Effect of zinc-treated Entamoeba histolytica on the human polymorphonuclear respiratory burst

One of the mechanisms that Entamoeba histolytica uses to evade host immune response is inhibition of the polymorphonuclear (PMN) leukocyte respiratory burst. In studies previously conducted in a model used in our laboratory, we observed that when treating trophozoites with different zinc concentrations certain amebic functions are inhibited while significantly limiting development of hepatic abscess in golden hamsters (Mesocricetus aureatus). We carried out an in vitro study using a chemoluminescent method to assess the effect zinc-treated amebic trophozoites exercise on respiratory burst in human PMNs. We measured response of PMNs incubated with E. histolytica trophozoites from cultures with TYI-S33 medium alone and with zinc. Zinc concentrations between 0.1 and 1.0 mM did not affect amebic trophozoite viability, and PMNs in contact with these in a zinc-free medium had an oxidative response similar to that obtained with zymosan and significantly greater (p <0.05) than that generated by cells co-incubated with amebas cultured in TYI-S33 medium alone. These results suggest that zinc alters the amebic mechanism that inhibits the oxidative function of human polymorphonuclear leukocyte.     

运用L-半胱氨酸维护维生素C效价的研究

目的:探讨维护维生素 C效价的方法.方法:采用碘量法测定维生素C和 L-半胱氨酸、L-半胱氨酸盐酸盐、L-半胱氨酸盐酸盐水合物单体及混合物氧化后的保存量,以保存的百分数为指标,比较研究它们的稳定性以及它们之间维护稳定的协同效应.结果:L-半胱氨酸、L-半胱氨酸盐酸盐、L-半胱氨酸盐酸盐水合物比维生素C稳定,与维生素C配伍,既保持L-半胱氨酸、L-半胱氨酸盐酸盐、L-半胱氨酸盐酸盐水合物的稳定,又维护了维生素C的效价.结论:运用L-半胱氨酸维护维生素C效价的方式,必定可以简化工艺、节省投资、降低成本、提高效益.     

IgA reactivity in patients with intestinal or hepatic amebiasis.

The antigens of E. histolytica recognized by IgA antibodies in sera from patients with either amebic liver abscess or intestinal amebiasis, as well as saliva from the latter, were determined in immunoblots of whole native trophozoite proteins. Results show that sera of patients with amebic liver abscess reacted mainly with amebic proteins of more than 200 kDa, whereas those of individuals with intestinal amebiasis recognized a protein of 145 kDa. The saliva of the latter detected a protein of 210 kDa which is not always seen when using the sera of patients with intestinal or hepatic amebiasis.     

Immediate autoproteolysis and new proteinases in Entamoeba invadens and Entamoeba moshkovskii trophozoites.

We have examined the sodium dodecyl sulfate (SDS)-induced autoproteolysis of E. invadens (PZ and IP101) and E. moshkovskii (FIC and Laredo) trophozoite lysates. Heat-treated lysates containing parahydroxy-mercuribenzoate (pHMB) of all four strains had undegraded protein patterns. Unheated pHMB-lacking lysates of PZ had two (99 and 90 kDa) and IP101 lysates had three (45, 99, 90 kDa) major proteins, whereas FIC and Laredo lysates had only one (90 kDa). Heat-treatment changed the remaining proteins to smaller ones: 37 in PZ and IP101, 33 and 37 kDa in FIC and Laredo. Unheated lysates run on gelatin-containing "substrate" gels had gelatinases whose sizes were higher in lysates containing pHMB and allowed us to detect a 200 kDa gelatinase in E. invadens strains. Our results indicate that SDS induces immediate autoproteolysis by CPs in non-histolytica trophozoites, whose proteinases appear to be processed by self-digestion, and Entamoeba proteinases vary considerably under the various conditions used to obtain and characterize them.     

