collagenase (EC 3.4.24.7) and plasminogen activator (EC 3.4.21.-). Cells treated for 2 to 24 hr with plasmin, trypsin, chymotrypsin, pancreatic elastase, papain, bromelain, thermolysin, or ..cap alpha..-protease but not with thrombin or neuraminidase secreted detectable amounts of collagenase within 16 to 48 hr. Treatment of fibroblasts with trypsin also induced secretion of plasminogen activator. Proteases initiated secretion of collagenase (up to 20 units per 10/sup 6/ cells per 24 hr) only when treatment produced decreased cell adhesion. Collagenase production did not depend on continuedmore » presence of proteolytic activity or on subsequent cell adhesion, spreading, or proliferation. Routine subculturing with crude trypsin also induced collagenase secretion by cells. Secretion of collagenase was prevented and normal spreading was obtained if the trypsinized cells were placed into medium containing fetal calf serum. Soybean trypsin inhibitor, ..cap alpha../sub 1/-antitrypsin, bovine serum albumin, collagen, and fibronectin did not inhibit collagenase production. Although proteases that induced collagenase secretion also removed surface glycoprotein, the kinetics of induction of cell protease secretion were different from those for removal of fibronectin. Physiological inducers of secretion of collagenase and plasminogen activator by cells have not been identified. These results suggest that extracellular proteases in conjunction with plasma proteins may govern protease secretion by cells.« less

Gene analysis and structure prediction for the cold-adaption mechanism of trypsin from the krill Euphausia superba (Dana, 1852).

PubMed

Zhou, Tingting; Wang, Xichang; Yan, Juan; Li, Yan

2018-06-01

The ability of Antarctic krill, Euphausia superba (Dana, 1852), to thrive in a cold environment comes from its capacity to synthesize cold-adapted enzymes. Its trypsin, as a main substance in the metabolic reactions, plays a key role in the adaption to low temperatures. However, the progress of research on its cold-adaption mechanism is being influenced due to the limited information on its gene and spatial structure. We studied the gene of E. superba trypsin with transcriptome sequencing first, and then discussed its cold-adaption mechanism with the full gene and predicted structure basing on bioinformatics. The results showed the proportion of certain residues played important roles in the cold-adaptation behavior for trypsin. Furthermore, a higher proportion of random coils and reduced steric hindrance might also be key factors promoting its cold adaption. This research aimed to reveal the cold-adaption mechanism of E. superba trypsin and provide support for basic research on molecular modification by site-directed mutagenesis of complementary DNA used to produce new and improved recombinant variants with cold adaption. Furthermore, it may broaden its commercial application on minimizing undesirable changes elevated at higher temperature in food processing and in treatment of trauma and inflammation in medicine. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Cloning, genetic engineering and characterization of TMOF expressed in Saccharomyces cerevisiae to control larval mosquitoes

USDA-ARS?s Scientific Manuscript database

Trypsin modulating oostatic factor (TMOF), a decapaptide isolated from the ovaries of Aedes aegypti, is the physiological factor that terminates the trypsin biosynthesis after the blood meal. Earlier results obtained from feeding mosquito larvae and injecting female mosquitoes with TMOF show that tr...

Establishment and optimization of NMR-based cell metabonomics study protocols for neonatal Sprague-Dawley rat cardiomyocytes.

PubMed

Zhang, Ming; Sun, Bo; Zhang, Qi; Gao, Rong; Liu, Qiao; Dong, Fangting; Fang, Haiqin; Peng, Shuangqing; Li, Famei; Yan, Xianzhong

2017-01-15

A quenching, harvesting, and extraction protocol was optimized for cardiomyocytes NMR metabonomics analysis in this study. Trypsin treatment and direct scraping cells in acetonitrile were compared for sample harvesting. The results showed trypsin treatment cause normalized concentration increasing of phosphocholine and metabolites leakage, since the trypsin-induced membrane broken and long term harvesting procedures. Then the intracellular metabolite extraction efficiency of methanol and acetonitrile were compared. As a result, washing twice with phosphate buffer, direct scraping cells and extracting with acetonitrile were chosen to prepare cardiomyocytes extracts samples for metabonomics studies. This optimized protocol is rapid, effective, and exhibits greater metabolite retention. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of a new soybean Kunitz trypsin inhibitor mutation and its effect on Bowman-Birk protease inhibitor content in soybean seed

USDA-ARS?s Scientific Manuscript database

Soybean seeds possess anti-nutritional compounds which inactivate digestive proteases, principally corresponding to two families: Kunitz Trypsin Inhibitors (KTi) and Bowman-Birk Inhibitors (BBI). High levels of raw soybeans/soybean meal in feed mixtures can cause poor weight gain and pancreatic abno...

Kunitz trypsin inhibitor in addition to Bowman-Birk inhibitor influence stability of lunasin against pepsin-pancreatin hydrolysis

USDA-ARS?s Scientific Manuscript database

Soybean contains several biologically active components and one of this belongs to the bioactive peptide group. The objectives of this study were to produce different lunasin-enriched preparations (LEP) and determine the effect of Bowman-Birk inhibitor and Kunitz trypsin concentrations on the stabil...

ACE Inhibitory and Antioxidant Activities of Collagen Hydrolysates from the Ribbon Jellyfish (Chrysaora sp.).

PubMed

Barzideh, Zoha; Latiff, Aishah Abd; Gan, Chee-Yuen; Abedin, Md Zainul; Alias, Abd Karim

2014-12-01

Collagen isolated from the ribbon jellyfish ( Chrysaora sp.) was hydrolysed using three different proteases ( i.e. trypsin, alcalase and Protamex) to obtain bioactive peptides. Angiotensin-I-converting enzyme (ACE) inhibitory activity and antioxidant activities ( i.e. ferric reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity) of the peptides were measured and compared, and the effect of the duration of hydrolysis on the bioactivity (ACE inhibitory and antioxidant activities) of peptides was also evaluated. FRAP activity was the highest in Protamex-induced (25-27 mM) and trypsin-induced hydrolysates (24-26 mM) at 7 and 9 h, respectively. Conversely, hydrolysates produced by trypsin for 1 and 3 h showed the highest DPPH radical scavenging activities (94 and 92%, respectively). Trypsin-induced hydrolysates (at 3 h) also showed the highest ACE inhibitory activity (89%). The peptide sequences with the highest activities were identified using tandem mass spectrometry, and the results show that the hydrolysates had a high content of hydrophobic amino acids as well as unique amino acid sequences, which likely contribute to their biological activities.

A diverse family of serine proteinase genes expressed in cotton boll weevil (Anthonomus grandis): implications for the design of pest-resistant transgenic cotton plants.

PubMed

Oliveira-Neto, Osmundo B; Batista, João A N; Rigden, Daniel J; Fragoso, Rodrigo R; Silva, Rodrigo O; Gomes, Eliane A; Franco, Octávio L; Dias, Simoni C; Cordeiro, Célia M T; Monnerat, Rose G; Grossi-De-Sá, Maria F

2004-09-01

Fourteen different cDNA fragments encoding serine proteinases were isolated by reverse transcription-PCR from cotton boll weevil (Anthonomus grandis) larvae. A large diversity between the sequences was observed, with a mean pairwise identity of 22% in the amino acid sequence. The cDNAs encompassed 11 trypsin-like sequences classifiable into three families and three chymotrypsin-like sequences belonging to a single family. Using a combination of 5' and 3' RACE, the full-length sequence was obtained for five of the cDNAs, named Agser2, Agser5, Agser6, Agser10 and Agser21. The encoded proteins included amino acid sequence motifs of serine proteinase active sites, conserved cysteine residues, and both zymogen activation and signal peptides. Southern blotting analysis suggested that one or two copies of these serine proteinase genes exist in the A. grandis genome. Northern blotting analysis of Agser2 and Agser5 showed that for both genes, expression is induced upon feeding and is concentrated in the gut of larvae and adult insects. Reverse northern analysis of the 14 cDNA fragments showed that only two trypsin-like and two chymotrypsin-like were expressed at detectable levels. Under the effect of the serine proteinase inhibitors soybean Kunitz trypsin inhibitor and black-eyed pea trypsin/chymotrypsin inhibitor, expression of one of the trypsin-like sequences was upregulated while expression of the two chymotrypsin-like sequences was downregulated. Copyright 2004 Elsevier Ltd.

Evaluation of hyaluronic acid-protein conjugates for polymer masked-unmasked protein therapy.

PubMed

Ferguson, Elaine L; Alshame, Alshame M J; Thomas, David W

2010-12-15

Bioresponsive polymers may effectively be utilized to enhance the circulation time and stability of biologically active proteins and peptides, while reducing their immunogenicity and toxicity. Recently, dextrin-epidermal growth factor (EGF) conjugates, which make use of the Polymer-masked UnMasked Protein Therapy (PUMPT) concept, have been developed and shown potential as modulators of impaired wound healing. This study investigated the potential of PUMPT using hyaluronic acid (HA) conjugates to mask activity and enhance protein stability, while allowing restoration of biological activity following triggered degradation. HA fragments (Mw ∼90,000g/mol), obtained by acid hydrolysis of Rooster comb HA, were conjugated to trypsin as a model enzyme or to EGF as a model growth factor. Conjugates contained 2.45 and 0.98% (w/w) trypsin or EGF, respectively, and contained <5% free protein. HA conjugation did not significantly alter trypsin's activity. However, incubation of the conjugate with physiological concentrations of HAase increased its activity to ∼145% (p<0.001) that of the free enzyme. In contrast, when HA-EGF conjugates were tested in vitro, no effect on cell proliferation was seen, even in the presence of HAase. HA conjugates did not display typical masking/unmasking behavior, HA-trypsin conjugates exhibited ∼52% greater stability in the presence of elastase, compared to free trypsin, demonstrating the potential of HA conjugates for further development as modulators of tissue repair. Copyright © 2010 Elsevier B.V. All rights reserved.

Alternative Enzymes Lead to Improvements in Sequence Coverage and PTM Analysis

PubMed Central

Hooper, Kyle; Rosenblatt, Michael; Urh, Marjeta; Saveliev, Sergei; Hosfield, Chris; Kobs, Gary; Ford, Michael; Jones, Richard; Amunugama, Ravi; Allen, David; Brazas, Robert

2013-01-01

The profiling of proteins using biological mass spectrometry (bottom up proteomics) most commonly requires trypsin. Trypsin is advantageous in that it produces peptides of optimal charge and size. However, for applications in which the proteins under investigation are part of a complex mixture or not isolated at high levels (i.e. low ng from an immunoprecipitation), sequence coverage is rarely complete. In addition, we have found that in several cases, like phosphorylation, acetylation, and methylation, alternative proteases are required to prepare peptides suitable for MS detection. This poster will provide specific examples which demonstrate this observation. For example, the application of a combined Trypsin/ Lys-C mixture reduces the number of missed cleavages by more than 3-fold producing samples with lower CV's (for biological replicates). The mixture is also well-suited for the complete proteolysis of hydrophobic, compact proteins. The addition of chymotrypsin and elastase has been found to be useful for identifying phosphorylation sites on proteins, especially on sequences where the site of phosphorylation inhibits trypsin (i.e. proximal to K or R). Many epigenetic applications have focused on histone modifications, like lysine acetylation and arginine methylation. Alternative proteases like Asp-N, Glu-C, and chymotrypsin have been especially useful given the fact that the modified K and R residues are resistant to c-terminal cleavage by trypsin. Finally, in the case of serum profiling, the addition of the endoglycosidase, PNGase F has been found to improve sequence coverage due to the removal of N-linked glycans.

Magnetic nanoparticles coated with polyaniline to stabilize immobilized trypsin

NASA Astrophysics Data System (ADS)

Maciel, J. C.; D. Mercês, A. A.; Cabrera, M.; Shigeyosi, W. T.; de Souza, S. D.; Olzon-Dionysio, M.; Fabris, J. D.; Cardoso, C. A.; Neri, D. F. M.; C. Silva, M. P.; Carvalho, L. B.

2016-12-01

It is reported the synthesis of magnetic nanoparticles via the chemical co-precipitation of Fe 3+ ions and their preparation by coating them with polyaniline. The electronic micrograph analysis showed that the mean diameter for the nanoparticles is ˜15 nm. FTIR, powder X-ray diffraction and Mössbauer spectroscopy were used to understand the chemical, crystallographic and 57Fe hyperfine structures for the two samples. The nanoparticles, which exhibited magnetic behavior with relatively high spontaneous magnetization at room temperature, were identified as being mainly formed by maghemite ( γFe2O3). The coated magnetic nanoparticles (sample labeled "mPANI") presented a real ability to bind biological molecules such as trypsin, forming the magnetic enzyme derivative (sample "mPANIG-Trypsin"). The amount of protein and specific activity of the immobilized trypsin were found to be 13±5 μg of protein/mg of mPANI (49.3 % of immobilized protein) and 24.1±0.7 U/mg of immobilized protein, respectively. After 48 days of storage at 4 ∘C, the activity of the immobilized trypsin was found to be 89 % of its initial activity. This simple, fast and low-cost procedure was revealed to be a promising way to prepare mPANI nanoparticles if technological applications addressed to covalently link biomolecules are envisaged. This route yields chemically stable derivatives, which can be easily recovered from the reaction mixture with a magnetic field and recyclable reused.

A colostrum trypsin inhibitor gene expressed in the Cape fur seal mammary gland during lactation.

PubMed

Pharo, Elizabeth A; Cane, Kylie N; McCoey, Julia; Buckle, Ashley M; Oosthuizen, W H; Guinet, Christophe; Arnould, John P Y

2016-03-01

The colostrum trypsin inhibitor (CTI) gene and transcript were cloned from the Cape fur seal mammary gland and CTI identified by in silico analysis of the Pacific walrus and polar bear genomes (Order Carnivora), and in marine and terrestrial mammals of the Orders Cetartiodactyla (yak, whales, camel) and Perissodactyla (white rhinoceros). Unexpectedly, Weddell seal CTI was predicted to be a pseudogene. Cape fur seal CTI was expressed in the mammary gland of a pregnant multiparous seal, but not in a seal in its first pregnancy. While bovine CTI is expressed for 24-48 h postpartum (pp) and secreted in colostrum only, Cape fur seal CTI was detected for at least 2-3 months pp while the mother was suckling its young on-shore. Furthermore, CTI was expressed in the mammary gland of only one of the lactating seals that was foraging at-sea. The expression of β-casein (CSN2) and β-lactoglobulin II (LGB2), but not CTI in the second lactating seal foraging at-sea suggested that CTI may be intermittently expressed during lactation. Cape fur seal and walrus CTI encode putative small, secreted, N-glycosylated proteins with a single Kunitz/bovine pancreatic trypsin inhibitor (BPTI) domain indicative of serine protease inhibition. Mature Cape fur seal CTI shares 92% sequence identity with Pacific walrus CTI, but only 35% identity with BPTI. Structural homology modelling of Cape fur seal CTI and Pacific walrus trypsin based on the model of the second Kunitz domain of human tissue factor pathway inhibitor (TFPI) and porcine trypsin (Protein Data Bank: 1TFX) confirmed that CTI inhibits trypsin in a canonical fashion. Therefore, pinniped CTI may be critical for preventing the proteolytic degradation of immunoglobulins that are passively transferred from mother to young via colostrum and milk. Copyright © 2015 Elsevier B.V. All rights reserved.

Characterization of a backbone cleavage product of BMS-196854 (Oncostatin M), a recombinant anti-inflammatory cytokine.

PubMed

Zhao, F; Stein, D J; Paborji, M; Cash, P W; Root, B J; Wei, Z; Knupp, C J

2001-01-01

BMS-196843 (Oncostatin M) is a therapeutic recombinant protein in development. Scale-up process changes led to unexpected instability of the bulk drug substance solution during storage. A product with an apparent higher MW than the parent protein was observed by the size-exclusion chromatography (SEC). This study was aimed to fully characterize the product and to identify a solution to stabilize the protein. SEC, SDS-PAGE, tryptic mapping, and N-terminal sequencing were performed to characterize the unknown product. The effect of pH, temperature, bulk concentration, and immobilized trypsin inhibitor on the degradation rate was studied to elucidate the mechanism and to identify stabilization strategies. Despite the apparent high MW indicated initially by SEC, the unknown was characterized to be a degradation product resulted from a backbone cleavage between residues Arg145-Gly146. The resulting fragments from the backbone cleavage were, however, still linked through an intramolecular disulfide bond. Thus, the final product had a more open structure with an increased hydrodynamic radius compared to the parent protein, which explains the initial SEC results. The site-specific backbone cleavage was suspected to be catalyzed by trypsin-like protease impurities in the bulk solution. The bulk drug substance solution was subsequently treated with immobilized soybean trypsin inhibitor, and the degradation rate was significantly reduced. Furthermore, increasing the solution pH from 5 to 8 led to an increase in the degradation rate, which was consistent with the expected pH dependency of trypsin activity. In addition, the effect of bulk concentration also supported the involvement of protease impurities rather than a spontaneous peptide bond hydrolysis reaction. Trace trypsin-like protease impurities led to an unusual site-specific backbone cleavage of BMS-196854. The proteolytic degradation can be minimized by treating the bulk solution with immobilized soybean trypsin inhibitor and/or controlling the solution pH and storage temperature.

[Effect of ERK/AP-1 signaling pathway on proliferation of hepatoma cells induced by PAR-2 agonists].

PubMed

Zheng, Yan-min; Xie, Li-qun; Li, Xuan; Zhao, Jun-yan; Chen, Xiao-yi; Chen, Li; Zhou, Jing; Li, Fei

2009-12-01

To investigate the expression of protease activated receptor-2 (PAR-2) in human HepG2 hepatoma cells and elucidate the effects of trypsin and PAR-2 agonist peptide SLIGKV-NH(2) upon the proliferation of hepatoma cells and its intracellular signaling mechanism. PAR-2 protein and mRNA expression were detected by immunofluorescence and RT-PCR. The cells were treated with SLIGKV-NH(2), trypsin, reverse PAR-2 agonist peptide VKGILS-NH(2) or PD98059. The changes of cell cycle distribution were evaluated by flow cytometry. The proliferative potential of HepG2 cells was estimated by MTT. The changes of PAR-2, c-fos and PCNA mRNA expression were detected by RT-PCR. The changes of c-fos and PCNA protein expression were detected by Western blotting. PAR-2 protein and mRNA were expressed in HepG2 cells. PAR-2 mRNA expression (PAR-2/beta-actin) were 0.70 +/- 0.04 and 0.99 +/- 0.05 respectively in cells treated with trypsin and SLIGKV-NH(2). They were both significantly higher than that in the control group (0.35 +/- 0.05, F = 135.534, P < 0.01). Percent G(0)/G(1) phase of HepG2 cells treated with trypsin or SLIGKV-NH(2) were significantly lower than those in the control group [(56.11 +/- 0.85)%, (57.85 +/- 0.46)% vs (79.12 +/- 0.67)%, both P < 0.01] Percent S phase, G(2)/M phase and proliferation index (PI) of HepG2 cells treated with trypsin or SLIGKV-NH(2) were significantly elevated (P < 0.01). The proliferation-enhancing effects and the up-regulation of mRNA and protein of c-fos and PCNA induced by trypsin or SLIGKV-NH(2) were significantly blocked by pretreatment with PD98059 (P < 0.01). There was no statistical significance in proliferation of HepG2 cells between the reverse PAR-2 agonist peptide VKGILS-NH(2) and control group (P > 0.05). PAR-2 is expressed in HepG2 hepatoma cells. PAR-2 activation induced by trypsin or SLIGKV-NH(2) promotes the proliferation of HepG2 cells partially via the ERK/AP-1 pathway.

The midgut transcriptome of Phlebotomus (Larroussius) perniciosus, a vector of Leishmania infantum: comparison of sugar fed and blood fed sand flies.

PubMed

Dostálová, Anna; Votýpka, Jan; Favreau, Amanda J; Barbian, Kent D; Volf, Petr; Valenzuela, Jesus G; Jochim, Ryan C

2011-05-10

Parasite-vector interactions are fundamental in the transmission of vector-borne diseases such as leishmaniasis. Leishmania development in the vector sand fly is confined to the digestive tract, where sand fly midgut molecules interact with the parasites. In this work we sequenced and analyzed two midgut-specific cDNA libraries from sugar fed and blood fed female Phlebotomus perniciosus and compared the transcript expression profiles. A total of 4111 high quality sequences were obtained from the two libraries and assembled into 370 contigs and 1085 singletons. Molecules with putative roles in blood meal digestion, peritrophic matrix formation, immunity and response to oxidative stress were identified, including proteins that were not previously reported in sand flies. These molecules were evaluated relative to other published sand fly transcripts. Comparative analysis of the two libraries revealed transcripts differentially expressed in response to blood feeding. Molecules up regulated by blood feeding include a putative peritrophin (PperPer1), two chymotrypsin-like proteins (PperChym1 and PperChym2), a putative trypsin (PperTryp3) and four putative microvillar proteins (PperMVP1, 2, 4 and 5). Additionally, several transcripts were more abundant in the sugar fed midgut, such as two putative trypsins (PperTryp1 and PperTryp2), a chymotrypsin (PperChym3) and a microvillar protein (PperMVP3). We performed a detailed temporal expression profile analysis of the putative trypsin transcripts using qPCR and confirmed the expression of blood-induced and blood-repressed trypsins. Trypsin expression was measured in Leishmania infantum-infected and uninfected sand flies, which identified the L. infantum-induced down regulation of PperTryp3 at 24 hours post-blood meal. This midgut tissue-specific transcriptome provides insight into the molecules expressed in the midgut of P. perniciosus, an important vector of visceral leishmaniasis in the Old World. Through the comparative analysis of the libraries we identified molecules differentially expressed during blood meal digestion. Additionally, this study provides a detailed comparison to transcripts of other sand flies. Moreover, our analysis of putative trypsins demonstrated that L. infantum infection can reduce the transcript abundance of trypsin PperTryp3 in the midgut of P. perniciosus.

Characterization of cDNAs encoding serine proteases and their transcriptional responses to Cry1Ab protoxin in the gut of Ostrinia nubilalis larvae

USDA-ARS?s Scientific Manuscript database

Serine proteases, such as trypsin and chymotrypsin, are the primary digestive enzymes in lepidopteran larvae, and are also involved in Bacillus thuringiensis (Bt) protoxin activation and protoxin/toxin degradation. We isolated and sequenced 34 cDNAs putatively encoding trypsins, chymotrypsins and th...

Influence of surface-imprinted nanoparticles on trypsin activity.

PubMed

Guerreiro, António; Poma, Alessandro; Karim, Kal; Moczko, Ewa; Takarada, Jessica; de Vargas-Sansalvador, Isabel Perez; Turner, Nicholas; Piletska, Elena; de Magalhães, Cristiana Schmidt; Glazova, Natalia; Serkova, Anastasia; Omelianova, Aleksandra; Piletsky, Sergey

2014-09-01

Here, the modulation of enzyme activity is presented by protein-imprinted nanoparticles produced using a solid-phase approach. Using trypsin as target, binding of the nanoparticles to the enzyme results in its inhibition or in stabilization, depending on the orientation of the immobilized enzyme used during imprinting. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

21 CFR 184.1914 - Trypsin.

Code of Federal Regulations, 2012 CFR

2012-04-01

... defined in § 170.3(o)(9) of this chapter to hydrolyze proteins or polypeptides. (2) The ingredient is used... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Trypsin. 184.1914 Section 184.1914 Food and Drugs... characterizing enzyme activity is that of a peptide hydrolase (EC 3.4.21.4). (b) The ingredient meets the general...

21 CFR 184.1914 - Trypsin.

Code of Federal Regulations, 2014 CFR

2014-04-01

...: (1) The ingredient is used as an enzyme as defined in § 170.3(o)(9) of this chapter to hydrolyze... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Trypsin. 184.1914 Section 184.1914 Food and Drugs... activity is that of a peptide hydrolase (EC 3.4.21.4). (b) The ingredient meets the general requirements...

21 CFR 184.1914 - Trypsin.

Code of Federal Regulations, 2010 CFR

2010-04-01

... defined in § 170.3(o)(9) of this chapter to hydrolyze proteins or polypeptides. (2) The ingredient is used... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Trypsin. 184.1914 Section 184.1914 Food and Drugs... characterizing enzyme activity is that of a peptide hydrolase (EC 3.4.21.4). (b) The ingredient meets the general...

Use of microwaves to improve nutritional value of soybeans for future space inhabitants

NASA Technical Reports Server (NTRS)

Singh, G.

1983-01-01

Whole soybeans from four different varieties at different moisture contents were microwaved for varying times to determine the conditions for maximum destruction of trypsin inhibitor and lipoxygenase activities, and optimal growth of chicks. Microwaving 150 gm samples of soybeans (at 14 to 28% moisture) for 1.5 min was found optimal for reduction of trypsin inhibitor and lipoxygenase activities. Microwaving 1 kgm samples of soybeans for 9 minutes destroyed 82% of the trypsin inhibitor activity and gave optimal chick growth. It should be pointed out that the microwaving time would vary according to the weight of the sample and the power of the microwave oven. The microwave oven used in the above experiments was rated at 650 watts 2450 MHz.

Methacrylate gels with epoxide groups as supports for immobilization of enzymes in pH range 3-12.

PubMed

Turková, J; Bláha, K; Malaníková, M; Vancurová, D; Svec, F; Kálal, J

1978-05-11

Glycidyl methacrylate gels are carriers suitable for attachment of enzymes and for use in affinity chromatography. Experiments on the coupling of glycyl-L-leucine and acetyl-L-leucine to these gels have shown a high pH-dependence of the bond formation between the support and the alpha-amino group (pH optimum 9.7); the coupling reaction between the epoxide group and the carboxyl group is practically pH-independent. Serum albumin and trypsin were attached to a greater extent in acidic than in alkaline media. The effects of time and temperature were also studied. The catalytic action of immobilized trypsin, as well as its use for affinity chromatography of trypsin inhibitor, were studied.

Oviductal protease and trypsin treatment enhance sperm-envelope interaction in Bufo arenarum coelomic eggs.

PubMed

Llanos, Ricardo J; Barrera, Daniel; Valz-Gianinet, Jorge N; Miceli, Dora C

2006-10-01

We describe the morphological and biochemical changes in Bufo arenarum coelomic egg envelopes (CE) following passage through the oviduct. In this species, the transformation of the CE into the vitelline envelope (VE) leads to the acquisition of fertilizability and involves the cleavage of a glycoprotein component. Electrophoretic patterns indicate that a pars recta oviductal protease selectively hydrolyzes in vitro the 84 and the 55 kDa glycoproteins of the CE. During the CE to VE transformation, the relative concentrations of gp48, 42 and 39 kDa also change. In in vitro tests, sperm binding to envelope glycoprotein occurs when they are exposed to VE but not when treated with CE, and VE labeled glycoproteins bind to the head and mid piece of the sperm. The gp39 VE component has 100% identity with internal domains of the sequence deduced from ovarian cDNA for the homologous zona pellucida glycoprotein type C (ZPC) protein precursor in B. arenarum. The effects of trypsin as a substitute for oviductal protease were also examined. Trypsin selectively attacks the 84 and the 55 kDa glycoproteins without hydrolyzing other components and renders coelomic eggs fertilizable in a jelly water preparation. Therefore, trypsin can mimic in vitro the biological action of the oviductal protease. However, it does not wholly mimic the biological action of the oviduct which, in B. arenarum at least, exceeds a mere proteolytic effect. This fact was verified by the lower fertility rates and the abnormal embryo development found when trypsin-treated coelomic eggs were fertilized in vitro. 2006 Wiley-Liss, Inc.

Clearance of bile and trypsin in rat lungs following aspiration of human gastric fluid

PubMed Central

Leung, Jason H.; Chang, Jui-Chih; Foltz, Emily; Bell, Sadé M.; Pi, Cinthia; Azad, Sassan; Everett, Mary Lou; Holzknecht, Zoie E.; Sanders, Nathan L.; Parker, William; Davis, R. Duane; Keshavjee, Shaf; Lin, Shu S.

2016-01-01

ABSTRACT Purpose: In the clinical setting, there is no reliable tool for diagnosing gastric aspiration. A potential way of diagnosing gastric fluid aspiration entails bronchoalveolar lavage (BAL) with subsequent examination of the BAL fluid for gastric fluid components that are exogenous to the lungs. The objective of this study was to determine the longevity of the gastric fluid components bile and trypsin in the lung, in order to provide an estimate of the time frame in which assessment of these components in the BAL might effectively be used as a measure of aspiration. Materials and Methods: Human gastric fluid (0.5 mg/kg) was infused in the right lung of intubated male Fischer 344 rats (n = 30). Animals were sacrificed at specified times following the experimentally induced aspiration, and bronchoalveolar lavage fluid (BALF) was collected. Bile concentrations were analyzed by an enzyme-linked chromatogenic method, and the concentration of trypsin was quantified using an ELISA. Data were analyzed using non-linear regression and a one-phase decay equation. Results: In this experimental model, the half-life of bile was 9.3 hours (r 2 = 0.81), and the half-life of trypsin was 9.0 hours (r 2 = 0.68). Conclusions: The half-lives of bile and trypsin in the rodent aspiration model suggest that the ability to detect aspiration may be limited to a few days post-aspiration. If studies using rats are any indication, it may be most effective to collect BAL samples within the first 24 hours of suspected aspiration events in order to detect aspiration. PMID:26873328

Clearance of bile and trypsin in rat lungs following aspiration of human gastric fluid.

PubMed

Leung, Jason H; Chang, Jui-Chih; Foltz, Emily; Bell, Sadé M; Pi, Cinthia; Azad, Sassan; Everett, Mary Lou; Holzknecht, Zoie E; Sanders, Nathan L; Parker, William; Davis, R Duane; Keshavjee, Shaf; Lin, Shu S

2016-01-01

In the clinical setting, there is no reliable tool for diagnosing gastric aspiration. A potential way of diagnosing gastric fluid aspiration entails bronchoalveolar lavage (BAL) with subsequent examination of the BAL fluid for gastric fluid components that are exogenous to the lungs. The objective of this study was to determine the longevity of the gastric fluid components bile and trypsin in the lung, in order to provide an estimate of the time frame in which assessment of these components in the BAL might effectively be used as a measure of aspiration. Human gastric fluid (0.5 mg/kg) was infused in the right lung of intubated male Fischer 344 rats (n = 30). Animals were sacrificed at specified times following the experimentally induced aspiration, and bronchoalveolar lavage fluid (BALF) was collected. Bile concentrations were analyzed by an enzyme-linked chromatogenic method, and the concentration of trypsin was quantified using an ELISA. Data were analyzed using non-linear regression and a one-phase decay equation. In this experimental model, the half-life of bile was 9.3 hours (r(2) = 0.81), and the half-life of trypsin was 9.0 hours (r(2) = 0.68). The half-lives of bile and trypsin in the rodent aspiration model suggest that the ability to detect aspiration may be limited to a few days post-aspiration. If studies using rats are any indication, it may be most effective to collect BAL samples within the first 24 hours of suspected aspiration events in order to detect aspiration.

Midgut Protease Activity During Larval Development of Anastrepha obliqua (Diptera: Tephritidae) Fed With Natural and Artificial Diet

PubMed Central

Rivera-Ciprian, José Pedro; Aceituno-Medina, Marysol; Guillen, Karina

2017-01-01

Abstract In this study, we examined the activity of two serine proteases (chymotrypsin and trypsin) and two metalloproteases (carboxypeptidases A and B) during larval development in Anastrepha obliqua fed natural (mango fruit) and artificial (formulation used in mass-rearing) diets. Proteolytic activity of chymotrypsin, trypsin, carboxypeptidase A, and carboxypeptidase B was detected in the midgut of different instars of A. obliqua and was strongly affected by the pH and diet type. The protein content of the natural and artificial diets was similar. Enzymatic activity was higher in the midgut of the larvae fed the natural diet than in larvae fed the artificial diet. The activity of the endopeptidases (chymotrypsin and trypsin) was lower than those of the exopeptidases (carboxypeptidases A and B). The pH of the midgut varied from acidic to neutral. The results indicate that in the midgut of the larvae reared on both types of diet, the level of carboxypeptidase activity was approximately 100-fold greater than the level of chymotrypsin activity and 10,000-fold greater than the level of trypsin. In conclusion, carboxypeptidase A and B are the main proteases involved in the digestion of proteins in the larvae of A. obliqua. The natural diet showed a high bioaccessibility. A clear tendency to express high activities of chymotrypsin and trypsin was observed by the third instar. Our research contributes to the planning and development of novel bioaccessibility assays to understand the nutrition processing of A. obliqua larvae under mass-rearing conditions for sterile insect technique.

Cell-free released components of Streptococcus sanguis inhibit human platelet aggregation.

PubMed Central

Herzberg, M C; Brintzenhofe, K L; Clawson, C C

1983-01-01

To study the role of surface components in the selective binding and aggregation of platelet-rich plasma (PRP) by strains of viridans streptococci, we treated the binding, aggregation strain Streptococcus sanguis I 2017-78 by sonication or trypsinization. Morphologically identifiable electron-dense fibrils were released from the cell wall, apparently from an inner electron-dense layer, under conditions that left cells intact. These controlled conditions were determined to cause submaximal loss in adhesion to platelet ghosts and PRP aggregation by treated, washed S. sanguis. Soluble components were recovered from the controlled sonic or L-(tosylamido 2-phenyl)ethyl chloromethyl ketone-trypsin treatments. Each showed dose-response inhibition of aggregation when preincubated with PRP before challenge with fresh, untreated S. sanguis. The time to onset of PRP aggregation was inhibited by 50% with 0.2 mg of TPCK-trypsin peptides or 1.0 mg of the sonicate per ml per 2 X 10(8) platelets. Components of both preparations were immunologically cross-reactive, but lipoteichoic acid was not a major antigen of either. By weight, the TPCK-trypsin peptides were virtually all protein; the sonicate residues identified were about 50% protein and 7% hexose. Each was a complex mixture of components as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. More than 8 TPCK-trypsin peptides and 16 sonicate components were so identified. In contrast, at least four or five components from either preparation were recognized as surface determinants by a rabbit antiserum to whole homologous microbes. Platelet-binding ligands of S. sanguis could be among these determinants. Images PMID:6618669

Kinetic characterization of factor Xa binding using a quenched fluorescent substrate based on the reactive site of factor Xa inhibitor from Bauhinia ungulata seeds.

PubMed

Oliva, M L V; Andrade, S A; Juliano, M A; Sallai, R C; Torquato, R J; Sampaio, M U; Pott, V J; Sampaio, C A M

2003-07-01

The specific Kunitz Bauhinia ungulata factor Xa inhibitor (BuXI) and the Bauhinia variegata trypsin inhibitor (BvTI) blocked the activity of trypsin, chymotrypsin, plasmin, plasma kallikrein and factor XIIa, and factor Xa inhibition was achieved only by BuXI (K(i) 14 nM). BuXI and BvTI are highly homologous (70%). The major differences are the methionine residues at BuXI reactive site, which are involved in the inhibition, since the oxidized protein no longer inhibits factor Xa but maintains the trypsin inhibition. Quenched fluorescent substrates based on the reactive site sequence of the inhibitors were synthesized and the kinetic parameters of the hydrolysis were determined using factor Xa and trypsin. The catalytic efficiency k(cat)/K(m) 4.3 x 10(7) M(-1)sec(>-1) for Abz-VMIAALPRTMFIQ-EDDnp (lead peptide) hydrolysis by factor Xa was 10(4)-fold higher than that of Boc-Ile-Glu-Gly-Arg-AMC, widely used as factor Xa substrate. Lengthening of the substrate changed its susceptibility to factor Xa hydrolysis. Both methionine residues in the substrate influence the binding to factor Xa. Serine replacement of threonine (P(1)') decreases the catalytic efficiency by four orders of magnitude. Factor Xa did not hydrolyze the substrate containing the reactive site sequence of BvTI, that inhibits trypsin inhibitor but not factor Xa. Abz-VMIAALPRTMFIQ-EDDnp prolonged both the prothrombin time and the activated partial thromboplastin time, and the other modified substrates used in this experiment altered blood-clotting assays.

Immunofluorescent staining of rabies virus antigen in formalin-fixed tissue after treatment with trypsin

PubMed Central

Umoh, J. U.; Blenden, D. C.

1981-01-01

Formalin-fixed central nervous system tissue from clinically rabid animals was treated with 0.25% trypsin and tested for the presence of rabies virus antigen by direct immunofluorescent (IF) staining. The results were comparable with those obtained from direct IF staining of acetone-fixed standard smears or fresh frozen-cut sections. Experiments were conducted using coded brain specimens (classified as IF-negative, weakly positive, or strongly positive) and showed a specificity of 100% for sections and 92% for smears; the latter figure was subsequently improved by modifying the preparation technique. The specificity of the technique was checked by standard virus neutralization of the conjugate, and by known antibody neutralization of the virus antigen in the specimens. The optimal duration for the trypsin digestion was found to be a minimum of 60 minutes at 37 °C or 120 minutes at 4 °C. The tissues could be held in buffered formalin for between 3 days and 7 weeks with no apparent difference in the results. Satisfactory concentrations of formalin were 0.125% or 0.25%. Trypsin was found to have no effect on non-formalinized tissues, with the exception that softening occurred making tissues harder to cut and process. The results suggest that trypsinization of formalin-fixed tissue is a valid procedure for the preparation of tissues for IF examination, which would be useful in cases where the current standard techniques cannot be used. However, further evaluation of the method is still required. ImagesFig. 3Fig. 1Fig. 2 PMID:6172212

Thylakoid membrane protein topography: transmembrane orientation of the chloroplast cytochrome b-559 psbE gene product.

PubMed

Tae, G S; Black, M T; Cramer, W A; Vallon, O; Bogorad, L

1988-12-27

Protease accessibility and antibody to a COOH-terminal peptide were used as probes for the in situ topography of the Mr 10,000 psbE gene product (alpha subunit) of the chloroplast cytochrome b-559. Exposure of thylakoid membranes to trypsin or Staphylococcus aureus V8 protease cleaved the alpha subunit to a slightly smaller polypeptide (delta Mr approximately -1000) as detected on Western blots, without loss of reactivity to COOH-terminal antibody. The disappearance of the parent Mr 10,000 polypeptide from thylakoids in the presence of trypsin correlated with the appearance of the smaller polypeptide with delta Mr = -750, the conversion having a half-time of approximately 15 min. Exposure of inside-out vesicles to trypsin resulted in almost complete loss of reactivity to the antibody, showing that the COOH terminus is exposed on the lumenal side of the membrane. Removal of the extrinsic polypeptides of the oxygen-evolving complex resulted in an increase of the accessibility of the alpha subunit to trypsin. These data establish that the alpha subunit of cytochrome b-559 crosses the membrane once, as predicted from its single, 26-residue, hydrophobic domain. The NH2 terminus of the alpha polypeptide is on the stromal side of the membrane, where it is accessible, most likely at Arg-7 or Glu-6/Asp-11, to trypsin or V8 protease, respectively. As a consequence of this orientation, the single histidine residue in the alpha subunit is located on the stromal side of the hydrophobic domain.(ABSTRACT TRUNCATED AT 250 WORDS)

21 CFR 184.1914 - Trypsin.

Code of Federal Regulations, 2013 CFR

2013-04-01

... practice conditions of use: (1) The ingredient is used as an enzyme as defined in § 170.3(o)(9) of this... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Trypsin. 184.1914 Section 184.1914 Food and Drugs... characterizing enzyme activity is that of a peptide hydrolase (EC 3.4.21.4). (b) The ingredient meets the general...

Enhanced proliferation of human hepatoma cells by PAR-2 agonists via the ERK/AP-1 pathway.

PubMed

Xie, Liqun; Zheng, Yanmin; Li, Xuan; Zhao, Junyan; Chen, Xiaoyi; Chen, Li; Zhou, Jing; Hai, Ou; Li, Fei

2012-11-01

To investigate the expression and role of PAR-2 in the proliferation of the human hepatoma cell line HepG2, PAR-2 protein and mRNA expression were evaluated by immuno-histochemistry, immunofluorescence and RT-PCR analysis. The signaling pathways downstream of PAR-2 activation that lead to hepatoma cell proliferation were analyzed. The results showed that PAR-2 is expressed in human hepatoma cells and PAR-2 mRNA expression was found to be upregulated in cells treated with trypsin or SLIGKV-NH2 (P<0.001). The proliferation rate of HepG2 cells treated with trypsin or SLIGKV-NH2 was significantly increased (P<0.001). The percentage of S phase, G2/M phase and the proliferation index (PI) of HepG2 cells treated with trypsin or SLIGKV-NH2 were significantly elevated (P<0.001). The proliferative responses of HepG2 to trypsin and SLIGKV-NH2 were associated with the upregulation of c-fos and PCNA, which were significantly blocked by PD98059 pretreatment. In conclusion, our results indicate that PAR-2 enhances proliferation of human hepatoma cells possibly via the ERK/AP-1 pathway.

Kunitz-type protease inhibitors group B from Solanum palustre.

PubMed

Speransky, Anna S; Cimaglia, Fabio; Krinitsina, Anastasya A; Poltronieri, Palmiro; Fasano, Pasqua; Bogacheva, Anna M; Valueva, Tatiana A; Halterman, Dennis; Shevelev, Alexei B; Santino, Angelo

2007-11-01

Five Kunitz protease inhibitor group B genes were isolated from the genome of the diploid non-tuber-forming potato species Solanum palustre. Three of five new genes share 99% identity to the published KPI-B genes from various cultivated potato accessions, while others exhibit 96% identity. Spls-KPI-B2 and Spls-KPI-B4 proteins contain unique substitutions of the most conserved residues usually involved to trypsin and chymotrypsin-specific binding sites of Kunitz-type protease inhibitor (KPI)-B, respectively. To test the inhibition of trypsin and chymotrypsin by Spls-KPI proteins, five of them were produced in E. coli purified using a Ni-sepharose resin and ion-exchange chromatography. All recombinant Spls-KPI-B inhibited trypsin; K(i) values ranged from 84.8 (Spls-KPI-B4), 345.5 (Spls-KPI-B1), and 1310.6 nM (Spls-KPI-B2) to 3883.5 (Spls-KPI-B5) and 8370 nM (Spls-KPI-B3). In addition, Spls-KPI-B1 and Spls-KPI-B4 inhibited chymotrypsin. These data suggest that regardless of substitutions of key active-center residues both Spls-KPI-B4 and Spls-KPI-B1 are functional trypsin-chymotrypsin inhibitors.

Improving off-line accelerated tryptic digestion. Towards fast-lane proteolysis of complex biological samples.

PubMed

Vukovic, Jadranka; Loftheim, Håvard; Winther, Bjørn; Reubsaet, J Léon E

2008-06-27

Off-line digestion of proteins using immobilized trypsin beads is studied with respect to the format of the digestion reactor, the digestion conditions, the comparison with in-solution digestion and its use in complex biological samples. The use of the filter vial as the most appropriate digestion reactor enables simple, efficient and easy-to-handle off-line digestion of the proteins on trypsin beads. It was shown that complex proteins like bovine serum albumin (BSA) need much longer time (89 min) and elevated temperature (37 degrees C) to be digested to an acceptable level compared to smaller proteins like cytochrome c (5 min, room temperature). Comparing the BSA digestion using immobilized trypsin beads with conventional in-solution digestion (overnight at 37 degrees C), it was shown that comparable results were obtained with respect to sequence coverage (>90%) and amount of missed cleavages (in both cases around 20 peptides with 1 or 2 missed cleavages were detected). However, the digestion using immobilized trypsin beads was considerable less time consuming. Good reproducibility and signal intensities were obtained for the digestion products of BSA in a complex urine sample. In addition to this, peptide products of proteins typically present in urine were identified.

Performance of bean bruchids Callosobruchus maculatus and Zabrotes subfasciatus (Coleoptera: Bruchidae) reared on resistant (IT81D-1045) and susceptible (Epace 10) Vigna unguiculata seeds: relationship with trypsin inhibitor and vicilin excretion.

PubMed

Sales, M P; Andrade, L B S; Ary, M B; Miranda, M R A; Teixeira, F M; Oliveira, A S; Fernandes, K V S; Xavier-Filho, J

2005-12-01

Callosobruchus maculatus (Cm) and Zabrotes subfasciatus (Zs) were reared on resistant (IT81D-1045) and on susceptible (Epace 10) cowpea seeds. The emergence of adult insects, total developmental period (TDP) and excretion of trypsin inhibitor and vicilin were determined for both bruchid populations. Parameter evaluation showed that the Zs populations emerged from both seeds had no significant differences in emergence and TDP. The Cm population raised from resistant seeds had lower emergence (5.6+/-1.3%) and delayed TDP (46+/-1.25 days) than those emerged from susceptible seeds. The excretion of defense proteins showed that Zs reared in resistant seeds excreted 1.7 times more trypsin inhibitor, but this did not affect emergence or TDP. Furthermore, Cm population emerged from resistant seeds excreted 7 times higher vicilin and 0.4 times less trypsin inhibitor than that emerged from susceptible seeds. These results indicate that vicilins from resistant seeds are involved to significantly longer TDP (46 days) and also drastic reduction of insect emergence ( approximately 5%) of C. maculatus.

Allergenicity, trypsin inhibitor activity and nutritive quality of enzymatically modified soy proteins.

PubMed

De La Barca, Ana María Calderón; Wall, Abraham; López-Díaz, José Alberto

2005-05-01

Two ultrafiltered soy flour protein fractions were evaluated; the first was obtained by hydrolysis (0.5-3 kDa, F(0.5-3)), and the second was an enzymatically methionine-enriched fraction (1-10 kDa, F(1-10)E). Amino acid profiles, protein quality, allergenicity (against soy-sensitive infant sera) and trypsin inhibitor activity were determined. Fraction F(1-10)E fulfilled amino acid requirements for infants, whereas the F(0.5-3) fraction was methionine deficient. Both fractions were similar in net protein utilization, and F(1-10)E digestibility was comparable with casein and higher (P?

Effects of soybean Kunitz trypsin inhibitor on the cotton boll weevil (Anthonomus grandis).

PubMed

Franco, Octávio L; Dias, Simoni C; Magalhães, Claudio P; Monteiro, Ana C S; Bloch, Carlos; Melo, Francislete R; Oliveira-Neto, Osmundo B; Monnerat, Rose G; Grossi-de-Sá, Maria Fátima

2004-01-01

The cotton boll weevil, Anthonomus grandis, is an economically important pest of cotton in tropical and subtropical areas of several countries in the Americas, causing severe losses due to their damage in cotton floral buds. Enzymatic assays using gut extracts from larval and adult boll weevil have demonstrated the presence of digestive serine proteinase-like activities. Furthermore, in vitro assays showed that soybean Kunitz trypsin inhibitor (SKTI) was able to inhibit these enzymes. Previously, in vivo effects of black-eyed pea trypsin chymotrypsin inhibitor (BTCI) have been demonstrated towards the boll weevil pest. Here, when neonate larvae were reared on an artificial diet containing SKTI at three different concentrations, a reduction of larval weight of up to 64% was observed for highest SKTI concentration 500 microM. The presence of SKTI caused an increase in mortality and severe deformities of larvae, pupae and adult insects. This work therefore represents the first observation of a Kunitz trypsin inhibitor active in vivo and in vitro against A. grandis. Bioassays suggested that SKTI could be used as a tool in engineering crop plants, which might exhibit increased resistance against cotton boll weevil.

Cholecystokinin-converting enzymes in brain.

PubMed Central

Malesci, A; Straus, E; Yalow, R S

1980-01-01

Crude extracts of porcine cerebral cortical tissue convert cholecystokinin (CCK) to its COOH-terminal fragments, the dodecapeptide (CCK-12) and the octapeptide (CCK-8). The Sephadex G-75 void volume eluate of the crude extract cleaves the arginine-isoleucine bond and effects conversion only to CCK-12; the Sephadex G-50 void volume eluate of the same extract cleaves the arginine-aspartate bond as well, so that both CCK-12 and CCK-8 are end products. Thus, there are at least two enzymes; the one involved in the conversion to CCK-12 is of larger molecular radius than the other. The Km for the cleavage of CCK at the arginine-isoleucine bond by the Sephadex G-75 void volume eluate enzyme is 1.1 X 10(-6) M; the Km for trypsin cleavage of the same bond is 4.7 x 10(-6) M. The lower Vmax for the brain enzyme (1.5 x 10(-11) mol/min per g of extract) compared with trypsin (66 x 10(-11) mol/min per g of trypsin) simply reflects the lesser degree of purify of the brain extract than of the highly purified trypsin. Images PMID:6987659

ACE Inhibitory and Antioxidant Activities of Collagen Hydrolysates from the Ribbon Jellyfish (Chrysaora sp.)

PubMed Central

Latiff, Aishah Abd; Gan, Chee-Yuen; Abedin, Md. Zainul; Alias, Abd Karim

2014-01-01

Summary Collagen isolated from the ribbon jellyfish (Chrysaora sp.) was hydrolysed using three different proteases (i.e. trypsin, alcalase and Protamex) to obtain bioactive peptides. Angiotensin-I-converting enzyme (ACE) inhibitory activity and antioxidant activities (i.e. ferric reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity) of the peptides were measured and compared, and the effect of the duration of hydrolysis on the bioactivity (ACE inhibitory and antioxidant activities) of peptides was also evaluated. FRAP activity was the highest in Protamex-induced (25–27 mM) and trypsin-induced hydrolysates (24–26 mM) at 7 and 9 h, respectively. Conversely, hydrolysates produced by trypsin for 1 and 3 h showed the highest DPPH radical scavenging activities (94 and 92%, respectively). Trypsin-induced hydrolysates (at 3 h) also showed the highest ACE inhibitory activity (89%). The peptide sequences with the highest activities were identified using tandem mass spectrometry, and the results show that the hydrolysates had a high content of hydrophobic amino acids as well as unique amino acid sequences, which likely contribute to their biological activities. PMID:27904323

Bauhinia variegata var. variegata trypsin inhibitor: From isolation to potential medicinal applications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fang, Evandro Fei; Wong, Jack Ho; Bah, Clara Shui Fern

Here we report for the first time of a new Kunitz-type trypsin inhibitor (termed BvvTI) from seeds of the Camel's foot tree, Bauhinia variegata var. variegata. BvvTI shares the same reactive site residues (Arg, Ser) and exhibits a homology of N-terminal amino acid sequence to other Bauhinia protease inhibitors. The trypsin inhibitory activity (K{sub i}, 0.1 x 10{sup -9} M) of BvvTI ranks the highest among them. Besides anti-HIV-1 reverse transcriptase activity, BvvTI could significantly inhibit the proliferation of nasopharyngeal cancer CNE-1 cells in a selective way. This may partially be contributed by its induction of cytokines and apoptotic bodies.more » These results unveil potential medicinal applications of BvvTI.« less

Bauhinia variegata var. variegata trypsin inhibitor: from isolation to potential medicinal applications.

PubMed

Fang, Evandro Fei; Wong, Jack Ho; Bah, Clara Shui Fern; Lin, Peng; Tsao, Sai Wah; Ng, Tzi Bun

2010-06-11

Here we report for the first time of a new Kunitz-type trypsin inhibitor (termed BvvTI) from seeds of the Camel's foot tree, Bauhinia variegata var. variegata. BvvTI shares the same reactive site residues (Arg, Ser) and exhibits a homology of N-terminal amino acid sequence to other Bauhinia protease inhibitors. The trypsin inhibitory activity (K(i), 0.1 x 10(-9)M) of BvvTI ranks the highest among them. Besides anti-HIV-1 reverse transcriptase activity, BvvTI could significantly inhibit the proliferation of nasopharyngeal cancer CNE-1 cells in a selective way. This may partially be contributed by its induction of cytokines and apoptotic bodies. These results unveil potential medicinal applications of BvvTI. (c) 2010 Elsevier Inc. All rights reserved.

Properties of a Kunitz-type trypsin inhibitor from Delonix regia seeds against digestive proteinases of Anagasta kuehniella (Z.) and Corcyra cephalonica (S.) (Lepidoptera: Pyralidae).

PubMed

Macedo, M L R; Pando, S C; Chevreuil, L R; Marangoni, S

2009-01-01

DrTI was effective against trypsin-like enzymes from A. kuehniella and C. cephalonica, however an artificial diet was insufficient to affect the survival and body weight of either insect. The inhibitor stimulated chymotrypsin-like enzymes and probably induced the synthesis of enzymes insensitive to TLCK in neonate larvae.

Isolation and identification of group A rotaviruses among neonatal diarrheic calves, Morocco.

PubMed

Ennima, Imane; Sebbar, Ghizlane; Harif, Bachir; Amzazi, Saaid; Loutfi, Chafiqa; Touil, Nadia

2016-05-05

Group A rotaviruses (RVA) are the main cause of neonatal calve diarrhea (NCD) in Morocco. In this study, we isolated RVA strains from NCD clinical samples in order to support RVA disease control in Morocco. This isolation process constitutes a first step toward vaccine development. Thirteen fecal samples were obtained from calves with a single episode of neonate calf diarrhea at three different dairies and two samples were collected from field during a severe NCD outbreak. Diagnosis of RVA infection was based on fecal immune-chromatographic rapid test and further evaluated for their hemagglutination (HA) activity. RVA isolation was carried out on MA104 cells after inoculates were treated with different concentrations of trypsin TPCK. All RVA isolates were confirmed by LSI VetMAX™ Triplex Ruminant Rotavirus & Coronavirus Real-Time PCR kit. G and P typing were determined by direct sequencing of the VP4 and VP7 amplicons. RVA isolation was achieved for nine clinical samples following one or two passages (60 %) and was properly depended on HA activity and trypsin treatment of inoculates. The first sign of CPE detected consisted of increased cell granularity, obscure cell boundaries, cell rounding, and eventual degeneration and detachment of cells. At lower TPCK concentration (3-10 μg/inoculum), no changes at the cellular level were observed, while cells activated with 25-30 μg of trypsin/inoculums, they degenerated and trypsin cytotoxicity was enhanced. Appreciable changes in cell's morphology were detected with optimal trypsin concentration of 15-20 μg trypsin/inoculums. Data from qRT-PCR confirmed that unsuccessful cultivations have No-Ct, and all nine isolates have Ct values ranged between 12.17 and 24.69. Analysis sequencing revealed that field isolates were of G6 P[5] serotype and isolates from the dairy NCD samples were of G10 P[14] serotype. To our knowledge, this is the first study in Morocco which reports the circulation of G10P[14] in NCD on dairy farms and G6P[5] in the field. Our study constitutes a crucial and a necessary step allowing preventive and veterinary medicine to support RVA disease controls in the country.

Methylene blue not ferrocene: Optimal reporters for electrochemical detection of protease activity.

PubMed

González-Fernández, Eva; Avlonitis, Nicolaos; Murray, Alan F; Mount, Andrew R; Bradley, Mark

2016-10-15

Electrochemical peptide-based biosensors are attracting significant attention for the detection and analysis of proteins. Here we report the optimisation and evaluation of an electrochemical biosensor for the detection of protease activity using self-assembled monolayers (SAMs) on gold surfaces, using trypsin as a model protease. The principle of detection was the specific proteolytic cleavage of redox-tagged peptides by trypsin, which causes the release of the redox reporter, resulting in a decrease of the peak current as measured by square wave voltammetry. A systematic enhancement of detection was achieved through optimisation of the properties of the redox-tagged peptide; this included for the first time a side-by-side study of the applicability of two of the most commonly applied redox reporters used for developing electrochemical biosensors, ferrocene and methylene blue, along with the effect of changing both the nature of the spacer and the composition of the SAM. Methylene blue-tagged peptides combined with a polyethylene-glycol (PEG) based spacer were shown to be the best platform for trypsin detection, leading to the highest fidelity signals (characterised by the highest sensitivity (signal gain) and a much more stable background than that registered when using ferrocene as a reporter). A ternary SAM (T-SAM) configuration, which included a PEG-based dithiol, minimised the non-specific adsorption of other proteins and was sensitive towards trypsin in the clinically relevant range, with a Limit of Detection (LoD) of 250pM. Kinetic analysis of the electrochemical response with time showed a good fit to a Michaelis-Menten surface cleavage model, enabling the extraction of values for kcat and KM. Fitting to this model enabled quantitative determination of the solution concentration of trypsin across the entire measurement range. Studies using an enzyme inhibitor and a range of real world possible interferents demonstrated a selective response to trypsin cleavage. This indicates that a PEG-based peptide, employing methylene blue as redox reporter, and deposited on an electrode as a ternary SAM configuration, is a suitable platform to develop clinically-relevant and quantitative electrochemical peptide-based protease biosensing. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Trypsin impaired epithelial barrier function and induced IL-8 secretion through basolateral PAR-2: a lesson from a stratified squamous epithelial model.

PubMed

Shan, Jing; Oshima, Tadayuki; Chen, Xin; Fukui, Hirokazu; Watari, Jiro; Miwa, Hiroto

2012-11-15

Immune-mediated injury by the protease-activated receptor-2-interleukin-8 (PAR-2-IL8) pathway may underlie the development of gastroesophageal reflux disease (GERD). However, the localization of PAR-2 and the mechanism of PAR-2 activation remain unclear. This study aimed to address these questions on an esophageal stratified squamous epithelial model and in the human esophageal mucosa of GERD patients. Normal human esophageal epithelial cells were cultured with the air-liquid interface system to establish the model. SLIGKV-NH2 (PAR-2 synthetic agonist), trypsin (PAR-2 natural activator), and weak acid (pH 4, 5, and 6) were added to either the apical or basolateral compartment to evaluate their effects on transepithelial electrical resistance (TEER) and IL-8 production. PAR-2 localization was examined both in the cell model and biopsies from GERD patients by immunohistochemistry. Apical trypsin stimulation induced IL-8 accompanied by decreased TEER in vitro, whereas the effective concentration from the basolateral side was 10 times lower. SLIGKV-NH2 from basolateral but not apical stimulation induced IL-8 production. Apical weak acid stimulation did not influence TEER or IL-8 production. Immunohistochemistry showed intense reactivity of PAR-2 in the basal and suprabasal layers after stimulation with trypsin. A similar PAR-2 reactivity that was mainly located at the basal and suprabasal layers was detected in GERD patients. In conclusion, the activation of the PAR-2-IL-8 pathway probably occurred at the basal and suprabasal layers, while the esophageal epithelial barrier may influence the activation of PAR-2. Under proton pump inhibitor therapy, refluxed trypsin may remain active and be a potential agent in the pathogenesis of refractory GERD.

