太湖蓝藻素—从太湖蓝藻水华中分离得到的天然胰蛋白酶抑制剂

通过各种层析方法,从太湖蓝藻水华中分离得到多种胰蛋白酶抑制剂。用质谱和核磁共振测定了其含量最多组分－－太湖蓝藻素的结构,确定与aeruginosin98-A同质。这是首次发现该物质在太湖蓝藻水华中存在。     

自制豆浆两注意

喜欢在家自制豆浆的人们,应注意以下2点充分浸泡大豆中含有丰富的蛋白质,但同时也含有胰蛋白酶抑制素,这种抑制素能抑制胰蛋白酶对蛋白质的作用,使大豆蛋白质不能被分解成人体可利用的氨基酸。要想充分利用大豆中的蛋白质,就必须通过充分浸泡、磨细、过滤、加热等处理方式,来消除胰蛋白酶抑     

口服含蛋自酶抑制剂的胰岛素微球及其降血糖作用

选用Eudragit L100作球材,分别将胰蛋白酶抑制剂、抑凝乳蛋白酶素、Bowman-Birk酶抑制剂和抑肽酶等与胰岛素共同包封于同一微球体系之中。实验结果表明,由于胰蛋白酶抑制剂和抑凝乳蛋白酶素仅能分别抑制胰蛋白酶和α-胰凝乳蛋白酶,它们对胰岛素的保护作用不明显,而抑酶谱广的Bowman-Birk酶抑制剂和抑肽酶能较好地保护胰岛素,使降血糖作用增加50％以上。     

HPLC法测定胰蛋白酶抑制剂及其在中药木鳖子提取液研究中的应用(英文)

本研究介绍了HPLC方法测定胰蛋白酶抑制剂活性及其在中药木鳖子胰蛋白酶抑制剂活性成分测定中的应用,由于分析样品中的胰蛋白酶抑制剂可以减低胰蛋白酶的活性,所以可以通过测定胰蛋白酶活性的下降来评价胰蛋白酶抑制剂的活性。测定原理是:胰蛋白酶能水解N-α-苯甲酰-DL-精氨酰-4-硝基苯胺盐酸盐生成产物4-硝基苯胺,采用HPLC方法对产物4-硝基苯胺进行分离和定量,最终测定样品中胰蛋白酶抑制剂的活性。该方法与一般的紫外分光光度法相比选择性更好。     

白杨素抗肿瘤衍生物的合成及其构效关系

目的合成一系列白杨素衍生物并结合体外实验探讨白杨素衍生物抗肿瘤作用的构效关系。方法以5,7-二羟基黄酮为原料,通过取代、水解、酰胺缩合等化学反应引入一些极性不同的小分子化合物,合成一系列白杨素的衍生物,然后采用MTT比色法检测白杨素及其衍生物对食管癌EC109细胞、肝癌HepG2细胞、宫颈癌Hela细胞的增殖抑制作用。结果化合物Ⅲa、Ⅲb、Ⅲc、Ⅲd、Ⅲf对抑制EC109增殖表现出的抗癌活性较白杨素有明显的提高,化合物Ⅲa、Ⅲd、Ⅲe、Ⅲf对抑制HepG2增殖表现出的抗癌活性较白杨素有明显的提高,其中Ⅲd和Ⅲf的抗肿瘤作用显著;白杨素及其衍生物对抑制Hela细胞的增殖均较高,但其衍生物抗肿瘤作用与白杨素相比没有提高。结论引入极性基团对白杨素的抗肿瘤活性有明显的提高,经过极性基团修饰后的白杨素衍生物能明显提高对EC109、HepG2细胞增殖抑制作用。     

一种酶反应器筛选胰蛋白酶抑制剂方法的建立

目的建立从黄酮化合物库中筛选胰蛋白酶抑制剂的新方法。方法利用毛细管固定化酶反应器技术,通过环氧基的开环反应,将胰蛋白酶与poly(GMA-co-PEGDA)有机聚合物整体材料共价键合制备毛细管固定化胰蛋白酶反应器;对酶反应浓度与反应时间等参数进行了系统研究,利用荧光显微镜、扫描电子显微镜和拉曼光谱对酶反应器进行了表征;采用微径液相色谱法对酶反应器的活性和动力学参数进行了系统评价;将制备的酶反应器用于20种中药黄酮化合物中胰蛋白酶抑制剂的筛选;通过分子对接手段进一步考察胰酶与抑制剂之间的作用机制。结果 16种黄酮化合物具有不同程度的胰蛋白酶抑制作用,其中,漆黄素的抑制效果最佳,抑制率为(62.9±1.2)%,分子对接结果进一步说明了漆黄素与胰蛋白酶之间的作用机制。结论建立了一种简单、有效的胰蛋白酶抑制剂的筛选方法,可进一步推广应用。     

