凝集素与细胞凝集 Ⅲ.放射受体法测定正常鼠肝及腹水型肝癌细胞的ConA受体

近年来,用放射受体法研究激素与受体的相互作用取得不少进展。用同位素标记的凝集素测定细胞表面膜的凝集素受体,文献也有不少报道,其中尤以ConA为材料者较多,但多数结果不能说明受体与凝集素强度之间有对应关系。不过,这些数据都是用饱和分析法测定细胞与ConA的最大结合量,而未作受体与ConA间结合常数(Ka)的测定,可是Ka是受体分析中最关重要的一项数据。因为专一     

烟草与蓝猪耳胚胎发生中细胞表面三种凝集素受体的动态分布

以异硫氰酸甲酯(FITC)标记的三种凝集素(伴刀豆凝集素, 麦芽凝集素和大豆凝集素)为荧光探针,对烟草及蓝猪耳各发育时期胚细胞表面的凝集素受体进行了定位.结果显示胚柄基部荧光信号最强,沿胚柄单列细胞向胚体方向渐次减弱.以后随着胚柄功能的逐渐丧失而改变.同时,三种凝集素受体集中分布于胚柄细胞间的分裂面;凝集素受体在原胚中分布的另一个特点是聚集于新形成的细胞壁上.随着胚胎发育至分化阶段,凝集素受体则主要分布在胚体细胞的外切向壁上;三种凝集素受体的动态分布显示了凝集素受体的分布与细胞分裂之间的密切关系及其调控胚胎发育的作用.     

几种植物生殖细胞质膜表面的凝集素受体荧光标记

为进一步探讨从生殖细胞到精子的发育过程中细胞质膜表面凝集素受体的可能变化,及其与两类对凝集素标记有不同结果的精子的关系,用异硫氰酸荧光素标记的伴刀豆凝集素(Con A)、麦芽凝集素(WGA)和大豆凝集素(SBA)对蚕豆(Vicia faba L.)、鸢尾(Iris tectorium Maxim.)和朱顶红(Hippeastrum vittatum Herb.)的生殖细胞质膜表面的凝集素受体进行标记.结果显示:在不同植物中均有部分生殖细胞不能被凝集素探针标记,且在保持尾状形态的生殖细胞的表面发现有凝集素受体的极性分布.这可能是导致部分精子表面不能被同种凝集素标记的重要原因.此外,同一种凝集素受体在不同物种的生殖细胞上分布不一致,不同的凝集素受体在同一种植物的生殖细胞上的分布模式亦有不同.在蚕豆和鸢尾的生殖细胞表面均有这三种凝集素的受体.在朱顶红生殖细胞的表面有前两种凝集素的受体,分布比较均一,但是没有大豆凝集素的受体.此外,在具尾生殖细胞表面发现有凝集素受体极性分布的现象,为探讨精细胞功能及其表面糖蛋白分布的可能差异提供了重要启示.     

牛精细胞表面凝集素受体的研究

外源凝集素能与细胞表面相应的受体特异性的结合,并导致细胞凝集,因而外源凝集素可用作细胞膜结构的探针,用来研究细胞膜的结构与功能。近年应用外源凝集素对精细胞膜上糖蛋白和凝集素受体的数目与功能状态的研究已取得很大进展,采用异硫氰基荧光素(FITC)或辣根过氧化物酶等标记凝集素来测定膜上凝集素受体的定位与分布,研究认为精细胞膜上受体与受精作用有密切关系。本文报道用具有血型专一性或无血型专一性或FITC标记的凝集素研究牛精细胞膜上凝集素受体的分布结果。     

几种植物生殖细胞质膜表面的凝集素受体荧光标记

为进一步探讨从生殖细胞到精子的发育过程中细胞质膜表面凝集素受体的可能变化,及其与两类对凝集素标记有不同结果的精子的关系,用异硫氰酸荧光素标记的伴刀豆凝集素(Con A)、麦芽凝集素(WGA)和大豆凝集素(SBA)对蚕豆(Vicia faba L．)、鸢尾(Iris tectorium Maxim．)和朱顶红(Hippeastrum vittatum Herb．)的生殖细胞质膜表面的凝集素受体进行标记。结果显示：在不同植物中均有部分生殖细胞不能被凝集素探针标记,且在保持尾状形态的生殖细胞的表面发现有凝集素受体的极性分布。这可能是导致部分精子表面不能被同种凝集素标记的重要原因。此外,同一种凝集素受体在不同物种的生殖细胞上分布不一致,不同的凝集素受体在同一种植物的生殖细胞上的分布模式亦有不同。在蚕豆和鸢尾的生殖细胞表面均有这三种凝集素的受体。在朱顶红生殖细胞的表面有前两种凝集素的受体,分布比较均一,但是没有大豆凝集素的受体。此外,在具尾生殖细胞表面发现有凝集素受体极性分布的现象,为探讨精细胞功能及其表面糖蛋白分布的可能差异提供了重要启示。     

