碱性磷酸酶在文昌鱼胚胎和幼体中表达图式的研究(英文)

研究组织特异性酶在胚胎发育中的表达图式对于研究细胞、组织和器官的分化具有重要意义。本文以 ＢＣＩＰ为底物研究了碱性磷酸酶在文昌鱼胚胎和幼体中的表达图式。在囊胚、原肠胚和神经胚早期（１２～１３小时）未检测到碱性磷酸酶的特异性表达;到神经胚中期（１５小时,约６个体节）,碱性磷酸酶在胚胎每一体节的中部开始表达,在胚胎中形成３～６个条纹（Ｆｉｇ．１）;在１８小时神经胚（９～１０体节）中,碱性磷酸酶在肌节中表达（Ｆｉｇ．２）;到２４小时在后部内胚层中也开始表达（Ｆｉｇ．３）;在３６～４８小时幼体中,碱性磷酸酶在消化道中强烈表达,但在咽鳃区不表达,同时在肌节中仍有弱的表达（Ｆｉｇ．４）。研究表明碱性磷酸酶可能在肌节的发育过程中具有一定作用。碱性磷酸酶在消化道中的表达可能与这一时期消化道开始行使功能有关。另外,Ｌ－苯丙氨酸只抑制３６、４８小时文昌鱼肠碱性磷酸酶活性,不抑制其体节肠碱性磷酸酶活性（Ｆｉｇ．５）、表明文昌鱼至少存在两种碱性磷酸酶：在消化道中表达的是一种肠型的,与脊椎动物碱性磷酸酶可能为同一类型;另一种主要在肌节中表达,可能是组织非特异性的破性磷酸酶。以上结果说明文昌鱼早期发育中碱性磷酸酶的表达与脊椎动物相似,具     

碱性磷酸酶在文昌鱼胚胎和幼体中表达图式的研究

研究组织特异性酶在胚胎发育中的表达图式对于研究细胞,组织和器官的分化具有重要意义。本文以BCIP为底物研究了碱性磷酸酶在文昌鱼胚胎和幼体中的表达图式,在囊胚、原肠胚和神经胚早期（12－13）未检测到碱性磷酸酶的特异性表达;到神经胚中期（15小时,约6个环节）,碱性磷酸酶在胚胎每一体节的中部开始表达,在胚胎中形成3－6个条纹（Fig.1);在18小时神经胚（9－10体节）中,碱性磷酸酶在肌节中表达（Fig.2);到24小时在后部内胚层中也开始表达（Fig.3）;在36－48小时幼体中,碱性磷酸酶在消化道中强烈表达,但在咽鳃区不表达,同时在肌节中仍有弱的表达（Fig.4)。研究表明碱性磷酸酶可能在肌节的发育过程中具有一定作用。碱性磷酸酶在消化道中的表达与这一时期消化道开始行使功能有关。另外,L－苯丙氨酸只抑制36、48小时文昌鱼肠碱性磷酸酶活性,不抑制其体节肠碱性磷酸酶活性（Fig.5)。表明文昌鱼至少存在两种碱性磷酸酶：在消化道中表达的是一种肠型的,与脊椎动物碱性磷酸酶可能为同一类型;另一种主要在肌节中表达,可能是组织非特异性的碱性酸酶。以上结果说明文昌鱼早期发育中碱性磷酸酶的表达与脊椎动物相似,具有时间和组织特异性。     

碱性磷酸酶同工酶研究进展与应用

碱性磷酸酶（ＡＬＰ）及其同工酶的研究已越来越为人们所重视,特别是目前对肾脏疾病及肿瘤方面的研究进展迅速,作为某些疾病的标志物为人们所认识,本文简要综述了在碱性磷酸酶同工酶的研究与应用的新进展。     

碱性磷酸酶同工酶研究进展与应用

碱性磷酸酶（ＡＬＰ）及其同工酶的研究已越来越为人们所重视,特别是目前对肾脏疾病及肿瘤方面的研究进展迅速,作为某些疾病的标志物正为人们所认识,本文简要综述了在碱性磷酸酶同工酶的研究与应用的新进展 。     

