10-formyltetrahydrofolate dehydrogenase, one of the major folate enzymes, is down-regulated in tumor tissues and possesses suppressor effects on cancer cells.
AuthorsKrupenko SA, Oleinik NV
JournalCell Growth Differ
PubMed ID12065246
Our studies showed that an abundant folate enzyme, 10-formyltetrahydrofolatedehydrogenase (FDH), is strongly down-regulated in several types of cancer on both the mRNA and the protein level. Transient expression of FDH in several human prostate cancer cell lines, a hepatocarcinoma cell line, HepG2, and a lung cancer cell line, A549, suppressed ... More
Primitive adult hematopoietic stem cells can function as osteoblast precursors.
AuthorsOlmsted-Davis EA, Gugala Z, Camargo F, Gannon FH, Jackson K, Kienstra KA, Shine HD, Lindsey RW, Hirschi KK, Goodell MA, Brenner MK, Davis AR
JournalProc Natl Acad Sci U S A
PubMed ID14673088
'Osteoblasts are continually recruited from stem cell pools to maintain bone. Although their immediate precursor is a plastic-adherent mesenchymal stem cell able to generate tissues other than bone, increasing evidence suggests the existence of a more primitive cell that can differentiate to both hematopoietic and mesenchymal cells. We show here ... More
Qualification of embryonal carcinoma 2102Ep as a reference for human embryonic stem cell research.
AuthorsJosephson R, Ording CJ, Liu Y, Shin S, Lakshmipathy U, Toumadje A, Love B, Chesnut JD, Andrews PW, Rao MS, Auerbach JM
JournalStem Cells
PubMed ID17284651
'As the number of human embryonic stem cell (hESC) lines increases, so does the need for systematic evaluation of each line''s characteristics and potential. Comparisons between lines are complicated by variations in culture conditions, feeders, spontaneous differentiation, and the absence of standardized assays. These difficulties, combined with the inability of ... More
Osteoclast responses to lipopolysaccharide, parathyroid hormone and bisphosphonates in neonatal murine calvaria analyzed by laser scanning confocal microscopy.
'Because the development and activity of osteoclasts in bone remodeling is critically dependent on cell-cell and cell-matrix interactions, we used laser confocal microscopy to study the response of osteoclasts to lipopolysaccharide (LPS; 10 microg/ml), parathyroid hormone (PTH; 10(-8) M), and bisphosphonates (BPs; 1-25 microM clodronate or 0.1-2.5 microM risedronate) in ... More
Heterogeneous proliferative potential in regenerative adult newt cardiomyocytes.
AuthorsBettencourt-Dias M, Mittnacht S, Brockes JP
JournalJ Cell Sci
PubMed ID12928330
'Adult newt cardiomyocytes, in contrast to their mammalian counterparts, can proliferate after injury and contribute to the functional regeneration of the heart. In order to understand the mechanisms underlying this plasticity we performed longitudinal studies on single cardiomyocytes in culture. We find that the majority of cardiomyocytes can enter S ... More
Visualization of mosaicism in tissues of normal and mismatch-repair-deficient mice carrying a microsatellite-containing transgene.
AuthorsHersh MN, Stambrook PJ, Stringer JR
JournalMutat Res
PubMed ID12175905
'To determine the frequency of mutation in different cell types of mammals, transgenic mice that allow mutant cells to be visualized in situ were used. These mice carry a defective allele of the human placental alkaline phosphatase (PLAP) gene. The allele does not produce enzyme because the reading frame is ... More
Quantitative differences in phase I and II metabolism between rat precision-cut liver slices and isolated hepatocytes.
'Testosterone (250 microM), 7-ethoxycoumarin (25 microM), and 1-chloro-2,4-dinitrobenzene (CDNB, 50 microM) were used as substrates to compare phase I and II metabolism in rat precision-cut liver slices and rat hepatocytes. Overall clearance to metabolites was significantly greater in hepatocytes for testosterone (1.9- to 16.9-fold), 7-ethoxycoumarin (O-deethylation, 14.8-fold; glucuronidation, 3.1-fold), and ... More
Alkaline phosphatase is involved in the control of adipogenesis in the murine preadipocyte cell line, 3T3-L1.
