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1.
目的制备一种低毒性的纳豆激酶(nattokinase,NK)注射液。方法在NK注射液中加入γ-聚谷氨酸(γ-polyglutamic acid,γ-PGA),制备NK-γ-PGA复合物注射液。采用急性毒性试验,以小鼠给药后的生存时间、生存状态和注射后48h存活小鼠尾部的状态作为毒性评价的指标,分别考察pH和γ-PGA浓度对NK与γ-PGA复合情况的影响。利用体外溶栓试验对NK-γ-PGA注射液毒性降低的原因进行分析。结果当以120 kU·kg^-1的剂量单次尾静脉注射NK-γ-PGA复合物注射液(NK 10 kU·mL^-1,γ-PGA 0.924 mg·mL^-1,pH 6.0)时,48 h后小鼠的死亡率为0,且尾部无红肿及坏死,说明在pH 6.0和γ-PGA的浓度为0.924mg·mL^-1时,NK与γ-PGA复合得最好。体外溶栓试验结果表明,γ-PGA的加入未降低NK活性,毒性的降低主要是因为NK与γ-PGA形成了复合物,起到了缓释作用。结论本研究中制备的NK-γ-PGA复合物注射液(NK 10 kU·mL^-1,γ-PGA 0.924 mg·mL^-1,pH 6.0)在不改变NK活性的前提下,降低了NK注射液毒性。  相似文献   

2.
目的:研究甲硫氨酸脑啡肽对机体免疫监督功能的影响。方法:测定met-enk对NK活性,抗癌细胞因子如:TNF-α和IL-12的产生和基因表达的影响。结果:Met-enk(1×10~(-8)-1×10~(-5)mol·L~(-1))能增强NK细胞活性。体外,体内均能刺激TNF-α的产生。ip 0.1及1 mg·kg~(-1)×6d可增强IL-12 p35 mRNA的表达。结论:Met-enk上调抗癌细胞因子及NK活性,在癌症监督中起一定的作用。  相似文献   

3.
胡静 《海峡药学》2013,25(5):47-48
建立快速、简单测定纳豆激酶活性的方法。本实验以TAME为底物测定纳豆激酶的效价,根据扫描发现蓝色复合物在574nm处有最大吸收,故在574nm处测定吸收度,依照公式,TAME(u.mL-1)=(甲醇微克分子数×样品稀释倍数)/(样品量mL×时间min),计算样品的TAME单位。结果 TAME法灵敏度高,操作简便,适合作为常规的分析检测手段。结论实验证明,TAME法能准确测定纳豆激酶的活性,且设计简单,操作方便,重现性好,是一个切实可行的测定纳豆激酶的方法。  相似文献   

4.
目的考察毛蚶多肽提取物(PEAS)的体外免疫活性。方法 MTT法测定PEAS对小鼠脾淋巴细胞增殖的影响及对小鼠NK细胞杀伤活性的影响;中性红吞噬法测定PEAS对小鼠腹腔巨噬细胞吞噬功能的影响;流式细胞术测定PEAS所致小鼠脾淋巴细胞周期的变化;双抗体夹心ELISA法测定PEAS对主要细胞因子IFN-γ和IL-4分泌水平的影响。结果 PEAS能够显著促进小鼠脾淋巴细胞增殖(P<0.05或0.01),能够增强小鼠腹腔巨噬细胞吞噬中性红活性和小鼠NK细胞杀伤活性(P<0.05或0.01);PEAS能够促进脾淋巴细胞G0/G1期向DNA合成期(S期)转化;PEAS协同Con A作用,能够增加IFN-γ的分泌(P<0.05或0.01),并且能够抑制IL-4的分泌(P<0.05或0.01)。结论 PEAS体外可促进小鼠脾淋巴细胞、腹腔巨噬细与NK细胞的免疫功能,其机制可能与促进脾淋巴细胞DNA合成,增加IFN-γ的分泌量有关。  相似文献   

