During postmortem storage,fluoride in Antarctic krill can be enriched in the muscle.Trypsin,as the most important digestive enzyme in Antarctic krill with a high activity in low temperature,plays a potential role in t...During postmortem storage,fluoride in Antarctic krill can be enriched in the muscle.Trypsin,as the most important digestive enzyme in Antarctic krill with a high activity in low temperature,plays a potential role in this process.In this study,endogenous trypsin was purified and its properties were investigated.The involvement of trypsin in the generation of free fluoride from Antarctic krill cuticle was explored.Cuticle microstructure before and after hydrolysis was compared with scanning electron microscopy,and the ash samples of the hydrolyzed Antarctic krill cuticle were analyzed with X-ray diffraction,Fourier transform infrared spectroscopy,and electron dispersive spectroscopy,respectively.Mass spectrometry analysis and inhibition tests confirmed that the purified enzyme was endogenous trypsin.Results of the present study indicated that trypsin digestion caused the increases of the concentrations of both fluoride ions and free amino N simultaneously,while the protein coated on the cuticle surface was dissolved too.However,no compositional change was detected in the cuticle inorganic salts.These findings suggest that trypsin triggered free fluoride release from Antarctic krill cuticle.In addition,the kinetics of free fluoride release could be described by the equation C_(W)=(1-0.97^(-0.006t)-0.03e^(0.0558t))x337.53+10.50.The present study improved the understanding of the role of trypsin in free fluoride release from Antarctic krill cuticle,facilitating future studies aimed at reducing the fluoride content in krill protein during Antarctic krill processing.展开更多
Phlorizin(PHL)is a natural compound with strong antioxidant properties mainly found in apples.In this paper,the interaction mechanism of PHL with pepsin and trypsin was comparatively evaluated by computer simulation,f...Phlorizin(PHL)is a natural compound with strong antioxidant properties mainly found in apples.In this paper,the interaction mechanism of PHL with pepsin and trypsin was comparatively evaluated by computer simulation,fluorescence spectra,circular dichroism(CD),and Fourier transform infrared(FT-IR)spectra at a molecular level.Fluorescence spectra showed that PHL quenches the pepsin/trypsin by static quenching.Thermodynamic parameters indicated that PHL binds to pepsin mainly through hydrogen bonds and van der Waals forces,and that of trypsin was electrostatic forces.The ground state complexes PHL and protease have a moderate affinity of 105 L/mol PHL binds more strongly to trypsin than to pepsin.CD and FT-IR spectra results showed that pepsin/trypsin decreased theβ-sheet content and slightly changed its secondary structure upon PHL.These experimental results are mutually verified with the predicted computer-aid simulation results.Upon PHL and trypsin binding,the antioxidant capacity of PHL was elevated.Nevertheless,the antioxidant capacity of PHL was decreased after binding to pepsin.This work elucidates the binding of PHL binding mechanisms to pepsin/trypsin and provides useful information for the digestion of PHL to improve the application of PHL in food processing.展开更多
Macroporous cross-linking chitosan layer coated on silica gel (CTS-SiO2) was prepared by phase inversion and polyethylene glycol (PEG)molecular imprinting methods. Formation of macroporous surtace was investigated...Macroporous cross-linking chitosan layer coated on silica gel (CTS-SiO2) was prepared by phase inversion and polyethylene glycol (PEG)molecular imprinting methods. Formation of macroporous surtace was investigated by scanning electron microscopy (SEM) and BET analysis.The prepared bead was activated by reacting with 1,2-ethylene digiycidyl ether for introducing epoxy groups, and trypsin could be efficiently immobilized on the bead as a biospecific ligand.The bead bearing trypsin was employed to purify trypsin inhibitor (TIs) from egg white as affinity adsorbent.展开更多
SDS-PAGE was applied to determine trypsin activity and inhibition. After the hydrolysis by trypsin to substrate bovine serum albulnin (BSA) under different temperatures and pH, the hydrolysis degree of BSA was conduct...SDS-PAGE was applied to determine trypsin activity and inhibition. After the hydrolysis by trypsin to substrate bovine serum albulnin (BSA) under different temperatures and pH, the hydrolysis degree of BSA was conducted using SDS-PAGE. From the quantitative analysis to the electrophoresis bands of BSA and its hydrolysis products in SDS-PAGE pattern, the change of trypsin activity was determined, and then the optimum temperature at 40°C and the optimum pH at pH 8.5 - 8.7 for trypsin activity were obtained. All the target bonds in BSA molecule could be hydrolyzed at the same time by trypsin. The inhibition was due to the binding of inhibitor to trypsin, which made it impossible for trypsin to touch the substrate protein. SDS-PAGE was demonstrated to be also an effect method for assaying the characteristics of trypsin activity and its inhibition.展开更多