小鼠腹腔感染中白色念珠菌芽管及蛋白酶活性与其毒力的相关性研究

目的 :探讨白色念珠菌与腹膜炎发生相关的毒力因子。方法 :40株临床分离菌分别以腹腔接种方式感染小鼠进行毒力试验 ,以血清中丙氨酸氨基转移酶 (ALT)和α -淀粉酶 (AM)活力为表示菌株毒力的指标。结果 :白念菌体外测得的芽管长度分别与受染动物血清中ALT、AM含量呈正相关 (rALT=0 . 893,rAM=0 . 80 1,P <0 . 0 1) ,菌株分泌型天冬氨酸蛋白酶 (SAP)活力与血中ALT、AM水平也存在显著的正相关关系 (P <0 .0 1) ,且经SAP特异抑制剂处理过的受染小鼠其ALT活性显著降低 (P <0 . 0 1)。结论 :白念菌芽管和蛋白酶均为其与腹膜炎发生有关的重要的毒力因子     

Fast estimation of Michaelis-Menten constant of arylesterase with a pair of medi-um concentrations of substrate

Objective: To investigate the reliability for fast estimation of Michaelis-Menten constant (Km) with calibra-ted specific activity at only two medium concentrations of substrate by both simulation and experimentation with aryles-terase (ArE)as model. Methods: Initial rates were simulated by randomly inserting uniform absolute error, and the experimental initial rates of ArE were determined by measuring the increaser of product absorbance. Calibrated specific activities at two substrate concentrations were obtained by regression analysis, and Km was calculated according to Michaelis-Menten equation. Results: By simulation with calibrated specific activities at two medium substrate concen-trations, Km could be calculated according to Michaelis-Menten equation with reasonable precision and accuracy. By experimentation with substrates of 2-naphthyl acetate, phenyl acetate, and p-nitrophenyl acetate, there were no differ-ences between the mean and SD of Km of ArE for either substrate by this linear kinetic method and the Lineweaver-Burk plot. Conclusion: This linear kinetic method was reliable for fast estimation of the Km of some specified enzyme on its substrate of lower solubility or lower sensitivity for quantification by common methods.     

霍乱毒素B亚单位基因的克隆和表达

用DNA重组技术构建CT-B表达性质粒pXB1及进一步修饰的pXB2,使CT-B基因在大肠杆菌中得到较高水平的表达(280～350ng/ml),且有71.5％分泌率。表达动力学研究阐明,克隆子培养24h产物收率较高。一定浓度的Mg~(2+)、L-组氨酸和天冬酰胺能刺激CT-B的表达。GM1-ELISA、CHO细胞测毒结合间接免疫荧光检测和家兔肠段结扎试验证实,表达的CT-B能与游离的或活细胞膜上的GM1受体结合,但无细胞和肠段毒性。这种细胞测毒结合免疫荧光序贯试验,为客观证实CT-B的生物学活性开拓了新途径。     

Bile salts promote adherence-decreasing effect of colonic luminal hydrolases on Entamoeba histolytica.

E. histolytica trophozoites cultivated for > 7 h in the presence of glycosidases, produced by a subset of the colonic anaerobic bacteria of healthy humans, and pancreatic proteases show decreased adherence to Chinese hamster ovary epithelial cells. Since activities of the glycosidases are enhanced by bile salts we investigated whether bile salts would enhance the E. histolytica-CHO cell adherence decreasing effects of the luminal hydrolases (glycosidases plus proteases). CHO cell adherence of control trophozoites was 78.4 +/- 1.2% (mean +/- SEM). Incubations with the hydrolases alone for 4 h did not change adherence. Addition of 5.0 mM sodium taurocholate to the hydrolases for 4 h decreased trophozoite adherence to 30.5 +/- 3.2% of that of control trophozoites (p < 0.05). The effect of sodium taurocholate was dose dependent over 0.5-5.0 mM. Four hour incubation with the hydrolases and sodium taurodeoxycholate at 2.0 and 5.0 mM also decreased trophozoite adherence to 25.1 +/- 2.9% and 29.4 +/- 1.7%, respectively, of that of control trophozoites. These findings show that bile salts enhance the effects of luminal hydrolases on E. histolytica trophozoites, decreasing their ability to adhere to epithelial cells.     