5.
目的 :研究大豆异黄酮对大鼠免疫细胞的体外作用。方法 :取大鼠抗凝血 ,密度梯度离心法分离外周血单个核细胞 ,以伴刀豆球蛋白A(ConA)和体内所能达到的不同浓度的金雀异黄素或大豆苷元共同孵育 6 8h后 ,四唑蓝还原法 (MTT)测定体外ConA诱导的淋巴细胞增殖 ;另取外周血单核细胞为效应细胞 ,YAC 1细胞为靶细胞 ,以乳酸脱氢酶法测定天然杀伤细胞 (NK )活性。结果 :在 0 .0 1~ 1μmol·L- 1浓度范围内 ,大豆苷元可剂量依赖性的增强体外ConA诱导的淋巴细胞增殖 (P <0 .0 1) ,1μmol·L- 1的大豆苷元可明显增强NK细胞活性 (P<0 .0 1)。而同样浓度范围内的金雀异黄素对淋巴细胞增殖及NK细胞活性均无明显作用。结论 :大豆苷元体外能剂量依赖性的刺激ConA诱导的淋巴细胞转化 ,增强NK细胞活性 ,这种增强的免疫功能可能是大豆制品抗肿瘤作用的机制之一  相似文献   

6.
目的在原核系统中表达基因重组人IL-21,研究其在体外对NK细胞杀伤活性的影响。方法利用基因工程技术,获得人IL-21的编码基因,重组于表达载体pGEX4T-2中,转化E.coliDH5α进行诱导表达,纯化获得重组人IL-21,分别将0、10、20、40μg/L的IL-21与健康人外周血单核细胞共同孵育,改良的MTT法检测NK细胞对靶细胞K562的杀伤活性。结果在原核表达系统中成功表达了GST-IL-21的融合蛋白,一定剂量的纯化后IL-21(40μg/L)在体外具有增强NK细胞杀伤活性的作用。结论在大肠杆菌中表达的基因重组人IL-21在体外可增强NK细胞杀伤活性,为系统研究新型的免疫调节因子IL-21奠定基础。  相似文献   

7.
水溶性壳聚糖抗肿瘤作用的实验研究   总被引:33,自引:8,他引:25  
目的研究水溶性壳聚糖对抗肿瘤活性因子和NK细胞活性的影响。方法分别用不同浓度水溶性壳聚糖腹腔注射荷瘤小鼠 ,然后测定巨噬细胞一氧化氮 (NO)、肿瘤坏死因子 (TNF)的生成 ,以及脾细胞γ 干扰素(IFN γ)和NK细胞活性。结果水溶性壳聚糖有显著地促进荷瘤小鼠NO、TNF、IFN γ的生成 ,提高NK细胞活性。与对照相比P <0 .0 1。结论水溶性壳聚糖能提高荷瘤小鼠的免疫功能。  相似文献   

8.
纳豆激酶的分离纯化及酶学性质研究   总被引:2,自引:0,他引:2  
目的研究纳豆激酶分离纯化工艺及酶学性质。方法纳豆激酶发酵液的粗提物经Superdex 75凝胶色谱和聚丙烯酰胺凝胶电泳(PAGE)分离纯化,采用TAME法测定酶的活性,通过SDS-PAGE对纯化结果进行了检验。结果SDS-PAGE中显示单一色带,相对分子质量28000,以TAME为底物时纳豆激酶的米氏常数(Km)为35.47mmol/L,最适宜的温度37℃,最适宜pH为8.6。结论该分离纯化方法可以得到较纯的纳豆激酶。  相似文献   

9.
利巴韦林在体外对小鼠脾细胞NK活性的影响   总被引:2,自引:0,他引:2  
采用125I-UdR同位素释放试验,观察利巴韦林(三氮唑核苷,RTC)在体外对小鼠脾细胞自然杀伤(Nk)活性的影响。结果表明,RTC的剂量与NK活性呈负相关,在5umol·L-1时RTC即可显著抑制NK活性。RTC与地塞米松合用对NK活性的抑制有相加作用,而RTC与γ干扰素合用则互为拮抗作用。此外,还发现RTC能明显降低刀豆蛋白A诱生的小鼠脾细胞干扰素及白细胞介素-2的产量,推测RTC对NK活性抑制的部分机理是阻碍了这些淋巴因子的生成。  相似文献   

10.
本文研究了阿克拉霉素B、阿霉素和小檗碱对人外周血NK细胞活性的影响.NK细胞活性由测定~(51)Cr从NK细胞作用的标记K562细胞的释放率来决定.阿克拉霉素B(0.5和1.Oμg/ml),阿霉素(0.5和1.0μg/ml)和小檗碱(1.0和10μg/ml)作用1小时后,未见对NK细胞活性有显著影响,提示三个药物可能即不明显抑制也不促进NK细胞的细胞毒作用.阿克拉霉素B和小檗碱在高浓度时表现轻微的促进作用,但阿霉素有轻微抑制作用.三个药物均有不同程度地增加靶细胞~(51)Cr的自然释放率,以阿霉素最强,提示有细胞膜毒性产生.因而尚难肯定阿克拉霉素B和小檗碱的NK细胞活性促进作用.  相似文献   