The aim of the present study was to optimize trypsin inhibitor degradation in soybean meal by solid-state fermentation (SSF) with Lactobacillus brevis and Aspergillus oryzae, and to determine the effect of SSF on ph...The aim of the present study was to optimize trypsin inhibitor degradation in soybean meal by solid-state fermentation (SSF) with Lactobacillus brevis and Aspergillus oryzae, and to determine the effect of SSF on phytic acid, crude protein, crude fat, and amino acid profile. Response surface methodology (RSM) with Box-Behnken design was used to optimize SSF. The optimal conditions derived from RSM for L. brevis fermentation were: pH=5. 1; inoculum size=10%; duration=72 h; substrate to water ratio=1.5. The minimum content of trypsin inhibitors was 6.4 mg g^-1 dry matter. The optimal conditions derived from RSM for A. oryzae fermentation were: substrate to water ratio= 0.8 1; inoculum size=4%; duration=120 h. The minimum content of trypsin inhibitors was 1.6 mg g^-1 dry matter. Both L. brevis and A. oryzae decreased trypsin inhibitors dramatically (57.1 and 89.2% respectively). L. brevis fermentation did not affect phytic acid (0.4%) and crude fat (5.2%) considerably, whereas A. oryzae fermentation degraded phytic acid (34.8%) and crude fat (22.0%) contents to a certain extent. Crude protein content was increased after both fermentation (6.4 and 12.9% for L. brevis and A. oryzae respectively). Urease activity was reduced greatly (83.3 and 58.3% for L. brevis and A. oryzae respectively). In conclusion, SSF with A. oryzae and L. brevis reduced trypsin inhibitor content and modified major macronutrients in soybean meal.展开更多
BACKGROUND: Urinary trypsin inhibitor (UTI) inhibits the inflammatory response and protects against ischemia-reperfusion (I/R) injury. The inflammatory response is mediated by nuclear factor-kappa B (NF-kappa B) and i...BACKGROUND: Urinary trypsin inhibitor (UTI) inhibits the inflammatory response and protects against ischemia-reperfusion (I/R) injury. The inflammatory response is mediated by nuclear factor-kappa B (NF-kappa B) and its related target genes and products such as vascular endothelial cell adhesion molecule and CXC chemokines. We aimed to assess the roles of those mediators in a UTI-treated mouse model of hepatic I/R injury. METHODS: Treatment group 1 (UTI given 5 minutes prior to liver ischemia), treatment group 2 (UTI given 5 minutes after the anhepatic phase) and a control group were investigated. Blood and liver samples were obtained and compared at 1, 3, 6 and 24 hours after reperfusion. RESULTS: Attenuation of pathological hepatocellular damage was greater in the treatment groups than in the control group (P < 0.05). Compared with the control group, the UTI treatment groups showed significantly lower serum alanine aminotransferase and aspartate aminotransferase levels, decreased myeloperoxidase activity, and reduced NF-kappa B activation. Also downregulated was the expression of tumor necrosis factor-alpha, cytokine-induced neutrophil chemoattractant, and macrophage inflammatory protein-2 at the mRNA level. P-selectin protein and intercellular adhesion molecule-1 protein expression were also downregulated. In addition, the treatment group I showed a better protective effect against I/R injury than the treatment group 2. CONCLUSIONS: UTI reduces NF-kappa B activation and downregulates the expression of its related mediators, followed by the inhibition of neutrophil aggregation and infiltration in hepatic I/R injury. The protective role of UTI is more effective in prevention than in treatment.展开更多
Kazal-type serine protease inhibitor is one of the most important and widely distributed protease inhibitor families.Pancreatic secretory trypsin inhibitor(PSTI),also known as serine protease inhibitor Kazal typeⅠ(SP...Kazal-type serine protease inhibitor is one of the most important and widely distributed protease inhibitor families.Pancreatic secretory trypsin inhibitor(PSTI),also known as serine protease inhibitor Kazal typeⅠ(SPINK1),binds rapidly to trypsin,inhibits its activity and is likely to protect the pancreas from prematurely activated trypsinogen.Therefore,it is an important factor in the onset of pancreatitis.Recent studies found that PSTI/SPINK1 is also involved in self-regulation of acinar cell phagocytosis,proliferation and growth of a variety of cell lines.In addition,it takes part in the response to inflammatory factor or injury and is highly related to adult type Ⅱ citrullinemia.展开更多
AIM: To investigate whether matrix metalloproteinases-9 (MMP-9) or trypsinogens could serve as histological markers for an aggressive disease course in pediatric ulcerative colitis (UC). METHODS: We identified 24 pati...AIM: To investigate whether matrix metalloproteinases-9 (MMP-9) or trypsinogens could serve as histological markers for an aggressive disease course in pediatric ulcerative colitis (UC). METHODS: We identified 24 patients with pediatric onset (≤ 16 years) UC who had undergone surgery during childhood/adolescence a median of 2.1 years (range 0.1-7.4 years) after the diagnosis (between 1990 and 2008) in Children's Hospital, Helsinki, Finland. We also identified 27 conservatively treated UC patients and matched them based on their age at the time of diagnosis and follow-up at a median of 6 years (range 3-11 years) to serve as disease controls. Twenty children for whom inflammatory bowel disease (IBD) had been excluded as a result of endoscopy served as non-IBD controls. Colon biopsies taken by diagnostic endoscopy before the onset of therapy were stained using immunohistochemistry to study the expression of MMP-9, trypsinogen-1 (Tryp-1), Tryp-2, and a trypsin inhibitor (TATI). The profiles of these proteases and inhibitor at diagnosis were compared between the surgery group, the conservatively treated UC patients and the non-IBD controls. RESULTS: The proportions of Tryp-1 and Tryp-2 positive samples in the colon epithelium and in the inflammatory cells of the colon stroma were comparable between the studied groups at diagnosis. Interestingly, the immunopositivity of Tryp-1 (median 1; range 0-3) was significantly lower in the epithelium of the colon in the pediatric UC patients undergoing surgery when compared to that of the conservatively treated UC patients (median 2; range 0-3; P = 0.03) and non-IBD controls (median 2; range 0-3; P = 0.04). For Tryp-2, there was no such difference. In the inflammatory cells of the colon stroma, the immunopositivities of Tryp-1 and Tryp-2 were comparable between the studied groups at diagnosis. Also, the proportion of samples