BACKGROUND Endogenous regeneration of pancreatic isletβ-cells is a path to cure both type 1 and advanced type 2 diabetes.Pancreatic cancer cell line-1(PANC-1),a human pancreatic islet progenitor cell line,can be indu...BACKGROUND Endogenous regeneration of pancreatic isletβ-cells is a path to cure both type 1 and advanced type 2 diabetes.Pancreatic cancer cell line-1(PANC-1),a human pancreatic islet progenitor cell line,can be induced by trypsin to differentiate into insulin-secreting islet-like aggregates(ILAs).However,the underlying mechanism has not been explored.AIM To explore the mechanism and signaling pathway of trypsin-induced differentiation of islet progenitor cells into insulin-secreting cells.METHODS PANC-1 cells were induced by trypsin to form ILAs and differentiate into insulinsecreting cells.Clustered regularly interspaced short palindromic repeats(CRISPR)-associated protein 9 knockout and small interfering RNA knockdown techniques were used to investigate membrane proteins and downstream signaling pathways involved in the process.RESULTS The extracellular domain of membrane receptor E-cadherin hydrolyzed by trypsin induced the aggregation of PANC-1 cells and stimulated E-cadherin-recruited casein kinase-1γ3,which specifically phosphorylated the Ser655/Thr658 site ofα-catenin in the cadherin-catenin complex,participating in the process of PANC-1 differentiation and affecting the maturation of differentiated ILAs.CONCLUSION The current study reveals the mechanism by which trypsin promotes PANC-1 cell differentiation into islet-like cells,providing a novel approach for endogenous isletβ-cell regeneration.展开更多
Targeting of the synthesized polypeptide in the cells is an important research field in modern cell biology. Cowpea trypsin inhibitor (cpti) gene has been modified and a fusion protein gene (sck) was produced by fusin...Targeting of the synthesized polypeptide in the cells is an important research field in modern cell biology. Cowpea trypsin inhibitor (cpti) gene has been modified and a fusion protein gene (sck) was produced by fusing a signal peptide sequence at cpti 5' end and an endoplasm reticulum (ER) retention signal peptide at cpti3' end respectively. The signal peptide can direct the newly synthesized polypeptide into ER, while ER retention signal can make the protein retained in the ER and its derivative protein body. ELISA test indicated that the accumulation level of foreign CpTI protein in sck transgenic tobacco (Nicotiana tabacum L.) was two times higher than cpti transgenic tobaccos and some individuals were four times higher. At the same time, sck transgenic tobacco has a high resistance to Lepidoptera pest due to the increased accumulation level of foreign CpTI protein. The strategy of foreign protein targeting can be used to increase the accumulation level of foreign protein in transgenic plants and can be widely applied to other related research field in plant genetic engineering.展开更多
A reliable and validated HPLC method was established for the assay of trypsin inhibitors（TI）and it was used in the investigation of the active TI components inMomordica cochinchinensis（Cucurbitaceae family）.The un...A reliable and validated HPLC method was established for the assay of trypsin inhibitors（TI）and it was used in the investigation of the active TI components inMomordica cochinchinensis（Cucurbitaceae family）.The underlying principle of the assay is the measurement of the decrease in trypsin activity due to the presence of TI in analyzed samples,which was achieved by using HPLC separation and quantification of p-nitroanilide that was generated by tryptic hydrolysis of N-α-benzoyl-DL-arginine -4-nitroanilide.The results showed that the HPLC method had higher selectivity than conventional spectrophotometric assay.展开更多
By 30% - 60% (NH4)(2)SO4 fractional precipitation, anion-exchange chromatography on DEAE-Sepharose CL-6B, gel filtration on Sephacryl S-200 and anion-exchange chromatography on Waters AP-1 column (Protein(PM)-Pak DEAE...By 30% - 60% (NH4)(2)SO4 fractional precipitation, anion-exchange chromatography on DEAE-Sepharose CL-6B, gel filtration on Sephacryl S-200 and anion-exchange chromatography on Waters AP-1 column (Protein(PM)-Pak DEAE 15HR), a proteinase which can inactivate soybean trypsin inhibitor (STI) was purified from mung bean ( Vigna rabiata (L.) Wilezek) sprouts. Its molecular weight was estimated to be 29.8 kD by SDS-PAGE, and its K-m and V-max for STI were 769.2 N-alpha -benzoyl-L-arginine ethyl ester BAEE/mL and 115.3 BAEE . mL(-1) . min(-1) respectively. This proteinase was stable at temperatures lower than 50 degreesC and pH 6.5 - 8.5, and 90.91% STI activity of defatted soybean powder was inactivated by this preparation, with proteolytic activity 5 000 BAEE/mL at 50 degreesC and pH 8.0 in 4 h.展开更多