The effect of axenic versus xenic culture conditions on the total and secreted proteolytic activity of Entamoeba histolytica strains.

Proteolytic activity was measured in lysates of four axenic and five xenic cultures of different strains of pathogenic E. histolytica, and in five non-pathogenic strains ("E. dispar") growing in xenic culture. There was no significant difference in total proteolytic activity, using either gelatin or albumin as substrate, between the two sets of xenic cultures; the axenic pathogens however had significantly higher levels of activity than the xenic pathogens, the levels appearing to correlate with virulence. An axenic strain (NIH 200) reassociated with mixed bacterial flora reverted to close to xenic levels of activity. There was no evidence of secretion of an elevated proportion of the total enzymic activity by pathogenic strains, and expression levels may owe more to culture conditions than to genotype.     

Further studies on the in vitro activity of gossypol as antiamebic agent.

Gossypol has an in vitro antiamebic effect, which is 11,39, and 980 times more potent than emetime, metronidazole and diiodohydroxyquinolein, respectively, on five Entamoeba histolytica axenic strains. The trophozoite NADP-dependent malic enzyme and alcohol dehydrogenase are inhibited by (+/-)-gossypol, in a noncompetitive manner with respect to the substrate. From the (-)- and (+)- enantiomers, which integrate the natural gossypol mixture, the first one is three and four times more active in inhibiting both the amebic culture growth and the mentioned oxidoreductases, respectively. The above results justify the analysis of gossypol as an antiamebic drug in experimentally infected animals.     

尿胰蛋白酶抑制剂对鼠溃疡性结肠炎结肠组织中蛋白酶活性的抑制作用

目的:确定尿胰蛋白酶抑制剂(UTI)对鼠溃疡性结肠炎(UC)结肠组织中蛋白酶的作用.方法:用18%的DNBS乙醇溶液0.25ml灌肠,在Wistar大鼠上建立UC模型;经尾静脉注射UTI;用明胶酶谱法测定病变结肠组织中的蛋白酶活性.结果:经UTI处理后,鼠UC结肠组织中蛋白酶活性仅为对照组的1/3.结论:UTI能显著降低鼠UC结肠组织中蛋白酶活性.     

肺炎克雷伯菌与双歧杆菌裂解物的免疫调节作用对比研究

目的:对肺炎丸雷伯菌与双歧杆菌裂解物的免疫调节作用做对比研究。方法:上述2种菌经培养后,采用超声破碎法获得其细菌裂解物。对细菌裂解物采用淋巴细胞转化试验和溶血空斑试验检测具对免疫功能的影响。结果:0.625g/L肺炎克雷伯菌和双歧杆菌裂解物对T细胞和B细胞的激活最显著,与对照组相比有显著性差别(P<0.01)。结论:肺炎克雷伯菌裂解物具有良好的免疫增强作用,在感染性疾病的防治中有良好的应用前景。     

重组MMP-14分解区蛋白诱导成骨样细胞凋亡的机制

目的：观察重组基质金属蛋白酶-14(matrix metalloproteinase-14,MMP-14)分解区蛋白（recombinant matrix metalloproteinase-14 catalytic domain, RMC）对人成骨样细胞SaOS-2凋亡的影响，探讨MMP-14分解区蛋白在骨代谢中的作用。 方法：MMP-14分解区蛋白在细胞培养液中的表达用Western blotting检测。RMC对SaOS-2细胞MMP-2酶原激活的影响用明胶酶谱检测，并检测其对SaOS-2细胞黏附功能的影响。细胞凋亡用吖啶橙/溴乙啶染色与ELISA检测。 结果：观察到MMP-14分解区蛋白在SaOS-2细胞培养液中56 kD的MMP-14表达。RMC促进细胞凋亡的作用呈剂量依赖性，且此作用可被EDTA阻断。RMC激活MMP-2酶原，抑制SaOS-2细胞在Ⅰ型胶原上的黏附。 结论：重组MMP-14分解区蛋白可通过降解骨基质蛋白，诱导人成骨样细胞SaOS-2凋亡。     