Bioinsecticidal activity of a novel Kunitz trypsin inhibitor from Catanduva (Piptadenia moniliformis) seeds.

PubMed

Cruz, Ana C B; Massena, Fábio S; Migliolo, Ludovico; Macedo, Leonardo L P; Monteiro, Norberto K V; Oliveira, Adeliana S; Macedo, Francisco P; Uchoa, Adriana F; Grossi de Sá, Maria F; Vasconcelos, Ilka M; Murad, Andre M; Franco, Octavio L; Santos, Elizeu A

2013-09-01

The present study aims to provide new in vitro and in vivo biochemical information about a novel Kunitz trypsin inhibitor purified from Piptadenia moniliformis seeds. The purification process was performed using TCA precipitation, Trypsin-Sepharose and reversed-phase C18 HPLC chromatography. The inhibitor, named PmTKI, showed an apparent molecular mass of around 19 kDa, visualized by SDS-PAGE, which was confirmed by mass spectrometry MALDI-ToF demonstrating a monoisotopic mass of 19.296 Da. The inhibitor was in vitro active against trypsin, chymotrypsin and papain. Moreover, kinetic enzymatic studies were performed aiming to understand the inhibition mode of PmTKI, which competitively inhibits the target enzyme, presenting Ki values of 1.5 × 10(-8) and 3.0 × 10(-1) M against trypsin and chymotrypsin, respectively. Also, the inhibitory activity was assayed at different pH ranges, temperatures and reduction environments (DTT). The inhibitor was stable in all conditions maintaining an 80% residual activity. N-terminal sequence was obtained by Edman degradation and the primary sequence presented identity with members of Kunitz-type inhibitors from the same subfamily. Finally after biochemical characterization the inhibitory effect was evaluated in vitro on insect digestive enzymes from different orders, PmTKI demonstrated remarkable activity against enzymes from Anthonomus grandis (90%), Plodia interpuncptella (60%), and Ceratitis capitata (70%). Furthermore, in vivo bioinsecticidal assays of C. capitata larvae were also performed and the concentration of PmTKI (w/w) in an artificial diet required to LD50 and ED50 larvae were 0.37 and 0.3% respectively. In summary, data reported here shown the biotechnological potential of PmTKI for insect pest control. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Molecular Genetic Analysis of Midgut Serine Proteases in Aedes aegypti Mosquitoes

PubMed Central

Isoe, Jun; Rascón, Alberto A.; Kunz, Susan; Miesfeld, Roger L.

2009-01-01

Digestion of blood meal proteins by midgut proteases provides anautogenous mosquitoes with the nutrients required to complete the gonotrophic cycle. Inhibition of protein digestion in the midgut of blood feeding mosquitoes could therefore provide a strategy for population control. Based on recent reports indicating that the mechanism and regulation of protein digestion in blood fed female Aedes aegypti mosquitoes is more complex than previously thought, we used a robust RNAi knockdown method to investigate the role of four highly expressed midgut serine proteases in blood meal metabolism. We show by Western blotting that the early phase trypsin protein (AaET) is maximally expressed at 3 h post blood meal (PBM), and that AaET is not required for the protein expression of three late phase serine proteases, AaLT (late trypsin), AaSPVI (5G1), and AaSPVII. Using the trypsin substrate analog BApNA to analyze in vitro enzyme activity in midgut extracts from single mosquitoes, we found that knockdown of AaSPVI expression caused a 77.6% decrease in late phase trypsin-like activity, whereas, knockdown of AaLT and AaSPVII expression had no significant effect on BApNA activity. In contrast, injection of AaLT, AaSPVI, and AaSPVII dsRNA inhibited degradation of endogenous serum albumin protein using an in vivo protease assay, as well as, significantly decreased egg production in both the first and second gonotrophic cycles (p<0.001). These results demonstrate that AaLT, AaSPVI, and AaSPVII all contribute to blood protein digestion and oocyte maturation, even though AaSPVI is the only abundant midgut late phase serine protease that appears to function as a classic trypsin enzyme. PMID:19883761

Quantifying Protein-Ligand Binding Constants using Electrospray Ionization Mass Spectrometry: A Systematic Binding Affinity Study of a Series of Hydrophobically Modified Trypsin Inhibitors

NASA Astrophysics Data System (ADS)

Cubrilovic, Dragana; Biela, Adam; Sielaff, Frank; Steinmetzer, Torsten; Klebe, Gerhard; Zenobi, Renato

2012-10-01

NanoESI-MS is used for determining binding strengths of trypsin in complex with two different series of five congeneric inhibitors, whose binding affinity in solution depends on the size of the P3 substituent. The ligands of the first series contain a 4-amidinobenzylamide as P1 residue, and form a tight complex with trypsin. The inhibitors of the second series have a 2-aminomethyl-5-chloro-benzylamide as P1 group, and represent a model system for weak binders. The five different inhibitors of each group are based on the same scaffold and differ only in the length of the hydrophobic side chain of their P3 residue, which modulates the interactions in the S3/4 binding pocket of trypsin. The dissociation constants (KD) for high affinity ligands investigated by nanoESI-MS ranges from 15 nM to 450 nM and decreases with larger hydrophobic P3 side chains. Collision-induced dissociation (CID) experiments of five trypsin and benzamidine-based complexes show a correlation between trends in KD and gas-phase stability. For the second inhibitor series we could show that the effect of imidazole, a small stabilizing additive, can avoid the dissociation of the complex ions and as a result increases the relative abundance of weakly bound complexes. Here the KD values ranging from 2.9 to 17.6 μM, some 1-2 orders of magnitude lower than the first series. For both ligand series, the dissociation constants (KD) measured via nanoESI-MS were compared with kinetic inhibition constants (Ki) in solution.

Insights into the Structure of the Vip3Aa Insecticidal Protein by Protease Digestion Analysis

PubMed Central

Bel, Yolanda; Banyuls, Núria; Chakroun, Maissa; Escriche, Baltasar; Ferré, Juan

2017-01-01

Vip3 proteins are secretable proteins from Bacillus thuringiensis whose mode of action is still poorly understood. In this study, the activation process for Vip3 proteins was closely examined in order to better understand the Vip3Aa protein stability and to shed light on its structure. The Vip3Aa protoxin (of 89 kDa) was treated with trypsin at concentrations from 1:100 to 120:100 (trypsin:Vip3A, w:w). If the action of trypsin was not properly neutralized, the results of SDS-PAGE analysis (as well as those with Agrotis ipsilon midgut juice) equivocally indicated that the protoxin could be completely processed. However, when the proteolytic reaction was efficiently stopped, it was revealed that the protoxin was only cleaved at a primary cleavage site, regardless of the amount of trypsin used. The 66 kDa and the 19 kDa peptides generated by the proteases co-eluted after gel filtration chromatography, indicating that they remain together after cleavage. The 66 kDa fragment was found to be extremely resistant to proteases. The trypsin treatment of the protoxin in the presence of SDS revealed the presence of secondary cleavage sites at S-509, and presumably at T-466 and V-372, rendering C-terminal fragments of approximately 29, 32, and 42 kDa, respectively. The fact that the predicted secondary structure of the Vip3Aa protein shows a cluster of beta sheets in the C-terminal region of the protein might be the reason behind the higher stability to proteases compared to the rest of the protein, which is mainly composed of alpha helices. PMID:28387713

Interaction between wheat alpha-amylase/trypsin bi-functional inhibitor and mammalian digestive enzymes: Kinetic, equilibrium and structural characterization of binding.

PubMed

Cuccioloni, Massimiliano; Mozzicafreddo, Matteo; Ali, Ishtiaq; Bonfili, Laura; Cecarini, Valentina; Eleuteri, Anna Maria; Angeletti, Mauro

2016-12-15

Alpha-amylase/trypsin bi-functional inhibitors (ATIs) are non-gluten protein components of wheat and other cereals that can hypersensitise the human gastrointestinal tract, eventually causing enteropathies in predisposed individuals. These inhibitory proteins can act both directly by targeting specific pro-inflammatory receptors, and indirectly by impairing the activity of digestive enzymes, the latter event causing the accumulation of undigested peptides with potential immunogenic properties. Herein, according to a concerted approach based on in vitro and in silico methods we characterized kinetics, equilibrium parameters and modes of binding of the complexes formed between wheat ATI and two representative mammalian digestive enzymes, namely trypsin and alpha-amylase. Interestingly, we demonstrated ATI to target both enzymes with independent binding sites and with moderately high affinity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Serum trypsin, alpha-amylase and lipase during bombesin stimulation in normal subjects and patients with pancreatic insufficiency.

PubMed

Hafkenscheid, J C; Hessels, M; Jansen, J B; Lamers, C B

1984-01-31

The effect of infusion of bombesin (60 pmol/kg 20 min) on pancreatic enzymes in serum was studied in 13 normal subjects and 12 patients with pancreatic insufficiency. In normal subjects administration of bombesin induced large increases in serum trypsin (p less than 0.01), while serum total alpha-amylase and pancreatic alpha-amylase did not change and serum lipase showed only a modest rise (0.01 less than p less than 0.05). Patients with pancreatic insufficiency had significantly lower serum concentrations of all enzymes studied (p less than 0.01) and in such patients bombesin did not change the concentrations of pancreatic enzymes in serum. It is concluded that determination of the serum trypsin response to bombesin may be of help in the diagnosis of pancreatic insufficiency.

Evidence for a latent form of protein phosphatase 1 associated with cardiac myofibrils.

PubMed

Schlender, K K; Wang, W; Wilson, S E

1989-02-28

Detergent-purified myofibrils from bovine heart contained very little spontaneously active protein phosphatase 1 activity. Phosphatase 1, extracted from the myofibrils by freeze-thawing in the presence of 500 mM KCl, was markedly activated by cobalt/trypsin treatment. Myofibril phosphatase 1 was separated from phosphatase 2A by chromatography on heparin-Sepharose. The phosphatase 1 was isolated in a latent form. Pretreatment with trypsin released free catalytic subunit and increased activity about 25-fold. Addition of cobalt with the trypsin increased activity another 2-fold. The latent myofibril phosphatase 1 did not appear to be the same as previously characterized forms of protein phosphatase 1. We suggest that cardiac myofibril phosphatase 1 contains a unique inhibitory subunit which directs the enzyme to the myofibril and regulates the dephosphorylation of myofibril phosphoproteins.

Combinatorial Enzyme Design Probes Allostery and Cooperativity in the Trypsin Fold

DOE Office of Scientific and Technical Information (OSTI.GOV)

Page, Michael J.; Di Cera, Enrico; St. Louis-MED)

2010-06-14

Converting one enzyme into another is challenging due to the uneven distribution of important amino acids for function in both protein sequence and structure. We report a strategy for protein engineering allowing an organized mixing and matching of genetic material that leverages lower throughput with increased quality of screens. Our approach successfully tested the contribution of each surface-exposed loop in the trypsin fold alone and the cooperativity of their combinations towards building the substrate selectivity and Na{sup +}-dependent allosteric activation of the protease domain of human coagulation factor Xa into a bacterial trypsin. As the created proteases lack additional proteinmore » domains and protein co-factor activation mechanism requisite for the complexity of blood coagulation, they are stepping-stones towards further understanding and engineering of artificial clotting factors.« less

Saponin content and trypsin inhibitor activity in processed and cooked pigeon pea cultivars.

PubMed

Duhan, A; Khetarpaul, N; Bishnoi, S

2001-01-01

Four high-yielding varieties of pigeon pea namely UPAS-120, Manak, JCPL-151. ICPL-87 had considerable amounts of antinutrients i.e. saponins and trypsin inhibitors. Saponin content of these unprocessed cultivars ranged from 2164 to 3494 mg/100 g. There were significant varietal variations in trypsin inhibitor activity (1007-1082 TIU/g) of these pigeon pea cultivars. Some simple, inexpensive and easy-to-use domestic processing and cooking methods, namely, soaking (6, 12, 18 h), soaking (12 h)-dehulling, ordinary cooking, pressure cooking and germination (24, 36, 48 h) were found to be quite effective in lowering the level of saponins and trypsin inhibitors in all the pigeon pea cultivars. Pressure cooking of soaked and dehulled seeds lowered the content of saponins to a maximum extent (28 to 38%) followed by ordinary cooking of soaked and dehulled seeds (28 to 35%), soaked dehulled raw seeds (22 to 27%) and 48 h germinated seeds (15 to 19%). Loss of TIA was marginal due to soaking but ordinary as well as pressure cooking of unsoaked and soaked-dehulled pigeon pea seeds reduced the TIA drastically. Pressure cooking of pigeon pea seeds completely destroyed the TIA while it was reduced to the extent of 86-88% against the control in 48 h pigeon pea sprouts.

Trypsin and MALDI matrix pre-coated targets simplify sample preparation for mapping proteomic distributions within biological tissues by imaging mass spectrometry

PubMed Central

Zubair, Faizan; Laibinis, Paul E.; Swisher, William G.; Yang, Junhai; Spraggins, Jeffrey M.; Norris, Jeremy L.; Caprioli, Richard M.

2017-01-01

Prefabricated surfaces containing α-cyano-4-hydroxycinnamic acid and trypsin have been developed to facilitate enzymatic digestion of endogenous tissue proteins prior to matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). Tissue sections are placed onto slides that were previously coated with α-cyano-4-hydroxycinnamic acid and trypsin. After incubation to promote enzymatic digestion, the tissue is analyzed by MALDI IMS to determine the spatial distribution of the tryptic fragments. The peptides detected in the MALDI IMS dataset were identified by Liquid chromatography-tandem mass spectrometry/mass spectrometry. Protein identification was further confirmed by correlating the localization of unique tryptic fragments originating from common parent proteins. Using this procedure, proteins with molecular weights as large as 300 kDa were identified and their distributions were imaged in sections of rat brain. In particular, large proteins such as myristoylated alanine-rich C-kinase substrate (29.8 kDa) and spectrin alpha chain, non-erythrocytic 1 (284 kDa) were detected that are not observed without trypsin. The pre-coated targets simplify workflow and increase sample throughput by decreasing the sample preparation time. Further, the approach allows imaging at higher spatial resolution compared with robotic spotters that apply one drop at a time. PMID:27676701

Expression of protease-activated-receptor 2 (PAR-2) in human esophageal mucosa.

PubMed

Inci, Kamuran; Edebo, Anders; Olbe, Lars; Casselbrant, Anna

2009-01-01

The role of duodenal reflux in gastroesophageal reflux disease (GERD) containing bile salts and pancreatic enzymes (with special attention to trypsin) is still under discussion. Proteinase-activated receptors (PARs) are a novel family and PAR-2 is a unique member of this family because it is activated by trypsin. The aim of the present study was to examine the presence and the position of the PAR-2 receptor in human esophageal mucosa in different subgroups of GERD. Distal biopsies taken from healthy controls, patients with erosive reflux disease (ERD), patients with specialized intestinal metaplasia (SIM) and adenocarcinoma were analyzed for the PAR-2 receptor with reverse-transcription polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry. Gene transcripts for the PAR-2 receptor were found in all groups, with increased levels in SIM patients compared to controls. However, this visual pattern was not seen for the protein expression of the PAR-2 receptor showing no apparent quantitative differences between the groups. Immunohistochemistry revealed distinct staining for the PAR-2 receptor in the luminal part of the esophageal epithelium. The localization of the PAR-2 receptor indicates that the receptor can be cleaved and activated by trypsin in duodenogastric esophageal refluxate. The data thus suggest that the trypsin-PAR-2 pathway may be involved in the pathogenesis of GERD.

Investigation of Possible Role of the PAR-2 Receptor in Intestinal Inflammation.

PubMed

Patel, Mb; Patel, Ma; Shah, Gb

2010-01-01

The present study was undertaken to study the role of PAR-2 receptor activation in pathophysiology of intestinal inflammation. Inflammatory bowel disease was induced in Wistar albino rats by intrarectal administration of 2, 4, 6 trinitrobenzenesulfonic acid (TNBS, 0.25 ml 120 mg/ml in 50% ethanol intrarectally, on 1(st) day only). Trypsin (500 μg/kg, 1 mg/kg, 5 mg/kg, intrarectal) was given from the same day up to 20 days. Various physical parameters including body weight, food and water intake were measured on 1(st) and 20(th) days. At end of the experiment, colon weight and various histopathological indexes were assessed. The colon homogenate malondialdehyde (MDA), myeloperoxidase (MPO), and superoxide dismutase (SOD) and % mast cell protection in mesentery were also measured. Trypsin at higher dose (5 mg/kg) showed the higher level of oxidative enzymes and lower level of protective enzymes as compared to the animals treated with only TNBS. Trypsin treatment produced significantly more mast cell degranulation. Finally in the histopathology, there was increased in severity of the disease in trypsin-treated animals. The role of PAR-2 (protease activated receptor-2) receptor in gut is pro-inflammatory and thus appears as a new potential therapeutic target for inflammatory bowel disease treatments.

Influence of carbohydrates on the interaction of procyanidin B3 with trypsin.

PubMed

Gonçalves, Rui; Mateus, Nuno; De Freitas, Victor

2011-11-09

The biological properties of procyanidins, in particular their inhibition of digestive enzymes, have received much attention in the past few years. Dietary carbohydrates are an environmental factor that is known to affect the interaction of procyanidins with proteins. This work aimed at understanding the effect of ionic food carbohydrates (polygalacturonic acid, arabic gum, pectin, and xanthan gum) on the interaction between procyanidins and trypsin. Physical-chemical techniques such as saturation transfer difference-NMR (STD-NMR) spectroscopy, fluorescence quenching, and nephelometry were used to evaluate the interaction process. Using STD-NMR, it was possible to identify the binding of procyanidin B3 to trypsin. The tested carbohydrates prevented the association of procyanidin B3 and trypsin by a competition mechanism in which the ionic character of carbohydrates and their ability to encapsulate procyanidins seem crucial leading to a reduction in STD signal and light scattering and to a recovery of the proteins intrinsic fluorescence. On the basis of these results, it was possible to grade the carbohydrates in their aggregation inhibition ability: XG > PA > AG ≫ PC. These effects may be relevant since the coingestion of procyanidins and ionic carbohydrates are frequent and furthermore since these might negatively affect the antinutritional properties ascribed to procyanidins in the past.

Proteolytic and Trypsin Inhibitor Activity in Germinating Jojoba Seeds (Simmondsia chinensis).

PubMed

Samac, D; Storey, R

1981-12-01

Changes in proteolytic activity (aminopeptidase, carboxypeptidase, endopeptidase) were followed during germination (imbibition through seedling development) in extracts from cotyledons of jojoba seeds (Simmondsia chinensis). After imbibition, the cotyledons contained high levels of sulfhydryl aminopeptidase activity (APA) but low levels of serine carboxypeptidase activity (CPA). CPA increased with germination through the apparent loss of a CPA inhibitor substance in the seed. Curves showing changes in endopeptidase activity (EPA) assayed at pH 4, 5, 6, 7, and 8 during germination were distinctly different. EPA at pH 4, 5, 6, and 7 showed characteristics of sulfhydryl enzymes while activity at pH 8 was probably due to a serine type enzyme. EPA at pH 6 was inhibited early in germination by one or more substances in the seed. Activities at pH 5 and later at pH 6 were the highest of all EPA throughout germination and increases in these activities were associated with a rapid loss of protein from the cotyledons of the developing seedling.Jojoba cotyledonary extracts were found to inhibit the enzymic activity of trypsin, chymotrypsin, and pepsin but not the protease from Aspergillus saotoi. The heat-labile trypsin inhibitor substance(s) was found in commercially processed jojoba seed meal and the albumin fraction of seed proteins. Trypsin inhibitor activity decreased with germination.

Proteolytic and Trypsin Inhibitor Activity in Germinating Jojoba Seeds (Simmondsia chinensis) 1

PubMed Central

Samac, Deborah; Storey, Richard

1981-01-01

Changes in proteolytic activity (aminopeptidase, carboxypeptidase, endopeptidase) were followed during germination (imbibition through seedling development) in extracts from cotyledons of jojoba seeds (Simmondsia chinensis). After imbibition, the cotyledons contained high levels of sulfhydryl aminopeptidase activity (APA) but low levels of serine carboxypeptidase activity (CPA). CPA increased with germination through the apparent loss of a CPA inhibitor substance in the seed. Curves showing changes in endopeptidase activity (EPA) assayed at pH 4, 5, 6, 7, and 8 during germination were distinctly different. EPA at pH 4, 5, 6, and 7 showed characteristics of sulfhydryl enzymes while activity at pH 8 was probably due to a serine type enzyme. EPA at pH 6 was inhibited early in germination by one or more substances in the seed. Activities at pH 5 and later at pH 6 were the highest of all EPA throughout germination and increases in these activities were associated with a rapid loss of protein from the cotyledons of the developing seedling. Jojoba cotyledonary extracts were found to inhibit the enzymic activity of trypsin, chymotrypsin, and pepsin but not the protease from Aspergillus saotoi. The heat-labile trypsin inhibitor substance(s) was found in commercially processed jojoba seed meal and the albumin fraction of seed proteins. Trypsin inhibitor activity decreased with germination. PMID:16662104

Protein cleavage strategies for an improved analysis of the membrane proteome

PubMed Central

Fischer, Frank; Poetsch, Ansgar

2006-01-01

Background Membrane proteins still remain elusive in proteomic studies. This is in part due to the distribution of the amino acids lysine and arginine, which are less frequent in integral membrane proteins and almost absent in transmembrane helices. As these amino acids are cleavage targets for the commonly used protease trypsin, alternative cleavage conditions, which should improve membrane protein analysis, were tested by in silico digestion for the three organisms Saccharomyces cerevisiae, Halobacterium sp. NRC-1, and Corynebacterium glutamicum as hallmarks for eukaryotes, archea and eubacteria. Results For the membrane proteomes from all three analyzed organisms, we identified cleavage conditions that achieve better sequence and proteome coverage than trypsin. Greater improvement was obtained for bacteria than for yeast, which was attributed to differences in protein size and GRAVY. It was demonstrated for bacteriorhodopsin that the in silico predictions agree well with the experimental observations. Conclusion For all three examined organisms, it was found that a combination of chymotrypsin and staphylococcal peptidase I gave significantly better results than trypsin. As some of the improved cleavage conditions are not more elaborate than trypsin digestion and have been proven useful in practice, we suppose that the cleavage at both hydrophilic and hydrophobic amino acids should facilitate in general the analysis of membrane proteins for all organisms. PMID:16512920

Docking, thermodynamics and molecular dynamics (MD) studies of a non-canonical protease inhibitor, MP-4, from Mucuna pruriens.

PubMed

Kumar, Ashish; Kaur, Harmeet; Jain, Abha; Nair, Deepak T; Salunke, Dinakar M

2018-01-12

Sequence and structural homology suggests that MP-4 protein from Mucuna pruriens belongs to Kunitz-type protease inhibitor family. However, biochemical assays showed that this protein is a poor inhibitor of trypsin. To understand the basis of observed poor inhibition, thermodynamics and molecular dynamics (MD) simulation studies on binding of MP-4 to trypsin were carried out. Molecular dynamics simulations revealed that temperature influences the spectrum of conformations adopted by the loop regions in the MP-4 structure. At an optimal temperature, MP-4 achieves maximal binding while above and below the optimum temperature, its functional activity is hampered due to unfavourable flexibility and relative rigidity, respectively. The low activity at normal temperature is due to the widening of the conformational spectrum of the Reactive Site Loop (RSL) that reduces the probability of formation of stabilizing contacts with trypsin. The unique sequence of the RSL enhances flexibility at ambient temperature and thus reduces its ability to inhibit trypsin. This study shows that temperature influences the function of a protein through modulation in the structure of functional domain of the protein. Modulation of function through appearance of new sequences that are more sensitive to temperature may be a general strategy for evolution of new proteins.

Selecting and expressing protective single-chain Fv fragment to stabilize L-asparaginase against inactivation by trypsin.

PubMed

Guo, L; Yan, X; Qian, S; Meng, G

2000-02-01

Four non-inhibitory specific single-chain Fv (sc Fv) fragments directed against L-asparaginase (ASNase) of Escherichia coli were selected from a synthetic phage-display scFv library. The scFv46 fragment could enhance the resistance of ASNase to trypsin proteolysis, with 70% of the initial ASNase activity present after the ASNase-scFv46 complex had been treated with trypsin for 30 min at 37 degrees C, whereas little residual activity was detected without the scFv46 fragment. The scFv46 gene was cloned to an expression vector pET-21a and expressed at high levels (about 45% of total cell protein) in E. coli BL21 (DE3) as inclusion bodies. The refolded and purified scFv46 fragment was proved to protect ASNase, and the protective effect was further confirmed by SDS/PAGE. It was found that under optimum conditions of molar ratio of scFv to ASNase, incubation time and temperature, the residual activity of the ASNase-scFv46 complex could reach about 78% after treatment with trypsin for 30 min at 37 degrees C. The results demonstrated that scFv fragments prepared by phage-antibody library technology could be used to protect target proteins.

Potentiometric determination of trypsin using a polymeric membrane polycation-sensitive electrode based on current-controlled reagent delivery.

PubMed

Chen, Yan; Ding, Jiawang; Qin, Wei

2012-12-01

A potentiometric biosensor for the determination of trypsin is described based on current-controlled reagent delivery. A polymeric membrane protamine-sensitive electrode with dinonylnaphthalene sulfonate as cation exchanger is used for in situ generation of protamine. Diffusion of protamine across the polymeric membrane can be controlled precisely by applying an external current. The hydrolysis catalyzed with trypsin in sample solution decreases the concentration of free protamine released at the sample-membrane interface and facilitates the stripping of protamine out of the membrane surface via the ion-exchange process with sodium ions from the sample solution, thus decreasing the membrane potential, by which the protease can be sensed potentiometrically. The influences of anodic current amplitude, current pulse duration and protamine concentration in the inner filling solution on the membrane potential response have been studied. Under optimum conditions, the proposed protamine-sensitive electrode is useful for continuous and reversible detection of trypsin over the concentration range of 0.5-5UmL(-1) with a detection limit of 0.3UmL(-1). The proposed detection strategy provides a rapid and reagentless way for the detection of protease activities and offers great potential in the homogeneous immunoassays using proteases as labels. Copyright © 2012 Elsevier B.V. All rights reserved.

Effect of pancreatic proteases on plasma cholecystokinin, secretin, and pancreatic exocrine secretion in response to sodium oleate.

PubMed

Li, P; Lee, K Y; Ren, X S; Chang, T M; Chey, W Y

1990-06-01

The effect of pancreatic proteases or juice on the sodium oleate-stimulated pancreatic secretion and plasma concentrations of secretin and cholecystokinin in anesthetized rats was investigated. Each rat received sodium oleate in a dose of 0.12 mmol.h-1 via a duodenal tube. Sodium oleate infusion significantly increased pancreatic secretion (volume and protein output) compared with the saline given the control group. The increase in pancreatic secretion paralleled significant elevations of plasma concentrations of secretin and cholecystokinin. To determine a possible role of pancreatic proteases on the responses induced by sodium oleate, saline, chymotrypsin, and trypsin, a combination of chymotrypsin and trypsin or pancreatic juice was infused into the duodenum. The pancreatic secretion was significantly reduced by pancreatic proteases or pancreatic juice, and the reduction paralleled the decreases in plasma concentrations of the two hormones. These agents suppressed both pancreatic secretion and plasma hormone levels in the following order of magnitude: (pancreatic juice or chymotrypsin + trypsin) greater than (trypsin) greater than (chymotrypsin). The reduction of pancreatic secretion by pancreatic proteases was reversed by intravenous administration of secretin and cholecystokinin in physiological doses. It is concluded that negative-feedback regulation of pancreatic secretion is operative in the intestinal phase in rats and that both secretin and cholecystokinin are involved in the regulation.

The human anterior lens capsule--an attempted chemical debridement of epithelial cells by ethylenediaminetetracetic acid (EDTA) and trypsin.

PubMed Central

Humphry, R C; Davies, E G; Jacob, T J; Thompson, G M

1988-01-01

The addition of edetic acid (EDTA) or trypsin to the infusion during a simulated extracapsular cataract extraction on cadaver eyes facilitates the removal of lens epithelial cells from the anterior capsule. Modification of the chemical composition of infusions used during extracapsular surgery may maximise lens epithelial cell removal and hence reduce the incidence of opacification of the posterior capsule after cataract extraction. Images PMID:3134044

Effects of enterally- and parenterally-administered bombesin on intestinal luminal tryptic activity and protein in the suckling rat.

PubMed

Pollack, P F; Adamson, C; Koldovsky, O

1989-04-15

Because of the presence of bombesin-like immunoreactivity in milk, we investigated if enteral administration of bombesin affects the intestinal luminal content of trypsin and protein in 12-14-day-old rats. Bombesin (40 micrograms/kg), given either orogastrically or subcutaneously, produced a significant elevation in the intestinal content of trypsin activity. Thus, enterally-administered bombesin can produce acute biologic effects in suckling rats.

sup 1 H assignments and secondary structure determination of the soybean trypsin/chymotrypsin Bowman-Birk inhibitor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Werner, M.H.; Wemmer, D.E.

1991-04-09

The {sup 1}H resonance assignments and secondary structure of the trypsin/chymotrypsin Bowman-Birk inhibitor from soybeans were determined by nuclear magnetic resonance spectroscopy (NMR) at 600 MHz in an 18% acetonitrile-d{sub 3}/aqueous cosolvent. Resonances from 69 to 71 amino acids were assigned sequence specifically. Residues Q11-T15 form an antiparallel {beta}-sheet with residues Q21-S25 in the tryptic inhibitory domain and an analogous region of antiparallel sheet forms between residues S38-A42 and Q48-V52 in the chymotryptic inhibitory domain. The inhibitory sites of each fragment (K16-S17 for trypsin, L43-S44 for chymotrypsin) are each part of a type VI like turn at one end ofmore » their respective region of the antiparallel {beta}-sheet. These structural elements are compared to those found in other Bowman-Birk inhibitors.« less

Screening of Caatinga plants as sources of lectins and trypsin inhibitors.

PubMed

Arcoverde, José Hélton Vasconcelos; Carvalho, Aline de Souza; Neves, Fernanda Pacífico de Almeida; Dionízio, Bianca Paiva; Pontual, Emmanuel Viana; Paiva, Patrícia Maria Guedes; Napoleão, Thiago Henrique; Correia, Maria Tereza dos Santos; da Silva, Márcia Vanusa; Carneiro-da-Cunha, Maria das Graças

2014-01-01

Although it is one of the most threatened areas in the Earth, there are few studies on the biotechnological potential of the Caatinga. This work evaluated 36 extracts from 27 Caatinga plants for lectin and trypsin inhibitor activities. The presence of lectin was detected in 77.7% of samples by haemagglutinating assay. The highest values of specific haemagglutinating activity were found in extracts of leaves from Mimosa lewesii, Bauhinia acuruana and Manilkara rufula and in branches from Myracrodruon urundeuva. Trypsin inhibitor activity was detected in 63.9% of the tested extracts, strong inhibitory effect (>70%) being found in 11 samples. This work demonstrates that Caatinga is a potential source of bioactive plant proteins that can be isolated and studied for several applications. The biochemical prospecting of Caatinga is essential for collection of bioactive principles so as to add conservation value to the region.

High feline trypsin-like immunoreactivity in a cat with pancreatitis and hepatic lipidosis.

PubMed

Bruner, J M; Steiner, J M; Williams, D A; Van Alstine, W G; Blevins, W

1997-06-15

A 1.5-year-old domestic shorthair cat was examined because of vomiting and icterus. Clinicopathologic abnormalities included high alanine transaminase, alkaline phosphatase, and gamma-glutamyltransferase activities and high total bilirubin concentration. During abdominal ultrasonography, the left limb and body of the pancreas appeared hypoechoic, and a small quantity of peritoneal effusion was seen. The liver was diffusely hyperechoic, with echogenicity similar to that of the spleen, indicating hepatic lipidosis. Feline trypsin-like immunoreactivity was high, suggesting that the cat also had pancreatitis. The cat was treated with crystalloid fluids and was fed a protein-restricted diet via a percutaneous endoscopically placed gastrostomy tube. The cat's condition continued to deteriorate despite medical treatment, and it was euthanatized. Necropsy confirmed the clinical suspicion of acute pancreatitis and hepatic lipidosis. This case suggests that measurement of trypsin-like immunoreactivity may be useful in cats suspected of having pancreatitis.

A Minimally Invasive Method for Retrieving Single Adherent Cells of Different Types from Cultures

PubMed Central

Zeng, Jia; Mohammadreza, Aida; Gao, Weimin; Merza, Saeed; Smith, Dean; Kelbauskas, Laimonas; Meldrum, Deirdre R.

2014-01-01

The field of single-cell analysis has gained a significant momentum over the last decade. Separation and isolation of individual cells is an indispensable step in almost all currently available single-cell analysis technologies. However, stress levels introduced by such manipulations remain largely unstudied. We present a method for minimally invasive retrieval of selected individual adherent cells of different types from cell cultures. The method is based on a combination of mechanical (shear flow) force and biochemical (trypsin digestion) treatment. We quantified alterations in the transcription levels of stress response genes in individual cells exposed to varying levels of shear flow and trypsinization. We report optimal temperature, RNA preservation reagents, shear force and trypsinization conditions necessary to minimize changes in the stress-related gene expression levels. The method and experimental findings are broadly applicable and can be used by a broad research community working in the field of single cell analysis. PMID:24957932

Quantitative measurement of negligible trypsin inhibitor activity and nutrient analysis of guar meal fractions.

PubMed

Lee, Jason T; Connor-Appleton, Stacey; Haq, Akram U; Bailey, Christopher A; Cartwright, Aubrey L

2004-10-20

A complete nutrient characterization of three possible products of guar bean processing does not apparently exist in the literature. Guar meal is a high-protein byproduct produced during extraction of galactomannan gum from the guar bean. During the extraction process, two fractions are produced (germ and hull). Germ and hull fractions are usually combined to form the marketed product, guar meal. Analyses characterized the nutrient, trypsin inhibitor, and galactomannan gum content of the three guar meal byproducts to determine which fraction is more valuable as an ingredient in poultry diets. Analyses indicated that the germ fraction is most appropriate for inclusion in poultry diets. Trypsin inhibitor activity previously reported as an antigrowth factor associated with guar meal was negligible and not considered to be a significant factor limiting its use in poultry feeds. Copyright 2004 American Chemical Society

A study on trypsin, Aspergillus flavus and Bacillus sp. protease inhibitory activity in Cassia tora (L.) syn Senna tora (L.) Roxb. seed extract.

PubMed

Tripathi, Vinayak R; Kumar, Shailendra; Garg, Satyendra K

2011-07-12

Proteases play an important role in virulence of many human, plant and insect pathogens. The proteinaceous protease inhibitors of plant origin have been reported widely from many plant species. The inhibitors may potentially be used for multiple therapeutic applications in viral, bacterial, fungal diseases and physiological disorders. In traditional Indian medicine system, Cassia tora (Senna tora) is reportedly effective in treatment of skin and gastrointestinal disorders. The present study explores the protease inhibitory activity of the above plant seeds against trypsin, Aspergillus flavus and Bacillus sp. proteases. The crushed seeds of Cassia tora were washed thoroughly with acetone and hexane for depigmentation and defatting. The proteins were fractionated by ammonium sulphate (0-30, 30-60, 60-90%) followed by dialysis and size exclusion chromatography (SEC). The inhibitory potential of crude seed extract and most active dialyzed fraction against trypsin and proteases was established by spot test using unprocessed x-ray film and casein digestion methods, respectively. Electrophoretic analysis of most active fraction (30-60%) and SEC elutes were carried employing Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Gelatin SDS-PAGE. Inhibition of fungal spore germination was studied in the presence of dialyzed active inhibitor fraction. Standard deviation (SD) and ANOVA were employed as statistical tools. The crude seeds' extract displayed strong antitryptic, bacterial and fungal protease inhibitory activity on x-ray film. The seed protein fraction 30-60% was found most active for trypsin inhibition in caseinolytic assay (P < 0.001). The inhibition of caseinolytic activity of the proteases increased with increasing ratio of seed extract. The residual activity of trypsin, Aspergillus flavus and Bacillus sp. proteases remained only 4, 7 and 3.1%, respectively when proteases were incubated with 3 mg ml-1 seed protein extract for 60 min. The inhibitory activity was evident in gelatin SDS-PAGE where a major band (~17-19 kD) of protease inhibitor (PI) was detected in dialyzed and SEC elute. The conidial germination of Aspergillus flavus was moderately inhibited (30%) by the dialyzed seed extract. Cassia tora seed extract has strong protease inhibitory activity against trypsin, Aspergillus flavus and Bacillus sp. proteases. The inhibitor in Cassia tora may attenuate microbial proteases and also might be used as phytoprotecting agent. © 2011 Tripathi et al; licensee BioMed Central Ltd.

Trypsin-protease activated receptor-2 signaling contributes to pancreatic cancer pain

PubMed Central

Zhu, Jiao; Miao, Xue-Rong; Tao, Kun-Ming; Zhu, Hai; Liu, Zhi-Yun; Yu, Da-Wei; Chen, Qian-Bo; Qiu, Hai-Bo; Lu, Zhi-Jie

2017-01-01

Pain treatment is a critical aspect of pancreatic cancer patient clinical care. This study investigated the role of trypsin-protease activated receptor-2 (PAR-2) in pancreatic cancer pain. Pancreatic tissue samples were collected from pancreatic cancer (n=22) and control patients (n=22). Immunofluorescence analyses confirmed colocalization of PAR-2 and neuronal markers in pancreatic cancer tissues. Trypsin levels and protease activities were higher in pancreatic cancer tissue specimens than in the controls. Supernatants from cultured human pancreatic cancer tissues (PC supernatants) induced substance P and calcitonin gene-related peptide release in dorsal root ganglia (DRG) neurons, and FS-NH2, a selective PAR-2 antagonist, inhibited this effect. A BALB/c nude mouse orthotopic tumor model was used to confirm the role of PAR-2 signaling in pancreatic cancer visceral pain, and male Sprague-Dawley rats were used to assess ambulatory pain. FS-NH2 treatment decreased hunch scores, mechanical hyperalgesia, and visceromotor reflex responses in tumor-bearing mice. In rats, subcutaneous injection of PC supernatant induced pain behavior, which was alleviated by treatment with FS-NH2 or FUT-175, a broad-spectrum serine protease inhibitor. Our findings suggest that trypsin-PAR-2 signaling contributes to pancreatic cancer pain in vivo. Treatment strategies targeting PAR-2 or its downstream signaling molecules might effectively relieve pancreatic cancer pain. PMID:28977906

Peptide code-on-a-microplate for protease activity analysis via MALDI-TOF mass spectrometric quantitation.

PubMed

Hu, Junjie; Liu, Fei; Ju, Huangxian

2015-04-21

A peptide-encoded microplate was proposed for MALDI-TOF mass spectrometric (MS) analysis of protease activity. The peptide codes were designed to contain a coding region and the substrate of protease for enzymatic cleavage, respectively, and an internal standard method was proposed for the MS quantitation of the cleavage products of these peptide codes. Upon the cleavage reaction in the presence of target proteases, the coding regions were released from the microplate, which were directly quantitated by using corresponding peptides with one-amino acid difference as the internal standards. The coding region could be used as the unique "Protease ID" for the identification of corresponding protease, and the amount of the cleavage product was used for protease activity analysis. Using trypsin and chymotrypsin as the model proteases to verify the multiplex protease assay, the designed "Trypsin ID" and "Chymotrypsin ID" occurred at m/z 761.6 and 711.6. The logarithm value of the intensity ratio of "Protease ID" to internal standard was proportional to trypsin and chymotrypsin concentration in a range from 5.0 to 500 and 10 to 500 nM, respectively. The detection limits for trypsin and chymotrypsin were 2.3 and 5.2 nM, respectively. The peptide-encoded microplate showed good selectivity. This proposed method provided a powerful tool for convenient identification and activity analysis of multiplex proteases.

Sporamin-mediated resistance to beet cyst nematodes (Heterodera schachtii Schm.) is dependent on trypsin inhibitory activity in sugar beet (Beta vulgaris L.) hairy roots.

PubMed

Cai, Daguang; Thurau, Tim; Tian, Yanyan; Lange, Tina; Yeh, Kai-Wun; Jung, Christian

2003-04-01

Sporamin, a sweet potato tuberous storage protein, is a Kunitz-type trypsin inhibitor. Its capability of conferring insect-resistance on transgenic tobacco and cauliflower has been confirmed. To test its potential as an anti-feedant for the beet cyst nematode (Heterodera schachtii Schm.), the sporamin gene SpTI-1 was introduced into sugar beet (Beta vulgaris L.) by Agrobacterium rhizogenes-mediated transformation. Twelve different hairy root clones expressing sporamin were selected for studying nematode development. Of these, 8 hairy root clones were found to show significant efficiency in inhibiting the growth and development of the female nematodes whereas 4 root clones did not show any inhibitory effects even though the SpTI-1 gene was regularly expressed in all of the tested hairy roots as revealed by northern and western analyses. Inhibition of nematode development correlated with trypsin inhibitor activity but not with the amount of sporamin expressed in hairy roots. These data demonstrate that the trypsin inhibitor activity is the critical factor for inhibiting growth and development of cyst nematodes in sugar beet hairy roots expressing the sporamin gene. Hence, the sweet potato sporamin can be used as a new and effective anti-feedant for controlling cyst nematodes offering an alternative strategy for establishing nematode resistance in crops.

A Novel Trypsin Inhibitor-Like Cysteine-Rich Peptide from the Frog Lepidobatrachus laevis Containing Proteinase-Inhibiting Activity.

PubMed

Wang, Yu-Wei; Tan, Ji-Min; Du, Can-Wei; Luan, Ning; Yan, Xiu-Wen; Lai, Ren; Lu, Qiu-Min

2015-08-01

Various bio-active substances in amphibian skins play important roles in survival of the amphibians. Many protease inhibitor peptides have been identified from amphibian skins, which are supposed to negatively modulate the activity of proteases to avoid premature degradation or release of skin peptides, or to inhibit extracellular proteases produced by invading bacteria. However, there is no information on the proteinase inhibitors from the frog Lepidobatrachus laevis which is unique in South America. In this work, a cDNA encoding a novel trypsin inhibitor-like (TIL) cysteine-rich peptide was identified from the skin cDNA library of L. laevis. The 240-bp coding region encodes an 80-amino acid residue precursor protein containing 10 half-cysteines. By sequence comparison and signal peptide prediction, the precursor was predicted to release a 55-amino acid mature peptide with amino acid sequence, IRCPKDKIYKFCGSPCPPSCKDLTPNCIAVCKKGCFCRDGTVDNNHGKCVKKENC. The mature peptide was named LL-TIL. LL-TIL shares significant domain similarity with the peptides from the TIL supper family. Antimicrobial and trypsin-inhibitory abilities of recombinant LL-TIL were tested. Recombinant LL-TIL showed no antimicrobial activity, while it had trypsin-inhibiting activity with a Ki of 16.5178 μM. These results suggested there was TIL peptide with proteinase-inhibiting activity in the skin of frog L. laevis. To the best of our knowledge, this is the first report of TIL peptide from frog skin.

Dietary protein hydrolysate and trypsin inhibitor effects on digestive capacities and performances during early-stages of spotted wolffish: suggested mechanisms.

PubMed

Savoie, A; Le François, N R; Lamarre, S G; Blier, P U; Beaulieu, L; Cahu, C

2011-04-01

Growth rate is dependent upon adequate provision of amino acids especially in newly-hatched fish which experience very high growth rate. The replacement of a fraction of protein content by partially hydrolyzed (pre-digested) proteins was carried out and the digestive capacities and performances of larval/juvenile spotted wolffish (Anarhichas minor) were measured. The goal of this study was to verify whether the scope for growth is principally dictated by the proteolytic capacity of the digestive system by examining the effect of protein hydrolysates (PH) and trypsin inhibitor dietary inclusion on protein digestion/assimilation capacities, growth and survival. Four experimental diets were examined: C (control) I (supplemented with 750 mg/kg soybean trypsin inhibitor (SBTI)) H (supplemented with 20% PH) and HI (supplemented with 20% PH and 750 mg/kg SBTI). Protein hydrolysate supplementation gave significantly higher body mass than control at day 15 post-hatching. Unexpectedly, at day 30 and 60, fish administered diet HI (containing trypsin inhibitor) were heavier than the other groups. Suggested mechanisms are presented and discussed. The main conclusions of this study are that wolffish larval stage lasts roughly 15 days and that juvenile growth is linked to proteolytic capacity, but also very likely to absorption capacity of peptides and amino acids. Copyright © 2011 Elsevier Inc. All rights reserved.

A Trypsin Inhibitor from Tamarind Reduces Food Intake and Improves Inflammatory Status in Rats with Metabolic Syndrome Regardless of Weight Loss.

PubMed

Carvalho, Fabiana M C; Lima, Vanessa C O; Costa, Izael S; Medeiros, Amanda F; Serquiz, Alexandre C; Lima, Maíra C J S; Serquiz, Raphael P; Maciel, Bruna L L; Uchôa, Adriana F; Santos, Elizeu A; Morais, Ana H A

2016-09-27

Trypsin inhibitors are studied in a variety of models for their anti-obesity and anti-inflammatory bioactive properties. Our group has previously demonstrated the satietogenic effect of tamarind seed trypsin inhibitors (TTI) in eutrophic mouse models and anti-inflammatory effects of other trypsin inhibitors. In this study, we evaluated TTI effect upon satiety, biochemical and inflammatory parameters in an experimental model of metabolic syndrome (MetS). Three groups of n = 5 male Wistar rats with obesity-based MetS received for 10 days one of the following: (1) Cafeteria diet; (2) Cafeteria diet + TTI (25 mg/kg); and (3) Standard diet. TTI reduced food intake in animals with MetS. Nevertheless, weight gain was not different between studied groups. Dyslipidemia parameters were not different with the use of TTI, only the group receiving standard diet showed lower very low density lipoprotein (VLDL) and triglycerides (TG) (Kruskal-Wallis, p < 0.05). Interleukin-6 (IL-6) production did not differ between groups. Interestingly, tumor necrosis factor-alpha (TNF-α) was lower in animals receiving TTI. Our results corroborate the satietogenic effect of TTI in a MetS model. Furthermore, we showed that TTI added to a cafeteria diet may decrease inflammation regardless of weight loss. This puts TTI as a candidate for studies to test its effectiveness as an adjuvant in MetS treatment.

A Trypsin Inhibitor from Tamarind Reduces Food Intake and Improves Inflammatory Status in Rats with Metabolic Syndrome Regardless of Weight Loss

PubMed Central

Carvalho, Fabiana M. C.; Lima, Vanessa C. O.; Costa, Izael S.; Medeiros, Amanda F.; Serquiz, Alexandre C.; Lima, Maíra C. J. S.; Serquiz, Raphael P.; Maciel, Bruna L. L.; Uchôa, Adriana F.; Santos, Elizeu A.; Morais, Ana H. A.

2016-01-01

Trypsin inhibitors are studied in a variety of models for their anti-obesity and anti-inflammatory bioactive properties. Our group has previously demonstrated the satietogenic effect of tamarind seed trypsin inhibitors (TTI) in eutrophic mouse models and anti-inflammatory effects of other trypsin inhibitors. In this study, we evaluated TTI effect upon satiety, biochemical and inflammatory parameters in an experimental model of metabolic syndrome (MetS). Three groups of n = 5 male Wistar rats with obesity-based MetS received for 10 days one of the following: (1) Cafeteria diet; (2) Cafeteria diet + TTI (25 mg/kg); and (3) Standard diet. TTI reduced food intake in animals with MetS. Nevertheless, weight gain was not different between studied groups. Dyslipidemia parameters were not different with the use of TTI, only the group receiving standard diet showed lower very low density lipoprotein (VLDL) and triglycerides (TG) (Kruskal–Wallis, p < 0.05). Interleukin-6 (IL-6) production did not differ between groups. Interestingly, tumor necrosis factor-alpha (TNF-α) was lower in animals receiving TTI. Our results corroborate the satietogenic effect of TTI in a MetS model. Furthermore, we showed that TTI added to a cafeteria diet may decrease inflammation regardless of weight loss. This puts TTI as a candidate for studies to test its effectiveness as an adjuvant in MetS treatment. PMID:27690087

Role of Proteases in Extra-Oral Digestion of a Predatory Bug, Andrallus spinidens

PubMed Central

Zibaee, Arash; Hoda, Hassan; Mahmoud, Fazeli-Dinan

2012-01-01

Roles of salivary proteases in the extra-oral digestion of the predatory bug, Andrallus spinidens Fabricius (Hemiptera: Pentatomidae) were studied by using 2% azocasein as a general substrate and specific protease substrates, as well as synthetic and endogenous inhibitors. It was found that salivary glands of A. spinidens have two anterior, two lateral, and two posterior lobes. Azocasein was used to measure the activity of general proteases in the salivary glands using different buffer solutions. The enzyme had the highest activity at pH 8. General protease activity was highest at 40 °C and was stable for 6–16 hours. The use of specific substrates showed that trypsin-like, chymotrypsin-like, aminopeptidase, and carboxypeptidase are the active proteases present in salivary glands, by the maximum activity of trypsin-like protease in addition to their optimal pH between 8–9. Ca2+ and Mg2+ increased proteolytic activity about 216%, while other ions decreased it. Specific inhibitors including SBTI, PMSF, TLCK, and TPCK significantly decreased enzyme activity, as well as the specific inhibitors of methalloproteases including phenanthroline, EGTA, and TTHA. Extracted endogenous trypsin inhibitors extracted from potential prey, Chilo suppressalis, Naranga aenescens, Pieris brassicae, Hyphantria cunea, and Ephestia kuhniella, had different effects on trypsin-like protease activity of A. spinidens salivary glands. With the exception of C. suppressalis, the endogenous inhibitors significantly decreased enzyme activity in A. spinidens. PMID:22954419

Comparison of Conventional and Microwave Treatment on Soymilk for Inactivation of Trypsin Inhibitors and In Vitro Protein Digestibility

PubMed Central

Vagadia, Brinda Harish; Raghavan, Vijaya

2018-01-01

Soymilk is lower in calories compared to cow’s milk, since it is derived from a plant source (no cholesterol) and is an excellent source of protein. Despite the beneficial factors, soymilk is considered as one of the most controversial foods in the world. It contains serine protease inhibitors which lower its nutritional value and digestibility. Processing techniques for the elimination of trypsin inhibitors and lipoxygenase, which have shorter processing time and lower production costs are required for the large-scale manufacturing of soymilk. In this study, the suitable conditions of time and temperature are optimized during microwave processing to obtain soymilk with maximum digestibility with inactivation of trypsin inhibitors, in comparison to the conventional thermal treatment. The microwave processing conditions at a frequency of 2.45 GHz and temperatures of 70 °C, 85 °C and 100 °C for 2, 5 and 8 min were investigated and were compared to conventional thermal treatments at the same temperature for 10, 20 and 30 min. Response surface methodology is used to design and optimize the experimental conditions. Thermal processing was able to increase digestibility by 7% (microwave) and 11% (conventional) compared to control, while trypsin inhibitor activity reduced to 1% in microwave processing and 3% in conventional thermal treatment when compared to 10% in raw soybean. PMID:29316679

Dynamic Seeding of Perfusing Human Umbilical Vein Endothelial Cells (HUVECs) onto Dual-Function Cell Adhesion Ligands: Arg-Gly-Asp (RGD)-Streptavidin and Biotinylated Fibronectin

PubMed Central

Anamelechi, Charles C.; Clermont, Edward C.; Novak, Matthew T.; Reichert, William M.

2014-01-01

Surfaces decorated with high affinity ligands can be used to facilitate rapid attachment of endothelial cells; however, standard equilibrium cell detachment studies are poorly suited for assessing these initial adhesion events. Here, a dynamic seeding and cell retention method was used to examine the initial attachment of perfusing human umbilical vein endothelial cells (HUVECs) to bare Teflon-AF substrates, substrates pre-adsorbed with fibronectin alone, or substrates co-pre-adsorbed with two dual-function cell-adhesion ligands: biotinylated fibronectin (bFN) and RGD-streptavidin mutant (RGD-SA). Cell attachment was evaluated as a function of cell trypsinization (integrin digestion), surface protein formulation, and cell perfusion rate. Surfaces co-pre-adsorbed with bFN and RGD-SA showed the highest density of attached cells after 8 min of perfusion and the highest percent retention when subjected to shear flow at 60 dynes/cm2 for 2 min. Surfaces with no ligand treatment showed the lowest cell attachment and retention under flow in all cases. HUVECs trypsinized with mild 0.025% trypsin/ethylenediaminetetraacetic acid (EDTA) showed greater cell adhesion after perfusion and higher percent retention after shear flow than those trypsinized using harsher 0.05% trypsin/EDTA. The preferential affinities of the two dual-function ligands for α5β1 and αvβ3 integrins were also examined by surface plasmon resonance (SPR) spectroscopy. The dynamic cell seeding studies confirmed that the dual-function ligand system promotes HUVEC adhesion and retention at short time points when tested using a perfusion assay. SPR studies showed that the two ligands exhibited equal affinity for both α5β1 and αvβ3 integrins but that the combined ligands bound more total integrins than the two ligands tested separately. PMID:19348476

Both epsilon-toxin and beta-toxin are important for the lethal properties of Clostridium perfringens type B isolates in the mouse intravenous injection model.

PubMed

Fernandez-Miyakawa, Mariano E; Fisher, Derek J; Poon, Rachael; Sayeed, Sameera; Adams, Vicki; Rood, Julian I; McClane, Bruce A; Uzal, Francisco A

2007-03-01

Clostridium perfringens is capable of producing up to 15 toxins, including alpha-toxin (CPA), beta-toxin (CPB), epsilon-toxin (ETX), enterotoxin, beta2-toxin (CPB2), and perfringolysin O. Type B isolates, which must produce CPA, CPB, and ETX, are associated with animal illnesses characterized by sudden death or acute neurological signs, with or without intestinal damage. Type B pathogenesis in ruminants is poorly understood, with some animals showing lesions and clinical signs similar to those caused by either type C or type D infections. It is unknown whether host or environmental conditions are dominant for determining the outcome of type B disease or if disease outcomes are determined by variable characteristics of type B isolates. To help clarify this issue, 19 type B isolates were evaluated for toxin production during late-log-phase growth via quantitative Western blotting and by biological activity assays. Most type B isolates produced CPB levels similar to those produced by type C isolates in vitro and have the potential to produce genotype C-like disease. The lethality of type B isolate supernatants administered intravenously to mice was evaluated with or without prior trypsin treatment, and monoclonal antibody neutralization studies also were performed. Correlation analyses comparing toxin levels in type B supernatants versus lethality and neutralization studies both found that the main contributor to lethality without pretreatment with trypsin was CPB, whereas neutralization studies indicated that CPB and ETX were both important after trypsin pretreatment. At least part of the CPB produced by type B isolates remained active after trypsin treatment. However, the overall lethalities of most supernatants were lower after trypsin pretreatment. Also, there was a significant association between ETX, CPB2, and CPA production in vitro among type B isolates. However, our results suggest that both CPB and ETX are likely the most important contributors to the pathogenesis of C. perfringens type B infections in domestic animals.

The spinal inhibition of N-type voltage-gated calcium channels selectively prevents scratching behavior in mice.

PubMed

Maciel, I S; Azevedo, V M; Pereira, T C; Bogo, M R; Souza, A H; Gomez, M V; Campos, M M

2014-09-26

The present study investigated the effects of pharmacological spinal inhibition of voltage-gated calcium channels (VGCC) in mouse pruritus. The epidural administration of P/Q-type MVIIC or PhTx3.3, L-type verapamil, T-type NNC 55-0396 or R-type SNX-482 VGCC blockers failed to alter the scratching behavior caused by the proteinase-activated receptor 2 (PAR-2) activator trypsin, injected into the mouse nape skin. Otherwise, trypsin-elicited pruritus was markedly reduced by the spinal administration of preferential N-type VGCC inhibitors MVIIA and Phα1β. Time-course experiments revealed that Conus magus-derived toxin MVIIA displayed significant effects when dosed from 1h to 4h before trypsin, while the anti-pruritic effects of Phα1β from Phoneutria nigriventer remained significant for up to 12h. In addition to reducing trypsin-evoked itching, MVIIA or Phα1β also prevented the itching elicited by intradermal (i.d.) injection of SLIGRL-NH2, compound 48/80 or chloroquine, although they did not affect H2O2-induced scratching behavior. Furthermore, the co-administration of MVIIA or Phα1β markedly inhibited the pruritus caused by the spinal injection of gastrin-releasing peptide (GRP), but not morphine. Notably, the epidural administration of MVIIA or Phα1β greatly prevented the chronic pruritus allied to dry skin model. However, either tested toxin failed to alter the edema formation or neutrophil influx caused by trypsin, whereas they significantly reduced the c-Fos activation in laminas I, II and III of the spinal cord. Our data bring novel evidence on itching transmission mechanisms, pointing out the therapeutic relevance of N-type VGCC inhibitors to control refractory pruritus. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

Studies on the regioselectivity and kinetics of the action of trypsin on proinsulin and its derivatives using mass spectrometry.

PubMed

Gardner, Qurra-tul-Ann Afza; Younas, Hooria; Akhtar, Muhammad

2013-01-01

Human M-proinsulin was cleaved by trypsin at the R(31)R(32)-E(33) and K(64)R(65)-G(66) bonds (B/C and C/A junctions), showing the same cleavage specificity as exhibited by prohormone convertases 1 and 2 respectively. Buffalo/bovine M-proinsulin was also cleaved by trypsin at the K(59)R(60)-G(61) bond but at the B/C junction cleavage occurred at the R(31)R(32)-E(33) as well as the R(31)-R(32)E(33) bond. Thus, the human isoform in the native state, with a 31 residue connecting C-peptide, seems to have a unique structure around the B/C and C/A junctions and cleavage at these sites is predominantly governed by the structure of the proinsulin itself. In the case of both the proinsulin species the cleavage at the B/C junction was preferred (65%) over that at the C/A junction (35%) supporting the earlier suggestion of the presence of some form of secondary structure at the C/A junction. Proinsulin and its derivatives, as natural substrates for trypsin, were used and mass spectrometric analysis showed that the k(cat.)/K(m) values for the cleavage were most favourable for the scission of the bonds at the two junctions (1.02±0.08×10(5)s(-1)M(-1)) and the cleavage of the K(29)-T(30) bond of M-insulin-RR (1.3±0.07×10(5)s(-1)M(-1)). However, the K(29)-T(30) bond in M-insulin, insulin as well as M-proinsulin was shielded from attack by trypsin (k(cat.)/K(m) values around 1000s(-1)M(-1)). Hence, as the biosynthetic path follows the sequence; proinsulin→insulin-RR→insulin, the K(29)-T(30) bond becomes shielded, exposed then shielded again respectively. Copyright © 2012 Elsevier B.V. All rights reserved.

