Repeated immunostaining of the same tissue section using alkaline phosphatase as a reporter

One can determine the best dilution of a primary antibody for immunohistochemistry that uses horseradish peroxidase conjugated to a secondary antibody by testing increasing concentrations sequentially on the same tissue section. When the same tissue section is incubated repeatedly with increasing concentrations of primary antibodies to epithelial membrane antigen, smooth muscle α-actin, or vimentin using alkaline phosphatase conjugated to a secondary antibody as the reporter, the best staining was obtained with a less concentrated primary antibody than was optimal for a single staining test. The best concentration of primary antibody for single run staining using an alkaline phosphatase reporting system is usually four times the best concentration for staining with multiple runs. The optimal concentration can be determined by denaturing the residual alkaline phosphatase and extracting residual stain by incubating the section in 4:1 diglyme:phosphate buffered saline for 20 min at 80^o C between tests of primary antibody concentrations. I tested the method for four chromogens from one supplier and one chromogen from a different supplier.     

报道基因SEAP的应用

分泌型碱性磷酸酶(secreted alkaline phosphatase,SEAP)由于既有很高的灵敏性,又可分泌至培养介质中,因而SEAP基因成为一种广泛使用的报道基因。目前,该基因在中和抗体检测试验、实时监测分析及工程抗体构建与检测方法有较多应用。该基因在双报道基因系统中运用,可同时起到定性定量作用。本文将对报道基因SEAP的使用原理与应用加以综述。     

Human placental alkaline phosphatase as a histochemical marker of gene expression in transgenic mice

The human placental alkaline phosphatase (PLAP) gene was analysed for its utility as a histochemically detectable reporter gene in transgenic mice. A reporter gene was made by linking the PLAP structural gene to an enhancerpromoter element from the human -actin gene. This gene was inserted into the mouse genome by transfection of embryonic stem cells, and by microinjection of fertilized eggs. Histochemical staining showed that the transgene was uniformly expressed in four of four stable ES cell lines, and in all ten tissues examined from adult animals from five lines of transgenic mice. Non-transgenic cells did not stain. These results suggest that the human PLAP gene will be of utility in studies requiring phenotypic marking of cells in tissues of mice.     

A new electrochemical assay method for gene expression using HeLa cells with a secreted alkaline phosphatase (SEAP) reporter system

A new electrochemical assay for the detection of secreted alkaline phosphatase (SEAP) from transfectant HeLa cells is proposed using a microarray device and scanning electrochemical microscopy (SECM). The assay consists of two steps: the first is the incubation of a transfected cell in a microarray culture device covered with a substrate modified with anti-SEAP under physiological conditions without any additives. The array device consists of a 4 × 4 array of microwells having a size of 100 μm × 100 μm (diameter × depth). The second step is SECM measurement of secreted SEAP at the antibody-immobilized substrate. This assay ensures accuracy and intactness because the undesired influence of endogeneous ALP is eliminated and the transfected cells are incubated in a culture device under suitable conditions. We successfully detected the expression of SEAP from intact cells at the single-cell level using this assay. The system is useful as a cell-based gene-expression assay.     

Alkaline phosphatase vs luciferase as secreted reporter molecules in vivo

Secreted alkaline phosphatase (SEAP) and Metridia luciferase (MLuc) are useful reporter molecules in vitro, but little is understood about their usefulness in vivo. In this study, we investigated in vivo activity of recombinant SEAP and MLuc in blood and urine. When SEAP-transfected cells or recombinant SEAP were injected into rats, substantial increase in the level of serum SEAP was observed. In contrast, activity of SEAP was not detected in urine of rats injected with either the SEAP-transfected cells or recombinant SEAP. SEAP activity was also undetectable in urine of SEAP-injected Nagase analbuminemic rats in which glomerular permeability to macromolecules is enhanced. When MLuc-transfected cells were implanted into rats, activity of MLuc was undetectable not only in urine but also in serum. Even immediately after intravenous injection of recombinant MLuc, activity of MLuc was not detected in serum. Subsequent experiments revealed that, in contrast to SEAP, MLuc was rapidly inactivated either by rat serum, fetal bovine serum, or human serum. Albumin was identified as the molecule responsible for the inhibition of MLuc activity. These data elucidated advantages and limitations of secreted reporter molecules SEAP and MLuc under in vivo situations.     