非诺贝特对TNF-α诱导的人脐静脉内皮细胞CD40表达和基质金属蛋白酶活性的抑制作用

目的研究非诺贝特对肿瘤坏死因子-α(TNF-α)诱导的人脐静脉内皮细胞(HUVECs)CD40表达和基质金属蛋白酶(MMP)活性的作用。方法应用RT-PCR和流式细胞仪分别检测非诺贝特对TNF-α诱导的HUVECs的CD40mRNA和细胞表面CD40表达的影响;用明胶酶谱法测定TNF-α对HUVECs的MMP-2、MMP-9活性的影响以及非诺贝特对它们的作用。结果非诺贝特在5×10-5,1×10-4和2×10-4mol/L的浓度范围内能显著降低CD40mRNA和细胞表面CD40的表达(P<0.01),以1×10-4mol/L的非诺贝特的效果最为明显;浓度为2×10-4mol/L时,非诺贝特并没有进一步降低CD40mRNA和细胞表面CD40的表达。非诺贝特能抑制TNF-α诱导的HUVECs中MMP-2和MMP-9活性的增加。结论非诺贝特能降低TNF-α诱导的HUVECs的CD40表达,并且能抑制TNF-α诱导的HUVECs中MMP-2和MMP-9活性的增加。     

大鼠心肌成纤维细胞合成金属硫蛋白

目的 :探讨脂多糖 (lipopolysaccharide ,LPS)、碱性成纤维细胞生长因子 (basicfibroblastgrowthfactor ,bFGF)、氧化型低密度脂蛋白 (oxidizedlow densitylipoproteins ,oxLDL)及重金属物质ZnCl2 等刺激因素对大鼠心肌成纤维细胞金属硫蛋白 (metallothionein ,MT)合成的影响。方法 :用10 9Cd Hb饱和法测定细胞MT含量 ,用RT PCR法测定细胞MTmRNA含量。结果 :本实验中各种处理因素均可刺激心肌成纤维细胞MT含量的增加。 1和5mg·L-1LPS处理组成纤维细胞MT含量与对照组相比分别增加 94 %和 130 % (P <0 .0 1)。 0 .2和 1.0mg·L-1bFGF处理组分别增加 4 1%和 5 9% (P <0 .0 1)。oxLDL亦可以促进心肌成纤维细胞MT的合成 ,2和 10mg·L-1oxLDL组MT含量较对照组分别增加 2 3% (P <0 .0 5 )和 4 1% (P <0 .0 1)。 1,2 ,5mg·L-1ZnCl2 呈浓度依赖性刺激心肌成纤维细胞MT含量的增加 ,与对照组相比分别增加 6 6 %、99%和 14 9% (P <0 .0 1) ,2～ 8mg·L-1ZnCl2 诱导MT1mRNA含量增加 6 9%～ 12 9% (P <0 .0 1) ,MT2mRNA含量增加 31%～ 6 0 % (P <0 .0 1)。但 10mg·L-1ZnCl2 不能进一步增加细胞MT含量 ,仅较对照组增加 91% ,可能与高浓度ZnCl2 致细胞损伤 ,细胞存活率降低有关。结论 :LPS、bFGF、oxLDL、ZnCl2 等多     

齿龈内阿米巴形态学的观察

目的 :观察齿龈内阿米巴滋养体的形态。方法 :从口腔疾病患者牙周组织或龈隙挑取渗出物 ,生理盐水涂片后镜下观察其形态并测量大小。结果 :齿龈内阿米巴滋养体活体大小平均为 14 .93× 12 .4 2μm ,形态呈圆形、长椭圆形及不规则葫芦形等。伪足透明 ,呈指状、舌状 ,球形及草帽形等不规则形态 ,其伪足伸缩变化较大 ,运动无一定方向。与溶组织内阿米巴滋养体形态有很多相似之处。结论 :全面观察了其运动方式及其形态变化特点 ,因取材方便 ,建议在寄生虫学教学实习中代替溶组织内阿米巴滋养体 ,观察活体阿米巴运动     

Model of intestinal amebiasis: structural and functional lesions to the rabbit colon mucosa by Entamoeba histolytica lysates.

