紫花芸豆胰蛋白酶抑制剂的分离和纯化

[目的]从紫花芸豆中分离纯化胰蛋白酶抑制剂。[方法]以紫花芸豆为材料,采用脱脂、酸抽提、热变性、硫酸铵分级沉淀及离子交换层析和凝胶过滤层析等方法,分离纯化胰蛋白酶抑制剂。[结果]胰蛋白酶抑制剂经PAGE和IEF电泳显示单一条带。凝胶过滤法测定其表观分子量约为59kD。SDS电泳结果显示它有3个亚基,分子量分别为34、16、15kD。等电聚焦法测定其等电点为5．25。动力学的方法检测PVTT与胰蛋白酶发生不可逆抑制作用。[结论]离子交换层析和凝胶过滤层析法能快速地分离纯化紫花芸豆中的胰蛋白酶抑制剂。     

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.     

紫花芸豆胰蛋白酶抑制剂部分性质研究

[目的]研究紫花芸豆（Phaseolus vulgaris）胰蛋白酶抑制剂（PVTI）的部分性质。[方法]探索了不同温度、不同还原剂和变性剂处理对PVTI抑制活性和蛋白构象的影响。[结果]PVTI是一种对热稳定蛋白;部分二硫键对活性中心有重要贡献;经尿素处理,PVTI的荧光光谱没有明显变化,但残留了较少的抑制活性,推测亚基的解离影响了抑制活性而每个亚基的内部结构未受到影响;经盐酸胍处理,Trp残基完全暴露于极性环境中,整个蛋白质分子处于一种松散伸展的状态,PVTI活性全部丧失。[结论]该研究可为PVTI的功能研究及其进一步应用奠定基础。     

樟树种子休眠机制初探

以樟树种子为试验材料,从种壳透水性和内源抑制物两方面探索种子休眠原因.结果表明:樟树种子坚硬的外壳影响种子的吸水透性;种壳、种仁中的萌发抑制物抑制了白菜种子的萌发率、淀粉酶活性以及白菜幼苗的鲜质量增加.种壳浸提液抑制强度顺序均为:种壳甲醇浸提液＞种壳乙醚浸提液＞种壳水浸提液,抑制物主要成分为极性物质,白菜种子萌发率依次...     

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.     

Response of broiler chicks to non-steam- or steam-pelleted diets containing raw,full-fat soybean meal

The objective of this study was to evaluate the effects of steam pelleting of diets containing graded levels of raw, full-fat soybean meal (RSBM) on the chemical properties and feeding values of the diets. Samples of diets with steam- or non-steam-pelleted as well as the mash containing varying levels of RSBM were subjected to detailed chemical analysis. As a result of this study, trypsin inhibitor (TI) concentrations in the diets ranged between 4,153 and 10,484 TIU/g. Amino acid concentrations were higher in the non-steam-pelleted and mash diets than the steam-pelleted diets. A 4 × 2 factorial arrangement (RSBM: zero, 10, 20 or 30%, equivalent to zero, 30, 60, and 90 g/kg of diet, respectively, and non-steam- or steam-pelleted diets) was used while feeding broiler chicks (zero to 14 d of age). Each treatment was replicated 6 times with 8 birds per replicate. As a result of this study, there was no difference (P > 0.05) in mortality of birds among the groups. Feed intake (FI) (P < 0.05) and body weight gain (BWG) (P < 0.001) decreased with increasing levels of RSBM. Birds fed on steam-pelleted diets gained less (P < 0.001) weight than birds on the non-steam-pelleted diets, but the FI was not significantly (P > 0.05) different. The FCR was negatively affected (P < 0.05) by increasing levels of RSBM. There was no interaction effect between RSBM and pelleting method on the FI, BWG, or FCR of birds. The weight of the pancreas (P < 0.001) and duodenum (P < 0.01) increased with a rise in the level of RSBM in diets. Non-steam pelleting increased (P < 0.05) the pancreatic protein content, whereas the activity of chymotrypsin was reduced (P < 0.01) when the RSBM level was increased. Birds fed with RSBM-free diets had thicker muscle, longer villi, wider villus surface area, and higher villus to crypt depth ratios than birds on the other diets, but these differences were not significant. It can be concluded that steam pelleting of diets containing RSBM is inadequate to reduce the adverse impact of TI on chicks.     