AuthorsAli AT, Penny CB, Paiker JE, van Niekerk C, Smit A, Ferris WF, Crowther NJ
JournalClin Chim Acta
PubMed ID15748605
'OBJECTIVE: As alkaline phosphatase may play a role in cell differentiation, our aim was to study the possible role of this enzyme in the differentiation of preadipocytes (3T3-L1 cells) into adipocytes. RESEARCH METHODS AND PROCEDURES: 3T3-L1 cells were grown in medium containing insulin, dexamethasone and IBMX to induce adipogenesis. Adipogenesis ... More
Fluorescence in situ hybridization of scarce leptin receptor mRNA using the enzyme-labeled fluorescent substrate method and tyramide signal amplification.
AuthorsBreininger JF, Baskin DG
JournalJ Histochem Cytochem
PubMed ID11101627
'To increase the sensitivity of fluorescence in situ hybridization (FISH) for detection of low-abundance mRNAs, we performed FISH on cryostat sections of rat hypothalamus with biotin-labeled riboprobes to leptin receptor (ObRb) and amplified the signal by combining tyramide signal amplification (TSA) and Enzyme-Labeled Fluorescent alkaline phosphatase substrate (ELF) methods. First, ... More
Enzymatic activity of alkaline phosphatase inside protein and polymer structures fabricated via multiphoton excitation.
AuthorsBasu S, Campagnola PJ
JournalBiomacromolecules
PubMed ID15003023
'We demonstrate micron scale control of bioactivity through the use of multiphoton excited photochemistry, where this technique has been used to cross-link three-dimensional matrixes of alkaline phosphatase, bovine serum albumin, and polyacrylamide and combinations therein. Using a fluorescence-based assay (ELF-97), the enzymatic activity has been studied using a Michaelis-Menten analysis, ... More
Sequence versus structure for the direct detection of 16S rRNA on planar oligonucleotide microarrays.
AuthorsChandler DP, Newton GJ, Small JA, Daly DS
JournalAppl Environ Microbiol
PubMed ID12732571
'A two-probe proximal chaperone detection system consisting of a species-specific capture probe for the microarray and a labeled, proximal chaperone probe for detection was recently described for direct detection of intact rRNAs from environmental samples on oligonucleotide arrays. In this study, we investigated the physical spacing and nucleotide mismatch tolerance ... More
Immunofluorescence assay for the quantitative and qualitative evaluation of intracellular interleukin-8 in microtiter plates.
AuthorsWolff B, Zsak M, Rabeck C
JournalAnal Biochem
PubMed ID9025904
'In order to monitor the effects of drugs on interleukin-8 (IL-8) production by cells, a microtiter plate assay that determines four parameters simultaneously was established (i) levels of secreted IL-8 (supernatant ELISA), (ii) levels of intracellular IL-8 (cell ELISA), (iii) intracellular localization (fluorescence microscopy), and (iv) the amount of cellular ... More
AuthorsMendoza LG, McQuary P, Mongan A, Gangadharan R, Brignac S, Eggers M
JournalBiotechniques
PubMed ID10524321
'A new generation biochip is described as capable of supporting high-throughput (HT), multiplexed enzyme-linked immunosorbent assays (ELISAs). These biochips consist of an optically flat, glass plate containing 96 wells formed by an enclosing hydrophobic Teflon mask. The footprint dimensions of each well and the plate precisely match those of a ... More
Affinity capture of proteins from solution and their dissociation by contact printing.
AuthorsBernard A, Fitzli D, Sonderegger P, Delamarche E, Michel B, Bosshard HR, Biebuyck H
JournalNat Biotechnol
PubMed ID11533647
'Biological experiments at the solid/liquid interface, in general, require surfaces with a thin layer of purified molecules, which often represent precious material. Here, we have devised a method to extract proteins with high selectivity from crude biological sample solutions and place them on a surface in a functional, arbitrary pattern. ... More
Confocal multilaser focusing and single-laser characterization of ultraviolet excitable stains of cellular preparations.
