'Other than stimulation of cell contractility, little is known about the potential metabolic effects induced by sulfonylureas, independently of insulin action. Previous studies from our laboratory demonstrated complete abrogation of glomerulosclerosis in an experimental model of type 1 diabetes chronically (9 mo) treated with low-dose sulfonylureas (Biederman JI, Vera E, ... More
Negative regulation of EGFR signalling through integrin-alpha1beta1-mediated activation of protein tyrosine phosphatase TCPTP.
AuthorsMattila E, Pellinen T, Nevo J, Vuoriluoto K, Arjonen A, Ivaska J
JournalNat Cell Biol
PubMed ID15592458
'Integrin-mediated cell adhesion regulates a multitude of cellular responses, including proliferation, survival and cross-talk between different cellular signalling pathways. So far, integrins have been mainly shown to convey permissive signals enabling anchorage-dependent receptor tyrosine kinase signalling. Here we show that a collagen-binding integrin alpha(1)beta(1) functions as a negative regulator of ... More
Functional screening of intracellular proteins in single cells and in patterned cell arrays using electroporation.
AuthorsNolkrantz K, Farre C, Hurtig KJ, Rylander P, Orwar O
JournalAnal Chem
PubMed ID12199607
'A tool for detection and characterization of intracellular enzyme-substrate and receptor-ligand interactions inside the cytoplasm of single targeted cells or small confined groups of cells is presented. Fluorogenic enzyme substrates and receptor ligands were rapidly delivered by electroosmosis and internalized by electroporation in cells using an electrolyte-filled capillary (EFC) biased ... More
'Array-based sensors provide an architecture for multianalyte sensing. In this paper, we report a new approach for array fabrication. Sensors are made by immobilizing different reactive chemistries on the surfaces of microspheres. Sensor arrays are prepared by randomly distributing a mixture of microsphere sensors on an optical substrate containing thousands ... More
In vivo imaging of tumors with protease-activated near-infrared fluorescent probes.
AuthorsWeissleder R, Tung CH, Mahmood U, Bogdanov A
JournalNat Biotechnol
PubMed ID10207887
'We have developed a method to image tumor-associated lysosomal protease activity in a xenograft mouse model in vivo using autoquenched near-infrared fluorescence (NIRF) probes. NIRF probes were bound to a long circulating graft copolymer consisting of poly-L-lysine and methoxypolyethylene glycol succinate. Following intravenous injection, the NIRF probe carrier accumulated in ... More
Alendronate inhibition of protein-tyrosine-phosphatase-meg1.
AuthorsOpas EE, Rutledge SJ, Golub E, Stern A, Zimolo Z, Rodan GA, Schmidt A
JournalBiochem Pharmacol
PubMed ID9310349
'Alendronate (4-amino-1-hydroxybutylidene-1,1-bisphosphonate) is a potent bisphosphonate that inhibits osteoclastic bone resorption and has proven effective for the treatment of osteoporosis. Its molecular mechanism of action, however, has not been defined precisely. Here we report that alendronate is a potent inhibitor of the protein-tyrosine-phosphatase-meg1 (PTPmeg1). Two substrates were employed in this ... More
Giant vesicles as models to study the interactions between membranes and proteins.
AuthorsFischer A, Oberholzer T, Luisi PL
JournalBiochim Biophys Acta
PubMed ID10930520
'The interaction between polypeptides and membranes is a fundamental aspect of cell biochemistry. Liposomes have been used in this context as in vitro systems to study such interactions. We present here the case of giant vesicles (GVs), which, due to their size (radius larger than 10 microns), mimic more closely ... More
A new microassay for the determination of alkaline phosphatase activity in early mouse concepti.
AuthorsAhumada A, Izquierdo L
JournalBiol Res
PubMed ID8728837
'A new enzymatic assay for the determination of alkaline phosphatase (APase) activity in preimplantation mouse concepti is described. This method allows estimation of APase activity of concepti extracts using the fluorogenic substrate, 3,6-fluorescein diphosphate (FDP). For measuring APase activity, 0.1% Nonidet P-40 was used to solubilize the enzyme. Control assays ... More
3,6-Fluorescein Diphosphate: A Sensitive Fluorogenic and Chromogenic Substrate for Protein Tyrosine Phosphatases*
AuthorsHuang Z, Wang Q, Ly HD, Gorvindarajan A, Scheigetz J, Zamboni R, Desmarais S, Ramachandran C
JournalJ Biomol Screen
PubMed ID10838430
'A highly sensitive and continuous protein tyrosine phosphatase (PTPase) assay using 3,6-fluorescein diphosphate (FDP) is described. Leukocyte phosphatase CD45 (leukocyte common antigen), protein tyrosine phosphatase-1B, and leukocyte common antigen-related protein LAR preferentially hydrolyze FDP to fluorescein monophosphate (FMP) with V(max) and K(m) values comparable with those of phosphotyrosine peptide substrates. ... More
Controlling the rates of biochemical reactions and signaling networks by shape and volume changes.