大鼠胆汁淤积型肝病碱性磷酸酶及其同工酶的测定

建立胆汁淤积型肝病大鼠动脉模型,用麦胚凝集素（WGA）亲和色谱法分离总碱性磷酸酶（TALP）中的肝型碱性磷酸酶（LALP）和骨型碱性磷酸酶（BALP）,然后用高效液相色谱法（HPLC）定量;结果显示胆汁淤积型肝病组大鼠TALP于结扎后第5天升高,其中LALP明显升高,且出现较早,BALP基因无变化。本研究检测了大鼠胆汁淤积型肝病过程中血清TALP及LALP的变化情况,探讨LALP作为胆汁淤积型肝病早期辅助诊断灵敏指标的重要意义。     

碱性磷酸酶钙-钴法染色的不同包埋方法比较

目的探讨石蜡切片、冰冻切片及碳蜡(聚乙二醇)包埋切片在碱性磷酸酶染色中的应用,综合分析几种组织处理方法的优缺点,以便在病理诊断及科研工作中能找到更适合的碱性磷酸酶染色方法。方法取新鲜乳腺组织及肝组织,每份标本各取3块组织,根据包埋方法的不同,分为3组:石蜡切片碱性磷酸酶染色、冰冻切片碱性磷酸酶染色、碳蜡包埋切片碱性磷酸酶染色。结果 3组切片均可见黑色碱性磷酸酶的存在,其中肝组织内显示碱性磷酸酶呈黑色,沿胆小管分布;乳腺组织内显示碱性磷酸酶呈黑色沉淀弥漫分布于乳腺间质中。结论碳蜡包埋切片在碱性磷酸酶染色中,具有冰冻切片和石蜡切片的优点,并弥补了常规冰冻切片和石蜡切片染色的缺点和局限性,在病理诊断及科研工作中具有一定的应用价值。     

一株产高酶活性碱性磷酸酶解淀粉芽孢杆菌的分离及其phoD碱性磷酸酶基因的克隆与表达

[背景]碱性磷酸酶作为工具酶被广泛应用于各个领域,在免疫学检测方面应用较多的是PhoA家族的碱性磷酸酶,尚无关于PhoD家族的碱性磷酸酶在免疫学检测方面的研究。[目的]筛选出一株产高酶活性PhoD家族碱性磷酸酶的细菌,并将其phoD基因进行克隆表达,研究PhoD的酶学性质,为PhoD家族的碱性磷酸酶在免疫学检测方面的应用奠定一定的基础。[方法]采取有机质丰富的土样在有机磷平板中进行细菌分离,以4-硝基苯磷酸二钠盐（4-nitrophenyl phosphate disodium salt hexahydrate,p-NPP）为底物测定有机磷平板中单菌落的酶活性,选取酶活性高的菌株作为目的菌株,克隆其phoD基因。[结果]筛选到一株产碱性磷酸酶酶活性高的菌株S2-4,通过16S rRNA基因序列同源性比较分析,鉴定该菌株为解淀粉芽孢杆菌,克隆了其phoD基因并进行诱导表达。研究了纯化后PhoD的酶学性质,PhoD的最适反应温度为70℃;最适反应pH为9.8;PhoD最适Ca²⁺浓度为3 mmol/L,Mg²⁺对PhoD的酶活性有抑制作用,K     

大瓶螺碱性磷酸酶的分离纯化及部分性质研究

大瓶螺碱性磷酸酶的分离纯化及部分性质研究李清漪,曾和期（西南师范大学生物系,重庆６３０７１５）碱性磷酸酶（ＥＣ３．１．３．１,简称ＡＫＰ）是广泛存在于动物组织中的水解酶。对软体动物中ＡＫＰ的研究仅有少数报道［１,２］,对属于单壳贝类的水生食用螺──大...     