Protection from oxidative inactivation of the 20S proteasome by heat-shock protein 90.

PubMed Central

Conconi, M; Petropoulos, I; Emod, I; Turlin, E; Biville, F; Friguet, B

1998-01-01

Heat-shock protein 90 (Hsp 90) has been implicated in both protection against oxidative inactivation and inhibition of the multicatalytic proteinase (MCP, also known as 20 S proteasome). We report here that the protective and inhibitory effects of Hsp 90 depend on the activation state of the proteasome. Hsp 90 (and also alpha-crystallin) inhibits the N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activity (Cbz=benzyloxycarbonyl; MCA=7-amido-4-methylcoumarin) when the rat liver MCP is in its latent form, but no inhibitory effects are observed when the MCP is in its active form. Metal-catalysed oxidation of the active MCP inactivates the Ala-Ala-Phe-MCA-hydrolysing (chymotrypsin-like), N-Boc-Leu-Ser-Thr-Arg-MCA-hydrolysing (trypsin-like; Boc=t-butyloxycarbonyl), N-Cbz-Leu-Leu-Glu-beta-naphthylamine-hydrolysing (peptidylglutamyl-peptide hydrolase) and N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activities, whereas these activities are actually increased when the MCP is in its latent form. Hsp 90 protects against oxidative inactivation of the trypsin-like and N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activities of the MCP active form, and alpha-crystallin protects the trypsin-like activity. The specificity of the Hsp 90-mediated protection was assessed by a quantitative analysis of the two-dimensional electrophoretic pattern of MCP subunits before and after oxidation of the MCP, in the presence or absence of Hsp 90. Treatment of the FAO hepatoma cell line with iron and ascorbate was found to inactivate the MCP. Hsp 90 overexpression obtained by challenging the cells with iron was associated with a decreased susceptibility to oxidative inactivation of the MCP trypsin-like activity. Depletion of Hsp 90 by using antisense oligonucleotides resulted in an increased susceptibility to oxidative inactivation of the MCP trypsin-like activity, providing evidence for the physiological relevance of Hsp 90-mediated protection of the MCP. PMID:9657982

Protection from oxidative inactivation of the 20S proteasome by heat-shock protein 90.

PubMed

Conconi, M; Petropoulos, I; Emod, I; Turlin, E; Biville, F; Friguet, B

1998-07-15

Heat-shock protein 90 (Hsp 90) has been implicated in both protection against oxidative inactivation and inhibition of the multicatalytic proteinase (MCP, also known as 20 S proteasome). We report here that the protective and inhibitory effects of Hsp 90 depend on the activation state of the proteasome. Hsp 90 (and also alpha-crystallin) inhibits the N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activity (Cbz=benzyloxycarbonyl; MCA=7-amido-4-methylcoumarin) when the rat liver MCP is in its latent form, but no inhibitory effects are observed when the MCP is in its active form. Metal-catalysed oxidation of the active MCP inactivates the Ala-Ala-Phe-MCA-hydrolysing (chymotrypsin-like), N-Boc-Leu-Ser-Thr-Arg-MCA-hydrolysing (trypsin-like; Boc=t-butyloxycarbonyl), N-Cbz-Leu-Leu-Glu-beta-naphthylamine-hydrolysing (peptidylglutamyl-peptide hydrolase) and N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activities, whereas these activities are actually increased when the MCP is in its latent form. Hsp 90 protects against oxidative inactivation of the trypsin-like and N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activities of the MCP active form, and alpha-crystallin protects the trypsin-like activity. The specificity of the Hsp 90-mediated protection was assessed by a quantitative analysis of the two-dimensional electrophoretic pattern of MCP subunits before and after oxidation of the MCP, in the presence or absence of Hsp 90. Treatment of the FAO hepatoma cell line with iron and ascorbate was found to inactivate the MCP. Hsp 90 overexpression obtained by challenging the cells with iron was associated with a decreased susceptibility to oxidative inactivation of the MCP trypsin-like activity. Depletion of Hsp 90 by using antisense oligonucleotides resulted in an increased susceptibility to oxidative inactivation of the MCP trypsin-like activity, providing evidence for the physiological relevance of Hsp 90-mediated protection of the MCP.

Growth of Foot-and-Mouth Disease Virus in Dispersed Tissue Cells

PubMed Central

Patty, R. E.; Tozzini, F.; Seibold, H. R.; Callis, J. J.

1962-01-01

Methods are described for rapid and economical production of large quantities of foot-and-mouth disease virus in stationary cultures of trypsin-dispersed bovine kidney cells in a simple medium. Yields of between 107 and 108 plaque-forming units per milliliter were obtained from serum-free cultures containing approximately a million and a half viable trypsin-dispersed cells per milliliter. Some of the advantages and disadvantages of these methods of virus production are discussed. ImagesFig. 1Fig. 2Fig. 3 PMID:17649388

Effects of gamma radiation on total phenolics, trypsin and tannin inhibitors in soybean grains

NASA Astrophysics Data System (ADS)

de Toledo, T. C. F.; Canniatti-Brazaca, S. G.; Arthur, V.; Piedade, S. M. S.

2007-10-01

The objective was determining possible radiation-induced alterations (with doses of 2, 4 and 8 kGy) in raw or cooked grains from five soybean cultivars through the analysis of some antinutrient. Total phenolic ranged from 2.46 to 10.83 mg/g, the trypsin inhibited from 18.19 to 71.64 UTI/g and tannins from 0.01 to 0.39 mg/g. All the antinutrient studied underwent reduction with increases in the doses and cooking process was effective too.

Mouse hepatitis virus immunofluorescence in formalin- or Bouin's-fixed tissues using trypsin digestion.

PubMed

Brownstein, D G; Barthold, S W

1982-02-01

Mouse hepatitis viral antigens were demonstrated by immunofluorescence in formalin- and Bouin's-fixed tissues processed routinely for histopathology followed by partial digestion with trypsin. Staining was superior in tissues fixed in formalin and was not diminished in tissue sections from paraffin blocks stored at room temperature as long as 2 years. The relative ease of this procedure and the commercial availability of reagents makes this a useful technique for the definitive diagnosis of mouse hepatitis virus infection.

Mechanisms implicated in the effects of boron on wound healing.

PubMed

Nzietchueng, Rosine Mayap; Dousset, Brigitte; Franck, Patricia; Benderdour, Mohamed; Nabet, Pierre; Hess, Ketsia

2002-01-01

Recently, we demonstrated that boron modulates the turnover of the extracellular matrix and increases TNFalpha release. In the present study, we used an in vitro test to investigate the direct effect of boron on specific enzymes (elastase, trypsin-like enzymes, collagenase and alkaline phosphatase) implicated in extracellular matrix turnover. Boron decreased the elastase and alkaline phosphatase activity, but had no effect on trypsin and collagenase activities. The effect of boron on the enzyme activities was also tested in fibroblasts considered as an in vivo test. In contrast to the results obtained in vitro, boron enhanced the trypsin-like, collagenase, and cathepsin D activities in fibroblasts. Boron did not modify the generation of free radicals compared to the control and did not seem to act on the intracellular alkaline phosphatase activity, However, as it did enhance phosphorylation, it can be hypothesized that boron may affect living cells via a mediator, which could be TNFalpha whose transduction signal involves a cascade of phosphorylations.

Isolation and identification of antioxidant peptides from enzymatically hydrolyzed rice bran protein.

PubMed

Wattanasiritham, Ladda; Theerakulkait, Chockchai; Wickramasekara, Samanthi; Maier, Claudia S; Stevens, Jan F

2016-02-01

Khao Dawk Mali 105 rice bran protein (RBP) was fractionated into albumin (12.5%), globulin (13.9%), glutelin (70.8%) and prolamine (2.9%). The native and denatured RBP fractions were hydrolyzed with papain and trypsin for 3h at optimum conditions. The RBP fractions and their hydrolysates were evaluated for their antioxidant activity by the Oxygen Radical Absorbance Capacity (ORAC) assay. The trypsin-hydrolyzed denatured albumin exhibited the highest antioxidant activity with an ORAC value of 4.07 μmol of Trolox equivalent (TE)/mg protein. This hydrolysate was separated by using RP-HPLC and three fractions with high antioxidant activity were examined by LTQ-FTICR ESI mass spectrometry. The MW of the peptides from these fractions were 800-2100 Da. and consisted of 6-21 amino acid residues. Most of the peptides from the fractions demonstrated typical characteristics of well-known antioxidant peptides. The results suggest that trypsin-hydrolyzed denatured rice bran albumin might be useful as a natural food antioxidant. Copyright © 2015 Elsevier Ltd. All rights reserved.

'Click Chemistry' in the preparation of porous polymer-basedparticulate stationary phases for mu-HPLC separation of peptides andproteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Slater, Michael; Snauko, Marian; Svec, Frantisek

With the use of the copper(I)-catalyzed (3 + 2) azide-alkynecycloaddition, an element of "click chemistry," stationary phasescarrying long alkyl chains or soybean trypsin inhibitor have beenprepared for use in HPLC separations in the reversed-phase and affinitymodes, respectively. The ligands were attached via a triazole ring tosize monodisperse porous beads containing either alkyne or azide pendantfunctionalities. Alkyne-containing beads prepared by directcopolymerization of propargyl acrylate with ethylene dimethacrylate wereallowed to react with azidooctadecane to give a reversed-phase sorbent.Azide-functionalized beads were prepared by chemical modification ofglycidyl methacrylate particles. Subsequent reaction with a terminalaliphatic alkyne produced a reversed-phase sorbent similar to thatobtained from themore » alkyne beads. Soybean trypsin inhibitor wasfunctionalized with N-(4-pentynoyloxy) succinimide to carry alkyne groupsand then allowed to react with the azide-containing beads to produce anaffinity sorbent for trypsin. The performance of these stationary phaseswas demonstrated with the HPLC separations of a variety of peptides andproteins.« less

Separation and purification of enzymes by continuous pH-parametric pumping

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, S.Y.; Lin, C.K.; Juang, L.Y.

1985-10-01

Trypsin and chymotrypsin were separated from porcine pancreas extract by continuous pH-parametric pumping. CHOM (chicken ovomucoid) was convalently bound to laboratory-prepared crab chitin with glutaraldehyde to form an affinity adsorbent of trypsin. The pH levels of top and bottom feeds were 8.0 and 2.5, respectively. Similar inhibitor, DKOM (duck ovomucoid), and pH levels 8.0 and 2.0 for top and bottom feeds, respectively, were used for separation and purification of chymotrypsin. e-Amino caproyl-D-tryptophan methyl ester was coupled to chitosan to form an affinity adsorbent for stem bromelain. The pH levels were 8.7 and 3.0. Separation continued fairly well with high yield,more » e.g., 95% recovery of trypsin after continuous pumping of 10 cycles. Optimum operational conditions for concentration and purification of these enzymes were investigated. The results showed that the continuous pH-parametric pumping coupled with affinity chromatography is effective for concentration and purification of enzymes. 19 references.« less

The intracellular carboxyl tail of the PAR-2 receptor controls intracellular signaling and cell death.

PubMed

Zhu, Zhihui; Stricker, Rolf; Li, Rong yu; Zündorf, Gregor; Reiser, Georg

2015-03-01

The protease-activated receptors are a group of unique G protein-coupled receptors, including PAR-1, PAR-2, PAR-3 and PAR-4. PAR-2 is activated by multiple trypsin-like serine proteases, including trypsin, tryptase and coagulation proteases. The clusters of phosphorylation sites in the PAR-2 carboxyl tail are suggested to be important for the binding of adaptor proteins to initiate intracellular signaling to Ca(2+) and mitogen-activated protein kinases. To explore the functional role of PAR-2 carboxyl tail in controlling intracellular Ca(2+), ERK and AKT signaling, a series of truncated mutants containing different clusters of serines/threonines were generated and expressed in HEK293 cells. Firstly, we observed that lack of the complete C-terminus of PAR-2 in a mutated receptor gave a relatively low level of localization on the cell plasma membrane. Secondly, the shortened carboxyl tail containing 13 amino acids was sufficient for receptor internalization. Thirdly, the cells expressing truncation mutants showed deficits in their capacity to couple to intracellular Ca(2+) and ERK and AKT signaling upon trypsin challenge. In addition, HEK293 cells carrying different PAR-2 truncation mutants displayed decreased levels of cell survival after long-lasting trypsin stimulation. In summary, the PAR-2 carboxyl tail was found to control the receptor localization, internalization, intracellular Ca(2+) responses and signaling to ERK and AKT. The latter can be considered to be important for cell death control.

ELISA analysis of soybean trypsin inhibitors in processed foods.

PubMed

Brandon, D L; Bates, A H; Friedman, M

1991-01-01

Soybean proteins are widely used in human foods in a variety of forms, including infant formulas, flour, protein concentrates, protein isolates, soy sauces, textured soy fibers, and tofu. The presence of inhibitors of digestive enzymes in soy proteins impairs the nutritional quality and possibly the safety of soybeans and other legumes. Processing, based on the use of heat or fractionation of protein isolates, does not completely inactivate or remove these inhibitors, so that residual amounts of inhibitors are consumed by animals and humans. New monoclonal antibody-based immunoassays can measure low levels of the soybean Kunitz trypsin inhibitor (KTI) and the Bowman-Birk trypsin and chymotrypsin inhibitor (BBI) and the Bowman-Birk foods. The enzyme-linked immunosorbent assay (ELISA) was used to measure the inhibitor content of soy concentrates, isolates, and flours, both heated and unheated; a commercial soy infant formula; KTI and BBI with rearranged disulfide bonds; browning products derived from heat-treatment of KTI with glucose and starch; and KTI exposed to high pH. The results indicate that even low inhibitor isolates contain significant amounts of specific inhibitors. Thus, infants on soy formula consume about 10 mg of KTI plus BBI per day. The immunoassays complement the established enzymatic assays of trypsin and chymotrypsin inhibitors, and have advantages in (a) measuring low levels of inhibitors in processed foods; and (b) differentiating between the Kunitz and Bowman-Birk inhibitors. The significance of our findings for food safety are discussed.

Effects of black-eyed pea trypsin/chymotrypsin inhibitor on proteolytic activity and on development of Anthonomus grandis.

PubMed

Franco, Octávio L; dos Santos, Roseane C; Batista, João A N; Mendes, Ana Cristina M; de Araújo, Marcus Aurélio M; Monnerat, Rose G; Grossi-de-Sá, Maria Fátima; de Freitas, Sonia M

2003-06-01

The cotton boll weevil Anthonomus grandis (Boheman) is one of the major pests of cotton (Gossypium hirsutum L.) in tropical and sub-tropical areas of the New World. This feeds on cotton floral fruits and buds causing severe crop losses. Digestion in the boll weevil is facilitated by high levels of serine proteinases, which are responsible for the almost all proteolytic activity. Aiming to reduce the proteolytic activity, the inhibitory effects of black-eyed pea trypsin/chymotrypsin inhibitor (BTCI), towards trypsin and chymotrypsin from bovine pancreas and from midguts of A. grandis larvae and adult insects were analyzed. BTCI, purified from Vigna unguiculata (L.) seeds, was highly active against different trypsin-like proteinases studied and moderately active against the digestive chymotrypsin of adult insects. Nevertheless, no inhibitory activity was observed against chymotrypsin from A. grandis larval guts. To test the BTCI efficiency in vivo, neonate larvae were reared on artificial diet containing BTCI at 10, 50 and 100 microM. A reduction of larval weight of up to approximately 54% at the highest BTCI concentration was observed. At this concentration, the insect mortality was 65%. This work constitutes the first observation of a Bowman-Birk type inhibitor active in vitro and in vivo toward the cotton boll weevil A. grandis. The results of bioassays strongly suggest that BTCI may have potential as a transgene protein for use in engineered crop plants modified for heightened resistance to the cotton boll weevil.

Proteinase-activated receptor-2 activation evokes oesophageal longitudinal smooth muscle contraction via a capsaicin-sensitive and neurokinin-2 receptor-dependent pathway.

PubMed

Liu, H; Miller, D V; Lourenssen, S; Wells, R W; Blennerhassett, M G; Paterson, W G

2010-02-01

Intraluminal acid evokes sustained oesophageal longitudinal smooth muscle (LSM) contraction and oesophageal shortening, which may play a role in oesophageal pain and the aetiology of hiatus hernia. In the opossum model, this reflex has been shown to involve mast cell activation and release of neurokinins from capsaicin-sensitive neurons. The aim of this study was to determine whether proteinase-activated receptor-2 (PAR-2) activation evokes reflex LSM contraction via similar mechanisms. Tension recording studies were performed using opossum oesophageal LSM strips in the presence and absence of pharmacological agents. In addition, the effect of trypsin on single isolated LSM cells was determined using videomicroscopy, and the expression of PAR-2 in oesophageal tissue was examined using immunohistochemistry. The PAR-2 agonist trypsin evoked sustained, concentration-dependent contraction of LSM muscle strips, but had no effect on isolated LSM cells. The trypsin-induced contraction was blocked by capsaicin desensitization, substance P (SP) desensitization or application of the selective neurokinin-2 (NK-2) receptor antagonist MEN 10376. Immunohistochemistry revealed co-localization of SP, calcitonin gene-related peptide and PAR-2 in axons of opossum oesophageal LSM. Longitudinal smooth muscle contraction induced by trypsin involves capsaicin-sensitive neurons and subsequent activation of NK-2, which is identical to the pathway involved in acid-induced LSM contraction and oesophageal shortening. This suggests that acid-induced LSM contraction may involve mast cell-derived mediators that activate capsaicin-sensitive neurons via PAR-2.

Cathepsin B Activity Initiates Apoptosis via Digestive Protease Activation in Pancreatic Acinar Cells and Experimental Pancreatitis*

PubMed Central

Sendler, Matthias; Maertin, Sandrina; John, Daniel; Persike, Maria; Weiss, F. Ulrich; Krüger, Burkhard; Wartmann, Thomas; Wagh, Preshit; Halangk, Walter; Schaschke, Norbert; Mayerle, Julia; Lerch, Markus M.

2016-01-01

Pancreatitis is associated with premature activation of digestive proteases in the pancreas. The lysosomal hydrolase cathepsin B (CTSB) is a known activator of trypsinogen, and its deletion reduces disease severity in experimental pancreatitis. Here we studied the activation mechanism and subcellular compartment in which CTSB regulates protease activation and cellular injury. Cholecystokinin (CCK) increased the activity of CTSB, cathepsin L, trypsin, chymotrypsin, and caspase 3 in vivo and in vitro and induced redistribution of CTSB to a secretory vesicle-enriched fraction. Neither CTSB protein nor activity redistributed to the cytosol, where the CTSB inhibitors cystatin-B/C were abundantly present. Deletion of CTSB reduced and deletion of cathepsin L increased intracellular trypsin activation. CTSB deletion also abolished CCK-induced caspase 3 activation, apoptosis-inducing factor, as well as X-linked inhibitor of apoptosis protein degradation, but these depended on trypsinogen activation via CTSB. Raising the vesicular pH, but not trypsin inhibition, reduced CTSB activity. Trypsin inhibition did not affect apoptosis in hepatocytes. Deletion of CTSB affected apoptotic but not necrotic acinar cell death. In summary, CTSB in pancreatitis undergoes activation in a secretory, vesicular, and acidic compartment where it activates trypsinogen. Its deletion or inhibition regulates acinar cell apoptosis but not necrosis in two models of pancreatitis. Caspase 3-mediated apoptosis depends on intravesicular trypsinogen activation induced by CTSB, not CTSB activity directly, and this mechanism is pancreas-specific. PMID:27226576

4-Phenylbutyric acid reduces endoplasmic reticulum stress, trypsin activation, and acinar cell apoptosis while increasing secretion in rat pancreatic acini.

PubMed

Malo, Antje; Krüger, Burkhard; Göke, Burkhard; Kubisch, Constanze H

2013-01-01

Endoplasmic reticulum (ER) stress leads to misfolded proteins inside the ER and initiates unfolded protein response (UPR). Unfolded protein response components are involved in pancreatic function and activated during pancreatitis. However, the exact role of ER stress in the exocrine pancreas is unclear. The present study examined the effects of 4-phenylbutyric acid (4-PBA), an ER chaperone, on acini and UPR components. Rat acini were stimulated with cholecystokinin (10 pmol/L to 10 nmol/L) with or without preincubation of 4-PBA. The UPR components were analyzed, including chaperone-binding protein, protein kinaselike ER kinase, X-box-binding protein 1, c-Jun NH(2)-terminal kinase, CCAAT/enhancer-binding protein homologous protein, caspase 3, and apoptosis. Effects of 4-PBA were measured on secretion, calcium, and trypsin activation. 4-Phenylbutyric acid led to an increase of secretion, whereas trypsin activation with supraphysiological cholecystokinin was significantly reduced. 4-Phenylbutyric acid prevented chaperone-binding protein up-regulation, diminished protein kinaselike ER kinase, and c-Jun NH2-terminal kinase phosphorylation, prohibited X-box-binding protein 1 splicing and CCAAT/enhancer-binding protein homologous protein expression, caspase 3 activation, and apoptosis caused by supraphysiological cholecystokinin. By incubation with 4-PBA, beneficial in urea cycle deficiency, it was possible to enhance enzyme secretion to suppress trypsin activation, UPR activation, and proapoptotic pathways. The data hint new perspectives for the use of chemical chaperones in pancreatic diseases.

Cathepsin B Activity Initiates Apoptosis via Digestive Protease Activation in Pancreatic Acinar Cells and Experimental Pancreatitis.

PubMed

Sendler, Matthias; Maertin, Sandrina; John, Daniel; Persike, Maria; Weiss, F Ulrich; Krüger, Burkhard; Wartmann, Thomas; Wagh, Preshit; Halangk, Walter; Schaschke, Norbert; Mayerle, Julia; Lerch, Markus M

2016-07-08

Pancreatitis is associated with premature activation of digestive proteases in the pancreas. The lysosomal hydrolase cathepsin B (CTSB) is a known activator of trypsinogen, and its deletion reduces disease severity in experimental pancreatitis. Here we studied the activation mechanism and subcellular compartment in which CTSB regulates protease activation and cellular injury. Cholecystokinin (CCK) increased the activity of CTSB, cathepsin L, trypsin, chymotrypsin, and caspase 3 in vivo and in vitro and induced redistribution of CTSB to a secretory vesicle-enriched fraction. Neither CTSB protein nor activity redistributed to the cytosol, where the CTSB inhibitors cystatin-B/C were abundantly present. Deletion of CTSB reduced and deletion of cathepsin L increased intracellular trypsin activation. CTSB deletion also abolished CCK-induced caspase 3 activation, apoptosis-inducing factor, as well as X-linked inhibitor of apoptosis protein degradation, but these depended on trypsinogen activation via CTSB. Raising the vesicular pH, but not trypsin inhibition, reduced CTSB activity. Trypsin inhibition did not affect apoptosis in hepatocytes. Deletion of CTSB affected apoptotic but not necrotic acinar cell death. In summary, CTSB in pancreatitis undergoes activation in a secretory, vesicular, and acidic compartment where it activates trypsinogen. Its deletion or inhibition regulates acinar cell apoptosis but not necrosis in two models of pancreatitis. Caspase 3-mediated apoptosis depends on intravesicular trypsinogen activation induced by CTSB, not CTSB activity directly, and this mechanism is pancreas-specific. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Two variants of the major serine protease inhibitor from the sea anemone Stichodactyla helianthus, expressed in Pichia pastoris.

PubMed

García-Fernández, Rossana; Ziegelmüller, Patrick; González, Lidice; Mansur, Manuel; Machado, Yoan; Redecke, Lars; Hahn, Ulrich; Betzel, Christian; Chávez, María de Los Ángeles

2016-07-01

The major protease inhibitor from the sea anemone Stichodactyla helianthus (ShPI-1) is a non-specific inhibitor that binds trypsin and other trypsin-like enzymes, as well as chymotrypsin, and human neutrophil elastase. We performed site-directed mutagenesis of ShPI-1 to produce two variants (rShPI-1/K13L and rShPI/Y15S) that were expressed in Pichia pastoris, purified, and characterized. After a single purification step, 65 mg and 15 mg of protein per liter of culture supernatant were obtained for rShPI-1/K13L and rShPI/Y15S, respectively. Functional studies demonstrated a 100-fold decreased trypsin inhibitory activity as result of the K13L substitution at the reactive (P1) site. This protein variant has a novel tight-binding inhibitor activity of pancreatic elastase and increased activity toward neutrophil elastase in comparison to rShPI-1A. In contrast, the substitution Y15S at P2' site did not affect the Ki value against trypsin, but did reduce activity 10-fold against chymotrypsin and neutrophil elastase. Our results provide two new ShPI-1 variants with modified inhibitory activities, one of them with increased biomedical potential. This study also offers new insight into the functional impact of the P1 and P2' sites on ShPI-1 specificity. Copyright © 2016 Elsevier Inc. All rights reserved.

Structural basis of trypsin inhibition and entomotoxicity of cospin, serine protease inhibitor involved in defense of Coprinopsis cinerea fruiting bodies.

PubMed

Sabotič, Jerica; Bleuler-Martinez, Silvia; Renko, Miha; Avanzo Caglič, Petra; Kallert, Sandra; Štrukelj, Borut; Turk, Dušan; Aebi, Markus; Kos, Janko; Künzler, Markus

2012-02-03

Cospin (PIC1) from Coprinopsis cinerea is a serine protease inhibitor with biochemical properties similar to those of the previously characterized fungal serine protease inhibitors, cnispin from Clitocybe nebularis and LeSPI from Lentinus edodes, classified in the family I66 of the MEROPS protease inhibitor classification. In particular, it exhibits a highly specific inhibitory profile as a very strong inhibitor of trypsin with K(i) in the picomolar range. Determination of the crystal structure revealed that the protein has a β-trefoil fold. Site-directed mutagenesis and mass spectrometry results have confirmed Arg-27 as the reactive binding site for trypsin inhibition. The loop containing Arg-27 is positioned between the β2 and β3 strands, distinguishing cospin from other β-trefoil-fold serine protease inhibitors in which β4-β5 or β5-β6 loops are involved in protease inhibition. Biotoxicity assays of cospin on various model organisms revealed a strong and specific entomotoxic activity against Drosophila melanogaster. The inhibitory inactive R27N mutant was not entomotoxic, associating toxicity with inhibitory activity. Along with the abundance of cospin in fruiting bodies of C. cinerea and the lack of trypsin-like proteases in the C. cinerea genome, these results suggest that cospin and its homologs are effectors of a fungal defense mechanism against fungivorous insects that function by specific inhibition of serine proteases in the insect gut.

Release of “neurokinin” on nervous and electrical stimulation of a frog stomach muscle preparation

PubMed Central

Singh, I.

1964-01-01

Activation of a frog stomach muscle preparation by electrical stimulation of a vagus nerve or by direct stimulation released two polypeptides. One was destroyed by trypsin or chymotrypsin in about 10 min; the activity of the other was enhanced by trypsin for about 10 min, but was destroyed by chymotrypsin. Similar stimulation of dog stomach muscle did not release these polypeptides. Correspondingly, the transmission from vagus nerve to stomach muscle in the frog was resistant to atropine, but was blocked by atropine in the dog. PMID:14190475

Studies on trypsin-like enzymes in sperm and early embryos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Penn, A.

1975-12-09

Results are reported from a study of acrosomal proteinase, a trypsin-like enzyme (TLE), found in the acrosome of all eutherian mammals studied to date. It has been implicated in the dissolution of a passage for the sperm through the zona pellucida of the egg, a step necessary for in vivo fertilization. A cytochemical procedure employing autoradiographic film as a gelatin substrate is described for in situ detection and localization of acrosomal proteolytic activity. A role for TLE in the early development of embryos is suggested. (CH)

Allium sativum Protease Inhibitor: A Novel Kunitz Trypsin Inhibitor from Garlic Is a New Comrade of the Serpin Family.

PubMed

Shamsi, Tooba Naz; Parveen, Romana; Amir, Mohd; Baig, Mohd Affan; Qureshi, M Irfan; Ali, Sher; Fatima, Sadaf

2016-01-01

This study was aimed to purify and characterize the Protease inhibitor (PI) from a plant Allium sativum (garlic) with strong medicinal properties and to explore its phytodrug potentials. Allium sativum Protease Inhibitor (ASPI) was purified using ammonium sulphate fractionation and Fast Protein Liquid Chromatography on anion exchanger Hi-Trap DEAE column. The purified protein was analyzed for its purity and molecular weight by SDS PAGE. The confirmation of presence of trypsin inhibiting PI was performed by MALDI TOF-TOF and analyzed by MASCOT database. The ASPI was further investigated for its kinetic properties and stability under extreme conditions of pH, temperature and chemical denaturants. Secondary structure was determined by Circular Dichorism (CD) spectroscopy. ASPI of ~15 kDa inhibited trypsin and matched "truncated kunitz Trypsin Inhibitor (Glycine max)" in MASCOT database. The purified ASPI showed 30376.1371 U/mg specific activity with a fold purity of 159.92 and yield ~93%. ASPI was quite stable in the range of pH 2-12 showing a decline in the activity around pH 4-5 suggesting that the pI value of the protein as ASPI aggregates in this range. ASPI showed stability to a broad range of temperature (10-80°C) but declined beyond 80°C. Further, detergents, oxidizing agents and reducing agents demonstrated change in ASPI activity under varying concentrations. The kinetic analysis revealed sigmoidal relationship of velocity with substrate concentration with Vmax 240.8 (μM/min) and Km value of 0.12 μM. ASPI showed uncompetitive inhibition with a Ki of 0.08±0.01 nM). The Far UV CD depicted 2.0% α -helices and 51% β -sheets at native pH. To conclude, purified ~15 kDa ASPI exhibited fair stability in wide range of pH and temperature Overall, there was an increase in purification fold with remarkable yield. Chemical modification studies suggested the presence of lysine and tryptophan residues as lead amino acids present in the reactive sites. Therefore, ASPI with trypsin inhibitory property has the potential to be used as a non-cytotoxic clinical agents.

JcTI-I: a novel trypsin inhibitor from Jatropha curcas seed cake with potential for bacterial infection treatment.

PubMed

Costa, Helen P S; Oliveira, Jose T A; Sousa, Daniele O B; Morais, Janne K S; Moreno, Frederico B; Monteiro-Moreira, Ana Cristina O; Viegas, Ricardo A; Vasconcelos, Ilka M

2014-01-01

Jatropha curcas seed cake is a low-value by-product resulting from biodiesel production. The seed cake is highly toxic, but it has great potential for biotechnology applications as it is a repository of biomolecules that could be important in agriculture, medicine, and industry. To explore this potential, a novel trypsin inhibitor called JcTI-I was purified by fractionation of the crude extract with trichloroacetic acid (2.5%, v/v) followed by affinity chromatography (Trypsin-Sepharose 4B) and molecular exclusion (Sephacryl S-200). Non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration showed that JcTI-I has approximately 20.0~kDa. Mass spectrometry analysis revealed that the intact molecular mass of JcTI-I is 10.252~kDa. Moreover, JcTI-I is a glycoprotein with 6.4% (m/m) carbohydrates, pI of 6.6, N-terminal sequence similarity around 60% to plant albumins and high stability to heat, pH, and salinity. JcTI-I presented antibacterial activity against the human pathogenic bacteria Salmonella enterica subspecies enterica serovar choleraesuis and Staphylococcus aureus, with minimum inhibitory concentration less than 5~μg/mL. Furthermore, JcTI-I did have inhibitory activity against the serine proteases from the tested bacteria. Otherwise, no hemolytic activity of human erythrocytes and signs of acute toxicity to mice were observed for JcTI-I. The results demonstrate the benefits of J. curcas seed cake as a source of trypsin inhibitor with potential for biotechnological application as a new antimicrobial agent against human pathogenic bacteria.

JcTI-I: a novel trypsin inhibitor from Jatropha curcas seed cake with potential for bacterial infection treatment

PubMed Central

Costa, Helen P. S.; Oliveira, Jose T. A.; Sousa, Daniele O. B.; Morais, Janne K. S.; Moreno, Frederico B.; Monteiro-Moreira, Ana Cristina O.; Viegas, Ricardo A.; Vasconcelos, Ilka M.

2014-01-01

Jatropha curcas seed cake is a low-value by-product resulting from biodiesel production. The seed cake is highly toxic, but it has great potential for biotechnology applications as it is a repository of biomolecules that could be important in agriculture, medicine, and industry. To explore this potential, a novel trypsin inhibitor called JcTI-I was purified by fractionation of the crude extract with trichloroacetic acid (2.5%, v/v) followed by affinity chromatography (Trypsin-Sepharose 4B) and molecular exclusion (Sephacryl S-200). Non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration showed that JcTI-I has approximately 20.0~kDa. Mass spectrometry analysis revealed that the intact molecular mass of JcTI-I is 10.252~kDa. Moreover, JcTI-I is a glycoprotein with 6.4% (m/m) carbohydrates, pI of 6.6, N-terminal sequence similarity around 60% to plant albumins and high stability to heat, pH, and salinity. JcTI-I presented antibacterial activity against the human pathogenic bacteria Salmonella enterica subspecies enterica serovar choleraesuis and Staphylococcus aureus, with minimum inhibitory concentration less than 5~μg/mL. Furthermore, JcTI-I did have inhibitory activity against the serine proteases from the tested bacteria. Otherwise, no hemolytic activity of human erythrocytes and signs of acute toxicity to mice were observed for JcTI-I. The results demonstrate the benefits of J. curcas seed cake as a source of trypsin inhibitor with potential for biotechnological application as a new antimicrobial agent against human pathogenic bacteria. PMID:24523715

The flow and fate of digestive enzymes in the field cricket, Gryllus bimaculatus.

PubMed

Woodring, Joseph

2017-07-01

The flow of enzymes, the ratio of bound to unbound enzymes, and their inactivation in the cricket Gryllus bimaculatus was studied. The digestive enzymes are forced forward into the crop by caecal contraction and then they are mixed with freshly chewed food and saliva, forming a crop-chyme. This chyme is blended by crop peristalsis, and periodic opening of the preproventricular valve (PPV) allows posterior movement into the proventriculus and further into the midgut. The contraction of the crop is modulated by Grybi-AST and Grybi-SK peptides, which are partially secreted by the caecal endocrine cells. Most of the aminopeptidase and the four disaccharidases examined are membrane bound (62-80%); the remaining (20-38%) as well all trypsin, chymotrypsin, lipase, and amylase are secreted free into the caecal lumen. Cricket trypsin loses only 30% of its activity in 4 h and very little thereafter. The presence of digestive products in the lumen appears to retard further trypsin autolysis. Cricket trypsin digests 42% of the chymotrypsin, 37% of the lipase, and 45% of the amylase in the caecal fluids over 24 h in vitro no significant difference. Without Ca ion amylase was almost completely digested. About 50% of the membrane bound and free aminopeptidase was digested in the caecal lumen, and about 30-38% of the bound and free maltase. This loss of digestive enzyme activity is possible, because enzyme secretion rates are high, the unbound enzymes are effectively recycled, and the time of nutrient passage is short. © 2017 Wiley Periodicals, Inc.

Effect of trypsin inhibitor from Crotalaria pallida seeds on Callosobruchus maculatus (cowpea weevil) and Ceratitis capitata (fruit fly).

PubMed

Gomes, Carlos E M; Barbosa, Aulus E A D; Macedo, Leonardo L P; Pitanga, Joelma C M; Moura, Fabiano T; Oliveira, Adeliana S; Moura, Raniere M; Queiroz, Alexandre F S; Macedo, Francisco P; Andrade, Lúcia B S; Vidal, Márcia S; Sales, Mauricio P

2005-12-01

A proteinaceous trypsin inhibitor was purified from Crotalaria pallida seeds by ammonium sulfate precipitation, affinity chromatography on immobilized trypsin-Sepharose and TCA precipitation. The trypsin inhibitor, named CpaTI, had M(r) of 32.5 kDa as determined by SDS-PAGE and was composed of two subunits with 27.7 and 5.6 kDa linked by disulfide bridges. CpaTI was stable at 50 degrees C and lost 40% of activity at 100 degrees C. CpaTI was also stable from pH 2 to 12 at 37 degrees C. CpaTI weakly inhibited chymotrypsin and elastase and its inhibition of papain, a cysteine proteinase, were indicative of its bi-functionality. CpaTI inhibited, in different degrees, digestive enzymes from Spodoptera frugiperda, Alabama argillacea, Plodiainterpunctella, Anthonomus grandis and Zabrotes subfasciatus guts. In vitro and in vivo susceptibility of Callosobruchus maculatus and Ceratitis capitata to CpaTI was evaluated. C. maculatus and C. capitata enzymes were strongly susceptible, 74.4+/-15.8% and 100.0+/-7.3%, respectively, to CpaTI. When CpaTI was added to artificial diets and offered to both insect larvae, the results showed that C. maculatus was more susceptible to CpaTI with an LD(50) of 3.0 and ED(50) of 2.17%. C. capitata larvae were more resistant to CpaTI, in disagreement with the in vitro effects. The larvae were more affected at lower concentrations, causing 27% mortality and 44.4% mass decrease. The action was constant at 2-4% (w/w) with 15% mortality and 38% mass decrease.

Effects of trypsin and low Ca2+ on zonulae adhaerentes between chick retinal pigment epithelial cells in organ culture.

PubMed

Sandig, M; Hergott, G J; Kalnins, V I

1990-01-01

The junctional complexes in chick retinal pigment epithelial (RPE) cells in situ contain unusually large zonulae adhaerentes (ZAs) composed of subunits termed zonula adhaerens complexes (ZACs). To determine whether the properties of the ZAs differ between RPE cells which contain ZACs, and MDCK cells which lack ZACs, we investigated the effects of treatment with trypsin and/or low Ca2+ by transmission electron microscopy and staining for F-actin. Treatment of RPE cells for 1 h with trypsin alone has no apparent effect on the morphology of the ZA in either MDCK or RPE cells. In contrast to the ZAs in MDCK cells, which split after 3 min in low Ca2+, the ZAs in chick RPE cells stay intact even after 2 h, although the intermembrane discs, i.e., the extracellular components of the ZACs, are no longer visible. After 30 min of treatment with trypsin and low Ca2+, the ZAs split in both cell types. The CMBs start to contract, translocate toward the cell interior, and eventually disappear. This process continues even when the RPE cells are returned to normal medium. New ZAs, composed of ZACs, form between RPE cells 3 h after return to normal medium. These findings suggest that the ZACs in the ZAs of RPE cells are not directly responsible for the increase in resistance to low Ca2+. They also show that the ZA-junctions in RPE cells are not only structurally different from those previously examined, but also behave differently in response to experimental manipulation.

PAR-2 agonists induce contraction of murine small intestine through neurokinin receptors.

PubMed

Zhao, Aiping; Shea-Donohue, Terez

2003-10-01

Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor and is expressed throughout the gut. It is well known that PAR-2 participates in the regulation of gastrointestinal motility; however, the results are inconsistent. The present study investigated the effect and mechanism of PAR-2 activation on murine small intestinal smooth muscle function in vitro. Both trypsin and PAR-2-activating peptide SLIGRL induced a small relaxation followed by a concentration-dependent contraction. The sensitivity to trypsin was greater than that to SLIGRL (EC50 = 0.03 vs. 40 microM), but maximal responses were similar (12.3 +/- 1.6 vs. 13.7 +/- 1.3 N/cm2). Trypsin-evoked contraction (1 microM) exhibited a rapid desensitization, whereas the desensitization of response to SLIGRL was less even at high concentration (50 microM). Atropine had no effect on PAR-2 agonist-induced contractions. In contrast, TTX and capsaicin significantly attenuated those contractions, implicating a neurogenic mechanism that may involve capsaicin-sensitive sensory nerves. Furthermore, contractions induced by trypsin and SLIGRL were reduced by neurokinin receptor NK1 antagonist SR-140333 or NK2 antagonist SR-48968 alone or were further reduced by combined application of SR-140333 and SR-48968, indicating the involvement of neurokinin receptors. In addition, desensitizing neurokinin receptors with substance P and/or neurokinin A decreased the PAR-2 agonist-evoked contraction. We concluded that PAR-2 agonists induced a contraction of murine intestinal smooth muscle that was mediated by nerves. The excitatory effect is also dependent on sensory neural pathways and requires both NK1 and NK2 receptors.

Phosphorylation of the rat hepatic polymeric IgA receptor.

PubMed Central

Larkin, J M; Sztul, E S; Palade, G E

1986-01-01

In vivo labeling with [35S]cysteine has identified three transmembrane forms of the rat hepatic polymeric IgA receptor: (i) a 105-kDa core glycosylated precursor; (ii) a terminally glycosylated 116-kDa intermediate; and (iii) a mature 120-kDa form. In the current study we show that the 120-kDa form is phosphorylated. After in vivo labeling with [32P]orthophosphate, all receptor forms were immunoprecipitated from hepatic total microsomes (TM) (with an antireceptor antiserum), separated by NaDodSO4/PAGE, and detected by autoradiography. The 120-kDa form was selectively phosphorylated, whereas the 116- and 105-kDa forms incorporated no detectable 32P. To determine the topology of the phosphorylation sites, hepatic TM isolated from rats labeled in vivo with either [35S]cysteine or [32P]orthophosphate were treated with trypsin. TM were solubilized and receptors were immunoprecipitated from lysates. With increasing trypsin concentrations, the [35S]cysteine-labeled receptor triplet was degraded to a trypsin-resistant doublet of approximately 95 and 85 kDa, indicating that approximately 20 kDa was removed from the receptor endodomain by trypsin. The same treatment removed all detectable 32P from labeled receptors. Furthermore, no 32P was detected in the 80-kDa biliary form of the receptor. Serine was identified as the only phosphorylated residue in acid hydrolysates of 32P-labeled immunoprecipitated receptor. These findings indicate that (i) the 120-kDa form is the only phosphorylated species of the receptor; and (ii) the phosphorylated residues are serine(s) located in the endodomain of the protein. Images PMID:3460069

Characteristics of the Mg2+-ATPase Activity Associated with the Membrane-Bound Maize Coupling Factor 1

PubMed Central

Cohen, William S.

1989-01-01

The membrane-bound coupling factor of maize mesophyll thylakoids is a latent ATPase. Mg2+-ATPase activity can be induced in the light with either dithiothreitol or low concentrations of trypsin. Maize thylakoids that are activated with light plus trypsin exhibit considerably higher levels of activity in Na2SO3-dependent Mg2+-ATPase assays compared to thylakoids that are light and dithiothreitol activated (1400 micromoles per milligram of chlorophyll per hour versus 200 micromoles per milligram of chlorophyll per hour). Treatment with light and dithiothreitol or light plus trypsin were also required to demonstrate high levels of octyl glucoside-dependent Mg2+-ATPase activity in maize mesophyll thylakoids. Only small differences in octyl glucoside-dependent Mg2+-ATPase activity were observed in preparations that were activated in the light with either trypsin or dithiothreitol. Mg2+-ATPase activity can also be induced in maize mesophyll chloroplasts by illuminating intact preparations under appropriate conditions. Little or no ATPase activity was observed in the absence of illumination or in the presence of light plus methyl viologen. The active state decayed in the dark with a t½ of 6 to 7 minutes at room temperature. Based on the effect of the thiol oxidant, o-iodosobenzoate, and the uncoupler, nigericin, on the kinetics of deactivation of ATPase activity in intact maize chloroplasts, it appears that the activation process requires a transmembrane proton gradient and reduction of a key disulfide bridge in the gamma of chloroplast coupling factor one. PMID:16667119

Spatial characterization of proteolytic enzyme activity in the foregut region of the adult necrophagous fly, Protophormia terraenovae.

PubMed

Rivers, David B; Acca, Gillian; Fink, Marc; Brogan, Rebecca; Schoeffield, Andrew

2014-08-01

The spatial distribution of proteolytic enzymes in the adult foregut of Protophormia terraenovae was studied in the context of protein digestion and regurgitation. Based on substrate specificity, pH optima, and use of specific protease inhibitors, all adults tested displayed enzyme activity in the foregut consistent with pepsin, trypsin and chymotrypsin. Chymotrypsin-like and trypsin-like enzyme activity were detected in all gut fluids and tissues tested, with chymotrypsin displaying the highest activity in saliva and salivary gland tissue, whereas maximal trypsin activity was evident in the crop. Pepsin-like activity was only evident in crop fluids and tissues. The activity of all three enzymes was low or undetectable (pepsin) in the fluids and tissue homogenates derived from the esophagus and cardia of any of the adults assayed. Fed adult females displayed higher enzyme activities than fed males, and the activity of all three enzymes were much more prevalent in fed adults than starved. The pH optimum of the trypsin-like enzyme was between pH 7.0 and 8.0; chymotrypsin was near pH 8.0; and maximal pepsin-like activity occurred between pH 1.0 and 2.0. Regurgitate from fed adult females displayed enzyme activity consistent with the proteolytic enzymes detected in crop gut fluids. Enzymes in regurgitate were not derived from food sources based on assays of bovine liver samples. These latter observations suggest that adult flies release fluids from foregut when encountering dry foods, potentially as a means to initiate extra-oral digestion. Copyright © 2014 Elsevier Ltd. All rights reserved.

Complete disassociation of adult pancreas into viable single cells through cold trypsin-EDTA digestion*

PubMed Central

Li, Dan; Peng, Shi-yun; Zhang, Zhen-wu; Feng, Rui-cheng; Li, Lu; Liang, Jie; Tai, Sheng; Teng, Chun-bo

2013-01-01

The in vitro isolation and analysis of pancreatic stem/progenitor cells are necessary for understanding their properties and function; however, the preparation of high-quality single-cell suspensions from adult pancreas is prerequisite. In this study, we applied a cold trypsin-ethylenediaminetetraacetic acid (EDTA) digestion method to disassociate adult mouse pancreata into single cells. The yield of single cells and the viability of the harvested cells were much higher than those obtained via the two commonly used warm digestion methods. Flow cytometric analysis showed that the ratio of ductal or BCRP1-positive cells in cell suspensions prepared through cold digestion was consistent with that found in vivo. Cell culture tests showed that pancreatic epithelial cells prepared by cold digestion maintained proliferative capacity comparable to those derived from warm collagenase digestion. These results indicate that cold trypsin-EDTA digestion can effectively disassociate an adult mouse pancreas into viable single cells with minimal cell loss, and can be used for the isolation and analysis of pancreatic stem/progenitor cells. PMID:23825145

The rapidly metabolized 32,000-dalton polypeptide of the chloroplast is the "proteinaceous shield" regulating photosystem II electron transport and mediating diuron herbicide sensitivity.

PubMed Central

Mattoo, A K; Pick, U; Hoffman-Falk, H; Edelman, M

1981-01-01

Mild trypsin treatment of Spirodela oligorrhiza thylakoid membranes leads to partial digestion of the rapidly metabolized, surface-exposed, 32,000-dalton protein. Under these conditions, photoreduction of ferricyanide becomes insensitive to diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea], an inhibitor of photosystem II electron transport. Preincubation of thylakoids with diuron leads to a conformational change in the 32,000-dalton protein, modifying its trypsin digestion and preventing expression of diuron insensitivity. Finally, light affects the susceptibility of the 32,000-dalton protein to digestion by trypsin. In other experiments, thylakoids specifically depleted in the 32,000-dalton protein were found to be deficient in electron transport at the reducing side of photosystem II but not at the oxidizing side or in photosystem I activities. Thus, the rapidly metabolized 32,000-dalton thylakoid protein in Spirodela chloroplasts fulfills the requirements of the hypothesized "proteinaceous shield" [Renger, G. (1976) Biochim. Biophys. Acta 440, 287-300] regulating electron flow through photosystem II and mediating diuron sensitivity. Images PMID:6940173

Nanobiocatalysis for protein digestion in proteomic analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Jungbae; Kim, Byoung Chan; Lopez-Ferrer, Daniel

2010-02-01

The process of protein digestion is a critical step for successful protein identification in the bottom-up proteomic analysis. To substitute the present practice of in-solution protein digestion, which is long, tedious, and difficult to automate, a lot of efforts have been dedicated for the development of a rapid, recyclable and automated digestion system. Recent advances of nanobiocatalytic approaches have improved the performance of protein digestion by using various nanomaterials such as nanoporous materials, magnetic nanoparticles, and polymer nanofibers. Especially, the unprecedented success of trypsin stabilization in the form of trypsin-coated nanofibers, showing no activity decrease under repeated uses for onemore » year and retaining good resistance to proteolysis, has demonstrated its great potential to be employed in the development of automated, high-throughput, and on-line digestion systems. This review discusses recent developments of nanobiocatalytic approaches for the improved performance of protein digestion in speed, detection sensitivity, recyclability, and trypsin stability. In addition, we also introduce the protein digestions under unconventional energy inputs for protein denaturation and the development of microfluidic enzyme reactors that can benefit from recent successes of these nanobiocatalytic approaches.« less

Effect of detergents, trypsin and unsaturated fatty acids on latent loquat fruit polyphenol oxidase: basis for the enzyme's activity regulation.

PubMed

Sellés-Marchart, Susana; Casado-Vela, Juan; Bru-Martínez, Roque

2007-08-15

The effects of detergents, trypsin and fatty acids on structural and functional properties of a pure loquat fruit latent polyphenol oxidase have been studied in relation to its regulation. Anionic detergents activated PPO at pH 6.0 below critical micelle concentration (cmc), but inhibited at pH 4.5 well above cmc. This behavior is due to a detergent-induced pH profile alkaline shift, accompanied by changes of intrinsic fluorescence of the protein. Gel filtration experiments demonstrate the formation of PPO-SDS mixed micelles. Partial PPO proteolysis suggest that latent PPO losses an SDS micelle-interacting region but conserves an SDS monomer-interacting site. Unsaturated fatty acids inhibit PPO at pH 4.5, the strongest being linolenic acid while the weakest was gamma-linolenic acid for both, the native and the trypsin-treated PPO. Down-regulation of PPO activity by anionic amphiphiles is discussed based on both, the pH profile shift induced upon anionic amphiphile binding and the PPO interaction with negatively charged membranes.

Study on great northern beans (Phaseolus vulgaris): effect of drum drying process on bean flour properties and effect on gamma radiation on bean starch properties

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rayas-Solis, P.

Great Northern bean (Phaseolus vulgaris L.) drum dried flours at native pH of 6.54, pH 6 and 7 showed reduced activities of trypsin inhibitor, ..cap alpha..-amylase inhibitor, hemagglutinating titer, and nitrogen solubility. Electrophoretic analyses showed a slight modification of the native bean proteins, and the presence of at least four trypsin inhibitors. The study of the effect of 2.5-20 kGy irradiation doses on Great Northern beans showed essentially no modification of the electrophoretic mobility of the storage proteins or the trypsin inhibitors. Nitrogen solubility and hemagglutinating activity were essentially unchanged. With the 20 kGy dose, decrease in ..cap alpha..-amylase inhibitormore » activity, decrease reactive/available lysine content, and decrease cooking time of the irradiated beans after 11 months of storage were observed. Taste panel results indicated that the control and 20 kGy irradiated bean were significantly different at 5% level. At 20 kGy dose, the beans developed a partially water soluble brown color.« less

Entomocidal effects of beech apricot, Labramia bojeri, seed extract on a soybean pest, the velvetbean moth, Anticarsia gemmatalis, and its enzymatic activity

PubMed Central

Macedo, Maria L. R.; Kubo, Carlos E. G.; Freire, Maria G. M.; Júnior, Roberto T. A.; Parra, José R. P.

2014-01-01

Abstract The effects of the beech apricot, Labramia bojeri A. de Candolle (Sapotales: Sapotaceae), seed aqueous extract on the larval development of the velvetbean moth, Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae), was evaluated. The extract inhibited larval development, pupal weight, and survival and emergence of adults. Digestive proteolytic activity in larval midgut and feces extracts was determined. Larvae fed 10 g/L of the aqueous extract showed a significant reduction in trypsin activity (~64%), when compared with control larvae. Trypsin and chymotrypsin activities were also detected in fecal material in aqueous-extract-fed larvae, with about ~4.5 times more trypsin activity than the controls. The results from dietary utilization experiments with A. gemmatalis larvae showed a reduction in the efficiency of conversion of ingested food and digested food and an increase in approximate digestibility and metabolic cost. The effect of the extract suggests the potential use of L. bojeri seeds to inhibit the development of A. gemmatalis via oral exposure. The L. bojeri extract can be an alternative to other methods of control. PMID:25373174

Separation and nanoencapsulation of antitumor polypeptide from Spirulina platensis.

PubMed

Zhang, Bochao; Zhang, Xuewu

2013-01-01

Spirulina platensis is a multicellular edible blue-green alga with abundant proteins (∼ 60%). No report is available on the antitumor polypeptides from the whole proteins of S. platensis. In this study, for the first time, an antitumor polypeptide Y2 from trypsin digest of S. platensis proteins was obtained by using freeze-thawing plus ultrasonication extraction, hydrolysis with four enzymes (trypsin, alcalase, papain, and pepsin), and gel filtration chromatography. The results showed that the degree of hydrolysis can be ordered as: trypsin (38.5%) > alcalase (31.2%) > papain (27.8%) > pepsin (7.1%). For MCF-7 and HepG2 cells, at 250 µg/mL, the maximum inhibitory rate of Y2 was 97%, while standard drug 5-FU was 55 and 97%, respectively. Furthermore, the nanoencapsulation of Y2 with chitosan (CS) was also investigated. After nanoencapsulation, the maximum encapsulation efficiency and polypeptides contents are 49 and 15%, respectively; and the antitumor activity is basically not lost. These data demonstrated the potential of nanopolypeptides (Y2-CS) in food and pharmaceutical applications. © 2013 American Institute of Chemical Engineers.

Low molecular weight squash trypsin inhibitors from Sechium edule seeds.

PubMed

Laure, Hélen J; Faça, Vítor M; Izumi, Clarice; Padovan, Júlio C; Greene, Lewis J

2006-02-01

Nine chromatographic components containing trypsin inhibitor activity were isolated from Sechium edule seeds by acetone fractionation, gel filtration, affinity chromatography and RP-HPLC in an overall yield of 46% of activity and 0.05% of protein. The components obtained with highest yield of total activity and highest specific activity were sequenced by Edman degradation and their molecular masses determined by mass spectrometry. The inhibitors contained 31, 32 and 27 residues per molecule and their sequences were: SETI-IIa, EDRKCPKILMRCKRDSDCLAKCTCQESGYCG; SETI-IIb, EEDRKCPKILMRCKRDSDCLAKCTCQESGYCG and SETI-V, CPRILMKCKLDTDCFPTCTCRPSGFCG. SETI-IIa and SETI-IIb, which differed by an amino-terminal E in the IIb form, were not separable under the conditions employed. The sequences are consistent with consensus sequences obtained from 37 other inhibitors: CPriI1meCk_DSDCla_C_C_G_CG, where capital letters are invariant amino acid residues and lower case letters are the most preserved in this position. SETI-II and SETI-V form complexes with trypsin with a 1:1 stoichiometry and have dissociation constants of 5.4x10(-11)M and 1.1x10(-9)M, respectively.

Effect of detergents, trypsin, and bivalent metal ions on interfacial activation and functioning of phospholipase D.

PubMed

Madyarov, Sh R

2014-07-01

The effects of detergents, trypsin, and bivalent metal ions on production of phosphatidic and lysophosphatidic acids by the action of phospholipase D (PLD) on lecithin and lysolecithin were studied. It was found that these reaction products and dodecyl sulfate ions activate PLD, whereas other anionic detergents are less effective. A protective effect of the functioning enzyme against its hydrolytic inactivation by trypsin was found. Bivalent metal ions can be arranged in the following sequence by their ability to activate PLD in the hydrolysis of lecithin and lysolecithin: Ca2+>Sr2+>Ba2+>Mg2+. These results are considered in relation to a proposed mechanism of activation and functioning of PLD with the participation of clusters of phosphatidates and lysophosphatidates. Such Me2+-induced formation of rafts or microdomains from the products of hydrolysis of phospholipids can rationalize not only PLD activation and self-regulation, but also the action of this mechanism on other components and properties of biomembranes. PLD and other lipolytic enzymes can be classified as lateral vector enzymes.

Gelatin hydrolysates from farmed Giant catfish skin using alkaline proteases and its antioxidative function of simulated gastro-intestinal digestion.

PubMed

Ketnawa, Sunantha; Martínez-Alvarez, Oscar; Benjakul, Soottawat; Rawdkuen, Saroat

2016-02-01

This work aims to evaluate the ability of different alkaline proteases to prepare active gelatin hydrolysates. Fish skin gelatin was hydrolysed by visceral alkaline-proteases from Giant catfish, commercial trypsin, and Izyme AL®. All antioxidant activity indices of the hydrolysates increased with increasing degree of hydrolysis (P<0.05). The hydrolysates obtained with Izyme AL® and visceral alkaline-proteases showed the highest and lowest radical scavenging capacity, while prepared with commercial trypsin was the most effective in reducing ferric ions and showed the best metal chelating properties. The hydrolysate obtained with Izyme AL® showed the lowest iron reducing ability, but provided the highest average molecular weight (⩾ 7 kDa), followed by commercial trypsin (2.2 kDa) and visceral alkaline-proteases (1.75 kDa). After in vitro gastrointestinal digestion, the hydrolysates showed significant higher radical scavenging, reducing ferric ions and chelating activities. Gelatin hydrolysates, from fish skin, could serve as a potential source of functional food ingredients for health promotion. Copyright © 2015 Elsevier Ltd. All rights reserved.

MOFzyme: Intrinsic protease-like activity of Cu-MOF.