positive for TATI, as well as the immunopositivity, was comparable between the studied groups in the colon epithelium. In the stromal inflammatory cells of the colon, TATI was not detected. In UC patients, there were significantly more MMP-9 positive samples and a higher immunopositivity in the stromal inflammatory cells of the colon when compared to the samples from the non-IBD patients (P = 0.006 and P = 0.002, respectively); the immunopositivity correlated with the histological grade of inflammation (95%CI: 0.22-0.62; P = 0.0002), but not with the other markers of active disease. There were no differences in the immunopositivity or in the proportions of MMP-9 positive samples when examined by epithelial staining. The staining profiles in the ileal biopsies were comparable between the studied groups for all of the studied markers.CONCLUSION: For pediatric UC patients who require surgery, the immunopositivity of Tryp-1 at diagnosis is lower when compared to that of patients with a more benign disease course.展开更多
Herein,we report a novel sensor to detect trypsin using a purpose-designed fluorescein-labelled peptide with negatively charged carbon nanoparticles(CNPs)modified by acid oxidation.The fluorescence of the fluorescein-...Herein,we report a novel sensor to detect trypsin using a purpose-designed fluorescein-labelled peptide with negatively charged carbon nanoparticles(CNPs)modified by acid oxidation.The fluorescence of the fluorescein-labelled peptide was quenched by CNPs.The sensor reacted with trypsin to cleave the peptide,resulting in the release of the dye moiety and a substantial increase in fluorescence intensity,which was dose-and time-dependent,and trypsin could be quantified accordingly.Correspondingly,the biosensor has led to the development of a convenient and efficient fluorescent method to measure trypsin activity,with a detection limit of 0.7 mg/mL.The method allows rapid determination of trypsin activity in the normal and acute pancreatitis range,suitable for point-of-care testing.Furthermore,the applicability of the method has been demonstrated by detecting trypsin in spiked urine samples.展开更多
A Bowman-Birk inhibitor with activity against gut proteases of Helicoverpa armigera was extracted in 0.I M sodium phosphate buffer from defatted seed flour of Albizia lebbeck. It was purified to 29.62 folds with 51.43...A Bowman-Birk inhibitor with activity against gut proteases of Helicoverpa armigera was extracted in 0.I M sodium phosphate buffer from defatted seed flour of Albizia lebbeck. It was purified to 29.62 folds with 51.43% recovery using ammonium sulfate precipitation, gel filtration chromatography on Sephadex G-100 column and ion ex- change chromatography on DEAE-Sephadex As0. The purified protein had a molecular weight of 12,303 daltons as determined by SDS-PAGE. It was found to be heat stable up to 60~C and had two pH optima of 7.5 and 9.0. The inhibitor exhibited non-competitive pattern of inhibition with a low Ki value of 0.2 ~tM. The inhibitoi- was found to be susceptible to varying concentrations of reducing agents like DTT and 2- mercaptoethanol, thereby indicating the role of disulphide bridges in maintaining its three dimensional structure and stability. The purified inhibitor caused mortality and suppressed larval growth ofPieris brassi- cae larvae. It was also found to be effective against gut trypsin extracted from Spodoptera littoralis. The sequence of the genes encoding for such inhibitors can be determined and the genes expressing protease inhibitors can be used in vegetable crops to confer resistance against insect pests and other plant pathogens.展开更多
Sporamin is a soluble protein in sweet potato, and falls into two distinct homology groups, subfamilies A and B. In this research, a sporamin B was purified and its amino acid sequences, trypsin inhibitor activity (T...Sporamin is a soluble protein in sweet potato, and falls into two distinct homology groups, subfamilies A and B. In this research, a sporamin B was purified and its amino acid sequences, trypsin inhibitor activity (Ti activity) were analyzed. This sporamin B was isolated from sweet potato tubers [Ipomoea batatas (L.) Lam cv. 55-2] through extraction of the water-soluble fraction, dialysis, ultrafiltration and ion-exchange chromatography. Homology determined by polyacrylamide gel electrophoresis showed that mainly one bond appeared in gel after being reduced by SDS (sodium dodecyl sulfate), or by SDS and 2-mercaptoethanol, or in native situation. By comparing the data of the polypeptide mass Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry with those of the mass of the theoretical amino acid sequences from NCBI protein database, it was revealed that it was Q40091|Q40091_IPOBA, sweet potato sporamin B - Ipomoea Batatas (sweet potato) (Batate). The sequence coverage was 70.6%. N-terminal sequence was SETPV (Ser-Glu-Thr-Pro-Val). There is a linear relationship between trypsin inhibitor activity (Ti activity) and amounts of this sporamin B (3-18 μg mL-1). The equation of linear regression was y = 2.5809x + 17.049 (r2 = 0.9966). There was a curvilinear relationship between Ti activity and amounts of this sporamin B (21-150 μg mL-1). The equation of curvilinear regression is y = 14.417ln(x) + 23.26 (r2 = 0.9924). The concentration of sporamin B with Ti activity after heating at 40°C may induce part denature of this sporamin B, and there was no statistic difference after heating at 40, 50, 60°C for 20 min. Heat treatment at more than 90°C leads to a dramatic decrease of trypsin inhibitor efficiency. The results suggested that Q40091|Q40091_IPOBA was the major sporamin B in sweet potato tubers [Ipomoea Batatas (L.) Lam cv. 55-2], which had strong Ti activity, and was stable to both thermal and DTT (DL-dithiothreitol) relatively.展开更多