A single secreted luciferase-based gene reporter assay

Promoter analysis typically employs a reporter gene fused to a test promoter combined with a second reporter fused to a control promoter that is used for normalization purposes. However, this approach is not valid when experimental conditions affect the control promoter. We have developed and validated a single secreted luciferase reporter (SSLR) assay for promoter analysis that avoids the use of a control reporter. The approach uses an early level of expression of a secreted luciferase linked to a test promoter as an internal normalization control for subsequent analysis of the same promoter. Comparison of the SSLR assay with the dual luciferase reporter (DLR) assay using HMGCR (3-hydroxy-3-methylglutaryl-coenzyme A reductase) and LDLR (low-density lipoprotein receptor) promoter constructs, which are down-regulated by 25-hydroxycholesterol, show that both assays yield similar results. Comparison of the response of the HMGCR promoter in SSLR transient assays compared very favorably with the response of the same promoter in the stable cell line. Overall, the SSLR assay proved to be a valid alternative to the DLR assay for certain applications and had significant advantages in that measurement of only one luciferase is required and monitoring can be continuous because cell lysis is not necessary.     

Properties of a repressible alkaline phosphatase secreted by the wild-type strain 74a of neurospora crassa

A repressible extracellular alkaline phosphatase (with activity increasing steadily even up to pH 10.5) was purified from cultures of the wild-type strain 74A of Neurospora crassa, after growth on acetate and under limiting amounts of inorganic phosphate for 72 hr at 30°. The enzyme was homogeneous on polyacrylamide gel electrophoresis (PAGE) with or without sodium dodecyl sulphate (SDS). The MW was ca 172 000 and 82 000 as determined by Sephadex G-200 gel filtration and SDS-PAGE, respectively. The enzyme contained 23.6% neutral sugars, cations were not required for activity, and it was not inactivated by 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) at pH 8. Kinetic data showed Michaelian behaviour for the enzymatic hydrolysis of 4-nitrophenyl disodium orthophosphate (PNP-P) at pH 9 (the K_m value and Hill coefficient were 2.2 × 10^?4 M and 0.95, respectively). It was also shown that, at pH 9, the apparent number of Pi bound per dimer molecule equalled one, with a K_i value of 7.0 × 10^?4 M. The secreted enzyme showed half-lives of 23.5, 49.0 and 23.5 min at, pH 5.4, 7.4 and 9.0, respectively, after thermal inactivation at 60°. At pH 5.4, the half-life value was quite similar, while the others were respectively 2 and 4 times greater than those previously described for the repressible alkaline phosphatase retained by the mycelium at pH 5.6 or secreted by ‘slime’ cells.     

Firefly luciferase as a reporter of regulated gene expression in higher plants

The firefly luciferase, assayedin vivo with a low-light video camera, acts as a non-invasive, real-time reporter of the temporal and spatial regulation of gene expression in single plants. Furthermore, the sensitivity of the luciferase assay in extracts of transformed plant tissue makes it a particularly useful marker in transient or stable transformation experiments.     