AuthorsKahn E, Frouin F, Souchier C, Bernengo JC, Bruzzoni-Giovanelli H, Clément O, Frija G, Di Paola R, Calvo F, Linares-Cruz G
JournalCytometry
PubMed ID10754516
'BACKGROUND: The aims of this study were (1) to realign cellular preparations when spots and structures are excited by different lasers of a confocal laser scanning microscope (multilaser studies); (2) to avoid the use of realigment methods by selecting fluorochromes that can be excited by only one laser (single-laser experiments). ... More
Fluorescence-based staining for tartrate-resistant acidic phosphatase (TRAP) in osteoclasts combined with other fluorescent dyes and protocols.
AuthorsFilgueira L
JournalJ Histochem Cytochem
PubMed ID14966208
Osteoclasts are the only bone-resorbing cells. In addition to other specific properties, osteoclasts are characterized by their expression of tartrate-resistant acidic phosphatase (TRAP), which is usually detected using a histochemical method for light microscopy. Using ELF97 phosphatase substrate, this study describes a new fluorescence-based method for TRAP detection. The fluorescence-based ... More
Enzymatic quantification of cell-matrix and cell-cell adhesion.
AuthorsLöster K, Horstkorte R
JournalMicron
PubMed ID10568230
Adhesion assays are powerful tools to investigate the adhesive properties of cells. The quantification of cell adhesion enables determination of the capacity of cells to stick to a target, screening for novel adhesion involved binding molecules, exploration of structure-function relationships of adhesion molecules, evaluation of adhesion targets, and examination of ... More
Novel method for screening bacterial colonies for phosphatase activity.
Current methods for screening large numbers of bacterial colonies for phosphatase activity, rely heavily on the use of colorimetric assays. While such methods have been applied extensively in the laboratory, they are not without their drawbacks. We here describe a precipitating fluorescent probe that can be used to screen phosphatase ... More
Functional dissociation of the basolateral transcytotic compartment from the apical phago-lysosomal compartment in human osteoclasts.
AuthorsMeagher J, Zellweger R, Filgueira L,
JournalJ Histochem Cytochem
PubMed ID15872059
Tartrate-resistant acid phosphatase (TRAP) is essential for elimination of Staphylococcus aureus, the main infectious agent responsible for osteomyelitis. This in vitro study investigated uptake and processing of fluorescence-labeled S. aureus by human osteoclasts and dendritic cells. The cells were stained for TRAP and the acidic compartment using a fluorescence-based protocol. ... More
Osteoclast size is controlled by Fra-2 through LIF/LIF-receptor signalling and hypoxia.
AuthorsBozec A, Bakiri L, Hoebertz A, Eferl R, Schilling AF, Komnenovic V, Scheuch H, Priemel M, Stewart CL, Amling M, Wagner EF,
JournalNature
PubMed ID18548006
Osteoclasts are multinucleated haematopoietic cells that resorb bone. Increased osteoclast activity causes osteoporosis, a disorder resulting in a low bone mass and a high risk of fractures. Increased osteoclast size and numbers are also a hallmark of other disorders, such as Paget's disease and multiple myeloma. The protein c-Fos, a ... More
Carbonic anhydrase isoform VII acts as a molecular switch in the development of synchronous gamma-frequency firing of hippocampal CA1 pyramidal cells.
AuthorsRuusuvuori E, Li H, Huttu K, Palva JM, Smirnov S, Rivera C, Kaila K, Voipio J
JournalJ Neurosci
PubMed ID15028762
Identification of the molecular mechanisms that enable synchronous firing of CA1 pyramidal neurons is central to the understanding of the functional properties of this major hippocampal output pathway. Using microfluorescence measurements of intraneuronal pH, in situ hybridization, as well as intracellular, extracellular, and K+-sensitive microelectrode recordings, we show now that ... More
Photobleaching of arterial autofluorescence for immunofluorescence applications.