The pepsin and trypsin activities and some of the properties of the two enzymes of southern sheatfish larvae were studied. The results were as follows: the highest level of trypsin activity is in the foregut in all...The pepsin and trypsin activities and some of the properties of the two enzymes of southern sheatfish larvae were studied. The results were as follows: the highest level of trypsin activity is in the foregut in all measured tissues; from foregut to hindgut, trypsin activities decrease; the pH optimum of trypsin activity is pH9.0; the strongest pepsin activity is in the stomach; the proper density of haemoglobin for detecting pepsin activity is 1.0%. These data are useful in solving applied nutritional problems, such as the adequacy of artificial food to the digestive abilities of the fish.展开更多
Animal-derived biological products, such as fetal bovine serum(FBS) and trypsin, are important supplements for scientific, pharmaceutical, and medical use. Although preventive guidelines and tests are implemented to r...Animal-derived biological products, such as fetal bovine serum(FBS) and trypsin, are important supplements for scientific, pharmaceutical, and medical use. Although preventive guidelines and tests are implemented to reduce potential viral contamination in these biologicals, they do not target unusual or emerging viruses, leading to safety concerns. Using unbiased metagenomics, we investigated the presence of viruses in recently collected commercial FBS and trypsin samples from different geographic regions. In total, we detected viralsequencesbelongingto Parvoviridae,Anelloviridae,Flaviviridae,Herpesviridae,Caliciviridae, Nodaviridae, Rhabdoviridae, and Paramyxoviridae, including several viruses related to bovine diseases, viruses of potential human and insect origin, and viruses of unknown origin. Bovine parvovirus 3 and bosavirus were detected with high frequency and abundance in FBS, necessitating more stringent testing for these parvoviruses during production. Both bovine norovirus and bovine viral diarrhea virus 1 displayed relatively high genetic distance to closest hits, indicating the presence of new genotypes in farm animals. While the origin of novel lyssavirus and Nipah virus is unclear, their presence raises the possibility of the introduction of pathogenic animal-derived viruses into biologicals.Our results showed relatively widespread contamination of different viruses in biologicals,underscoring the need for robust safety protocol alternatives, such as metagenomic sequencing, to monitor emerging viruses.展开更多
The proteins and trypsin inhibitors were isolated from the seeds of different varieties/accessions of an underutilized legume, Mucuna. The crude protein content of all the germplasms of Mucuna is varied from 15% - 26%...The proteins and trypsin inhibitors were isolated from the seeds of different varieties/accessions of an underutilized legume, Mucuna. The crude protein content of all the germplasms of Mucuna is varied from 15% - 26%, showed little variation and contain higher crude protein when compared with other Mucuna species reported earlier and the pulse crops commonly consumed in India. The seeds of all the varieties of Mucuna exhibited trypsin inhibitor activity. The trypsin inhibitor activity varied from 11 - 14 TIA/mg of protein. Not much variation was observed in trypsin inhibitory activities in soaked seeds compared to dry seeds. Germination of Mucuna pruriens has been carried out and the change in the protein content and trypsin inhibitors were monitored. The protein content of the endosperm increased up to 72 hrs of germination and then decreased. The trypsin inhibitory activity decreased with increase in germination time. The trypsin inhibitor activity was decreased from 14.81 TIA/mg to 2.62 TIA/mg (82% reduction in the trypsin inhibitor activity) after 144 hrs germination.展开更多
Objective:The inter-α-trypsin inhibitor heavy chain 4(ITIH4)protein is involved in the development of tumors.However,the relationship between ITIH4 and ovarian cancer(OC)has not been extensively examined.This study a...Objective:The inter-α-trypsin inhibitor heavy chain 4(ITIH4)protein is involved in the development of tumors.However,the relationship between ITIH4 and ovarian cancer(OC)has not been extensively examined.This study aimed to explore the effect of ITIH4 on OC and to identify its underlying mechanism.Methods:Expressions of ITIH4 in OC tissues and cells were determined using quantitative reverse transcription polymerase chain reaction(RT-qPCR)and western blots.The function of ITIH4 in the OC cell line HO8910 pm was tested via ITIH4 knockdown.The cell growth rate was measured using MTT and colony formation assays.Flow cytometry was performed to evaluate cell cycle progression.Cell migration and invasion abilities were observed using the transwell migration assay.Results:ITIH4 was downregulated in OC tissues and cells.ITIH4 knockdown promoted cell growth and cell cycle progression.Consistent with these results,inhibition of ITIH4 in OC cells significantly increased cell migration and invasion abilities.Cox regression analysis suggests that ITIH4 expression alone is not a good predictor of the prognosis of malignant ovarian tumors in patients.Conclusions:ITIH4 inhibits the progression of OC,suggesting that ITIH4 may be a useful biomarker for OC.This study may provide a potential novel target for the treatment of OC.展开更多
基金supported by the Guangdong Provincial Key Laboratory of Aquatic Product Processing and Safety(No.GDPKLAPPS2005)the School Level Talent Project of Lingnan Normal University(No.ZL2021009)+2 种基金the Science and Technology Planning Project of Zhanjiang(No.2020A01040)the Study on the Preparation of Active Polypeptide from the Processing Waste of White Shrimp and its Fatigue Resistance(No.2021E05022)the Scientific Research Capacity Improvement Project of the Key Construction Discipline of Guangdong Province(No.2022ZD JS079).