昆虫碱性磷酸酶的研究进展

碱性磷酸酶存在于昆虫的头、唾液腺（唾液）、肠道、马氏管、表皮、血淋巴、脂肪体、生殖系统、附肢等部位,广泛参与了昆虫的发育、神经传导、激素合成、物质代谢、滞育、社会型昆虫亚种形成等过程。同时碱性磷酸酶与昆虫抗性有关,特别涉及到对Bt制剂的阻滞作用,其本身也是某些农药的靶标酶,某些生物源化合物及病毒、真菌也可以影响其活性。昆虫碱性磷酸酶的研究,将有助于提高对昆虫生化机制及代谢过程的认识,并为害虫治理和资源昆虫饲养提供新的思路。本文综述了国内外对昆虫碱性磷酸酶的研究状况,并描述了昆虫碱性磷酸酶的生化性质及其与生理功能的关系。     

丹皮酚对过氧亚硝基阴离子致大鼠成骨细胞分化抑制的影响

探讨丹皮酚（Pae）拮抗过氧亚硝基阴离子（ONOO^-）对体外培养大鼠成骨细胞分化的影响。用改良的组织块法分离培养新生大鼠颅骨成骨细胞,采用淬灭流动反应方法体外制备ONOO^-,以不同终浓度加入成骨细胞培养体系,在作用不同时间后,用对硝基苯二钠动力学（PNPP）法检测细胞内碱性磷酸酶（ALP）的活性,用Lowry法测定蛋白含量,并以不同终浓度Pae消除ONOO^-（1000μmoL/L）对成骨细胞分化的影响。结果显示,不同浓度的ONOO^-（50-1000μmoL/L）均能抑制碱性磷酸酶的活性,影响分化;高浓度的丹皮酚（10^-3-10^-6mol/L）能消除ONOO^-（1000μmoL/L）对碱性磷酸酶活性的抑制,拮抗ONOO^-抑制成骨细胞分化的作用。     

Effect of magnesium on ATP labelling by kidney brush border membrane

1. Brush border membranes purified from rat kidney cortex were incubated in the presence of ATP and analysed by SDS polyacrylamide gel electrophoresis. 2. Quantitative analysis of phosphorylation was performed with a calibration curve obtained by autoradiography. 3. The presence of magnesium was required for the phosphorylation of membrane proteins. 4. EDTA completely inhibited the labelling of all bands, except for the alkaline phosphatase band. 5. In contrast, alkaline phosphatase was inhibited by 52, 65 and 85% in the presence of 1 mM bromotetramisole, 10 mM NaF and 10 mM Na arsenate respectively. 6. However these inhibitors had only minor effects on the labelling of other proteins. 7. High concentrations of magnesium caused a pronounced inhibition on the labelling of the alkaline phosphatase band but had no effect on the phosphorylation of other proteins.     

CELL CYCLE EVENTS IN THE HYDROCORTISONE REGULATION OF ALKALINE PHOSPHATASE IN HELA S3 CELLS

The increase in alkaline phosphatase in asynchronous cultures of HeLa S₃ cells grown in medium supplemented with hydrocortisone is characterized by a lag period of 10–12 hr. Present studies utilizing synchronous cell populations indicate: (a) a minimum of 8–10 hr of incubation with hydrocortisone is necessary for maximum induction of alkaline phosphatase; (b) the increase in enzyme activity produced by hydrocortisone is initiated exclusively in the synthetic phase of the cell cycle; (c) alkaline phosphatase activity does not vary appreciably over a normal control cell cycle. Radioactive hydrocortisone is rapidly distributed into HeLa cells irrespective of their position in the cell cycle, indicating that inductive effects are not governed by selective permeability during the cell cycle. Hydrocortisone-1,2-[³H] diffuses back from the cell into the medium when the cells are incubated in fresh medium containing no hydrocortisone, and the alkaline phosphatase induction, under these conditions, is completely reversible.     

Mechanism of action of Zn2+ and Mg2+ on rat placenta alkaline phosphatase. II. Studies on membrane-bound phosphatase in tissue sections and in whole placenta.

