Development and characterization of monoclonal antibodies against canine trypsin

Canine cationic trypsin was purified by salting-out, gel filtration and affinity chromatography. Purity was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was ca. 28 kDa by SDS-PAGE.

Thirty hybridomas were obtained which produced mAb to canine cationic trypsin by the cell fusion technique. Twenty-two of these recognized cationic trypsin only, while eight hybridomas recognized both cationic and anionic trypsin. Several of the anti-canine cationic trypsin mAb were purified by salting-out and DEAE ion-change chromatography using ascites fluid of immunized BALB/c mice. The mAb proved to have very high specificity to canine cationic trypsin as shown by immunoblotting and it may be possible to use them to develop clinical assays.     

Variability in the antinutritional constituents in greengram Vigna radiata

Five greengram varieties, viz. Mg 161, M₃, Co₂, Pusa 8793 and Pusa baisakhi, were analyzed for phytic acid, tannic acid and trypsin activity. Significant varietal variation in tannin (310 to 400 mg percent), phytic acid (201.33 to 265.33 mg percent) and trypsin inhibitor activity (55.74 to 97.70 TIU/mg) were observed.     

Comparison of two methods for culturing neonatal rat cardiac fibroblasts

AIM:To establish and compare the methods for culturing neonatal rat cardiac fibroblasts culture. METHODS:Neonatal rat hearts were isolated by collagenase+trypsin or trypsin digestion. The cardiomyocytes and cardiac fibroblasts were isolated by different attachment techniques. The cellular morphology, purity and reactions to the reagent were tested. RESULTS:For morphology and purity, no difference between the 2 methods was observed, though more myocytes were harvested by the method of collagenase+trypsin digestion. For the cellular responses to reagent, the cardiac fibroblasts harvested with trypsin digestion had more potent proliferative ability than those with collagenase+trypsin digestion. CONCLUSION:There is no difference of cellular morphology and purity in the cardiac fibroblasts isolated by collagenase+trypsin digestion and trypsin digestion, but the fibroblasts with trypsin digestion have more potent proliferative ability.     

大豆抗营养因子对生长蛋鸡生产性能、氮代谢和小肠内胰蛋白酶活性的影响

为了观察不同梯度大豆抗营养因子（Ａｎｔｉｎｕｔｒｉｔｉｏｎａｌｆａｃｔｏｒｓ、简称ＡＮＦｓ）对不同生理阶段的生长蛋鸡的影响,本试验采用４８０只海兰褐蛋用雏鸡,以熟豆饼日粮为对照,从３或９周开始给饲３个水平的生大豆日粮（４％,９％和１３％）,每期每个日粮４个重复。在３～８周和９～１８周观察雏鸡的采食量,日增重和饲料效率。在第８和１７周分别测定各组的粗蛋白质和干物质的代谢率。并于８、１３和１８周末每个重复随机抽取试鸡两只屠宰,观察小肠内胰蛋白酶活性的变化。结果表明,３个水平的大豆ＡＮＦｓ对育雏期雏鸡的增重没有影响,育成期对照组的增重明显高于采食生大豆组（Ｐ＜０．０５）,大豆ＡＮＦｓ使饲料效率降低,但对氮和干物质的代谢影响不显著;８、１３和１８周龄雏鸡小肠内胰蛋白酶活性亦无显著变化。     

Proteolytic activity in soil: A review

The aim of this work is to review current knowledge on inputs, sources and regulation of protease activities in soils from different ecosystems, while exploring limitations to proteolysis and N mineralisation. Extracellular proteases enter the soil via microbial production and other sources, including plant root exudates, animal excrements, decomposition processes and leaching from agro-industrial fertilisers. The synthesis and activities of proteases in soil are regulated by many factors, including climate, soil properties and the presence of organic compounds of plant and microbial origin. Two particularly important areas for future research are the regulation of proteolysis by low-molecular-weight organic compounds, including amino acids, sugars, flavonoids, plant hormones and siderophores, as well as the identification and characterisation of proteinaceous protease inhibitors of plant and microbial origin in the soil. Despite all the work that has been performed on soil proteases, our understanding of the roles of extracellular plant root proteases in N nutrition is weak. Furthermore, the regulation of soil proteolytic activities of different ecosystems, especially in terms of pollutant inputs and the impact of climate change, requires investigation. Other areas that pose important questions for the future include assessments of protease inhibitor inputs to the soil, regulation of these inhibitors via naturally occurring soil organic compounds and the interactions between soil organisms.     