文摘During postmortem storage,fluoride in Antarctic krill can be enriched in the muscle.Trypsin,as the most important digestive enzyme in Antarctic krill with a high activity in low temperature,plays a potential role in this process.In this study,endogenous trypsin was purified and its properties were investigated.The involvement of trypsin in the generation of free fluoride from Antarctic krill cuticle was explored.Cuticle microstructure before and after hydrolysis was compared with scanning electron microscopy,and the ash samples of the hydrolyzed Antarctic krill cuticle were analyzed with X-ray diffraction,Fourier transform infrared spectroscopy,and electron dispersive spectroscopy,respectively.Mass spectrometry analysis and inhibition tests confirmed that the purified enzyme was endogenous trypsin.Results of the present study indicated that trypsin digestion caused the increases of the concentrations of both fluoride ions and free amino N simultaneously,while the protein coated on the cuticle surface was dissolved too.However,no compositional change was detected in the cuticle inorganic salts.These findings suggest that trypsin triggered free fluoride release from Antarctic krill cuticle.In addition,the kinetics of free fluoride release could be described by the equation C_(W)=(1-0.97^(-0.006t)-0.03e^(0.0558t))x337.53+10.50.The present study improved the understanding of the role of trypsin in free fluoride release from Antarctic krill cuticle,facilitating future studies aimed at reducing the fluoride content in krill protein during Antarctic krill processing.
基金supported by the National Natural Science Foundation of China(21808020)the Applied Basic Research Program of Science&Technology Department of Sichuan Province(2018JY0151)。
文摘Phlorizin(PHL)is a natural compound with strong antioxidant properties mainly found in apples.In this paper,the interaction mechanism of PHL with pepsin and trypsin was comparatively evaluated by computer simulation,fluorescence spectra,circular dichroism(CD),and Fourier transform infrared(FT-IR)spectra at a molecular level.Fluorescence spectra showed that PHL quenches the pepsin/trypsin by static quenching.Thermodynamic parameters indicated that PHL binds to pepsin mainly through hydrogen bonds and van der Waals forces,and that of trypsin was electrostatic forces.The ground state complexes PHL and protease have a moderate affinity of 105 L/mol PHL binds more strongly to trypsin than to pepsin.CD and FT-IR spectra results showed that pepsin/trypsin decreased theβ-sheet content and slightly changed its secondary structure upon PHL.These experimental results are mutually verified with the predicted computer-aid simulation results.Upon PHL and trypsin binding,the antioxidant capacity of PHL was elevated.Nevertheless,the antioxidant capacity of PHL was decreased after binding to pepsin.This work elucidates the binding of PHL binding mechanisms to pepsin/trypsin and provides useful information for the digestion of PHL to improve the application of PHL in food processing.
基金The work is partially supported by the National Natural Science Foundation of China(20277031).
文摘Macroporous cross-linking chitosan layer coated on silica gel (CTS-SiO2) was prepared by phase inversion and polyethylene glycol (PEG)molecular imprinting methods. Formation of macroporous surtace was investigated by scanning electron microscopy (SEM) and BET analysis.The prepared bead was activated by reacting with 1,2-ethylene digiycidyl ether for introducing epoxy groups, and trypsin could be efficiently immobilized on the bead as a biospecific ligand.The bead bearing trypsin was employed to purify trypsin inhibitor (TIs) from egg white as affinity adsorbent.
文摘SDS-PAGE was applied to determine trypsin activity and inhibition. After the hydrolysis by trypsin to substrate bovine serum albulnin (BSA) under different temperatures and pH, the hydrolysis degree of BSA was conducted using SDS-PAGE. From the quantitative analysis to the electrophoresis bands of BSA and its hydrolysis products in SDS-PAGE pattern, the change of trypsin activity was determined, and then the optimum temperature at 40°C and the optimum pH at pH 8.5 - 8.7 for trypsin activity were obtained. All the target bonds in BSA molecule could be hydrolyzed at the same time by trypsin. The inhibition was due to the binding of inhibitor to trypsin, which made it impossible for trypsin to touch the substrate protein. SDS-PAGE was demonstrated to be also an effect method for assaying the characteristics of trypsin activity and its inhibition.