AuthorsKingsley K, Carroll K, Huff JL, Plopper GE
JournalBiotechniques
PubMed ID11314262
Immunohistochemical localization of low-level antigens in the arterial vasculature is complicated by the presence of complex molecules such as collagen, elastin, cholesterol, and fluorescent lipids that exhibit autofluorescence over a wide spectrum of wavelengths. UV irradiation of arterial vasculature has remained ineffective in preparing samples for immunofluorescent staining because of ... More
Laminin-5 induces osteogenic gene expression in human mesenchymal stem cells through an ERK-dependent pathway.
AuthorsKlees RF, Salasznyk RM, Kingsley K, Williams WA, Boskey A, Plopper GE
JournalMol Biol Cell
PubMed ID15574877
The laminin family of proteins is critical for managing a variety of cellular activities including migration, adhesion, and differentiation. In bone, the roles of laminins in controlling osteogenic differentiation of human mesenchymal stem cells (hMSC) are unknown. We report here that laminin-5 is found in bone and expressed by hMSC. ... More
Activation of NF-kappa B by XIAP, the X chromosome-linked inhibitor of apoptosis, in endothelial cells involves TAK1.
AuthorsHofer-Warbinek R, Schmid JA, Stehlik C, Binder BR, Lipp J, de Martin R
JournalJ Biol Chem
PubMed ID10807933
Exposure of endothelial and many other cell types to tumor necrosis factor alpha generates both apoptotic and anti-apoptotic signals. The anti-apoptotic pathway leads to activation of the transcription factor NF-kappaB that regulates the expression of genes such as A20 or members of the IAP gene family that protect cells from ... More
AuthorsMcBeath R, Pirone DM, Nelson CM, Bhadriraju K, Chen CS
JournalDev Cell
PubMed ID15068789
Commitment of stem cells to different lineages is regulated by many cues in the local tissue microenvironment. Here we demonstrate that cell shape regulates commitment of human mesenchymal stem cells (hMSCs) to adipocyte or osteoblast fate. hMSCs allowed to adhere, flatten, and spread underwent osteogenesis, while unspread, round cells became ... More
Laser interference pattern ablation of a carbon fiber microelectrode: biosensor signal enhancement after enzyme attachment.
AuthorsRosenwald SE, Nowall WB, Dontha N, Kuhr WG
JournalAnal Chem
PubMed ID11055709
Fluorescence microscopy was used to visualize the accumulated fluorescent product of the enzyme alkaline phosphatase to indicate where active covalently bound enzyme remained on the surface after application of a Nd: YAG laser interference pattern to a surface that was first globally derivatized with the covalently bound enzyme. The electrochemical ... More
Elevated expression of endoglin, a component of the TGF-beta-receptor complex, correlates with proliferation of tumor endothelial cells.
AuthorsMiller DW, Graulich W, Karges B, Stahl S, Ernst M, Ramaswamy A, Sedlacek HH, Müller R, Adamkiewicz J
JournalInt J Cancer
PubMed ID10225446
Endoglin/CD105 is a membrane protein involved in the TGF-beta receptor signalling pathway. Endoglin expression has been reported to be selective for a few cell types, in particular endothelial cells, although a number of conflicting reports have been published. In this study, we performed a detailed analysis of endoglin expression in ... More
Enzyme cytochemical techniques for metabolic mapping in living cells, with special reference to proteolysis.
AuthorsBoonacker E, Van Noorden CJ
JournalJ Histochem Cytochem
PubMed ID11724895
Specific enzymes play key roles in many pathophysiological processes and therefore are targets for therapeutic strategies. The activity of most enzymes is largely determined by many factors at the post-translational level. Therefore, it is essential to study the activity of target enzymes in living cells and tissues in a quantitative ... More
A central role for the notochord in vertebral patterning.
AuthorsFleming A, Keynes R, Tannahill D
JournalDevelopment
PubMed ID14736741
The vertebrates are defined by their segmented vertebral column, and vertebral periodicity is thought to originate from embryonic segments, the somites. According to the widely accepted 'resegmentation' model, a single vertebra forms from the recombination of the anterior and posterior halves of two adjacent sclerotomes on both sides of the ... More
In vivo targeted repair of a point mutation in the canine dystrophin gene by a chimeric RNA/DNA oligonucleotide.