灯盏乙素衍生物的合成及其生物活性

目的为提高灯盏乙素的生物利用度,设计合成了一系列灯盏乙素衍生物,评价其抗脂质过氧化和抗肿瘤的生物活性。方法通过羟乙基化、羟丙基化、酯化反应合成了一系列灯盏乙素衍生物,采用生化法测试部分衍生物对小鼠脑脂质过氧化产物丙二醛(malondialdehyde,MDA)的抑制作用,采用台盼蓝法测试体外抑制白血病细胞HL-60生长活性。结果合成了13个灯盏乙素衍生物,其中7个为未见文献报道的新化合物,其结构经1H-NMR和MS确证;化合物1、13对MDA的抑制作用显著,6-9具有中等强度的抗肿瘤活性。结论灯盏乙素结构中的酚羟基是其抑制脂质过氧化产物MDA的活性基团,葡萄糖醛酸的6-位游离羧基能够增强抗肿瘤活性。     

异丙酰基为桥合成白杨素氨基酸衍生物及其抗癌活性研究

目的　为改善天然产物白杨素的活性,利用氨基酸类物质特性,以异丙酰基为桥合成一系列白杨素氨基酸类衍生物,以期得到活性更高的衍生物。方法　采用烷基化、水解、酰胺缩合等药物合成方法在白杨素的7 位引入不同类型的氨基酸,得到一系列衍生物,运用1H-NMR 和13C-NMR 等方法对其结构进行确证;采用MTT 法,以白杨素母体、5-FU 为对照,检测其抗人肝癌细胞HepG2 和人乳腺癌细胞MCF-7 活性。结果　1H-NMR 和13C-NMR 等数据显示,烷酰基、氨基酸等基团被成功地引入到白杨素分子的7 位。活性测试结果显示,化合物2,5b,5d 抑制HepG2 细胞的IC50 值低于母体化合物白杨素,化合物2,3,4b,4c,4d,5a 和5c 抑制MCF-7 细胞的IC50 值低于白杨素。结论　通过实验能成功地将烷酰基、氨基酸等基团引入白杨素的7 位,且得到的大多数衍生物,相较于天然产物白杨素母体,具有更好的抗人肝癌细胞HepG2 和人乳腺癌细胞MCF-7 的活性。     

紫花芸豆胰蛋白酶抑制剂的分离纯化及降糖作用研究

目的：探讨紫花芸豆胰蛋白酶抑制剂的分离纯化及降糖作用。方法通过分离提纯得到紫花芸豆胰蛋白酶抑制剂,后建立四氧嘧啶糖尿病动物模型,取雄性昆明种小鼠15例,随机分为正常组、四氧嘧啶糖尿病模型组与胰蛋白酶抑制剂组,各5例。四氧嘧啶糖尿病模型组小鼠腹腔注射四氧嘧啶后予以紫花芸豆胰蛋白酶抑制,胰蛋白酶抑制剂组小鼠予以紫花芸豆胰蛋白酶抑制,正常组小鼠注射0.9%氯化钠溶液。观察3组小鼠的血糖变化情况。结果当pH值为4.5和7.5时,紫花芸豆胰蛋白酶抑制剂的提取率高,75℃下紫花芸豆胰蛋白酶抑制剂的活性回收率和纯化倍数最高,pH=8,馏分比例30%~60%时的活性强,又因收集的样品应为酸性蛋白,故后续纯化过程中考虑加硫酸铵饱和30%后,继续加至60%,收集此时沉淀进行溶解透析为下一步离子交换所用;紫花芸豆胰蛋白酶抑制剂可降低四氧嘧啶诱导的血糖升高,同时降低总胆固醇（ TC）,但对高密度脂蛋白胆固醇（ HDL-C）和三酰甘油（ TG）的效果不明显。结论紫花芸豆胰蛋白酶抑制剂治疗糖尿病有一定的临床效果。     