DRESSA: biosensing of dioxin and dioxin-like chemicals using secreted alkaline phosphatase

In this article, we describe a highly sensitive biosensing system, DRESSA, for detection of dioxin and dioxin-like chemicals. Tandem copies of the dioxin-responsive element (DRE) fused to a minimal viral promoter were subcloned into an expression plasmid upstream of a secreted alkaline phosphatase (SEAP) gene. When murine hepatoma cell line Hepa-1c1c7 was stably transfected with this construct, established sensor clones secreted SEAP following stimulation with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). A clone HeDS49 was found to be extremely sensitive; it secreted SEAP in response to TCDD in dose- and time-dependent manners, and the minimal detection limit was 100 fM. To detect more than 6 pM of TCDD, the whole assay time (from cell seeding to measurement of SEAP activity) could be reduced to 4h. Secretion of SEAP was induced selectively by other activators of DRE (3-methylcholanthrene, benzo[a]pyrene, and beta-naphthoflavone) but not by activators of unrelated responsive elements. These data suggested that because of the rapidity, easiness, specificity, and high sensitivity of DRESSA, it is more suitable than currently available detection systems for dioxin and dioxin-like chemicals and would be of great advantage to high-throughput screening of these pollutants in environmental samples.     

Catalytic properties and stability of three common variants of placental alkaline phosphatase

Three placental alkaline phosphatases purified to homogeneity, i.e., the F, I, and S variants, were investigated for catalytic and stability properties. All three forms of the enzyme were found to have almost identical pH optima (10.7–10.8), similar sensitivity to the uncompetitive inhibitors L-phenylalanine (70%) and L-leucine (30%), and identical K_m values against p-nitrophenylphosphate, -glycerophosphate, and -naphthylphosphate. Significant differences among the three types were observed in thermal stability. The F variant was found to be most stable and the I variant most labile at 79 C. At 70 C all three forms were stable.This investigation was supported by grants from the Swedish Medical Research Council (Projects No. 4217 and 03X-2725), from the Medical Faculty, University of Umeå, and Jubileumsklinikens i Umeå forskningsfond.     

Genetic variability of alkaline phosphatase expression in inbred mouse tissues

Quantitative alkaline phosphatase (ALP; EC 3.1.3.1) expression varies among various tissues and among inbred mouse strains. There is about a 20-fold difference in ALP activity in lungs from CBA/J and C57L/J inbred strains and this difference is inherited additively with a heritability of 0.84. Studies of thermostability at 56 and 65° C and sensitivity toward inhibitors (l-phenylalanine, l-homoarginine, l-phenylalanylglycylglycine, and levamisole) do not demonstrate differences in the ALP from lungs or liver of the CBA/J and C57L/J strains. The ALP activity in intestine expressed by the intestinal locus varies over 100-fold between A/J and DBA/1J strains. Further studies of the mechanisms resulting in this difference in ALP activity should help elucidate the mechanisms for aberrant expression of ALP in malignancy and for manipulation of low ALP activity in hypophosphatasia.This work has partially supported by NIH Grants GM-27018, GM-20138, GM-07511.     

Secretory fluorescent protein,a secretion green fluorescent fusion protein with alkaline phosphatase activity as a sensitive and traceable reporter in baculovirus expression system

The advantages of using traceable fluorescent protein (enhanced green fluorescent protein; EGFP) and a secretory alkaline phosphatase (SEAP) have been used to generate a reporter gene: the secretory fluorescent protein (SEFP). Sf21 cells, infected with the recombinant baculovirus containing the SEFP gene, revealed both traceable fluorescence and easily detectable alkaline phosphatase activity in the culture medium. The distribution of SEFP within the cells revealed that it was excluded from the nucleus, implying that the accumulation of SEFP in a secretory pathway, similar to that of the secretion signal-tagged FPs. Furthermore, the time- and dose-dependent release from the blockage of brefeldin A (BFA) confirmed that the secretion of SEFP was mediated by the secretion pathway and excluded leakage from viral infection. This SEFP reporter gene with traceable fluorescence and alkaline phosphatase activity may become a useful tool for studies on secretory protein production.     