Both non-transgenic hybrid triploid poplars [ (Populus tomentosa×P.bolleana)×P.tomentosa ] and transgenic ones expressing cowpea trypsin inhibitor were cut at the base of the stem to produce auxoblasts, an...Both non-transgenic hybrid triploid poplars [ (Populus tomentosa×P.bolleana)×P.tomentosa ] and transgenic ones expressing cowpea trypsin inhibitor were cut at the base of the stem to produce auxoblasts, and used as source of leaves for insect feeding trials performed on 3 major insect species of poplar, the forest tent caterpillar ( Malacosoma disstria L.), gypsy moth ( Lymantria dispar L.) and willow moth ( Stilpnotia candida Staudinger). The height and basal diameter of trees were measured by the end of that year (2000). The results indicated that the growth elements of transgenic poplars were not interfered by the incorporation of the CpTI gene. Intriguingly, the height and basal diameter of the clone TG04 were much greater than that of the control. The transgenic foliage consumed by insects induced the increase of larval mortality, and decrease of larval wet weight gain, faecal output, pupal weight and egg deposition. Among them 3 transgenic clones, TG04, TG07 and TG71 received special attention for their outstanding insect resistance compared with other transgenic clones, which showed that the CpTI gene in them was expressed more actively and stably than in others.展开更多
AIM:To study two methods for culturing and purifying Sprague-Dawley(SD)rat retinal Muller cells and determine which one is better.METHODS:The passage culture method of Muller cells was respectively carried out by comp...AIM:To study two methods for culturing and purifying Sprague-Dawley(SD)rat retinal Muller cells and determine which one is better.METHODS:The passage culture method of Muller cells was respectively carried out by complete pancreatic enzyme digestion method and repeated incomplete pancreatic enzyme digestion method.After culturing retinal cells for one month through these two methods,fluorescence-activated cell sorter(FACS),RT-PCR,and immunohistochemistry technology were performed to examine the enrichment and purity of Muller glial cells,and carried out two-sample approximate t test using SSPS 13.0 to further compare the Muller cell positive rate in both methods.RESULTS:The statistical results showed that the purity of Muller cells was 83.2%±5.16%in group A,and the purity was 98.5%±1.08%in group B.The two-sample approximate t test analysis demonstrated that the difference between group A and group B was statistically significant(t=-9.178,P【0.005).The results clearly exhibited a difference between the purity of Muller cells cultured by the complete pancreatic enzyme digestion method(group A)and the repeated incomplete pancreatic enzyme digestion method(group B).CONCLUSION:Compared with the complete pancreatic enzyme digestion method,this novel method was more efficient and a higher purity of Muller cells could be obtained using this approach.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
AIM： To investigate the role of cryofibrinogen （CF） in active inflammatory bowel disease （IBD）. METHODS： CF was assayed in 284 subjects： 61 with active and 63 with inactive ulcerative colitis （UC）, 45 who had...AIM： To investigate the role of cryofibrinogen （CF） in active inflammatory bowel disease （IBD）. METHODS： CF was assayed in 284 subjects： 61 with active and 63 with inactive ulcerative colitis （UC）, 45 who had proctocolectomy, 35 with active and 20 with inactive Crohn＇s disease （CD）, 40 with other diseases and 20 healthy controls. Trypsin inhibitor （TI） and TI antibody （TI-Ab） were measured in plasma and CF complex by ELISA. RESULTS： CF in active UC was strikingly high compared with all other groups （x^2〈0.001）. Similarly, CF was significantly higher in active CD than in inactive CD or in controls （x^2〈0.01）. In UC, high CF and TI-Ab were associated with the need for operations. Further, high CF, CF/fibrinogen ratio, low TI and high TI-Ab in plasma were associated with disease activity or refractoriness to medication. Elevated CF was not associated with acute reactants like C-reactive protein and white blood cell counts except for erythrocyte sedimentation rate, suggesting that elevated CF was not a consequence of acute inflammation. CONCLUSION： Elevated CF in active IBD appears to be morbigenous. CF promotes IBD via two main mechanisms, quenching of TI （an anti-inflammatory substance） and impairing microvascular perfusion by forming protein aggregates. CF may also serve as a biomarker of chronic IBD. Additional studies are warranted to fully evaluate the role of CF in IBD and the outcome should contribute to a better understanding of the pathogenesis of IBD.展开更多
AIM: To examine the role of p38 during acute experimental cerulein pancreatitis. METHODS: Rats were treated with cerulein with or without a specific JNK inhibitor (CEP1347) and/or a specific p38 inhibitor (SB203580) a...AIM: To examine the role of p38 during acute experimental cerulein pancreatitis. METHODS: Rats were treated with cerulein with or without a specific JNK inhibitor (CEP1347) and/or a specific p38 inhibitor (SB203580) and pancreatic stress kinase activity was determined. Parameters to assess pancreatitis included trypsin, amylase, lipase, pancreatic weight and histology. RESULTS: JNK inhibition with CEP1347 ameliorated pancreatitis, reducing pancreatic edema. In contrast, p38 inhibition with SB203580 aggravated pancreatitis with higher trypsin levels and, with induction of acinar necrosis not normally found after cerulein hyperstimulation. Simultaneous treatment with both CEP1347 and SB203580 mutually abolished the effects of either compound on cerulein pancreatitis. CONCLUSION: Stress kinases modulate pancreatitis differentially. JNK seems to promote pancreatitis development, possibly by supporting inflammatory reactions such as edema formation while its inhibition ameliorates pancreatitis. In contrast, p38 may help reduce organ destruction while inhibition of p38 during induction of cerulein pancreatitis leads to the occurrence of acinar necrosis.展开更多