PubMed

Li, Bin; Chen, Daomei; Wang, Jiaqiang; Yan, Zhiying; Jiang, Liang; Deliang Duan; He, Jiao; Luo, Zhongrui; Zhang, Jinping; Yuan, Fagui

2014-10-24

The construction of efficient enzyme mimetics for the hydrolysis of peptide bonds in proteins is challenging due to the high stability of peptide bonds and the importance of proteases in biology and industry. Metal-organic frameworks (MOFs) consisting of infinite crystalline lattices with metal clusters and organic linkers may provide opportunities for protease mimic which has remained unknown. Herein, we report that Cu₂(C₉H₃O₆)₄/₃ MOF (which is well known as HKUST-1 and denoted as Cu-MOF here), possesses an intrinsic enzyme mimicking activity similar to that found in natural trypsin to bovine serum albumin (BSA) and casein. The Michaelis constant (Km) of Cu-MOF is about 26,000-fold smaller than that of free trypsin indicating a much higher affinity of BSA for Cu-MOF surface. Cu-MOF also exhibited significantly higher catalytic efficiency than homogeneous artificial metalloprotease Cu(II) complexes and could be reused for ten times without losing in its activity. Moreover, Cu-MOF was successfully used to simulate trypsinization in cell culture since it dissociated cells in culture even without EDTA.

MOFzyme: Intrinsic protease-like activity of Cu-MOF

NASA Astrophysics Data System (ADS)

Li, Bin; Chen, Daomei; Wang, Jiaqiang; Yan, Zhiying; Jiang, Liang; Deliang Duan; He, Jiao; Luo, Zhongrui; Zhang, Jinping; Yuan, Fagui

2014-10-01

The construction of efficient enzyme mimetics for the hydrolysis of peptide bonds in proteins is challenging due to the high stability of peptide bonds and the importance of proteases in biology and industry. Metal-organic frameworks (MOFs) consisting of infinite crystalline lattices with metal clusters and organic linkers may provide opportunities for protease mimic which has remained unknown. Herein, we report that Cu2(C9H3O6)4/3 MOF (which is well known as HKUST-1 and denoted as Cu-MOF here), possesses an intrinsic enzyme mimicking activity similar to that found in natural trypsin to bovine serum albumin (BSA) and casein. The Michaelis constant (Km) of Cu-MOF is about 26,000-fold smaller than that of free trypsin indicating a much higher affinity of BSA for Cu-MOF surface. Cu-MOF also exhibited significantly higher catalytic efficiency than homogeneous artificial metalloprotease Cu(II) complexes and could be reused for ten times without losing in its activity. Moreover, Cu-MOF was successfully used to simulate trypsinization in cell culture since it dissociated cells in culture even without EDTA.

MOFzyme: Intrinsic protease-like activity of Cu-MOF

PubMed Central

Li, Bin; Chen, Daomei; Wang, Jiaqiang; Yan, Zhiying; Jiang, Liang; Deliang Duan; He, Jiao; Luo, Zhongrui; Zhang, Jinping; Yuan, Fagui

2014-01-01

The construction of efficient enzyme mimetics for the hydrolysis of peptide bonds in proteins is challenging due to the high stability of peptide bonds and the importance of proteases in biology and industry. Metal-organic frameworks (MOFs) consisting of infinite crystalline lattices with metal clusters and organic linkers may provide opportunities for protease mimic which has remained unknown. Herein, we report that Cu2(C9H3O6)4/3 MOF (which is well known as HKUST-1 and denoted as Cu-MOF here), possesses an intrinsic enzyme mimicking activity similar to that found in natural trypsin to bovine serum albumin (BSA) and casein. The Michaelis constant (Km) of Cu-MOF is about 26,000-fold smaller than that of free trypsin indicating a much higher affinity of BSA for Cu-MOF surface. Cu-MOF also exhibited significantly higher catalytic efficiency than homogeneous artificial metalloprotease Cu(II) complexes and could be reused for ten times without losing in its activity. Moreover, Cu-MOF was successfully used to simulate trypsinization in cell culture since it dissociated cells in culture even without EDTA. PMID:25342169

Purification and characterization of native and recombinant SaPIN2a, a plant sieve element-localized proteinase inhibitor.

PubMed

Wang, Zhen-Yu; Ding, Ling-Wen; Ge, Zhi-Juan; Wang, Zhaoyu; Wang, Fanghai; Li, Ning; Xu, Zeng-Fu

2007-01-01

SaPIN2a encodes a proteinase inhibitor in nightshade (Solanum americanum), which is specifically localized to the enucleate sieve elements. It has been proposed to play an important role in phloem development by regulating proteolysis in sieve elements. In this study, we purified and characterized native SaPIN2a from nightshade stems and recombinant SaPIN2a expressed in Escherichia coli. Purified native SaPIN2a was found as a charge isomer family of homodimers, and was weakly glycosylated. Native SaPIN2a significantly inhibited serine proteinases such as trypsin, chymotrypsin, and subtilisin, with the most potent inhibitory activity on subtilisin. It did not inhibit cysteine proteinase papain and aspartic proteinase cathepsin D. Recombinant SaPIN2a had a strong inhibitory effect on chymotrypsin, but its inhibitory activities toward trypsin and especially toward subtilisin were greatly reduced. In addition, native SaPIN2a can effectively inhibit midgut trypsin-like activities from Trichoplusia ni and Spodoptera litura larvae, suggesting a potential for the production of insect-resistant transgenic plants.

Characterization of the molecular features and expression patterns of two serine proteases in Hermetia illucens (Diptera: Stratiomyidae) larvae.

PubMed

Kim, Wontae; Bae, Sungwoo; Kim, Ayoung; Park, Kwanho; Lee, Sangbeom; Choi, Youngcheol; Han, Sangmi; Park, Younghan; Koh, Youngho

2011-06-01

To investigate the molecular scavenging capabilities of the larvae of Hermetia illucens, two serine proteases (SPs) were cloned and characterized. Multiple sequence alignments and phylogenetic tree analysis of the deduced amino acid sequences of Hi-SP1 and Hi-SP2 were suggested that Hi-SP1 may be a chymotrypsin- and Hi-SP2 may be a trypsin-like protease. Hi-SP1 and Hi-SP2 3-D homology models revealed that a catalytic triad, three disulfide bonds, and a substrate-binding pocket were highly conserved, as would be expected of a SP. E. coli expressed Hi-SP1 and Hi-SP2 showed chymotrypsin or trypsin activities, respectively. Hi-SP2 mRNAs were consistently expressed during larval development. In contrast, the expression of Hi-SP1 mRNA fluctuated between feeding and molting stages and disappeared at the pupal stages. These expression pattern differences suggest that Hi-SP1 may be a larval specific chymotrypsin-like protease involved with food digestion, while Hi-SP2 may be a trypsin-like protease with diverse functions at different stages.

Different thermostabilities of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPases from rabbit and trout muscles.

PubMed

de Toledo, F G; Albuquerque, M C; Goulart, B H; Chini, E N

1995-05-01

Trout and rabbit (Ca2+ + Mg2+)-ATPases from sarcoplasmic reticulum were compared for differences in thermal inactivation and susceptibility to trypsin digestion. The trout ATPase is more heat-sensitive than the rabbit ATPase and is stabilized by Ca2+, Na+, K+ and nucleotides. Solubilization of both ATPases shows that the two ATPases have different protein-intrinsic inactivation kinetics. When digested by trypsin, the two ATPases display different cleavage patterns. The present results indicate that the trout and rabbit ATPases have dissimilarities in protein structure that may explain the differences in thermal inactivation kinetics.

Calcium sensitivity of residual force enhancement in rabbit skinned fibers.

PubMed

Joumaa, V; Herzog, W

2014-08-15

Isometric force after active stretch of muscles is higher than the purely isometric force at the corresponding length. This property is termed residual force enhancement. Active force in skeletal muscle depends on calcium attachment characteristics to the regulatory proteins. Passive force has been shown to influence calcium attachment characteristics, specifically the sarcomere length dependence of calcium sensitivity. Since one of the mechanisms proposed to explain residual force enhancement is the increase in passive force that results from engagement of titin upon activation and stretch, our aim was to test if calcium sensitivity of residual force enhancement was different from that of its corresponding purely isometric contraction and if such a difference was related to the molecular spring titin. Force-pCa curves were established in rabbit psoas skinned fibers for reference and residual force-enhanced states at a sarcomere length of 3.0 μm 1) in a titin-intact condition, 2) after treatment with trypsin to partially eliminate titin, and 3) after treatment with trypsin and osmotic compression with dextran T-500 to decrease the lattice spacing in the absence of titin. The force-pCa curves of residual force enhancement were shifted to the left compared with their corresponding controls in titin-intact fibers, indicating increased calcium sensitivity. No difference in calcium sensitivity was observed between reference and residual force-enhanced contractions in trypsin-treated and osmotically compressed trypsin-treated fibers. Furthermore, calcium sensitivity after osmotic compression was lower than that observed for residual force enhancement in titin-intact skinned fibers. These results suggest that titin-based passive force regulates the increase in calcium sensitivity of residual force enhancement by a mechanism other than reduction of the myofilament lattice spacing. Copyright © 2014 the American Physiological Society.

Effect of trypsin inhibitor activity in soya bean on growth performance, protein digestibility and incidence of sub-clinical necrotic enteritis in broiler chicken flocks.

PubMed

Palliyeguru, M W C D; Rose, S P; Mackenzie, A M

2011-06-01

1. The effect of three different levels of dietary trypsin inhibitor activity (achieved by varying the amount of non-toasted full fat soya bean in replacement for toasted full fat soya bean) on the incidence of spontaneously-occurring sub-clinical necrotic enteritis (NE) in broiler chickens was compared. A fourth dietary treatment compared the effect of a diet that used potato protein concentrate as the major protein source. The determined trypsin inhibitor activity increased with the increasing content of non-toasted soya bean: 1·90, 6·21, 8·46 and 3·72 mg/g for the three soya bean diets (0, 100 and 200 g of non-toasted soya bean/kg) and the potato protein diet respectively. 2. Although increasing amounts of the non-toasted full-fat soya bean increased the feed intakes of the birds, there was a marked reduction in protein digestibility, weight gain and feed conversion efficiency. 3. There was a linear increase in sub-clinical NE lesions in the duodenum, jejunum, mid small intestine and ileum with increasing non-toasted soya bean. Caecal Clostridium perfringens counts increased with the increasing dietary content of non-toasted soya bean. Serum α-toxin antibodies were higher in the birds fed the 200 g non-toasted soya bean/kg diet compared with the other diets. 4. The results demonstrated that variation in the amount of non-toasted dietary soya bean not only affects growth performance of broilers but also affects the incidence of sub-clinical necrotic enteritis in the flock. Ensuring the lowest possible trypsin-inhibitor activity in soya bean samples is a valuable tool to improve the health and welfare of birds and in reducing the financial losses from this disease.

Effects of Different Cell-Detaching Methods on the Viability and Cell Surface Antigen Expression of Synovial Mesenchymal Stem Cells.

PubMed

Tsuji, Kunikazu; Ojima, Miyoko; Otabe, Koji; Horie, Masafumi; Koga, Hideyuki; Sekiya, Ichiro; Muneta, Takeshi

2017-06-09

Flow cytometric analysis of cell surface antigens is a powerful tool for the isolation and characterization of stem cells residing in adult tissues. In contrast to the collection of hematopoietic stem cells, the process of enzymatic digestion is usually necessary to prepare mesenchymal stem cells (MSCs) suspensions, which can influence the expression of cell surface markers. In this study, we examined the effects of various cell-detaching reagents and digestion times on the expression of stem cell-related surface antigens and MSC functions. Human MSCs were detached from dishes using four different reagents: trypsin, TrypLE, collagenase, and a nonenzymatic cell dissociation reagent (C5789; Sigma-Aldrich). Following dissociation reagent incubations ranging from 5 to 120 min, cell surface markers were analyzed by flow cytometry. Trypsin and TrypLE quickly dissociated the cells within 5 min, while collagenase and C5789 required 60 min to obtain maximum cell yields. C5789 significantly decreased cell viability at 120 min. Trypsin treatment significantly reduced CD44+, CD55+, CD73+, CD105+, CD140a+, CD140b+, and CD201+ cell numbers within 30 min. Collagenase treatment reduced CD140a expression by 30 min. In contrast, TrypLE treatment did not affect the expression of any cell surface antigens tested by 30 min. Despite the significant loss of surface antigen expression after 60 min of treatment with trypsin, adverse effects of enzymatic digestion on multipotency of MSCs were limited. Overall, our data indicated that TrypLE is advantageous over other cell dissociation reagents tested for the rapid preparation of viable MSC suspensions.

Secretion of Proteases by an Opportunistic Fungal Pathogen Scedosporium aurantiacum

PubMed Central

Kautto, Liisa; Nevalainen, Helena

2017-01-01

Scedosporium aurantiacum is an opportunistic filamentous fungus increasingly isolated from the sputum of cystic fibrosis patients, and is especially prevalent in Australia. At the moment, very little is known about the infection mechanism of this fungus. Secreted proteases have been shown to contribute to fungal virulence in several studies with other fungi. Here we have compared the profiles of proteases secreted by a clinical isolate Scedosporium aurantiacum (WM 06.482) and an environmental strain (WM 10.136) grown on a synthetic cystic fibrosis sputum medium supplemented with casein or mucin. Protease activity was assessed using class-specific substrates and inhibitors. Subtilisin-like and trypsin-like serine protease activity was detected in all cultures. The greatest difference in the secretion of proteases between the two strains occurred in mucin-supplemented medium, where the activities of the elastase-like, trypsin-like and aspartic proteases were, overall, 2.5–75 fold higher in the clinical strain compared to the environmental strain. Proteases secreted by the two strains in the mucin-supplemented medium were further analyzed by mass spectrometry. Six homologs of fungal proteases were identified from the clinical strain and five from the environmental strain. Of these, three were common for both strains including a subtilisin peptidase, a putative leucine aminopeptidase and a PA-SaNapH-like protease. Trypsin-like protease was identified by mass spectrometry only in the clinical isolate even though trypsin-like activity was present in all cultures. In contrast, high elastase-like activity was measured in the culture supernatant of the clinical strain but could not be identified by mass spectrometry searching against other fungi in the NCBI database. Future availability of an annotated genome will help finalise identification of the S. aurantiacum proteases. PMID:28060882

Secretion of Proteases by an Opportunistic Fungal Pathogen Scedosporium aurantiacum.

PubMed

Han, Zhiping; Kautto, Liisa; Nevalainen, Helena

2017-01-01

Scedosporium aurantiacum is an opportunistic filamentous fungus increasingly isolated from the sputum of cystic fibrosis patients, and is especially prevalent in Australia. At the moment, very little is known about the infection mechanism of this fungus. Secreted proteases have been shown to contribute to fungal virulence in several studies with other fungi. Here we have compared the profiles of proteases secreted by a clinical isolate Scedosporium aurantiacum (WM 06.482) and an environmental strain (WM 10.136) grown on a synthetic cystic fibrosis sputum medium supplemented with casein or mucin. Protease activity was assessed using class-specific substrates and inhibitors. Subtilisin-like and trypsin-like serine protease activity was detected in all cultures. The greatest difference in the secretion of proteases between the two strains occurred in mucin-supplemented medium, where the activities of the elastase-like, trypsin-like and aspartic proteases were, overall, 2.5-75 fold higher in the clinical strain compared to the environmental strain. Proteases secreted by the two strains in the mucin-supplemented medium were further analyzed by mass spectrometry. Six homologs of fungal proteases were identified from the clinical strain and five from the environmental strain. Of these, three were common for both strains including a subtilisin peptidase, a putative leucine aminopeptidase and a PA-SaNapH-like protease. Trypsin-like protease was identified by mass spectrometry only in the clinical isolate even though trypsin-like activity was present in all cultures. In contrast, high elastase-like activity was measured in the culture supernatant of the clinical strain but could not be identified by mass spectrometry searching against other fungi in the NCBI database. Future availability of an annotated genome will help finalise identification of the S. aurantiacum proteases.

Trypsin pre-treatment corrects SRID over-estimation of immunologically active, pre-fusion HA caused by mixed immunoprecipitin rings.

PubMed

Wen, Yingxia; Palladino, Giuseppe; Xie, Yuhong; Ferrari, Annette; Ma, Xiuwen; Han, Liqun; Dormitzer, Philip R; Settembre, Ethan C

2016-06-17

Influenza vaccines are the primary intervention to prevent the substantial health burden of seasonal and pandemic influenza. Subunit and split influenza vaccines are formulated, released for clinical use, and tested for stability based on their content of immunologically active (capable of eliciting functional antibodies) hemagglutinin (HA). Single-radial immunodiffusion (SRID), the standard in vitro potency assay in the field, is believed to specifically detect immunologically active HA. We confirmed that, with conformationally homogeneous HA preparations, SRID specifically detected native, pre-fusion HA, which elicited influenza neutralizing and hemagglutination inhibiting (HI) antibodies in mice, and it did not detect low-pH stressed, post-fusion HA, which was selectively removed from the SRID gel during a blotting step and was significantly less immunologically active. This selective detection was due to the SRID format, not a conformational specificity of the sheep antiserum used in the SRID, as the same antiserum detected non-stressed and low-pH stressed HA similarly when used in an ELISA format. However, when low-pH stressed HA was mixed with non-stressed HA, SRID detected both forms in mixed immunoprecipitin rings, leading to over-quantification of pre-fusion HA. We previously reported that trypsin digestion of antigen samples selectively degrade stressed HA, so that an otherwise conformationally insensitive biophysical quantification technique, reversed-phase high pressure liquid chromatography (RP-HPLC), can specifically quantify trypsin-resistant, immunologically active, pre-fusion HA. Here, we report that trypsin digestion can also improve the specificity of SRID so that it can quantify immunologically active, pre-fusion HA when it is mixed with less immunologically active, post-fusion HA. Copyright © 2016 Elsevier Ltd. All rights reserved.

Higher Activities of Defense-Associated Enzymes may Contribute to Greater Resistance of Manchurian Ash to Emerald Ash Borer Than A closely Related and Susceptible Congener.

PubMed

Rigsby, Chad M; Herms, Daniel A; Bonello, Pierluigi; Cipollini, Don

2016-08-01

Emerald ash borer (EAB) is an invasive beetle native to Asia that infests and kills ash (Fraxinus spp.) in North America. Previous experiments indicated that larvae feeding on co-evolved, resistant Manchurian ash (F. mandshurica) have increased antioxidant and quinone-protective enzyme activities compared to larvae feeding on susceptible North American species. Here, we examined mechanisms of host-generated oxidative and quinone-based stress and other putative defenses in Manchurian ash and the closely related and chemically similar, but susceptible, black ash (F. nigra), with and without exogenous application of methyl jasmonate (MeJA) to induce resistance mechanisms. Peroxidase activities were 4.6-13.3 times higher in Manchurian than black ash, although both species appeared to express the same three peroxidase isozymes. Additionally, peroxidase-mediated protein cross-linking activity was stronger in Manchurian ash. Polyphenol oxidase, β-glucosidase, chitinase, and lipoxygenase activities also were greater in Manchurian ash, but only lipoxygenase activity increased with MeJA application. Phloem H 2 O 2 levels were similar and were increased by MeJA application in both species. Lastly, trypsin inhibitor activity was detected in methanol and water extracts that were not allowed to oxidize, indicating the presence of phenolic-based trypsin inhibitors. However, no proteinaceous trypsin inhibitor activity was detected in either species. In response to MeJA application, Manchurian ash had higher trypsin inhibitor activity than black ash using the unoxidized water extracts, but no treatment effects were detected using methanol extracts. Based on these results we hypothesize that peroxidases, lignin polymerization, and quinone generation contribute to the greater resistance to EAB displayed by Manchurian ash.

Molecular ontogenesis of digestive capability and associated endocrine control in Atlantic cod (Gadus morhua) larvae.

PubMed

Kortner, Trond M; Overrein, Ingrid; Oie, Gunvor; Kjørsvik, Elin; Bardal, Tora; Wold, Per-Arvid; Arukwe, Augustine

2011-10-01

We have profiled the expression of twelve genes, in order to provide an overview on the molecular ontogeny of digestive capability with the associated endocrine control during Atlantic cod (Gadus morhua) larval development. Enzyme activity levels for the key digestive enzyme, trypsin, was also measured. Specifically, transcripts for trypsin, amylase, lipolytic enzymes: bile salt activated lipase (BAL), phospholipase A2 (PLA2) and Acyl CoA dehydrogenase (ACADM), regulatory peptides: neuropeptide Y (NPY), orexin (OX) cholecystokinin (CCK) and cocaine and amphetamine-related transcript (CART), the somatotropic factors: growth hormone (GH), preprosomatostatin 1 (PPSS1) and thyroid hormone receptors (TRα and TRβ) were analyzed using quatitative (real-time) polymerase chain reaction (qPCR). Trypsin and BAL mRNA levels peaked at approximately day 17 and 25 post-hatch, respectively, and thereafter displayed a decreasing pattern until metamorphosis. GH mRNA levels decreased moderately from 3 to 33dph, and thereafter, an increase was observed until 46dph. TRα mRNA levels showed a fluctuating pattern peaking at day 39 post-hatch. TRβ mRNA levels were too low to obtain quantitative measurements. Amylase mRNA slightly increased from day 3 to 17 post-hatch, and thereafter showed a steady decrease until day 60. Interestingly, PLA2 mRNA expression showed a consistent increase throughout the study period, indicating an increasingly important role during larval development. Overall, data from this study indicate that cod larvae show differential developmental mode of expression patterns for key genes and endocrine factors that regulate digestive capability, growth and development. These data are discussed in relation to larval trypsin enzyme activity and previous reports for other teleost species. Copyright © 2011 Elsevier Inc. All rights reserved.

Trypsin from the processing waste of the lane snapper (Lutjanus synagris) and its compatibility with oxidants, surfactants and commercial detergents.

PubMed

Espósito, Talita S; Marcuschi, Marina; Amaral, Ian P G; Carvalho, Luiz B; Bezerra, Ranilson S

2010-05-26

A trypsin from the viscera of the lane snapper (Lutjanus synagris) was purified by heat treatment, fractionation with ammonium sulfate and affinity chromatography. The molecular weight of the enzyme was estimated to be 28.4 kDa (SDS-PAGE). The purified enzyme was capable of hydrolyzing the specific substrate for trypsin benzoyl-arginine-p-nitroanilide (BApNA) and was inhibited by benzamidine and tosyl lysine chloromethyl ketone (TLCK), synthetic trypsin inhibitors and phenylmethylsulfonyl fluoride (PMSF), which is a serine-protease inhibitor. The enzyme exhibited maximal activity at pH 9.0 and 45 degrees C and retained 100% of the activity after incubation at the optimal temperature for 30 min. At a concentration of 10 mM, activity was slightly activated by Ca(2+) and inhibited by the following ions in decreasing order: Cd(2+) > Hg(2+) > Cu(2+) > Zn(2+) > Al(3+). The effects of Ba(2+), K(1+) and Li(1+) proved to be less intensive. Using 1% (w/v) azocasein as substrate, the enzyme revealed high resistance (60% residual activity) when incubated with 10% H(2)O(2) for 75 min. The enzyme retained more than 80% activity after 60 min in the presence of different surfactants (Tween 20, Tween 80 and sodium choleate). The alkaline protease demonstrated compatibility with commercial detergents (7 mg/mL), such as Bem-te-vi, Surf and Ala, retaining more than 50% of initial activity after 60 min at 25 degrees C and 30 min at 40 degrees C. The thermostability and compatibility of this enzyme with commercial detergents suggest a good potentiality for application in the detergent industry.

Identification and Characterization of a New Enterotoxin Produced by Clostridium perfringens Isolated from Food Poisoning Outbreaks.

PubMed

Irikura, Daisuke; Monma, Chie; Suzuki, Yasunori; Nakama, Akiko; Kai, Akemi; Fukui-Miyazaki, Aya; Horiguchi, Yasuhiko; Yoshinari, Tomoya; Sugita-Konishi, Yoshiko; Kamata, Yoichi

2015-01-01

There is a strain of Clostridium perfringens, W5052, which does not produce a known enterotoxin. We herein report that the strain W5052 expressed a homologue of the iota-like toxin components sa and sb of C. spiroforme, named Clostridium perfringens iota-like enterotoxin, CPILE-a and CPILE-b, respectively, based on the results of a genome sequencing analysis and a systematic protein screening. In the nicotinamide glyco-hydrolase (NADase) assay the hydrolysis activity was dose-dependently increased by the concentration of rCPILE-a, as judged by the mass spectrometry analysis. In addition, the actin monomer of the lysates of Vero and L929 cells were radiolabeled in the presence of [32P]NAD and rCPILE-a. These findings indicated that CPILE-a possesses ADP-ribosylation activity. The culture supernatant of W5052 facilitated the rounding and killing of Vero and L929 cells, but the rCPILE-a or a non-proteolyzed rCPILE-b did not. However, a trypsin-treated rCPILE-b did. Moreover, a mixture of rCPILE-a and the trypsin-treated rCPILE-b enhanced the cell rounding and killing activities, compared with that induced by the trypsin-treated rCPILE-b alone. The injection of the mixture of rCPILE-a and the trypsin-treated rCPILE-b into an ileum loop of rabbits evoked the swelling of the loop and accumulation of the fluid dose-dependently, suggesting that CPILE possesses enterotoxic activity. The evidence presented in this communication will facilitate the epidemiological, etiological, and toxicological studies of C. perfringens food poisoning, and also stimulate studies on the transfer of the toxins' gene(s) among the Genus Clostridium.

Identification and Characterization of a New Enterotoxin Produced by Clostridium perfringens Isolated from Food Poisoning Outbreaks

PubMed Central

Suzuki, Yasunori; Nakama, Akiko; Kai, Akemi; Fukui-Miyazaki, Aya; Horiguchi, Yasuhiko; Yoshinari, Tomoya; Sugita-Konishi, Yoshiko; Kamata, Yoichi

2015-01-01

There is a strain of Clostridium perfringens, W5052, which does not produce a known enterotoxin. We herein report that the strain W5052 expressed a homologue of the iota-like toxin components sa and sb of C. spiroforme, named Clostridium perfringens iota-like enterotoxin, CPILE-a and CPILE-b, respectively, based on the results of a genome sequencing analysis and a systematic protein screening. In the nicotinamide glyco-hydrolase (NADase) assay the hydrolysis activity was dose-dependently increased by the concentration of rCPILE-a, as judged by the mass spectrometry analysis. In addition, the actin monomer of the lysates of Vero and L929 cells were radiolabeled in the presence of [32P]NAD and rCPILE-a. These findings indicated that CPILE-a possesses ADP-ribosylation activity. The culture supernatant of W5052 facilitated the rounding and killing of Vero and L929 cells, but the rCPILE-a or a non-proteolyzed rCPILE-b did not. However, a trypsin-treated rCPILE-b did. Moreover, a mixture of rCPILE-a and the trypsin-treated rCPILE-b enhanced the cell rounding and killing activities, compared with that induced by the trypsin-treated rCPILE-b alone. The injection of the mixture of rCPILE-a and the trypsin-treated rCPILE-b into an ileum loop of rabbits evoked the swelling of the loop and accumulation of the fluid dose-dependently, suggesting that CPILE possesses enterotoxic activity. The evidence presented in this communication will facilitate the epidemiological, etiological, and toxicological studies of C. perfringens food poisoning, and also stimulate studies on the transfer of the toxins’ gene(s) among the Genus Clostridium. PMID:26584048

Mechanical loading of bovine pericardium accelerates enzymatic degradation.

PubMed

Ellsmere, J C; Khanna, R A; Lee, J M

1999-06-01

Bioprosthetic heart valves fail as the result of two simultaneous processes: structural deterioration and calcification. Leaflet deterioration and perforation have been correlated with regions of highest stress in the tissue. The failures have long been assumed to be due to simple mechanical fatigue of the collagen fibre architecture; however, we have hypothesized that local stresses-and particularly dynamic stresses-accelerate local proteolysis, leading to tissue failure. This study addresses that hypothesis. Using a novel, custom-built microtensile culture system, strips of bovine pericardium were subjected to static and dynamic loads while being exposed to solutions of microbial collagenase or trypsin (a non-specific proteolytic enzyme). The time to extend to 30% strain (defined here as time to failure) was recorded. After failure, the percentage of collagen solubilized was calculated based on the amount of hydroxyproline present in solution. All data were analyzed by analysis of variance (ANOVA). In collagenase, exposure to static load significantly decreased the time to failure (P < 0.002) due to increased mean rate of collagen solubilization. Importantly, specimens exposed to collagenase and dynamic load failed faster than those exposed to collagenase under the same average static load (P = 0.02). In trypsin, by contrast, static load never led to failure and produced only minimal degradation. Under dynamic load, however, specimens exposed to collagenase, trypsin, and even Tris/CaCl2 buffer solution, all failed. Only samples exposed to Hanks' physiological solution did not fail. Failure of the specimens exposed to trypsin and Tris/CaCl2 suggests that the non-collagenous components and the calcium-dependent proteolytic enzymes present in pericardial tissue may play roles in the pathogenesis of bioprosthetic heart valve degeneration.

Secretagogues differentially activate endoplasmic reticulum stress responses in pancreatic acinar cells.

PubMed

Kubisch, Constanze H; Logsdon, Craig D

2007-06-01

Endoplasmic reticulum (ER) stress leads to the accumulation of misfolded proteins in the ER lumen and initiates the unfolded protein response (UPR). Components of the UPR are important in pancreatic development, and recent studies have indicated that the UPR is activated in the arginine model of acute pancreatitis. However, the effects of secretagogues on UPR components in the pancreas are unknown. The present study aimed to examine the effects of different types and concentrations of secretagogues on acinar cell function and specific components of the UPR. Rat pancreatic acini were stimulated with the CCK analogs CCK8 (10 pM-10 nM) or JMV-180 (10 nM-10 microM) or with bombesin (1-100 nM). Components of the UPR, including chaperone BiP expression, PKR-like ER kinase (PERK) phosphorylation, X box-binding protein 1 (XBP1) splicing, and CCAAT/enhancer binding protein homologous protein (CHOP) expression, were measured, as were effects on amylase secretion and intracellular trypsin activation. CCK8 generated a biphasic secretion dose-response curve, and high concentrations increased intracellular active trypsin levels. In contrast, JMV-180 and bombesin secretion dose-response curves were monophasic, and high concentrations did not increase intracellular trypsin activity. All three secretagogues increased BiP levels and XBP1 splicing. However, only supraphysiological levels of CCK8 associated with inhibited amylase secretion and trypsin activation stimulated PERK phosphorylation and expression of CHOP. The effects of CCK8 on UPR components were rapid, occurring within 5-20 min. In conclusion, ER stress response mechanisms appear to be involved in both pancreatic physiology and pathophysiology, and future efforts should be directed at understanding the roles of these mechanisms in the pancreas.

Trypsin digestion for determining orientation of ATPase in Halobacterium saccharovorum membrane vesicles

NASA Technical Reports Server (NTRS)

Kristjansson, H.; Hochstein, L. I.

1986-01-01

Membranes prepared by low pressure disruption of cells exhibited no ATPase activity in the absence of Triton X-100, although 43% of the total menadione reductase activity was detected. Trypsin digestion reduced menadione reductase activity by 45% whereas ATPase activity was not affected. Disruption of the membrane fraction at higher pressure solubilized about 45% of the ATPase activity. The soluble activity was still enhanced by Triton X-100, suggesting that the detergent, besides disrupting membrane vesicles, also activated the ATPase. The discrepancy in localization of menadione reductase and ATPase activities raised questions regarding the reliability of using a single marker enzyme as an indicator of vesicle orientation.

Structures and Activity of New Anabaenopeptins Produced by Baltic Sea Cyanobacteria.

PubMed

Spoof, Lisa; Błaszczyk, Agata; Meriluoto, Jussi; Cegłowska, Marta; Mazur-Marzec, Hanna

2015-12-30

Anabaenopeptins, bioactive cyclic hexapeptides, were isolated by preparative reversed-phase high performance liquid chromatography from an extract of Baltic Sea cyanobacterial bloom material composed of Nodularia spumigena (50%), Aphanizomenon flos-aquae (40%) and Dolichospermum spp. (10%). Five new anabaenopeptins and nine previously known anabaenopeptins were isolated, and their putative structures were determined by tandem mass spectrometry. The activity of the peptides against carboxypeptidase A and protein phosphatase 1 as well as chymotrypsin, trypsin and thrombin was tested. All anabaenopeptins inhibited carboxypeptidase A (apart from one anabaenopeptin variant) and protein phosphatase 1 with varying potency, but no inhibition against chymotrypsin, trypsin and thrombin was observed.

Synthesis and biochemical evaluation of triazole/tetrazole-containing sulfonamides against thrombin and related serine proteases

PubMed Central

Siles, Rogelio; Kawasaki, Yuko; Ross, Patrick; Freire, Ernesto

2011-01-01

A small library of 25 triazole/tetrazole-based sulfonamides have been synthesized and further evaluated for their inhibitory activity against thrombin, trypsin, tryptase and chymase. In general, the triazole-based sulfonamides inhibited thrombin more efficiently than the tetrazole counterparts. Particularly, compound 26 showed strong thrombin inhibition (Ki =880 nM) and significant selectivity against other human related serine proteases like trypsin (Ki =729 µM). Thrombin binding affinity of the same compound was determined by ITC and demonstrated that the binding of this new triazole-based scaffold is enthalpically driven, making it a good candidate for further development. PMID:21807511

Involvement of the N-terminal region in alpha-crystallin-lens membrane recognition

NASA Technical Reports Server (NTRS)

Ifeanyi, F.; Takemoto, L.; Spooner, B. S. (Principal Investigator)

1991-01-01

Previous studies have demonstrated that alpha-crystallin binds specifically, in a saturable manner, to lens membrane. To determine the region of the alpha-crystallin molecule that might be involved in this binding, native alpha-crystallin from the bovine lens has been treated by limited digestion with trypsin, to produce alpha-A molecules with an intact C-terminal region, and a nicked N-terminal region. Compared to intact alpha-crystallin, trypsin-treated alpha-crystallin binds less avidly to lens membrane, suggesting that the N-terminal region of the alpha-A molecule may play a key role in the recognition between lens membrane and crystallin.

In Situ Caging of Biomolecules in Graphene Hybrids for Light Modulated Bioactivity.

PubMed

Cheng, Gong; Han, Xiao-Hui; Hao, Si-Jie; Nisic, Merisa; Zheng, Si-Yang

2018-01-31

Remote and noninvasive modulation of protein activity is essential for applications in biotechnology and medicine. Optical control has emerged as the most attractive approach owing to its high spatial and temporal resolutions; however, it is challenging to engineer light responsive proteins. In this work, a near-infrared (NIR) light-responsive graphene-silica-trypsin (GST) nanoreactor is developed for modulating the bioactivity of trypsin molecules. Biomolecules are spatially confined and protected in the rationally designed compartment architecture, which not only reduces the possible interference but also boosts the bioreaction efficiency. Upon NIR irradiation, the photothermal effect of the GST nanoreactor enables the ultrafast in situ heating for remote activation and tuning of the bioactivity. We apply the GST nanoreactor for remote and ultrafast proteolysis of proteins, which remarkably enhances the proteolysis efficiency and reduces the bioreaction time from the overnight of using free trypsin to seconds. We envision that this work not only provides a promising tool of ultrafast and remotely controllable proteolysis for in vivo proteomics in study of tissue microenvironment and other biomedical applications but also paves the way for exploring smart artificial nanoreactors in biomolecular modulation to gain insight in dynamic biological transformation.

Digestive enzymes and gut morphometric parameters of threespine stickleback (Gasterosteus aculeatus): Influence of body size and temperature

PubMed Central

Marchand, Adrien; Turies, Cyril; Kerambrun, Elodie; Palluel, Olivier; Bado-Nilles, Anne; Beaudouin, Rémy; Porcher, Jean-Marc; Geffard, Alain; Dedourge-Geffard, Odile

2018-01-01

Determining digestive enzyme activity is of potential interest to obtain and understand valuable information about fish digestive physiology, since digestion is an elementary process of fish metabolism. We described for the first time (i) three digestive enzymes: amylase, trypsin and intestinal alkaline phosphatase (IAP), and (ii) three gut morphometric parameters: relative gut length (RGL), relative gut mass (RGM) and Zihler’s index (ZI) in threespine stickleback (Gasterosteus aculeatus), and we studied the effect of temperature and body size on these parameters. When mimicking seasonal variation in temperature, body size had no effect on digestive enzyme activity. The highest levels of amylase and trypsin activity were observed at 18°C, while the highest IAP activity was recorded at 20°C. When sticklebacks were exposed to three constant temperatures (16, 18 and 21°C), a temporal effect correlated to fish growth was observed with inverse evolution patterns between amylase activity and the activities of trypsin and IAP. Temperature (in both experiments) had no effect on morphometric parameters. However, a temporal variation was recorded for both RGM (in the second experiment) and ZI (in both experiments), and the later was correlated to fish body mass. PMID:29614133

Effects of acylation on the functional properties and in vitro trypsin digestibility of red kidney bean (Phaseolus vulgaris L.) protein isolate.

PubMed

Yin, Shou-Wei; Tang, Chuan-He; Wen, Qi-Biao; Yang, Xiao-Quan

2009-01-01

The effects of succinylation and acetylation on some functional properties and the in vitro trypsin digestibility of kidney bean protein isolate (KPI) were investigated. The extent of succinylation or acetylation progressively increased from 0% to 96% to 97%, as the anhydride-to-protein ratio increased from 0 to 1 g/g. Polyacrylamide gel electrophoresis (PAGE) and zeta potential analyses indicated that acylation, especially succinylation, considerably increased the net charge and hydrodynamic radius of the proteins in KPI, especially vicilin. Acylation treatment at various anhydride-to-protein ratios (0.05 to 1 g/g) remarkably improved the protein solubility (PS) and emulsifying activity index (EAI) at neutral pH, but the improvement by succinylation was much better than that by acetylation. Succinylation resulted in a marked decrease in mechanical moduli of heat-induced gels of KPI, while the mechanical moduli were, on the contrary, increased by acetylation. Additionally, in vitro trypsin digestibility was improved by the acylation in an anhydride-type and level-dependent manner. The results suggest that the functional properties of KPI could be modulated by the chemical acylation treatment, using succinic or acetic anhydride at appropriate anhydride-to-protein ratios.

The modifier effects of chymotrypsin and trypsin enzymes on fluorescence lifetime distribution of "N-(1-pyrenyl)maleimide-bovine serum albumin" complex

NASA Astrophysics Data System (ADS)

Özyiğit, İbrahim Ethem; Karakuş, Emine; Pekcan, Önder

2016-02-01

Chymotrypsin and trypsin are the well known proteolytic enzymes, both of which are synthesized in the pancreas as their precursors - the inactive forms; chymotrypsinogen and trypsinogen - and then are released into the duodenum to cut proteins into smaller peptides. In this paper, the effects of activities of chymotrypsin and trypsin enzymes on fluorescence lifetime distributions of the substrat bovine serum albumin (BSA) modified with N-(1-pyrenyl)maleimide (PM) were examined. In the labeling study of BSA with PM, it is aimed to attach PM to the single free thiol (Cys34) and to all the free amine groups in accessible positions in order to produce excimers of pyrene planes of the possible highest amount to form the lifetime distributions in the widest range, that may show specifically distinguishing changes resulting from the activities of the proteases. The time resolved spectrofluorometer was used to monitor fluorescence decays, which were analyzed by using the exponential series method (ESM) to obtain the changes of lifetime distributions. After the exposure of the synthesized substrat PM-BSA to the enzymes, the fluorescence lifetime distributions exhibited different structures which were attributed to the different activities of the proteases.

Compositions, Protease Inhibitor and Gelling Property of Duck Egg Albumen as Affected by Salting

PubMed Central

2018-01-01

Chemical compositions, trypsin inhibitory activity, and gelling properties of albumen from duck egg during salting of 30 days were studied. As the salting time increased, moisture content decreased, the salt content and surface hydrophobicity increased (p<0.05). Trypsin inhibitory activity and specific activity were continuously decreased throughout the salting time of 30 days (p<0.05). This coincided with the decrease in band intensity of inhibitor with molecular weight of 44 kDa as examined by inhibitory activity staining. Nevertheless, no differences in protein patterns were observed in albumen during the salting of 30 days. Based on texture profile analysis, hardness, springiness, gumminess, chewiness, and resilience of albumen gel decreased with increasing salting time. Conversely, salted albumen gels exhibited higher cohesiveness and adhesiveness, compared to those of fresh albumen. Scanning electron microscopic study revealed that gel of salted albumen showed the larger voids and less compactness. In general, salting lowered trypsin inhibitory activity and gelling property of albumen from duck egg to some extent. Nevertheless, the salted albumen with the remaining inhibitor could be an alternative additive for surimi or other meat products to prevent proteolysis. PMID:29725221

Identification of novel serine proteinase gene transcripts in the midguts of two tropical insect pests, Scirpophaga incertulas (Wk.) and Helicoverpa armigera (Hb.).

PubMed

Mazumdar-Leighton, S; Babu, C R; Bennett, J

2000-01-01

We have used RT PCR and 3'RACE to identify diverse serine proteinase genes expressed in the midguts of the rice yellow stem borer (Scirpophaga incertulas) and Asian corn borer (Helicoverpa armigera). The RT-PCR primers encoded the conserved regions around the active site histidine57 and serine195 of Drosophila melanogaster alpha trypsin, including aspartate189 of the specificity pocket. These primers amplified three transcripts (SiP1-3) from midguts of S. incertulas, and two transcripts (HaP1-2) from midguts of H. armigera. The five RT PCR products were sequenced to permit design of gene-specific forward primers for use with anchored oligo dT primers in 3'RACE. Sequencing of the 3'RACE products indicated that SiP1, SiP2 and HaP1 encoded trypsin-like serine proteinases, while HaP2 encoded a chymotrypsin-like serine proteinases. The SiP3 transcript proved to be an abundant 960 nt mRNA encoding a trypsin-like protein in which the active site serine195 was replaced by aspartate. The possible functions of this unusual protein are discussed.

Effect of urinary trypsin inhibitor on potassium currents: fetus modulates membrane excitability by production of UTI.

PubMed

Takeuchi, Kinya; Fukuda, Atsuo; Kanayama, Naohiro

2004-01-01

Amniotic fluid contains a significant level of urinary trypsin inhibitor (UTI). Previously, we reported that UTI inhibits calcium influx of myometrium and it is effective in preventing uterine contraction. This study examined the effects of UTI upon potassium channels, which is important for membrane excitability. Whole-cell patch-clamp recordings were performed in fibroblasts derived from human fetal skin. Potassium currents were recorded and the effects of exogenous UTI and/or cadmium determined. Tetraethylammonium sensitive potassium currents were elicited by step or ramp stimulations at depolarized membrane potentials (over +30 mV). Administration of 1 micro M UTI significantly increased these potassium currents by 16.9%. When calcium channels were blocked by the administration of cadmium, UTI increased the rest of the potassium currents by 4.8%. This indicates that UTI increased calcium-dependent potassium currents by 94.8% but only increased voltage-dependent potassium currents by 4.8%. Urinary trypsin inhibitor is a physiological substance of fetal origin that modulates calcium-dependent and voltage-dependent potassium channels. These data suggest that UTI is capable of regulating the membrane properties of the fetal and myometrial cells in contact with amniotic fluid.

A potential human hepatocellular carcinoma inhibitor from Bauhinia purpurea L. seeds: from purification to mechanism exploration.

PubMed

Fang, Evandro Fei; Bah, Clara Shui Fern; Wong, Jack Ho; Pan, Wen Liang; Chan, Yau Sang; Ye, Xiu Juan; Ng, Tzi Bun

2012-02-01

A 20-kDa Kunitz-type trypsin-chymotrypsin inhibitor, Bauhinia purpurea trypsin inhibitor (BPLTI), has been isolated from the seeds of B. purpurea L. by using liquid chromatography procedures that involved ion exchange chromatography on Sp-Sepharose and Mono S and gel filtration on Superdex 75. BPLTI demonstrated protease inhibitory activities of 7226 BAEE units/mg and 65 BTEE units/mg toward trypsin and α-chymotrypsin, respectively. BPLTI was relatively thermal (0-60°C) and pH (3-10) stable and its activity could be decreased by dithiothreitol treatment. BPLTI exhibited a wide spectrum of anti-proliferative and pro-apoptotic activities especially on human hepatocellular carcinoma Hep G2 cells. However, it was devoid of a significant antiproliferative effect on immortal human hepatic WRL 68 cells. We show here that BPLTI stimulates apoptosis in Hep G2 cells, including (1) evoking DNA damage including the production of chromatin condensation and apoptotic bodies; (2) induction of cell apoptosis/necrosis; (3) mitochondrial membrane depolarization; and (4) increasing the production of cytokines. Taken together, our findings show for the first time that purified protease inhibitor from B. purpurea L. seeds is a promising candidate for the treatment of human hepatocellular carcinoma.

Digestive enzymes and gut morphometric parameters of threespine stickleback (Gasterosteus aculeatus): Influence of body size and temperature.

PubMed

Hani, Younes Mohamed Ismail; Marchand, Adrien; Turies, Cyril; Kerambrun, Elodie; Palluel, Olivier; Bado-Nilles, Anne; Beaudouin, Rémy; Porcher, Jean-Marc; Geffard, Alain; Dedourge-Geffard, Odile

2018-01-01

Determining digestive enzyme activity is of potential interest to obtain and understand valuable information about fish digestive physiology, since digestion is an elementary process of fish metabolism. We described for the first time (i) three digestive enzymes: amylase, trypsin and intestinal alkaline phosphatase (IAP), and (ii) three gut morphometric parameters: relative gut length (RGL), relative gut mass (RGM) and Zihler's index (ZI) in threespine stickleback (Gasterosteus aculeatus), and we studied the effect of temperature and body size on these parameters. When mimicking seasonal variation in temperature, body size had no effect on digestive enzyme activity. The highest levels of amylase and trypsin activity were observed at 18°C, while the highest IAP activity was recorded at 20°C. When sticklebacks were exposed to three constant temperatures (16, 18 and 21°C), a temporal effect correlated to fish growth was observed with inverse evolution patterns between amylase activity and the activities of trypsin and IAP. Temperature (in both experiments) had no effect on morphometric parameters. However, a temporal variation was recorded for both RGM (in the second experiment) and ZI (in both experiments), and the later was correlated to fish body mass.

Allium sativum Protease Inhibitor: A Novel Kunitz Trypsin Inhibitor from Garlic Is a New Comrade of the Serpin Family

PubMed Central

Shamsi, Tooba Naz; Parveen, Romana; Amir, Mohd.; Baig, Mohd. Affan; Qureshi, M. Irfan; Ali, Sher; Fatima, Sadaf

2016-01-01

Purpose This study was aimed to purify and characterize the Protease inhibitor (PI) from a plant Allium sativum (garlic) with strong medicinal properties and to explore its phytodrug potentials. Methods Allium sativum Protease Inhibitor (ASPI) was purified using ammonium sulphate fractionation and Fast Protein Liquid Chromatography on anion exchanger Hi-Trap DEAE column. The purified protein was analyzed for its purity and molecular weight by SDS PAGE. The confirmation of presence of trypsin inhibiting PI was performed by MALDI TOF-TOF and analyzed by MASCOT database. The ASPI was further investigated for its kinetic properties and stability under extreme conditions of pH, temperature and chemical denaturants. Secondary structure was determined by Circular Dichorism (CD) spectroscopy. Results ASPI of ~15 kDa inhibited trypsin and matched "truncated kunitz Trypsin Inhibitor (Glycine max)" in MASCOT database. The purified ASPI showed 30376.1371 U/mg specific activity with a fold purity of 159.92 and yield ~93%. ASPI was quite stable in the range of pH 2–12 showing a decline in the activity around pH 4–5 suggesting that the pI value of the protein as ASPI aggregates in this range. ASPI showed stability to a broad range of temperature (10–80°C) but declined beyond 80°C. Further, detergents, oxidizing agents and reducing agents demonstrated change in ASPI activity under varying concentrations. The kinetic analysis revealed sigmoidal relationship of velocity with substrate concentration with Vmax 240.8 (μM/min) and Km value of 0.12 μM. ASPI showed uncompetitive inhibition with a Ki of 0.08±0.01 nM). The Far UV CD depicted 2.0% α -helices and 51% β -sheets at native pH. Conclusions To conclude, purified ~15 kDa ASPI exhibited fair stability in wide range of pH and temperature Overall, there was an increase in purification fold with remarkable yield. Chemical modification studies suggested the presence of lysine and tryptophan residues as lead amino acids present in the reactive sites. Therefore, ASPI with trypsin inhibitory property has the potential to be used as a non-cytotoxic clinical agents. PMID:27846232

Identification of Analytical Factors Affecting Complex Proteomics Profiles Acquired in a Factorial Design Study with Analysis of Variance: Simultaneous Component Analysis.

PubMed

Mitra, Vikram; Govorukhina, Natalia; Zwanenburg, Gooitzen; Hoefsloot, Huub; Westra, Inge; Smilde, Age; Reijmers, Theo; van der Zee, Ate G J; Suits, Frank; Bischoff, Rainer; Horvatovich, Péter

2016-04-19

Complex shotgun proteomics peptide profiles obtained in quantitative differential protein expression studies, such as in biomarker discovery, may be affected by multiple experimental factors. These preanalytical factors may affect the measured protein abundances which in turn influence the outcome of the associated statistical analysis and validation. It is therefore important to determine which factors influence the abundance of peptides in a complex proteomics experiment and to identify those peptides that are most influenced by these factors. In the current study we analyzed depleted human serum samples to evaluate experimental factors that may influence the resulting peptide profile such as the residence time in the autosampler at 4 °C, stopping or not stopping the trypsin digestion with acid, the type of blood collection tube, different hemolysis levels, differences in clotting times, the number of freeze-thaw cycles, and different trypsin/protein ratios. To this end we used a two-level fractional factorial design of resolution IV (2(IV)(7-3)). The design required analysis of 16 samples in which the main effects were not confounded by two-factor interactions. Data preprocessing using the Threshold Avoiding Proteomics Pipeline (Suits, F.; Hoekman, B.; Rosenling, T.; Bischoff, R.; Horvatovich, P. Anal. Chem. 2011, 83, 7786-7794, ref 1) produced a data-matrix containing quantitative information on 2,559 peaks. The intensity of the peaks was log-transformed, and peaks having intensities of a low t-test significance (p-value > 0.05) and a low absolute fold ratio (<2) between the two levels of each factor were removed. The remaining peaks were subjected to analysis of variance (ANOVA)-simultaneous component analysis (ASCA). Permutation tests were used to identify which of the preanalytical factors influenced the abundance of the measured peptides most significantly. The most important preanalytical factors affecting peptide intensity were (1) the hemolysis level, (2) stopping trypsin digestion with acid, and (3) the trypsin/protein ratio. This provides guidelines for the experimentalist to keep the ratio of trypsin/protein constant and to control the trypsin reaction by stopping it with acid at an accurately set pH. The hemolysis level cannot be controlled tightly as it depends on the status of a patient's blood (e.g., red blood cells are more fragile in patients undergoing chemotherapy) and the care with which blood was sampled (e.g., by avoiding shear stress). However, its level can be determined with a simple UV spectrophotometric measurement and samples with extreme levels or the peaks affected by hemolysis can be discarded from further analysis. The loadings of the ASCA model led to peptide peaks that were most affected by a given factor, for example, to hemoglobin-derived peptides in the case of the hemolysis level. Peak intensity differences for these peptides were assessed by means of extracted ion chromatograms confirming the results of the ASCA model.

An improved approach to the analysis of drug-protein binding by distance geometry

NASA Technical Reports Server (NTRS)

Goldblum, A.; Kieber-Emmons, T.; Rein, R.

1986-01-01

The calculation of side chain centers of coordinates and the subsequent generation of side chain-side chain and side chain-backbone distance matrices is suggested as an improved method for viewing interactions inside proteins and for the comparison of protein structures. The use of side chain distance matrices is demonstrated with free PTI, and the use of difference distance matrices for side chains is shown for free and trypsin-bound PTI as well as for the X-ray structures of trypsin complexes with PTI and with benzamidine. It is found that conformational variations are reflected in the side chain distance matrices much more than in the standard C-C distance representations.

Lectin Activation in Giardia lamblia by Host Protease: A Novel Host-Parasite Interaction

NASA Astrophysics Data System (ADS)

Lev, Boaz; Ward, Honorine; Keusch, Gerald T.; Pereira, Miercio E. A.

1986-04-01

A lectin in Giardia lamblia was activated by secretions from the human duodenum, the environment where the parasite lives. Incubation of the secretions with trypsin inhibitors prevented the appearance of lectin activity, implicating proteases as the activating agent. Accordingly, lectin activation was also produced by crystalline trypsin and Pronase; other proteases tested were ineffective. When activated, the lectin agglutinated intestinal cells to which the parasite adheres in vivo. The lectin was most specific to mannose-6-phosphate and apparently was bound to the plasma membrane. Activation of a parasite lectin by a host protease represents a novel mechanism of hostparasite interaction and may contribute to the affinity of Giardia lamblia to the infection site.

Bauhinia proteinase inhibitor-based synthetic fluorogenic substrates for enzymes isolated from insect midgut and caterpillar bristles.

PubMed

Andrade, Sonia A; Santomauro-Vaz, Eugênio M; Lopes, Adriana R; Chudzinski-Tavassi, Ana M; Juliano, Maria A; Terra, Walter R; Sampaio, Misako U; Sampaio, Claudio A M; Oliva, Maria Luiza V

2003-03-01

Bauhinia ungulata factor Xa inhibitor (BuXI) inactivates factor Xa and LOPAP, a prothrombin activator proteinase isolated from the venom of Lonomia obliqua caterpillar bristles. The reactive site of the enzyme-inhibitor interaction was explored to design specific substrates for both enzymes. Methionine is crucial for LOPAP and factor Xa substrate interaction, since the change of both Met residues in the substrates abolished the hydrolysis. Synthetic substrates containing the sequence around the reactive site of BbKI, a plasma kallikrein inhibitor, were shown to be specific for trypsin hydrolysis. Therefore, these substrates may be an alternative in studies aiming at a characterization of trypsin-like enzyme activities, especially non-mammalian enzymes.

Expansion and Harvesting of hMSC-TERT

PubMed Central

Weber, Christian; Pohl, Sebastian; Pörtner, Ralf; Wallrapp, Christine; Kassem, Moustapha; Geigle, Peter; Czermak, Peter

2007-01-01

The expansion of human mesenchymal stem cells as suspension culture by means of spinner flasks and microcarriers, compared to the cultivation in tissue culture flasks, offers the advantage of reducing the requirements of large incubator capacities as well as reducing the handling effort during cultivation and harvesting. Nonporous microcarriers are preferable when the cells need to be kept in viable condition for further applications like tissue engineering or cell therapy. In this study, the qualification of Biosilon, Cytodex 1, Cytodex 3, RapidCell and P102-L for expansion of hMSC-TERT with an associated harvesting process using either trypsin, accutase, collagenase or a trypsin-accutase mixture was investigated. A subsequent adipogenic differentiation of harvested hMSC-TERT was performed in order to observe possible negative effects on their (adipogenic) differentiation potential as a result of the cultivation and harvesting method. The cultivated cells showed an average growth rate of 0.52 d-1. The cells cultivated on Biosilon, RapidCell and P102-L were harvested succesfully achieving high cell yield and vitalities near 100%. This was not the case for cells on Cytodex 1 and Cytodex 3. The trypsin-accutase mix was most effective. After spinner expansion and harvesting the cells were successfully differentiated to adipocytes. PMID:19662126

Edible provenances of Jatropha curcas from Quintana Roo state of Mexico and effect of roasting on antinutrient and toxic factors in seeds.

PubMed

Makkar, H P; Becker, K; Schmook, B

1998-01-01

Seven seed samples of J. curcas, both in raw and roasted state, sold in some villages in Quintana Roo state, Mexico for human consumption were analyzed for physical characteristics, nutrients and antinutrients. The average seed weight varied from 0.53 to 0.74 g and kernel weight as proportion of raw seed weight was from 61 to 66%. The contents of crude protein, lipid and ash of kernels from raw seeds were 27-30%, 55-62% and 3.7-5.2% respectively. The levels of antinutrients in meal from the raw seeds were: trypsin inhibitor activity (14.6-28.7 mg trypsin inhibited/g), lectin (25.6-52.2 unit; one unit is the reverse of minimum amount of mg meal/ml assay which produced haemagglutination), saponins (1.9-2.3% as diosgenin equivalent) and phytate (8.4-10%). Phorbol esters in kernels from raw seeds were not detected in four samples and in other three samples it ranged from 0.01 to 0.02 mg/g as phorbol-12-myristate 13-acetate equivalent. Roasting of seeds inactivated almost 100% of trypsin inhibitor activity. Although lectin activity reduced on roasting, it was still present in high amounts. Saponins, phytate and phorbol esters were not affected by roasting.

One-pot preparation of a sulfamethoxazole functionalized affinity monolithic column for selective isolation and purification of trypsin.

PubMed

Xiao, Yuan; Guo, Jialiang; Ran, Danni; Duan, Qianqian; Crommen, Jacques; Jiang, Zhengjin

2015-06-26

A facile and efficient "one-pot" copolymerization strategy was used for the preparation of sulfonamide drug (SA) functionalized monolithic columns. Two novel SA-immobilized methacrylate monolithic columns, i.e. poly(GMA-SMX-co-EDMA) and poly(GMA-SAA-co-EDMA) were prepared by one-pot in situ copolymerization of the drug ligand (sulfamethoxazole (SMX) or sulfanilamide (SAA)), the monomer (glycidyl methacrylate, GMA) and the cross-linker (ethylene dimethacrylate, EDMA) within 100 μm i.d. capillaries under optimized polymerization conditions. The physicochemical properties and column performance of the fabricated monolithic columns were characterized by elemental analysis, scanning electron microscopy and micro-HPLC. Satisfactory column permeability, efficiency and separation performance were obtained on the optimized poly(GMA-SMX-co-EDMA) monolithic column for small molecules, such as a standard test mixture and eight aromatic ketones. Notably, it was found that the poly(GMA-SMX-co-EDMA) monolith showed a selective affinity to trypsin, while the poly(GMA-SAA-co-EDMA) monolith containing sulfanilamide did not exhibit such affinity at all. This research not only provides a novel monolith for the selective isolation and purification of trypsin, but it also offers the possibility to easily prepare novel drug functionalized methacrylate monoliths through a one-pot copolymerization strategy. Copyright © 2015 Elsevier B.V. All rights reserved.

