植物原生质体质膜表面电荷性质的研究——Ⅰ.pH及某些酶制剂对元麦叶肉原生质体电泳率的影响

用细胞电泳方法研究了pH 及胰蛋白酶,脂—蛋白脂酶、碱性磷酸酶等酶制剂对元麦叶肉原生质体电泳率的影响。结果表明,新鲜制备的元麦叶肉原生质体表面具负电性,但在低pH(<2.8)条件下其表面具正电荷;当pH>2.8时,其表面电性才由具正电性转变为具负电性。上述三种酶制剂处理原生质体后,均能改变原生质体的电泳率,随酶浓度增高均使原生质体的电泳速度减慢。文章并对所获得的结果在阐明质膜表面电荷的化学特性上的意义进行了讨论。     

基于微电极阵列的微生物电融合

利用自主研制的梳状交叉微电极阵列细胞电融合芯片系统,研究微生物电融合。在酶解浓度1．5％、酶解温度33℃、酶解时间3h、酶解pH值6．0、0．8mol／L山梨醇的渗透压条件下,获得生成率和再生率分别为95．2％和8．9％的微生物细胞原生质体。在脉冲峰值电压50V、持续时间80μs、8个脉冲、间隔1s的电融合条件下,该原生质体通过电融合获得一株菌株其乳化性能从62％提高到85％。     

洋葱内表皮细胞中的网状结构物

使用 Triton 抽提和卡马氏亮蓝染色方法,在洋葱鳞茎内表皮细胞中观察到网状结构物的存在。秋水仙素和细胞松弛素 B 的处理,进一步区分出两种不同的网状结构:近质膜较细的网状结构能被秋水仙素破坏;核周围与核成切线状结合的较粗的网状结构,经细胞松弛素 B 处理后,其形象更为突出。本文讨论了卡马氏亮蓝方法所揭示的带壁细胞的网状结构物。     

表面活性剂对大肠杆菌胞外生产α-环糊精葡萄糖基转移酶的影响

研究了不同浓度表面活性剂Tween-80,Triton X-100,SDS对大肠杆菌生产α-环糊精葡萄糖基转移酶（α-CGT酶）的影响。结果表明：发酵初始添加Tween-80和Triton X-100的最适浓度分别为2％,0.5％,最终胞外酶活分别达2.03U/ml和4.92U/ml,相对于未添加表面活性剂时提高4.6倍和12.67倍,且改变添加时间不能提高酶的产量;发酵36 h添加0.02％SDS对α-CGT酶产量促进最大,最终胞外酶活达5.31U/ml,较对照组提高12.75倍。表面活性剂对α-CGT酶生产的促进作用可能是由大肠杆菌细胞内外膜渗透性增加所致,使细胞周质空间中α-CGT酶能更加快速地渗透到胞外。     

DMSO、Tween-20和Triton X-100对野葛悬浮细胞异黄酮生产的影响

二甲基亚砜(DMSO)、Tween-20和Triton X-100可以增大悬浮培养细胞的细胞膜和液泡膜的透性,促进细胞内次生代谢物的释放,从而影响这些次生代谢产物的产量。为了提高野葛悬浮细胞中异黄酮类化合物的产量,以1％、3％和5％的DMSO、Tween-20和Triton X-100分别处理野葛叶悬浮细胞,结果显示,Tween-20和Triton X-100皆明显促进细胞生物量和葛根素和异黄酮化合物的释放。5％的Triton X-100处理3d,促进细胞产生总异黄酮化合物,增产率达40．6％。     

嗜热四膜虫接合生殖后期皮层细胞骨架蛋白的研究

用秋水仙素和细胞松驰素B处理接合期的嗜热四膜虫,以观察其对接合生殖期,尤其是接合后期的嗜热四膜虫皮层细胞骨架蛋白的影响,用秋水仙素处理的试验组的皮层细胞骨架蛋白组分中34KD、37KD、46KD和57KD蛋白的含量有明显改变,而用细胞松驰素B处理的试验组中40KD和74KD蛋白的含量改变较大。根据相关文献,作者推测34KD、37KD、46KD、57KD蛋白是微管蛋白,而40KD和74KD可能是微丝蛋白。这些蛋白对嗜热四膜虫接合过程中的形态发生的重要作用有等进一步研究。     