Hereditary pancreatitis is an autosomal dominant condition,which results in recurrent attacks of acute pancreatitis,progressing to chronic pancreatitis often at a young age.The majority of patients with hereditary pan...Hereditary pancreatitis is an autosomal dominant condition,which results in recurrent attacks of acute pancreatitis,progressing to chronic pancreatitis often at a young age.The majority of patients with hereditary pancreatitis expressone of two mutations (R122H or N29I) in the cationictrypsinogen gene (PRSS1 gene). It has been hypothesisedthat one of these mutations, the R122H mutation causespancreatitis by altering a trypsin recognition site sopreventing deactivation of trypsin within the pancreas andprolonging its action, resulting in autodigestion. Families withthese two mutations have been identified in many countriesand there are also other rarer mutations, which have alsobeen linked to hereditary pancreatitis.Patients with hereditary pancreatitis present in the sameway as those with sporadic pancreatitis but at an earlierage. It is common for patients to remain undiagnosed formany years, particularly ifthey present with non-specificsymptoms. Hereditary pancreatitis should always beconsidered in patients who present with recurrent pancreatitiswith a family history of pancreatic disease. If patients withthe 2 common mutations are compared, those with theR122H mutation are more likely to present at a younger ageand are more likely to require surgical intervention than thosewith N29I. Hereditary pancreatitis carries a 40 % lifetimerisk of pancreatic cancer with those patients aged between50 to 70 being most at risk in whom screening tests maybecome important.展开更多
AIM: To assess the effect of non-selective ETA/B (LU 302872)and selective ETA (LU 302146) antagonist on pancreatic histology and ultrastructure of acinar cells in connection with trypsinogen activation in early caerul...AIM: To assess the effect of non-selective ETA/B (LU 302872)and selective ETA (LU 302146) antagonist on pancreatic histology and ultrastructure of acinar cells in connection with trypsinogen activation in early caerulein-induced AP.METHODS: Male Wistar rats with caerulein-induced AP,lasting 4 h, were treated i.p. with 10 and 20 mg/kg b.w.of each antagonist. Edema, inflammatory infiltration,necrosis and vacuolization of acinar cells in the pancreas were scored at 0-3 scale. Free active trypsin (FAT), total potential trypsin (TPT) after activation with enterokinase,and index of trypsinogen activation (%FAT/TPT) were assayed in pancreatic homogenates.RESULTS: In untreated AP, the edema, inflammatory infiltration, necrosis and vacuolization increased as compared to control healthy rats (P＜0.01). None of the treatment exerted any meaningful effect on the edema and inflammatory infiltration. The selective antagonist increased slightly the necrosis score to 0.82±0.06 at higher dose (P＜0.05) vs 0.58±0.06 in untreated AP. The nonselective antagonist increased slightly the vacuolization score to 2.41±0.07 at higher dose (P＜0.01) vs 1.88±0.08in untreated AP. The decrease in the number of zymogen granules, disorganization of endoplasmic reticulum,autophagosomes and cytoplasmic vacuoles were more prominent in treated AP than in untreated AP groups.%FAT/TPT in untreated AP increased about four times (18.4±3.8 vs4.8±1.3 in control group without AP, P＜0.001).Treatment of AP with both antagonists did not affect significantly augmented trypsinogen activation.CONCLUSION: The treatment with endothelin-1 receptors (non-selective ETA/B and selective ETA) antagonists has essential effect neither on the edema and inflammatory infiltration nor on trypsinogen activation observed in the early course of caerulein-induced AP. Nevertheless a slight increase of the necrosis and vacuolization score and some of the ultrastructural data could suggest the possibility of their undesired effects in caerulein-induced AP at investigated doses.展开更多
In this article, the influence factors of trypsin extracted from crude pancreatin was investigated, and scanning turmeling microscope（STM） was used to observe the image of trypsin in butane-diacid-2-ethyl-hexyl-este...In this article, the influence factors of trypsin extracted from crude pancreatin was investigated, and scanning turmeling microscope（STM） was used to observe the image of trypsin in butane-diacid-2-ethyl-hexyl-ester-sulfonic sodium （AOT）/iso-octane reversed micelles. The STM image showed that trypsins bounded in reversed micelles was rigid, which weakened its conjugative effect and caused maximum ultraviolet absorption and fluorescence emissive absorption moving toward blue waves. AOT concentration, pH and cations were the main influence factors of extraction. Specifically, extraction percentage of trypsin decreased with the increase of AOT concentration from 0.01 to 0.1mol·L^-1. When pH value is from 5.30 to 10.0, i.e. less than pI of trypsin, the extraction percentage is raised with the different increase of pI-pH, but when the pH value is less than 5.20, the extraction percentage is decreased