Len Pagliaro - Academia.edu (original) (raw)
Papers by Len Pagliaro
Experimental Cell Research, Apr 30, 1989
The lectin concanavalin A (Con A) inhibits adhesion of cells of complementary mating types into p... more The lectin concanavalin A (Con A) inhibits adhesion of cells of complementary mating types into pairs during conjugation in the ciliate Tetrahymena thermophila.
J Biol Chem, 1996
We used site-directed mutagenesis of rabbit muscle aldolase, falling ball viscometry, co-sediment... more We used site-directed mutagenesis of rabbit muscle aldolase, falling ball viscometry, co-sedimentation binding assays, and negative stain electron microscopy, to identify specific residues involved in the aldolase-actin interaction. Three mutants, R42A (Arg --> Ala), K107A (Lys --> Ala), and R148A (Arg --> Ala), had minimal actin binding activity relative to wild type (wt) aldolase, and one mutant, K229A (Lys --> Ala), had intermediate actin binding activity. A mutant with approximately 4,000-fold reduced catalytic activity, D33S (Asp --> Ser), had normal actin binding activity. The aldolase substrates and product, fructose 1,6-bisphosphate, fructose 1-phosphate, and dihydroxyacetone phosphate, reversed the gelling of wt aldolase and F-actin, consistent with at least partial overlap of catalytic and actin-binding sites on aldolase. Molecular modeling reveals that the actin-binding residues we have identified are clustered in or around the catalytic pocket of the molecule. These data confirm that the aldolase-actin interaction is due to specific binding, and they suggest that electrostatic interactions between specific residues, rather than net charge, mediate this interaction. Low concentration of wt and D33S aldolase caused formation of high viscosity actin gel networks, while high concentrations of wt and D33S aldolase resulted in solation of the gel by bundling actin filaments, consistent with a potential role for this enzyme in the regulation of cytoplasmic structure.
Undersea Hyperbaric Medicine Journal of the Undersea and Hyperbaric Medical Society Inc, Jul 1, 1995
We have developed an optical pressure chamber designed for use with high numerical aperture oil i... more We have developed an optical pressure chamber designed for use with high numerical aperture oil immersion microscope objectives at working pressures up to 1,000 psi (67 atm abs). The chamber is optimized for studies of living, adherent, cultured mammalian cells using high resolution epifluorescence and phase contrast microscopy, and biophysical techniques such as fluorescence redistribution after photobleaching and optical trapping. The primary optical window assembly of the chamber can be removed and placed into a standard 35-mm tissue culture dish, allowing for culture, microinjection, and micromanipulation of adherent cells before they are loaded into the chamber. The chamber is designed to fit into a commercially available stage heater for temperature control, and we used a computer-controlled high pressure liquid chromatography pump for pressure control. A graphic software interface allows the user to program "dive" profiles and to link temperature and pressure data with digital image files of specimens under study. A minor modification of the present design will allow perfusion at high pressure.
Journal of Nanomaterials, 2014
Photothermal therapy (PTT) treatments have shown strong potential in treating tumors through thei... more Photothermal therapy (PTT) treatments have shown strong potential in treating tumors through their ability to target destructive heat preferentially to tumor regions. In this paper we demonstrate that PTT in a murine melanoma model using gold nanorods (GNRs) and near-infrared (NIR) light decreases tumor volume and increases animal survival to an extent that is comparable to the current generation of melanoma drugs. GNRs, in particular, have shown a strong ability to reach ablative temperatures quickly in tumors when exposed to NIR light. The current research tests the efficacy of GNRs PTT in a difficult and fast growing murine melanoma model using a NIR light-emitting diode (LED) light source. LED light sources in the NIR spectrum could provide a safer and more practical approach to photothermal therapy than lasers. We also show that the LED light source can effectively and quickly heat in vitro and in vivo models to ablative temperatures when combined with GNRs. We anticipate that this approach could have significant implications for human cancer therapy.