AuthorsLizana L, Bauer B, Orwar O,
JournalProc Natl Acad Sci U S A
PubMed ID18337513
'In biological systems, chemical activity takes place in micrometer- and nanometer-sized compartments that constantly change in shape and volume. These ever-changing cellular compartments embed chemical reactions, and we demonstrate that the rates of such incorporated reactions are directly affected by the ongoing shape reconfigurations. First, we show that the rate ... More
Protein-tyrosine phosphatase activity regulates osteoclast formation and function: inhibition by alendronate.
AuthorsSchmidt A, Rutledge SJ, Endo N, Opas EE, Tanaka H, Wesolowski G, Leu CT, Huang Z, Ramachandaran C, Rodan SB, Rodan GA
JournalProc Natl Acad Sci U S A
PubMed ID8610169
'Alendronate (ALN), an aminobisphosphonate used in the treatment of osteoporosis, is a potent inhibitor of bone resorption. Its molecular target is still unknown. This study examines the effects of ALN on the activity of osteoclast protein-tyrosine phosphatase (PTP; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48), called PTPepsilon. Using osteoclast-like cells generated by coculturing ... More
Genetic selection for dissociative inhibitors of designated protein-protein interactions.
AuthorsPark SH, Raines RT
JournalNat Biotechnol
PubMed ID10932153
'Many biological processes rely on protein-protein interactions. These processes include signal transduction, cell cycle regulation, gene regulation, and viral assembly and replication. Moreover, many proteins and enzymes manifest their function as oligomers. We describe here an efficient means to sift through large combinatorial libraries and identify molecules that block the ... More
Nuclear organization studied with the help of a hypotonic shift: its use permits hydrophilic molecules to enter into living cells.
AuthorsKoberna K, Stanek D, Malínský J, Eltsov M, Pliss A, Ctrnáctá V, Cermanová S, Raska I
JournalChromosoma
PubMed ID10525969
'A new procedure for introduction of hydrophilic molecules into living cells based on efficient uptake of these molecules into the cells during hypotonic treatment is presented and its use is demonstrated by a variety of applications. Experiments with cultured vertebrate and Drosophila cells and various animal tissues demonstrated that the ... More
Fluorogenic substrates for beta-D-galactosidases and phosphatases derived from fluorescein (3,6-dihydroxyfluoran) and its monomethylether.
AuthorsROTMAN B, ZDERIC JA, EDELSTEIN M,
JournalProc Natl Acad Sci U S A
PubMed ID13975398
Syntheses of several new biochemically useful fluorogenic substrates for beta-D-galactosidases and phosphatases are described.
Controlling enzymatic reactions by geometry in a biomimetic nanoscale network.
AuthorsSott K, Lobovkina T, Lizana L, Tokarz M, Bauer B, Konkoli Z, Orwar O
JournalNano Lett
PubMed ID16464036
We demonstrate that a transition from a compact geometry (sphere) to a structured geometry (several spheres connected by nanoconduits) in nanotube-vesicle networks (NVNs) induces an ordinary enzyme-catalyzed reaction to display wavelike properties. The reaction dynamics can be controlled directly by the geometry of the network, and such networks can be ... More
On-chip capillary electrophoresis for alkaline phosphatase testing.
AuthorsMurakami Y, Morita T, Kanekiyo T, Tamiya E
JournalBiosens Bioelectron
PubMed ID11679282
We fabricated an on-chip capillary electrophoresis device for blood analysis. An on-chip capillary electrophoresis device was photolithographically fabricated on a glass chip. Alkaline phosphatase (ALP) was employed as a sample enzyme. Small amounts of enzyme in the mixture of other proteins were detected with the electrophoretically mediated microanalysis (EMMA) method. ... 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
Protein tyrosine phosphatase: enzymatic assays.
AuthorsMontalibet J, Skorey KI, Kennedy BP
JournalMethods
PubMed ID15588980
Activity assays for tyrosine phosphatases are based on the hydrolysis of a arylphosphate moiety from a synthetic substrate yielding a spectroscopically active product. Many different substrates can be used for these assays with p-nitrophenyl phosphate (pNPP), fluorescein diphosphate (FDP), and 6,8-difluoro-4-methylumbellyferyl phosphate (DiFMUP) being the most efficient and versatile. Equally, ... More
Development of a method for evaluating protein tyrosine phosphatase CD45 inhibitors using Jurkat cell membrane.