with the acidity added. Besides, the extraction efficiency is negative, related with the concentrations of Ca^2＋, Na^＋, K^＋ which were in the range of 0.2-1.0mol.L^-1, and influence of concentration of Ca^2＋ is greater than that of Na^＋, and K^＋ which has the minimum impact with the same concentration. Finally, optimum conditions to extract trypsin were： AOT reversed micelles 0.05mol·L^-1, trypsin concentration in crude pancreatin solution 3mg·ml^-1, pH 5.2-- 5.3, ratio （by volume） of extraction phase to strip-extraction phase 1 ： 1, and time of 5min. The corresponding percentage of extraction was 22.7% and specific activity was 78.9 N-benzoyl-L-arginlne ethyl ester （BAEE） U·mg^-1 protein, three times than that in crude pancreatin. There was no lipase and amylopsin activity was decreased to 1/5 of crude pancreatin. Partly purifying solution was treated by condition mentioned above with 0.05mol·L^-1 ceryl-trimethyl-ammonium bromide （CTAB）, total extraction percentage of trypsin was 74.18% and specific activity was 3148.3 BAEE U·mg^-1, i.e. 48.16 times purer than that in crude pancreatin. Through sodium dodecyl sulfate-polyacryl amide gel electrophoresis （SDS-PAGE） and image analysis of extracted product, there were only three bands in the trypsin, while seven in crude pancreatin, and electrophoresis location of main bend was almost identical with the standard enzyme.展开更多
In the present study, the cleavage activities of both recombinant and bovine enzymes （trypsin and carboxy peptidase B） were determined using proinsulin fusion protein. The proteolysis products were analyzed with HPL...In the present study, the cleavage activities of both recombinant and bovine enzymes （trypsin and carboxy peptidase B） were determined using proinsulin fusion protein. The proteolysis products were analyzed with HPLC. We found that recombinant enzymes had stronger activities and produced fewer by-products. In addition, recombinant enzymes were more efficient in the production of human insulin than bovine enzymes.展开更多
Conserved dopamine neurotrophic factor protects and rescues dopaminergic neurodegeneration induced by 6-hydroxydopamine in vivo,but its potential value in treating Parkinson＇s disease remains controversial.Here,we us...Conserved dopamine neurotrophic factor protects and rescues dopaminergic neurodegeneration induced by 6-hydroxydopamine in vivo,but its potential value in treating Parkinson＇s disease remains controversial.Here,we used the proteasome inhibitors lactacystin and MG132 to induce neurodegeneration of PC12 cells.Afterwards,conserved dopamine neurotrophic factor was administrated as a therapeutic factor,both pretreatment and posttreatment.Our results showed that（1）conserved dopamine neurotrophic factor enhanced lactacystin/MG132-induced cell viability and morphology,and attenuated alpha-synuclein accumulation in differentiated PC12 cells.（2）Enzyme linked immunosorbent assay showed up-regulated 26S proteasomal activity in MG132-induced PC12 cells after pre-and posttreatment with conserved dopamine neurotrophic factor.Similarly,26S proteasome activity was upregulated in lactacystin-induced PC12 cells pretreated with conserved dopamine neurotrophic factor.（3）With regard proteolytic enzymes（specifically,glutamyl peptide hydrolase,chymotrypsin,and trypsin）,glutamyl peptide hydrolase activity was up-regulated in lactacystin/MG132-administered PC12 cells after pre-and posttreatment with conserved dopamine neurotrophic factor.However,upregulation of chymotrypsin activity was only observed in MG132-administered PC12 cells pretreated with conserved dopamine neurotrophic factor.There was no change in trypsin expression.We conclude that conserved dopamine neurotrophic factor develops its neurotrophic effects by modulating proteasomal activities,and thereby protects and rescues PC12 cells against neurodegeneration.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
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
Abstract The digestive enzyme activity and mRNA level of trypsin during the embryonic development of Cherax quadricarinatus were analyzed using biochemical and Fluorogenic Quantitative PCR （FQ-PCR） methods. The resu...Abstract The digestive enzyme activity and mRNA level of trypsin during the embryonic development of Cherax quadricarinatus were analyzed using biochemical and Fluorogenic Quantitative PCR （FQ-PCR） methods. The results show that the activities of trypsin and chymotrypsin had two different change patterns. Trypsin specific activity increased rapidly in the early stages of development and still remained high in preparation for the hatch stage. However, chymotrypsin activity peaked in stage 4 of embryonic development and decreased significantly in the last stage. The mRNA level of trypsin was elevated in all stages and two peak values were observed in stages 2 and 5 respectively. The results indicate that trypsin is very important for the utilization of the yolk during embryonic development and for the assimilation of dietary protein for larvae. The gene of trypsin is probably regulated at transcriptional level. The mRNA levels of trypsin can reflect not only trypsin activity, but also the regulatory mechanism for expression of trypsin gene to a certain degree.展开更多
基金Supported by the National Natural Science Foundation of China,No.82073908.