Use of oligonucleotide-alkaline phosphatase conjugates as non-radioactive probes for rapid analysis of a proteinase inhibitor gene fromZea mays

Conjugates of oligonucleotides and alkaline phosphatase have been prepared and used as nonradioactive hybridization probes for the study ofPis3 (=MPI) a gene encoding a proteinase inhibitor fromZea mays. Attachment of the alkaline phosphatase was carried out either at the 5′ or 3′ end of two 25-bp oligonucleotides. Sensitivity of each alkaline phosphatase-oligonucleotide probe was assessed using a chemiluminescent substrate for detection of alkaline phosphatase activity. This sensitive method allows the rapid analysis of genomic clones isolated from aZea mays library and the subsequent characterization of the completePis3 gene without the need for construction of restriction maps for the cloned DNA fragments. This general strategy may be valuable for the identification of any gene for which a limited sequence is known and for location of specific DNA sequences that represent a small region within a larger DNA fragment.     

Expression and characterization of recombinant thermostable alkaline phosphatase from a novel thermophilic bacterium Thermus thermophilus XM

A gene （tap） encoding a thermostable alkaline phosphatase from the thermophilic bacterium Thermus thermophilus XM was cloned and sequenced. It is 1506 bp long and encodes a protein of 501 amino acid residues with a calculated molecular mass of 54.7 kDa. Comparison of the deduced amino acid sequence with other alkaline phosphatases showed that the regions in the vicinity of the phosphorylation site and metal binding sites are highly conserved. The recombinant thermostable alkaline phosphatase was expressed as a His6-tagged fusion protein in Escherichia coli and its enzymatic properties were characterized after purification. The pH and temperature optima for the recombinant thermostable alkaline phosphatases activity were pH 12 and 75 ℃. As expected, the enzyme displayed high thermostability, retaining more than 50% activity after incubating for 6 h at 80 ℃. Its catalytic function was accelerated in the presence of 0.1 mM Co^2＋, Fe^2＋, Mg^2＋, or Mn^2＋ but was strongly inhibited by 2.0 mM Fe^2＋. Under optimal conditions, the Michaelis constant （Kin） for cleavage of p-nitrophenyl-phosphate was 0.034 mM. Although it has much in common with other alkaline phosphatases, the recombinant thermostable alkaline phosphatase possesses some unique features, such as high optimal pH and good thermostability.     

Integration of alkaline phosphatase in the intestinal brush border membrane

Alkaline phosphatase has been solubilized from porcine intestinal mucosa by two different methods: treatment of the mucosa by Emulphogen BC 720 and papain hydrolysis of enterocyte brush border membrane vesicles. Two different enzyme forms have been obtained by these methods.The two enzyme forms (‘detergent form’ and ‘papain form’) have been purified to homogeneity by similar techniques and exhibit closely related molecular characteristics. However, the detergent form displays a hydrophobic behaviour and aggregates in media free of detergent. The two forms can be differentiated by their electrophoretic mobility on polyacrylamide gel in the absence of sodium dodecyl sulphate.By electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulphate, it has been shown that the detergent and papain forms of alkaline phophatase are dimers consisting of two apparently identical subunits whose molecular weights are 64 000 and 61 000, respectively. The difference between these molecular weights has been attributed to the existence of a hydrophobic region in the detergent form which is present on each subunit.     

Purification and partial characterization of the I variant of placental alkaline phosphatase

The I variant of placental alkaline phosphatase was purified to homogeneity by means of DEAE-cellulose chromatography, isoelectric focusing, and gel filtration on AcA-34. The specific activity of the I variant was found to be 3.33 kat/mg. The enzyme is a dimer with an isoelectric point of 4.6 and a molecular weight of 120,000 as determined by sodium dodecylsulfate electrophoresis. The amino acid composition and other physicochemical properties of the I variant were compared with those of the more common F and S variants. The low activity associated with the I variant is apparently not due to a low specific activity, but to decreased molecular stability. The behavior in the ultracentrifuge and other observations suggest that the I variant differs from the F and S variants in surface charge distribution.This investigation was supported by grants from the Swedish Medical Research Council (projects No. 4217 and No. 03X-2725) and from the Medical Faculty, University of Umeå.     