Functional properties and in vitro trypsin digestibility of red kidney bean (Phaseolus vulgaris L.) protein isolate: Effect of high-pressure treatment.

PubMed

Yin, Shou-Wei; Tang, Chuan-He; Wen, Qi-Biao; Yang, Xiao-Quan; Li, Lin

2008-10-15

The effects of high-pressure (HP) treatment at 200-600MPa, prior to freeze-drying, on some functional properties and in vitro trypsin digestibility of vicilin-rich red kidney bean (Phaseolus vulgaris L.) protein isolate (KPI) were investigated. Surface hydrophobicity and free sulfhydryl (SH) and disulfide bond (SS) contents were also evaluated. HP treatment resulted in gradual unfolding of protein structure, as evidenced by gradual increases in fluorescence strength and SS formation from SH groups, and decrease in denaturation enthalpy change. The protein solubility of KPI was significantly improved at pressures of 400MPa or higher, possibly due to formation of soluble aggregate from insoluble precipitate. HP treatment at 200 and 400MPa significantly increased emulsifying activity index (EAI) and emulsion stability index (ESI); however, EAI was significantly decreased at 600MPa (relative to untreated KPI). The thermal stability of the vicilin component was not affected by HP treatment. Additionally, in vitro trypsin digestibility of KPI was decreased only at a pressure above 200MPa and for long incubation time (e.g., 120min). The data suggest that some physiochemical and functional properties of vicilin-rich kidney proteins can be improved by means of high-pressure treatment. Copyright © 2008 Elsevier Ltd. All rights reserved.

Evidence for Ancient Origins of Bowman-Birk Inhibitors from Selaginella moellendorffii

PubMed Central

James, Amy M.; Jayasena, Achala S.; Zhang, Jingjing; Secco, David; Knott, Gavin J.; Whelan, James

2017-01-01

Bowman-Birk Inhibitors (BBIs) are a well-known family of plant protease inhibitors first described 70 years ago. BBIs are known only in the legume (Fabaceae) and cereal (Poaceae) families, but peptides that mimic their trypsin-inhibitory loops exist in sunflowers (Helianthus annuus) and frogs. The disparate biosynthetic origins and distant phylogenetic distribution implies these loops evolved independently, but their structural similarity suggests a common ancestor. Targeted bioinformatic searches for the BBI inhibitory loop discovered highly divergent BBI-like sequences in the seedless, vascular spikemoss Selaginella moellendorffii. Using de novo transcriptomics, we confirmed expression of five transcripts in S. moellendorffii whose encoded proteins share homology with BBI inhibitory loops. The most highly expressed, BBI3, encodes a protein that inhibits trypsin. We needed to mutate two lysine residues to abolish trypsin inhibition, suggesting BBI3’s mechanism of double-headed inhibition is shared with BBIs from angiosperms. As Selaginella belongs to the lycopod plant lineage, which diverged ∼200 to 230 million years before the common ancestor of angiosperms, its BBI-like proteins imply there was a common ancestor for legume and cereal BBIs. Indeed, we discovered BBI sequences in six angiosperm families outside the Fabaceae and Poaceae. These findings provide the evolutionary missing links between the well-known legume and cereal BBI gene families. PMID:28298518

The modifier effects of chymotrypsin and trypsin enzymes on fluorescence lifetime distribution of "N-(1-pyrenyl)maleimide-bovine serum albumin" complex.

PubMed

Özyiğit, İbrahim Ethem; Karakuş, Emine; Pekcan, Önder

2016-02-05

Chymotrypsin and trypsin are the well known proteolytic enzymes, both of which are synthesized in the pancreas as their precursors - the inactive forms; chymotrypsinogen and trypsinogen - and then are released into the duodenum to cut proteins into smaller peptides. In this paper, the effects of activities of chymotrypsin and trypsin enzymes on fluorescence lifetime distributions of the substrat bovine serum albumin (BSA) modified with N-(1-pyrenyl)maleimide (PM) were examined. In the labeling study of BSA with PM, it is aimed to attach PM to the single free thiol (Cys34) and to all the free amine groups in accessible positions in order to produce excimers of pyrene planes of the possible highest amount to form the lifetime distributions in the widest range, that may show specifically distinguishing changes resulting from the activities of the proteases. The time resolved spectrofluorometer was used to monitor fluorescence decays, which were analyzed by using the exponential series method (ESM) to obtain the changes of lifetime distributions. After the exposure of the synthesized substrat PM-BSA to the enzymes, the fluorescence lifetime distributions exhibited different structures which were attributed to the different activities of the proteases. Copyright © 2015 Elsevier B.V. All rights reserved.

Effects of Trypsinization and Mineralization on Intrasynovial Tendon Allograft Healing to Bone

PubMed Central

Qu, Jin; van Alphen, Nick A.; Thoreson, Andrew R.; Chen, Qingshan; An, Kai-Nan; Amadio, Peter C.; Schmid, Thomas M.; Zhao, Chunfeng

2014-01-01

The purpose of the current study was to develop a novel technology to enhance tendon-to-bone interface healing by trypsinizing and mineralizing (TM) an intrasynovial tendon allograft in a rabbit bone tunnel model. Eight rabbit flexor digitorum profundus (FDP) tendons were used to optimize the trypsinization process. An additional 24 FDP tendons were stratified into control and TM groups; in each group, 4 tendons were used for in vitro evaluation of TM and 8 were transplanted into proximal tibial bone tunnels in rabbits. The samples were evaluated histologically and with mechanical testing at postoperative week 8. Maximum failure strength and linear stiffness were not significantly different between the control and TM tendons. A thin fibrous band of scar tissue formed at the graft-to-bone interface in the control group. However, only the TM group showed obvious new bone formation inside the tendon graft and a visible fibrocartilage layer at the bone tunnel entrance. This study is the first to explore effects of TM on the intrasynovial allograft healing to a bone tunnel. TM showed beneficial effects on chondrogenesis, osteogenesis, and integration of the intrasynovial tendon graft, but mechanical strength was the same as the control tendons in this short-term in vivo study. PMID:25611186

Protease-activated receptor 2 agonist increases cell proliferation and invasion of human pancreatic cancer cells

PubMed Central

XIE, LIQUN; DUAN, ZEXING; LIU, CAIJU; ZHENG, YANMIN; ZHOU, JING

2015-01-01

The aim of this study was to determine the expression of protease-activated receptor 2 (PAR-2) in the human pancreatic cancer cell line SW1990, and to evaluate its effect on cell proliferation and invasion. The expression of PAR-2 protein and mRNA in SW1990 cells was determined by immunocytochemistry and reverse transcription polymerase chain reaction (PCR), respectively. MTT and cell invasion and migration assays, as well as semi-quantitative PCR and zymography analysis, were additionally performed. PAR-2 mRNA was significantly upregulated in the cells treated with trypsin or the PAR-2 activating peptide Ser-Leu-Ile-Gly-Lys-Val (SLIGKV) (P<0.01), but not in the Val-Lys-Gly-Ile-Leu-Ser group (P>0.05). Trypsin and SLIGKV significantly promoted SW1990 cell proliferation in a dose- and time-dependent manner (P<0.05). Compared with the control group, trypsin and SLIGKV significantly increased the mRNA expression (P<0.01) and gelatinolytic activity (P<0.01) of matrix metalloproteinase (MMP)-2. In conclusion, PAR-2 is expressed in SW1990 cells. PAR-2 activation may promote the invasion and migration of human pancreatic cancer cells by increasing MMP-2 expression. PMID:25452809

Entomocidal effects of beech apricot, Labramia bojeri, seed extract on a soybean pest, the velvetbean moth, Anticarsia gemmatalis, and its enzymatic activity.

PubMed

Macedo, Maria L R; Kubo, Carlos E G; Freire, Maria G M; Júnior, Roberto T A; Parra, José R P

2014-02-26

The effects of the beech apricot, Labramia bojeri A. de Candolle (Sapotales: Sapotaceae), seed aqueous extract on the larval development of the velvetbean moth, Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae), was evaluated. The extract inhibited larval development, pupal weight, and survival and emergence of adults. Digestive proteolytic activity in larval midgut and feces extracts was determined. Larvae fed 10 g/L of the aqueous extract showed a significant reduction in trypsin activity (~64%), when compared with control larvae. Trypsin and chymotrypsin activities were also detected in fecal material in aqueous-extract-fed larvae, with about ~4.5 times more trypsin activity than the controls. The results from dietary utilization experiments with A. gemmatalis larvae showed a reduction in the efficiency of conversion of ingested food and digested food and an increase in approximate digestibility and metabolic cost. The effect of the extract suggests the potential use of L. bojeri seeds to inhibit the development of A. gemmatalis via oral exposure. The L. bojeri extract can be an alternative to other methods of control. This is an open access paper. We use the Creative Commons Attribution 3.0 license that permits unrestricted use, provided that the paper is properly attributed.

Inhibition of proteases activity in intestine needs a sustainable acidic environment rather than a transient.

PubMed

Xing, Chang; Xing, Jin-Feng; Ge, Zhi-Qiang

2017-10-01

α-Chymotrypsin (α-CT) and trypsin are important components of the enzymatic barrier. They could degrade the therapeutic proteins and peptides, inhibit their activity consequently, and thereby reduce their oral bioavailability. Acidic agents, as one type of indirect protease inhibitors, have shown proof of concept in clinical trials. We report here the inactivated proteases due to acid influence can be reactivated immediately by environmental pH recovery regardless of how long the inactivation last. To keep the inactivation time of proteases for 4-5 h, we designed and prepared a sustained-release tablet containing citric acid (CA) which can effectively reduce the pH below 5.0 and maintain it for 5 h in the dissolution-reaction medium. The activity of α-CT and trypsin was quantified by analyzing the residual amount of their respective substrates BTEE and TAME. More than 80% of the substrates were survived in 5.0 h of incubation, whereas the common tablet inhibited the proteases activity for only two hours in the same experimental medium. It indicates that the sustained-release tablet loaded with CA can efficiently inhibit the α-CT and trypsin activity longer than the common tablet. The results will be beneficial for designing and formulating the peroral administration of peptide and protein drugs.

Optimization of the Enzymatic Hydrolysis of Lupin (Lupinus) Proteins for Producing ACE-Inhibitory Peptides.

PubMed

Boschin, Giovanna; Scigliuolo, Graziana Maria; Resta, Donatella; Arnoldi, Anna

2014-02-26

Recently, the enzymatic hydrolysis of Lupinus albus and Lupinus angustifolius proteins with pepsin was showed to produce peptides able to inhibit the angiotensin-converting enzyme (ACE). The objective of the present work was to test different hydrolytic enzymes and to investigate three lupin species (L. albus, L. angustifolius, Lupinus luteus) with the final goal of selecting the best enzyme/species combination for an efficient production of ACE-inhibitory peptide mixtures. Pepsin gave peptides with the best IC50 values (mean value on three species 186 ± 10 μg/mL), followed by pepsin + trypsin (198 ± 16 μg/mL), chymotrypsin (213 ± 83 μg/mL), trypsin (405 ± 54 μg/mL), corolase PP (497 ± 32 μg/mL), umamizyme (865 ± 230 μg/mL), and flavourzyme (922 ± 91 μg/mL). The three species showed similar activity scales, but after pepsin + trypsin and chymotrypsin treatments, L. luteus peptide mixtures resulted to be significantly the most active. This investigation indicates that lupin proteins may be a valuable source of ACE-inhibitory peptides, which may explain the activity observed in experimental and clinical studies and foresee the application of lupin proteins into functional foods or dietary supplements.

New Insights into the Pathogenesis of Pancreatitis

PubMed Central

Sah, Raghuwansh P.; Dawra, Rajinder K.; Saluja, Ashok K.

2014-01-01

Purpose of review In this article, we review important advances in our understanding of the mechanisms of pancreatitis. Recent Findings The relative contribution of intra-pancreatic trypsinogen activation and NFκB activation, the two major early independent cellular events in the etiology of pancreatitis, have been investigated using novel genetic models. Trypsinogen activation has traditionally held the spotlight for many decades as it is believed to be the central pathogenic event of pancreatitis However, recent experimental evidence points to the role of trypsin activation in early acinar cell damage but not in the inflammatory response of acute pancreatitis through NFκB activation. Further, chronic pancreatitis in the caerulein model develops independently of typsinogen activation. Sustained activation of the NFκB pathway, but not persistent intra-acinar expression of active trypsin, was shown to result in chronic pancreatitis. Calcineurin-NFAT signaling was shown to mediate downstream effects of pathologic rise in intracellular calcium. IL-6 was identified as a key cytokine mediating pancreatitis-associated lung injury. Summary Recent advances challenge the long-believed trypsin-centered understanding of pancreatitis. It is becoming increasingly clear that activation of intense inflammatory signaling mechanisms in acinar cells is crucial to the pathogenesis of pancreatitis, which may explain the strong systemic inflammatory response in pancreatitis. PMID:23892538

Wolbachia infection in Aedes aegypti mosquitoes alters blood meal excretion and delays oviposition without affecting trypsin activity.

PubMed

Pimenta de Oliveira, Sofia; Dantas de Oliveira, Caroline; Viana Sant'Anna, Mauricio Roberto; Carneiro Dutra, Heverton Leandro; Caragata, Eric Pearce; Moreira, Luciano Andrade

2017-08-01

Blood feeding in Aedes aegypti is essential for reproduction, but also permits the mosquito to act as a vector for key human pathogens such as the Zika and dengue viruses. Wolbachia pipientis is an endosymbiotic bacterium that can manipulate the biology of Aedes aegypti mosquitoes, making them less competent hosts for many pathogens. Yet while Wolbachia affects other aspects of host physiology, it is unclear whether it influences physiological processes associated with blood meal digestion. To that end, we examined the effects of wMel Wolbachia infection in Ae. aegypti, on survival post-blood feeding, blood meal excretion, rate of oviposition, expression levels of key genes involved in oogenesis, and activity levels of trypsin blood digestion enzymes. We observed that wMel infection altered the rate and duration of blood meal excretion, delayed the onset of oviposition and was associated with a greater number of eggs being laid later. wMel-infected Ae. aegypti also had lower levels of key yolk protein precursor genes necessary for oogenesis. However, all of these effects occurred without a change in trypsin activity. These results suggest that Wolbachia infection may disrupt normal metabolic processes associated with blood feeding and reproduction in Ae. aegypti. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effects of pristine polyvinyl chloride fragments on whole body histology and protease activity in silver barb Barbodes gonionotus fry.

PubMed

Romano, Nicholas; Ashikin, Munirah; Teh, Jun Chin; Syukri, Fadhil; Karami, Ali

2018-06-01

Silver barb Barbodes gonionotus fry were exposed to polyvinyl chloride (PVC) fragments at increasing concentrations of 0.2, 0.5 and 1.0 mg/L for 96 h, following which whole body histological evaluation and analysis of the digestive enzymes trypsin and chymotrypsin were performed. Whole body trypsin and chymotrypsin activities increased significantly in fish exposed to 0.5 and 1.0 mg/L PVC as compared those exposed to zero or 0.2 mg/L PVC. In fish exposed to all tested concentrations, PVCs were observed in both the proximal and distal intestine, and fish exposed to 0.5-1.0 and 1.0 mg/L PVC, respectively, and these particles were associated with localized thickening of the mucosal epithelium. No tissue damage was evident in any other internal organs or gills. This lack of damage may be attributed to the absence of contaminants associated with the PVC fragments and their relatively smooth surface. The increased whole body trypsin and chymotrypsin activities may indicate an attempt to enhance digestion to compensate for epithelial thickening of the intestine and/or to digest the plastics. Copyright © 2017 Elsevier Ltd. All rights reserved.

Extraction of collagenase from the involuting rat uterus.

PubMed

Weeks, J G; Halme, J; Woessner, J F

1976-08-12

Collagenase (EC 3.4.24.3) activity can be measured directly in homogenates of the involuting rat uterus. Latent forms of collagenase are activated by a brief exposure to trypsin; trypsin activity is then blocked with soybean trypsin inhibitor. Homogenizing conditions have been developed that permit 90-95% recovery of the total active and latent collagenase activity in a 6000 X g pellet, where it is presumably bound to its collagen substrate. This insoluble activity can then be extracted by heating to 60 degrees C for 4 min in 0.04 M Tris - HCl buffer, pH 7.5, containing 0.1 M CaCl2. Methods are presented for the estimation of the recovery of collagenase in the extracts; this approximates 65-70% of the total. Small amounts of activity can also be extracted from rat liver and kidney. This extraction procedure should be of use in purifying collagenase without culturing the enzyme-producing tissue and in the direct assay of tissue collagenase activity. The activity extracted from rat uterus has been proven to be collagenase by its characteristic pattern of collagen breakdown products on disc electrophoresis and by the split of tropocollagen at interband 41 as shown by electron microscopy of reconstituted fragments. The activity is inhibited by EDTA, and this inhibition is not reversed by calcium or zinc ions.

Crystallization and preliminary X-ray diffraction studies of trypsin-like proteases from the gastric fluid of the marine crab Cancer pagurus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hehemann, Jan-Hendrik; Redecke, Lars; Perbandt, Markus

2007-03-01

Two trypsins from the gastric fluid of the marine crab C. pagurus were purified and crystallized and X-ray data were collected to 0.97 and 3.2 Å resolution. The digestive fluid of the marine crab Cancer pagurus (Decapoda, Brachyura) contains highly stable proteases which display enhanced activity in aqueous mixtures of organic solvents. Three trypsins were isolated from the gastric fluid and two of them, C.p.TryII and C.p.TryIII, were purified to homogeneity by anion-exchange chromatography and crystallized by hanging-drop vapour diffusion. Diffraction data were collected at a synchrotron to 0.97 and 3.2 Å resolution, respectively. The crystal of C.p.TryII belongs tomore » the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 52.06, b = 62.00, c = 71.66 Å. Based on the Matthews coefficient, one protein molecule per asymmetric unit is suggested. In contrast, crystals of C.p.TryIII, which belong to the cubic space group P2{sub 1}3 with unit-cell parameters a = b = c = 215.4 Å, are assumed to contain 12 molecules per asymmetric unit.« less

Physiology of digestion and the molecular characterization of the major digestive enzymes from Periplaneta americana.

PubMed

Tamaki, Fábio K; Pimentel, André C; Dias, Alcides B; Cardoso, Christiane; Ribeiro, Alberto F; Ferreira, Clélia; Terra, Walter R

2014-11-01

Cockroaches are among the first insects to appear in the fossil record. This work is part of ongoing research on insects at critical points in the evolutionary tree to disclose evolutionary trends in the digestive characteristics of insects. A transcriptome (454 Roche platform) of the midgut of Periplanetaamericana was searched for sequences of digestive enzymes. The selected sequences were manually curated. The complete or nearly complete sequences showing all characteristic motifs and highly expressed (reads counting) had their predicted sequences checked by cloning and Sanger sequencing. There are two chitinases (lacking mucin and chitin-binding domains), one amylase, two α- and three β-glucosidases, one β-galactosidase, two aminopeptidases (none of the N-group), one chymotrypsin, 5 trypsins, and none β-glucanase. Electrophoretic and enzymological data agreed with transcriptome data in showing that there is a single β-galactosidase, two α-glucosidases, one preferring as substrate maltase and the other aryl α-glucoside, and two β-glucosidases. Chromatographic and enzymological data identified 4 trypsins, one chymotrypsin (also found in the transcriptome), and one non-identified proteinase. The major digestive trypsin is identifiable to a major P. americana allergen (Per a 10). The lack of β-glucanase expression in midguts was confirmed, thus lending support to claims that those enzymes are salivary. A salivary amylase was molecularly cloned and shown to be different from the one from the midgut. Enzyme distribution showed that most digestion occurs under the action of salivary and midgut enzymes in the foregut and anterior midgut, except the posterior terminal digestion of proteins. A counter-flux of fluid may be functional in the midgut of the cockroach to explain the low excretory rate of digestive enzymes. Ultrastructural and immunocytochemical localization data showed that amylase and trypsin are released by both merocrine and apocrine secretion mainly from gastric caeca. Finally, a discussion on Polyneoptera digestive physiology is provided. Copyright © 2014 Elsevier Ltd. All rights reserved.

Structural and functional characterization of proteinase inhibitors from seeds of Cajanus cajan (cv. ICP 7118).

PubMed

Swathi, Marri; Lokya, Vadthya; Swaroop, Vanka; Mallikarjuna, Nalini; Kannan, Monica; Dutta-Gupta, Aparna; Padmasree, Kollipara

2014-10-01

Proteinase inhibitors (C11PI) from mature dry seeds of Cajanus cajan (cv. ICP 7118) were purified by chromatography which resulted in 87-fold purification and 7.9% yield. SDS-PAGE, matrix assisted laser desorption ionization time-of-flight (MALDI-TOF/TOF) mass spectrum and two-dimensional (2-D) gel electrophoresis together resolved that C11PI possessed molecular mass of 8385.682 Da and existed as isoinhibitors. However, several of these isoinhibitors exhibited self association tendency to form small oligomers. All the isoinhibitors resolved in Native-PAGE and 2-D gel electrophoresis showed inhibitory activity against bovine pancreatic trypsin and chymotrypsin as well as Achaea janata midgut trypsin-like proteases (AjPs), a devastating pest of castor plant. Partial sequences of isoinhibitor (pI 6.0) obtained from MALDI-TOF/TOF analysis and N-terminal sequencing showed 100% homology to Bowman-Birk Inhibitors (BBIs) of leguminous plants. C11PI showed non-competitive inhibition against trypsin and chymotrypsin. A marginal loss (<15%) in C11PI activity against trypsin at 80 (°)C and basic pH (12.0) was associated with concurrent changes in its far-UV CD spectra. Further, in vitro assays demonstrated that C11PI possessed significant inhibitory potential (IC50 of 78 ng) against AjPs. On the other hand, in vivo leaf coating assays demonstrated that C11PI caused significant mortality rate with concomitant reduction in body weight of both larvae and pupae, prolonged the duration of transition from larva to pupa along with formation of abnormal larval-pupal and pupal-adult intermediates. Being smaller peptides, it is possible to express C11PI in castor to protect them against its devastating pest A. janata. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Protein-directed synthesis of Mn-doped ZnS quantum dots: a dual-channel biosensor for two proteins.

PubMed

Wu, Peng; Zhao, Ting; Tian, Yunfei; Wu, Lan; Hou, Xiandeng

2013-06-03

Proteins typically have nanoscale dimensions and multiple binding sites with inorganic ions, which facilitates the templated synthesis of nanoparticles to yield nanoparticle-protein hybrids with tailored functionality, water solubility, and tunable frameworks with well-defined structure. In this work, we report a protein-templated synthesis of Mn-doped ZnS quantum dots (QDs) by exploring bovine serum albumin (BSA) as the template. The obtained Mn-doped ZnS QDs give phosphorescence emission centered at 590 nm, with a decay time of about 1.9 ms. A dual-channel sensing system for two different proteins was developed through integration of the optical responses (phosphorescence emission and resonant light scattering (RLS)) of Mn-doped ZnS QDs and recognition of them by surface BSA phosphorescent sensing of trypsin and RLS sensing of lysozyme. Trypsin can digest BSA and remove BSA from the surface of Mn-doped ZnS QDs, thus quenching the phosphorescence of QDs, whereas lysozyme can assemble with BSA to lead to aggregation of QDs and enhanced RLS intensity. The detection limits for trypsin and lysozyme were 40 and 3 nM, respectively. The selectivity of the respective channel for trypsin and lysozyme was evaluated with a series of other proteins. Unlike other protein sensors based on nanobioconjugates, the proposed dual-channel sensor employs only one type of QDs but can detect two different proteins. Further, we found the RLS of QDs can also be useful for studying the BSA-lysozyme binding stoichiometry, which has not been reported in the literature. These successful biosensor applications clearly demonstrate that BSA not only serves as a template for growth of Mn-doped ZnS QDs, but also impacts the QDs for selective recognition of analyte proteins. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Matrix metalloproteinase-2: A key regulator in coagulation proteases mediated human breast cancer progression through autocrine signaling.

PubMed

Das, Kaushik; Prasad, Ramesh; Ansari, Shabbir Ahmed; Roy, Abhishek; Mukherjee, Ashis; Sen, Prosenjit

2018-06-02

Cell invasion is attributed to the synthesis and secretion of proteolytically active matrix-metalloproteinases (MMPs) by tumor cells to degrade extracellular matrix (ECM) and promote metastasis. The role of protease-activated receptor 2 (PAR2) in human breast cancer migration/invasion via MMP-2 up-regulation remains ill-defined; hence we investigated whether TF-FVIIa/trypsin-mediated PAR2 activation induces MMP-2 expression in human breast cancer. MMP-2 expression and the signaling mechanisms were analyzed by western blotting and RT-PCR. MMP-2 activity was measured by gelatin zymography. Cell invasion was analyzed by transwell invasion assay whereas; wound healing assay was performed to understand the cell migratory potential. Here, we highlight that TF-FVIIa/trypsin-mediated PAR2 activation leads to enhanced MMP-2 expression in human breast cancer cells contributing to tumor progression. Knock-down of PAR2 abrogated TF-FVIIa/trypsin-induced up-regulation of MMP-2. Again, genetic manipulation of AKT or inhibition of NF-ĸB suggested that PAR2-mediated enhanced MMP-2 expression is dependent on the PI3K-AKT-NF-ĸB pathway. We also reveal that TF, PAR2, and MMP-2 are over-expressed in invasive breast carcinoma tissues as compared to normal. Knock-down of MMP-2 significantly impeded TF-FVIIa/trypsin-induced cell invasion. Further, we report that MMP-2 activates p38 MAPK-MK2-HSP27 signaling axis that leads to actin polymerization and induces cell migration. Pharmacological inhibition of p38 MAPK or MK2 attenuates MMP-2-induced cell migration. The study delineates a novel signaling pathway by which PAR2-induced MMP-2 expression regulates human breast cancer cell migration/invasion. Understanding these mechanistic details will certainly help to identify crucial targets for therapeutic interventions in breast cancer metastasis. Copyright © 2018. Published by Elsevier Masson SAS.

Determination of molecular weight of a purified fraction of colloidal calcium phosphate derived from the casein micelles of bovine milk.

PubMed

Choi, J; Horne, D S; Lucey, J A

2011-07-01

Colloidal calcium phosphate (CCP) plays a key role in the formation and integrity of casein (CN) micelles. However, limited information is available on the molecular weight (M(w)) of CCP. Recently, we theoretically derived the M(w) of CCP and the objectives of this study were to experimentally determine the M(w) of CCP. We used 2 methods to prepare CCP fractions: skim milk was enzymatically digested with either trypsin or a combination of papain and proteinase enzymes to remove most CN. The CN phosphopeptides are resistant to trypsin hydrolysis. Digestion was carried out in a membrane tube that was dialyzed against the same bulk milk used in sample preparation to remove small peptides and to minimize perturbation of CCP. After digestion, the protein contents of the enzyme-treated milks were 0.92 and 0.36% for the trypsin and papain-proteinase treatments, respectively. Size-exclusion chromatography, coupled with multi-angle laser light scattering, was used to separate the CCP-phosphopeptide fraction from the digested mixture. Simulated milk ultrafiltrate was used as a mobile phase during size-exclusion chromatography separation to try to preserve the integrity of CCP. Size-exclusion chromatography peaks, which had higher Ca and P contents than the baseline, were identified as the likely fractions containing the phosphopeptide-stabilized CCP; this peak eluted with retention times of 100 to approximately 110 min for trypsinated samples. The papain-proteinase treatment caused excessive loss of CN that were needed to stabilize CCP, which resulted in no obvious peak that had elevated Ca and P contents. Debye plots at these retention times indicated that the weight-average M(w) for the fraction prepared by trypsin was 17,450 g/mol. Attempts to estimate the M(w) of the phosphopeptides associated with CCP using sodium dodecyl sulfate-PAGE were not successful, as we did not observe any peptide bands in these gels, presumably because of their low concentration in the isolated, unconcentrated fraction. Assuming that 4 CN phosphopeptides stabilized each CCP and if the M(w) of each of these phosphopeptides was about 2,500 g/mol, then the M(w) of CCP would be around 7,450 g/mol. This experimental value was close to the theoretically-derived M(w) of 4,897 and 9,757 g/mol for tetrahedron and bi-pyramid shaped objects, respectively, when using the brushite form of calcium phosphate. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Effects of protease and non-starch polysaccharide enzyme on performance, digestive function, activity and gene expression of endogenous enzyme of broilers.

PubMed

Yuan, Lin; Wang, Mingfa; Zhang, Xiaotu; Wang, Zhixiang

2017-01-01

Three hundred one-day-old male broiler chickens (Ross-308) were fed corn-soybean basal diets containing non-starch polysaccharide (NSP) enzyme and different levels of acid protease from 1 to 42 days of age to investigate the effects of exogenous enzymes on growth performance, digestive function, activity of endogenous digestive enzymes in the pancreas and mRNA expression of pancreatic digestive enzymes. For days 1-42, compared to the control chickens, average daily feed intake (ADFI) and average daily gain (ADG) were significantly enhanced by the addition of NSP enzyme in combination with protease supplementation at 40 or 80 mg/kg (p<0.05). Feed-to-gain ratio (FGR) was significantly improved by supplementation with NSP enzymes or NSP enzyme combined with 40 or 80 mg/kg protease compared to the control diet (p<0.05). Apparent digestibility of crude protein (ADCP) was significantly enhanced by the addition of NSP enzyme or NSP enzyme combined with 40 or 80 mg/kg protease (p<0.05). Cholecystokinin (CCK) level in serum was reduced by 31.39% with NSP enzyme combined with protease supplementation at 160 mg/kg (p<0.05), but the CCK level in serum was increased by 26.51% with NSP enzyme supplementation alone. After 21 days, supplementation with NSP enzyme and NSP enzyme combined with 40 or 80 mg/kg protease increased the activity of pancreatic trypsin by 74.13%, 70.66% and 42.59% (p<0.05), respectively. After 42 days, supplementation with NSP enzyme and NSP enzyme combined with 40 mg/kg protease increased the activity of pancreatic trypsin by 32.45% and 27.41%, respectively (p<0.05). However, supplementation with NSP enzyme and 80 or 160 mg/kg protease decreased the activity of pancreatic trypsin by 10.75% and 25.88%, respectively (p<0.05). The activities of pancreatic lipase and amylase were significantly higher in treated animals than they were in the control group (p<0.05). Supplementation with NSP enzyme, NSP enzyme combined with 40 or 80 mg/kg protease increased pancreatic trypsin mRNA levels by 40%, 44% and 28%, respectively. Supplementation with NSP enzyme and 160 mg/kg protease decreased pancreatic trypsin mRNA levels by 13%. Pancreatic lipase and amylase mRNA expression were significantly elevated in treated animals compared to the control group (p<0.05). These results suggest that the amount of NSP enzyme and acid protease in the diet significantly affects digestive function, endogenous digestive-enzyme activity and mRNA expression in broilers.

Cytometric analysis of retinopathies in retinal trypsin digests

NASA Astrophysics Data System (ADS)

Ghanian, Zahra; Staniszewski, Kevin; Sorenson, Christine M.; Sheibani, Nader; Ranji, Mahsa

2014-03-01

The objective of this work was to design an automated image cytometry tool for determination of various retinal vascular parameters including extraction of features that are relevant to postnatal retinal vascular development, and the progression of diabetic retinopathy. To confirm the utility and accuracy of the software, retinal trypsin digest from TSP1-/- and diabetic Akita/+; TSP1-/- mice were analyzed. TSP1 is a critical inhibitor of development of retinopathies and lack of TSP1 exacerbates progression of early diabetic retinopathies. Loss of vascular cells of and gain more acellular capillaries as two major signs of diabetic retinopathies were used to classify a retina as normal or injured. This software allows quantification and high throughput assessment of retinopathy changes associated with diabetes.

[Comparative analysis of sensitivity of proteases (chymotrypsin and trypsin) and cholinesterases of different origin to some organophosphorus inhibitors].

PubMed

Rozengart, E V

2009-01-01

The antichymotrypsin, antitrypsin, and anticholinesterase efficiencies of four homologous series of organophosphorus inhibitors are compared: O-ethyl-S-(n-alkyl)methylthiophosphonates, O-(n-alkyl)-S-(n-butyl)methylthiophosphonates, O-(n-alkyl)-S-beta-(ethylmercaptoethylene)methylthiophosphonates, and their methylsulfomethylates. As sources of a-chymotrypsin and trypsin, commercial compounds of Worthington Biochemical Corporation and Leningrad Myasokombinat were tested. Bimolecular constant of the reaction rate was used as the measure of antienzyme efficiency. In all cases, the antichymotrypsin efficiency was lower, while the antitrypsin--essentially higher than the anticholinesterase activity of the studied inhibitors. These differences were found to much depend both on the inhibitor structure and on nature of the cholinesterase compounds.

Evaluation of a High Intensity Focused Ultrasound-Immobilized Trypsin Digestion and 18O-Labeling Method for Quantitative Proteomics

PubMed Central

López-Ferrer, Daniel; Hixson, Kim K.; Smallwood, Heather; Squier, Thomas C.; Petritis, Konstantinos; Smith, Richard D.

2009-01-01

A new method that uses immobilized trypsin concomitant with ultrasonic irradiation results in ultra-rapid digestion and thorough 18O labeling for quantitative protein comparisons. The reproducible and highly efficient method provided effective digestions in <1 min with a minimized amount of enzyme required compared to traditional methods. This method was demonstrated for digestion of both simple and complex protein mixtures, including bovine serum albumin, a global proteome extract from the bacteria Shewanella oneidensis, and mouse plasma, as well as 18O labeling of such complex protein mixtures, which validated the application of this method for differential proteomic measurements. This approach is simple, reproducible, cost effective, rapid, and thus well-suited for automation. PMID:19555078

Total Synthesis and Stereochemical Assignment of Nostosin B

PubMed Central

Wang, Xiaoji; Feng, Junmin; Xu, Zhengshuang; Ye, Tao; Meng, Yi; Zhang, Zhiyu

2017-01-01

Nostosins A and B were isolated from a hydrophilic extract of Nostoc sp. strain from Iran, which exhibits excellent trypsin inhibitory activity. Nostosin A was the most potent natural tripeptide aldehyde as trypsin inhibitor up to now. Both r- and s-2-hydroxy-4-(4-hydroxy-phenyl)butanoic acid (Hhpba) were prepared and incorporated into the total synthesis of nostosin B, respectively. Careful comparison of the NMR spectra and optical rotation data of synthetic nostosin B (1a and 1b) with the natural product led to the unambiguous identification of the r-configuration of the Hhpba fragment, which was further confirmed by co-injection with the authentic sample on HPLC using both reversed phase column and the chiral AD-RH column. PMID:28264450

RAMA casein zymography: Time-saving and highly sensitive casein zymography for MMP7 and trypsin.

PubMed

Yasumitsu, Hidetaro; Ozeki, Yasuhiro; Kanaly, Robert A

2016-11-01

To detect metalloproteinase-7 (MMP7), zymography is conducted using a casein substrate and conventional CBB stain. It has disadvantages because it is time consuming and has low sensitivity. Previously, a sensitive method to detect MMP7 up to 30 pg was reported, however it required special substrates and complicated handlings. RAMA casein zymography described herein is rapid, sensitive, and reproducible. By applying high-sensitivity staining with low substrate conditions, the staining process is completed within 1 h and sensitivity was increased 100-fold. The method can detect 10 pg MMP7 by using commercially available casein without complicated handlings. Moreover, it increases detection sensitivity for trypsin. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A ratiometric fluorescent quantum dots based biosensor for organophosphorus pesticides detection by inner-filter effect.

PubMed

Yan, Xu; Li, Hongxia; Han, Xiaosong; Su, Xingguang

2015-12-15

In this work, we develop a novel and sensitive sensor for the detection of organophosphorus pesticides based on the inner-filter effect (IFE) between gold nanoparticles (AuNPs) and ratiometric fluorescent quantum dots (RF-QDs). The RF-QDs has been designed by hybridizing two differently colored CdTe QDs, in which the red emissive QDs entrapped in the silica sphere acting as the reference signal, and the green emissive QDs covalently attached on the silica surface serving as the response signal.The fluorescence of RF-QDs could be quenched by AuNPs based on IFE. Protamine could effectively turn on the fluorescence due to the electrostatic attraction between protamine and AuNPs. Trypsin can easily hydrolyze protamine, leading to the quench of the fluorescence. Then, the fluorescence could be recovered again by the addition of parathion-methyl (PM) which could inhibit the activity of trypsin. By measuring the fluorescence of RF-QDs, the inhibition efficiency of PM to trypsin activity was evaluated. Under the optimized conditions, the inhibition efficiency was proportional to the logarithm of PM concentration in the range of 0.04-400 ng mL(-1), with a detection limit of 0.018 ng mL(-1). Furthermore, the simple and convenient method had been used for PM detection in environmental and agricultural samples with satisfactory results. Copyright © 2015 Elsevier B.V. All rights reserved.

Size-exclusion chromatography of tea tannins and intercepting potentials of peptides for the inhibition of trypsin-caseinolytic activity by tea tannins.

PubMed

Kasai, Naoya; Nakatsubo, Genki

2006-07-12

Molecular-weight distribution and characterization of tea tannin were investigated by high-performance liquid chromatography and the equivalent preparative exclusion gel chromatography using Sephadex G-25. The characteristics of the fractions were studied regarding the amounts of terminal catechin, sugar, and gallic acid, the color reaction of the Folin-Chiocalteu reagent, the UV absorbance, and the inhibition activity for the trypsin-caseinolytic activity per weight. Furthermore, we investigated the intercepting activities of the inhibition by the amino acids, peptides, their analogues, poly(ethylene glycol)s (PEGs), and histatin 5 using the inhibition of trypsin-caseinolytic activity by tea. Arg, Lys, and their peptides had strong intercepting activities for the inhibition, but only a weak activity was detected in the Pro peptides or gelatin-like peptides of (Pro-Pro-Gly)(n) (n = 5 or 10). The guanidyl group of Arg and the amino methylene group of Lys were important for the intercepting activity, but the activity was weakly dependent upon the peptide bond formation. The intercepting activity of the peptides or PEG exponentially increased with the number of polymerizations. Histatin 5 did not have a remarkably strong intercepting activity considering the peptide length. The activity of the synthetic histatin 5 in which all of the Lys and Arg were substituted by Ala was at the same level as histatin 5.

Potential Activities of Freshwater Exo- and Endo-Acting Extracellular Peptidases in East Tennessee and the Pocono Mountains.

PubMed

Mullen, Lauren; Boerrigter, Kim; Ferriero, Nicholas; Rosalsky, Jeff; Barrett, Abigail van Buren; Murray, Patrick J; Steen, Andrew D

2018-01-01

Proteins constitute a particularly bioavailable subset of organic carbon and nitrogen in aquatic environments but must be hydrolyzed by extracellular enzymes prior to being metabolized by microorganisms. Activities of extracellular peptidases (protein-degrading enzymes) have frequently been assayed in freshwater systems, but such studies have been limited to substrates for a single enzyme [leucyl aminopeptidase (Leu-AP)] out of more than 300 biochemically recognized peptidases. Here, we report kinetic measurements of extracellular hydrolysis of five substrates in 28 freshwater bodies in the Delaware Water Gap National Recreation Area in the Pocono Mountains (PA, United States) and near Knoxville (TN, United States), between 2013 and 2016. The assays putatively test for four aminopeptidases (arginyl aminopeptidase, glyclyl aminopeptidase, Leu-AP, and pyroglutamyl aminopeptidase), which cleave N -terminal amino acids from proteins, and trypsin, an endopeptidase, which cleaves proteins mid-chain. Aminopeptidase and the trypsin-like activity were observed in all water bodies, indicating that a diverse set of peptidases is typical in freshwater. However, ratios of peptidase activities were variable among sites: aminopeptidases dominated at some sites and trypsin-like activity at others. At a given site, the ratios remained fairly consistent over time, indicating that they are driven by ecological factors. Studies in which only Leu-AP activity is measured may underestimate the total peptidolytic capacity of an environment, due to the variable contribution of endopeptidases.

Preparation and evaluation of dual-enzyme microreactor with co-immobilized trypsin and chymotrypsin.

PubMed

Meller, Kinga; Pomastowski, Paweł; Grzywiński, Damian; Szumski, Michał; Buszewski, Bogusław

2016-04-01

The preparation of capillary microfluidic reactor with co-immobilized trypsin and chymotrypsin with the use of a low-cost commercially available enzymatic reagent (containing these proteases) as well as the evaluation of its usefulness in proteomic research were presented. The monolithic copolymer synthesized from glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) was used as a support. Firstly, the polymerization conditions were optimized and the monolithic bed was synthesized in the fused silica capillary modified with 3-(trimethoxysilyl)propyl methacrylate (γ-MAPS). The polymer containing epoxy groups was then modified with 1,6-diaminohexane, followed by the attachment of glutaraldehyde and immobilization of enzymes. The efficiency of the prepared monolithic Immobilized Enzyme Microreactor (μ-IMER) with regard to trypsin activity was evaluated using the low-molecular mass compound (Nα-benzoyl-l-arginine ethyl ester, BAEE). The activities of both enzymes were investigated using a macromolecular protein (human transferrin, Tf) as a substrate. In the case of BAEE, the reaction product was separated from the substrate using the capillary liquid chromatography and the efficiency of the reaction was determined by the peak area of the substrate. The hydrolysis products of transferrin were analyzed with MALDI-TOF which allows for the verification of the prepared enzymatic system applicability in the field of proteomic research. Copyright © 2016 Elsevier B.V. All rights reserved.

Sarcocystis neurona infection in gamma interferon gene knockout (KO) mice: comparative infectivity of sporocysts in two strains of KO mice, effect of trypsin digestion on merozoite viability, and infectivity of bradyzoites to KO mice and cell culture.

PubMed

Dubey, J P; Sundar, N; Kwok, O C H; Saville, W J A

2013-09-01

The protozoan Sarcocystis neurona is the primary cause of Equine Protozoal Myeloencephalitis (EPM). EPM or EPM-like illness has been reported in horses, sea otters, and several other mammals. The gamma interferon gene knockout (KO) mouse is often used as a model to study biology and discovery of new therapies against S. neurona because it is difficult to induce clinical EPM in other hosts, including horses. In the present study, infectivity of three life cycle stages (merozoites, bradyzoites, sporozoites) to KO mice and cell culture was studied. Two strains of KO mice (C57-black, and BALB/c-derived, referred here as black or white) were inoculated orally graded doses of S. neurona sporocysts; 12 sporocysts were infective to both strains of mice and all infected mice died or became ill within 70 days post-inoculation. Although there was no difference in infectivity of sporocysts to the two strains of KO mice, the disease was more severe in black mice. S. neurona bradyzoites were not infectious to KO mice and cell culture. S. neurona merozoites survived 120 min incubation in 0.25% trypsin, indicating that trypsin digestion can be used to recover S. neurona from tissues of acutely infected animals. Published by Elsevier B.V.

Conformationally selective biophysical assay for influenza vaccine potency determination.

PubMed

Wen, Yingxia; Han, Liqun; Palladino, Giuseppe; Ferrari, Annette; Xie, Yuhong; Carfi, Andrea; Dormitzer, Philip R; Settembre, Ethan C

2015-10-05

Influenza vaccines are the primary intervention for reducing the substantial health burden from pandemic and seasonal influenza. Hemagglutinin (HA) is the most important influenza vaccine antigen. Subunit and split influenza vaccines are formulated, released for clinical use, and tested for stability based on an in vitro potency assay, single-radial immunodiffusion (SRID), which selectively detects HA that is immunologically active (capable of eliciting neutralizing or hemagglutination inhibiting antibodies in an immunized subject). The time consuming generation of strain-specific sheep antisera and calibrated antigen standards for SRID can delay vaccine release. The limitation in generating SRID reagents was evident during the early days of the 2009 pandemic, prompting efforts to develop more practical, alternative, quantitative assays for immunologically active HA. Here we demonstrate that, under native conditions, trypsin selectively digests HA produced from egg or mammalian cell in monovalent vaccines that is altered by stress conditions such as reduced pH, elevated temperature, or deamidation, leaving native, pre-fusion HA, intact. Subsequent reverse-phase high pressure liquid chromatography (RP-HPLC) can separate trypsin-resistant HA from the digested HA. Integration of the resulting RP-HPLC peak yields HA quantities that match well the values obtained by SRID. Therefore, trypsin digestion, to pre-select immunologically active HA, followed by quantification by RP-HPLC is a promising alternative in vitro potency assay for influenza vaccines. Copyright © 2015 Elsevier Ltd. All rights reserved.

Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2

PubMed Central

Reinke, Lennart Michel; Hartleib, Anika; Nehlmeier, Inga; Gierer, Stefanie; Hoffmann, Markus; Hofmann-Winkler, Heike; Winkler, Michael

2017-01-01

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) mediates viral entry into target cells. Cleavage and activation of SARS S by a host cell protease is essential for infectious viral entry and the responsible enzymes are potential targets for antiviral intervention. The type II transmembrane serine protease TMPRSS2 cleaves and activates SARS S in cell culture and potentially also in the infected host. Here, we investigated which determinants in SARS S control cleavage and activation by TMPRSS2. We found that SARS S residue R667, a previously identified trypsin cleavage site, is also required for S protein cleavage by TMPRSS2. The cleavage fragments produced by trypsin and TMPRSS2 differed in their decoration with N-glycans, suggesting that these proteases cleave different SARS S glycoforms. Although R667 was required for SARS S cleavage by TMPRSS2, this residue was dispensable for TMPRSS2-mediated S protein activation. Conversely, residue R797, previously reported to be required for SARS S activation by trypsin, was dispensable for S protein cleavage but required for S protein activation by TMPRSS2. Collectively, these results show that different residues in SARS S control cleavage and activation by TMPRSS2, suggesting that these processes are more complex than initially appreciated. PMID:28636671

Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2.

PubMed

Reinke, Lennart Michel; Spiegel, Martin; Plegge, Teresa; Hartleib, Anika; Nehlmeier, Inga; Gierer, Stefanie; Hoffmann, Markus; Hofmann-Winkler, Heike; Winkler, Michael; Pöhlmann, Stefan

2017-01-01

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) mediates viral entry into target cells. Cleavage and activation of SARS S by a host cell protease is essential for infectious viral entry and the responsible enzymes are potential targets for antiviral intervention. The type II transmembrane serine protease TMPRSS2 cleaves and activates SARS S in cell culture and potentially also in the infected host. Here, we investigated which determinants in SARS S control cleavage and activation by TMPRSS2. We found that SARS S residue R667, a previously identified trypsin cleavage site, is also required for S protein cleavage by TMPRSS2. The cleavage fragments produced by trypsin and TMPRSS2 differed in their decoration with N-glycans, suggesting that these proteases cleave different SARS S glycoforms. Although R667 was required for SARS S cleavage by TMPRSS2, this residue was dispensable for TMPRSS2-mediated S protein activation. Conversely, residue R797, previously reported to be required for SARS S activation by trypsin, was dispensable for S protein cleavage but required for S protein activation by TMPRSS2. Collectively, these results show that different residues in SARS S control cleavage and activation by TMPRSS2, suggesting that these processes are more complex than initially appreciated.

EDTA-insoluble, calcium-binding proteoglycan in bovine bone

NASA Technical Reports Server (NTRS)

Hashimoto, Y.; Lester, G. E.; Caterson, B.; Yamauchi, M.

1995-01-01

A calcium ion precipitable, trypsin-generated proteoglycan fragment has been isolated from the demineralized, EDTA-insoluble matrices of bone. The demineralized matrix was completely digested with trypsin, increasing concentrations of CaCl2 were added to the supernatant, and the resulting precipitates were analyzed. The amount of precipitate gradually increased with higher concentrations of calcium and was reversibly solubilized by EDTA. After molecular sieve and anion exchange chromatography, a proteoglycan-containing peak was obtained. Immunochemical analysis showed that this peak contained chondroitin 4-sulfate and possibly keratan sulfate. Amino acid analysis showed that this proteoglycan contained high amounts of aspartic acid/asparagine (Asx), serine (Ser), glutamic acid/glutamine (Glx), proline (Pro), and glycine (Gly); however, it contained little leucine (Leu) which suggests that it is not a member of the leucine-rich small proteoglycan family. In addition, significant amounts of phosphoserine (P-Ser) and hydroxyproline (Hyp) were identified in hydrolysates of this fraction. A single band (M(r) 59 kDa) was obtained on SDS-PAGE that stained with Stains-all but not with Coomassie Brilliant Blue R-250. If bone powder was trypsinized prior to demineralization, this proteoglycan-containing fraction was not liberated. Collectively, these results indicate that a proteoglycan occurs in the demineralized matrix that is precipitated with CaCl2 and is closely associated with both mineral and collagen matrices. Such a molecule might facilitate the structural network for the induction of mineralization in bone.

New Kunitz-Type HCRG Polypeptides from the Sea Anemone Heteractis crispa

PubMed Central

Gladkikh, Irina; Monastyrnaya, Margarita; Zelepuga, Elena; Sintsova, Oksana; Tabakmakher, Valentin; Gnedenko, Oksana; Ivanov, Alexis; Hua, Kuo-Feng; Kozlovskaya, Emma

2015-01-01

Sea anemones are a rich source of Kunitz-type polypeptides that possess not only protease inhibitor activity, but also Kv channels toxicity, analgesic, antihistamine, and anti-inflammatory activities. Two Kunitz-type inhibitors belonging to a new Heteractis crispa RG (HCRG) polypeptide subfamily have been isolated from the sea anemone Heteractis crispa. The amino acid sequences of HCRG1 and HCRG2 identified using the Edman degradation method share up to 95% of their identity with the representatives of the HCGS polypeptide multigene subfamily derived from H. crispa cDNA. Polypeptides are characterized by positively charged Arg at the N-terminus as well as P1 Lys residue at their canonical binding loop, identical to those of bovine pancreatic trypsin inhibitor (BPTI). These polypeptides are shown by our current evidence to be more potent inhibitors of trypsin than the known representatives of the HCGS subfamily with P1Thr. The kinetic and thermodynamic characteristics of the intermolecular interactions between inhibitors and serine proteases were determined by the surface plasmon resonance (SPR) method. Residues functionally important for polypeptide binding to trypsin were revealed using molecular modeling methods. Furthermore, HCRG1 and HCRG2 possess anti-inflammatory activity, reducing tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) secretions, as well as proIL-1β expression in lipopolysaccharide (LPS)-activated macrophages. However, there was no effect on nitric oxide (NO) generation. PMID:26404319

Evaluation of the Human Host Range of Bovine and Porcine Viruses that may Contaminate Bovine Serum and Porcine Trypsin Used in the Manufacture of Biological Products

PubMed Central

Marcus-Sekura, Carol; Richardson, James C.; Harston, Rebecca K.; Sane, Nandini; Sheets, Rebecca L.

2011-01-01

Current U.S. requirements for testing cell substrates used in production of human biological products for contamination with bovine and porcine viruses are U.S. Department of Agriculture (USDA) 9CFR tests for bovine serum or porcine trypsin. 9CFR requires testing of bovine serum for seven specific viruses in six families (immunofluorescence) and at least 2 additional families non-specifically (cytopathicity and hemadsorption). 9CFR testing of porcine trypsin is for porcine parvovirus. Recent contaminations suggest these tests may not be sufficient. Assay sensitivity was not the issue for these contaminations that were caused by viruses/virus families not represented in the 9CFR screen. A detailed literature search was undertaken to determine which viruses that infect cattle or swine or bovine or porcine cells in culture also have human host range [ability to infect humans or human cells in culture] and to predict their detection by the currently used 9CFR procedures. There are more viruses of potential risk to biological products manufactured using bovine or porcine raw materials than are likely to be detected by 9CFR testing procedures; even within families, not all members would necessarily be detected. Testing gaps and alternative methodologies should be evaluated to continue to ensure safe, high quality human biologicals. PMID:22000165

Digestive evaluation of soy isolate protein as affected by heat treatment and soy oil inclusion in broilers at an early age.

PubMed

Zhang, Xianglun; Lu, Peng; Xue, Wenyue; Wu, Dawei; Wen, Chao; Zhou, Yanmin

2016-10-01

Soy protein isolate (SPI) mixed with soybean oil (SPIO) incubated at 100°C for 8 h was used to evaluate changes of solubility and digestibility of SPI in vitro and digestive function in broilers at an early age. Arbor Acres broilers were allocated to three groups with six replicates of 12 birds, receiving basal diet (CON), 8 h heat-oxidized SPI diet (HSPI) and 8 h heat-oxidized mixture of SPI and 2% soybean oil diet (HSPIO) for 21 days, respectively. Nitrogen solubility index (NSI) declined and soybean oil accelerated the decline of NSI during incubation (P < 0.05). Decreased in vitro digestibility of dry matter (DM) and crude protein (CP) were observed in SPIO (P < 0.05). HSPI and HSPIO decreased body weight gain, relative jejunum weight and pancreatic trypsin activity at day 21 (P < 0.05). HSPIO decreased anterior intestinal trypsin activity at day 14 and amylase and trypsin activity at day 21, pancreatic amylase activity at day 21 and apparent digestibility of DM, organic matter and CP of broilers from days 18 to 20 (P < 0.05). Heat treatment and soybean oil could induce oxidative modification of SPI, and oxidized SPI negatively affected growth and digestion of broilers. © 2016 Japanese Society of Animal Science. © 2016 Japanese Society of Animal Science.

Adsorption induced enzyme denaturation: the role of protein surface in adsorption induced protein denaturation on allyl glycidyl ether (AGE)-ethylene glycol dimethacrylate (EGDM) copolymers.

PubMed

Thudi, Lahari; Jasti, Lakshmi S; Swarnalatha, Y; Fadnavis, Nitin W; Mulani, Khudbudin; Deokar, Sarika; Ponrathnam, Surendra

2012-02-01

The effects of protein size on adsorption and adsorption-induced denaturation of proteins on copolymers of allyl glycidyl ether (AGE)-ethylene glycol dimethacrylate (EGDM) have been studied. Different responses were observed for the amount of protein adsorbed and denatured on the polymer surface for different proteins (trypsin, alchol dehydrogenase from baker's yeast (YADH), glucose dehydrogenase (GDH) from Gluconobacter cerinus, and alkaline phosphates from calf intestinal mucosa (CIAP). Protein adsorption on the copolymer with 25% crosslink density (AGE-25) was dependent not only on the size of the protein but also on the presence of glycoside residues on the protein surface. Adsorption and denaturation of proteins follows the order YADH>trypsin>GDH>CIAP although the molecular weights of the proteins follow the order YADH>CIAP>GDH>trypsin. The lack of correlation between amount of adsorbed protein and its molecular weight was due to the presence of glycoside residues on CIAP and GDH which protect the enzyme surface from denaturation. Enzyme stabilities in aqueous solutions of 1-cyclohexyl-2-pyrrolidinone (CHP) correlate well with the trend in denaturation by the copolymer, strongly suggesting that hydrophobic interactions play a major role in protein binding and the mechanism of protein denaturation is similar to that for water-miscible organic solvents. Copyright © 2011 Elsevier B.V. All rights reserved.

Effect of acute hyperglycemia on basal and bombesin-stimulated pancreaticobiliary secretion in humans.

PubMed

Lam, W F; Masclee, A A; Muller, E S; Souverijn, J H; Lamers, C B

1998-08-01

This study was undertaken to investigate the effect of acute hyperglycemia on basal and bombesin-stimulated pancreaticobiliary secretion. Seven healthy subjects participated in two experiments performed in random order during normoglycemia and hyperglycemic clamping at 15 mM. Duodenal outputs of bilirubin, trypsin, amylase, and bicarbonate were measured by aspiration with a recovery marker under basal conditions for 60 min and during continuous infusion of bombesin (1 ng/kg x min) for 60 min. Plasma cholecystokinin (CCK) and pancreatic polypeptide (PP) levels were determined at regular intervals. Compared to normoglycemia, during hyperglycemia basal outputs of bilirubin (17 +/- 3 vs. 0.9 +/- 0.4 micromol/60 min), trypsin (24 +/- 4 vs. 4 +/- 1 U/60 min), amylase (12 +/- 1 vs. 3 +/- 1 kU/60 min), and bicarbonate (2.9 +/- 0.5 vs. 1.2 +/- 0.2 mmol/60 min) were significantly p < 0.05) reduced. Bombesin significantly (p < 0.05) increased pancreaticobiliary output during both normo- and hyperglycemia. During hyperglycemia bombesin-stimulated 60-min outputs of bilirubin, trypsin, amylase, and bicarbonate were not significantly different compared to those during normoglycemia. Basal and bombesin-stimulated plasma PP concentrations were significantly (p < 0.05) reduced during hyperglycemia, but plasma CCK levels were not significantly different. It is concluded that acute hyperglycemia reduces basal but does not affect bombesin-induced pancreaticobiliary secretion.

A Serpin Released by an Entomopathogen Impairs Clot Formation in Insect Defense System

PubMed Central

Hao, YouJin; Balasubramanian, Natesan; Jing, Yingjun; Montiel, Rafael; Faria, Tiago Q.; Brito, Rui M.; Simões, Nelson

2013-01-01

Steinernema carpocapsae is an entomopathogenic nematode widely used for the control of insect pests due to its virulence, which is mainly attributed to the ability the parasitic stage has to overcome insect defences. To identify the mechanisms underlying such a characteristic, we studied a novel serpin-like inhibitor (sc-srp-6) that was detected in a transcriptome analysis. Recombinant Sc-SRP-6 produced in Escherichia coli had a native fold of serpins belonging to the α-1-peptidase family and exhibited inhibitory activity against trypsin and α-chymotrypsin with Ki of 0.42×10−7 M and 1.22×10−7 M, respectively. Functional analysis revealed that Sc-SRP-6 inhibits insect digestive enzymes, thus preventing the hydrolysis of ingested particles. Moreover, Sc-SRP-6 impaired the formation of hard clots at the injury site, a major insect defence mechanism against invasive pathogens. Sc-SRP-6 does not prevent the formation of clot fibres and the activation of prophenoloxidases but impairs the incorporation of the melanin into the clot. Binding assays showed a complex formation between Sc-SRP-6 and three proteins in the hemolymph of lepidopteran required for clotting, apolipophorin, hexamerin and trypsin-like, although the catalytic inhibition occurred exclusively in trypsin-like. This data allowed the conclusion that Sc-SRP-6 promotes nematode virulence by inhibiting insect gut juices and by impairing immune clot reaction. PMID:23874900

Fully human antibodies against the Protease-Activated Receptor-2 (PAR-2) with anti-inflammatory activity.