AuthorsWaddleton D, Ramachandran C, Wang Q
JournalAnal Biochem
PubMed ID10998263
A simple, high-throughput fluorescent assay was developed to measure the inhibition of membrane-bound CD45 from Jurkat cells. This assay is based on the fact that approximately 64% of PTP activity from Jurkat cell membrane is contributed by CD45. This has been proven by comparing the activity in membrane protein from ... More
[Difluro(phosphono)methyl]phenylalanine-containing peptide inhibitors of protein tyrosine phosphatases.
AuthorsDesmarais S, Friesen RW, Zamboni R, Ramachandran C
JournalBiochem J
PubMed ID9882618
Peptides containing the non-hydrolysable phosphotyrosine analogue 4-[difluro(phosphono)methyl]phenylalanine [Phe(CF2P)] were synthesized and tested as inhibitors of the protein tyrosine phosphatases (PTPs) PTP1B, CD45, PTPbeta, LAR and SHP-1. We have identified peptides containing two adjacent Phe(CF2P) residues as potent inhibitors of PTPs. The tripeptide having the sequence Glu-Phe(CF2P)-Phe(CF2P) is a potent and ... More
Chemical transformations in individual ultrasmall biomimetic containers.
AuthorsChiu DT, Wilson CF, Ryttsén F, Strömberg A, Farre C, Karlsson A, Nordholm S, Gaggar A, Modi BP, Moscho A, Garza-López RA, Orwar O, Zare RN
JournalScience
PubMed ID10082457
Individual phospholipid vesicles, 1 to 5 micrometers in diameter, containing a single reagent or a complete reaction system, were immobilized with an infrared laser optical trap or by adhesion to modified borosilicate glass surfaces. Chemical transformations were initiated either by electroporation or by electrofusion, in each case through application of ... More
Significance of protein tyrosine kinase/protein tyrosine phosphatase balance in the regulation of NF-kappaB signaling in the inflammatory process and aging.
AuthorsJung KJ, Lee EK, Yu BP, Chung HY,
JournalFree Radic Biol Med
PubMed ID19596065
Protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) are well recognized for the essential roles they play in signal transduction by maintaining proper balance under redox status. One of the major transcription factors known to be involved in aging is the redox-sensitive proinflammatory NF-kappaB, which could be modulated by ... More
Development of fluorescence-based selective assays for serine/threonine and tyrosine phosphatases.
AuthorsPastula C, Johnson I, Beechem JM, Patton WF
JournalComb Chem High Throughput Screen
PubMed ID12769677
A number of aromatic substrates were evaluated for their ability to detect tyrosine phosphatase and serine/threonine phosphatase activity. Results demonstrated that the fluorinated coumarin DiFMUP is the most sensitive substrate for detecting LAR and PP-2A activity. Using this substrate, selective high-throughput screening assays for serine/threonine and tyrosine phosphatases were developed. ... More
A sensitive competitive ELISA for 2,4-dinitrophenol using 3,6-fluorescein diphosphate as a fluorogenic substrate.
AuthorsHuang Z, Olson NA, You W, Haugland RP
JournalJ Immunol Methods
PubMed ID1301064
A competitive ELISA for sensitive detection of 2,4-dinitrophenol (DNP) was established. Certain amounts of bovine serum albumin conjugate of DNP were readily coated on a polystyrene microplate. Free DNP was then quantitated by its competition with the coated DNP for binding to anti-DNP (antibody)-alkaline phosphatase conjugate. The enzyme conjugate remaining ... More
A nutrient uptake role for bacterial cell envelope extensions.
Bacteria exist in a variety of morphologies, but the relationship between cellular forms and biological functions remains poorly understood. We show that stalks (prosthecae), cylindrical extensions of the Caulobacter crescentus cell envelope, can take up and hydrolyze organic phosphate molecules and contain the high-affinity phosphate-binding protein PstS, but not PstA, ... More
A fluorescent microplate assay for diarrheic shellfish toxins.
AuthorsVieytes MR, Fontal OI, Leira F, Baptista de Sousa JM, Botana LM
JournalAnal Biochem
PubMed ID9177752
A fluorescent enzyme inhibition assay for okadaic acid using 4-methylumbelliferyl phosphate and fluorescein diphosphate as substrates for the enzyme phosphatase 2A was developed. In the inhibition assay, performed in a microtiter plate, the PP2A was inhibited by adding okadaic acid and the resulting fluorescence enhancement derived from enzymatic hydrolysis of ... More
Siglec-7 undergoes a major conformational change when complexed with the alpha(2,8)-disialylganglioside GT1b.
AuthorsAttrill H, Imamura A, Sharma RS, Kiso M, Crocker PR, van Aalten DM
JournalJ Biol Chem
PubMed ID16895906
The siglecs are a group of mammalian sialic acid binding receptors expressed predominantly in the immune system. The CD33-related siglecs show complex recognition patterns for sialylated glycans. Siglec-7 shows a preference for alpha(2,8)-disialylated ligands and provides a structural template for studying the key interactions that drive this selectivity. We have ... More
A fluorogenic assay of endogenous phosphatase for assessment of cell adhesion.