Although the events occurring during the initial interaction of E. histolytica trophozoites with the mucosa of the large intestine probably determine the invasion by the parasites, an appropriate experimental model does not exist. To develop such a model we used full-thickness rabbit colon preparations (0.28 cm2) mounted in Ussing-type chambers. Untreated preparations had electrophysiological properties (potential difference, short-circuit current and electrical resistance) similar in magnitude and duration to those reported for stripped colonic mucosa. Exposure to E. histolytica trophozoite lysates for up to 80 min produced dose-dependent lesions in the colon, consisting of: (a) increased decay rates for potential difference, short-circuit current and transmural resistance, and (b) mucosal lesions involving vacuolation at the bases and shortening of epithelial cells, loss of intercellular junctions, destruction of microvilli, and necrosis of interglandular epithelial zones. The specificity and speed of the electrophysiologic effects and their correlation with the microscopic lesions suggest that this new model will help to understand the initial pathogenic events of intestinal amebiasis.     

问号钩端螺旋体胶原酶体内和体外的活性研究

目的通过检测致病性钩端螺旋体（钩体）胶原酶在体内、外的活性,探讨问号钩体黄疸出血群赖型赖株（赖株）假定的胶原酶（LA0872）在钩体病出血中的可能作用。方法在大肠杆菌中克隆、表达重组赖株la0872,并行相关鉴定及蛋白酶学活性检测。比较不同毒力菌株赖株、赖株减毒株和双曲钩体三宝垄群montevalerio型Monte Valerio株钩体胶原酶的基因序列特异性及转录和酶活水平的差异。测定豚鼠和沙鼠赖株感染模型的血清胶原酶活性水平变化。结果成功构建的重组质粒经酶切和测序鉴定显示位点连接正确,插入序列与GenBank公布的la0872序列完全一致,并证实其具有胶原酶活性;不同毒力菌株中的钩体胶原酶基因序列一致,转录和酶活水平均无显著性差异;豚鼠和沙鼠感染赖株后,宿主体内血清胶原酶活性水平与未感染赖株动物比较差异无统计学意义（P〉0.05）。结论首次表达和鉴定了有活性的钩体胶原酶,但其在钩体病出血中的作用尚待证实和探讨。     

问号钩端螺旋体胶原酶体内和体外的活性研究

目的 通过检测致病性钩端螺旋体(钩体)胶原酶在体内、外的活性,探讨问号钩体黄疸出血群赖型赖株(赖株)假定的胶原酶(LA0872)在钩体病出血中的可能作用.方法 在大肠杆菌中克隆、表达重组赖株la0872,并行相关鉴定及蛋白酶学活性检测.比较不同毒力菌株赖株、赖株减毒株和双曲钩体三宝垄群montevalerio型Monte Valerio株钩体胶原酶的基因序列特异性及转录和酶活水平的差异.测定豚鼠和沙鼠赖株感染模型的血清胶原酶活性水平变化.结果 成功构建的重组质粒经酶切和测序鉴定显示位点连接正确,插入序列与GenBank公布的la0872序列完全一致,并证实其具有胶原酶活性;不同毒力菌株中的钩体胶原酶基因序列一致,转录和酶活水平均无显著性差异;豚鼠和沙鼠感染赖株后,宿主体内血清胶原酶活性水平与未感染赖株动物比较差异无统计学意义(P>0.05).结论 首次表达和鉴定了有活性的钩体胶原酶,但其在钩体病出血中的作用尚待证实和探讨.