液质联用鉴定重组人血管内皮抑制素

目的：用液质联用技术鉴定重组人血管内皮抑制素（rhEndostatin）。方法：利用ESI—Q—TOF—MS法测定rhEndostatin的精确相对分子质量，通过HPLC—ESI—Q—TOF—MS／MS法测定其胰蛋白酶酶切后肽段的部分氨基酸序列并结合数据库检索进行结构鉴定。结果：重组人血管内皮抑制素的实测相对分子质量为21114．5，与理论相对分子质量21113．8相比非常接近。HPLC—ESI—Q—TOF—MS／MS测定m／z802．0的肽段的部分氨基酸序列为Ala—Pro—Ser—Ala-Thr—Gly—Gin—Ala—Ser—Ser—Leu—Leu。将其m／z和氨基酸序列在MASCOT数据库检索，结果表明重组人血管内皮抑制素的结构正确。结论：液质联用是鉴定蛋白质的灵敏、快速、准确的新方法。     

Effects of netropsin and pentamidine amino analogues on the amidolytic activity of plasmin, trypsin and urokinase

Inhibitory effects of nine carbocyclic DNA minor groove binders on amidolytic activities of plasmin, trypsin and urokinase were examined. Some of the studied compounds affected plasmin or trypsin activity, but not urokinase activity. One of the pentamidine analogues (5) and two bis-netropsin like compounds (6, 8) were potent inhibitors of plasmin (IC50 equals 90 and 100 microM), whereas an analogue of netropsin (2) was trypsin inhibitor (IC50 = 100 microM).     

Amino acid sequence of a thrombin like enzyme, elegaxobin, from the venom of Trimeresurus elegans (Sakishima-habu).

The amino acid sequence of a thrombin like enzyme, named elegaxobin, isolated from the venom of Trimeresurus elegans (Sakishima-habu) was determined by Edman sequencing of the peptides, derived from digests with cyanogen bromide, hydroxylamine, achromobacter protease I, trypsin, V8 protease, and chymotrypsin. Elegaxobin showed conservation of the catalytic amino acid residues (His, Asp, and Ser) of trypsin like serine protease in the amino acid sequence. The carboxy-terminal amino acid, Leu, was determined using carboxypeptidase Y. Elegaxobin consisted of 233 amino acids and had a calculated molecular weight of 25,439. Elegaxobin was 53, 59, 26 and 40% homologous in sequence to ancrod, flavoxobin, bovine thrombin and trypsin, respectively.     

Activity of recombinant trypsin isoforms on human proteinase-activated receptors (PAR): mesotrypsin cannot activate epithelial PAR-1, -2, but weakly activates brain PAR-1

Trypsin-like serine proteinases trigger signal transduction pathways through proteolytic cleavage of proteinase-activated receptors (PARs) in many tissues. Three members, PAR-1, PAR-2 and PAR-4, are trypsin substrates, as trypsinolytic cleavage of the extracellular N terminus produces receptor activation. Here, the ability of the three human pancreatic trypsin isoforms (cationic trypsin, anionic trypsin and mesotrypsin (trypsin IV)) as recombinant proteins was tested on PARs.Using fura 2 [Ca(2+)](i) measurements, we analyzed three human epithelial cell lines, HBE (human bronchial epithelial), A549 (human pulmonary epithelial) and HEK (human embryonic kidney)-293 cells, which express functional PAR-1 and PAR-2. Human mesotrypsin failed to induce a PAR-mediated Ca(2+) response in human epithelial cells even at high concentrations. In addition, mesotrypsin did not affect the magnitude of PAR activation by subsequently added bovine trypsin. In HBE cells, which like A549 cells express high PAR-2 levels with negligible PAR-1 levels (<11%), half-maximal responses were seen for both cationic and anionic trypsins at about 5 nM. In the epithelial cells, mesotrypsin did not activate PAR-2 or PAR-1, whereas both anionic and cationic trypsins were comparable activators.We also investigated human astrocytoma 1321N1cells, which express PAR-1 and some PAR-3, but no PAR-2. High concentrations (>100 nM) of mesotrypsin produced a relatively weak Ca(2+) signal, apparently through PAR-1 activation. Half-maximal responses were observed at 60 nM mesotrypsin, and at 10-20 nM cationic and anionic trypsins.Using a desensitization assay with PAR-2-AP, we confirmed that both cationic and anionic trypsin isoforms cause [Ca(2+)](i) elevation in HBE cells mainly through PAR-2 activation. Desensitization of PAR-1 with thrombin receptor agonist peptide in 1321N1 cells demonstrated that all three recombinant trypsin isoforms act through PAR-1.Thus, the activity of human cationic and anionic trypsins on PARs was comparable to that of bovine pancreatic trypsin. Mesotrypsin (trypsin IV), in contrast to cationic and anionic trypsin, cannot activate or disable PARs in human epithelial cells, demonstrating that the receptors are no substrates for this isoenzyme. On the other hand, mesotrypsin activates PAR-1 in human astrocytoma cells. This might play a role in protection/degeneration or plasticity processes in the human brain.     