基金supported by a research project of the Science and Technology Key Group in Zhejiang Provincethe research projects from the Science and Technology Department of Zhejiang Province,China (2009C12068)
文摘The aim of the present study was to optimize trypsin inhibitor degradation in soybean meal by solid-state fermentation (SSF) with Lactobacillus brevis and Aspergillus oryzae, and to determine the effect of SSF on phytic acid, crude protein, crude fat, and amino acid profile. Response surface methodology (RSM) with Box-Behnken design was used to optimize SSF. The optimal conditions derived from RSM for L. brevis fermentation were: pH=5. 1; inoculum size=10%; duration=72 h; substrate to water ratio=1.5. The minimum content of trypsin inhibitors was 6.4 mg g^-1 dry matter. The optimal conditions derived from RSM for A. oryzae fermentation were: substrate to water ratio= 0.8 1; inoculum size=4%; duration=120 h. The minimum content of trypsin inhibitors was 1.6 mg g^-1 dry matter. Both L. brevis and A. oryzae decreased trypsin inhibitors dramatically (57.1 and 89.2% respectively). L. brevis fermentation did not affect phytic acid (0.4%) and crude fat (5.2%) considerably, whereas A. oryzae fermentation degraded phytic acid (34.8%) and crude fat (22.0%) contents to a certain extent. Crude protein content was increased after both fermentation (6.4 and 12.9% for L. brevis and A. oryzae respectively). Urease activity was reduced greatly (83.3 and 58.3% for L. brevis and A. oryzae respectively). In conclusion, SSF with A. oryzae and L. brevis reduced trypsin inhibitor content and modified major macronutrients in soybean meal.
文摘BACKGROUND: Urinary trypsin inhibitor (UTI) inhibits the inflammatory response and protects against ischemia-reperfusion (I/R) injury. The inflammatory response is mediated by nuclear factor-kappa B (NF-kappa B) and its related target genes and products such as vascular endothelial cell adhesion molecule and CXC chemokines. We aimed to assess the roles of those mediators in a UTI-treated mouse model of hepatic I/R injury. METHODS: Treatment group 1 (UTI given 5 minutes prior to liver ischemia), treatment group 2 (UTI given 5 minutes after the anhepatic phase) and a control group were investigated. Blood and liver samples were obtained and compared at 1, 3, 6 and 24 hours after reperfusion. RESULTS: Attenuation of pathological hepatocellular damage was greater in the treatment groups than in the control group (P < 0.05). Compared with the control group, the UTI treatment groups showed significantly lower serum alanine aminotransferase and aspartate aminotransferase levels, decreased myeloperoxidase activity, and reduced NF-kappa B activation. Also downregulated was the expression of tumor necrosis factor-alpha, cytokine-induced neutrophil chemoattractant, and macrophage inflammatory protein-2 at the mRNA level. P-selectin protein and intercellular adhesion molecule-1 protein expression were also downregulated. In addition, the treatment group I showed a better protective effect against I/R injury than the treatment group 2. CONCLUSIONS: UTI reduces NF-kappa B activation and downregulates the expression of its related mediators, followed by the inhibition of neutrophil aggregation and infiltration in hepatic I/R injury. The protective role of UTI is more effective in prevention than in treatment.
基金Supported by the National Basic Research 973 Pre-research Program of China,No.2006CB708506
文摘Kazal-type serine protease inhibitor is one of the most important and widely distributed protease inhibitor families.Pancreatic secretory trypsin inhibitor(PSTI),also known as serine protease inhibitor Kazal typeⅠ(SPINK1),binds rapidly to trypsin,inhibits its activity and is likely to protect the pancreas from prematurely activated trypsinogen.Therefore,it is an important factor in the onset of pancreatitis.Recent studies found that PSTI/SPINK1 is also involved in self-regulation of acinar cell phagocytosis,proliferation and growth of a variety of cell lines.In addition,it takes part in the response to inflammatory factor or injury and is highly related to adult type Ⅱ citrullinemia.