11.
Beta-fibrinogenase from the venom of Vipera lebetina   总被引:5,自引:0,他引:5  
E Siigur  A M?har  J Siigur 《Toxicon》1991,29(1):107-118
An arginine esterase was purified from the venom of Vipera lebetina by gel filtration on Sephadex G-100 and by affinity and DEAE-cellulose chromatography. The enzyme has a mol. wt of 52,500 and pI approximately 3. It is a glycoprotein containing 23% of neutral sugars, and has extremely high thermostability. The esterase activity is inhibited by diisopropylfluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF). The Km and kcat values are for alpha-N-benzoyl-L-arginine ethyl ester (BAEE) 7.7 x 10(-5) M and 43.8 sec-1, for p-tosyl-L-arginine methyl ester (TAME) 3.6 x 10(-4) M and 39.8 sec-1 (pH 8.5, 25 degrees C, and for alpha-N-benzoyl-DL-arginine-4-nitroanilide (BAPNA) 1.8 x 10(-4) M and 0.94 sec-1 (pH 8.3, 25 degrees C), respectively. Lysine esters are not hydrolyzed. The enzyme has weak caseinolytic activity and hydrolyzes glucagon at the sites Lys12-Tyr13, Arg17-Arg18 and Arg18-Ala19. In fibrinogen it cleaves B beta-chain first and later also the A alpha-chain.  相似文献   

12.
tert-Amyl methyl ether (TAME) may be widely used as an additive to gasoline in the future. The presence of this ether in gasoline reduces the tail pipe emission of pollutants. Therefore, widespread human exposure to TAME may occur. To contribute to the characterization of potential adverse effects of TAME, its biotransformation was compared in humans and rats after inhalation exposure. Human volunteers (three males and three females) and rats (five males and five females) were exposed to 4 (3.8 +/- 0.2) and 40 (38.4 +/- 1.7) ppm TAME for 4 h in a dynamic exposure system. Urine samples were collected for 72 h in 6-h intervals and blood samples were taken at regular intervals for 48 h in humans. In urine, the TAME metabolites tert-amyl alcohol (t-amyl alcohol), 2-methyl-2, 3-butane diol, 2-hydroxy-2-methylbutyric acid, and 3-hydroxy-3-methylbutyric acid were quantified. TAME and t-amyl alcohol were determined in blood samples. After the end of the exposure period, blood concentrations of TAME were 4.4 +/- 1.7 microM in humans and 9.6 +/- 1.4 microM in rats after 40 ppm TAME, and 0.6 +/- 0.1 microM in humans and 1.4 +/- 0.8 microM in rats after 4 ppm. TAME was rapidly cleared from blood in both rats and humans. The blood concentrations of t-amyl alcohol were 9.2 +/- 1.8 microM in humans and 8.1 +/- 1.5 microM in rats after 40 ppm TAME, and 1.0 +/- 0.3 microM in humans and 1.8 +/- 0.2 microM in rats after 4 ppm TAME. t-Amyl alcohol was also rapidly cleared from blood. In urine of humans, 2-methyl-2,3-butane diol, 2-hydroxy-2-methylbutyric acid, and 3-hydroxy-3-methylbutyric acid were recovered as major excretory products in urine. In rats, 2-methyl-2,3-butane diol and its glucuronide were major TAME metabolites. t-Amyl alcohol and its glucuronide were minor TAME metabolites in both species. All metabolites of TAME excreted with urine in rats were rapidly eliminated, with elimination half-lives of less than 6 h. Metabolite excretion in humans was slower and elimination half-lives of the different metabolites were between 6 and 40 h in humans. The obtained data indicate differences in TAME biotransformation and excretion between rats and humans. In rats, TAME metabolites are rapidly excreted. In humans, metabolic pathways are different and metabolite excretion is slower. Recovery of TAME metabolites in urine was higher in humans as compared to rats, suggesting more intensive biotransformation of TAME in humans.  相似文献   