A Randomized Controlled Trial of Trypsin to Treat Brown Recluse Spider Bites in Guinea Pigs

Brown recluse spider bites result in necrotic skin lesions for which there is no known antidote. Since venom toxins are proteins, a proteolytic enzyme like trypsin might be effective in reducing toxicity. The aim of this study was to conduct a randomized controlled trial of trypsin to treat brown recluse spider bites in guinea pigs. Subjects were 18 female guinea pigs. Anesthesia for injections was inhaled isoflurane. Analgesia was 0.05 mg/kg of buprenorphine twice a day as needed. Intervention was intradermal injection of 30 μg of brown recluse venom (Spider Pharm, Yarnell, AZ). Immediately after envenomation, subjects were randomized to two groups of nine: trypsin 10 μg in 1 mL normal saline and 1 mL of normal saline. The primary outcome was lesion area over a 10-day time period. Statistical analysis was performed with repeated measures ANOVA. Mean lesion area was smaller but not statistically different in the placebo group. Maximum lesion size occurred at day 4 in both groups, when lesion area was 76.1 ± 108.2 mm² in the placebo group and 149.7 ± 127.3 mm² in the treatment group. P value was 0.15 for placebo vs. treatment. This study did not establish a role for trypsin as a treatment for brown recluse spider bites in a guinea pig model.     

化学胶和纤维蛋白胶栓塞兔肺支气管的初步对照研究

目的 研究α-氰基丙烯酸烷基酯类化学胶(化学胶)和纤维蛋白胶栓塞支气管的效果差异以及胰蛋白酶在其中的辅助作用.方法 30只新西兰兔完全随机分为化学胶组、纤维蛋白胶组、胰蛋白酶+化学胶组、胰蛋白酶+纤维蛋白胶组和单纯胰蛋白酶组.化学胶组、纤维蛋白胶组分别使用相应粘接剂栓塞兔肺支气管;胰蛋白酶+化学胶组、胰蛋白酶+纤维蛋白胶组先用猪胰蛋白酶预处理支气管,然后分别采用相应粘接剂栓塞兔肺支气管;单纯胰蛋白酶组只用猪胰蛋白酶处理支气管而不进行支气管栓塞.栓塞操作在X线透视下进行,通过CT扫描观察兔肺不张的发生发展.2周后处死动物,取出兔肺标本进行病理研究.结果 化学胶组全部动物出现肺不张且持续到2周.纤维蛋白胶组栓塞后第2天4只动物出现短期的肺不张,10 d时已经全部复通.联合使用胰蛋白酶组肺组织病理有纤维组织增生.结论 化学胶栓塞支气管效果优于纤维蛋白胶效果.栓塞前用胰蛋白酶预处理支气管能够使肺不张更加稳定,具有很好的协同作用.     