不同方法制备大鼠肝脏脱细胞支架及其免疫原性研究

目的:肝脏脱细胞支架(decellularized liver bioscaffold,DLB)在组织工程研究中具有良好前景,但对DLB的免疫原性尚未见探讨.本研究利用不同方案制备大鼠DLB,检测支架成分和体内重塑反应,为制备出低免疫原性的DLB提供改进依据.方法:选取文献报道的三种方案(分别以SDS,Triton X-100和NP-40为主要洗脱成分),制备F344大鼠DLB,进行HE和Masson染色,检测DLB中DNA、氨基葡聚糖(GAG)以及羟脯氨酸(HYP)含量;再将DLB埋植于C57BL/6小鼠的背部皮下,于术后第3、7和14天取出后进行形态学观察和组织学评分.结果:三种方案均成功制备出符合脱细胞标准的大鼠DLB;形态学结果提示TritonX-100和NP-40方案较SDS方案能更好地保护肝脏超微结构;成分检测结果发现NP-40方案去除DNA的能力显著强于SDS和Triton X-100方案(P＜0.05),而对GAG含量的洗脱作用最弱(P＜0.05);异种体内埋置实验提示NP-40方案制备的DLB引起的免疫反应强度明显弱于SDS和Triton X-100方案,体内重塑结局评分显著高于SDS和Triton X-100方案.结论:与SDS和TritonX-100方案相比,NP-40方案能更有效地去除肝脏DNA,较好地保留GAG,诱导移植受体对DLB的良性重塑反应,为进一步的体内研究提供更优化的支架.     

一种抗不同类型中等纤维的混合型兔自发抗体

本文报道了存在于未免疫家兔血清中的一种自发抗体(SRI)。使用间接免疫荧光法,这种自发抗体能在鸡肌原纤维上染出-线和Z-盘,在CHO细胞和HeLa细胞中能染出不同排列式样的胞质纤维结构。在秋水仙素处理的CHO细胞中,SRI血清仅能染出具有典型凝聚式样的波形纤维。而在秋水仙素处理的:HeLa细胞中,SRI血清同时能染出对秋水仙素不敏感的前角质蛋白纤维和对该药敏感的波形纤维。细胞松弛素B处理和Trito X-100抽提对:HeLa细胞的纤维染色无影响。根据所染纤维的特征,我们认为SRI自体免疫抗血清是一个至少含有抗前角质蛋白抗体和抗波形纤维蛋白抗体的混合抗体。     

牛心包瓣叶组织碱性磷酸酶与其钙化关系的初探

近几年来,尽管已研制出许多新型机械瓣但终究未能解决瓣膜置换术后终身抗凝这一难题。故生物瓣的研究再度受到重视。目前所用异种人工心脏瓣膜材料均为戊二醛(GA)处理的生物组织(牛心包或猪主脉瓣)。其植入体内后所发生的钙化一直是生物瓣过早损坏的最主要原因之一。文献报道这种钙化最初多发生在细胞成分较多的组织部位。考虑到碱性磷酸酶在正常骨骼钙化过程中起重要作用,我们推测,生物瓣的钙化很可能与其组织碱性磷酸酶有关。故对GA处理的牛心包片和经抗钙化处理的牛心包片(Triton X-     

南极产低温脂肪酶菌株Psychrobacter sp. 7195 的 选育、发酵条件及酶学性质研究

从南极普里兹湾深海沉积物中筛选到一株产低温脂肪酶的菌株7195,细菌学形态鉴定及16S rDNA序列分析表明该菌株属于嗜冷杆菌属 (Psychrobacter). 生长特性研究表明该菌株属于耐冷菌,其最适生长温度范围为5～15°C, 7195菌株能利用多种碳、氮源产酶.粗酶液经硫酸铵盐析、DEAE cellulose-52 柱层析进行初步分离纯化后进行酶学性质的研究. 该菌株所分泌的脂肪酶最适作用温度为30°C,最适pH值为9.0,对热敏感,60°C热处理10min剩余酶活为30%,是典型的低温酶. Ca2+、Mn2+、Cu2+对该酶有较为明显的激活作用,而Co2+、Zn2+、Hg2+、Rb2+、Cd2+、Fe3+、EDTA则能抑制酶活,此外该脂肪酶能在高浓度的SDS、CHAPS、Triton X-100、Tween 80、Tween20等变性剂中表现出较好的稳定性.     

