Immunohistochemical Double Staining of Microwave Enhanced and Nonenhanced Nuclear and Cytoplasmic Antigens

Many of the antigens commonly investigated in histopathology can be enhanced by microwave pretreatment (MWPT) of formalin fixed, paraffin embedded tissue sections. We developed a double labeling method using microwave heating to detect otherwise undetectable nuclear antigens combined.with immunohisto-chemistry (IHC) of cytoplasmic or membranous antigens that do not benefit from MWPT. We used the same primary antibody solutions used in single antibody IHC. The staining technique is based on the alkaline phosphatase anti-alkaline phosphatase (APAAP) and the labeled avidin-biotin (LSAB) methods. Four different protocols were tested, each modifying the sequence of MWPT, APAAP and LSAB staining. In this study Ki67, estrogen receptor, progesterone receptor, c-neu, CD68 and desmin primary antibodies were used in routinely formalin fixed, paraffin embedded tissues of 50 tumor specimens. MWPT followed by LSAB for microwave enhanced antigens and APAAP for antigens that cannot be enhanced by MWPT gave the best double staining results. This method improves characterization of tumor cell features from paraffin embedded tissue and should aid analysis of tumor differentiation, receptor status and nuclear proteins in the single cells in archival tissues.     

Enhancement of Antigenic Site Detection with Gold Labeled Secondary and Tertiary Antibodies Using the Immunogold-Silver Staining Method

We report a modification of the immunogold-silver staining method (IGSS) for localizing hepatic phosphoenolpyruvate carboxykinase (PEPCK) in tissue sections, and we compare the efficacy of localizing the primary antibody with either a 5 nm gold labeled secondary antibody or 5 nm gold labeled secondary and tertiary antibodies. Light microscope examination of 10 μm frozen sections demonstrated that the use of combined secondary and tertiary gold labeled antibodies was superior to using a secondary gold labeled antibody alone. The increased labeling density (number of colloidal gold particles/antigenic site/cell) achieved by combined gold labeled antibodies was confirmed by electron microscopy. The increased labeling density resulted in a two-thirds reduction in the time needed for the IGSS physical development of the silver shells and less background. We achieved intense specific staining of hepatocytes expressing PEPCK while minimizing background staining. The use of combined secondary and tertiary gold labeled antibodies enhances the signal-to-noise ratio, achieves high resolution and is a suitable method for use in both light and electron microscopy.     

Effects of Polyethylene Glycol on Bovine Intestine Alkaline Phosphatase Activity and Stability

In this study, we evaluated the effects of polyethylene glycol (PEG) on bovine intestine alkaline phosphatase (BIALP) activity and stability. In the hydrolysis of p-nitrophenylphosphate (pNPP) at pH 9.8 at 20 °C, the k _cat?K _m values of BIALP plus 5–15% w/v free PEG with molecular masses of 1, 2, 6, and 20 kDa (PEG1000, PEG2000, PEG6000, and PEG20000 respectively) were 120–140%, 180–300%, 130–170%, and 110–140% respectively of that of BIALP without free PEG (1.8 μM^?1 s^?1), indicating that activation by PEG2000 was the highest. Unmodified BIALP plus 5% PEG2000 and BIALP pegylated with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine exhibited 1.3-fold higher activity on average than that of BIALP without free PEG under various conditions, including pH 7.0–10.0 and 20–65 °C. The temperatures reducing initial activity by 50% in 30-min incubation of unmodified BIALP plus 5% PEG2000 and pegylated BIALP were 51 and 47 °C respectively, similar to that of BIALP without free PEG (49 °C). These results indicate that the addition of PEG2000 and pegylation increase BIALP activity without affecting its stability, suggesting that they can be used in enzyme immunoassay with BIALP to increase sensitivity and rapidity.     