AuthorsBartlett RJ, Stockinger S, Denis MM, Bartlett WT, Inverardi L, Le TT, thi Man N, Morris GE, Bogan DJ, Metcalf-Bogan J, Kornegay JN
JournalNat Biotechnol
PubMed ID10835598
In the canine model of Duchenne muscular dystrophy in golden retrievers (GRMD), a point mutation within the splice acceptor site of intron 6 leads to deletion of exon 7 from the dystrophin mRNA, and the consequent frameshift causes early termination of translation. We have designed a DNA and RNA chimeric ... More
Macrophage-independent fungicidal action of the pulmonary collectins.
Histoplasma capsulatum (Hc) is a facultative intracellular fungal pathogen that causes acute and chronic pneumonia. In this study, we investigated the role of the pulmonary collectins, surfactant proteins (SP) A and D, in the clearance of Hc yeast from the lung. Exposure of yeast to either collectin induced a dose-dependent ... More
Fluorimetric determination of theophylline in serum by inhibition of bovine alkaline phosphatase in AOT based water/in oil microemulsion.
AuthorsJourquin G, Kauffmann JM
JournalJ Pharm Biomed Anal
PubMed ID9919959
Theophylline is an effective bronchodilatator used in the treatment of asthma which requires frequent control because of its narrow therapeutic index. Over the past decade much attention has been dedicated to the peculiar properties of the inner water pools of AOT (sodium 2-bishexyl-ethyl sulfosuccinate) microemulsions as enzyme microreactors, yet few ... More
Detecting and genotyping Escherichia coli O157:H7 using multiplexed PCR and nucleic acid microarrays.
AuthorsCall DR, Brockman FJ, Chandler DP
JournalInt J Food Microbiol
PubMed ID11482571
Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of ... More
Semi-automated positional analysis using laser scanning microscopy of cells transfected in a regenerating newt limb.
AuthorsPecorino LT, Brockes JP, Entwistle A
JournalJ Histochem Cytochem
PubMed ID8666741
Limb regeneration in urodele amphibians such as the newt is a key system for investigating the positional identity of cells. The regenerate arises locally from blastemal cells, mesenchymal progenitors that normally give rise to structures distal to the amputation plane but which can be respecified (proximalized) by treatment with retinoic ... More
2-(2'-phosphoryloxyphenyl)-4(3H)-quinazolinone derivatives as fluorogenic precipitating substrates of phosphatases.
AuthorsHuang Z, Terpetschnig E, You W, Haugland RP
JournalAnal Biochem
PubMed ID1336935
Characterization of 2-(2'-phosphoryloxyphenyl)-4(3H)-quinazolinone (PPQ) derivatives as fluorogenic precipitating substrates of phosphatases is reported in this work. Soluble and colorless PPQ derivatives can be specifically hydrolyzed by acid and alkaline phosphatases into insoluble products, 2-(2'-hydroxyphenyl)-4(3H)-quinazolinone (HPQ) derivatives which appear as fluorescent precipitates in water. The fluorescence and precipitation of HPQ depend ... More
Roles of substrate availability and infection of resting and activated CD4+ T cells in transmission and acute simian immunodeficiency virus infection.
AuthorsZhang ZQ, Wietgrefe SW, Li Q, Shore MD, Duan L, Reilly C, Lifson JD, Haase AT
JournalProc Natl Acad Sci U S A
PubMed ID15064398
In studies of sexual mucosal transmission and early stages of simian immunodeficiency virus (SIV) and HIV infections, productive infection predominates in CD4(+) T cell populations, in both ostensibly resting and activated cells. The surprising ability of SIV and HIV to replicate in resting cells in vivo, in contrast to propagation ... More
HGF inhibits BMP-induced osteoblastogenesis: possible implications for the bone disease of multiple myeloma.