PubMed

Giblin, Patricia; Boxhammer, Rainer; Desai, Sudha; Kroe-Barrett, Rachel; Hansen, Gale; Ksiazek, John; Panzenbeck, Maret; Ralph, Kerry; Schwartz, Racheline; Zimmitti, Clare; Pracht, Catrin; Miller, Sandra; Magram, Jeanne; Litzenburger, Tobias

2011-01-01

PAR-2 belongs to a family of G-protein coupled Protease-Activated Receptors (PAR) which are activated by specific proteolytic cleavage in the extracellular N-terminal region. PAR-2 is activated by proteases such as trypsin, tryptase, proteinase 3, factor VIIa, factor Xa and is thought to be a mediator of inflammation and tissue injury, where elevated levels of proteases are found. Utilizing the HuCAL GOLD® phage display library we generated fully human antibodies specifically blocking the protease cleavage site in the N-terminal domain. In vitro affinity optimization resulted in antibodies with up to 1000-fold improved affinities relative to the original parental antibodies with dissociation constants as low as 100 pM. Corresponding increases in potency were observed in a mechanistic protease cleavage assay. The antibodies effectively inhibited PAR-2 mediated intracellular calcium release and cytokine secretion in various cell types stimulated with trypsin. In addition, the antibodies demonstrated potent inhibition of trypsin induced relaxation of isolated rat aortic rings ex vivo. In a short term mouse model of inflammation, the trans vivo DTH model, anti-PAR-2 antibodies showed inhibition of the inflammatory swelling response. In summary, potent inhibitors of PAR-2 were generated which allow further assessment of the role of this receptor in inflammation and evaluation of their potential as therapeutic agents.

Potential Activities of Freshwater Exo- and Endo-Acting Extracellular Peptidases in East Tennessee and the Pocono Mountains

PubMed Central

Mullen, Lauren; Boerrigter, Kim; Ferriero, Nicholas; Rosalsky, Jeff; Barrett, Abigail van Buren; Murray, Patrick J.; Steen, Andrew D.

2018-01-01

Proteins constitute a particularly bioavailable subset of organic carbon and nitrogen in aquatic environments but must be hydrolyzed by extracellular enzymes prior to being metabolized by microorganisms. Activities of extracellular peptidases (protein-degrading enzymes) have frequently been assayed in freshwater systems, but such studies have been limited to substrates for a single enzyme [leucyl aminopeptidase (Leu-AP)] out of more than 300 biochemically recognized peptidases. Here, we report kinetic measurements of extracellular hydrolysis of five substrates in 28 freshwater bodies in the Delaware Water Gap National Recreation Area in the Pocono Mountains (PA, United States) and near Knoxville (TN, United States), between 2013 and 2016. The assays putatively test for four aminopeptidases (arginyl aminopeptidase, glyclyl aminopeptidase, Leu-AP, and pyroglutamyl aminopeptidase), which cleave N-terminal amino acids from proteins, and trypsin, an endopeptidase, which cleaves proteins mid-chain. Aminopeptidase and the trypsin-like activity were observed in all water bodies, indicating that a diverse set of peptidases is typical in freshwater. However, ratios of peptidase activities were variable among sites: aminopeptidases dominated at some sites and trypsin-like activity at others. At a given site, the ratios remained fairly consistent over time, indicating that they are driven by ecological factors. Studies in which only Leu-AP activity is measured may underestimate the total peptidolytic capacity of an environment, due to the variable contribution of endopeptidases. PMID:29559961

Activation of Bt Protoxin Cry1Ac in Resistant and Susceptible Cotton Bollworm.

PubMed

Wei, Jizhen; Liang, Gemei; Wang, Bingjie; Zhong, Feng; Chen, Lin; Khaing, Myint Myint; Zhang, Jie; Guo, Yuyuan; Wu, Kongming; Tabashnik, Bruce E

2016-01-01

Crystalline (Cry) proteins from Bacillus thuringiensis (Bt) are used extensively for insect control in sprays and transgenic plants, but their efficacy is reduced by evolution of resistance in pests. Here we evaluated reduced activation of Cry1Ac protoxin as a potential mechanism of resistance in the invasive pest Helicoverpa armigera. Based on the concentration killing 50% of larvae (LC50) for a laboratory-selected resistant strain (LF120) divided by the LC50 for its susceptible parent strain (LF), the resistance ratio was 1600 for Cry1Ac protoxin and 1200 for trypsin-activated Cry1Ac toxin. The high level of resistance to activated toxin as well as to protoxin indicates reduced activation of protoxin is not a major mechanism of resistance to Cry1Ac in LF120. For both insect strains, treatment with either the trypsin inhibitor N-a-tosyl-L-lysine chloromethyl ketone (TLCK) or the chymotrypsin inhibitor N-a-tosyl-L-phenylalanine chloromethyl ketone (TPCK) did not significantly affect the LC50 of Cry1Ac protoxin. Enzyme activity was higher for LF than LF120 for trypsin-like proteases, but did not differ between strains for chymotrypsin-like proteases. The results here are consistent with previous reports indicating that reduced activation of protoxin is generally not a major mechanism of resistance to Bt proteins.

Activation of Bt Protoxin Cry1Ac in Resistant and Susceptible Cotton Bollworm

PubMed Central

Liang, Gemei; Wang, Bingjie; Zhong, Feng; Chen, Lin; Khaing, Myint Myint; Zhang, Jie; Guo, Yuyuan; Wu, Kongming; Tabashnik, Bruce E.

2016-01-01

Crystalline (Cry) proteins from Bacillus thuringiensis (Bt) are used extensively for insect control in sprays and transgenic plants, but their efficacy is reduced by evolution of resistance in pests. Here we evaluated reduced activation of Cry1Ac protoxin as a potential mechanism of resistance in the invasive pest Helicoverpa armigera. Based on the concentration killing 50% of larvae (LC50) for a laboratory-selected resistant strain (LF120) divided by the LC50 for its susceptible parent strain (LF), the resistance ratio was 1600 for Cry1Ac protoxin and 1200 for trypsin-activated Cry1Ac toxin. The high level of resistance to activated toxin as well as to protoxin indicates reduced activation of protoxin is not a major mechanism of resistance to Cry1Ac in LF120. For both insect strains, treatment with either the trypsin inhibitor N-a-tosyl-L-lysine chloromethyl ketone (TLCK) or the chymotrypsin inhibitor N-a-tosyl-L-phenylalanine chloromethyl ketone (TPCK) did not significantly affect the LC50 of Cry1Ac protoxin. Enzyme activity was higher for LF than LF120 for trypsin-like proteases, but did not differ between strains for chymotrypsin-like proteases. The results here are consistent with previous reports indicating that reduced activation of protoxin is generally not a major mechanism of resistance to Bt proteins. PMID:27257885

Detection of bovine viral diarrhoea virus nucleic acid, but not infectious virus, in bovine serum used for human vaccine manufacture.

PubMed

Laassri, Majid; Mee, Edward T; Connaughton, Sarah M; Manukyan, Hasmik; Gruber, Marion; Rodriguez-Hernandez, Carmen; Minor, Philip D; Schepelmann, Silke; Chumakov, Konstantin; Wood, David J

2018-06-22

Bovine viral diarrhoea virus (BVDV) is a cattle pathogen that has previously been reported to be present in bovine raw materials used in the manufacture of biological products for human use. Seven lots of trivalent measles, mumps and rubella (MMR) vaccine and 1 lot of measles vaccine from the same manufacturer, together with 17 lots of foetal bovine serum (FBS) from different vendors, 4 lots of horse serum, 2 lots of bovine trypsin and 5 lots of porcine trypsin were analysed for BVDV using recently developed techniques, including PCR assays for BVDV detection, a qRT-PCR and immunofluorescence-based virus replication assays, and deep sequencing to identify and genotype BVDV genomes. All FBS lots and one lot of bovine-derived trypsin were PCR-positive for the presence of BVDV genome; in contrast all vaccine lots and the other samples were negative. qRT-PCR based virus replication assay and immunofluorescence-based infection assay detected no infectious BVDV in the PCR-positive samples. Complete BVDV genomes were generated from FBS samples by deep sequencing, and all were BVDV type 1. These data confirmed that BVDV nucleic acid may be present in bovine-derived raw materials, but no infectious virus or genomic RNA was detected in the final vaccine products. Copyright © 2018 International Alliance for Biological Standardization. All rights reserved.

Immobilized Pepsin Microreactor for Rapid Peptide Mapping with Nanoelectrospray Ionization Mass Spectrometry

NASA Astrophysics Data System (ADS)

Long, Ying; Wood, Troy D.

2015-01-01

Most enzymatic microreactors for protein digestion are based on trypsin, but proteins with hydrophobic segments may be difficult to digest because of the paucity of Arg and Lys residues. Microreactors based on pepsin, which is less specific than trypsin, can overcome this challenge. Here, an integrated immobilized pepsin microreactor (IPMR)/nanoelectrospray emitter is examined for its potential for peptide mapping. For myoglobin, equivalent sequence coverage is obtained in a thousandth the time of solution digestion with better sequence coverage. While sequence coverage of cytochrome c is lesser than solution in this short duration, more highly-charged peptic peptides are produced and a number of peaks are unidentified at low-resolution, suggesting that high-resolution mass spectrometry is needed to take full advantage of integrated IPMR/nanoelectrospray devices.

Role of inhibitors of serine peptidases in protecting Leishmania donovani against the hydrolytic peptidases of sand fly midgut.

PubMed

Verma, Sudha; Das, Sushmita; Mandal, Abhishek; Ansari, Md Yousuf; Kumari, Sujata; Mansuri, Rani; Kumar, Ajay; Singh, Ruby; Saini, Savita; Abhishek, Kumar; Kumar, Vijay; Sahoo, Ganesh Chandra; Das, Pradeep

2017-06-23

In vector-borne diseases such as leishmaniasis, the sand fly midgut is considered to be an important site for vector-parasite interaction. Digestive enzymes including serine peptidases such as trypsin and chymotrypsin, which are secreted in the midgut are one of the obstacles for Leishmania in establishing a successful infection. The presence of some natural inhibitors of serine peptidases (ISPs) has recently been reported in Leishmania. In the present study, we deciphered the role of these ISPs in the survival of Leishmania donovani in the hostile sand fly midgut environment. In silico and co-immunoprecipitation studies were performed to observe the interaction of L. donovani ISPs with trypsin and chymotrypsin. Zymography and in vitro enzyme assays were carried out to observe the inhibitory effect of purified recombinant ISPs of L. donovani (rLdISPs) on trypsin, chymotrypsin and the sand fly midgut peptidases. The expression of ISPs in the amastigote to promastigote transition stages were studied by semi-quantitative RT-PCR and Western blot. The role of LdISP on the survival of ISP overexpressed (OE) and ISP knocked down (KD) Leishmania parasites inside the sand fly gut was investigated by in vitro and in vivo cell viability assays. We identified two ecotin-like genes in L. donovani, LdISP1 and LdISP2. In silico and co-immunoprecipitation results clearly suggest a strong interaction of LdISP molecules with trypsin and chymotrypsin. Zymography and in vitro enzyme assay confirmed the inhibitory effect of rLdISP on trypsin, chymotrypsin and the sand fly midgut peptidases. The expression of LdISP2 was found to be strongly associated with the amastigote to promastigote phase transition. The activities of the digestive enzymes were found to be significantly reduced in the infected sand flies when compared to uninfected. To our knowledge, our study is the first report showing the possible reduction of chymotrypsin activity in L. donovani infected sand flies compared to uninfected. Interestingly, during the early transition stage, substantial killing was observed in ISP2 knocked down (ISP2KD) parasites compared to wild type (WT), whereas ISP1 knocked down (ISP1KD) parasites remained viable. Therefore, our study clearly indicates that LdISP2 is a more effective inhibitor of serine peptidases than LdISP1. Our results suggest that the lack of ISP2 is detrimental to the parasites during the early transition from amastigotes to promastigotes. Moreover, the results of the present study demonstrated for the first time that LdISP2 has an important role in the inhibition of peptidases and promoting L. donovani survival inside the Phlebotomus argentipes midgut.

Calcium signalling in the acinar environment of the exocrine pancreas: physiology and pathophysiology.

PubMed

Gryshchenko, Oleksiy; Gerasimenko, Julia V; Peng, Shuang; Gerasimenko, Oleg V; Petersen, Ole H

2018-02-09

Ca 2+ signalling in different cell types in exocrine pancreatic lobules was monitored simultaneously and signalling responses to various stimuli were directly compared. Ca 2+ signals evoked by K + -induced depolarization were recorded from pancreatic nerve cells. Nerve cell stimulation evoked Ca 2+ signals in acinar but not in stellate cells. Stellate cells are not electrically excitable as they, like acinar cells, did not generate Ca 2+ signals in response to membrane depolarization. The responsiveness of the stellate cells to bradykinin was markedly reduced in experimental alcohol-related acute pancreatitis, but they became sensitive to stimulation with trypsin. Our results provide fresh evidence for an important role of stellate cells in acute pancreatitis. They seem to be a critical element in a vicious circle promoting necrotic acinar cell death. Initial trypsin release from a few dying acinar cells generates Ca 2+ signals in the stellate cells, which then in turn damage more acinar cells causing further trypsin liberation. Physiological Ca 2+ signals in pancreatic acinar cells control fluid and enzyme secretion, whereas excessive Ca 2+ signals induced by pathological agents induce destructive processes leading to acute pancreatitis. Ca 2+ signals in the peri-acinar stellate cells may also play a role in the development of acute pancreatitis. In this study, we explored Ca 2+ signalling in the different cell types in the acinar environment of the pancreatic tissue. We have, for the first time, recorded depolarization-evoked Ca 2+ signals in pancreatic nerves and shown that whereas acinar cells receive a functional cholinergic innervation, there is no evidence for functional innervation of the stellate cells. The stellate, like the acinar, cells are not electrically excitable as they do not generate Ca 2+ signals in response to membrane depolarization. The principal agent evoking Ca 2+ signals in the stellate cells is bradykinin, but in experimental alcohol-related acute pancreatitis, these cells become much less responsive to bradykinin and then acquire sensitivity to trypsin. Our new findings have implications for our understanding of the development of acute pancreatitis and we propose a scheme in which Ca 2+ signals in stellate cells provide an amplification loop promoting acinar cell death. Initial release of the proteases kallikrein and trypsin from dying acinar cells can, via bradykinin generation and protease-activated receptors, induce Ca 2+ signals in stellate cells which can then, possibly via nitric oxide generation, damage more acinar cells and thereby cause additional release of proteases, generating a vicious circle. © 2018 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

Effects of protease and non-starch polysaccharide enzyme on performance, digestive function, activity and gene expression of endogenous enzyme of broilers

PubMed Central

Wang, Mingfa; Zhang, Xiaotu; Wang, Zhixiang

2017-01-01

Three hundred one-day-old male broiler chickens (Ross-308) were fed corn-soybean basal diets containing non-starch polysaccharide (NSP) enzyme and different levels of acid protease from 1 to 42 days of age to investigate the effects of exogenous enzymes on growth performance, digestive function, activity of endogenous digestive enzymes in the pancreas and mRNA expression of pancreatic digestive enzymes. For days 1-42, compared to the control chickens, average daily feed intake (ADFI) and average daily gain (ADG) were significantly enhanced by the addition of NSP enzyme in combination with protease supplementation at 40 or 80 mg/kg (p<0.05). Feed-to-gain ratio (FGR) was significantly improved by supplementation with NSP enzymes or NSP enzyme combined with 40 or 80 mg/kg protease compared to the control diet (p<0.05). Apparent digestibility of crude protein (ADCP) was significantly enhanced by the addition of NSP enzyme or NSP enzyme combined with 40 or 80 mg/kg protease (p<0.05). Cholecystokinin (CCK) level in serum was reduced by 31.39% with NSP enzyme combined with protease supplementation at 160 mg/kg (p<0.05), but the CCK level in serum was increased by 26.51% with NSP enzyme supplementation alone. After 21 days, supplementation with NSP enzyme and NSP enzyme combined with 40 or 80 mg/kg protease increased the activity of pancreatic trypsin by 74.13%, 70.66% and 42.59% (p<0.05), respectively. After 42 days, supplementation with NSP enzyme and NSP enzyme combined with 40 mg/kg protease increased the activity of pancreatic trypsin by 32.45% and 27.41%, respectively (p<0.05). However, supplementation with NSP enzyme and 80 or 160 mg/kg protease decreased the activity of pancreatic trypsin by 10.75% and 25.88%, respectively (p<0.05). The activities of pancreatic lipase and amylase were significantly higher in treated animals than they were in the control group (p<0.05). Supplementation with NSP enzyme, NSP enzyme combined with 40 or 80 mg/kg protease increased pancreatic trypsin mRNA levels by 40%, 44% and 28%, respectively. Supplementation with NSP enzyme and 160 mg/kg protease decreased pancreatic trypsin mRNA levels by 13%. Pancreatic lipase and amylase mRNA expression were significantly elevated in treated animals compared to the control group (p<0.05). These results suggest that the amount of NSP enzyme and acid protease in the diet significantly affects digestive function, endogenous digestive-enzyme activity and mRNA expression in broilers. PMID:28323908

Ipomoea pes-caprae (L.) R. Br (Convolvulaceae) relieved nociception and inflammation in mice - A topical herbal medicine against effects due to cnidarian venom-skin contact.

PubMed

da Silva Barth, Cristiane; Tolentino de Souza, Hugo Guilherme; Rocha, Lilian W; da Silva, Gislaine Francieli; Dos Anjos, Mariana Ferreira; Pastor, Veronica D'Avila; Belle Bresolin, Tania Mari; Garcia Couto, Angelica; Roberto Santin, José; Meira Quintão, Nara Lins

2017-03-22

Ipomoea pes-caprae is known as bayhops, beach morning glory or goat's foot, and in Brazil as salsa-de-praia. Its leaves are used in worldwide folk medicine for the relief of jellyfish-stings symptoms. The literature only reports the neutralizing effects of nonpolar plant derived over jellyfish venoms, without validating the popular use or exploring the mechanism of action. This study aimed to evaluate and validate the topical effects of a semisolid containing hydroethanolic extract obtained from the aerial parts of I. pes-caprae using different models of paw- and ear-oedema and spontaneous nociception in mice, attempting to better understand the mechanism involved in its effect. Leaf and stem of I. pes-caprae were extracted by ethanol 50% (v/v) and the soft-extract was incorporated in Hostacerin® cream base at 0.5%, 1.0% and 2% (w/w). The anti-hypersensitivity effects were evaluated by injecting the Physalia physalis venom into the right mice's hindpaw pre-treated either with the semisolid containing the I. pes-caprae extract or with the isolated majority compound 3,5-Di-O-caffeoylquinic acid (ISA). The topical anti-inflammatory activity was investigated using both preclinical models: paw oedema induced by trypsin, bradykinin (BK), histamine and carrageenan, and ear oedema induced by capsaicin. Additionally, the model of spontaneous nociception induced by trypsin and capsaicin were used to verify the topical antinociceptive activity. The animals pre-treated with the semisolid containing I. pes-caprae extract or with the intraplantar injection of the major component (ISA) had the mechanical hypersensitivity induced by P. physalis venom significantly reduced. Significant inhibition was also observed in paw-oedema induced by trypsin, histamine and BK, and in a less extent in carrageenan-induced paw oedema. Similar effect was observed in mice challenged to the capsaicin-induced ear-oedema. Besides the vascular effects, the extract interfered with leukocyte migration induced by histamine injection. Finally, the semisolid presented significant inhibition in trypsin- and capsaicin-induced spontaneous nociception. The hydroethanolic extract of I. pes-caprae showed compliance with the topical popular use of the herbal product to relieve the symptoms evoked by the cnidarian venom-skin contact, such as neurogenic oedema and nociception. The extract components seem to interfere with the effects resulting from the TRPV1, B 2 R and PAR-2 activation, once it interfered with painful-behaviour and oedema induced by capsaicin, BK and trypsin, pointing the histaminergic system as the main target, once it is an important mediator in the signalling pathway of the aforementioned receptors. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

21 CFR 862.1725 - Trypsin test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

...) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862... other body fluids and in feces. Measurements obtained by this device are used in the diagnosis and...

21 CFR 862.1725 - Trypsin test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

...) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862... other body fluids and in feces. Measurements obtained by this device are used in the diagnosis and...

21 CFR 862.1725 - Trypsin test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

...) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862... other body fluids and in feces. Measurements obtained by this device are used in the diagnosis and...

21 CFR 862.1725 - Trypsin test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

...) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862... other body fluids and in feces. Measurements obtained by this device are used in the diagnosis and...

21 CFR 862.1725 - Trypsin test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

...) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862... other body fluids and in feces. Measurements obtained by this device are used in the diagnosis and...

Science Notes.

ERIC Educational Resources Information Center

Thurman, Shirley; And Others

1988-01-01

Describes 36 science activities. Topics include: osmosis, fermentation, anhydrobiotic organisms, breathing monitors, trypsin, weeds, amyloplasts, electrolysis, polarimeters, ethene ripening of fruit, colorimetry, diffusion, redox reactions, equilibria, acid-base relationships, electricity, power, resonance, measurement, parallax, amplifiers,…

LC/MS/MS Bioanalysis of Protein-Drug Conjugates-The Importance of Incorporating Succinimide Hydrolysis Products.

PubMed

Shi, Chuan; Goldberg, Shalom; Lin, Tricia; Dudkin, Vadim; Widdison, Wayne; Harris, Luke; Wilhelm, Sharon; Jmeian, Yazen; Davis, Darryl; O'Neil, Karyn; Weng, Naidong; Jian, Wenying

2018-04-17

Bioanalysis of antibody-drug conjugates (ADCs) is challenging due to the complex, heterogeneous nature of their structures and their complicated catabolism. To fully describe the pharmacokinetics (PK) of an ADC, several analytes are commonly quantified, including total antibody, conjugate, and payload. Among them, conjugate is the most challenging to measure, because it requires detection of both small and large molecules as one entity. Existing approaches to quantify the conjugated species of ADCs involve a ligand binding assay (LBA) for conjugated antibody or hybrid LBA/liquid chromatography/tandem mass spectrometry (LC/MS/MS) for quantitation of conjugated drug. In our current work for a protein-drug conjugate (PDC) using the Centyrin scaffold, a similar concept to ADCs but with smaller protein size, an alternative method to quantify the conjugate by using a surrogate peptide approach, was utilized. The His-tagged proteins were isolated from biological samples using immobilized metal affinity chromatography (IMAC), followed by trypsin digestion. The tryptic peptide containing the linker attached to the payload was used as a surrogate of the conjugate and monitored by LC/MS/MS analysis. During method development and its application, we found that hydrolysis of the succinimide ring of the linker was ubiquitous, taking place at many stages during the lifetime of the PDC including in the initial drug product, in vivo in circulation in the animals, and ex vivo during the trypsin digestion step of the sample preparation. We have shown that hydrolysis during trypsin digestion is concentration-independent and consistent during the work flow-therefore, having no impact on assay performance. However, for samples that have undergone extensive hydrolysis prior to trypsin digestion, significant bias could be introduced if only the non-hydrolyzed form is considered in the quantitation. Therefore, it is important to incorporate succinimide hydrolysis products in the quantitation method in order to provide an accurate estimation of the total conjugate level. More importantly, the LC/MS/MS-based method described here provides a useful tool to quantitatively evaluate succinimide hydrolysis of ADCs in vivo, which has been previously reported to have significant impact on their stability, exposure, and efficacy.

Thesis Abstract Bean (Phaseolus vulgaris L.) lines: chemical composition and protein digestibility.

PubMed

Mesquita, F R; Silva, M I A; Corrêa, A D

2016-05-09

The bean represents the main source of proteins for the low income populations, although the digestibility of those proteins is relatively low. Consequently, the programs of plant genetic breeding have been working on the search for new lines with higher protein levels. Thus, with the purpose of supplying information to the researchers, in this study, 21 bean (Phaseolus vulgaris L.) lines were analyzed for the centesimal and mineral composition, protein digestibility, phenolic compounds, and trypsin inhibitor. The entirely randomized experimental design was used with 21 treatments (lines) and three repetitions. All values were within the following ranges: 22.34 to 36.28 g crude protein/100 g dry matter (DM); 7.56 to 20.91 g neutral detergent fiber/100 g DM; 0.53 to 2.55 g fat/100 g DM and 2.97 to 4.87 g ashes/100 g DM. The levels of phosphorus, potassium, calcium, magnesium, and sulfur, in g/100 g DM, varied from 0.45 to 0.72; 1.51 to 2.48; 0.03 to 0.28; 0.18 to 0.34 and 0.28 to 0.45, respectively. Regarding copper, manganese, zinc and iron, the levels, in mg/kg DM, varied from 11.37 to 17.73; 14.93 to 28.90; 36.67 to 69.90 and 71.37 to 126.90, respectively. The in vitro protein digestibility varied from 18.03 to 48.32%. The levels of phenolic compounds varied from 0.28 to 1.08 mg acid tanic/100 g DM and the one of trypsin inhibitor from 59.93 to 151.07 trypsin inhibited units/mg DM. Among the lines with higher protein contents, "ESAL 569" (beige with brown stripe) presented the largest protein digestibility and considerable levels of minerals. "P-180" (beige with brown stripe) was one of the lines with higher crude protein contents and digestibilities, and also presented high levels for most of the minerals. No relation between protein digestibility and the contents of phenolic compounds or trypsin inhibitor was observed.

Reduction of spiked porcine circovirus during the manufacture of a Vero cell-derived vaccine.

PubMed

Lackner, Cornelia; Leydold, Sandra M; Modrof, Jens; Farcet, Maria R; Grillberger, Leopold; Schäfer, Birgit; Anderle, Heinz; Kreil, Thomas R

2014-04-11

Porcine circovirus-1 (PCV1) was recently identified as a contaminant in live Rotavirus vaccines, which was likely caused by contaminated porcine trypsin. The event triggered the development of new regulatory guidance on the use of porcine trypsin which shall ensure that cell lines and porcine trypsin in use are free from PCV1. In addition, manufacturing processes of biologicals other than live vaccines include virus clearance steps that may prevent and mitigate any potential virus contamination of product. In this work, artificial spiking of down-scaled models for the manufacturing process of an inactivated pandemic influenza virus vaccine were used to investigate inactivation of PCV1 and the physico-chemically related porcine parvovirus (PPV) by formalin and ultraviolet-C (UV-C) treatment as well as removal by the purification step sucrose gradient ultracentrifugation. A PCV1 infectivity assay, using a real-time PCR infectivity readout was established. The formalin treatment (0.05% for 48h) showed substantial inactivation for both PCV1 and PPV with reduction factors of 3.0log10 and 6.8log10, respectively, whereas UV-C treatment resulted in complete PPV (≥5.9log10) inactivation already at a dose of 13mJ/cm but merely 1.7log10 at 24mJ/cm(2) for PCV1. The UV-C inactivation results with PPV were confirmed using minute virus of mice (MVM), indicating that parvoviruses are far more sensitive to UV-C than PCV1. The sucrose density gradient ultracentrifugation also contributed to PCV1 clearance with a reduction factor of 2log10. The low pH treatment during the production of procine trypsin was investigated and showed effective inactivation for both PCV1 (4.5log10) and PPV (6.4log10). In conclusion, PCV1 in general appears to be more resistant to virus inactivation than PPV. Still, the inactivated pandemic influenza vaccine manufacturing process provides for robust virus reduction, in addition to the already implemented testing for PCV1 to avoid any contaminations. Copyright © 2014 Elsevier Ltd. All rights reserved.

A Randomized, Clinical Trial to Evaluate Efficacy and Tolerability of Trypsin:Chymotrypsin as Compared to Serratiopeptidase and Trypsin:Bromelain:Rutoside in Wound Management.

PubMed

Chandanwale, Ajay; Langade, Deepak; Sonawane, Dheeraj; Gavai, Piyush

2017-01-01

Systemic enzyme therapy can play an important role in maintaining normal inflammatory processes within the body and thereby helps support and speed up healing. In the course of the anti-inflammatory action, enzymes degrade damaged cells and necrotic material and, through the inactivation of mediators and toxic products, they restrict the edema and pain. The study conducted at Grant Medical College, Mumbai, India was a clinical trial comparing the efficacy and tolerability of three oral enzyme treatment groups-oral tablets containing trypsin:chymotrypsin (TC) (Chymoral Forte ® ), serratiopeptidase (S) 5 mg oral tablets, and oral enzyme tablets containing trypsin 48 mg, bromelain 90 mg, and rutoside 100 mg (TBR)-to evaluate their healing potential in surgical wounds after orthopedic surgery. A total of 75 patients were screened, randomized, and divided into three groups in 1:1:1 ratio receiving either of the three treatments. In the TC group, erythema was significantly reduced from 3.44 on day 3 to 1.16 on day 10 (p < 0.01). There was significantly better reduction in erythema scores in the TC group as compared to S and TBR groups (p < 0.05) at each follow-up visit. Similarly reduction in the local irritation, wound discharge, edema, induration, and tenderness score with TC treatment at the end of the study was significantly higher than that observed in the other two groups. In addition TC showed significant reduction in pain on the VAS scale (p < 0.01). Global assessment of response to therapy for efficacy and tolerability was reported to be good to excellent in 88% and 92% of the patients on TC as compared to 12% and 8% with S and 12% and 8% with TBR. TC provides a better resolution of symptoms of inflammation after orthopedic surgery as compared to S and TBR, thus facilitating better wound healing. Further studies are warranted to confirm the findings. Clinical Trial Registry of India (Reg. No. CTRI/2011/07/001920).

Trypsin and chymotrypsin in stool

MedlinePlus

... the stool. You can catch the stool on plastic wrap that is loosely placed over the toilet bowl ... child wears a diaper, line the diaper with plastic wrap. Place the plastic wrap so that urine and ...

Revealing of Saccharomyces cerevisiae yeast cell wall proteins capable of binding thioflavin T, a fluorescent dye specifically interacting with amyloid fibrils.

PubMed

Gorkovskii, A A; Bezsonov, E E; Plotnikova, T A; Kalebina, T S; Kulaev, I S

2009-11-01

Proteins binding thioflavin T leading to its specific fluorescence were discovered in a fraction of noncovalently bound Saccharomyces cerevisiae yeast cell wall mannoproteins. Thioflavin-binding proteins display high resistance to trypsin digestion in solution. These data are the first experimental evidence for the presence of proteins whose properties are characteristic of amyloids in yeast cell wall, except for data on glucanotransferase Bgl2p that has amyloid properties. Our data suggest the anchoring of these proteins in the cell wall by a trypsin-sensitive part of the protein molecule. Experiments with a mutant strain devoid of the BGL2 gene suggest the compensation of absent amyloid-like protein Bgl2p by increase in contents of thioflavin-binding proteins in the cell wall.

QUANTITATIVE STUDIES ON THE TOTAL PLASMIN AND THE TRYPSIN INHIBITOR OF HUMAN BLOOD SERUM

PubMed Central

Todd, Edgar W.

1949-01-01

The total plasmin and the trypsin inhibitor were titrated separately in samples of serum taken at weekly intervals from three different groups of scarlet fever patients: (a) those who did not develop any complications, (b) those who developed purulent complications, and (c) those who developed rheumatic fever. When these determinations were plotted, it was found that the resulting curves showed characteristic patterns for each of the diseases investigated. The uncomplicated cases had plasmin curves which were considerably higher on the charts than the inhibitor curves. The septic cases had plasmin and inhibitor curves which were closer together on the charts. The rheumatic cases had plasmin and inhibitor curves which were close together and which crossed at the time of rheumatic activity so that the inhibitor curve reached a higher level than the plasmin curve. PMID:18110885

Four rice seed cDNA clones belonging to the alpha-amylase/trypsin inhibitor gene family encode potential rice allergens.

PubMed

Alvarez, A M; Fukuhara, E; Nakase, M; Adachi, T; Aoki, N; Nakamura, R; Matsuda, T

1995-07-01

Four rice seed proteins encoded by cDNAs belonging to the alpha-amylase/trypsin inhibitor gene family were overexpressed as TrpE-fusion proteins in E. coli. The expressed rice proteins were detected by SDS-PAGE as major proteins in bacterial cell lysates. Western blot analyses showed that all the recombinant proteins were immunologically reactive to rabbit polyclonal antibodies and to a mouse monoclonal antibody (25B9) specific for a previously isolated rice allergen of 16 kDa. Some truncated proteins from deletion mutants of the cDNAs retained their reactivity to the specific antibodies. These results suggest that the cDNAs encode potential rice allergens and that some epitopes of the recombinant proteins are still immunoreactive when they are expressed as their fragments.

Heated oligonucleotide ligation assay (HOLA): an affordable single nucleotide polymorphism assay.

PubMed

Black, W C; Gorrochotegui-Escalante, N; Duteau, N M

2006-03-01

Most single nucleotide polymorphism (SNP) detection requires expensive equipment and reagents. The oligonucleotide ligation assay (OLA) is an inexpensive SNP assay that detects ligation between a biotinylated "allele-specific detector" and a 3' fluorescein-labeled "reporter" oligonucleotide. No ligation occurs unless the 3' detector nucleotide is complementary to the SNP nucleotide. The original OLA used chemical denaturation and neutralization. Heated OLA (HOLA) instead uses a thermal stable ligase and cycles of denaturing and hybridization for ligation and SNP detection. The cost per genotype is approximately US$1.25 with two-allele SNPs or approximately US$1.75 with three-allele SNPs. We illustrate the development of HOLA for SNP detection in the Early Trypsin and Abundant Trypsin loci in the mosquito Aedes aegypti (L.) and at the a-glycerophosphate dehydrogenase locus in the mosquito Anopheles gambiae s.s.

Effects of method of detachment on electrophoretic mobility of mammalian cells grown in monolayer culture

NASA Technical Reports Server (NTRS)

Plank, L. D.; Kunze, M. E.; Todd, P. W.

1985-01-01

A variety of proteolytic and micolytic enzumes, mechanical procedures, and changes in the ionic environment, especially Ca chelation, are used for dispersal of monolayer grown cells. If either chelating agents or mechanical dispersion are used alone, the cell yield is often low and suspensions of single cells are difficult to obtain. Confluent monolayers treated with EDTA tend to be released from their surfaces in sheets, and clumps of cells remain even after further incubation in EDTA. Crude trypsin is the most popular dispersal agent and is known to contain a variety of contaminating enzymes which contribute to the dispersal of cells. A variety of cell injuries resulting from the activity of proteolytic enzymes are reported. It is shown that crystalline trypsin is least harmful to cell integrity as judged by trypan blue uptake.

Relationship between proportion and composition of albumins, and in vitro protein digestibility of raw and cooked pea seeds (Pisum sativum L.).

PubMed

Park, Sei Joon; Kim, Tae Wan; Baik, Byung-Kee

2010-08-15

Peas provide an excellent plant protein resource for human diets, but their proteins are less readily digestible than animal proteins. To identify the relationship between composition and in vitro digestibility of pea protein, eight pea varieties with a wide range of protein content (157.3-272.7 g kg(-1)) were determined for the proportion of albumins and globulins, their compositions using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and in vitro protein digestibility (IVPD) before and after heat treatment using a multi-enzyme (trypsin, chymotrypsin and peptidase) method. The proportion of albumins based on total seed protein content decreased from 229 to 147 g kg(-1) as seed protein content increased from 157.3 to 272.7 g kg(-1), while the proportion of globulins increased from 483 to 590 g kg(-1). The IVPDs of eight raw pea seeds were 79.9-83.5%, with significant varietal variations, and those were improved to 85.9-86.8% by cooking. Albumins, including (pea albumins 2) PA2, trypsin inhibitor, lectin and lipoxygenase, were identified as proteolytic resistant proteins. Globulins were mostly digested by protease treatment after heating. The quantitative ratio of albumins and globulins, and the quantitative variations of albumin protein components, including lipoxygenase, PA2, lectins and trypsin inhibitors, appear to influence the protein digestibility of both raw and cooked pea seeds. Copyright (c) 2010 Society of Chemical Industry.

Bio-detoxification of phorbol esters and other anti-nutrients of Jatropha curcas seed cake by fungal cultures using solid-state fermentation.

PubMed

Sharath, B S; Mohankumar, B V; Somashekar, D

2014-03-01

Jatropha seed cake, a byproduct after biodiesel extraction, has several anti-nutrients and toxins. Solid-state fermentation was carried out for the detoxification of the Jatropha seed cake (JSC) using different fungal cultures. The reduction in the anti-nutritional components such as tannins, phytates, saponins, lectin and protease inhibitor, and phorbol esters on 6th, 9th, and 12th day of fermentation was analyzed. The phorbol ester content in the unfermented JSC was 0.83 mg/g, and the maximum degradation of phorbol esters to the extent of 75% was observed in the case of JSC fermented with Cunninghamella echinulata CJS-90. The phytate degradation in the fermented JSC was in the range of 65-96%. There was a gradual reduction of saponin content in the JSC from 6th to 12th day, and the reduction of saponin was in the range of 55-99% after solid-state fermentation. The trypsin inhibitor activity and lectin were 1,680 trypsin inhibitor units (TIU) per gram and 0.32 hemagglutinating unit in the unfermented JSC, respectively. Trypsin inhibitor activity and lectin could not be detected in JSC after 12th day of solid-state fermentation. Tannins accounted for 0.53% in unfermented JSC, and there was a marginal increase of tannins after solid-state fermentation. The results indicate that biological detoxification could be a promising method to reduce anti-nutritional compounds and toxins in the JSC.

Proteinase-activated receptor 2 (PAR-2) in gastrointestinal and pancreatic pathophysiology, inflammation and neoplasia.

PubMed

Søreide, Kjetil

2008-08-01

Of all the body systems, the gastrointestinal (GI) tract is the most exposed to proteinases. Proteolytic activity must thus be tightly regulated in the face of diverse environmental challenges, because unrestrained or excessive proteolysis leads to pathological GI conditions. The protease-activated receptor-2 (PAR-2) is expressed in numerous cell types within the GI tract, suggesting both multiple functions and numerous modes of receptor activation. Although best known as a pancreatic digestive enzyme, trypsin has also been found in other tissues and various cancers. Of interest, trypsin and PAR-2 act together in an autocrine loop that promotes proliferation, invasion and metastasis in neoplasia through various mechanisms. Trypsin and PAR-2 seem to act both directly and indirectly through activation of other proteinase cascades, including metalloproteinases. PAR-2 activation can participate in inflammatory reactions, be protective to mucosal surfaces, send or inhibit nociceptive messages, modify gut motility or secretory functions, and stimulate cell proliferation and motility. Several studies point to a role for the PARs in disease processes of the GI tract and pancreas ranging from inflammatory bowel disease, symptoms associated with irritable bowel syndrome, pain in pancreatitis, development of colon and other GI cancers, and even infectious colitis. Proteinases should not only be considered from the traditional view as digestive or degradative enzymes in the gut, but additionally as signalling molecules that actively participate in the spectrum of physiology and diseased states of the GI tract.

Aspergillus oryzae GB-107 fermentation improves nutritional quality of food soybeans and feed soybean meals.

PubMed

Hong, Kee-Jong; Lee, Chan-Ho; Kim, Sung Woo

2004-01-01

This study evaluated the effect of fermentation on the nutritional quality of food-grade soybeans and feed-grade soybean meals. Soybeans and soybean meals were fermented by Aspergillus oryzae GB-107 in a bed-packed solid fermentor for 48 hours. After fermentation, their nutrient contents as well as trypsin inhibitor were measured and compared with those of raw soybeans and soybean meals. Proteins were extracted from fermented and non-fermented soybeans and soybean meals, and the peptide characteristics were evaluated after electrophoresis. Fermented soybeans and fermented soybean meals contained 10% more (P < .05) crude protein than raw soybeans and soybean meals. The essential amino acid profile was unchanged after fermentation. Fermentation eliminated (P < .05) most of the trypsin inhibitor from both soybeans and soybean meals. Fermentation increased the amount of small-size peptides (<20 kDa) (P < .05) compared with raw soybeans, while significantly decreasing large-size peptides (>60 kDa) (P < .05). Fermented soybean meal contained more (P < .01) small-size peptides (<20 kDa) than soybean meal. Fermented soybean meal did not contain large-size peptides (>60 kDa), whereas 22.1% of peptides in soybean meal were large-size (>60 kDa). Collectively, fermentation increased protein content, eliminated trypsin inhibitors, and reduced peptide size in soybeans and soybean meals. These effects of fermentation might make soy foods more useful in human diets as a functional food and benefit livestock as a novel feed ingredient.

Protein purification by aminosquarylium cyanine dye-affinity chromatography.

PubMed

Silva, M S; Graça, V C; Reis, L V; Santos, P F; Almeida, P; Queiroz, J A; Sousa, F

2013-12-01

The most selective purification method for proteins and other biomolecules is affinity chromatography. This method is based on the unique biological-based specificity of the biomolecule-ligand interaction and commonly uses biological ligands. However, these ligands may present some drawbacks, mainly because of their cost and lability. Dye-affinity chromatography overcomes the limitations of biological ligands and is widely used owing to the low cost of synthetic dyes and to their resistance to biological and chemical degradation. In this work, immobilized aminosquarylium cyanine dyes are used in order to exploit affinity interactions with standard proteins such as lysozyme, α-chymotrypsin and trypsin. These studies evaluate the affinity interactions occurring between the immobilized ligand and the different proteins, as a reflection of the sum of several molecular interactions, namely ionic, hydrophobic and van der Waals, spread throughout the structure, in a defined spatial manner. The results show the possibility of using an aminosquarylium cyanine dye bearing a N-hexyl pendant chain, with a ligand density of 1.8 × 10(-2) mmol of dye/g of chromatographic support, to isolate lysozyme, α-chymotrypsin and trypsin from a mixture. The application of a decreasing ammonium sulfate gradient resulted in the recovery of lysozyme in the flowthrough. On the other hand, α-chymotrypsin and trypsin were retained, involving different interactions with the ligand. In conclusion, this study demonstrates the potential applicability of ligands such as aminosquarylium cyanine dyes for the separation and purification of proteins by affinity chromatography. Copyright © 2013 John Wiley & Sons, Ltd.

Metabolite extraction from adherently growing mammalian cells for metabolomics studies: optimization of harvesting and extraction protocols.

PubMed

Dettmer, Katja; Nürnberger, Nadine; Kaspar, Hannelore; Gruber, Michael A; Almstetter, Martin F; Oefner, Peter J

2011-01-01

Trypsin/ethylenediaminetetraacetic acid (EDTA) treatment and cell scraping in a buffer solution were compared for harvesting adherently growing mammalian SW480 cells for metabolomics studies. In addition, direct scraping with a solvent was tested. Trypsinated and scraped cell pellets were extracted using seven different extraction protocols including pure methanol, methanol/water, pure acetone, acetone/water, methanol/chloroform/water, methanol/isopropanol/water, and acid-base methanol. The extracts were analyzed by GC-MS after methoximation/silylation and derivatization with propyl chloroformate, respectively. The metabolic fingerprints were compared and 25 selected metabolites including amino acids and intermediates of energy metabolism were quantitatively determined. Moreover, the influence of freeze/thaw cycles, ultrasonication and homogenization using ceramic beads on extraction yield was tested. Pure acetone yielded the lowest extraction efficiency while methanol, methanol/water, methanol/isopropanol/water, and acid-base methanol recovered similar metabolite amounts with good reproducibility. Based on overall performance, methanol/water was chosen as a suitable extraction solvent. Repeated freeze/thaw cycles, ultrasonication and homogenization did not improve overall metabolite yield of the methanol/water extraction. Trypsin/EDTA treatment caused substantial metabolite leakage proving it inadequate for metabolomics studies. Gentle scraping of the cells in a buffer solution and subsequent extraction with methanol/water resulted on average in a sevenfold lower recovery of quantified metabolites compared with direct scraping using methanol/water, making the latter one the method of choice to harvest and extract metabolites from adherently growing mammalian SW480 cells.

Effects of Bacterial Microflora of the Lower Digestive Tract of Free-Range Waterfowl on Influenza Virus Activation ▿

PubMed Central

King, Marcus D.; Guentzel, M. Neal; Arulanandam, Bernard P.; Bodour, Adria A.; Brahmakshatriya, Vinayak; Lupiani, Blanca; Chambers, James P.

2011-01-01

Proteolytic cleavage activation of influenza virus hemagglutinin (HA0) is required for cell entry via receptor-mediated endocytosis. Despite numerous studies describing bacterial protease-mediated influenza A viral activation in mammals, very little is known about the role of intestinal bacterial flora of birds in hemagglutinin cleavage/activation. Therefore, the cloaca of wild waterfowl was examined for (i) representative bacterial types and (ii) their ability to cleave in a “trypsin-like” manner the precursor viral hemagglutinin molecule (HA0). Using radiolabeled HA0, bacterial secretion-mediated trypsin-like conversion of HA0 to HA1 and HA2 peptide products was observed to various degrees in 42 of 44 bacterial isolates suggestive of influenza virus activation in the cloaca of wild waterfowl. However, treatment of uncleaved virus with all bacterial isolates gave rise to substantially reduced emergent virus progeny compared with what was expected. Examination of two isolates exhibiting pronounced trypsin-like conversion of HA0 to HA1 and HA2 peptide products and low infectivity revealed lipase activity to be present. Because influenza virus possesses a complex lipid envelope, the presence of lipid hydrolase activity could in part account for the observed less-than-expected level of viable progeny. A thorough characterization of respective isolate protease HA0 hydrolysis products as well as other resident activities (i.e., lipase) is ongoing such that the role of these respective contributors in virus activation/inactivation can be firmly established. PMID:21531837

Toxicity to cotton boll weevil Anthonomus grandis of a trypsin inhibitor from chickpea seeds.

PubMed

de P G Gomes, Angélica; Dias, Simoni C; Bloch, Carlos; Melo, Francislete R; Furtado, José R; Monnerat, Rose G; Grossi-de-Sá, Maria F; Franco, Octávio L

2005-02-01

Cotton (Gossypium hirsutum L.) is an important agricultural commodity, which is attacked by several pests such as the cotton boll weevil Anthonomus grandis. Adult A. grandis feed on fruits and leaf petioles, reducing drastically the crop production. The predominance of boll weevil digestive serine proteinases has motivated inhibitor screenings in order to discover new ones with the capability to reduce the digestion process. The present study describes a novel proteinase inhibitor from chickpea seeds (Cicer arietinum L.) and its effects against A. grandis. This inhibitor, named CaTI, was purified by using affinity Red-Sepharose Cl-6B chromatography, followed by reversed-phase HPLC (Vydac C18-TP). SDS-PAGE and MALDI-TOF analyses, showed a unique monomeric protein with a mass of 12,877 Da. Purified CaTI showed significant inhibitory activity against larval cotton boll weevil serine proteinases (78%) and against bovine pancreatic trypsin (73%), when analyzed by fluorimetric assays. Although the molecular mass of CaTI corresponded to alpha-amylase/trypsin bifunctional inhibitors masses, no inhibitory activity against insect and mammalian alpha-amylases was observed. In order to observe CaTI in vivo effects, an inhibitor rich fraction was added to an artificial diet at different concentrations. At 1.5% (w/w), CaTI caused severe development delay, several deformities and a mortality rate of approximately 45%. These results suggested that CaTI could be useful in the production of transgenic cotton plants with enhanced resistance toward cotton boll weevil.

Digestive enzyme as benchmark for insecticide resistance development in Culex pipiens larvae to chemical and bacteriologic insecticides.

PubMed

Kamel, Nashwa H; Bahgat, Iman M; El Kady, Gamal A

2013-04-01

This work monitored changes in some digestive enzymes (trypsin and aminopeptidase) associated with the building up of resistance in Cx. pipiens larvae to two chemical insecticides (methomyl and/or malathion) and one biological insecticide (Bacillus thuringiensis-H14 or B.t H 14). The LC50 value of methomyl for both field- and the 12th generation (F12) of the selected strain was 1.789 ppm and 8.925 ppm respectively. The LC50 value of malathion for both field and the F12 of the selected strain was 0.082 ppm and 0.156 ppm respectively, and those of B.t H14 of field strain and the F12 was 2.550ppm & 2.395ppm respectively. The specific activity of trypsin enzyme in control susceptible colony was 20.806 +/- 0.452micromol/min/mg protein; but at F4 and F8 for malathion and methomyl treated larvae were 10.810 +/- 0.860 & 15.616+/-0.408 micromol/min/mg protein, respectively. Trypsin activity of F12 in treated larvae with B.t.H14 was 2.097 +/- 0.587 microiol/min/mg protein. Aminopeptidase specific activity for susceptible control larvae was 173.05 +/- 1.3111 micromol/min/mg protein. This activity decreased to 145.15 +/- 4.12, 152.497 +/- 6.775 & 102.04 +/- 3.58a micromol/min/mg protein after larval (F 12) treatment with methomyl, malathion and B.t H 14 respectively.

Trypsin Inhibitors from Cajanus cajan and Phaseolus limensis Possess Antioxidant, Anti-Inflammatory, and Antibacterial Activity.

PubMed

Shamsi, Tooba Naz; Parveen, Romana; Afreen, Sumbul; Azam, Mudasser; Sen, Priyankar; Sharma, Yamini; Haque, Qazi Mohd Rizwanul; Fatma, Tasneem; Manzoor, Nikhat; Fatima, Sadaf

2018-01-18

Protease inhibitors are one of the most promising and investigated subjects for their role in pharmacognostic and pharmacological studies. This study aimed to investigate antioxidant, anti-inflammatory, and antimicrobial activities of trypsin inhibitors (TIs) from two plant sources (Cajanus cajan and Phaseolus limensis). TI was purified from C. cajan (PUSA-992) by ammonium sulfate precipitation followed by ion exchange chromatography. TI from Phaseolus limensis (lima bean trypsin inhibitor; LBTI) was procured from Sigma-Aldrich, St. Louis, Missouri, United States. The antioxidant activity was analyzed by ferric ion reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH). The anti-inflammatory property of TIs was determined by inhibition of albumin denaturation assay. Ascorbic acid and aspirin were used as standards for antioxidant and anti-inflammatory assays, respectively. These TIs were tested against various bacterial and fungal strains. The TIs showed DPPH radical-scavenging activity in a concentration-dependent manner with IC 50 values comparable to ascorbic acid. The FRAP values were also observed comparable to ascorbic acid and followed the trend of dose-dependent manner. The half maximal inhibitory concentration (IC 50 ) values of CCTI and LBTI in anti-inflammatory test showed that LBTI is more potent than CCTI. The TIs showed potent antibacterial activity, but apparently no action against fungi. This study has reported the biological properties of CCTI and LBTI for the first time. The results show that TIs possess the ability to inhibit diseases caused by oxidative stress, inflammation, and bacterial infestation.

Human Langerhans cells express E-cadherin.

PubMed

Blauvelt, A; Katz, S I; Udey, M C

1995-02-01

Murine Langerhans cells (LC) synthesize and express E-cadherin, a Ca(++)-dependent homophilic cell adhesion molecule that mediates LC-keratinocyte (KC) binding in vitro. In vivo, E-cadherin expression by LC may promote localization and persistence of LC within the epidermis through LC-KC adhesion. In addition, changes in LC E-cadherin expression or affinity may be an important factor in the egress of LC from the epidermis after exposure to antigen. The aim of the present study was to determine if human LC also express E-cadherin. Suction blister roofs were obtained from normal volunteers and epidermal cell (EC) suspensions were prepared by limited trypsinization in the presence of 1 mM Ca++. EC were then incubated with antibodies to E-cadherin and CD1a or HLA-DR, and examined by two-color analytical flow cytometry or immunofluorescence microscopy. Most (82.9% +/- 7.4% [mean +/- SD], range 67-89%, n = 7) freshly prepared human LC expressed E-cadherin, as did the majority of KC. The amount of E-cadherin (as determined by mean fluorescence intensity) expressed by LC and KC was similar. Trypsin/EDTA treatment of freshly prepared EC abrogated expression of E-cadherin by LC and KC, whereas E-cadherin was not degraded by trypsin in the presence of Ca++. LC expressed lower levels of E-cadherin after 3 d in culture. Thus, human LC, like murine LC, express the homophilic adhesion molecule E-cadherin, which may be important in establishing and maintaining interactions between LC and KC in mammalian epidermis.

Quantitative Assessment of In-solution Digestion Efficiency Identifies Optimal Protocols for Unbiased Protein Analysis*

PubMed Central

León, Ileana R.; Schwämmle, Veit; Jensen, Ole N.; Sprenger, Richard R.

2013-01-01

The majority of mass spectrometry-based protein quantification studies uses peptide-centric analytical methods and thus strongly relies on efficient and unbiased protein digestion protocols for sample preparation. We present a novel objective approach to assess protein digestion efficiency using a combination of qualitative and quantitative liquid chromatography-tandem MS methods and statistical data analysis. In contrast to previous studies we employed both standard qualitative as well as data-independent quantitative workflows to systematically assess trypsin digestion efficiency and bias using mitochondrial protein fractions. We evaluated nine trypsin-based digestion protocols, based on standard in-solution or on spin filter-aided digestion, including new optimized protocols. We investigated various reagents for protein solubilization and denaturation (dodecyl sulfate, deoxycholate, urea), several trypsin digestion conditions (buffer, RapiGest, deoxycholate, urea), and two methods for removal of detergents before analysis of peptides (acid precipitation or phase separation with ethyl acetate). Our data-independent quantitative liquid chromatography-tandem MS workflow quantified over 3700 distinct peptides with 96% completeness between all protocols and replicates, with an average 40% protein sequence coverage and an average of 11 peptides identified per protein. Systematic quantitative and statistical analysis of physicochemical parameters demonstrated that deoxycholate-assisted in-solution digestion combined with phase transfer allows for efficient, unbiased generation and recovery of peptides from all protein classes, including membrane proteins. This deoxycholate-assisted protocol was also optimal for spin filter-aided digestions as compared with existing methods. PMID:23792921

Impact of lipases on the protective effect of SEDDS for incorporated peptide drugs towards intestinal peptidases.

PubMed

Leonaviciute, Gintare; Zupančič, Ožbej; Prüfert, Felix; Rohrer, Julia; Bernkop-Schnürch, Andreas

2016-07-11

The aim of this study is the development of self-emulsifying drug delivery systems (SEDDS) differing in amounts of ester substructures and to evaluate their stability in presence of pancreatic lipase and protective effect against luminal enzymatic metabolism using leuprorelin as model peptide drug. Hydrophobic leuprolide oleate was incorporated into three different SEDDS formulations and their stability towards pancreatic lipases was investigated utilizing a dynamic in vitro digestion model. Protective effect of SEDDS in respect to peptide drug stability against proteolytic enzymes, trypsin and α-chymotrypsin, was determined via HPLC. Results of in vitro digestion demonstrated that 80% of SEDDS containing the highest amount of ester linkages was degraded within 60min. In comparison to that, SEDDS without ester bonds showed no degradation. With increasing oil droplets hydrolysis the remaining amount of peptide encapsulated into formulation decreased. Furthermore, after 180min incubation with trypsin up to 33.5% and with α-chymotrypsin up to 60.5% of leuprolide oleate was intact while leuprorelin acetate aqueous solution was completely metabolized by trypsin within 120min and by α-chymotrypsin within 5min. Protective effect in environment containing lipases was lower due to oil phase degradation, however, the amount of peptide in ester-free SEDDS was remarkably higher compared to SEDDS susceptible to lipases. The present study revealed that SEDDS stable towards hydrolysis is able to exhibit a protective effect for oral peptide delivery. Copyright © 2016 Elsevier B.V. All rights reserved.

Differences in Grain Ultrastructure, Phytochemical and Proteomic Profiles between the Two Contrasting Grain Cd-Accumulation Barley Genotypes

PubMed Central

Sun, Hongyan; Cao, Fangbin; Wang, Nanbo; Zhang, Mian; Mosaddek Ahmed, Imrul; Zhang, Guoping; Wu, Feibo

2013-01-01

To reveal grain physio-chemical and proteomic differences between two barley genotypes, Zhenong8 and W6nk2 of high- and low- grain-Cd-accumulation, grain profiles of ultrastructure, amino acid and proteins were compared. Results showed that W6nk2 possesses significantly lower protein content, with hordein depicting the greatest genotypic difference, compared with Zhenong8, and lower amino acid contents with especially lower proportion of Glu, Tyr, Phe and Pro. Both scanning and transmission electron microscopy observation declared that the size of A-type starch molecule in W6nk2 was considerably larger than that of Zhenong8. Grains of Zhenong8 exhibited more protein-rich deposits around starch granules, with some A-type granules having surface pits. Seventeen proteins were identified in grains, using 2-DE coupled with mass spectrometry, with higher expression in Zhenong8 than that in W6nk2; including z-type serpin, serpin-Z7 and alpha-amylase/trypsin inhibitor CM, carbohydrate metabolism, protein synthesis and signal transduction related proteins. Twelve proteins were less expressed in Zhenong8 than that in W6nk2; including barley trypsin inhibitor chloroform/methanol-soluble protein (BTI-CMe2.1, BTI-CMe2.2), trypsin inhibitor, dehydroascorbate reductase (DHAR), pericentrin, dynein heavy chain and some antiviral related proteins. The data extend our understanding of mechanisms underlying Cd accumulation/tolerance and provides possible utilization of elite genetic resources in developing low-grain-Cd barley cultivars. PMID:24260165

Kinetic Dissection of the Pre-existing Conformational Equilibrium in the Trypsin Fold*

PubMed Central

Vogt, Austin D.; Chakraborty, Pradipta; Di Cera, Enrico

2015-01-01

Structural biology has recently documented the conformational plasticity of the trypsin fold for both the protease and zymogen in terms of a pre-existing equilibrium between closed (E*) and open (E) forms of the active site region. How such plasticity is manifested in solution and affects ligand recognition by the protease and zymogen is poorly understood in quantitative terms. Here we dissect the E*-E equilibrium with stopped-flow kinetics in the presence of excess ligand or macromolecule. Using the clotting protease thrombin and its zymogen precursor prethrombin-2 as relevant models we resolve the relative distribution of the E* and E forms and the underlying kinetic rates for their interconversion. In the case of thrombin, the E* and E forms are distributed in a 1:4 ratio and interconvert on a time scale of 45 ms. In the case of prethrombin-2, the equilibrium is shifted strongly (10:1 ratio) in favor of the closed E* form and unfolds over a faster time scale of 4.5 ms. The distribution of E* and E forms observed for thrombin and prethrombin-2 indicates that zymogen activation is linked to a significant shift in the pre-existing equilibrium between closed and open conformations that facilitates ligand binding to the active site. These findings broaden our mechanistic understanding of how conformational transitions control ligand recognition by thrombin and its zymogen precursor prethrombin-2 and have direct relevance to other members of the trypsin fold. PMID:26216877

CLONING AND EXPRESSING TRYPSIN MODULATING OOSTATIC FACTOR IN Chlorella desiccata TO CONTROL MOSQUITO LARVAE.