文摘BACKGROUND Endogenous regeneration of pancreatic isletβ-cells is a path to cure both type 1 and advanced type 2 diabetes.Pancreatic cancer cell line-1(PANC-1),a human pancreatic islet progenitor cell line,can be induced by trypsin to differentiate into insulin-secreting islet-like aggregates(ILAs).However,the underlying mechanism has not been explored.AIM To explore the mechanism and signaling pathway of trypsin-induced differentiation of islet progenitor cells into insulin-secreting cells.METHODS PANC-1 cells were induced by trypsin to form ILAs and differentiate into insulinsecreting cells.Clustered regularly interspaced short palindromic repeats(CRISPR)-associated protein 9 knockout and small interfering RNA knockdown techniques were used to investigate membrane proteins and downstream signaling pathways involved in the process.RESULTS The extracellular domain of membrane receptor E-cadherin hydrolyzed by trypsin induced the aggregation of PANC-1 cells and stimulated E-cadherin-recruited casein kinase-1γ3,which specifically phosphorylated the Ser655/Thr658 site ofα-catenin in the cadherin-catenin complex,participating in the process of PANC-1 differentiation and affecting the maturation of differentiated ILAs.CONCLUSION The current study reveals the mechanism by which trypsin promotes PANC-1 cell differentiation into islet-like cells,providing a novel approach for endogenous isletβ-cell regeneration.
文摘Targeting of the synthesized polypeptide in the cells is an important research field in modern cell biology. Cowpea trypsin inhibitor (cpti) gene has been modified and a fusion protein gene (sck) was produced by fusing a signal peptide sequence at cpti 5' end and an endoplasm reticulum (ER) retention signal peptide at cpti3' end respectively. The signal peptide can direct the newly synthesized polypeptide into ER, while ER retention signal can make the protein retained in the ER and its derivative protein body. ELISA test indicated that the accumulation level of foreign CpTI protein in sck transgenic tobacco (Nicotiana tabacum L.) was two times higher than cpti transgenic tobaccos and some individuals were four times higher. At the same time, sck transgenic tobacco has a high resistance to Lepidoptera pest due to the increased accumulation level of foreign CpTI protein. The strategy of foreign protein targeting can be used to increase the accumulation level of foreign protein in transgenic plants and can be widely applied to other related research field in plant genetic engineering.
基金National Natural Science Foundation of China (Grant No 30873405)National Drug Innovation Program(Grant No.2009ZX09301-011)
文摘A reliable and validated HPLC method was established for the assay of trypsin inhibitors（TI）and it was used in the investigation of the active TI components inMomordica cochinchinensis（Cucurbitaceae family）.The underlying principle of the assay is the measurement of the decrease in trypsin activity due to the presence of TI in analyzed samples,which was achieved by using HPLC separation and quantification of p-nitroanilide that was generated by tryptic hydrolysis of N-α-benzoyl-DL-arginine -4-nitroanilide.The results showed that the HPLC method had higher selectivity than conventional spectrophotometric assay.
文摘By 30% - 60% (NH4)(2)SO4 fractional precipitation, anion-exchange chromatography on DEAE-Sepharose CL-6B, gel filtration on Sephacryl S-200 and anion-exchange chromatography on Waters AP-1 column (Protein(PM)-Pak DEAE 15HR), a proteinase which can inactivate soybean trypsin inhibitor (STI) was purified from mung bean ( Vigna rabiata (L.) Wilezek) sprouts. Its molecular weight was estimated to be 29.8 kD by SDS-PAGE, and its K-m and V-max for STI were 769.2 N-alpha -benzoyl-L-arginine ethyl ester BAEE/mL and 115.3 BAEE . mL(-1) . min(-1) respectively. This proteinase was stable at temperatures lower than 50 degreesC and pH 6.5 - 8.5, and 90.91% STI activity of defatted soybean powder was inactivated by this preparation, with proteolytic activity 5 000 BAEE/mL at 50 degreesC and pH 8.0 in 4 h.