AuthorsTolosa E, Shaw S
JournalJ Immunol Methods
PubMed ID8699013
Assays of cell adhesion generally require prelabeling of cells with radioactive or fluorescent probes. A new fluorogenic phosphatase assay requiring no prelabeling has been developed to quantitate cell number, which can thus serve as the basis for quantitating cell adhesion or migration. The assay uses the non-fluorescent substrate 3,6-fluorescein diphosphate ... More
Fluorescein monophosphates as fluorogenic substrates for protein tyrosine phosphatases.
AuthorsWang Q, Scheigetz J, Gilbert M, Snider J, Ramachandran C
JournalBiochim Biophys Acta
PubMed ID10209274
A series of novel fluorescein monophosphates aimed as substrates for protein tyrosine phosphatases (PTPs) were synthesized and evaluated against fluorescein diphosphate (FDP), the currently used fluorescent substrate for PTPs. In contrast to FDP, which is dephosphorylated to monophosphate and then to fluorescein in a sequential reaction, these monophosphates are dephosphorylated ... More
Characterization of 9H-(1,3-dichlor-9, 9-dimethylacridin-2-ona-7-yl)-phosphate (DDAO) as substrate of PP-2A in a fluorimetric microplate assay for diarrhetic shellfish toxins (DSP).
AuthorsLeira F, Vieites JM, Vieytes MR, Botana LM
JournalToxicon
PubMed ID10858520
Specific inhibition of protein-phosphatases by diarrhetic shellfish toxins (DSP) of the okadaic acid group, has led to the development of a fluorescent enzyme inhibition assay for these toxins using protein-phosphatase 2A (PP-2A) and fluorogenic substrates of the enzyme. Two different substrates of PP-2A have been previously used in this microplate ... More
Observation and quantification of ultraviolet-induced reactive oxygen species in ex vivo human skin.
AuthorsHanson KM, Clegg RM
JournalPhotochem Photobiol
PubMed ID12126308
Two-photon fluorescence imaging is used to detect UV-induced reactive oxygen species (ROS) in ex vivo human skin in this study. ROS (potentially H202, singlet oxygen or peroxynitrite [or all]) are detected after reaction with nonfluorescent dihydrorhodamine-123 (DHR) and the consequent formation of fluorescent rhodamine-123 (R123). The cellular regions at each ... More
Measuring the specific activity of the CD45 protein tyrosine phosphatase.
AuthorsRider DA, Young SP
JournalJ Immunol Methods
PubMed ID12799045
Many assays for protein tyrosine phosphatases (PTPs) rely on colorimetric substrates or more recently developed fluorescent substrates. Enzyme activity is measured but no assessment of specific activity is made, which is important since these enzymes can exist in an enzymatically inactive form. In this study, we have directly compared the ... More
Mechanism of inhibition of protein-tyrosine phosphatases by disodium aurothiomalate.
AuthorsWang Q, Janzen N, Ramachandran C, Jirik F
JournalBiochem Pharmacol
PubMed ID9310347
Disodium aurothiomalate (AuTM) has been used successfully in the treatment of various autoimmune and inflammatory disorders; however, the molecular target(s) for this agent remains unknown. The aim of this study was to investigate whether the activity of CD45, a protein-tyrosine phosphatase (PTP, EC 3.1.3.48) essential for antigen-receptor-mediated lymphocyte signaling, was ... 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
How does alendronate inhibit protein-tyrosine phosphatases?
AuthorsSkorey K, Ly HD, Kelly J, Hammond M, Ramachandran C, Huang Z, Gresser MJ, Wang Q
JournalJ Biol Chem
PubMed ID9278398
Alendronate (4-amino-1-hydroxybutylidene 1,1-bisphosphonate) is a drug used in the treatment of osteoporosis and other bone diseases. The inhibition of protein-tyrosine phosphatases (PTPs) by alendronate suggests that PTPs may be molecular targets. As a clear understanding of the inhibition mechanism is lacking, our aim was to analyze the mechanism to provide ... More
Residues distant from the active site influence protein-tyrosine phosphatase 1B inhibitor binding.
AuthorsMontalibet J, Skorey K, McKay D, Scapin G, Asante-Appiah E, Kennedy BP
JournalJ Biol Chem
PubMed ID16332678
Regions of protein-tyrosine phosphatase (PTP) 1B that are distant from the active site yet affect inhibitor binding were identified by a novel library screen. This screen was based on the observation that expression of v-Src in yeast leads to lethality, which can be rescued by the coexpression of PTP1B. However, ... More
A generally applicable, high-throughput screening-compatible assay to identify, evaluate, and optimize antimicrobial agents for drug therapy.