胃癌细胞PHA、DBA、WGA受体的电镜定位

应用自制的Ｓｔｒｅｐｔａｖｉｄｉｎ－ｇｏｌｄ探针,在胃癌和非癌胃粘膜组织的超薄冰冻切片上,进行ＰＨＡ、ＤＢＡ、ＷＧＡ受体的电镜定位。结果显示,三种凝集素受体主要定位于胃癌细胞的粘液泡、细胞膜和内微囊腔面微绒毛,以及非癌胃粘膜细胞的膜相结构内。证实凝集素能与细胞内特定糖基结合,且胃癌细胞内某些凝集素受体结合位点明显多于非癌胃粘膜细胞。本文结果可为进一步探讨凝集素用于胃癌导向诊断和治疗的可行性,提供超微形态学依据。     

凝集素与细胞凝集 Ⅱ细胞膜在凝集反应中的作用

凝集素对细胞的凝集作用可被秋水仙硷、长春新硷和松胞素B所抑制;已知前两种药物能破坏微管,而松胞素B可分解微丝。因此认为凝集作用是和微丝、微管相关。此外,用荧光素、酶、铁蛋白或血蓝蛋白标记的凝集素与细胞作用,发现凝集素在正常细胞表面是随机分布的;在肿瘤细胞表面则是成簇分布的。许多实验证明细胞表面受体的成簇或成帽需要有完整的微丝及微管。这就是说凝集素在微丝和微管的作用下,使表面受体成簇,造成局部受体的浓聚,终于发生细胞凝集。这些观察结     

中性红染色后细胞对凝集素的反应性

中性红染色后的小鼠胸腺细胞。肝、脾和肾细胞,人扁桃体细胞,以及体外培养的不同肿瘤细胞(HL60,K562,Molt-4,MLA和L1210)都能够被凝集素凝集。除小鼠肝细胞和肾细胞外,它们的凝集特征都与红细胞的凝集特征相同。不同来源的细胞,其对凝集素的反应性不同,其中,以肿瘤细胞对凝集素的反应最敏感。结果说明,中性红染色法可以作为用凝集素初步检测一些无色细胞表面凝集素受体变化的一种简便方法。     

凝集素受体在正常及缺氧—再给氧脑微血管内皮细胞的表达

本文应用凝集素亲和细胞化学的方法,通过荧光标记的麦胚凝集素（ＦＩＴＣ－ＷＧＡ）对培养的大鼠脑微血管内皮细胞进行染色,观察了ＷＧＡ凝集素受体在缺氧－再给氧后脑微血管内皮细胞上的表达。通过图像分析定量细胞表面的荧光强度,发现脑微血管内皮细胞单纯缺氧６小时后凝集素受体表达与正常对照组相比无明显变化;其显著改变发生于再给氧１小时后,ＷＧＡ的结合位点明显增加,荧光强度达峰值;再给氧３小时及６小时后有所下降,但与正常对照组相比仍具有显著性差异。上述结果与我们以往的动物在体实验,即大鼠脑缺血－再灌注后内皮细胞ＷＧＡ受体的表达趋势相吻合。说明脑微血管内皮细胞凝集素受体在细胞损伤后发生改变,并显示了在单纯缺血损伤和再灌注损伤的表达有所不同,它与其它内皮损伤后的变化,如细胞因子及其受体、粘附分子等的关系有待于深入研究     

若干外源凝集素对胰岛素与其受体相互作用的影响

本文研究了若干外源凝集素对胰岛素与其脂肪细胞胰岛素受体相互作用的影响。结果表明与D-甘露糖和D-葡萄糖专一结合的外源凝集素,如ConA(伴刀豆球蛋白A)、豌豆及蚕豆凝集素可抑制~(125)Ⅰ-胰岛素与其受体的结合。与D-半乳糖专一结合的外源凝集素,如蓖麻凝集素Ⅰ和天花粉凝集素基本上不抑制~(125)Ⅰ-胰岛素与其受体的结合,在低浓度时有一定程度的促进胰岛素与受体结合的能力。另外两个和低聚糖专一结合的半夏凝集素和PHA(菜豆凝集素)的效应和蓖麻凝集素Ⅰ等类似。这些结果说明在胰岛素受体分子表面可能存在着与ConA等专一结合的糖,而且它们在胰岛素受体与胰岛素结合部位的附近。而和其他几个外源凝集素专一结合的糖或低聚糖链则和胰岛素受体与胰岛素结合的部位相距较远,并就这些凝集素对胰岛素与受体结合的促进作用进行了讨论。     