文摘Both non-transgenic hybrid triploid poplars [ (Populus tomentosa×P.bolleana)×P.tomentosa ] and transgenic ones expressing cowpea trypsin inhibitor were cut at the base of the stem to produce auxoblasts, and used as source of leaves for insect feeding trials performed on 3 major insect species of poplar, the forest tent caterpillar ( Malacosoma disstria L.), gypsy moth ( Lymantria dispar L.) and willow moth ( Stilpnotia candida Staudinger). The height and basal diameter of trees were measured by the end of that year (2000). The results indicated that the growth elements of transgenic poplars were not interfered by the incorporation of the CpTI gene. Intriguingly, the height and basal diameter of the clone TG04 were much greater than that of the control. The transgenic foliage consumed by insects induced the increase of larval mortality, and decrease of larval wet weight gain, faecal output, pupal weight and egg deposition. Among them 3 transgenic clones, TG04, TG07 and TG71 received special attention for their outstanding insect resistance compared with other transgenic clones, which showed that the CpTI gene in them was expressed more actively and stably than in others.
基金National Natural Scientific Foundation of China(No.81170844)all our experiments in the State Key Lab of Medical Genetics of China(Grant number 1989DA105084)
文摘AIM:To study two methods for culturing and purifying Sprague-Dawley(SD)rat retinal Muller cells and determine which one is better.METHODS:The passage culture method of Muller cells was respectively carried out by complete pancreatic enzyme digestion method and repeated incomplete pancreatic enzyme digestion method.After culturing retinal cells for one month through these two methods,fluorescence-activated cell sorter(FACS),RT-PCR,and immunohistochemistry technology were performed to examine the enrichment and purity of Muller glial cells,and carried out two-sample approximate t test using SSPS 13.0 to further compare the Muller cell positive rate in both methods.RESULTS:The statistical results showed that the purity of Muller cells was 83.2%±5.16%in group A,and the purity was 98.5%±1.08%in group B.The two-sample approximate t test analysis demonstrated that the difference between group A and group B was statistically significant(t=-9.178,P【0.005).The results clearly exhibited a difference between the purity of Muller cells cultured by the complete pancreatic enzyme digestion method(group A)and the repeated incomplete pancreatic enzyme digestion method(group B).CONCLUSION:Compared with the complete pancreatic enzyme digestion method,this novel method was more efficient and a higher purity of Muller cells could be obtained using this approach.
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.
文摘AIM： To investigate the role of cryofibrinogen （CF） in active inflammatory bowel disease （IBD）. METHODS： CF was assayed in 284 subjects： 61 with active and 63 with inactive ulcerative colitis （UC）, 45 who had proctocolectomy, 35 with active and 20 with inactive Crohn＇s disease （CD）, 40 with other diseases and 20 healthy controls. Trypsin inhibitor （TI） and TI antibody （TI-Ab） were measured in plasma and CF complex by ELISA. RESULTS： CF in active UC was strikingly high compared with all other groups （x^2〈0.001）. Similarly, CF was significantly higher in active CD than in inactive CD or in controls （x^2〈0.01）. In UC, high CF and TI-Ab were associated with the need for operations. Further, high CF, CF/fibrinogen ratio, low TI and high TI-Ab in plasma were associated with disease activity or refractoriness to medication. Elevated CF was not associated with acute reactants like C-reactive protein and white blood cell counts except for erythrocyte sedimentation rate, suggesting that elevated CF was not a consequence of acute inflammation. CONCLUSION： Elevated CF in active IBD appears to be morbigenous. CF promotes IBD via two main mechanisms, quenching of TI （an anti-inflammatory substance） and impairing microvascular perfusion by forming protein aggregates. CF may also serve as a biomarker of chronic IBD. Additional studies are warranted to fully evaluate the role of CF in IBD and the outcome should contribute to a better understanding of the pathogenesis of IBD.