电气石处理水对Caco-2细胞生长和碱性磷酸酶活性的影响

水是由若干水分子通过氢键结合形成的水分子团簇结构,水分子团簇的改变能产生多种生物学效应。研究了电气石对液态水团簇的影响,并以Caco-2细胞培养模型,探讨了电气石处理DMEM培养液对细胞生长和碱性磷酸酶活性的影响。研究结果显示：电气石使蒸馏水^17O核磁共振(^17O nucleaur magnetic resonance,^17O NMR)半高幅宽变窄,降低了水分子缔合度;电气石处理的DMEM培养液培养Caco-2细胞促进了细胞生长,提高了细胞碱性磷酸酶活性。结果表明电气石可降低水分子缔合度,促进细胞生长和分化。     

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     

定向合成化学配基亲和层析纯化碱性磷酸酶

设计合成一种含有碱性磷酸酶抑制剂－对氨基苄基磷酸的化学配基并将其耦联到琼脂糖凝胶上,制备出新型的亲和层析介质,分离纯化从小牛肠中提取的碱性磷酸酶。经过对不同配基浓度吸附剂碱性酸酶吸附容量的考察,确定配基浓度为６μｍｏｌ／ｇ的吸附剂具有最高的酰选择性。     

Staining of alkali-labile phosphoproteins and alkaline phosphatases on polyacrylamide gels

A sensitive staining method for alkali-labile phosphoproteins has been developed. As little as 0.2 nmol bound P/mm2 can be detected. The procedure is based on alkaline hydrolysis, phosphate capture, and formation of an insoluble rhodamine B-phosphomolybdate complex. A further modification for the qualitative detection of alkaline phosphatase activity on polyacrylamide gels is proposed. During incubation, the released Pi is precipitated as lead phosphate and subsequently stained with rhodamine B.     

大凉疣螈碱性磷酸酶的分离纯化及部分性质

碱性磷酸酶 (alkaline phosphatase,AKP)在生物界的分布很广 ,动物、植物、微生物中均广泛存在 .提纯的 AKP常被应用于对核酸等的研究 ,是基因工程常用的工具酶 ,也是酶标免疫测定技术的常用工具酶之一 .人类血清中的 AKP在不同疾病状态下有显著变化 ,临床上将血清 AKP变化指标作为诊断某些疾病的依据 .对于细菌和高等动物的 AKP已有广泛的研究 ,但国内外对两栖爬行类动物 AKP的研究报导却很少 ,仅有蛇毒中 AKP的研究报导 [1,2 ] .本文对大凉疣螈皮肤的 AKP进行了分离纯化 ,并对其部分性质进行了初步研究 .1　材料和方法1 .1　材…     

信号肽去除的耐热碱性磷酸酯酶FD-TAP在大肠杆菌中的亚克隆和高表达

通过计算机辅助分析,发现在耐热碱性磷酸酯酶（简称ＦＤ－ＴＡＰ）的Ｎ端存在２６个氨基酸的信号肽序列。但一般信号肽切点并非是完全专一的,所以利用基因工程手段将ＦＤ－ＴＡＰ的Ｎ端分别缺失２４、２５、２６和２７个氨基酸,得到了Ｎ端分别缺失２４、２５、２６和２７个氨基酸的克隆子ｐＴＡＰＮＤ２４、ｐＴＡＰＮＤ２５、ｐＴＡＰＮＤ２６和ｐＴＡＰＮＤ２７。考虑到这样的无隆子其翻译起始区所形成的能量较低结构稳定的二组     

耐热碱性磷酸酯酶的基因克隆及在大肠杆菌中的表达

以pUC118质粒为载体,以E.coil TGl为受体,构建了产耐热碱性磷酸酯酶(FD-TAP)的菌株Thermus sp.FD3041的染色体基因文库。在包含1.2万个转化子的文库中,90％的转化子插入有3～10kb的外源DNA片段。用菌落原位碱性磷酸酯酶显色法从文库中筛选到5个阳性克隆。对其中的1个克隆(pTAP362)所产的TAP进行了研究,发现克隆的TAP与出发菌株所产的TAP的热稳定性、最适反应温度和最适pH等性质都相同。通过做物理图谱和测定部分缺失的质粒的TAP活性,将TAP基因定位于2kb的BamHⅠ-HindⅢ DNA片段上。模拟PCR实验表明该酶可用于PCR扩增产物的检出。     