Alkaline phosphatase (EC 3.1.3.1) bound to trophoblastic cells in rat placenta is activated by Mg2+ and inhibited by Zn2+ in the same way as is found with partially purified soluble alkaline phosphatase in the same tissue (PetitClerc, C., Delisle, M., Martel, M., Fecteau, C. & Brière, N. (1975) Can. J. Biochem. 53, 1089-1100). In studies done with tissue sections (6-10 micron), it is shown that alkaline phosphatase activity and labelling of active sites by orthophosphate are lost during incubation with ethanolamine at pH 9.0. Addition of Mg2+ causes total recovery of catalytic activity and active sites labelling. Zn2+ displaces and replaces at the Mg2+ binding sites. The affinity for both ions is similar, and dissociation of Zn2+ from the enzyme is a very slow process, even in the presence of Mg2+. The Zn2+-alkaline phosphatase and Mg2+-alkaline phosphatase, which only differ by the ion bound to an apparent modulator site, have the same catalytic activity at pH less than 7.0, but the Zn2+ species has little activity at alkaline pH. Phosphorylation of the enzyme by orthophosphate indicates that with both enzyme species phosphoryl intermediate does not accumulate at alkaline pH. These results suggest that with orthophosphate, the phosphorylation step is rate determining for both enzymes, and that Zn2+ affects this step to a much greater extent. It is proposed that Zn2+ and Mg2+ regulate alkaline phosphatase in rat placenta. The concentration of both ions in maternal serum and placenta suggest that such a mechanism could exist in vivo.     

CHARACTERISTICS OF PHOSPHORUS DEFICIENCY IN ANABAENA1

Several aspects of the metabolism and composition of a strain of Anabaena have been studied during phosphorus deficiency. The effects of medium composition, substrate concentration, temperature, pH, and illumination on alkaline phosphatase activity and phosphate uptake have been examined. Of particular interest among these results was the dependence of maximum alkaline phosphatase activity on Ca and of phosphate uptake on Mg. Depletion of dissolved phosphate from the culture medium runs accompanied by a marked increase in alkaline phosphatase activity, initial rate of phosphate uptake, and total amount of phosphate taken up to satisfaction of the phosphorus debt. Readdition of phosphate to a phosphorus-deficient culture resulted in a rapid decline in the ability to take up phosphate but no loss of alkaline phosphatase beyond dilution of activity already present. Entry into phophorus deficiency was accompanied by a loss of heterocysts, a decline in chlorophyll a, protein, RNA, and cellular phosphorus, and an increase in carbohydrate per unit dry weight. The possible use of these changes as physiological indicators of phosphorus limitation in natural situations is discussed.     

STUDIES OF TWO TYPES OF ALKALINE PHOSPHATASE IN NUCLEI ISOLATED FROM THE LIVERS OF FED AND FASTED RATS BY A MODIFICATION OF THE BEHRENS TECHNIQUE

1. Rat liver nuclei were isolated from normal rats and rats fasted for 36 hours by a slight modification of the Behrens technique. 2. The nucleus of the rat liver cell contains two types of alkaline phosphatase. This confirms the previous findings on rat liver nuclei isolated in aqueous media. 3. The one type of alkaline phosphatase is not activated by magnesium ions, and this enzyme is very strongly bound to structural material of the nucleus. The other type of alkaline phosphatase is activated by magnesium ions, and this enzyme is probably free to diffuse from cytoplasm to nucleus and vice versa through the nuclear membrane. 4. Fasting caused a pronounced decrease of protein in general and of the alkaline phosphatase which is activated by magnesium ions from the nucleus of the rat liver cell, while the alkaline phosphatase that is not activated by magnesium was less affected.     

电镜图像定量分析肺毛细血管碱性磷酸酶在高压氧研究中的应用

采用碱性磷酸酶（ＡＬＰ）的电镜细胞化学方法,观察在高压氧条件下小鼠肺毛细血管ＡＬＰ活性的分布、活性强度与超微结构改变之间的联系,并用计算机图像定量测量方法对酶活性变化进行了２１个参数的定量分析。文中介绍了多参数图像定量分析方法及计算公式。     