文摘AIM: To examine the role of p38 during acute experimental cerulein pancreatitis. METHODS: Rats were treated with cerulein with or without a specific JNK inhibitor (CEP1347) and/or a specific p38 inhibitor (SB203580) and pancreatic stress kinase activity was determined. Parameters to assess pancreatitis included trypsin, amylase, lipase, pancreatic weight and histology. RESULTS: JNK inhibition with CEP1347 ameliorated pancreatitis, reducing pancreatic edema. In contrast, p38 inhibition with SB203580 aggravated pancreatitis with higher trypsin levels and, with induction of acinar necrosis not normally found after cerulein hyperstimulation. Simultaneous treatment with both CEP1347 and SB203580 mutually abolished the effects of either compound on cerulein pancreatitis. CONCLUSION: Stress kinases modulate pancreatitis differentially. JNK seems to promote pancreatitis development, possibly by supporting inflammatory reactions such as edema formation while its inhibition ameliorates pancreatitis. In contrast, p38 may help reduce organ destruction while inhibition of p38 during induction of cerulein pancreatitis leads to the occurrence of acinar necrosis.
文摘Hereditary pancreatitis is an autosomal dominant condition,which results in recurrent attacks of acute pancreatitis,progressing to chronic pancreatitis often at a young age.The majority of patients with hereditary pancreatitis expressone of two mutations (R122H or N29I) in the cationictrypsinogen gene (PRSS1 gene). It has been hypothesisedthat one of these mutations, the R122H mutation causespancreatitis by altering a trypsin recognition site sopreventing deactivation of trypsin within the pancreas andprolonging its action, resulting in autodigestion. Families withthese two mutations have been identified in many countriesand there are also other rarer mutations, which have alsobeen linked to hereditary pancreatitis.Patients with hereditary pancreatitis present in the sameway as those with sporadic pancreatitis but at an earlierage. It is common for patients to remain undiagnosed formany years, particularly ifthey present with non-specificsymptoms. Hereditary pancreatitis should always beconsidered in patients who present with recurrent pancreatitiswith a family history of pancreatic disease. If patients withthe 2 common mutations are compared, those with theR122H mutation are more likely to present at a younger ageand are more likely to require surgical intervention than thosewith N29I. Hereditary pancreatitis carries a 40 % lifetimerisk of pancreatic cancer with those patients aged between50 to 70 being most at risk in whom screening tests maybecome important.
基金Supported by the Medical University of Bialystok within the Project #30-12770
文摘AIM: To assess the effect of non-selective ETA/B (LU 302872)and selective ETA (LU 302146) antagonist on pancreatic histology and ultrastructure of acinar cells in connection with trypsinogen activation in early caerulein-induced AP.METHODS: Male Wistar rats with caerulein-induced AP,lasting 4 h, were treated i.p. with 10 and 20 mg/kg b.w.of each antagonist. Edema, inflammatory infiltration,necrosis and vacuolization of acinar cells in the pancreas were scored at 0-3 scale. Free active trypsin (FAT), total potential trypsin (TPT) after activation with enterokinase,and index of trypsinogen activation (%FAT/TPT) were assayed in pancreatic homogenates.RESULTS: In untreated AP, the edema, inflammatory infiltration, necrosis and vacuolization increased as compared to control healthy rats (P＜0.01). None of the treatment exerted any meaningful effect on the edema and inflammatory infiltration. The selective antagonist increased slightly the necrosis score to 0.82±0.06 at higher dose (P＜0.05) vs 0.58±0.06 in untreated AP. The nonselective antagonist increased slightly the vacuolization score to 2.41±0.07 at higher dose (P＜0.01) vs 1.88±0.08in untreated AP. The decrease in the number of zymogen granules, disorganization of endoplasmic reticulum,autophagosomes and cytoplasmic vacuoles were more prominent in treated AP than in untreated AP groups.%FAT/TPT in untreated AP increased about four times (18.4±3.8 vs4.8±1.3 in control group without AP, P＜0.001).Treatment of AP with both antagonists did not affect significantly augmented trypsinogen activation.CONCLUSION: The treatment with endothelin-1 receptors (non-selective ETA/B and selective ETA) antagonists has essential effect neither on the edema and inflammatory infiltration nor on trypsinogen activation observed in the early course of caerulein-induced AP. Nevertheless a slight increase of the necrosis and vacuolization score and some of the ultrastructural data could suggest the possibility of their undesired effects in caerulein-induced AP at investigated doses.