An enzyme-linked lectin binding assay for quantitative determination of lectin receptors

An enzyme-linked lectin binding assay (ELBA) has been developed for the detection of soluble lectin binding substances (receptors) and the determination of their relative affinity for the lectin. The assay is based on competitive binding to enzyme-labeled lectin of a known lectin receptor, bound to a solid phase, and unknown sample receptors. In this paper the assay is exemplified with the mannose/glucose-specific pea lectin, with the glycoprotein ovalbumin as its receptor, and with horseradish peroxidase (EC 1.11.1.7) as the enzyme used for labeling. Also a method was developed for the preparation of peroxidase-labeled lectin. Labeling was started by mixing equimolar amounts of lectin and periodate-oxidized enzyme at pH 4.5 at a final concentration of 10(-4)M, after which conjugation was started by raising the pH to 9.5. This resulted in complete conjugation, after which the product could be diluted 50-500 times for application in ELBA. For the ELBA ovalbumin was adsorbed onto polystyrene microtiter plates. Sample receptors, added together with the enzyme-labeled lectin, inhibited binding of the latter to ovalbumin. Bound enzyme activity was colorimetrically determined after addition of o-phenylenediamine. Relative lectin affinity (KL) was expressed as (formula; see text) in which [X]50% is the concentration of sample receptor necessary to inhibit 50% of the binding of a certain amount of lectin, and [M]50% is the concentration of D-mannose necessary to inhibit 50% binding of the same amount of lectin. With this technique lectin affinity of both monovalent and polyvalent lectin binding substances can be estimated: low KL values mean high lectin affinity.     

Activation of phospholipase C and protein kinase C is required for expression of anthrax lethal toxin cytotoxicity in J774A.1 cells

Anthrax lethal toxin (LT) comprises two proteins: the protective antigen (PA) and the lethal factor (LF). The LT is cytotoxic to macrophage-like cell line J774A.1. Pre-treatment of these cells with neomycin, a phospholipase C inhibitor, protected them against anthrax LT cytotoxicity. Protection obtained with neomycin indicated that LT stimulates phospholipase C in these cells. It was found that levels of inositol 1,4,5-triphosphate (IP3) dramatically increased in toxin-treated cells. The rise in IP3 levels was proportional to the dose of LF that was allowed to bind to receptor-bound PA. By using protein kinase C (PKC) inhibitors, we found that the activation of PKC is required for mediating anthrax LT cytotoxicity. Activation of phospholipase C or PKC is not required for the binding of PA to the cell surface receptors or for the uptake or internalisation of the toxin. In this study, we demonstrate that the IP3 signalling cascade is initiated by anthrax lethal toxin in J774A.1 cells. The second messengers generated during the cascade aid LF in mediating lethality only after its translocation into the cytosol.     

Ultrastructural detection of lectin receptors by cytochemical affinity reaction using mannan-iron complex.

A two-step affinity reaction is described for electron microscopic demonstration of the Concanavalin A as well as the Lens culinaris lectin receptors by means of the yeast mannan-iron complex. First the tissue was incubated in the lectin. Afterwards the incubation in the yeast mannan-iron complex was performed and reaction takes place between the still free second sugar binding site of membrane bound lectin molecules and the polysaccharides. This membrane receptor-lectin-polysaccharide complex is revealed by the electron dense iron core of the yeast mannan-iron complex. The specificity of the reactions could be demonstrated by addition of the hapten or by incubation in the yeast mannan-iron complex only. The proposed technique has proved useful for demonstration of lectin receptors in the small intestine.     

Anthrax Lethal Factor Cleaves Mouse Nlrp1b in Both Toxin-Sensitive and Toxin-Resistant Macrophages

Anthrax lethal factor (LF) is the protease component of anthrax lethal toxin (LT). LT induces pyroptosis in macrophages of certain inbred mouse and rat strains, while macrophages from other inbred strains are resistant to the toxin. In rats, the sensitivity of macrophages to toxin-induced cell death is determined by the presence of an LF cleavage sequence in the inflammasome sensor Nlrp1. LF cleaves rat Nlrp1 of toxin-sensitive macrophages, activating caspase-1 and inducing cell death. Toxin-resistant macrophages, however, express Nlrp1 proteins which do not harbor the LF cleavage site. We report here that mouse Nlrp1b proteins are also cleaved by LF. In contrast to the situation in rats, sensitivity and resistance of Balb/cJ and NOD/LtJ macrophages does not correlate to the susceptibility of their Nlrp1b proteins to cleavage by LF, as both proteins are cleaved. Two LF cleavage sites, at residues 38 and 44, were identified in mouse Nlrp1b. Our results suggest that the resistance of NOD/LtJ macrophages to LT, and the inability of the Nlrp1b protein expressed in these cells to be activated by the toxin are likely due to polymorphisms other than those at the LF cleavage sites.     