MODULATION OF OSTEOGENIC DIFFERENTIATION IN HUMAN SKELETAL CELLS IN VITRO BY 5-AZACYTIDINE

Cellular differentiation is controlled by a variety of factors including gene methylation, which represses particular genes as cell fate is determined. The incorporation of 5-azacytidine (5azaC) into DNA in vitro prevents methylation and thus can alter cellular differentiation pathways. Human bone marrow fibroblasts and MG63 cells treated with 5azaC were used as models of osteogenic progenitors and of a more mature osteoblast phenotype, respectively. The capacity for differentiation of these cells following treatment with glucocorticoids was investigated. 5azaC treatment led to significant expression of the osteoblastic marker alkaline phosphatase in MG63 osteosarcoma cells, which was further augmented by glucocorticoids; however, in human marrow fibroblasts alkaline phosphatase activity was only observed in glucocorticoid-treated cultures. MG63 cells represent a phenotype late in the osteogenic lineage in which demethylation is sufficient to induce alkaline phosphatase activity. Marrow fibroblasts are at an earlier stage of differentiation and require stimulation with glucocorticoids. In contrast, the expression of osteocalcin, an osteoblastic marker, was unaffected by 5azaC treatment, suggesting that regulation of expression of the osteocalcin gene does not involve methylation. These models provide novel approaches to the study of the control of differentiation in the marrow fibroblastic system.     

Poly-l -lysine-gold complexes used at different pH are probes for differential detection of glycosaminoglycans and phosphoproteins in the predentine and dentine of rat incisor

Summary At neutral pH, poly-l-lysine-gold complexes labelled the predentine extensively, whereas in dentine the number of gold complexes was reduced by half. Hyaluronidase pretreatment of the section at pH 6.8, prior to labelling, suppressed most of the staining in predentine and did not affect dentine. In contrast, alkaline phosphatase pretreatment at pH 9 enhanced the gold complex labelling in predentine and removed most of the labelling in dentine. This proves that at pH 7.2, the polyanions which are stained include a heterogeneous population of glycosaminoglycans, located in predentine, and phosphoproteins, visualized in dentine. At acidic pH levels (2.9 and 1.1), the number of scored gold complexes decreased, but the ratio between predentine and dentine labelling remained constant. Hyaluronidase pretreatment removed or firmly reduced the gold complex labelling both in predentine and dentine, whereas alkaline phosphatase pretreatment of the sections at pH 9 prior to labelling did not induce any change. This argues in favour of an increased specificity of polylysine-gold complex staining for glycosaminoglycans, stained at low pH in both predentine and dentine. Differential staining of glycosaminoglycans and phosphoproteins according to the pH provides a useful tool for studying the role played respectively by the two matrix components in dentine mineralization.     

EFFECT OF THYROID DEFICIENCY ON THE LEVELS OF SEVERAL ENZYMES IN RAT BRAIN

—Activities of acid phosphatase, alkaline phosphatase and β-glucuronidase have been estimated in the brain tissues, using various subcellular particles, in growing thyroidectomized rats and also using cytoplasmic extracts free from debris and nuclear fraction in young hypothyroid animals. Hepatic glucose-6-phosphate dehydrogenase activity was markedly reduced after thyroidectomy but the enzyme was brought back to normal levels by thyroxine treatment. There was no change, however, in the activity of neural glucose-6-phosphate dehydrogenase after thyroidectomy. In the thyroidectomized animals an increase only in the free acid phosphatase activity in the neural synaptosomes was found and this increase in activity was not counteracted by administration of thyroxine. In the hypothyroid young animal β-glucuronidase, acid phosphatase and alkaline phosphatase activities were found to be affected during development.     

灭幼脲Ⅰ号对致倦库蚊幼虫磷酸酶活力的影响

本文用稍加改良的磷酸苯二钠法,测定了经灭幼脲Ⅰ号处理的致倦库蚊4龄期幼虫的酸性磷酸酶(ACP)和碱性磷酸酶(AKP)活力的变化,发现被处理幼虫的这两种磷酸酶活力均受到影响。在整个4龄期,灭幼脲Ⅰ号对AKP活力的抑制都比较明显,抑制强度甚至达80％,对ACP活力的抑制则比较弱;但在化蛹前,当对照幼虫的ACP活力增高时,被处理幼虫的ACP活力却下降,致使两者活力也相差一倍以上。由于灭幼脲Ⅰ号对幼虫两种磷酸酶产生明显的抑制作用,从而导致幼虫的生长发育缓慢,幼虫4龄期延长,幼虫不能化蛹而终于死亡。