PubMed

Borovsky, Dov; Sterner, Andeas; Powell, Charles A

2016-01-01

The insect peptide hormone trypsin modulating oostatic factor (TMOF), a decapeptide that is synthesized by the mosquito ovary and controls the translation of the gut's trypsin mRNA was cloned and expressed in the marine alga Chlorella desiccata. To express Aedes aegypti TMOF gene (tmfA) in C. desiccata cells, two plasmids (pYES2/TMOF and pYDB4-tmfA) were engineered with pKYLX71 DNA (5 Kb) carrying the cauliflower mosaic virus (CaMV) promoter 35S(2) and the kanamycin resistant gene (neo), as well as, a 8 Kb nitrate reductase gene (nit) from Chlorella vulgaris. Transforming C. desiccata with pYES2/TMOF and pYDB4-tmfA show that the engineered algal cells express TMOF (20 ± 4 μg ± SEM and 17 ± 3 μg ± SEM, respectively in 3 × 10(8) cells) and feeding the cells to mosquito larvae kill 75 and 60% of Ae. aegypti larvae in 4 days, respectively. Southern and Northern blots analyses show that tmfA integrated into the genome of C. desiccata by homologous recombination using the yeast 2 μ circle of replication and the nit in pYES2/TMOF and pYDB4-tmfA, respectively, and the transformed algal cells express tmfA transcript. Using these algal cells it will be possible in the future to control mosquito larvae in the marsh. © 2015 Wiley Periodicals, Inc.

Change of digestive physiology in sea cucumber Apostichopus japonicus (Selenka) induced by corn kernels meal and soybean meal in diets

NASA Astrophysics Data System (ADS)

Yu, Haibo; Gao, Qinfeng; Dong, Shuanglin; Hou, Yiran; Wen, Bin

2016-08-01

The present study was conducted to determine the change of digestive physiology in sea cucumber Apostichopus japonicus (Selenka) induced by corn kernels meal and soybean meal in diets. Four experimental diets were tested, in which Sargassum thunbergii was proportionally replaced by the mixture of corn kernels meal and soybean meal. The growth performance, body composition and intestinal digestive enzyme activities in A. japonicus fed these 4 diets were examined. Results showed that the sea cucumber exhibited the maximum growth rate when 20% of S. thunbergii in the diet was replaced by corn kernels meal and soybean meal, while 40% of S. thunbergii in the diet can be replaced by the mixture of corn kernels meal and soybean meal without adversely affecting growth performance of A. japonicus. The activities of intestinal trypsin and amylase in A. japonicus can be significantly altered by corn kernels meal and soybean meal in diets. Trypsin activity in the intestine of A. japonicus significantly increased in the treatment groups compared to the control, suggesting that the supplement of corn kernels meal and soybean meal in the diets might increase the intestinal trypsin activity of A. japonicus. However, amylase activity in the intestine of A. japonicus remarkably decreased with the increasing replacement level of S. thunbergii by the mixture of corn kernels meal and soybean meal, suggesting that supplement of corn kernels meal and soybean meal in the diets might decrease the intestinal amylase activity of A. japonicus.

Effects of processing and in vitro proteolytic digestion on soybean and yambean hemagglutinins.

PubMed

Ojimelukwe, P C; Onuoha, C C; Obanu, Z A

1995-06-01

Some conventional processing methods were applied on yambean and soybean seeds and flour samples. They include soaking fermentation, cooking whole seeds in the presence and absence of trona, autoclaving and dry heat treatment of flour samples. Hemagglutinating activity was assayed for after processing treatments. The hemagglutinating proteins from these seeds were classified based on their solubility properties. Effects of the presence of 0.01% concentration of trypsin, pepsin and proteases on agglutination of human red blood cells were also evaluated. Most processing methods, particularly cooking whole seeds for 1-2 h, soaking and fermentation, reduced hemagglutinating activity on cow red blood cells. Size reduction accompanied by heat treatment was effective in eliminating hemagglutination. Both the albumin and globulin fractions of the soybean showed hemagglutinating activity but only the albumin fraction of the yambean had agglutinating properties. Proteolytic action of proteases was more effective in reduction of hemagglutinating activity than that of trypsin and pepsin.

Tryptic peptides of canine thyroglobulin reactive with sera of patients with canine hypothyroidism caused by autoimmune thyroiditis.

PubMed

Lee, J-Y; Uzuka, Y; Tanabe, S; Takasawa, T; Sarashina, T; Nachreiner, R F

2004-10-01

Canine thyroglobulin (cTg) was treated with trypsin at a ratio of trypsin to cTg of 1:100 (w/w). Tryptic peptides of cTg were analysed by Western immunoblotting for their reactivity to serum thyroglobulin autoantibodies (TgAA) from patients with TgAA-positive hypothyroidism and normal individuals. The sera of patients with TgAA-positive hypothyroidism reacted with several peptides: 43, 32.5 and 31 kDa; the sera of normal individuals did not bind these tryptic peptides. Some of the TgAA-positive sera of patients reacted with 25 kDa peptide in addition to three tryptic peptides above. This experiment was the first report about antigenic epitopes of cTg. These small tryptic peptides recognized by TgAA may be related with the induction of TgAA and may be useful as markers for autoimmune thyroid diseases in dog.

The Protective Effects of Trypsin Inhibitor on Hepatic Ischemia-Reperfusion Injury and Liver Graft Survival

PubMed Central

Guan, Lianyue; Liu, Hongyu; Fu, Peiyao; Li, Zhuonan; Li, Peidong; Xie, Lijuan; Xin, Mingang; Wang, Zhanpeng

2016-01-01

The aim of this study was to explore the protective effects of ulinastatin (urinary trypsin inhibitor, UTI) on liver ischemia-reperfusion injury (IRI) and graft survival. We employed mouse liver cold IRI and orthotopic liver transplantation (OLTx) models. UTI was added to lactated Ringer's (LR) solution for liver perfusion and preservation in vitro or combined with UTI injection intraperitoneally to the liver graft recipient. Our results indicated that UTI supplementation protected the liver from cold IRI in a dose-dependent manner and prolonged liver graft survival from extended cold preserved liver donors significantly. The underlying mechanism of UTI on liver IRI may be mediated by inhibition of proinflammatory cytokine release, increasing the expression of the antiapoptotic gene Bcl-2 and decreasing the expression of the proapoptosis genes of Caspase-3 and Bax, and further protects hepatocytes from apoptotic death and improves liver function. PMID:26783413

Activity of trypsin-like enzymes and gelatinases in rats with doxorubicin cardiomyopathy.

PubMed

Gordiienko, Iu A; Babets, Ya V; Kulinich, A O; Shevtsova, A I; Ushakova, G O

2014-01-01

Activity of trypsin-like enzymes (ATLE) and gelatinases A and B were studied in the blood plasma and extracts from cardiac muscle, cerebral cortex and cerebellum of rats with cardiomyopathy caused by anthracycline antibiotic doxorubicin against the background of preventive application of corvitin and α-ketoglutarate. ATLE significantly increased in blood plasma and extracts from cerebral cortex but decreased in extracts from cardiac muscle and cerebellum in doxorubicin cardiomyopathy (DCMP). In addition, a significant increase of activity of both gelatinases in plasma and tissue extracts was observed. Preventive administration of corvitin and α-ketoglutarate resulted in differently directed changes of activity of the above mentioned enzymes in heart and brain tissues. Obtained data confirm the hypothesis about activation of proteolysis under the influence of anthracycline antibiotics and testify to selective effect of corvitin and α-ketoglutarate on ATLE and gelatinases.

Rational design of functional and tunable oscillating enzymatic networks

NASA Astrophysics Data System (ADS)

Semenov, Sergey N.; Wong, Albert S. Y.; van der Made, R. Martijn; Postma, Sjoerd G. J.; Groen, Joost; van Roekel, Hendrik W. H.; de Greef, Tom F. A.; Huck, Wilhelm T. S.

2015-02-01

Life is sustained by complex systems operating far from equilibrium and consisting of a multitude of enzymatic reaction networks. The operating principles of biology's regulatory networks are known, but the in vitro assembly of out-of-equilibrium enzymatic reaction networks has proved challenging, limiting the development of synthetic systems showing autonomous behaviour. Here, we present a strategy for the rational design of programmable functional reaction networks that exhibit dynamic behaviour. We demonstrate that a network built around autoactivation and delayed negative feedback of the enzyme trypsin is capable of producing sustained oscillating concentrations of active trypsin for over 65 h. Other functions, such as amplification, analog-to-digital conversion and periodic control over equilibrium systems, are obtained by linking multiple network modules in microfluidic flow reactors. The methodology developed here provides a general framework to construct dissipative, tunable and robust (bio)chemical reaction networks.

Purification of a novel myofibril-bound serine proteinase inhibitor (MBSPI) from the skeletal muscle of lizard fish.

PubMed

Cao, M J; Osatomi, K; Hara, K; Ishihara, T

2001-01-01

A novel myofibril-bound serine proteinase inhibitor (MBSPI) was purified to homogeneity from the skeletal muscle of lizard fish (Saurida wanieso). Purification was carried out by ammonium sulfate fractionation, followed by column chromatographies on DEAE-Sephacel, SP-Sepharose and Sephadex G-150. MBSPI was purified 7.7-fold starting from the DEAE-Sephacel fraction, with a yield of 0.2%. It is a monomeric protein with the molecular mass of 50 kDa as estimated by SDS-PAGE and gel filtration. MBSPI reveals high inhibition specificity toward a myofibril-bound serine proteinase (MBSP) purified from lizard fish muscle. No inhibition is detected toward bovine trypsin, bovine chymotrypsin, two trypsins from carp hepatopancreas and a serine proteinase isolated from the sarcoplasmic fraction of white croaker muscle. It does not exert any inhibitory activity toward a myofibril-bound serine proteinase from carp muscle.

The Protective Effects of Trypsin Inhibitor on Hepatic Ischemia-Reperfusion Injury and Liver Graft Survival.

PubMed

Guan, Lianyue; Liu, Hongyu; Fu, Peiyao; Li, Zhuonan; Li, Peidong; Xie, Lijuan; Xin, Mingang; Wang, Zhanpeng; Li, Wei

2016-01-01

The aim of this study was to explore the protective effects of ulinastatin (urinary trypsin inhibitor, UTI) on liver ischemia-reperfusion injury (IRI) and graft survival. We employed mouse liver cold IRI and orthotopic liver transplantation (OLTx) models. UTI was added to lactated Ringer's (LR) solution for liver perfusion and preservation in vitro or combined with UTI injection intraperitoneally to the liver graft recipient. Our results indicated that UTI supplementation protected the liver from cold IRI in a dose-dependent manner and prolonged liver graft survival from extended cold preserved liver donors significantly. The underlying mechanism of UTI on liver IRI may be mediated by inhibition of proinflammatory cytokine release, increasing the expression of the antiapoptotic gene Bcl-2 and decreasing the expression of the proapoptosis genes of Caspase-3 and Bax, and further protects hepatocytes from apoptotic death and improves liver function.

Understanding the Effects of Genotype, Growing Year, and Breeding on Tunisian Durum Wheat Allergenicity. 1. The Baker's Asthma Case.

PubMed

Boukid, Fatma; Prandi, Barbara; Sforza, Stefano; Sayar, Rhouma; Seo, Yong Weon; Mejri, Mondher; Yacoubi, Ines

2017-07-19

Baker's asthma is a serious airway disease triggered by wheat protein CM3 α-amylase/trypsin inhibitor. The purpose of the present study was to investigate the impact of genotype and crop year on allergen CM3 α-amylase/trypsin inhibitor associated with baker's asthma. A historical series of Tunisian durum wheat (100 accessions), derived from three crop years, was used to compare the amount of CM3 from landraces to advanced cultivars. CM3 protein quantification was assessed after an enzymatic cleavage of the soluble protein extracts on a UPLC/ESI-MS system, using a marker peptide for its quantification. Combined data analysis of variance revealed an important effect of genotype, crop year, and their interaction. The CM3 allergenic proteins were found to significantly vary among studied genotypes, as confirmed by genetic variability, coefficient of variance, heritability, and genetic advance.

A new family of cystine knot peptides from the seeds of Momordica cochinchinensis.

PubMed

Chan, Lai Yue; He, Wenjun; Tan, Ninghua; Zeng, Guangzhi; Craik, David J; Daly, Norelle L

2013-01-01

Momordica cochinchinensis, a Cucurbitaceae plant commonly found in Southeast Asia, has the unusual property of containing both acyclic and backbone-cyclized trypsin inhibitors with inhibitor cystine knot (ICK) motifs. In the current study we have shown that M. cochinchinensis also contains another family of acyclic ICK peptides. We recently reported two novel peptides from M. cochinchinensis but have now discovered four additional peptides (MCo-3-MCo-6) with related sequences. Together these peptides form a novel family of M. cochinchinensis ICK peptides (MCo-ICK) that do not have sequence homology with other known peptides and are not potent trypsin inhibitors. Otherwise these new peptides MCo-3 to MCo-6 were evaluated for antimalarial activity against Plasmodium falciparum, and cytotoxic activity against the cancer cell line MDA-MB-231. But these peptides were not active. Copyright © 2012 Elsevier Inc. All rights reserved.

Effects of non-starch polysaccharides enzymes on pancreatic and small intestinal digestive enzyme activities in piglet fed diets containing high amounts of barley.

PubMed

Li, Wei-Fen; Feng, Jie; Xu, Zi-Rong; Yang, Cai-Mei

2004-03-15

To investigate effects of non-starch polysaccharides(NSP) enzymes on pancreatic and small intestinal digestive enzyme activities in piglet fed diets containing high amounts of barley. Sixty crossbred piglets averaging 13.5 kg were randomly assigned to two treatment groups with three replications (pens) based on sex and mass. Each group was fed on the diet based on barley with or without added NSP enzymes (0.15%) for a 40-d period. At the end of the experiment the pigs were weighed. Three piglets of each group were chosen and slaughtered. Pancreas, digesta from the distal end of the duodenum and jejunal mucosa were collected for determination. Activities of the digestive enzymes trypsin, chymotrypsin, amylase and lipase were determined in the small intestinal sections as well as in homogenates of pancreatic tissue. Maltase, sucrase, lactase and gamma-glutamyl transpeptidase (gamma-GT) activities were analyzed in jejunal mucosa. Supplementation with NSP enzymes improved growth performance of piglets. It showed that NSP enzymes had no effect on digestive enzyme activities in pancreas, but decreased the activities of proteolytic enzyme, trypsin, amylase and lipase in duodenal contents by 57.56%, 76.08%, 69.03% and 40.22%(P<0.05) compared with control, and increased gamma-GT activities in jejunal mucosa by 118.75%(P<0.05). Supplementation with NSP enzymes in barley based diets could improve piglets' growth performance, decrease activities of proteolytic enzyme, trypsin, amylase and lipase in duodenal contents and increase gamma-GT activities in jejunal mucosa.

Biopotency of serine protease inhibitors from cowpea (Vigna unguiculata) seeds on digestive proteases and the development of Spodoptera littoralis (Boisduval).

PubMed

Abd El-latif, Ashraf Oukasha

2015-05-01

Serine protease inhibitors (PIs) have been described in many plant species and are universal throughout the plant kingdom, where trypsin inhibitors is the most common type. In the present study, trypsin and chymotrypsin inhibitory activity was detected in the seed flour extracts of 13 selected cultivars/accessions of cowpea. Two cowpea cultivars, Cream7 and Buff, were found to have higher trypsin and chymotrypsin inhibitory potential compared to other tested cultivars for which they have been selected for further purification studies using ammonium sulfate fractionation and DEAE-Sephadex A-25 column. Cream7-purified proteins showed two bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) corresponding to molecular mass of 17.10 and 14.90 kDa, while the purified protein from Buff cultivar showed a single band corresponding mass of 16.50 kDa. The purified inhibitors were stable at temperature below 60°C and were active at wide range of pH from 2 to 12. The kinetic analysis revealed noncompetitive type of inhibition for both inhibitors against both enzymes. The inhibitor constant (Ki ) values suggested high affinity between inhibitors and enzymes. Purified inhibitors were found to have deep and negative effects on the mean larval weight, larval mortality, pupation, and mean pupal weight of Spodoptera littoralis, where Buff PI was more effective than Cream7 PI. It may be concluded that cowpea PI gene(s) could be potential insect control protein for future studies in developing insect-resistant transgenic plants. © 2014 Wiley Periodicals, Inc.

BG-4, a novel anticancer peptide from bitter gourd (Momordica charantia), promotes apoptosis in human colon cancer cells.

PubMed

Dia, Vermont P; Krishnan, Hari B

2016-09-15

Momordica charantia is a perennial plant with reported health benefits. BG-4, a novel peptide from Momordica charantia, was isolated, purified and characterized. The trypsin inhibitory activity of BG-4 is 8.6 times higher than purified soybean trypsin inhibitor. The high trypsin inhibitory activity of BG-4 may be responsible for its capability to cause cytotoxicity to HCT-116 and HT-29 human colon cancer cells with ED50 values of 134.4 and 217.0 μg/mL after 48 h of treatment, respectively. The mechanism involved in the cytotoxic effect may be associated with induction of apoptosis as evidenced by increased percentage of HCT-116 and HT-29 colon cancer cells undergoing apoptosis from 5.4% (untreated) to 24.8% (BG-4 treated, 125 μg/mL for 16 h) and 8.5% (untreated) to 31.9% (BG-4 treated, 125 μg/mL for 16 h), respectively. The molecular mechanistic explanation in the apoptosis inducing property of BG-4 is due to reduced expression of Bcl-2 and increased expression of Bax leading to increased expression of caspase-3 and affecting the expression of cell cycle proteins p21 and CDK2. This is the first report on the anti-cancer potential of a novel bioactive peptide isolated from Momordica charantia in vitro supporting the potential therapeutic property of BG-4 against colon cancer that must be addressed using in vivo models of colon carcinogenesis.

BG-4, a novel anticancer peptide from bitter gourd (Momordica charantia), promotes apoptosis in human colon cancer cells

PubMed Central

Dia, Vermont P.; Krishnan, Hari B.

2016-01-01

Momordica charantia is a perennial plant with reported health benefits. BG-4, a novel peptide from Momordica charantia, was isolated, purified and characterized. The trypsin inhibitory activity of BG-4 is 8.6 times higher than purified soybean trypsin inhibitor. The high trypsin inhibitory activity of BG-4 may be responsible for its capability to cause cytotoxicity to HCT-116 and HT-29 human colon cancer cells with ED50 values of 134.4 and 217.0 μg/mL after 48 h of treatment, respectively. The mechanism involved in the cytotoxic effect may be associated with induction of apoptosis as evidenced by increased percentage of HCT-116 and HT-29 colon cancer cells undergoing apoptosis from 5.4% (untreated) to 24.8% (BG-4 treated, 125 μg/mL for 16 h) and 8.5% (untreated) to 31.9% (BG-4 treated, 125 μg/mL for 16 h), respectively. The molecular mechanistic explanation in the apoptosis inducing property of BG-4 is due to reduced expression of Bcl-2 and increased expression of Bax leading to increased expression of caspase-3 and affecting the expression of cell cycle proteins p21 and CDK2. This is the first report on the anti-cancer potential of a novel bioactive peptide isolated from Momordica charantia in vitro supporting the potential therapeutic property of BG-4 against colon cancer that must be addressed using in vivo models of colon carcinogenesis. PMID:27628414

Protective immunity against tick infestation in cattle vaccinated with recombinant trypsin inhibitor of Rhipicephalus microplus.

PubMed

Andreotti, Renato; Cunha, Rodrigo Casquero; Soares, Mariana Aparecida; Guerrero, Felix D; Leite, Fábio P Leivas; de León, Adalberto A Pérez

2012-10-19

The cattle tick, Rhipicephalus microplus, is regarded as the most economically important ectoparasite of livestock globally. Control is achieved primarily through the use of acaricides. This approach is hampered by the development of resistance to commercial acaricides among cattle tick populations. Vaccination against R. microplus infestation is another technology that can be integrated for effective cattle tick control. Proteins belonging to the Kunitz-BPTI family are abundant in cattle tick salivary glands, midgut, and ovaries. These organs are attractive targets for the development of a novel cattle tick vaccine. Efficacy assessment against cattle tick infestation in bovines using a vaccine containing the recombinant form of a member of the Kunitz family from R. microplus produced in a yeast expression system is reported for the first time here. The yeast Pichia pastoris was bioengineered to produce the recombinant version of a trypsin inhibitor that is expressed in cattle tick larvae (rRmLTI). Immunization with rRmLTI afforded 32% efficacy against R. microplus. The estimated molecular weight of rRmLTI was 46 kDa. Structural homology to the native form of the larval trypsin inhibitor was documented by recognition of rRmLTI in Western-blots using polyclonal antibodies from mice immunized with cattle tick larval extract or rRmLTI. Bioinformatics analysis of the partial nucleotide and deduced amino acid sequences indicated that the rRmLTI closely resembles BmTI-6, which is a three-headed Kunitz protein present in cattle tick ovary and fat tissue. Published by Elsevier Ltd.

In Vitro and In Vivo Evaluation of Casein as a Drug Carrier for Enzymatically Triggered Dissolution Enhancement from Solid Dispersions.

PubMed

Bani-Jaber, Ahmad; Alshawabkeh, Iyad; Abdullah, Samaa; Hamdan, Imad; Ardakani, Adel; Habash, Maha

2017-07-01

Due to its unique properties, such as biodegradability, biocompatibility, high amphiphilic property, and micelle formation, casein (CS) has been increasingly studied for drug delivery. We used CS as a drug carrier in solid dispersions (SDs) and evaluated the effect of its degradation by trypsin on drug dissolution from the dispersions. SDs of CS and mefenamic acid (MA) were prepared by physical mixing, kneading, and coprecipitation methods. In comparison to pure MA, the dispersions were evaluated for drug-protein interaction, loss of drug crystalinity, and drug morphology by differential scanning calorimetry, X-ray diffractometry, Fourier transform infrared spectroscopy, and scanning electron microscopy. Drug dissolution from the dispersions was evaluated in simulated intestinal fluid as enzyme free and trypsin-enriched media. Furthermore, in vivo drug absorption of MA from CS-MA coprecipitate was evaluated in rats, in comparison with a reference SD of polyethylene glycol and MA (PEG-MA SD). Relative to other CS preparations, CS-MA coprecipitate showed the highest loss of drug crystallinity, drug micronization, and CS-MA interaction. CS remarkably enhanced the dissolution rate and extent of MA from the physical and kneaded mixtures. However, the highest dissolution enhancement was obtained when MA was coprecipitated with CS. Trypsin that can hydrolyze CS during dissolution resulted in further enhancement of MA dissolution from the physical and kneaded mixtures. However, a corresponding retardation effect was obtained for the coprecipitate. In correlation with in vitro drug release, CS-MA coprecipitate also showed significantly higher MA bioavailability in rats than PEG-MA SD.

Application and bioactive properties of CaTI, a trypsin inhibitor from Capsicum annuum seeds: membrane permeabilization, oxidative stress and intracellular target in phytopathogenic fungi cells.

PubMed

Silva, Marciele S; Ribeiro, Suzanna Ff; Taveira, Gabriel B; Rodrigues, Rosana; Fernandes, Katia Vs; Carvalho, André O; Vasconcelos, Ilka Maria; Mello, Erica Oliveira; Gomes, Valdirene M

2017-08-01

During the last few years, a growing number of antimicrobial peptides have been isolated from plants and particularly from seeds. Recent results from our laboratory have shown the purification of a new trypsin inhibitor, named CaTI, from chilli pepper (Capsicum annuum L.) seeds. This study aims to evaluate the antifungal activity and mechanism of action of CaTI on phytopathogenic fungi and detect the presence of protease inhibitors in other species of this genus. Our results show that CaTI can inhibit the growth of the phytopathogenic fungi Colletotrichum gloeosporioides and C. lindemuthianum. CaTI can also permeabilize the membrane of all tested fungi. When testing the inhibitor on its ability to induce reactive oxygen species, an induction of reactive oxygen species (ROS) and nitric oxide (NO) particularly in Fusarium species was observed. Using CaTI coupled to fluorescein isothiocyanate (FITC), it was possible to determine the presence of the inhibitor inside the hyphae of the Fusarium oxysporum fungus. The search for protease inhibitors in other Capsicum species revealed their presence in all tested species. This paper shows the antifungal activity of protease inhibitors such as CaTI against phytopathogenic fungi. Antimicrobial peptides, among which the trypsin protease inhibitor family stands out, are present in different species of the genus Capsicum and are part of the chemical arsenal that plants use to defend themselves against pathogens. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Characterization of Clostridium perfringens TpeL Toxin Gene Carriage, Production, Cytotoxic Contributions, and Trypsin Sensitivity

PubMed Central

Chen, Jianming

2015-01-01

Large clostridial toxins (LCTs) are produced by at least four pathogenic clostridial species, and several LCTs are proven pivotal virulence factors for both human and veterinary diseases. TpeL is a recently identified LCT produced by Clostridium perfringens that has received relatively limited study. In response, the current study surveyed carriage of the tpeL gene among different C. perfringens strains, detecting this toxin gene in some type A, B, and C strains but not in any type D or E strains. This study also determined that all tested strains maximally produce, and extracellularly release, TpeL at the late-log or early-stationary growth stage during in vitro culture, which is different from the maximal late-stationary-phase production reported previously for other LCTs and for TpeL production by C. perfringens strain JIR12688. In addition, the present study found that TpeL levels in culture supernatants can be repressed by either glucose or sucrose. It was also shown that, at natural production levels, TpeL is a significant contributor to the cytotoxic activity of supernatants from cultures of tpeL-positive strain CN3685. Lastly, this study identified TpeL, which presumably is produced in the intestines during diseases caused by TpeL-positive type B and C strains, as a toxin whose cytotoxicity decreases after treatment with trypsin; this finding may have pathophysiologic relevance by suggesting that, like beta toxin, TpeL contributes to type B and C infections in hosts with decreased trypsin levels due to disease, diet, or age. PMID:25824828

Manduca sexta recognition and resistance among allopolyploid Nicotiana host plants

PubMed Central

Lou, Yonggen; Baldwin, Ian T.

2003-01-01

Allopolyploid speciation occurs instantly when the genomes of different species combine to produce self-fertile offspring and has played a central role in the evolution of higher plants, but its consequences for adaptive responses are unknown. We compare herbivore-recognition and -resistance responses of the diploid species and putative ancestral parent Nicotiana attenuata with those of the two derived allopolyploid species Nicotiana clevelandii and Nicotiana bigelovii. Manduca sexta larvae attack all three species, and in N. attenuata attack is recognized when larval oral secretions are introduced to wounds during feeding, resulting in a jasmonate burst, a systemic amplification of trypsin inhibitor accumulation, and a release of volatile organic compounds, which function as a coordinated defense response that slows caterpillar growth and increases the probability of their being attacked. Most aspects of this recognition response are retained with modifications in one allotetraploid (N. bigelovii) but lost in the other (N. clevelandii). Differences between diploid and tetraploid species were apparent in delays (maximum 1 and 0.5 h, respectively) in the jasmonate burst, the elicitation of trypsin inhibitors and release of volatile organic compounds, and the constitutive levels of nicotine, trypsin inhibitors, diterpene glycosides, rutin, and caffeoylputrescine in the leaves. Resistance to M. sexta larvae attack was most strongly associated with diterpene glycosides, which were higher in the diploid than in the two allotetraploid species. Because M. sexta elicitors differentially regulate a large proportion of the N. attenuata transcriptome, we propose that these species are suited for the study of the evolution of adaptive responses requiring trans-activation mechanisms. PMID:14530394

Phenotypic flexibility of digestive system in Atlantic cod (Gadus morhua).

PubMed

Blier, P U; Dutil, J-D; Lemieux, H; Bélanger, F; Bitetera, L

2007-02-01

This study examined the restoration of the digestive capacity of Atlantic cod (Gadus morhua Linnaeus) following a long period of food deprivation. Fifty cod (48 cm, 1 kg) were food-deprived for 68 days and then fed in excess with capelin (Mallotus villosus Müller) on alternate days. Ten fish were sampled after 0, 2, 6, 14 and 28 days and the mass of the pyloric caeca, intestine and carcass determined. Two metabolic enzymes (cytochrome c oxidase and citrate synthase) were assayed in white muscle, pyloric caeca and intestine, and trypsin activity was measured in the pyloric caeca. A delay of 14 days was required before body mass started to increase markedly, whereas most of the increase in mass of both the pyloric caeca and intestine relative to fish length occurred earlier in the experiment. By day 14, the activities of trypsin and citrate synthase in the pyloric caeca as well as citrate synthase in the intestine had reached maxima. The growth of the digestive tissues and restoration of their metabolic capacities thus occur early upon refeeding and are likely required for recovery growth to take place. The phenotypic flexibility of the cod digestive system is therefore remarkable: increases in trypsin activity and size of pyloric caeca resulted in a combined 29-fold increase in digestive capacity of the fish during the refeeding period. Our study suggests that Atlantic cod are able to cope with marked fluctuations in food availability in their environment by making a rapid adjustment of their digestive capacity as soon as food availability increases.

Bowman-Birk proteinase inhibitor from Cajanus cajan seeds: purification, characterization, and insecticidal properties.

PubMed

Prasad, Elaprolu R; Merzendorfer, H; Madhurarekha, C; Dutta-Gupta, A; Padmasree, K

2010-03-10

A red gram proteinase inhibitor (RgPI) was purified from red gram ( Cajanus cajan ) seeds by using ammonium sulfate precipitation and ion-exchange, affinity, and gel filtration chromatography. SDS-PAGE under nonreducing condition revealed two protein bands with molecular masses of approximately 8.5 and approximately 16.5 kDa corresponding to monomeric and dimeric forms of RgPI, respectively. Similarly, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry also confirmed the presence of dimer as well as other oligomeric forms: trimer, tetramer, and pentamer. Reduction of RgPI with dithiothreitol (DTT) led to the dissociation of the dimeric and oligomeric forms. Native-PAGE and two-dimensional gel electrophoresis indicated the existence of isoinhibitors with pI values of 5.95, 6.25, 6.50, 6.90, and 7.15, respectively. The MALDI-TOF-TOF mass spectrum and N-terminal sequence 'DQHHSSKACC' suggested that the isolated RgPI is a member of the Bowman-Birk inhibitor family. RgPI exhibited noncompetitive type inhibitory activity against bovine pancreatic trypsin and chymotrypsin, with inhibition constants of 292 and 2265 nM, respectively. It was stable up to a temperature of 80 degrees C and was active over a wide pH range between 2 and 12. However, reduction with DTT or 2-mercaptoethanol resulted in loss of inhibitory activity against trypsin and chymotrypsin. It also decreased the activity of larval midgut trypsin-like proteinases in Manduca sexta . Its insecticidal property was further confirmed by reduction in the growth and development of these larvae, when supplemented in the diet.

Temporal synthesis of proteins and RNAs during human astrovirus infection of cultured cells.

PubMed Central

Monroe, S S; Stine, S E; Gorelkin, L; Herrmann, J E; Blacklow, N R; Glass, R I

1991-01-01

Astroviruses are nonenveloped particles with a distinctive star-shaped surface structure that have been detected by electron microscopy in stool samples from humans and animals with gastroenteritis. We examined the patterns of macromolecular synthesis in astrovirus-infected cells with a goal of establishing a molecular basis for taxonomic classification. Trypsin is required for continuous replication of astrovirus in cultured cells; however, during a single cycle of infection, astrovirus antigen was synthesized earlier and at higher levels when serum, rather than trypsin, was included in the growth medium. This enhanced production of antigen, as measured by enzyme immunoassay, was accompanied by the appearance of aggregates of virus particles in the cytoplasm of infected cells. During astrovirus replication in cells cultured in the presence of serum, we detected a single infection-specific protein (90 kDa) beginning at 12 h postinfection. This protein was recognized by antiastrovirus rabbit serum and was sensitive to trypsin digestion in vitro, with the concomitant appearance of three smaller immunoreactive proteins (31, 29, and 20 kDa). We also detected two dactinomycin-resistant RNAs (7.2 and 2.8 kb), both of which were polyadenylated, in the cytoplasm of astrovirus-infected cells. The larger of these two RNAs is presumably the viral genome, whereas the smaller species may be a subgenomic messenger. Comparison of the proteins and RNAs synthesized in astrovirus-infected cells with those of the recognized families of nonenveloped single-stranded RNA animal viruses suggests that astroviruses should not be classified as members of either Caliciviridae or Picornaviridae. Images PMID:1987373

Opposite Electron-Transfer Dissociation and Higher-Energy Collisional Dissociation Fragmentation Characteristics of Proteolytic K/R(X)n and (X)nK/R Peptides Provide Benefits for Peptide Sequencing in Proteomics and Phosphoproteomics.

PubMed

Tsiatsiani, Liana; Giansanti, Piero; Scheltema, Richard A; van den Toorn, Henk; Overall, Christopher M; Altelaar, A F Maarten; Heck, Albert J R

2017-02-03

A key step in shotgun proteomics is the digestion of proteins into peptides amenable for mass spectrometry. Tryptic peptides can be readily sequenced and identified by collision-induced dissociation (CID) or higher-energy collisional dissociation (HCD) because the fragmentation rules are well-understood. Here, we investigate LysargiNase, a perfect trypsin mirror protease, because it cleaves equally specific at arginine and lysine residues, albeit at the N-terminal end. LysargiNase peptides are therefore practically tryptic-like in length and sequence except that following ESI, the two protons are now both positioned at the N-terminus. Here, we compare side-by-side the chromatographic separation properties, gas-phase fragmentation characteristics, and (phospho)proteome sequence coverage of tryptic (i.e., (X) n K/R) and LysargiNase (i.e., K/R(X) n ) peptides using primarily electron-transfer dissociation (ETD) and, for comparison, HCD. We find that tryptic and LysargiNase peptides fragment nearly as mirror images. For LysargiNase predominantly N-terminal peptide ions (c-ions (ETD) and b-ions (HCD)) are formed, whereas for trypsin, C-terminal fragment ions dominate (z-ions (ETD) and y-ions (HCD)) in a homologous mixture of complementary ions. Especially during ETD, LysargiNase peptides fragment into low-complexity but information-rich sequence ladders. Trypsin and LysargiNase chart distinct parts of the proteome, and therefore, the combined use of these enzymes will benefit a more in-depth and reliable analysis of (phospho)proteomes.

Anti-fibrinolytic and anti-microbial activities of a serine protease inhibitor from honeybee (Apis cerana) venom.

PubMed

Yang, Jie; Lee, Kwang Sik; Kim, Bo Yeon; Choi, Yong Soo; Yoon, Hyung Joo; Jia, Jingming; Jin, Byung Rae

2017-10-01

Bee venom contains a variety of peptide constituents, including low-molecular-weight protease inhibitors. While the putative low-molecular-weight serine protease inhibitor Api m 6 containing a trypsin inhibitor-like cysteine-rich domain was identified from honeybee (Apis mellifera) venom, no anti-fibrinolytic or anti-microbial roles for this inhibitor have been elucidated. In this study, we identified an Asiatic honeybee (A. cerana) venom serine protease inhibitor (AcVSPI) that was shown to act as a microbial serine protease inhibitor and plasmin inhibitor. AcVSPI was found to consist of a trypsin inhibitor-like domain that displays ten cysteine residues. Interestingly, the AcVSPI peptide sequence exhibited high similarity to the putative low-molecular-weight serine protease inhibitor Api m 6, which suggests that AcVSPI is an allergen Api m 6-like peptide. Recombinant AcVSPI was expressed in baculovirus-infected insect cells, and it demonstrated inhibitory activity against trypsin, but not chymotrypsin. Additionally, AcVSPI has inhibitory effects against plasmin and microbial serine proteases; however, it does not have any detectable inhibitory effects on thrombin or elastase. Consistent with these inhibitory effects, AcVSPI inhibited the plasmin-mediated degradation of fibrin to fibrin degradation products. AcVSPI also bound to bacterial and fungal surfaces and exhibited anti-microbial activity against fungi as well as gram-positive and gram-negative bacteria. These findings demonstrate the anti-fibrinolytic and anti-microbial roles of AcVSPI as a serine protease inhibitor. Copyright © 2017 Elsevier Inc. All rights reserved.

Analysis of Glucose Transporter Topology and Structural Dynamics*

PubMed Central

Blodgett, David M.; Graybill, Christopher; Carruthers, Anthony

2008-01-01

Homology modeling and scanning cysteine mutagenesis studies suggest that the human glucose transport protein GLUT1 and its distant bacterial homologs LacY and GlpT share similar structures. We tested this hypothesis by mapping the accessibility of purified, reconstituted human erythrocyte GLUT1 to aqueous probes. GLUT1 contains 35 potential tryptic cleavage sites. Fourteen of 16 lysine residues and 18 of 19 arginine residues were accessible to trypsin. GLUT1 lysine residues were modified by isothiocyanates and N-hydroxysuccinimide (NHS) esters in a substrate-dependent manner. Twelve lysine residues were accessible to sulfo-NHS-LC-biotin. GLUT1 trypsinization released full-length transmembrane helix 1, cytoplasmic loop 6–7, and the long cytoplasmic C terminus from membranes. Trypsin-digested GLUT1 retained cytochalasin B and d-glucose binding capacity and released full-length transmembrane helix 8 upon cytochalasin B (but not d-glucose) binding. Transmembrane helix 8 release did not abrogate cytochalasin B binding. GLUT1 was extensively proteolyzed by α-chymotrypsin, which cuts putative pore-forming amphipathic α-helices 1, 2, 4, 7, 8, 10, and 11 at multiple sites to release transmembrane peptide fragments into the aqueous solvent. Putative scaffolding membrane helices 3, 6, 9, and 12 are strongly hydrophobic, resistant to α-chymotrypsin, and retained by the membrane bilayer. These observations provide experimental support for the proposed GLUT1 architecture; indicate that the proposed topology of membrane helices 5, 6, and 12 requires adjustment; and suggest that the metastable conformations of transmembrane helices 1 and 8 within the GLUT1 scaffold destabilize a sugar translocation intermediate. PMID:18981181

21 CFR 524.2620 - Liquid crystalline trypsin, Peru balsam, castor oil.

Code of Federal Regulations, 2014 CFR

2014-04-01

... paragraph (a)(2). (c) Conditions of use—(1) Amount. Apply directly to the wound site. (2) Indications for use. As an aid in the treatment of external wounds and assists healing by facilitating the removal of...

Multiple Diphtheria Antigen-Antibody Systems Investigated by Passive Haemagglutination Techniques and Other Methods

PubMed Central

Fulthorpe, A. J.

1962-01-01

A fair degree of correlation has been found between the in vivo antitoxin content of sera from horses immunized with crude Corynebacterium diphtheriae culture filtrates and the direct agglutinin titre of the sera when tested with sheep cells sensitized with diphtheria toxoid. Haemagglutination inhibition tests at the LA level of test with the same sera showed some rather large discrepancies from the in vivo and further tests with special agglutinin inhibiting toxins suggested that specific antitoxin free from other accessory antibodies might be non-agglutinating, and therefore not titratable by haemagglutination inhibition. The phosphate-stable, pepsin-stable and trypsin-stable antigens isolated from culture filtrates of C. diphtheriae were found to contain extremely small quantities of specific toxoid, and cross titration of each of the three antigen preparations showed that there was very little contamination by other antigens within the group. Absorption of diphtheria antiserum with red cells sensitized with each of the three accessory antigens individually, showed that the antibodies were highly specific and distinct. Absorption of diphtheria antiserum with a mixture of red cells sensitized with the three different antigens removed all demonstrable accessory antibodies, and the absorbed serum would no longer agglutinate cells sensitized with complete diphtheria toxoid. The absorbed serum, however, retained a large proportion of its neutralizing capacity for diphtheria toxin, when titrated in vivo. Titration of each of the accessory antibodies in a number of horse sera by haemagglutination inhibition demonstrated a correlation between the values for the accessory antibodies to the phosphate-stable and pepsin-stable antigens, but no correlation with the values for the antibody to the trypsin-stable antigen, when compared with results of the flocculation test. The relative proportions of diphtheria toxin and of the phosphate-stable and pepsin-stable antigens remained constant at all stages of purification of a filtrate including several recrystallization procedures. However, the concentration of the trypsin-stable antigen declined steadily during the process. The relative proportions of toxin, phosphate-stable and pepsin-stable antigens in several crude culture filtrates were constant under reasonable conditions of storage and very varied where deterioration had occurred. These three antigens also maintained a constant ratio in growing culture, the trypsin-stable antigen did not. PMID:13895880

Measuring peptide translocation into large unilamellar vesicles.

PubMed

Spinella, Sara A; Nelson, Rachel B; Elmore, Donald E

2012-01-27

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors(1). Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors(1-3). Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms(4,5), particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes(6,7). In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides(1,8). A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs)(9-13). LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies(14,15). Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II(16,17). In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties(18), to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell-penetrating peptides are cleaved as they encounter uninhibited trypsin encapsulated with the LUVs, leading to disassociation from the LUV membrane and a drop in FRET signal. The drop in FRET signal observed for a translocating peptide is significantly greater than that observed for the same peptide when the LUVs contain both trypsin and trypsin inhibitor, or when a peptide that does not spontaneously cross lipid membranes is exposed to trypsin-containing LUVs. This change in fluorescence provides a direct quantification of peptide translocation over time.

Studies on Proteinases from Some Blood-Sucking Insects,

DTIC Science & Technology

ovinus, and Pediculus humanus, but not in those of Cimex lectularius or Rhodnius prolixus. The trypsin and chymotrypsin have been partially... Cimex and Rhodnius appear to have a high molecular weight proteinase with optimal activity at pH 5 in their midguts. (Author)

Digestive enzymes of the Californian two-spot octopus, Octopus bimaculoides (Pickford and McConnaughey, 1949).

PubMed

Ibarra-García, Laura Elizabeth; Tovar-Ramírez, Dariel; Rosas, Carlos; Campa-Córdova, Ángel Isidro; Mazón-Suástegui, José Manuel

2018-01-01

Octopus bimaculoides is an important commercially fished species in the California Peninsula with aquaculture potential; however, to date limited information is available regarding its digestive physiology. The objective of this study was focused on biochemically characterizing the main enzymes involved in the digestion of O. bimaculoides. Optimum pH, temperature and thermostability were determined for amylases, lipases, trypsin and chymotrypsin; optimum pH and protease inhibitor effect were assessed for acidic and alkaline proteases, and the effect of divalent ions on trypsin and chymotrypsin activity was evaluated in enzymatic extracts from the digestive (DG) and salivary glands (SG) and crop gastric juices (GJ). High amylase activity was detected in GD and GJ whereas this activity is very low in other cephalopods. Salivary glands had the greatest activity in most of the enzyme groups, showing the importance of this organ in digestion. Optimum pH was different depending on the organ and enzyme analyzed. The optimum pH in DG was 3 showing the predominance of acidic proteases in the digestion process. All enzymes were resistant and stable at high temperatures in contrast with other marine species. Trypsin and chymotrypsin activity were highly incremented with the presence of Mg 2+ , Co 2+ , Cu 2+ and Zn 2+ in some tissues. The inhibitor assay showed the importance of serine proteases, metalloproteases and aspartic proteases in the digestive process of this species. This study is the first in assessing the main digestive enzymes of O. bimaculoides and in remarking the importance of other digestive enzyme groups besides proteases in octopuses. Copyright © 2017 Elsevier Inc. All rights reserved.

Occupational Exposure to Bioaerosols in Norwegian Crab Processing Plants.

PubMed

Thomassen, Marte R; Kamath, Sandip D; Lopata, Andreas L; Madsen, Anne Mette; Eduard, Wijnand; Bang, Berit E; Aasmoe, Lisbeth

2016-08-01

Aerosolization of components when processing king crab (Paralithodes camtschaticus) and edible crab (Cancer pagurus) may cause occupational health problems when inhaled by workers. A cross-sectional study was carried out in three king crab plants and one edible crab plant. Personal exposure measurements were performed throughout work shifts. Air was collected for measurement of tropomyosin, total protein, endotoxin, trypsin, and N-acetyl-β-d-glucosaminidase (NAGase). T-tests and ANOVAs were used to compare the levels of exposure in the different plants and areas in the plants. Total protein and tropomyosin levels were highest in the edible crab plant, endotoxin levels were highest in king crab plants. King crab exposure levels were highest during raw processing. Tropomyosin levels were highest during raw king crab processing with geometric mean (GM) 9.6 versus 2.5ng m(-3) during cooked processing. Conversely, edible crab tropomyosin levels were highest during cooked processing with GM 45.4 versus 8.7ng m(-3) during raw processing. Endotoxin levels were higher in king crab plants than in the edible crab plant with GM = 6285.5 endotoxin units (EU) m(-3) versus 72 EU m(-3). In the edible crab plant, NAGase levels were highest during raw processing with GM = 853 pmol4-methylumbelliferone (MU) m(-3) versus 422 pmol4-MU m(-3) during cooked processing. Trypsin activity was found in both king crab and edible crab plants and levels were higher in raw than cooked processing. Differences in exposure levels between plants and worker groups (raw and cooked processing) were identified. Norwegian crab processing workers are exposed to airborne proteins, tropomyosin, endotoxins, trypsin, and NAGase in their breathing zone. Levels vary between worker groups and factories. © The Author 2016. Published by Oxford University Press on behalf of the British Occupational Hygiene Society.

Corn Silk Extract and Its Bioactive Peptide Ameliorated Lipopolysaccharide-Induced Inflammation in Mice via the Nuclear Factor-κB Signaling Pathway.

PubMed

Ho, Tin-Yun; Li, Chia-Cheng; Lo, Hsin-Yi; Chen, Feng-Yuan; Hsiang, Chien-Yun

2017-02-01

Bioactive peptides derived from foods have shown beneficial anti-inflammatory potential. Inhibitory κB kinase-β (IKKβ) plays a crucial role in the activation of nuclear factor-κB (NF-κB), a transcription factor involved in inflammation. Here we applied proteomic and bioinformatics approaches to identify anti-inflammatory peptides that target IKKβ from corn silk. Corn silk extract significantly suppressed lipopolysaccharide (LPS)-induced NF-κB activities [(1.7 ± 0.2)-fold vs (3.0 ± 0.6)-fold, p < 0.05] in cells. Trypsin hydrolysate of corn silk also suppressed LPS-induced NF-κB activities [(1.1 ± 0.3)-fold vs 3.3 ± 0.5 fold, p < 0.01]. In addition, both corn silk extract and trypsin hydrolysate significantly inhibited LPS-induced interleukin-1β (IL-1β) production by 58.3 ± 4.5 and 55.1 ± 7.4%, respectively. A novel peptide, FK2, docked into the ATP-binding pocket of IKKβ, was further identified from trypsin hydrolysis of corn silk. FK2 inhibited IKKβ activities, IκB phosphorylation, and subsequent NF-κB activation [(2.3 ± 0.4)-fold vs (5.5 ± 0.4)-fold, p < 0.001]. Moreover, FK2 significantly reduced NF-κB-driven luminescent signals in organs by 5-11-fold and suppressed LPS-induced NF-κB activities and IL-β production in tissues. In conclusion, our findings indicated that corn silk displayed anti-inflammatory abilities. In addition, we first identified an anti-inflammatory peptide FK2 from corn silk. Moreover, the anti-inflammatory effect of FK2 might be through IKKβ-NF-κB signaling pathways.

Depth-Dependent Glycosaminoglycan Concentration in Articular Cartilage by Quantitative Contrast-Enhanced Micro–Computed Tomography

PubMed Central

Mittelstaedt, Daniel

2015-01-01

Objective A quantitative contrast-enhanced micro–computed tomography (qCECT) method was developed to investigate the depth dependency and heterogeneity of the glycosaminoglycan (GAG) concentration of ex vivo cartilage equilibrated with an anionic radiographic contrast agent, Hexabrix. Design Full-thickness fresh native (n = 19 in 3 subgroups) and trypsin-degraded (n = 6) articular cartilage blocks were imaged using micro–computed tomography (μCT) at high resolution (13.4 μm3) before and after equilibration with various Hexabrix bathing concentrations. The GAG concentration was calculated depth-dependently based on Gibbs-Donnan equilibrium theory. Analysis of variance with Tukey’s post hoc was used to test for statistical significance (P < 0.05) for effect of Hexabrix bathing concentration, and for differences in bulk and zonal GAG concentrations individually and compared between native and trypsin-degraded cartilage. Results The bulk GAG concentration was calculated to be 74.44 ± 6.09 and 11.99 ± 4.24 mg/mL for native and degraded cartilage, respectively. A statistical difference was demonstrated for bulk and zonal GAG between native and degraded cartilage (P < 0.032). A statistical difference was not demonstrated for bulk GAG when comparing Hexabrix bathing concentrations (P > 0.3214) for neither native nor degraded cartilage. Depth-dependent GAG analysis of native cartilage revealed a statistical difference only in the radial zone between 30% and 50% Hexabrix bathing concentrations. Conclusions This nondestructive qCECT methodology calculated the depth-dependent GAG concentration for both native and trypsin-degraded cartilage at high spatial resolution. qCECT allows for more detailed understanding of the topography and depth dependency, which could help diagnose health, degradation, and repair of native and contrived cartilage. PMID:26425259

PAR-2 activation enhances weak acid-induced ATP release through TRPV1 and ASIC sensitization in human esophageal epithelial cells.

PubMed

Wu, Liping; Oshima, Tadayuki; Shan, Jing; Sei, Hiroo; Tomita, Toshihiko; Ohda, Yoshio; Fukui, Hirokazu; Watari, Jiro; Miwa, Hiroto

2015-10-15

Esophageal visceral hypersensitivity has been proposed to be the pathogenesis of heartburn sensation in nonerosive reflux disease. Protease-activated receptor-2 (PAR-2) is expressed in human esophageal epithelial cells and is believed to play a role in inflammation and sensation. PAR-2 activation may modulate these responses through adenosine triphosphate (ATP) release, which is involved in transduction of sensation and pain. The transient receptor potential vanilloid receptor 1 (TRPV1) and acid-sensing ion channels (ASICs) are both acid-sensitive nociceptors. However, the interaction among these molecules and the mechanisms of heartburn sensation are still not clear. We therefore examined whether ATP release in human esophageal epithelial cells in response to acid is modulated by TRPV1 and ASICs and whether PAR-2 activation influences the sensitivity of TRPV1 and ASICs. Weak acid (pH 5) stimulated the release of ATP from primary human esophageal epithelial cells (HEECs). This effect was significantly reduced after pretreatment with 5-iodoresiniferatoxin (IRTX), a TRPV1-specific antagonist, or with amiloride, a nonselective ASIC blocker. TRPV1 and ASIC3 small interfering RNA (siRNA) transfection also decreased weak acid-induced ATP release. Pretreatment of HEECs with trypsin, tryptase, or a PAR-2 agonist enhanced weak acid-induced ATP release. Trypsin treatment led to the phosphorylation of TRPV1. Acid-induced ATP release enhancement by trypsin was partially blocked by IRTX, amiloride, or a PAR-2 antagonist. Conversely, acid-induced ATP release was augmented by PAR-2 activation through TRPV1 and ASICs. These findings suggested that the pathophysiology of heartburn sensation or esophageal hypersensitivity may be associated with the activation of PAR-2, TRPV1, and ASICs. Copyright © 2015 the American Physiological Society.

Digestive proteinases of red shrimp Pleoticus muelleri (Decapoda, Penaeoidea): partial characterization and relationship with molting.

PubMed

Fernández Gimenez, A V; García-Carreño, F L; Navarrete del Toro, M A; Fenucci, J L

2001-10-01

The present study describes the activity and some characteristics of proteinases in the hepatopancreas of red shrimp Pleoticus muelleri during the different stages of the molting cycle. Proteolytic activity was highest between pH 7.5 and 8. The hepatopancreatic protein content in the premolt stage was higher than in the other stages of the molting cycle (P<0.05). No significant differences were found in total proteolytic activity in the hepatopancreas when comparing molting stages. The proteolytic activity of the P. muelleri hepatopancreas enzyme preparations is the main responsibility of serine proteinases. TLCK, a trypsin inhibitor, reduced azocasein hydrolysis between 26% (intermolt) and 37% (premolt). TPCK, a chymotrypsin inhibitor, did not decrease hydrolytic activity, except for in postmolt. Low trypsin and chymotrypsin activities were found during intermolt, and increased in postmolt. The electrophoretogram of the enzyme extracts shows 12 bands of activity during intermolt (from 16.6 to 53.1 kDa). Some fractions were not detected in the postmolt and premolt stages. Three low molecular weight trypsin forms (17.4, 19.1 and 20 kDa) were found in all molting stages. One band of chymotrypsin (21.9 kDa) was observed in all molting stages. High molecular mass active bands (66-205 kDa) could not be characterized with inhibitors. Comparison of the protease-specific activity of the hepatopancreas of some species indicated a relationship between digestive enzyme activity and feeding habits of the shrimp. Omnivorous shrimp, such as Penaeus vannamei (syn: Litopenaeus vannamei) and Penaeus monodon, showed higher protease activity than the carnivorous shrimp, Penaeus californiensis (syn: Farfantepenaeus californiensis) and P. muelleri. In fact, the enzymatic activity in the hepatopancreas of P. muelleri showed variations in relation to feeding habit and molting cycle.

Detoxification and anti-nutrients reduction of Jatropha curcas seed cake by Bacillus fermentation.

PubMed

Phengnuam, Thanyarat; Suntornsuk, Worapot

2013-02-01

Jatropha curcas seed cake is a by-product generated from oil extraction of J. curcas seed. Although it contains a high amount of protein, it has phorbol esters and anti-nutritional factors such as phytate, trypsin inhibitor, lectin and saponin. It cannot be applied directly in the food or animal feed industries. This investigation was aimed at detoxifying the toxic and anti-nutritional compounds in J. curcas seed cake by fermentation with Bacillus spp. Two GRAS (generally recognized as safe) Bacillus strains used in the study were Bacillus subtilis and Bacillus licheniformis with solid-state and submerged fermentations. Solid-state fermentation was done on 10 g of seed cake with a moisture content of 70% for 7 days, while submerged fermentation was carried out on 10 g of seed cake in 100 ml distilled water for 5 days. The fermentations were incubated at the optimum condition of each strain. After fermentation, bacterial growth, pH, toxic and anti-nutritional compounds were determined. Results showed that B. licheniformis with submerged fermentation were the most effective method to degrade toxic and anti-nutritional compounds in the seed cake. After fermentation, phorbol esters, phytate and trypsin inhibitor were reduced by 62%, 42% and 75%, respectively, while lectin could not be eliminated. The reduction of phorbol esters, phytate and trypsin inhibitor was related to esterase, phytase and protease activities, respectively. J. curcas seed cake could be mainly detoxified by bacterial fermentation and the high-protein fermented seed cake could be potentially applied to animal feed. Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Proteolytic enzymes in seawater: contribution of prokaryotes and protists

NASA Astrophysics Data System (ADS)

Obayashi, Y.; Suzuki, S.

2016-02-01

Proteolytic enzyme is one of the major catalysts of microbial processing of organic matter in biogeochemical cycle. Here we summarize some of our studies about proteases in seawater, including 1) distribution of protease activities in coastal and oceanic seawater, 2) responses of microbial community and protease activities in seawater to organic matter amending, and 3) possible contribution of heterotrophic protists besides prokaryotes to proteases in seawater, to clarify cleared facts and remaining questions. Activities of aminopeptidases, trypsin-type and chymotrypsin-type proteases were detected from both coastal and oceanic seawater by using MCA-substrate assay. Significant activities were detected from not only particulate (cell-associated) fraction but also dissolved fraction of seawater, especially for trypsin-type and chymotrypsin-type proteases. Hydrolytic enzymes in seawater have been commonly thought to be mainly derived from heterotrophic prokaryotes; however, it was difficult to determine actual source organisms of dissolved enzymes in natural seawater. Our experiment with addition of dissolved protein to subtropical oligotrophic Pacific water showed drastically enhancement of the protease activities especially aminopeptidases in seawater, and the prokaryotic community structure simultaneously changed to be dominant of Bacteroidetes, indicating that heterotrophic bacteria were actually one of the sources of proteases in seawater. Another microcosm experiment with free-living marine heterotrophic ciliate Paranophrys marina together with an associated bacterium showed that extracellular trypsin-type activity was mainly attributed to the ciliate. The protist seemed to work in organic matter digestion in addition to be a grazer. From the results, we propose a system of organic matter digestion by prokaryotes and protists in aquatic environments, although their actual contribution in natural environments should be estimated in future studies.

High Hydrostatic Pressure-Assisted Enzymatic Treatment Improves Antioxidant and Anti-inflammatory Properties of Phosvitin.