Cytochrome P-450-dependent 14 alpha-demethylation of lanosterol in Candida albicans.

Analysis of receptor-ligand binding characteristics can be greatly hampered by the presence of non-specific binding, defined as low-affinity binding to non-receptor domains which is not saturable within the range of ligand concentrations used. Conventional binding analyses, e.g. according to the methods described by Scatchard or Klotz, relate the amount of specific receptor-ligand binding to the concentration of free ligand, and therefore require assumptions on the amount of non-specific binding. In this paper a method is described for determining the parameters of specific receptor-ligand interaction which does not require any assumption or separate determination of the amount of non-specific binding. If the concentration of labelled free ligand is constant, a plot of Fu/(B0*-B*) versus Fu yields a linear relationship, in the case of a single receptor class, in which Fu is the concentration of unlabelled free ligand, B0* is the total amount of labelled bound ligand in the absence of unlabelled ligand and B* is the total amount of labelled bound ligand in the presence of an unlabelled ligand concentration Fu; all of these data are readily obtained from binding studies. This linear relationship holds irrespective of the amount of non-specific binding, and the values for receptor density, ligand dissociation constant and a constant for non-specific binding can be readily obtained from it. If the concentration of labelled free ligand is not a constant for all data points, data are first converted according to a straightforward normalization procedure to permit the use of this relationship. The presence of multiple receptor classes with dissociation constants in the range of the ligand concentrations used results in a negative deviation from this linearity, and therefore the presence of multiple receptor classes can be discriminated unequivocally from non-specific binding. Both theoretical and practical advantages of the present method are described. The method, which will be referred to as the linear subtraction method, is illustrated using the binding of tumour promoters and polypeptide growth factors to their specific cellular receptors.     

Half-time analysis of the kinetics of irreversible enzyme inhibition by an unstable site-specific reagent

The half-time method for the determination of Michaelis parameters from enzyme progress-curve data (Wharton, C.W. and Szawelski, R.J. (1982) Biochem. J. 203, 351-360) has been adapted for analysis of the kinetics of irreversible enzyme inhibition by an unstable site-specific inhibitor. The method is applicable to a model in which a product (R) of the decomposition of the site-specific reagent, retaining the chemical moiety responsible for inhibitor specificity, binds reversibly to the enzyme with dissociation constant Kr: (formula; see text). Half-time plots of simulated enzyme inactivation time-course data are shown to be unbiased, and excellent estimates of the apparent second-order rate constant for inactivation (k +2/Ki) and Kr can be obtained from a series of experiments with varying initial concentrations of inhibitor. Reliable estimates of k +2 and Ki individually are dependent upon the relative magnitudes of the kinetic parameters describing inactivation. The special case, Kr = Ki, is considered in some detail, and the integrated rate equation describing enzyme inactivation shown to be analogous to that for a simple bimolecular reaction between enzyme and an unstable irreversible inhibitor without the formation of a reversible enzyme-inhibitor complex. The half-time method can be directly extended to the kinetics of enzyme inactivation by an unstable mechanism-based (suicide) inhibitor, provided that the inhibitor is not also a substrate for the enzyme.     

Development of support matrices for affinity chromatography of thyroid hormone receptors

Certain cellular responses to thyroid hormones appear to be mediated by non-histone chromatin protein receptors. Purification of these proteins is important for an investigation of the detailed mechanisms of their regulatory role. In the present studies, we report the development of an affinity chromatographic system that can be used to purify thyroid hormone receptors solubilized from nuclei. Amine-substituted hormone analogs were prepared with D and L isomers of T3; these bind to the receptor. This finding supports the hypothesis that thyroid hormones fit into the receptor with the amino groups accessible from outside the binding site. Although L-triiodothyronine (LT3) (the naturally occurring isomer) binds more tightly (relative Kd = 1.0 nM) to the nuclear receptor than D-triiodothyronine (DT3) (relative Kd = 2.0 nM), the amine-substituted analog of DT3 binds more tightly than the LT3 analog (DT3 analog, relative Kd = 40 nM; LT3 analog, relative Kd = 1500 nM). Agarose-based gels containing DT3 and LT3 covalently coupled by their amino groups were also prepared. Binding of receptor to these gels was biospecific in that it could be inhibited by prior incubation of the receptors with LT3. In addition, as predicted by the analog studies, the DT3 affinity gels were more effective than LT3 gels in adsorbing receptor. Elution of receptor from the LT3-derived gels was achieved in a predicted volume and concentration of counter-ligand in elution buffer. These results suggest that affinity chromatography can be applied to the purification of thyroid hormone receptors.