重组牛胰蛋白酶抑制剂对小鼠急性肝损伤的保护作用

目的观察重组牛胰蛋白酶抑制剂(rBPTI)对小鼠急性肝损伤的保护作用。方法小鼠60只随机分成6组:正常对照组、模型组、rBPTI的3个剂量组(分别为3、6、12×104kIU·kg-1)、抑肽酶组(6×104kIU·kg-1)。每天腹腔注射给药,7d后体外注射CCl4建立小鼠急性肝损伤模型。末次给药后眼眶取血测定小鼠血清ALT、AST活性,观察肝脏组织病理改变。结果各剂量rBPTI可不同程度降低小鼠血清ALT、AST活性,降低肝组织MDA含量,与模型组比较差异有统计学意义(P<0·05、P<0·01),同时不同程度减轻肝组织病理损伤。结论rBPTI对体外CCl4诱导的小鼠急性肝损伤具有保护作用,作用效果同天然BPTI相当。     

Stability of Bovine Lactoferrin in Luminal Extracts and Mucosal Homogenates from Rat Intestine: A Prelude to Oral Absorption

Oral delivery is the most common method for bovine lactoferrin (bLf) administration. However, the presence of proteolytic enzymes in the stomach and intestine limits the effective absorption of bLf within the gastrointestinal (GI) tract. To determine the extent of bLf proteolysis, several digestion models were developed using luminal extracts and mucosal homogenates isolated from four regions of rat intestine: duodenum, jejunum, ileum, and proximal colon. The kinetics of bLf degradation followed a pseudo‐first‐order rate, and almost complete hydrolysis of bLf was observed in the luminal extracts, indicating that bLf is more susceptive to luminal peptidases rather than mucosal enzymes. Moreover, a significant reduction in bLf proteolysis was observed in the presence of soybean trypsin inhibitor (SBTI), bestatin, and bacitracin, suggesting that there exist trypsin‐like and aminopeptidase‐like proteases, which play a key role in the degradation of bLf in the intestine. Lactoferrin was then encapsulated in several lipid‐based delivery systems including liposomes and solid lipid particles (SLPs) with polymer modification, showing at least 50% of intact bLf remaining after 6 h of digestion compared with native bLf. These findings suggest that particle encapsulation may modulate protein digestion and possibly achieve sufficient oral bioavailability of bLf.     

Primary structures of four trypsin inhibitor E homologs from venom of Dendroaspis angusticeps: structure-function comparisons with other dendrotoxin homologs.

Four trypsin inhibitor homologs, the first known from Dendroaspis angusticeps venom, were characterized using a combination of gel filtration, cation exchange, reverse-phase liquid chromatography, Edman degradation and mass spectrometry. The four toxins comprise two 57 residue and two 59 residue isoforms. The long toxins possess a Lys-Gln N-terminal extension lacked by the short toxins. The only other structural difference is an Arg/His replacement at position 55. The long Arg55 variant is identical to trypsin inhibitor E from the venom of Dendroaspis polylepis. The name epsilon-dendrotoxin is suggested so as to follow the nomenclature of Benishin, C.G., Sorensen, R.G., Brown, W.E., Krueger, B.K., Blaustein, M.P., 1988. Four polypeptide components of green mamba venom selectively block certain potassium channels in rat brain synaptosomes. Mol. Pharmacol. 34, 152-159. Among snake venom protease inhibitors, the epsilon-dendrotoxins are structurally most like the delta-dendrotoxins, with which they share only 64% of their residues. In addition, the epsilon-dendrotoxins display hydropathy profiles more like those of the alpha- and delta-dendrotoxins, than those of the trypsin inhibitors from snake venoms. Given the strong protease inhibitory activity of trypsin inhibitor E and the recently demonstrated weak K(+) channel inhibitory activity of two of these variants (Tytgat, J., Vandenberghe, I., Ulens, C., Van Beeumen, J., 2001. New polypeptide components purified from mamba venom. FEBS Lett. 491, 217-221), the epsilon-dendrotoxins represent structural and functional intermediates between the facilitatory toxins and the protease inhibitors.     

胰蛋白酶对Madin-Darby犬肾细胞消化方法的研究

目的:考察不同胰蛋白酶及消化后是否弃去胰蛋白酶对Madin-Darby犬肾(Madin-Darby canine kidney,MDCK)细胞消化效果的影响,并评价培养基中添加胎牛血清对减轻胰蛋白酶细胞毒性的作用。方法:选用5种胰蛋白酶分别消化MDCK细胞,消化5 min后弃去胰蛋白酶为弃去组,不弃去为保留组。观察消化...