脲酶测定法判定豆浆抗营养因子热失活情况研究

大豆抗营养因子的主要成分是胰蛋白酶抑制剂,在大豆制品加工过程中,通常采用高温加热钝化的方法使胰蛋白酶抑制剂失去活性。豆浆是人们日常生活中喜欢的饮品之一,因为脲酶测定方法比较简单,目前多以加热钝化后豆浆脲酶活性来表示抗营养因子的去除情况。本研究通过测定豆浆中胰蛋白酶抑制剂和脲酶的活性研究豆浆加热过程中二者失活是否具有一致性,判定以脲酶活性来体现豆浆安全的科学性,同时通过豆浆加热程度与胰蛋白酶抑制剂失活情况的关系,对家庭豆浆制作提供一种安全的加热模式。     

Effect of Acibenzolar-S-methyl (ASM) pre-treatment in inducing resistance against Pythium aphanidermatum infection in Curcuma longa

In vitro experiments were carried out to test the efficacy of plant activator Acibenzolar-S-methyl (ASM, a benzothiadiazole derivative; trade name Bion 50WG) against rhizome rot disease of turmeric (Curcuma longa L.) caused by Pythium aphanidermatum. The plant activator was applied as a liquid rhizome pre-treatment followed by inoculation with P. aphanidermatum. Cell death, activities of pathogenesis related (PR) proteins such as cysteine protease (EC 3.4.22), peroxidases (EC 1.11.1.7) both soluble and ionically bound (IB), trypsin inhibitor (EC 3.4.21.1) and chymotrypsin inhibitor (EC 3.4.21.4) were monitored. Rhizome pre-treatment was effective in controlling P. aphanidermatum infection. Anatomical observation of turmeric rhizomes indicated the presence of calcium oxalate deposits in infected tissue and an accumulation of starch grains in response to infection by P. aphanidermatum. Pathogen infection also induced new basic polypeptides corresponding to 18.0 and 41.0 kDa. Induction of protease, protease inhibitors, soluble and ionically bound peroxidase activity was observed after ASM pre-treatment and P. aphanidermatum infection. ASM treatment also enhanced activities of proteases and peroxidase in rhizomes already infected with P. aphanidermatum. Increases in enzyme activities and protease inhibitors occurred much more rapidly and were enhanced in P. aphanidermatum infected rhizomes that were previously treated with ASM suggesting that increased activities of peroxidases and protease inhibitors may play a key role in restricting the development of disease symptoms on the rhizomes infected with P. aphanidermatum as evidenced by a reduction in cell death. Hence, pretreatment with ASM suppress the P. aphanidermatum induced oxidative damage through higher accumulation of peroxidases and induced defense through activities of protease inhibitors thereby, protected turmeric rhizomes from rhizome rot disease.     

The tryptophan-rich domain of puroindoline is directly associated with the starch granule surface as judged by tryptic shaving and mass spectrometry

The starch granule surface is a frontline of microbial attack and defence, operating in the background of normal starch granule metabolism. Puroindoline, a wheat protein which binds starch granule surfaces, contains a unique tryptophan-rich domain likely responsible for this property, though direct evidence is lacking. To test puroindoline’s tight association, prime starch granule extracts were water-washed 8 or 20 times and residual puroindoline removed using a solution of 50% isopropanol/50 mM NaCl. We found that this solvent was consistent in the amount of protein extracted from wheat flour and washed starch, regardless of initial protein content. Relative quantification of puroindoline following water-washing was performed using dot blot. Washing more than 8 times did not further reduce puroindoline content of starch granules suggesting a strong association with the starch granule surface. To identify the tryptophan-rich domain tightly associated with the starch granule surface, a combination of in situ tryptic digestion and mass spectrometry was used. Following digestion and water-washing, 50% isopropanol/50 mM NaCl was used to remove tightly-associated peptides for identification by mass spectrometry. Using this method, we identified the tryptophan-rich domain of puroindoline directly bound to the starch granule surface of wheat.     

Changes in trypsin and chymotrypsin inhibitory activity on soaking of sorghum (Sorghum bicolor L. Moench)

Changes in trypsin and chymotrypsin inhibitory activity of sorghum (Sorghum bicolor L. Moench) seeds on soaking in distilled water, different salt solutions and mixed salt solution were investigated. A greater reduction in proteinase inhibitory activity was observed on soaking in salt solution than on soaking in distilled water. Maximum loss of trypsin and chymotrypsin inhibitory activity was observed on soaking seeds in mixed salt solution.     

黑眶蟾蜍皮肤白蛋白的纯化及胰蛋白酶抑制活性

[目的]探讨黑眶蟾蜍皮肤白蛋白的纯化及胰蛋白酶抑制活性。[方法]通过离子交换层析和凝胶过滤层析,从黑眶蟾蜍皮肤中得到其白蛋白,命名为BufomA-skin。65nmol／L的BufomA-skin分别与30nmol／Ltrypsin在25℃不同保温时间（0．25—24h）下测定胰蛋白酶抑制活性,计算剩余酶活力。[结果]BufomA-skin为单链蛋白,分子量约为66．7kDa。但在非还原状态下,50～66kDa条带呈弥散样。该蛋白有抑制胰蛋白酶水解小肽底物的活性,但对其他丝氨酸蛋白酶如凝血酶、糜蛋白酶、弹性蛋白酶及枯草杆菌蛋白酶无抑制。BufomA-skin与胰蛋白酶保温24h后,胰蛋白酶活性无恢复。[结论]黑眶蟾蜍皮肤白蛋白具有稳定的胰蛋白酶抑制活性,在黑眶蟾蜍防御天敌捕食的过程中发挥重要作用.