13.
纳豆激酶活性测定方法的研究   总被引:4,自引:0,他引:4  
从专属性、灵敏度及实际操作的可行性等几个方面,对TAME法和纤维蛋白平板法进行了比较研究.结果:TAME法灵敏度高,操作简便,适合作为常规的分析检测手段,而平板法虽专属性高,却操作繁琐,适宜于定性分析.结论:利用TAME法与平板法各自的优点,将两者结合起来作为纳豆激酶的活性检测方法,可达到高效灵敏、专属性强的目的.  相似文献   

14.
1. The effects of the competitive proteinase inhibitor TAME on isolated human umbilical and basilar arteries were studied. 2. Most experiments were performed on umbilical arteries and showed that 5 x 10(-4) M or 5 x 10(-3) M TAME significantly inhibited contractions elicited by prostaglandin F2 alpha, bradykinin, serotonin, and histamine. The inhibition was not endothelium-dependent. 3. The contraction elicited by prostaglandin E2 in basilar arteries was inhibited by TAME in a dose-dependent manner indicating that inhibition did not depend on the origin of the vessel. 4. Inhibition by 5 x 10(-7) M TAME of the contraction produced by KCl in the basilar artery was limited to low concentrations of KCl (10 and 30 mM). TAME did not significantly inhibit contractions of umbilical arteries to KCl. 5. At 5 U/ml (approximately 8.6 x 10(-8) M) antithrombin III markedly inhibited contractions of basilar arteries to prostaglandin E2 but had no effect of contractions elicited by 10(-6) M serotonin in the umbilical artery, nor did aprotinin (5 x 10(-4) M). 6. Although vessels of different origin may differ in sensitivity, the manifest effect of antiproteinases on arteries is inhibition.  相似文献   

15.
To reduce the production of pollutants in motor vehicle exhaust, methyl tert-butyl ether (MTBE) and other ethers such as ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME) are added to gasoline as oxygenates for more complete combustion. Metabolism of these gasoline ethers is catalyzed by cytochrome P450 (P450) enzymes. P450 2E1, which metabolizes diethyl ether, was suggested to be an enzyme involved. The present study used 2E1 knock-out mice (2E1-/-) to assess the contribution of 2E1 to the metabolism of MTBE, ETBE and TAME. Liver microsomes prepared from the 2E1 knock-out mice lacked 2E1 activity (assayed as N-nitrosodimethylamine demethylation), but were still active in metabolizing all three gasoline ethers. The levels of ether-metabolizing activity (nmol/min per mg) in the liver microsomes from 7 week old female 2E1 knock-out mice were 0.54+/-0.17 for MTBE, 0.51+/-0.24 for ETBE and 1.14+/-0.25 for TAME at a 1 mM substrate concentration. These activity levels were not significantly different from those of the sex- and age-matched C57BL/6N and 129/Sv mice, which are the parental lineage strains of the 2E1 knock-out mice and are both 2E1+/+. Our results clearly demonstrate that 2E1 plays a negligible role in the metabolism of MTBE, ETBE and TAME in mouse livers.  相似文献   

16.
Methyl t-butyl ether (MTBE), ethyl t-butyl ether (ETBE), and t-amyl methyl ether (TAME) are three alkoxyethers added to gasoline to improve combustion and thereby to reduce the level of carbon monoxide and aromatic hydrocarbons in automobile exhaust. Oxidative demethylation of MTBE and TAME and deethylation of ETBE by CYP enzymes results in the formation of tertiary alcohols and aldehydes, both potentially toxic. The metabolism of these three alkoxyethers was studied in a panel of 12 human liver microsomes. The relatively low apparent Km(1) was 0.25+/-0.17 (mean+/-SD), 0.11+/-0.08 and 0.10+/-0.07 mM and the high apparent Km(2) was 2.9+/-1.8, 5.0+/-2.7 and 1.7+/-1.0 mM for MTBE, ETBE and TAME, respectively. Kinetic data, correlation studies, chemical inhibition and metabolism by heterologously expressed human CYPs support the assertion that the major enzyme involved in MTBE, ETBE and TAME metabolisms is CYP2A6, with a minor contribution of CYP3A4 at low substrate concentration.  相似文献   