牙鲆碱性磷酸酶cDNA序列分析与蛋白质高级结构预测

为研究碱性磷酸酶(EC 3.1.3.1; alkaline phosphatase,ALP)在牙鲆(Paralichthys Olivaceus)发育和变态中的作用,采用RACE的方法克隆了牙鲆ALP基因cDNA全长,通过生物信息学分析了核苷酸序列并进行蛋白结构预测. 结果表明,牙鲆ALP cDNA全长为1 811bp,能编码476个氨基酸的蛋白质,分子量为52 293.1,等电点为7.67. 编码区核苷酸GC含量在ALP同源基因中差异比较大,脊椎动物明显高于非脊椎动物和细菌. 分子系统分析显示,牙鲆ALP和青黑斑河豚(Tetraodon nigroviridis)、斑马鱼（Danio rerio）的组织非特异性ALP有较高的同源性,分子进化树和物种进化树是一致的. 在蛋白序列中的一些重要的功能位点,包括金属离子结合位点、N糖基化位点和丝氨酸磷酸化位点等表现了较高的保守性. 牙鲆ALP和人胎盘ALP(PALP)在蛋白序列上有43%的相似性,其3D结构非常接近.通过氨基酸空间位置比较发现,牙鲆ALP中141和203位半胱氨酸对应于人PALP的121和183位半胱氨酸,推测能形成一个二硫键. 在两者酶活性中心,3个金属离子结合的氨基酸残基非常保守,Zn离子周围的9个氨基酸中有2个不同;Mg离子周围的7个氨基酸也只有2个不同,包括一对类似的丝氨酸155和苏氨酸175.     

耐热碱性磷酸酶突变体耐热性变化机理研究

从耐热碱性磷酸酶（TAP）200多个随机突变体的克隆库中选出耐热性明显下降的4株突变体,进行全序列及表达产物的最高耐受温度和最适反应温度测定和酶分子高级结构的模拟,分析突变位点、高级结构和耐热性表现三者的关系,探讨引起耐热性变化的机理。结构模拟显示所有突变位点都仅能引起细微的、局部的结构变化,除T320→I外都未直接触及酶的活性中心;结构上的细微改变虽然对最适反应温度影响不明显,但却使最高耐受温度降低了10℃左右;T320→I靠近酶的活性中心,尽管未能引起结构的较大变化,但却使最高耐受温度和最适反应温度同时显著降低。可见,多数点突变对高级结构的影响都不剧烈,但对耐热性尤其是最高耐受温度的影响却比较明显,一般地,在非活性区的突变通常只能引起最高耐受温度的降低,靠近活性区的突变则能同时引起最适反应温度和最高耐受温度的降低。     

Comparison of Luminescent Immunoassays Using Biotinylated Proteins of Aequorin,Alkaline Phosphatase and Horseradish Peroxidase as Reporters

We compare aequorin, alkaline phosphatase and horseradish peroxidase as reporters for luminescent immunoassays using α-fetoprotein (AFP) as a model analyte. Biotinylated aequorin was prepared from mutated aequorin containing a reactive cysteine residue by chemical conjugation. The measurable range of AFP using this biotinylated aequorin was 0.02–200 ng/ml, with a lower background level than the other biotinylated enzymes tested.     

通过定点诱变法研究耐热碱性磷酸酯酶的活性位点、耐热性和亲热性

通过对耐热碱性磷酸酯酶ＴＡＰＮＤ２７活性位点Ｓ（６９）两侧的Ｅ（６８）和Ｓ（７０）的定点突变,得到了３个突变子Ｅ６８Ｓ、Ｓ７０Ａ和Ｅ６８ＳＳ７０Ａ。在蛋白纯化的基础上测定了３个变体的一些酶学性质,与ＴＡＰＮＤ２７相比,Ｅ６８Ｓ的比活力上升８倍,Ｔｍ下降了３℃,最适反应温度上升了２０℃;Ｓ７０Ａ的比活力上升了１部,Ｔｍ下降了２℃,最适反应温度上升了５℃;Ｅ６８ＳＳ７０Ａ的比活力下降了５０％,Ｔｍ下降     