Furanoditerpene ester and thiocarbonyldioxy derivatives inhibit photosynthesis

6α,7β-Dihydroxyvouacapan-17β-oic acid (1) and methyl 6α,7β-dihydroxyvouacapan-17β-oate (8) were isolated from Pterodon polygalaeflorus Benth. 1 was modified to obtain 6α-hydroxyvouacapan-7-β,17β lactone (2). Then, 6-oxovouacapan-7β,17β lactone (3) was obtained from 2. The furanoditerpene ester derivatives: propyl 7β-hydroxy-6-oxovouacapan-17β-oate (4), butyl 7β-hydroxy-6-oxovouacapan-17β-oate (5), 2-methoxyethyl 7β-hydroxy-6-oxovouacapan-17β-oate (6) and 3-methylbut-2-enyl 7β-hydroxy-6-oxovouacapan-17β-oate (7) were synthesized from (3) and methyl 6α,7β-thiocarbonyldioxyvouacapan-17β-oate (9) was obtained from (8). In this work, the lactone ester derivatives 4-7 and 9 were tested on photosynthetic activities in an attempt to search for new compounds as potential herbicide agents that affect photosynthesis. All compounds inhibited ATP synthesis and electron flow from water to MV, therefore, they act as Hill reaction inhibitors, being 4- to 9-fold more potent than 2 and 3 as inhibitors of ATP synthesis. Their interaction site was located at PSII in a similar way to diuron. Furthermore, furanoditerpene esters 6 and 7 act as uncouplers, and were corroborated by enhancement of the light-activated Mg²⁺-ATPase, while 5 act as an energy transfer inhibitor. Finally 5-7 behave as herbicides, since they inhibit the biomass production of weeds assay.     

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.     

舒巴坦、利血平、乙二胺四乙酸二钠对鸭源大肠杆菌抗菌药物敏感性的影响

本试验旨在研究β-内酰胺酶抑制剂舒巴坦、外排泵抑制剂利血平及膜通透促进剂乙二胺四乙酸二钠(EDTA)对鸭源大肠杆菌抗菌药物敏感性的影响。采用微量肉汤稀释法测定8株多重耐药鸭源大肠杆菌对头孢噻呋钠、恩诺沙星、阿米卡星、氟苯尼考、多西环素及其与舒巴坦、利血平和EDTA联用的最小抑菌浓度(MIC)。结果表明,舒巴坦能使5株鸭源大肠杆菌对头孢噻呋钠的MIC值下降4倍以上;利血平仅能使3株菌株对头孢噻呋钠的MIC值下降4倍以上;而EDTA能使7、5、3、6、8株菌对以上5种药物的MIC下降4倍以上;舒巴坦+利血平使4株菌株对头孢噻呋钠的MIC下降4倍以上,使5株菌株对氟苯尼考的MIC下降4倍以上;舒巴坦+EDTA使鸭源大肠杆菌对以上5类药物的MIC大幅度下降,分别使8、5、2、8、8株菌MIC下降4倍以上;利血平+EDTA使7株菌株对恩诺沙星MIC下降4倍以上,使所有分离株对其余4种药物的MIC显著下降;舒巴坦+利血平+EDTA和5类药物联用后,所用分离株的MIC下降4倍以上。由此可见,引起鸭源大肠杆菌对以上抗菌药物的耐药原因主要包括细胞膜通透性的下降、药物的主动外排作用和产生β-内酰胺酶的破坏作用,三者相协同提高了鸭源大肠杆菌的耐药水平。