17.
A fibrinogen-clotting enzyme from the venom of the Peruvian bushmaster snake was purified to homogeneity by gel filtration on Sephadex G-100 followed by DEAE-cellulose ion-exchange chromatography using a linear ionic strength gradient with NaCl. The specific activity of the enzyme was 866 NIH U/mg, representing a 55-fold purification, with a recovery of 45%. The amino acid composition was Asx30, Thr14, Ser15, Glx33, Pro23, Gly22, Ala15, Val22, Cys18, Met3, Ile18, Leu23, Tyr2, Phe13, His8, Lys11, Arg11. The total carbohydrate content was 13.4%, comprised of 3.4% hexose, 8.7% hexosamine and 1.3% sialic acid. The enzyme was active against the synthetic amide substrate alpha-N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) and against the ester substrates alpha-N-benzoyl-L-arginine ethyl ester (BAEE) and tosyl-L-arginine methyl ester (TAME). Kinetic parameters for TAME esterolysis were: Vmax, 135 mumoles/min/mg and Km, 2.5 x 10(-4) M. The pH optimum was 8.0. Vmax for BAPNA amidolysis was 0.363 mumoles/min/mg and Km, 7.5 x 10(-5) M. Enzyme activity was reduced by diethylpyrocarbonate and by photo-oxidation, suggesting that the enzyme is a serine protease with a histidine residue involved in the active site. The enzyme released fibrinopeptide A rapidly from purified human fibrinogen and fibrinopeptide B more slowly. Factor XIII was not activated and the clotting activity was not inhibited by heparin. A dose of 50 micrograms/kg brought about defibrinogenation in anaesthetized rats but rabbits were unaffected. A dose of 80 micrograms/kg defibrinogenated conscious rats after 5 hr. There were no hypotensive or haemorrhagic effects.  相似文献   

18.
Activation of dog plasma kininogenase with glass   总被引:1,自引:0,他引:1  
The activity of glass-activated kallikreins in dog plasma was determined by measuring both kininogenase and p-toluensulphonyl-l-arginine methyl ester (TAME) esterase activities. Non-contact plasma, after being shaken with glass beads, was centrifuged immediately at 4° to sediment the glass beads and the supernatant was used for the experiments. Glass activation of dog plasma was more effective on TAME esterase activity. When the supernatant alone was preincubated at 37°, its kininogenase activity was suppressed about 50 per cent, while TAME esterase activity was unchanged. High kininogenase activity in the supernatant could be induced by treatment with 50% ammonium sulphate, suggesting the presence of peculiar inhibitors for kininogenase activity in glass-activated dog plasma. Kininogenase inhibitors in plasma were separated by gel filtration on Sephadex G-200; there were two inhibitors which were able to suppress kininogenase activity in glass-activated dog plasma. When dog plasma was preincubated with lima bean trypsin inhibitor, generation of both kininogenase and TAME esterase was significantly suppressed during contact with glass beads. Acetone-activated dog plasma could be reactivated by glass contact, although the level of kininogenase activity was lower than that by glass activation alone. The non-absorbed supernatant of glass-activated dog plasma formed kinins from rat kininogen. The present results suggest that the kinin-forming system by glass activation in dog plasma is not qualitatively different from that in human plasma with the exception of the presence of potent kininogenase inhibitors in the plasma.  相似文献   

19.
An arginine ester hydrolase (ME-5) was isolated from the venom of Trimeresurus mucrosquamatus by column chromatography on Sephadex G-100, CM-Sephadex C-50, DEAE-Sephacel and by isoelectric focusing, obtaining 1.4 mg of purified enzyme from 1 g of crude venom. The enzyme was homogeneous by SDS and non SDS disc electrophoresis on polyacrylamide gel at pH 8.3. ME-5 is a glycoprotein which possesses both TAME hydrolase and capillary permeability-increasing activity, but it did not show clotting or bradykinin-releasing activities. Its molecular weight is approximately 33,000 and its isoelectric point is 6.48. The enzyme is stable to heat treatment and to pH changes between 5 and 9. Trimeresurus mucrosquamatus venom contains five arginine ester hydrolases, designated as ME-1, 2, 3, 4 and 5. Three of the five (ME-3, 4 and 5) are inactivated by DFP, suggesting that the serine hydroxyl group is involved in enzymatic activity. All five arginine ester hydrolases showed capillary permeability-increasing activity, but none of the enzymes showed clotting activity. Their amino acid compositions were determined and all appear to be unique and distinct from those of other snake venoms.  相似文献   

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