基金Supported by Pivikki and Sakari Sohlberg Foundation, to Piekkala MHelsinki University Central Hospital Grant, to Kolho KLthe Finnish Pediatric Research Foundation, to Kolho KL
文摘AIM: To investigate whether matrix metalloproteinases-9 (MMP-9) or trypsinogens could serve as histological markers for an aggressive disease course in pediatric ulcerative colitis (UC). METHODS: We identified 24 patients with pediatric onset (≤ 16 years) UC who had undergone surgery during childhood/adolescence a median of 2.1 years (range 0.1-7.4 years) after the diagnosis (between 1990 and 2008) in Children's Hospital, Helsinki, Finland. We also identified 27 conservatively treated UC patients and matched them based on their age at the time of diagnosis and follow-up at a median of 6 years (range 3-11 years) to serve as disease controls. Twenty children for whom inflammatory bowel disease (IBD) had been excluded as a result of endoscopy served as non-IBD controls. Colon biopsies taken by diagnostic endoscopy before the onset of therapy were stained using immunohistochemistry to study the expression of MMP-9, trypsinogen-1 (Tryp-1), Tryp-2, and a trypsin inhibitor (TATI). The profiles of these proteases and inhibitor at diagnosis were compared between the surgery group, the conservatively treated UC patients and the non-IBD controls. RESULTS: The proportions of Tryp-1 and Tryp-2 positive samples in the colon epithelium and in the inflammatory cells of the colon stroma were comparable between the studied groups at diagnosis. Interestingly, the immunopositivity of Tryp-1 (median 1; range 0-3) was significantly lower in the epithelium of the colon in the pediatric UC patients undergoing surgery when compared to that of the conservatively treated UC patients (median 2; range 0-3; P = 0.03) and non-IBD controls (median 2; range 0-3; P = 0.04). For Tryp-2, there was no such difference. In the inflammatory cells of the colon stroma, the immunopositivities of Tryp-1 and Tryp-2 were comparable between the studied groups at diagnosis. Also, the proportion of samples positive for TATI, as well as the immunopositivity, was comparable between the studied groups in the colon epithelium. In the stromal inflammatory cells of the colon, TATI was not detected. In UC patients, there were significantly more MMP-9 positive samples and a higher immunopositivity in the stromal inflammatory cells of the colon when compared to the samples from the non-IBD patients (P = 0.006 and P = 0.002, respectively); the immunopositivity correlated with the histological grade of inflammation (95%CI: 0.22-0.62; P = 0.0002), but not with the other markers of active disease. There were no differences in the immunopositivity or in the proportions of MMP-9 positive samples when examined by epithelial staining. The staining profiles in the ileal biopsies were comparable between the studied groups for all of the studied markers.CONCLUSION: For pediatric UC patients who require surgery, the immunopositivity of Tryp-1 at diagnosis is lower when compared to that of patients with a more benign disease course.
文摘Herein,we report a novel sensor to detect trypsin using a purpose-designed fluorescein-labelled peptide with negatively charged carbon nanoparticles(CNPs)modified by acid oxidation.The fluorescence of the fluorescein-labelled peptide was quenched by CNPs.The sensor reacted with trypsin to cleave the peptide,resulting in the release of the dye moiety and a substantial increase in fluorescence intensity,which was dose-and time-dependent,and trypsin could be quantified accordingly.Correspondingly,the biosensor has led to the development of a convenient and efficient fluorescent method to measure trypsin activity,with a detection limit of 0.7 mg/mL.The method allows rapid determination of trypsin activity in the normal and acute pancreatitis range,suitable for point-of-care testing.Furthermore,the applicability of the method has been demonstrated by detecting trypsin in spiked urine samples.
文摘A Bowman-Birk inhibitor with activity against gut proteases of Helicoverpa armigera was extracted in 0.I M sodium phosphate buffer from defatted seed flour of Albizia lebbeck. It was purified to 29.62 folds with 51.43% recovery using ammonium sulfate precipitation, gel filtration chromatography on Sephadex G-100 column and ion ex- change chromatography on DEAE-Sephadex As0. The purified protein had a molecular weight of 12,303 daltons as determined by SDS-PAGE. It was found to be heat stable up to 60~C and had two pH optima of 7.5 and 9.0. The inhibitor exhibited non-competitive pattern of inhibition with a low Ki value of 0.2 ~tM. The inhibitoi- was found to be susceptible to varying concentrations of reducing agents like DTT and 2- mercaptoethanol, thereby indicating the role of disulphide bridges in maintaining its three dimensional structure and stability. The purified inhibitor caused mortality and suppressed larval growth ofPieris brassi- cae larvae. It was also found to be effective against gut trypsin extracted from Spodoptera littoralis. The sequence of the genes encoding for such inhibitors can be determined and the genes expressing protease inhibitors can be used in vegetable crops to confer resistance against insect pests and other plant pathogens.
基金supported by the Green-Agricultural Science Research and Demonstration Project (2007-1-2)the National High Technology Research andDevelopment Program of China (863 Program,20060110Z3051)
文摘Sporamin is a soluble protein in sweet potato, and falls into two distinct homology groups, subfamilies A and B. In this research, a sporamin B was purified and its amino acid sequences, trypsin inhibitor activity (Ti activity) were analyzed. This sporamin B was isolated from sweet potato tubers [Ipomoea batatas (L.) Lam cv. 55-2] through extraction of the water-soluble fraction, dialysis, ultrafiltration and ion-exchange chromatography. Homology determined by polyacrylamide gel electrophoresis showed that mainly one bond appeared in gel after being reduced by SDS (sodium dodecyl sulfate), or by SDS and 2-mercaptoethanol, or in native situation. By comparing the data of the polypeptide mass Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry with those of the mass of the theoretical amino acid sequences from NCBI protein database, it was revealed that it was Q40091|Q40091_IPOBA, sweet potato sporamin B - Ipomoea Batatas (sweet potato) (Batate). The sequence coverage was 70.6%. N-terminal sequence was SETPV (Ser-Glu-Thr-Pro-Val). There is a linear relationship between trypsin inhibitor activity (Ti activity) and amounts of this sporamin B (3-18 μg mL-1). The equation of linear regression was y = 2.5809x + 17.049 (r2 = 0.9966). There was a curvilinear relationship between Ti activity and amounts of this sporamin B (21-150 μg mL-1). The equation of curvilinear regression is y = 14.417ln(x) + 23.26 (r2 = 0.9924). The concentration of sporamin B with Ti activity after heating at 40°C may induce part denature of this sporamin B, and there was no statistic difference after heating at 40, 50, 60°C for 20 min. Heat treatment at more than 90°C leads to a dramatic decrease of trypsin inhibitor efficiency. The results suggested that Q40091|Q40091_IPOBA was the major sporamin B in sweet potato tubers [Ipomoea Batatas (L.) Lam cv. 55-2], which had strong Ti activity, and was stable to both thermal and DTT (DL-dithiothreitol) relatively.