PubMed

Yoo, Heejoo; Bamdad, Fatemeh; Gujral, Naiyana; Suh, Joo-Won; Sunwoo, Hoon

2017-01-01

Phosvitin (PV) is a highly-phosphorylated metal-binding protein in egg yolk. Phosphoserine clusters make PV resistant to enzymatic digestion, which might be nutritionally undesirable. This study was designed to determine the effects of high hydrostatic pressure and enzymatic hydrolysis (HHP-EH) on the antioxidant and anti-inflammatory properties of PV hydrolysates (PVHs). PV was hydrolyzed by alcalase, elastase, savinase, thermolysin, and trypsin at 0.1, 50, and 100 MPa pressure levels. PVHs were evaluated for degree of hydrolysis, molecular weight distribution patterns, antioxidant and anti-inflammatory properties in chemical and cellular models. The effect of PVH on gene expression of pro-inflammatory cytokines (TNF-α and IL-1β) was also evaluated using real time-PCR. The hydrolysate with most potent antioxidant and anti-inflammatory properties was subjected to LC-MS/MS analysis to identify the peptide sequence. Hydrolysates produced at 100 MPa exhibited higher degree of hydrolysis and greater reducing power and free radical scavenging activity compared to those obtained at atmospheric pressure. After adjusting the phosphate content, alcalase- and trypsin-digested PVHs showed superior iron chelation capacity (69-73%), regardless of pressure. Both alcalase- and trypsin-digested PVHs significantly inhibited nitric oxide production by RAW264.7 macrophage cells. LPS-stimulated up-regulation of proinflammatory cytokines was also suppressed by alcalase-digested PVH. The HHP-EH method could play a promising role in the production of bioactive peptides from hydrolysis-resistant proteins. HHP-assisted PVH may be useful in preparing a potential pharmaceutical with antioxidant and anti-inflammatory properties. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Integrated CZE-ESI-MS/MS system with an immobilized trypsin microreactor for online digestion and analysis of picogram amounts of RAW 264.7 cell lysate

PubMed Central

Sun, Liangliang; Zhu, Guijie; Dovichi, Norman J.

2013-01-01

A capillary zone electrophoresis (CZE) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) system was integrated with an immobilized trypsin microreactor. The system was evaluated and then applied for online digestion and analysis of picogram loadings of RAW 264.7 cell lysate. Protein samples were dissolved in a buffer containing 50% (v/v) acetonitrile (ACN), and then directly loaded into the capillary for digestion, followed by CZE separation and MS/MS identification. The organic solvent (50% (v/v) ACN) assisted the immobilized trypsin digestion and simplified the protein sample preparation protocol. Neither protein reduction nor alkylation steps were employed, which minimized sample loss and contamination. The integrated CZE-ESI-MS/MS system generated confident identification of bovine serum albumin (BSA) with 19% sequence coverage and 14 peptide IDs when 20 fmole was loaded. When only 1 fmole BSA was injected, one BSA peptide was consistently detected. For the analysis of a standard protein mixture, the integrated system produced efficient protein digestion and confident identification for proteins with different molecular weights and isoelectric points when low fmole amount was loaded for each protein. We further applied the system for triplicate analysis of a RAW 264.7 cell lysate; 2 ± 1 and 7 ± 2 protein groups were confidently identified from only 300 pg and 3 ng loadings, respectively. The 300 pg sample loading corresponds to the protein content of three RAW 264.7 cells. In addition to high sensitivity analysis, the integrated CZE-ESI-MS/MS system produces good reproducibility in terms of peptide and protein IDs, peptide migration time, and peptide intensity. PMID:23510126

Using nonlinear ac electrokinetics vortex flow to enhance catalytic activities of sol-gel encapsulated trypsin in microfluidic devices

PubMed Central

Wang, Shau-Chun; Chen, Hsiao-Ping; Lai, Yi-Wen; Chau, Lai-Kwan; Chuang, Yu-Chun; Chen, Yi-Jie

2007-01-01

A novel microstirring strategy is applied to accelerate the digestion rate of the substrate Nα-benzoyl-L-arginine-4-nitroanilide (L-BAPA) catalyzed by sol-gel encapsulated trypsin. We use an ac nonlinear electrokinetic vortex flow to stir the solution in a microfluidic reaction chamber to reduce the diffusion length between the immobilized enzyme and substrate in the solution. High-intensity nonlinear electroosmotic microvortices, with angular speeds in excess of 1 cm∕s, are generated around a small (∼1.2 mm) conductive ion exchange granule when ac electric fields (133 V∕cm) are applied across a miniature chamber smaller than 10 μl. Coupling between these microvortices and the on-and-off electrophoretic motion of the granule in low frequency (0.1 Hz) ac fields produces chaotic stream lines to stir substrate molecules sufficiently. We demonstrate that, within a 5-min digestion period, the catalytic reaction rate of immobilized trypsin increases almost 30-fold with adequate reproducibility (15%) due to sufficient stirring action through the introduction of the nonlinear electrokinetic vortices. In contrast, low-frequency ac electroosmotic flow without the granule, provides limited stirring action and increases the reaction rate approximately ninefold with barely acceptable reproducibility (30%). Dye molecules are used to characterize the increases in solute diffusivity in the reaction reservoir in which sol-gel particles are placed, with and without the presence of granule, and compared with the static case. The solute diffusivity enhancement data show respective increases of ∼30 and ∼8 times, with and without the presence of granule. These numbers are consistent with the ratios of the enhanced reaction rate. PMID:19693360

Using nonlinear ac electrokinetics vortex flow to enhance catalytic activities of sol-gel encapsulated trypsin in microfluidic devices.

PubMed

Wang, Shau-Chun; Chen, Hsiao-Ping; Lai, Yi-Wen; Chau, Lai-Kwan; Chuang, Yu-Chun; Chen, Yi-Jie

2007-09-04

A novel microstirring strategy is applied to accelerate the digestion rate of the substrate N(alpha)-benzoyl-L-arginine-4-nitroanilide (L-BAPA) catalyzed by sol-gel encapsulated trypsin. We use an ac nonlinear electrokinetic vortex flow to stir the solution in a microfluidic reaction chamber to reduce the diffusion length between the immobilized enzyme and substrate in the solution. High-intensity nonlinear electroosmotic microvortices, with angular speeds in excess of 1 cms, are generated around a small ( approximately 1.2 mm) conductive ion exchange granule when ac electric fields (133 Vcm) are applied across a miniature chamber smaller than 10 mul. Coupling between these microvortices and the on-and-off electrophoretic motion of the granule in low frequency (0.1 Hz) ac fields produces chaotic stream lines to stir substrate molecules sufficiently. We demonstrate that, within a 5-min digestion period, the catalytic reaction rate of immobilized trypsin increases almost 30-fold with adequate reproducibility (15%) due to sufficient stirring action through the introduction of the nonlinear electrokinetic vortices. In contrast, low-frequency ac electroosmotic flow without the granule, provides limited stirring action and increases the reaction rate approximately ninefold with barely acceptable reproducibility (30%). Dye molecules are used to characterize the increases in solute diffusivity in the reaction reservoir in which sol-gel particles are placed, with and without the presence of granule, and compared with the static case. The solute diffusivity enhancement data show respective increases of approximately 30 and approximately 8 times, with and without the presence of granule. These numbers are consistent with the ratios of the enhanced reaction rate.

MOLECULAR IDENTIFICATION OF CYSTEINE AND TRYPSIN PROTEASE, EFFECT OF DIFFERENT HOSTS ON PROTEASE EXPRESSION, AND RNAI MEDIATED SILENCING OF CYSTEINE PROTEASE GENE IN THE SUNN PEST.

PubMed

Amiri, Azam; Bandani, Ali Reza; Alizadeh, Houshang

2016-04-01

Sunn pest, Eurygaster integriceps, is a serious pest of cereals in the wide area of the globe from Near and Middle East to East and South Europe and North Africa. This study described for the first time, identification of E. integriceps trypsin serine protease and cathepsin-L cysteine, transcripts involved in digestion, which might serve as targets for pest control management. A total of 478 and 500 base pair long putative trypsin and cysteine gene sequences were characterized and named Tryp and Cys, respectively. In addition, the tissue-specific relative gene expression levels of these genes as well as gluten hydrolase (Gl) were determined under different host kernels feeding conditions. Result showed that mRNA expression of Cys, Tryp, and Gl was significantly affected after feeding on various host plant species. Transcript levels of these genes were most abundant in the wheat-fed E. integriceps larvae compared to other hosts. The Cys transcript was detected exclusively in the gut, whereas the Gl and Tryp transcripts were detectable in both salivary glands and gut. Also possibility of Sunn pest gene silencing was studied by topical application of cysteine double-stranded RNA (dsRNA). The results indicated that topically applied dsRNA on fifth nymphal stage can penetrate the cuticle of the insect and induce RNA interference. The Cys gene mRNA transcript in the gut was reduced to 83.8% 2 days posttreatment. Also, it was found that dsRNA of Cys gene affected fifth nymphal stage development suggesting the involvement of this protease in the insect growth, development, and molting. © 2015 Wiley Periodicals, Inc.

A carboxy-terminal fragment of protein mu 1/mu 1C is present in infectious subvirion particles of mammalian reoviruses and is proposed to have a role in penetration.

PubMed Central

Nibert, M L; Fields, B N

1992-01-01

Penetration of a cell membrane as an early event in infection of cells by mammalian reoviruses appears to require a particular type of viral particle, the infectious subvirion particle (ISVP), which is generated from an intact virion by proteolytic cleavage of the outer capsid proteins sigma 3 and mu 1/mu 1C. Characterizations of the structural components and properties of ISVPs are thus relevant to attempts to understand the mechanism of penetration by reoviruses. In this study, a novel, approximately 13-kDa carboxy-terminal fragment (given the name phi) was found to be generated from protein mu 1/mu 1C during in vitro treatments of virions with trypsin or chymotrypsin to yield ISVPs. With trypsin treatment, both the carboxy-terminal fragment phi and the amino-terminal fragment mu 1 delta/delta were shown to be generated and to remain attached to ISVPs in stoichiometric quantities. Sites of protease cleavage were identified in the deduced amino acid sequence of mu 1 by determining the amino-terminal sequences of phi proteins: trypsin cleaves between arginine 584 and isoleucine 585, and chymotrypsin cleaves between tyrosine 581 and glycine 582. Findings in this study indicate that sequences in the phi portion of mu 1/mu 1C may participate in the unique functions attributed to ISVPs. Notably, the delta-phi cleavage junction was predicted to be flanked by a pair of long amphipathic alpha-helices. These amphipathic alpha-helices, together with the myristoyl group at the extreme amino terminus of mu 1/mu 1N, are proposed to interact directly with the lipid bilayer of a cell membrane during penetration by mammalian reoviruses. Images PMID:1328674

Kunitzins: Prototypes of a new class of protease inhibitor from the skin secretions of European and Asian frogs.

PubMed

Chen, Xiaole; Wang, He; Shen, Yue; Wang, Lei; Zhou, Mei; Chen, Tianbao; Shaw, Chris

2016-08-19

Amphibian skin secretions contain biologically-active compounds, such as anti-microbial peptides and trypsin inhibitors, which are used by biomedical researchers as a source of potential novel drug leads or pharmacological agents. Here, we report the application of a recently developed technique within our laboratory to "shotgun" clone the cDNAs encoding two novel but structurally-related peptides from the lyophilised skin secretions of one species of European frog, Rana esculenta and one species of Chinese frog, Odorrana schmackeri. Bioanalysis of the peptides established the structure of a 17-mer with an N-terminal Ala (A) residue and a C-terminal Cys (C) residue with a single disulphide bridge between Cys 12 and 17, which is a canonical Kunitz-type protease inhibitor motif (-CKAAFC-). Due to the presence of this structural attribute, these peptides were named kunitzin-RE (AAKIILNPKFRCKAAFC) and kunitzin-OS (AVNIPFKVHLRCKAAFC). Synthetic replicates of these two novel peptides were found to display a potent inhibitory activity against Escherichia coli but were ineffective at inhibiting the growth of Staphylococcus aureus and Candida albicans at concentrations up to 160 μM, and both showed little haemolytic activity at concentrations up to 120 μM. Subsequently, kunitzin-RE and kunitzin-OS were found to be a potent inhibitor of trypsin with a Ki of 5.56 μM and 7.56 μM that represent prototypes of a novel class of highly-attenuated amphibian skin protease inhibitor. Substitution of Lys-13, the predicted residue occupying the P1 position within the inhibitory loop, with Phe (F) resulted in decrease in trypsin inhibitor effectiveness and antimicrobial activity against Esherichia coli, but exhibits a potential inhibition activity against chymotrypsin. Copyright © 2016 Elsevier Inc. All rights reserved.

Defense response in non-genomic model species: methyl jasmonate exposure reveals the passion fruit leaves' ability to assemble a cocktail of functionally diversified Kunitz-type trypsin inhibitors and recruit two of them against papain.

PubMed

Botelho-Júnior, Sylvio; Machado, Olga L T; Fernandes, Kátia V S; Lemos, Francisco J A; Perdizio, Viviane A; Oliveira, Antônia E A; Monteiro, Leandro R; Filho, Mauri L; Jacinto, Tânia

2014-08-01

Multiplicity of protease inhibitors induced by predators may increase the understanding of a plant's intelligent behavior toward environmental challenges. Information about defense mechanisms of non-genomic model plant passion fruit (Passiflora edulis Sims) in response to predator attack is still limited. Here, via biochemical approaches, we showed its flexibility to build-up a broad repertoire of potent Kunitz-type trypsin inhibitors (KTIs) in response to methyl jasmonate. Seven inhibitors (20-25 kDa) were purified from exposed leaves by chromatographic techniques. Interestingly, the KTIs possessed truncated Kunitz motif in their N-terminus and some of them also presented non-consensus residues. Gelatin-Native-PAGE established multiple isoforms for each inhibitor. Significant differences regarding inhibitors' activity toward trypsin and chymotrypsin were observed, indicating functional polymorphism. Despite its rarity, two of them also inhibited papain, and such bifunctionality suggests a recruiting process onto another mechanistic class of target protease (cysteine-type). All inhibitors acted strongly on midgut proteases from sugarcane borer, Diatraea saccharalis (a lepidopteran insect) while in vivo assays supported their insecticide properties. Moreover, the bifunctional inhibitors displayed activity toward midgut proteases from cowpea weevil, Callosobruchus maculatus (a coleopteran insect). Unexpectedly, all inhibitors were highly effective against midgut proteases from Aedes aegypti a dipteran insect (vector of neglected tropical diseases) opening new avenues for plant-derived PIs for vector control-oriented research. Our results reflect the KTIs' complexities in passion fruit which could be wisely exploited by influencing plant defense conditions. Therefore, the potential of passion fruit as source of bioactive compounds with diversified biotechnological application was strengthened.

Proteomic analysis of Lupinus angustifolius (var. Zeus and Bojar) and Lupinus luteus (var. Lord and Parys) seed proteins and their hydrolysates.

PubMed

Czubinski, Jaroslaw; Montowska, Magdalena; Pospiech, Edward; Lampart-Szczapa, Eleonora

2017-12-01

Proteins enzymatic digestion is a very complex process, during which some components are degraded, whereas others remain in an unchanged form. Moreover, enzymatic hydrolysis is one of the most popular methods used to reduce the allergenicity of food proteins. In the present study, the efficiency of enzymatic hydrolysis of lupin seed proteins was assessed by proteomic analysis as performed by two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry identification. Two digestion systems were used: oriented digestion carried out by trypsin and model in vitro digestion mimicking the conditions present in the gastrointestinal tract. The comparisons of 2-DE maps of proteins isolated form different lupin seed species revealed that the differences in proteins expression were observed mainly in the central parts of gels (i.e. in the molecular weight range from 20 to 70 kDa, and the pH range 5-7). In total, 27 differentially expressed proteins spots were successfully identified by mass spectrometry analysis. An important reduction in the number of proteins spots on 2-DE maps was observed when trypsin and the in vitro digestion model were applied. The protein spot insensitive to digestion in both hydrolysis systems was identified as β-conglutin. The results of the present study provide insight into the nature of the digestion process that may take place after lupin seed protein intake and highlight the important fact that some of the proteins are insensitive to digestive enzyme activity. Moreover, evaluation of digestion activity of trypsin towards lupin seed proteins may be used for the development of specific processes with respect to hypoallergenic food production. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Mesotrypsin Signature Mutation in a Chymotrypsin C (CTRC) Variant Associated with Chronic Pancreatitis.

PubMed

Szabó, András; Ludwig, Maren; Hegyi, Eszter; Szépeová, Renata; Witt, Heiko; Sahin-Tóth, Miklós

2015-07-10

Human chymotrypsin C (CTRC) protects against pancreatitis by degrading trypsinogen and thereby curtailing harmful intra-pancreatic trypsinogen activation. Loss-of-function mutations in CTRC increase the risk for chronic pancreatitis. Here we describe functional analysis of eight previously uncharacterized natural CTRC variants tested for potential defects in secretion, proteolytic stability, and catalytic activity. We found that all variants were secreted from transfected cells normally, and none suffered proteolytic degradation by trypsin. Five variants had normal enzymatic activity, whereas variant p.R29Q was catalytically inactive due to loss of activation by trypsin and variant p.S239C exhibited impaired activity possibly caused by disulfide mispairing. Surprisingly, variant p.G214R had increased activity on a small chromogenic peptide substrate but was markedly defective in cleaving bovine β-casein or the natural CTRC substrates human cationic trypsinogen and procarboxypeptidase A1. Mutation p.G214R is analogous to the evolutionary mutation in human mesotrypsin, which rendered this trypsin isoform resistant to proteinaceous inhibitors and conferred its ability to cleave these inhibitors. Similarly to the mesotrypsin phenotype, CTRC variant p.G214R was inhibited poorly by eglin C, ecotin, or a CTRC-specific variant of SGPI-2, and it readily cleaved the reactive-site peptide bonds in eglin C and ecotin. We conclude that CTRC variants p.R29Q, p.G214R, and p.S239C are risk factors for chronic pancreatitis. Furthermore, the mesotrypsin-like CTRC variant highlights how the same natural mutation in homologous pancreatic serine proteases can evolve a new physiological role or lead to pathology, determined by the biological context of protease function. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Changes in digestive enzyme activities during larval development of leopard grouper (Mycteroperca rosacea).

PubMed

Martínez-Lagos, R; Tovar-Ramírez, D; Gracia-López, V; Lazo, J P

2014-06-01

The leopard grouper is an endemic species of the Mexican Pacific with an important commercial fishery and good aquaculture potential. In order to assess the digestive capacity of this species during the larval period and aid in the formulation of adequate weaning diets, this study aimed to characterize the ontogeny of digestive enzymes during development of the digestive system. Digestive enzymes trypsin, chymotrypsin, acid protease, leucine-alanine peptidase, alkaline phosphatase, aminopeptidase N, lipase, amylase and maltase were quantified in larvae fed live prey and weaned onto a formulated microdiet at 31 days after hatching (DAH) and compared with fasting larvae. Enzyme activity for trypsin, lipase and amylase were detected before the opening of the mouth and the onset of exogenous feeding, indicating a precocious development of the digestive system that has been described in many fish species. The intracellular enzyme activity of leucine-alanine peptidase was high during the first days of development, with a tendency to decrease as larvae developed, reaching undetectable levels at the end of the experimental period. In contrast, activities of enzymes located in the intestinal brush border (i.e., aminopeptidase and alkaline phosphatase) were low at the start of exogenous feeding but progressively increased with larval development, indicating the gradual maturation of the digestive system. Based on our results, we conclude that leopard grouper larvae possess a functional digestive system at hatching and before the onset of exogenous feeding. The significant increase in the activity of trypsin, lipase, amylase and acid protease between 30 and 40 DAH suggests that larvae of this species can be successfully weaned onto microdiets during this period.

Characterization of the Mamestra configurata (Lepidoptera: Noctuidae) larval midgut protease complement and adaptation to feeding on artificial diet, Brassica species, and protease inhibitor.

PubMed

Erlandson, Martin A; Hegedus, Dwayne D; Baldwin, Douglas; Noakes, Amy; Toprak, Umut

2010-10-01

The midgut protease profiles from 5th instar Mamestra configurata larvae fed various diets (standard artificial diet, low protein diet, low protein diet with soybean trypsin inhibitor [SBTI], or Brassica napus) were characterized by one-dimensional enzymography in gelatin gels. The gut protease profile of larvae fed B. napus possessed protease activities of molecular masses of approximately 33 and 55 kDa, which were not present in the guts of larvae fed artificial diet. Similarly, larvae fed artificial diet had protease activities of molecular masses of approximately 21, 30, and 100 kDa that were absent in larvae fed B. napus. Protease profiles changed within 12 to 24 h after switching larvae from artificial diet to plant diet and vice versa. The gut protease profiles from larvae fed various other brassicaceous species and lines having different secondary metabolite profiles did not differ despite significant differences in larval growth rates on the different host plants. Genes encoding putative digestive proteolytic enzymes, including four carboxypeptidases, five aminopeptidases, and 48 serine proteases, were identified in cDNA libraries from 4th instar M. configurata midgut tissue. Many of the protease-encoding genes were expressed at similar levels on all diets; however, three chymoptrypsin-like genes (McSP23, McSP27, and McSP37) were expressed at much higher levels on standard artificial diet and diet containing SBTI as was the trypsin-like gene McSP34. The expression of the trypsin-like gene McSP50 was highest on B. napus. The adaptation of M. configurata digestive biochemistry to different diets is discussed in the context of the flexibility of polyphagous insects to changing diet sources.

Effects of different dietary conditions on the expression of trypsin- and chymotrypsin-like protease genes in the digestive system of the migratory locust, Locusta migratoria.

PubMed

Spit, Jornt; Zels, Sven; Dillen, Senne; Holtof, Michiel; Wynant, Niels; Vanden Broeck, Jozef

2014-05-01

While technological advancements have recently led to a steep increase in genomic and transcriptomic data, and large numbers of protease sequences are being discovered in diverse insect species, little information is available about the expression of digestive enzymes in Orthoptera. Here we describe the identification of Locusta migratoria serine protease transcripts (cDNAs) involved in digestion, which might serve as possible targets for pest control management. A total of 5 putative trypsin and 15 putative chymotrypsin gene sequences were characterized. Phylogenetic analysis revealed that these are distributed among 3 evolutionary conserved clusters. In addition, we have determined the relative gene expression levels of representative members in the gut under different feeding conditions. This study demonstrated that the transcript levels for all measured serine proteases were strongly reduced after starvation. On the other hand, larvae of L. migratoria displayed compensatory effects to the presence of Soybean Bowman Birk (SBBI) and Soybean Trypsin (SBTI) inhibitors in their diet by differential upregulation of multiple proteases. A rapid initial upregulation was observed for all tested serine protease transcripts, while only for members belonging to class I, the transcript levels remained elevated after prolonged exposure. In full agreement with these results, we also observed an increase in proteolytic activity in midgut secretions of locusts that were accustomed to the presence of protease inhibitors in their diet, while no change in sensitivity to these inhibitors was observed. Taken together, this paper is the first comprehensive study on dietary dependent transcript levels of proteolytic enzymes in Orthoptera. Our data suggest that compensatory response mechanisms to protease inhibitor ingestion may have appeared early in insect evolution. Copyright © 2014 Elsevier Ltd. All rights reserved.

Identification of a serine protease as a major allergen (Per a 10) of Periplaneta americana.

PubMed

Sudha, V T; Arora, N; Gaur, S N; Pasha, S; Singh, B P

2008-06-01

Cockroach allergens are associated with the development of asthma, but none of these has been characterized for proteolytic activity. This study was undertaken to isolate and characterize a protease from Periplaneta americana and determine its allergenicity. A serine protease was isolated from P. americana extract using benzamidine sepharose column and characterized by immunobiochemical methods. Allergenicity of the protease was assessed by enzyme-linked immunosorbent assay, immunoblot, intradermal testing, histamine release and peripheral blood mononuclear cells (PBMCs) proliferation. Affinity purified protein of approximately 28 kDa (Per a 10) showed a single band of activity in gelatin zymogram and agarose plate assay. N-terminal sequence (IVGGRPAQI) revealed similarity with mite serine protease allergens and insect trypsins. It demonstrated proteolytic activity with azocollagen > gelatin > defatted-milk > casein including serine protease specific substrate, N-benzoyl-arginine-ethyl-ester-hydrochloride. It was inhibited by serine protease inhibitors, namely aprotinin > pefabloc > AEBSF > PMSF > benzamidine > antipain > leupeptin and trypsin-specific inhibitor (tosyl-lysyl-chloromethyl-ketone) suggesting it to be a trypsin-like serine protease. Per a 10 was recognized as a major allergen, showing IgE reactivity with >80% of cockroach sensitized patients by skin tests and immunoblot. It could induce significant histamine release (P < 0.05) in blood and secretion of interleukin-4 (IL-4) (P < 0.05) and IL-5 (P < 0.05) in culture supernatant of PBMCs from cockroach hypersensitive patients, suggesting a strong allergenic potency. A serine protease isolated from P. americana was demonstrated to be a major allergen (Per a 10). It has a potential for component-based diagnosis of allergy and will be useful in elucidating the mechanism of allergy.

Modulation of serpin reaction through stabilization of transient intermediate by ligands bound to alpha-helix F.

PubMed

Komissarov, Andrey A; Zhou, Aiwu; Declerck, Paul J

2007-09-07

Mechanism-based inhibition of proteinases by serpins involves enzyme acylation and fast insertion of the reactive center loop (RCL) into the central beta-sheet of the serpin, resulting in mechanical inactivation of the proteinase. We examined the effects of ligands specific to alpha-helix F (alphaHF) of plasminogen activator inhibitor-1 (PAI-1) on the stoichiometry of inhibition (SI) and limiting rate constant (k(lim)) of RCL insertion for reactions with beta-trypsin, tissue-type plasminogen activator (tPA), and urokinase. The somatomedin B domain of vitronectin (SMBD) did not affect SI for any proteinase or k(lim) for tPA but decreased the k(lim) for beta-trypsin. In contrast to SMBD, monoclonal antibodies MA-55F4C12 and MA-33H1F7, the epitopes of which are located at the opposite side of alphaHF, decreased k(lim) and increased SI for every enzyme. These effects were enhanced in the presence of SMBD. RCL insertion for beta-trypsin and tPA is limited by different subsequent steps of PAI-1 mechanism as follows: enzyme acylation and formation of a loop-displaced acyl complex (LDA), respectively. Stabilization of LDA through the disruption of the exosite interactions between PAI-1 and tPA induced an increase in the k(lim) but did not affect the SI. Thus it is unlikely that LDA contributes significantly to the outcome of the serpin reaction. These results demonstrate that the rate of RCL insertion is not necessarily correlated with SI and indicate that an intermediate, different from LDA, which forms during the late steps of PAI-1 mechanism, and could be stabilized by ligands specific to alphaHF, controls bifurcation between the inhibitory and the substrate pathways.

Characterization of digestive enzymes from de-oiled mackerel (Scomber japonicus) muscle obtained by supercritical carbon dioxide and n-hexane extraction as a comparative study.

PubMed

Asaduzzaman, A K M; Chun, Byung-Soo

2015-06-01

The oil in mackerel muscle was extracted using an environmental friendly solvent, supercritical carbon dioxide (SC-CO2) at a semi-batch flow extraction process and an n-hexane. The SC-CO2 was carried out at temperature 45 °C and pressures ranging from 15 to 25 MPa. The flow rate of CO2 (27 g/min) was constant at the entire extraction period of 2 h. The highest oil extracted residues after SC-CO2 extraction was used for activity measurement of digestive enzymes. Four digestive enzymes were found in water soluble extracts after n-hexane and SC-CO2 treated samples. Amylase, lipase and trypsin activities were higher in water soluble extracts after SC-CO2 treated samples except protease. Among the four digestive enzymes, the activity of amylase was highest and the value was 44.57 uM/min/mg of protein. The water soluble extracts of SC-CO2 and n-hexane treated mackerel samples showed same alkaline optimum pH and pH stability for each of the digestive enzymes. Optimum temperature of amylase, lipase, protease and trypsin was 40, 50, 60 and 30 °C, respectively of both extracts. More than 80 % temperature stability of amylase, lipase, protease and trypsin were retained at mentioned optimum temperature in water soluble extracts of both treated samples. Based on protein patterns, prominent protein band showed in water soluble extracts after SC-CO2 treated samples indicates no denaturation of protein than untreated and n-hexane.

Thrombin like activity of Asclepias curassavica L. latex: action of cysteine proteases.

PubMed

Shivaprasad, H V; Rajesh, R; Nanda, B L; Dharmappa, K K; Vishwanath, B S

2009-05-04

To validate the scientific basis of plant latex to stop bleeding on fresh cuts. Cysteine protease(s) from Asclepias curassavica (Asclepiadaceae) plant latex was assessed for pro-coagulant and thrombin like activities. A waxy material from the latex of Asclepias curassavica latex was removed by freezing and thawing. The resulted latex enzyme fraction was assayed for proteolytic activity using denatured casein as substrate. Its coagulant activity and thrombin like activity were determined using citrated plasma and pure fibrinogen, respectively. Inhibition studies were performed using specific protease inhibitors to know the type of protease. The latex enzyme fraction exhibited strong proteolytic activity when compared to trypsin and exerted pro-coagulant action by reducing plasma clotting time from 195 to 58 s whereas trypsin reduced clotting time marginally from 195 to 155 s. The pro-coagulant activity of this enzyme fraction was exerted by selectively hydrolyzing A alpha and B beta subunits of fibrinogen to form fibrin clot when pure fibrinogen was used as substrate as assessed by fibrinogen-agarose plate method and fibrinogen polymerization assay. Trypsin failed to induce any fibrin clot under similar conditions. The electrophoretic pattern of latex enzyme fraction-induced fibrin clot was very much similar to that of thrombin-induced fibrin clot and mimic thrombin like action. The proteolytic activity including thrombin like activity of Asclepias curassavica latex enzyme fraction was completely inhibited by iodoaceticacid (IAA). Cysteine proteases from Asclepias curassavica latex exhibited strong pro-coagulant action and were found to be specific in its action (Thrombin like). This could be the basis for the use of plant latex in pharmacological applications that justify their use as folk medicine.

Glandular kallikrein in the innate immune system of Atlantic salmon (Salmo salar).

PubMed

Haussmann, D; Figueroa, J

2011-02-15

Glandular Kallikrein is a serine-protease with trypsin-like activity and is able to generate bioactive peptides from inactive precursors. We have evaluated the presence of this protease in the different organs of the Atlantic salmon (Salmo salar). The results clearly indicate that GK and PRL are generated in the same pituitary cells based on a co-localization by confocal microscopy. Based on probed cross-reactivity between C. striata and C. carpio glandular anti-GK antibodies, we used a homologous antibody to detect the presence of GK in several salmon tissues. We have evaluated the GK expression in healthy and defied fish. P. salmonis and V. ordalii. The GK immunoreaction in organs such as leukocytes, gills and skin is considerably increased in defied fish compared to healthy fish. This increase was present in the cells of the excretory kidney and in the intercellular tissue, where the development of hematopoietic and lymphocytic lines in fish take place. One of the most interesting organs to study was the skin, bearing in mind that this is a primary barrier to all pathogens. The skin of the defied fish exhibited an increase in immunoreactivity for glandular kallikrein similar to the protease found in mucus. An immunoreactive tissue kallikrein-like protein was identified and partially separated by perfusion chromatography. Enzymatic activity of salmon muscle prokallikrein was determined before and after trypsin activation. Kallikrein activity was characterized with respect to their ability to cleave the chromogenic leaving group, p-nitroanilide, from the peptidyl kallikrein and trypsin substrate. These findings constitute a important contribution to reveal the role of kallikrein in the innate immune system of fish. Copyright © 2010 Elsevier B.V. All rights reserved.

A Direct Cell Quenching Method for Cell-Culture Based Metabolomics

EPA Science Inventory

A crucial step in metabolomic analysis of cellular extracts is the cell quenching process. The conventional method first uses trypsin to detach cells from their growth surface. This inevitably changes the profile of cellular metabolites since the detachment of cells from the extr...

21 CFR 864.9400 - Stabilized enzyme solution.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Stabilized enzyme solution. 864.9400 Section 864... and Blood Products § 864.9400 Stabilized enzyme solution. (a) Identification. A stabilized enzyme... enzyme solutions include papain, bromelin, ficin, and trypsin. (b) Classification. Class II (performance...

21 CFR 864.9400 - Stabilized enzyme solution.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Stabilized enzyme solution. 864.9400 Section 864... and Blood Products § 864.9400 Stabilized enzyme solution. (a) Identification. A stabilized enzyme... enzyme solutions include papain, bromelin, ficin, and trypsin. (b) Classification. Class II (performance...

Characterization of tail sheath protein of giant bacteriophage phiKZ Pseudomonas aeruginosa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kurochkina, Lidia P., E-mail: lpk@ibch.r; Aksyuk, Anastasia A.; Sachkova, Maria Yu.

2009-12-20

The tail sheath protein of giant bacteriophage phiKZ Pseudomonas aeruginosa encoded by gene 29 was identified and its expression system was developed. Localization of the protein on the virion was confirmed by immunoelectron microscopy. Properties of gene product (gp) 29 were studied by electron microscopy, immunoblotting and limited trypsinolysis. Recombinant gp29 assembles into the regular tubular structures (polysheaths) of variable length. Trypsin digestion of gp29 within polysheaths or extended sheath of virion results in specific cleavage of the peptide bond between Arg135 and Asp136. However, this cleavage does not affect polymeric structure of polysheaths, sheaths and viral infectivity. Digestion bymore » trypsin of the C-truncated gp29 mutant, lacking the ability to self-assemble, results in formation of a stable protease-resistant fragment. Although there is no sequence homology of phiKZ proteins to proteins of other bacteriophages, some characteristic biochemical properties of gp29 revealed similarities to the tail sheath protein of bacteriophage T4.« less

Food for thought: Selecting the right enzyme for the digestion of gluten.

PubMed

Colgrave, Michelle L; Byrne, Keren; Howitt, Crispin A

2017-11-01

Gluten describes a complex mixture of proteins found in wheat, rye, barley and oats that pose a health risk to people affected by conditions such as coeliac disease and non-coeliac gluten sensitivity. Complete digestion of gluten proteins is of critical importance during quantitative analysis. To this end, chymotrypsin was investigated for its ability to efficiently and reproducibly digest specific classes of gluten in barley. Using proteomics a chymotryptic peptide marker panel was elucidated and subjected to relative quantification using LC-MRM-MS. Thorough investigation of peptide markers revealed robust and reproducible quantification with CVs <15% was possible, however a greater proportion of non-specific cleavage variants were observed relative to trypsin. The selected peptide markers were assessed to ensure their efficient liberation from their parent proteins. While trypsin remains the preferred enzyme for quantification of the avenin-like A proteins, the B-, D- and γ-hordeins, chymotrypsin was the enzyme of choice for the C-hordeins. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

Physicochemical properties of extrudates from white yam and bambara nut blends

NASA Astrophysics Data System (ADS)

Oluwole, O. B.; Olapade, A. A.; Awonorin, S. O.; Henshaw, F. O.

2013-01-01

This study was conducted to investigate effects of extrusion conditions on physicochemical properties of blend of yam and bambara nut flours. A blend of white yam grit (750 μm) and Bambara nut flour (500 μm) in a ratio of 4:1, respectively was extrusion cooked at varying screw speeds 50-70 r.p.m., feed moisture 12.5-17.5% (dry basis) and barrel temperatures 130-150°C. The extrusion variables employed included barrel temperature, screw speed, and feed moisture content, while the physicochemical properties of the extrudates investigated were the expansion ratio, bulk density, and trypsin inhibition activity. The results revealed that all the extrusion variables had significant effects (p<0.05) on the product properties considered in this study. The expansion ratio values ranged 1.55-2.06, bulk density values ranged 0.76-0.94 g cm-3, while trypsin inhibition activities were 1.01-8.08 mg 100 g-1 sample.

A multi-protease, multi-dissociation, bottom-up-to-top-down proteomic view of the Loxosceles intermedia venom

PubMed Central

Trevisan-Silva, Dilza; Bednaski, Aline V.; Fischer, Juliana S.G.; Veiga, Silvio S.; Bandeira, Nuno; Guthals, Adrian; Marchini, Fabricio K.; Leprevost, Felipe V.; Barbosa, Valmir C.; Senff-Ribeiro, Andrea; Carvalho, Paulo C.

2017-01-01

Venoms are a rich source for the discovery of molecules with biotechnological applications, but their analysis is challenging even for state-of-the-art proteomics. Here we report on a large-scale proteomic assessment of the venom of Loxosceles intermedia, the so-called brown spider. Venom was extracted from 200 spiders and fractioned into two aliquots relative to a 10 kDa cutoff mass. Each of these was further fractioned and digested with trypsin (4 h), trypsin (18 h), pepsin (18 h), and chymotrypsin (18 h), then analyzed by MudPIT on an LTQ-Orbitrap XL ETD mass spectrometer fragmenting precursors by CID, HCD, and ETD. Aliquots of undigested samples were also analyzed. Our experimental design allowed us to apply spectral networks, thus enabling us to obtain meta-contig assemblies, and consequently de novo sequencing of practically complete proteins, culminating in a deep proteome assessment of the venom. Data are available via ProteomeXchange, with identifier PXD005523. PMID:28696408

Investigating protein-protein interaction surfaces using a reduced stereochemical and electrostatic model.

PubMed

Warwicker, J

1989-03-20

A method of calculating the electrostatic potential energy between two molecules, using finite difference potential, is presented. A reduced charge set is used so that the interaction energy can be calculated as the two static molecules explore their full six-dimensional configurational space. The energies are contoured over surfaces fixed to each molecule with an interactive computer graphics program. For two crystal structures (trypsin-trypsin inhibitor and anti-lysozyme Fab-lysozyme), it is found that the complex corresponds to highly favourable interacting regions in the contour plots. These matches arise from a small number of protruding basic residues interacting with enhanced negative potential in each case. The redox pair cytochrome c peroxidase-cytochrome c exhibits an extensive favourably interacting surface within which a possible electron transfer complex may be defined by an increased electrostatic complementarity, but a decreased electrostatic energy. A possible substrate transfer configuration for the glycolytic enzyme pair glyceraldehyde phosphate dehydrogenase-phosphoglycerate kinase is presented.

Effect of gamma irradiation on lipoxygenases, trypsin inhibitor, raffinose family oligosaccharides and nutritional factors of different seed coat colored soybean (Glycine max L.)

NASA Astrophysics Data System (ADS)

Kumar Dixit, Amit; Kumar, Vineet; Rani, Anita; Manjaya, J. G.; Bhatnagar, Deepak

2011-04-01

Three soybean genotypes Kalitur, Hara soya and NRC37 with black, green and yellow seed coat color, respectively, were gamma irradiated at 0.5, 2.0 and 5.0 kGy and tested for antinutritional and nutritional factors. Gamma irradiation at all doses reduced the level of lipoxygenase isomers, trypsin inhibitor (TI) and ascorbic acid in all the 3 soybean genotypes as compared to the unirradiated control. However, irradiation dose of 5.0 kGy increased the sucrose content of the soybean genotypes. No significant change was observed in oil, protein fatty acids and total tocopherol content of the 3 genotypes at any irradiation dose. It is suggested that inhibition of lipoxygenase, reduction in TI and ascorbic acid may be due to the breakage or oxidation of protein structure by the gamma irradiation. Similarly, gamma irradiation at higher doses may break glycosidic linkages in oligosaccharides to produce more sucrose and decrease the content of flatulence causing oligosaccharides.

Inactivation Methods of Trypsin Inhibitor in Legumes: A Review.

PubMed

Avilés-Gaxiola, Sara; Chuck-Hernández, Cristina; Serna Saldívar, Sergio O

2018-01-01

Seed legumes have played a major role as a crop worldwide, being cultivated on about 12% to 15% of Earth's arable land; nevertheless, their use is limited by, among other things, the presence of several antinutritional factors (ANFs - naturally occurring metabolites that the plant produces to protect itself from pest attacks.) Trypsin inhibitors (TIs) are one of the most relevant ANFs because they reduce digestion and absorption of dietary proteins. Several methods have been developed in order to inactivate TIs, and of these, thermal treatments are the most commonly used. They cause loss of nutrients, affect functional properties, and require high amounts of energy. Given the above, new processes have emerged to improve the nutritional quality of legumes while trying to solve the problems caused by the use of thermal treatments. This review examines and discusses the methods developed by researchers to inactivate TI present in legumes and their effects over nutritional and functional properties. © 2017 Institute of Food Technologists®.

Evaluation of a toxoid for protection of rabbits against enterotoxaemia experimentally induced by trypsin-activated supernatant of Clostridium spiroforme.

PubMed

Ellis, T M; Gregory, A R; Logue, G D

1991-06-01

Investigations were conducted into an enterotoxaemia caused by Clostridium spiroforme responsible for significant losses in commercial rabbit farms in Western Australia. Two trials using laboratory and farm bred rabbits were performed to evaluate the protective value of a toxoid prepared from the supernatant of C. spiroforme cultures against intraperitoneal challenge with the trypsin-activated toxin of C. spiroforme. The trials showed clearly that a single vaccination at weaning (four weeks) was protective against toxin but more complete and lasting protection was conferred following a second vaccination administered 14 days after the first. Adults likewise showed similar levels of protective antibodies but did not appear to pass on this protection to their kits although ELISA results indicated levels of antibody in kits from unvaccinated mother to be lower than progeny from vaccinated mothers. However antibody levels in kits from vaccinated mothers were very low and did not protect against challenge with toxin.

Biochemical consequences of alginate encapsulation: a NMR study of insulin-secreting cells.

PubMed

Simpson, Nicholas E; Grant, Samuel C; Gustavsson, Lenita; Peltonen, Vilje-Mia; Blackband, Stephen J; Constantinidis, Ioannis

2006-04-01

In this study we explore the biochemical consequences of alginate encapsulation on betaTC3 cells. (13)C NMR spectroscopy and isotopomer analysis were used to investigate the effects of encapsulation on several enzymatic processes associated with the TCA cycle. Our data show statistically significant differences in various enzymatic fluxes related to the TCA cycle and insulin secretion between monolayer and alginate-encapsulated cultures. The principal cause for these effects was the process of trypsinization. Embedding the trypsinized cells in alginate beads did not have a compounded effect on the enzymatic fluxes of entrapped cells. However, an additional small but statistically significant decrease in insulin secretion was measured in encapsulated cells. Finally, differences in either enzymatic fluxes or glucose consumption as a function of bead diameter were not observed. However, differences in T(2), assessed by (1)H NMR microimaging, were observed as a function of bead diameter, suggesting that smaller beads became more organized with time in culture, while larger beads displayed a looser organization.

Peptide affinity labels for thrombin and other trypsin-like proteases

DOEpatents

Shaw, Elliott N.; Kettner, Charles A.

1982-03-09

A peptide affinity label of the formula (I): ##STR1## wherein X is a radical capable of acting as a leaving group in a nucleophilic substitution reaction; A is an aromatic amino acid residue; B is H, or a C.sub.1 -C.sub.4 alkyl group, or aryl; Y is selected from the group consisting of hydrogen, aroyl, C.sub.1 -C.sub.6 acyl, and Q--(A)--.sub.n, wherein Q=hydrogen, aroyl, or C.sub.1 -C.sub.6 acyl, n=1-10, A is an amino acid residue selected from the aliphatic, hydroxy-containing, carboxylic acid group, and amide-thereof-containing, aromatic, sulfur-containing and imino-containing amino acids; and wherein J is selected from the group consisting of --CH.sub.2 --, --CH.sub.2 --CH.sub.2 --,--CH.sub.2 --CH.sub.2 --CH.sub.2 --, --CH.dbd.CH-- and --CH(OH)--CH.sub.2. The affinity label is useful for irreversibly inactivating thrombin and trypsin-like enzymes and may be used as a potential anticlotting agent.

Development, acceptability and nutritional evaluation of 'Doli Ki Roti'--an indigenously fermented bread.

PubMed

Bhatia, A; Khetarpaul, N

2001-01-01

'Doli Ki Roti'-an indigenously fermented bread popular among the Indian Punjabi community who migrated from Pakistan during partition, is a wheat based product having spiced chickpea as stuffing. It contains a good blend of cereal and legume protein (14.5 to 17.1%), fat (7.3 to 9.2%) and ash (3.8 to 4.7%). It is a good source of dietary essential minerals, i.e. calcium (52.7 to 62.6 mg/100 g), iron (8.7 to 10.6 mg/100 g) and phosphorus (313.8 to 346.7 mg/100 g). The antinutrients like phytic acid and trypsin inhibitors are present in considerable amounts in the unfermented bread but are reduced to the extent of 5 to 18% (phytic acid) and 49 to 70% (trypsin inhibitors) due to the fermentation carried out at 35 and 40 degrees C for varying time periods. The products developed were organoleptically acceptable in terms of colour, taste, texture, flavour, etc.

Novel Peptidase Kunitz Inhibitor from Platypodium elegans Seeds Is Active against Spodoptera frugiperda Larvae.

PubMed

Ramalho, Suellen Rodrigues; Bezerra, Cézar da Silva; Lourenço de Oliveira, Daniella Gorete; Souza Lima, Letícia; Maria Neto, Simone; Ramalho de Oliveira, Caio Fernando; Valério Verbisck, Newton; Rodrigues Macedo, Maria Lígia

2018-02-14

A novel Kunitz-type inhibitor from Platypodium elegans seeds (PeTI) was purified and characterized. The mass spectrometry analyses of PeTI indicated an intact mass of 19 701 Da and a partial sequence homologous to Kunitz inhibitors. PeTI was purified by ion exchange and affinity chromatographies. A complex with a 1:1 ratio was obtained only for bovine trypsin, showing a K i = 0.16 nM. Stability studies showed that PeTI was stable over a wide range of temperature (37-80 °C) and pH (2-10). The inhibitory activity of PeTI was affected by dithiothreitol (DTT). Bioassays of PeTI on Spodoptera frugiperda showed negative effects on larval development and weight gain, besides extending the insect life cycle. The activities of digestive enzymes, trypsin and chymotrypsin, were reduced by feeding larvae with 0.2% PeTI in an artificial diet. In summary, we describe a novel Kunitz inhibitor with promising biotechnological potential for pest control.

Functional analysis of the interactions between reovirus particles and various proteases in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sargent, M.D.; Long, D.G.; Borsa, J.

1977-01-01

The digestion of purified reovirus particles by various proteases including chymotrypsin, trypsin, pronase, papain, bromelain, proteinase K, and fibrinolysin has been examined as it relates to virion transcriptase activation and alteration of infectivity. In every case uncoating to the level of active transcriptase proceeds via two mechanistically distinct steps. All the proteases tested serve to mediate only the first of the two steps, converting intact virions to intermediate subviral particles (ISVP) in which the transcriptase is retained in a latent state. The second step of the uncoating process is mediated by a K/sup +/ ion-triggered, endogenous mechanism and results inmore » conversion of ISVP to cores, concomitant with transcriptase activation and loss of infectivity. All of the tested enzymes, except trypsin, reversibly block the second step of uncoating. These results indicate the generality, with respect to protease employed, of the two-step process for reovirus uncoating and transcriptase activation demonstrated previously with chymotrypsin.« less

On-column trypsinization allows for re-use of matrix in modified multiplexed inhibitor beads assay.

PubMed

Petrovic, Voin; Olaisen, Camilla; Sharma, Animesh; Nepal, Anala; Bugge, Steffen; Sundby, Eirik; Hoff, Bård Helge; Slupphaug, Geir; Otterlei, Marit

2017-04-15

The Multiplexed Inhibitor Bead (MIB) assay is a previously published quantitative proteomic MS-based approach to study cellular kinomes. A rather extensive procedure, need for multiple custom-made kinase inhibitors and an inability to re-use the MIB-columns, has limited its applicability. Here we present a modified MIB assay in which elution of bound proteins is facilitated by on-column trypsinization. We tested the modified MIB assay by analyzing extract from three human cancer cell lines treated with the cytotoxic drugs cisplatin or docetaxel. Using only three immobilized kinase inhibitors, we were able to detect about 6000 proteins, including ∼40% of the kinome, as well as other signaling, metabolic and structural proteins. The method is reproducible and the MIB-columns are re-usable without loss of performance. This makes the MIB assay a simple, affordable, and rapid assay for monitoring changes in cellular signaling. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

GREEN: A program package for docking studies in rational drug design

NASA Astrophysics Data System (ADS)

Tomioka, Nobuo; Itai, Akiko

1994-08-01

A program package, GREEN, has been developed that enables docking studies between ligand molecules and a protein molecule. Based on the structure of the protein molecule, the physical and chemical environment of the ligand-binding site is expressed as three-dimensional grid-point data. The grid-point data are used for the real-time evaluation of the protein-ligand interaction energy, as well as for the graphical representation of the binding-site environment. The interactive docking operation is facilitated by various built-in functions, such as energy minimization, energy contribution analysis and logging of the manipulation trajectory. Interactive modeling functions are incorporated for designing new ligand molecules while considering the binding-site environment and the protein-ligand interaction. As an example of the application of GREEN, a docking study is presented on the complex between trypsin and a synthetic trypsin inhibitor. The program package will be useful for rational drug design, based on the 3D structure of the target protein.

Two-dimensional proteome reference maps for the soybean cyst nematode Heterodera glycines

USDA-ARS?s Scientific Manuscript database

Two-dimensional electrophoresis (2-DE) reference maps of Heterodera glycines were constructed. After in-gel digestion with trypsin, 803 spots representing 426 proteins were subsequently identified by LC-MS/MS. Proteins with annotated function were further categorized by Gene Ontology. Results showed...

[The efficacy of the combined use of 5-fluorouracil electrophoresis and magnetotherapy in experimental pancreatitis].

PubMed

Kents, V V; Tsympilova, T A; Mavrodiĭ, V M; Godlevskiĭ, L S

1994-01-01

As shown on the experimental model of rat acute pancreatitis, an intensive 5-fluorouracil electrophoresis course in combination with magnetotherapy significantly reduces the activity of blood trypsin, amylase, lipase and corticosterone. The treatment is thought effective in experimental pancreatitis.

Affinity and specificity of serine endopeptidase-protein inhibitor interactions. Empirical free energy calculations based on X-ray crystallographic structures.

PubMed

Krystek, S; Stouch, T; Novotny, J

1993-12-05

An empirical function was used to calculate free energy change (delta G) of complex formation between the following inhibitors and enzymes: Kunitz inhibitor (BPTI) with trypsin, trypsinogen and kallikrein; turkey ovomucoid 3rd domain (OMTKY3) with alpha-chymotrypsin and the Streptomyces griseus protease B; the potato chymotrypsin inhibitor with the protease B; and the barely chymotrypsin inhibitor and eglin-c with subtilisin and thermitase. Using X-ray coordinates of the nine complexes, we estimated the contributions that hydrophobic effect, electrostatic interactions and side-chain conformational entropy make towards the stability of the complexes. The calculated delta G values showed good agreement with the experimentally measured ones, the only exception being the kallikrein/BPTI complex whose X-ray structure was solved at an exceptionally low pH. In complexes with different enzymes, the same inhibitor residues contributed identically towards complex formation (delta G(residue) Spearman rank correlation coefficient 0.7 to 1.0). The most productive enzyme-contacting residues in OMTKY3, eglin-c, and the chymotrypsin inhibitors were found in analogous positions on their respective binding loops; thus, our calculations identified a functional (energetic) motif that parallels the well-known structural similarity of the binding loops. The delta G values calculated for BPTI complexed with trypsin (-21.7 kcal) and trypsinogen (-23.4 kcal) were similar and close to the experimental delta G value of the trypsin/BPTI complex (-18.1 kcal), lending support to the suggestion that the 10(7) difference in the observed stabilities (KA) of these two complexes reflects the energetic cost of conformational changes induced in trypsinogen during the pre-equilibrium stages of complex formation. In almost all of the complexes studied, the stabilization free energy contributed by the inhibitors was larger than that donated by the enzymes. In the trypsin-BPTI complex, the calculated delta G contribution of the amino group from the BPTI residue Lys15 (9.7 kcal) was somewhat higher than that arrived at in experiments with semisynthetic inhibitor analogs (7.5 kcal). In OMTKY3, different binding loop residues are known to affect differently the binding (delta delta G) to alpha-chymotrypsin and protease B; a good qualitative agreement was found between the calculated delta G(residue) estimates and the experimental delta delta G data (correlation coefficient 0.7). Large variations were observed in local surface complementarity and related interfacial volume in the two OMTKY3 complexes (by 20 to 60% for some side-chains).(ABSTRACT TRUNCATED AT 400 WORDS)

Digestive efficiency, free amino acid pools and quality of growth performance in Atlantic salmon (Salmo salar L.) affected by light regimes and vaccine types.

PubMed

Rungruangsak-Torrissen, Krisna; Sunde, Jan; Berg, Arne Erik; Nordgarden, Ulla; Fjelldal, Per Gunnar; Oppedal, Frode

2009-06-01

This study comprised the results of three different seawater trials using unique combination of techniques to study protease digestive efficiency and growth performance quality to illustrate the effects of light regimes and vaccine types in Atlantic salmon (Salmo salar L.). Fish with higher growth had higher trypsin (T) and chymotrypsin (C) specific activities with higher T/C ratio or slope T/C ratio [calculated from the regression between trypsin (y) and chymotrypsin (x) specific activities] in the pyloric caeca. The T/C ratios indicated fish growth rates over a period of 1-2 months, while the slope T/C ratios indicated fish growth rates at sampling. Adaptation period for adjustment to the new environment of continuous light was 70 days, indicated by the differences in trypsin specific activities and the crossing of slope T/C ratio regressions following with the changes in growth rate directions between the control and the treated group. Vaccine types affected fish vertebral growth, and additional continuous light enhanced the impact of vaccines on fish growth during springtime, indicated by differences in slope T/C ratios. Continuous light stimulated fish growth during winter to spring, when the natural day length was short, without significantly changing white muscle and oocyte qualities in the fish of about 500 g, except for significantly increased white muscle RNA concentration. Continuous light also reduced fish growth rate later during summer, when the natural day length was long, by precedently decreasing the T/C ratio in late spring. Interestingly, plasma levels of free lysine related to tryptic digestion were correlated with trypsin specific activity levels. Continuous light caused higher levels of most free amino acids (FAA) involved in nitrogen metabolism, higher incorporation of essential FAA for protein synthesis, and higher protein turnover rate (free hydroxyproline levels) in both plasma and white muscle. However, continuous light did not affect higher protein content, intracellular buffering capacity and RNA levels in the white muscle of the fish of about 1 kg, probably due to limitation of FAA available for protein synthesis. It is therefore suggested that enhancing fish growth by continuous light stimulation should be accompanied by increasing availability or content of dietary protein (and probably minerals), which in turn would improve the quality of fish growth performance through increasing fillet protein concentration, strengthening vertebral growth, and delaying oocyte development.

Cis-mediated down-regulation of a trypsin gene associated with Bt resistance in cotton bollworm

USDA-ARS?s Scientific Manuscript database

Transgenic plants producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are useful for pest control, but their efficacy is reduced when pests evolve resistance. Previously identified mechanisms of resistance to Bt toxins include reduced binding of activated Bt toxins to m...

Short peptides containing L-lysine and epsilon-aminocaproic acid as potential plasmin inhibitors.

PubMed

Purwin, M; Bruzgo, I; Markowska, A; Midura-Nowaczek, K

2009-11-01

Eight short peptides containing L-lysine and epsilon-aminocaproic acid were obtained and their effect on the amidolytic activities of plasmin, thrombin and trypsin was examined. Tripeptide amide Boc-EACA-L-Lys-EACA-NH2 was the most effective and specific plasmin inhibitor.

Bowman-Birk inhibitor affects pathways associated with energy metabolism in Drosophila melanogaster

USDA-ARS?s Scientific Manuscript database

Bowman-Birk inhibitor (BBI) is toxic when fed to certain insects, including the fruit fly, Drosophila melanogaster. Dietary BBI has been demonstrated to slow growth and increase insect mortality by inhibiting the digestive enzymes trypsin and chymotrypsin, resulting in a reduced supply of amino acid...

Three sorghum serpin recombinant proteins inhibit midgut trypsin activity and growth of corn earworm

USDA-ARS?s Scientific Manuscript database

The sorghum (Sorghum bicolor) genome contains at least 17 putative serpin (serine protease inhibitor) open reading frames, some of which are induced by pathogens. Recent transcriptome studies found that most of the putative serpins are expressed but their roles are unknown. Four sorghum serpins were...

In vitro digestion of soluble cashew proteins and characterization of surviving IgE-reactive peptides

USDA-ARS?s Scientific Manuscript database

The stability of food allergens to digestion varies; and the ability of food proteins to cause an allergic reaction may be affected by the susceptibility of the allergen to digestion by proteases, including pepsin and trypsin. Recent studies have demonstrated that cashew nut allergens are often a ca...

21 CFR 310.543 - Drug products containing active ingredients offered over-the-counter (OTC) for human use in...

Code of Federal Regulations, 2013 CFR

2013-04-01

... pancreatic insufficiency drug products. Pancreatin and pancrelipase are composed of enzymes: amylase, trypsin... potential for serious risk to patients using these drug products. The bioavailability of pancreatic enzymes... included in an OTC drug monograph. Therefore, the safe and effective use of these enzymes for treating...

21 CFR 310.543 - Drug products containing active ingredients offered over-the-counter (OTC) for human use in...

Code of Federal Regulations, 2014 CFR

2014-04-01

... pancreatic insufficiency drug products. Pancreatin and pancrelipase are composed of enzymes: amylase, trypsin... potential for serious risk to patients using these drug products. The bioavailability of pancreatic enzymes... included in an OTC drug monograph. Therefore, the safe and effective use of these enzymes for treating...

21 CFR 310.543 - Drug products containing active ingredients offered over-the-counter (OTC) for human use in...

Code of Federal Regulations, 2010 CFR

2010-04-01

... pancreatic insufficiency drug products. Pancreatin and pancrelipase are composed of enzymes: amylase, trypsin... potential for serious risk to patients using these drug products. The bioavailability of pancreatic enzymes... included in an OTC drug monograph. Therefore, the safe and effective use of these enzymes for treating...

Shelf Stable Egg-Based Products Processed By Ultra High Pressure Technology

DTIC Science & Technology

2008-07-03

nonpathogenic flat-sour thermophilic organism have been used as one of the surrogate organisms for Clostridium botulinum in thermal processing (IFT, 2000...treated with lysozyme (100 μg/ml for 30 min) and trypsin (200 μg/ml for 2 h) to minimize interfering cell debris. After enzyme treatment the spore