AuthorsStandal T, Abildgaard N, Fagerli UM, Stordal B, Hjertner O, Borset M, Sundan A
JournalBlood
PubMed ID17138824
The bone disease in multiple myeloma is caused by an uncoupling of bone formation from bone resorption. A key difference between patients with and patients without osteolytic lesion is that the latter have fewer and less active osteoblasts. Hepatocyte growth factor (HGF) is often produced by myeloma cells and is ... More
A high-resolution, fluorescence-based method for localization of endogenous alkaline phosphatase activity.
AuthorsCox WG, Singer VL
JournalJ Histochem Cytochem
PubMed ID10544217
We describe a high-resolution, fluorescence-based method for localizing endogenous alkaline phosphatase in tissues and cultured cells. This method utilizes ELF (Enzyme-Labeled Fluorescence)-97 phosphate, which yields an intensely fluorescent yellow-green precipitate at the site of enzymatic activity. We compared zebrafish intestine, ovary, and kidney cryosections stained for endogenous alkaline phosphatase using ... More
Simultaneous multianalyte ELISA performed on a microarray platform.
AuthorsWiese R, Belosludtsev Y, Powdrill T, Thompson P, Hogan M
JournalClin Chem
PubMed ID11468236
BACKGROUND: A logical progression of the widely used microtiter plate ELISA is toward a protein array format that allows simultaneous detection of multiple analytes at multiple array addresses within a single well. Here we describe the construction and use of such a multiplex ELISA to measure prostate-specific antigen (PSA), alpha1-antichymotrypsin-bound ... More
The ELF -97 phosphatase substrate provides a sensitive, photostable method for labelling cytological targets.
AuthorsParagas VB, Kramer JA, Fox C, Haugland RP, Singer VL
JournalJ Microsc
PubMed ID12000550
We compared fluorescent signals obtained with fluorescein conjugates and the ELF-97 (enzyme-labelled fluorescence) phosphatase substrate [2-(5'-chloro-2-phosphoryloxyphenyl)-6-chloro-4(3H)-quinazolinone] in labelling cytological structures requiring high spatial resolution. Enzymatic cleavage of the ELF-97 phosphatase substrate yields an extremely fine precipitate that remains well localized to the site of enzymatic activity. This precipitate fluoresces bright ... More
Extracellular phosphatase activity of natural plankton studied with ELF97 phosphate: fluorescence quantification and labelling kinetics.
AuthorsNedoma J, Strojsová A, Vrba J, Komárková J, Simek K
JournalEnviron Microbiol
PubMed ID12755713
ELF(R)97 phosphate (ELFP) is a phosphatase substrate which produces ELF(R)97 alcohol (ELFA), a fluorescent water-insoluble product, upon hydrolysis. We studied the kinetics of ELFA precipitation in freshwater samples at levels of total plankton and single phytoplankton cells, and tested the suitability of ELFP for measurement of surface-bound algal extracellular phosphatases. ... More
Fingerprinting closely related xanthomonas pathovars with random nonamer oligonucleotide microarrays.
Current bacterial DNA-typing methods are typically based on gel-based fingerprinting methods. As such, they access a limited complement of genetic information and many independent restriction enzymes or probes are required to achieve statistical rigor and confidence in the resulting pattern of DNA fragments. Furthermore, statistical comparison of gel-based fingerprints is ... More
A novel glucan-binding protein with lipase activity from the oral pathogen Streptococcus mutans.
AuthorsShah DS, Russell RR
JournalMicrobiology
PubMed ID15184580
Streptococcus mutans produces extracellular glucosyltransferases (GTFs) that synthesize glucans from sucrose. These glucans are important in determining the permeability properties and adhesiveness of dental plaque. GTFs and the GbpA glucan-binding protein are characterized by a binding domain containing a series of 33-amino-acid repeats, called 'A' repeats. The S. mutans genome ... More
Functional expression and characterization of the wild-type mammalian renal cortex sodium/phosphate cotransporter and an 215R mutant in Saccharomyces cerevisiae.