文摘In this article, the influence factors of trypsin extracted from crude pancreatin was investigated, and scanning turmeling microscope（STM） was used to observe the image of trypsin in butane-diacid-2-ethyl-hexyl-ester-sulfonic sodium （AOT）/iso-octane reversed micelles. The STM image showed that trypsins bounded in reversed micelles was rigid, which weakened its conjugative effect and caused maximum ultraviolet absorption and fluorescence emissive absorption moving toward blue waves. AOT concentration, pH and cations were the main influence factors of extraction. Specifically, extraction percentage of trypsin decreased with the increase of AOT concentration from 0.01 to 0.1mol·L^-1. When pH value is from 5.30 to 10.0, i.e. less than pI of trypsin, the extraction percentage is raised with the different increase of pI-pH, but when the pH value is less than 5.20, the extraction percentage is decreased with the acidity added. Besides, the extraction efficiency is negative, related with the concentrations of Ca^2＋, Na^＋, K^＋ which were in the range of 0.2-1.0mol.L^-1, and influence of concentration of Ca^2＋ is greater than that of Na^＋, and K^＋ which has the minimum impact with the same concentration. Finally, optimum conditions to extract trypsin were： AOT reversed micelles 0.05mol·L^-1, trypsin concentration in crude pancreatin solution 3mg·ml^-1, pH 5.2-- 5.3, ratio （by volume） of extraction phase to strip-extraction phase 1 ： 1, and time of 5min. The corresponding percentage of extraction was 22.7% and specific activity was 78.9 N-benzoyl-L-arginlne ethyl ester （BAEE） U·mg^-1 protein, three times than that in crude pancreatin. There was no lipase and amylopsin activity was decreased to 1/5 of crude pancreatin. Partly purifying solution was treated by condition mentioned above with 0.05mol·L^-1 ceryl-trimethyl-ammonium bromide （CTAB）, total extraction percentage of trypsin was 74.18% and specific activity was 3148.3 BAEE U·mg^-1, i.e. 48.16 times purer than that in crude pancreatin. Through sodium dodecyl sulfate-polyacryl amide gel electrophoresis （SDS-PAGE） and image analysis of extracted product, there were only three bands in the trypsin, while seven in crude pancreatin, and electrophoresis location of main bend was almost identical with the standard enzyme.
文摘In the present study, the cleavage activities of both recombinant and bovine enzymes （trypsin and carboxy peptidase B） were determined using proinsulin fusion protein. The proteolysis products were analyzed with HPLC. We found that recombinant enzymes had stronger activities and produced fewer by-products. In addition, recombinant enzymes were more efficient in the production of human insulin than bovine enzymes.
基金supported by the Natural Science Foundation of Anhui Province of China,No.11040606Q11the National Natural Science Foundation of China,No.81100960
文摘Conserved dopamine neurotrophic factor protects and rescues dopaminergic neurodegeneration induced by 6-hydroxydopamine in vivo,but its potential value in treating Parkinson＇s disease remains controversial.Here,we used the proteasome inhibitors lactacystin and MG132 to induce neurodegeneration of PC12 cells.Afterwards,conserved dopamine neurotrophic factor was administrated as a therapeutic factor,both pretreatment and posttreatment.Our results showed that（1）conserved dopamine neurotrophic factor enhanced lactacystin/MG132-induced cell viability and morphology,and attenuated alpha-synuclein accumulation in differentiated PC12 cells.（2）Enzyme linked immunosorbent assay showed up-regulated 26S proteasomal activity in MG132-induced PC12 cells after pre-and posttreatment with conserved dopamine neurotrophic factor.Similarly,26S proteasome activity was upregulated in lactacystin-induced PC12 cells pretreated with conserved dopamine neurotrophic factor.（3）With regard proteolytic enzymes（specifically,glutamyl peptide hydrolase,chymotrypsin,and trypsin）,glutamyl peptide hydrolase activity was up-regulated in lactacystin/MG132-administered PC12 cells after pre-and posttreatment with conserved dopamine neurotrophic factor.However,upregulation of chymotrypsin activity was only observed in MG132-administered PC12 cells pretreated with conserved dopamine neurotrophic factor.There was no change in trypsin expression.We conclude that conserved dopamine neurotrophic factor develops its neurotrophic effects by modulating proteasomal activities,and thereby protects and rescues PC12 cells against neurodegeneration.
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.
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.
基金Supported by the NNSF of China (No.30670227)hanghai Agricultural Science & Technology Key Grant [6-1(2006)].
文摘Abstract The digestive enzyme activity and mRNA level of trypsin during the embryonic development of Cherax quadricarinatus were analyzed using biochemical and Fluorogenic Quantitative PCR （FQ-PCR） methods. The results show that the activities of trypsin and chymotrypsin had two different change patterns. Trypsin specific activity increased rapidly in the early stages of development and still remained high in preparation for the hatch stage. However, chymotrypsin activity peaked in stage 4 of embryonic development and decreased significantly in the last stage. The mRNA level of trypsin was elevated in all stages and two peak values were observed in stages 2 and 5 respectively. The results indicate that trypsin is very important for the utilization of the yolk during embryonic development and for the assimilation of dietary protein for larvae. The gene of trypsin is probably regulated at transcriptional level. The mRNA levels of trypsin can reflect not only trypsin activity, but also the regulatory mechanism for expression of trypsin gene to a certain degree.