Membrane IgM: interactions with the cortical cytoskeleton in the human lymphoblastoid cell line WiL2

Cell-surface IgM (antigen receptor) sediments with the membrane fraction following osmotic lysis and homogenization of cells of the human lymphoblastoid cell line WiL2. In nonreducing buffers, SDS PAGE analysis of membrane pellets demonstrates that "native" membrane IgM exists as a dimer. In contrast to osmotic lysis, lysis of cells with the nonionic detergent Triton X-100 releases approximately 90% of the membrane-bound IgM into the supernatant; approximately 10% of the IgM pellets with the cytoskeletal fraction on centrifugation. Ligand challenge with either mu-chain-specific antibodies or concanavalin A induces a change in the state of membrane IgM making it refractory to detergent extraction, such that 43% of the IgM pellets during centrifugation. This ligand-induced retention of IgM is significantly diminished by the microfilament-disrupting agent cytochalasin D, whereas pretreatment of cells with sodium azide or colchicine results in no significant change in the percentage of membrane IgM retained by Triton X-100 residues. These results indicate that retention of IgM involves an association with the cortical actin-based cytoskeleton. Investigation of the structural basis for ligand-induced Triton X-100 retention of membrane IgM by using ferritin-conjugated antibodies, myosin subfragment S1, and stereo-imaging electron microscopy has revealed linkages between ligand-receptor (antigen-IgM) complexes and elements of the cortical actin-based cytoskeleton.     

The detergent solubility properties of a malarial (Plasmodium knowlesi) variant antigen expressed on the surface of infected erythrocytes

Four detergents have been compared for identification of the Plasmodium knowlesi variant antigen on infected erythrocytes by immunoprecipitation analysis. Erythrocytes infected with late trophozoite and schizont forms of cloned asexual parasites were labeled by lactoperoxidase-catalyzed radioiodination and extracted either with the anionic detergents sodium dodecyl sulfate (SDS) or cholate, the neutral detergent Triton X-100, or the zwitterion 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS). After addition of Triton X-100 to SDS and cholate extracts, parallel immunoprecipitations of the four extracts were performed using rhesus monkey antisera of defined agglutinability. Identical results were obtained with clone Pk1(A+), which has 125I-variant antigens of Mr 210,000 and 190,000, and with clone Pk1(B+)1+, which has variant antigens of Mr 200,000-205,000. SDS yielded maximal levels of immunoprecipitated 125I-variant antigens. Variant-specific immunoprecipitation was detected in some experiments with Triton X-100 and cholic acid but with significantly lower recovery than with SDS. CHAPS extraction did not yield the variant antigens on immunoprecipitation. The variant antigens could also be identified in Triton X-100-insoluble material by subsequent extraction with SDS, indicating that failure to recover these proteins in the Triton X-100-soluble fraction is due to failure of this detergent to extract the variant antigens rather than to degradation during extraction. We suggest that the 125I-variant antigens either have a structure that renders them intrinsically insoluble in Triton X-100, cholate, or CHAPS, or that they are associated in some way with host cell membrane components that also resist solubilization by these detergents.     

The acid phosphatase positive organelles of the Giardia lamblia trophozoite contain a membrane bound cathepsin C activity

We found a dipeptidyl aminopeptidase activity in the parasitic protozoan Giardia lamblia with properties similar to the lysosomal cathepsin C of rat-liver lysosomes. Subcellular fractionation of this parasite indicated that the cathepsin C activity is located in organelles not distinguishable from the ones containing acid phosphatase, a known marker enzyme of Giardia lysosome-like peripheral vesicles. Contrary to the rat lysosomal enzyme, Giardia cathepsin C behaved like a membrane protein. Moreover, the enzyme was not solubilized by Triton X-100 or Triton X-100/SDS at 0 degrees C but could be substantially solubilized by octylglucoside, Triton X-100 at 37 degrees C or by a pretreatment with the cholesterol complexing agent beta-cyclodextrin before the Triton/SDS treatment carried out at 0 degrees C. These observations suggest that binding/anchorage of this enzyme to membranes occurs in cholesterol-rich microdomains.     