Prediction and Molecular Modeling of T-cell Epitopes Derived from Placental Alkaline Phosphatase for use in Cancer Immunotherapy

Abstract

In our ongoing efforts to combat cancer, peptide-based tumor vaccines are promising as one of the several alternatives used for cancer immunotherapy and immunoprevention. We have attempted to identify T-cell epitopes suitable for the development of a peptide-based cancer vaccine directed towards placental isozyme of alkaline phosphatase (PLAP), an oncofetal antigen. After identifying amino acid residues specific to PLAP and distinct from other close PLAP homologs, we have used sequence-based immunoinformatics tools (BIMAS and SYF- PEITHI) and conducted molecular modeling studies using InsightII to investigate the binding affinity of the epitopes containing the unique residues with respective MHC class I molecules. Promiscuous epitopes binding to different alleles of different class I HLA loci were analyzed to get a population coverage that is widespread. Binding affinity deduced from the modeling studies corroborated the status of most of the epitopes scoring high in BIMAS and SYFPEITHI. We have thus identified specific epitopes from PLAP that have a potential for binding to their respective MHC class I alleles with high affinity. These peptides would be analysed in experiments to demonstrate their involvement in the induction of primary cytotoxic T-cell responses in vitro, using respective HLA-restricted T-cells in our way towards the development of an effective anti-cancer vaccine in a background of diverse MHC haplotypes.     

Interaction of SecB and SecA with the N-Terminal Region of Mature Alkaline Phosphatase on Its Secretion in Escherichia coli

Export-specific chaperone SecB and translocational ATPase SecA catalyze the cytoplasmic steps of Sec-dependent secretion in Escherichia coli. Their effects on secretion of periplasmic alkaline phosphatase (PhoA) were shown to depend on the N-terminal region of the mature PhoA sequence contained in the PhoA precursor. Amino acid substitutions in the vicinity of the signal peptide (positions +2, +3) not only dramatically inhibited secretion, but they also reduced its dependence on SecB. Immunoprecipitation reported their impaired binding with mutant prePhoA. The results testified that SecB and SecA interact with the mature PhoA region located close to the signal peptide in prePhoA.     

Inactivation of the pgsA Gene Affects Biosynthesis and Secretion of Alkaline Phosphatase in Escherichia coli

Inactivation of pgsA, which is responsible for biosynthesis of anionic phospholipid phosphatidyl-glycerol (PG), was shown to affect biosynthesis and secretion of alkaline phosphatase (PhoA) in Escherichia coli. A decrease in PG, but not in total anionic phospholipids, correlated with reduction of PhoA secretion, suggesting the role of PG in this process. A dramatic decrease in PG (from 18 to 3, but not 8, percent of the total phospholipids) inhibited not only secretion, but also synthesis of PhoA. In addition, pgsA inactivation expedited repression of PhoA synthesis by exogenous orthophosphate.     

An Automated Technique for Double Staining Rat and Rabbit Fetal Skeletal Specimens to Differentiate Bone and Cartilage

Assessment of chemicals for their potential to cause developmental toxicity must include evaluation of the development of the fetal skeleton. The method described here is an improved and fully automated double staining method using alizarin red S to stain bone and alcian blue to stain cartilage. The method was developed on the enclosed Shandon PathcentreTM, and the quality of specimens reported here will be reproduced only if carried out on a similar processor under the same environmental conditions. The staining, maceration and clearing process takes approximately 6 days. The personnel time, however, is minimal since solutions are changed automatically and the fetuses are not examined or removed from the processor until the procedure is completed. Upon completion of processing, the bone and cartilage assessment of the specimens can be carried out immediately if required. Full evaluation of skeletal development in both the rat and the rabbit is necessary to meet the requirements of safety assessment studies. This method allows this to be accomplished on a large scale with consistently clear specimens and in a realistic time.