文摘The pepsin and trypsin activities and some of the properties of the two enzymes of southern sheatfish larvae were studied. The results were as follows: the highest level of trypsin activity is in the foregut in all measured tissues; from foregut to hindgut, trypsin activities decrease; the pH optimum of trypsin activity is pH9.0; the strongest pepsin activity is in the stomach; the proper density of haemoglobin for detecting pepsin activity is 1.0%. These data are useful in solving applied nutritional problems, such as the adequacy of artificial food to the digestive abilities of the fish.
基金supported by the National Natural Science Foundation of China (32170147,31900158)。
文摘Animal-derived biological products, such as fetal bovine serum(FBS) and trypsin, are important supplements for scientific, pharmaceutical, and medical use. Although preventive guidelines and tests are implemented to reduce potential viral contamination in these biologicals, they do not target unusual or emerging viruses, leading to safety concerns. Using unbiased metagenomics, we investigated the presence of viruses in recently collected commercial FBS and trypsin samples from different geographic regions. In total, we detected viralsequencesbelongingto Parvoviridae,Anelloviridae,Flaviviridae,Herpesviridae,Caliciviridae, Nodaviridae, Rhabdoviridae, and Paramyxoviridae, including several viruses related to bovine diseases, viruses of potential human and insect origin, and viruses of unknown origin. Bovine parvovirus 3 and bosavirus were detected with high frequency and abundance in FBS, necessitating more stringent testing for these parvoviruses during production. Both bovine norovirus and bovine viral diarrhea virus 1 displayed relatively high genetic distance to closest hits, indicating the presence of new genotypes in farm animals. While the origin of novel lyssavirus and Nipah virus is unclear, their presence raises the possibility of the introduction of pathogenic animal-derived viruses into biologicals.Our results showed relatively widespread contamination of different viruses in biologicals,underscoring the need for robust safety protocol alternatives, such as metagenomic sequencing, to monitor emerging viruses.
文摘The proteins and trypsin inhibitors were isolated from the seeds of different varieties/accessions of an underutilized legume, Mucuna. The crude protein content of all the germplasms of Mucuna is varied from 15% - 26%, showed little variation and contain higher crude protein when compared with other Mucuna species reported earlier and the pulse crops commonly consumed in India. The seeds of all the varieties of Mucuna exhibited trypsin inhibitor activity. The trypsin inhibitor activity varied from 11 - 14 TIA/mg of protein. Not much variation was observed in trypsin inhibitory activities in soaked seeds compared to dry seeds. Germination of Mucuna pruriens has been carried out and the change in the protein content and trypsin inhibitors were monitored. The protein content of the endosperm increased up to 72 hrs of germination and then decreased. The trypsin inhibitory activity decreased with increase in germination time. The trypsin inhibitor activity was decreased from 14.81 TIA/mg to 2.62 TIA/mg (82% reduction in the trypsin inhibitor activity) after 144 hrs germination.
基金supported by Guangxi Scientific Research and Technology Development Project (No. 14124004-1-24)
文摘Objective:The inter-α-trypsin inhibitor heavy chain 4(ITIH4)protein is involved in the development of tumors.However,the relationship between ITIH4 and ovarian cancer(OC)has not been extensively examined.This study aimed to explore the effect of ITIH4 on OC and to identify its underlying mechanism.Methods:Expressions of ITIH4 in OC tissues and cells were determined using quantitative reverse transcription polymerase chain reaction(RT-qPCR)and western blots.The function of ITIH4 in the OC cell line HO8910 pm was tested via ITIH4 knockdown.The cell growth rate was measured using MTT and colony formation assays.Flow cytometry was performed to evaluate cell cycle progression.Cell migration and invasion abilities were observed using the transwell migration assay.Results:ITIH4 was downregulated in OC tissues and cells.ITIH4 knockdown promoted cell growth and cell cycle progression.Consistent with these results,inhibition of ITIH4 in OC cells significantly increased cell migration and invasion abilities.Cox regression analysis suggests that ITIH4 expression alone is not a good predictor of the prognosis of malignant ovarian tumors in patients.Conclusions:ITIH4 inhibits the progression of OC,suggesting that ITIH4 may be a useful biomarker for OC.This study may provide a potential novel target for the treatment of OC.