TritonX-100对含桐酸的卵磷脂脂质体的作用

本文研究了非离子型表面活性剂TritonX-100对含桐酸的卵磷脂脂质体的作用,结果表明,在TritonX-100对含桐酸的脂质体的作用中,存在一个TritonX-100的临界浓度,低于这个临界浓度时,TritonX-100的加入对脂质体的尺寸影响很小;当TritonX-100的浓度超过临界浓度时,脂质体迅速聚集成大团粒.     

Effect of Surfactants on Activity and Stability of Native and Chemically Modified Lipases A and B from Candida Rugosa

The effect of sodium dodecyl sulfate (SDS) and Triton X-100 on the hydrolytic activity of lipases A and B from Candida rugosa has been studied. Lipase B is significantly more affected than lipase A by the presence of both surfactants; Triton X-100 produces a more deleterious effect than SDS with both isoenzymes. In addition, the stability of lipases A and B in the presence of different concentrations of SDS was investigated; lipase A was more stable than isoform B. Both isoenzymes were chemically modified by reaction of their amino groups with octanoyl chloride or activated polyethylene glycol (PEG, mol. wt. 5000). In all cases the modification produced a protective effect against denaturation by SDS. In particular, PEG₅₀₀₀-liPases A and B were significantly more stable (stabilization factor: 3-4) than the native enzymes at the surfactant concentrations tested.     

PEG和表面活性剂对棉纤维细胞合成葡聚糖的影响

以陆地棉岱字-15号棉纤维细胞为材料,用3H-葡聚糖示踪方法测定β-1,3-葡聚糖和纤维素的合成。PEG4000促进β-1,3-葡聚糖和纤维素的合成,对刺激纤维素的合成更有效;随着非离子型表面活性剂 Trion X-100和Tween 20浓度的升高,抑制β-1,3-葡聚糖和纤维素的合成程度也增加,但抑制纤维素的合成更为强烈;而阴离子表面活性剂SDS则有所不同,在较高浓度下,又出现对β-1,3-葡聚糖合成抑制的减弱,这可能与SDS载负电荷的缘故有关。结果提示,完整的细胞膜有利于纤维素的合成,细胞膜损伤则利于β-1,3-葡聚糖的合成。     

Liposomes as a model for the study of the mechanism of fish toxicity of sodium dodecyl sulfate in sea water.

The mechanism underlying the shark repellency of SDS was studied by comparing it with the shark nonrepelling detergent, Triton X-100. The findings can be summarized as follows: (1) The effective concentration of SDS for termination of shark tonic immobility (an immediate and fast response) was close to its critical micellar concentration in sea water (70 microM). The fish lethal concentrations (LD50) were far below the CMC value for SDS, and at CMC level for Triton X-100. (2) In sea water SDS possesses a strong affinity for lipid membranes, expressed in a lipid sea water partition coefficient (Kp) of about 3000. (3) In liposomal systems examined by assays of turbidity, fluorescence resonance energy transfer and kinetics of carboxyfluorescein (CF) release, the pattern of SDS induced changes in the phospholipid bilayer suggests: (a) absence of vesicle-vesicle fusion; (b) occurrence of vesicle size increase, and (c) nonlytic gradual release of CF above and below its CMC values. In contrast, Triton X-100 above its CMC induces membrane solubilization. (4) Assays coupling CF release from liposomes to potassium diffusion potential induced by valinomycin indicate that SDS related CF release can also be attributed to a specific mechanism such as cation pore formation and not only to membrane solubilization. The hypothesis of pore formation by SDS is discussed.     

Quantitation of human immunoglobulin G and albumin in electroimmunodiffusion gels containing ionic and nonionic detergents

Quantitation of human immunoglobulin G (IgG) and albumin by agarose electroimmunodiffusion is influenced by the incorporation of ionic and nonionic detergents in the gel. The highest concentrations of each detergent at which human IgG and albumin determinations could be performed without perturbing the quantitations were 4% Triton X-100, 4% Tween 80, 1% NP-40, 0.5% sodium deoxycholate (SDOC), 0.5% Zwittergent, and 0.1% sodium dodecyl sulfate (SDS), and mixtures of Triton X-100, SDOC, and SDS. These detergent combinations all resulted in greater perturbations of albumin quantitation than of IgG. Immunoprecipitation of human IgG was quantitated in the absence and presence of Triton X-100, Zwittergent, and SDS. SDS was shown to cause nonspecific precipitation, whereas below 1% Triton X-100 or 0.5% Zwittergent no effects upon the immunoprecipitations were observed.     