Characterization of the mutant (A115V) tissue-nonspecific alkaline phosphatase gene from adult-type hypophosphatasia

Hypophosphatasia (HOPS) is a clinically heterogeneous heritable disorder characterized by defective skeletal mineralization, deficiency of tissue-nonspecific alkaline phosphatase (TNSALP) activity, and premature loss of deciduous teeth. To date, various mutations in the TNSALP gene have been identified. Especially, A115V located in exon 5 has been detected in a Japanese patient with severe periodontitis and adult-type HOPS. In this study, we have characterized the protein translated from the mutant A115V gene. Wild-type and A115V mutant-type TNSALP cDNA expression vector pcDNA3 have been constructed and transfected to COS-1 cells by lipofectin technique. After 48-h transfection, the cells were subjected to assay ALP activity. In order to identify possible dominant effect of the mutation, we performed co-transfections of wild-type and mutated cDNA, and evaluated the residual activities of each mutation. Detection of TNSALP synthesized by COS-1 cells transfected with the wild- or the mutated-type was also performed by using an immunofluorescent method. ALP activity of cell transfected with the mutant cDNA (A115V) plasmid after 48-h transfection exhibited 0.399+/-0.021 U/mg. As the enzymatic activity of the wild type was taken as 100%, the value of the mutant was estimated as 16.9%. When co-transfected this mutant showed no inhibition of the wild-type enzyme. TNSALP in COS-1 cells with transfected with the mutant exhibited strong fluorescence at the surface of cells as wild-type. This study indicated that the mutant (A115V) TNSALP gene produced the defective ALP enzyme and it could be recessively transmitted and be a disease-causing mutation of the adult-type hypophosphatasia.     

Nucleotide sequence of the gene for alkaline phosphatase of Thermus caldophilus GK24 and characteristics of the deduced primary structure of the enzyme

The gene encoding Thermus caldophilus GK24 (Tca) alkaline phosphatase was cloned into Escherichia coli. The primary structure of Tca alkaline phosphatase was deduced from its nucleotide sequence. The Tca alkaline phosphatase precursor, including the signal peptide sequence, was comprised of 501 amino acid residues. Its molecular mass was determined to be 54? omitted?760 Da. On the alignment of the amino acid sequence, Tca alkaline phosphatase showed sequence homology with the microbial alkaline phosphatases, 20% identity with E. coli alkaline phosphatase and 22% Bacillus subtilis (Bsu) alkaline phosphatases. High sequence identity was observed in the regions containing the Ser-102 residue of the active site, the zinc and magnesium binding sites of E. coli alkaline phosphatase. Comparison of Tca alkaline phosphatase and E. coli alkaline phosphatase structures suggests that the reduced activity of the Tca alkaline phosphatase, in the presence of zinc, is directly involved in some of the different metal binding sites. Heat-stable Tca alkaline phosphatase activity was detected in E. coli YK537, harboring pJRAP.     

Facile dimethyl amino group triggered cyclic sulfonamides synthesis and evaluation as alkaline phosphatase inhibitors

Owing to the biological importance of cyclic sulfonamides (sultams), herein we report a new, facile and cost-effective method for the synthesis of sultams that makes use of a reaction between dansyl amide and easily accessible benzaldehydes under mildly acidic conditions. All compounds were obtained in good yields (69–96%). Consequently a series of cyclic sulfonamides (7a–7n) was synthesized and characterized using FTIR, MS and NMR spectroscopy, crystal structure of compound 7b has also been determined. All compounds were evaluated for their potential to inhibit alkaline phosphatase (bTNAP and bIAP). All compounds were found to be excellent inhibitors of bTNAP with IC₅₀ values in lower micro-molar range (0.11–6.63 μM). Most of the compounds were selective inhibitors of bTNAP over bIAP. Only six compounds were found to be active against bIAP (IC₅₀ values in the range 0.38–3.48 μM). Molecular docking studies were carried out to identify and rationalize the structural elements necessary for efficient AP inhibition.