AuthorsBernhardt F, Schoner W, Schroeder B, Breves G, Scheiner-Bobis G
JournalBiochemistry
PubMed ID10521262
The wild-type and an R215E mutant of the rat renal cortex sodium/phosphate cotransporter type 2 (NaPi-2) were functionally expressed in the yeast Saccharomyces cerevisiae strain MB192, a cell line lacking the high-affinity endogenous H+/P(i) cotransporter. The expression of the mRNA molecules and corresponding proteins was confirmed by Northern and Western ... More
Fluid shear stress induces less calcium response in a single primary osteocyte than in a single osteoblast: implication of different focal adhesion formation.
AuthorsKamioka H, Sugawara Y, Murshid SA, Ishihara Y, Honjo T, Takano-Yamamoto T
JournalJ Bone Miner Res
PubMed ID16813522
The immediate calcium response to fluid shear stress was compared between osteocytes and osteoblasts on glass using real-time calcium imaging. The osteoblasts were responsive to fluid shear stress of up to 2.4 Pa, whereas the osteocytes were not. The difference in flow-induced calcium may be related to differences in focal ... More
Detection of endogenous and antibody-conjugated alkaline phosphatase with ELF-97 phosphate in multicolor flow cytometry applications.
AuthorsTelford W, Cox W, Singer V
JournalCytometry
PubMed ID11169576
BACKGROUND: The fluorogenic alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF(R)-97 phosphate, for Enzyme-Labeled Fluorescence) has been used primarily in microscope-based imaging applications to detect endogenous AP activity, antigens and various ligands in cells and tissues, and nucleic acid hybridization. In a previous study, we demonstrated the applicability of ELF-97 phosphate for ... More
Spatial patterns of alkaline phosphatase expression within bacterial colonies and biofilms in response to phosphate starvation.
AuthorsHuang CT, Xu KD, McFeters GA, Stewart PS
JournalAppl Environ Microbiol
PubMed ID9546188
The expression of alkaline phosphatase in response to phosphate starvation was shown to be spatially and temporally heterogeneous in bacterial biofilms and colonies. A commercial alkaline phosphatase substrate that generates a fluorescent, insoluble product was used in conjunction with frozen sectioning techniques to visualize spatial patterns of enzyme expression in ... More
Phosphate stress in cultures and field populations of the dinoflagellate prorocentrum minimum detected by a single-cell alkaline phosphatase assay
AuthorsDyhrman ST, Palenik B
JournalAppl Environ Microbiol
PubMed ID10388722
Alkaline phosphatase activity is a common marker of phosphate stress in many phytoplankton, but it has been difficult to attribute alkaline phosphatase activity to specific organisms or groups of phytoplankton in the field with traditional biochemical procedures. A new alkaline phosphatase substrate, ELF-97 (enzyme-labeled fluorescence), shows promise in this regard. ... More
Detection of endogenous alkaline phosphatase activity in intact cells by flow cytometry using the fluorogenic ELF-97 phosphatase substrate.
BACKGROUND: The alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF((R))-97 for enzyme-labeled fluorescence) has been found useful for the histochemical detection of endogenous AP activity and AP-tagged proteins and oligonucleotide probes. In this study, we evaluated its effectiveness at detecting endogenous AP activity by flow cytometry. METHODS: The ELF-97 phosphatase substrate was ... More
Evidence that the caudal brainstem is a target for the inhibitory effect of leptin on food intake.
Three experiments were performed to investigate the hypothesis that leptin action within the caudal brain stem (CBS) contributes to its intake inhibitory effects. The first experiment evaluated the anatomical distribution of leptin receptor mRNA in rat CBS using a sensitive fluorescence in situ hybridization method with a riboprobe specific for ... More
The ELF-97 alkaline phosphatase substrate provides a bright, photostable, fluorescent signal amplification method for FISH.
We used the ELF-97 (Enzyme-Labeled Fluorescence) phosphatase substrate, 2-(5'-chloro-2-phosphoryloxyphenyl)-6-chloro-4(3H)-quinazolinone, with alkaline phosphatase conjugates of streptavidin and appropriate antibodies to amplify signals from biotinylated and haptenylated hybridization probes. The dephosphorylated product, ELF-97 alcohol, is a bright yellow-green fluorescent precipitate optimally excited at approximately 360 nm, with emission centered at approximately 530 ... More