Stimulation by chemotactic factor of actin association with the cytoskeleton in rabbit neutrophils. Effects of calcium and cytochalasin B

The amounts of actin and myosin in rabbit neutrophils expressed as micrograms/10(6) cells are 5.6 +/- 0.75 and 0.56 +/- 0.08, respectively. The average value of the total actin in rabbit neutrophils under unstimulated conditions is distributed between Triton X-100 soluble fraction (74 +/- 7%) and Triton X-100 insoluble fraction (26 +/- 3%). The Triton X-100 soluble and insoluble fractions will be referred to as the cytoplasmic and the cytoskeletal components. When the cells are stimulated by the chemotactic factor formyl-Met-Leu-Phe the amount of actin associated with the cytoskeleton increases to 73.7 +/- 6% of the total cell actin. This increase is rapid, dose-dependent and mediated through fMet-Leu-Phe receptors. Neither the time course of the response nor the dose-response curve is affected by the removal of calcium from the suspending medium. Calcium ions at concentrations greater than 10(-7) M added after Triton X-100 extraction dissociate actin from the cytoskeleton. Calcium at 1.9 microM added after Triton X-100 extraction reduces the amount of cytoskeletal actin under control and stimulated conditions to 10.3 +/- 0.9 and 33 +/- 1.5% of the total cell actin, respectively. The average value of the total myosin in rabbit neutrophils under unstimulated conditions is distributed between the cytosol (32 +/- 10%) and the cytoskeleton (68 +/- 18%). When neutrophils are stimulated with the chemotactic factor fMet-Leu-Phe the amount of myosin associated with the cytoskeleton does not increase significantly. Cytochalasin B decreases cytoskeletal actin and myosin and causes a shift in the amount of actin and myosin from the cytoskeleton to the cytoplasm both under fMet-Leu-Phe-stimulated and control conditions. In the presence of 1.6 mM extracellular Ca2+ and cytochalasin B (5 micrograms/ml) the amount of actin associated with the cytoskeleton under control and stimulated conditions is reduced to 13 +/- 2.2 and 10.2 +/- 3.5% of total cell actin, and that of myosin is reduced to 50.2 +/- 14 and 2.3 +/- 0.8% of the total cell myosin. The effect of cytochalasin B on actin does not depend on the time of its addition relative to that of fMet-Leu-Phe and is more pronounced in the presence of Ca2+. These results are discussed in terms of the roles of cytochalasin B and calcium in the overall mechanism of neutrophil degranulation induced by chemotactic factors.     

Effects of ionic and nonionic detergents on antigen-antibody reactions.

Using highly sensitive and quantitative radioimmunoassay procedures we have measured the effects of different concentrations of three commonly used detergents, SDS, DOC, and Triton X-100, on antibody-antigen reactions. Triton X-100, had a relatively mild effect on primary antigen-antibody bindings, the precipitin reaction, and a double antibody RIA as evidenced by only an 8 to 10% inhibition of binding or precipitation. These results were not detergent concentration dependent, as Triton concentrations ranging from 5 to 0.1% had virtually no differential effects. Sodium deoxycholate (DOC) had a more profound effect on both primary antigen-antibody binding and the precipitin reaction than did Triton X-100, and its effects, unlike those of Triton X-100, were concentration dependent. There was a direct relationship between concentration of DOC and degree of inhibition of both primary binding and immune precepitation especially in antigen excess. Sodium dodecylsulfate (SDS), at concentrations 10- to 100-fold less than either Triton X-100 or DOC, had profound inhibitory effects on primary antigen-antibody binding, the precipitin reaction, and a double antibody radioimmunoassay. Generally, at concentrations greater that 0.01% SDS, almost all immunochemical reactivity is destroyed.