Journal of Microscopy, 1994
Optical fibres bent in two mutually perpendicular planes have proven useful for randomizing illum... more Optical fibres bent in two mutually perpendicular planes have proven useful for randomizing illumination in light microscopes. These optical scramblers can increase the resolution and/or contrast obtained with several modes of light microscopy. Here, computer simulations are used to investigate several parameters affecting light randomization in curved optical fibres in order to further the theoretical basis for scrambler design. Light passing through 90" bends of optical fibre of varying radii of curvature was modelled by ray tracing in two dimensions, and scrambling mechanisms were observed. The effects of varying the position and angle of entry of light on the phase and direction of propagation of the emergent light were determined. It was found that (a) thorough scrambling does not necessarily require high numerical aperture (NA) entry of light into the fibre, (b) considerable order persists after a single 90" bend of an idealized fibre and (c) a higher degree of scrambling (at the cost of transmission efficiency) is achieved in more tightly curved fibres. The pathlength variations introduced by scrambling proved smaller than typical laser coherence lengths, requiring temporal scrambling (vibrating the fibre).
Hormone and Metabolic Research, 1997
Experimental Cell Research, 1997
plasm has been accumulating for many years, but mod-The glycolytic enzyme aldolase is concentrate... more plasm has been accumulating for many years, but mod-The glycolytic enzyme aldolase is concentrated in a els for metabolic organization have been difficult to test domain around stress fibers in living Swiss 3T3 cells, directly, due to the complex nature of cytoplasm. Glybut the mechanism by which aldolase is localized has colysis has been studied extensively in vitro using solunot been revealed. We have recently identified a molection biochemistry, but there is growing evidence that ular binding site for F-actin on aldolase, and we hyglycolysis in vivo can not be explained by simple extrappothesized that this specific binding interaction, olation of in vitro data.
Experimental Cell Research, 1987
The lectin concanavalin A (conA; 25 ug/ml) inhibits conjugation in the ciliate Tetrahymenu, and b... more The lectin concanavalin A (conA; 25 ug/ml) inhibits conjugation in the ciliate Tetrahymenu, and binds to receptors localized at the junction between conjugating cells. We report here that succinyl+onA (30 &ml) has similar activity, but that two other mannosespecific lectins, lentil and pea lectins, have inhibitory activities more than tenfold lower in this system, indicating that factors other than mannose specificity are essential for biological activity. By using fluorescein-isothiocyanate (FITC)-conA, we have found that extraction of cells with the detergent Triton X-100 removes conA receptors from the extractionresistant cytoskeleton, but that the binding of conA to its receptor before extraction associates the ligand-receptor complex with the cytoskeleton. Under the hypothesis that the conA receptor may be a mating type receptor, we have used this ligand-induced differential cytoskeletal association, in conjunction with electrophoresis and Western blotting, to identify a glycoprotein with an apparent molecular weight (MW) of 23000 D which may be a mating type receptor. Our data are consistent with a model in which a direct interaction between the conA receptor and the cytoskeleton, rather than receptor cross-linking, is the biologically significant activity of ligand binding. 0 1987 Acade.nic PES.
Experimental Cell Research, 1989
The lectin concanavalin A (Con A) inhibits adhesion of cells of complementary mating types into p... more The lectin concanavalin A (Con A) inhibits adhesion of cells of complementary mating types into pairs during conjugation in the ciliate Tetrahymena thermophila.
Combinatorial Chemistry & High Throughput Screening, 2005
G protein-coupled receptors (GPCRs) have been one of the most productive classes of drug targets ... more G protein-coupled receptors (GPCRs) have been one of the most productive classes of drug targets for several decades, and new technologies for GPCR-based discovery promise to keep this field active for years to come. While molecular screens for GPCR receptor agonist- and antagonist-based drugs will continue to be valuable discovery tools, the most exciting developments in the field involve cell-based assays for GPCR function. Some cell-based discovery strategies, such as the use of beta-arrestin as a surrogate marker for GPCR function, have already been reduced to practice, and have been used as valuable discovery tools for several years. The application of high content cell-based screening to GPCR discovery has opened up additional possibilities, such as direct tracking of GPCRs, G proteins and other signaling pathway components using intracellular translocation assays. These assays provide the capability to probe GPCR function at the cellular level with better resolution than has previously been possible, and offer practical strategies for more definitive selectivity evaluation and counter-screening in the early stages of drug discovery. The potential of cell-based translocation assays for GPCR discovery is described, and proof-of-concept data from a pilot screen with a CXCR4 assay are presented. This chemokine receptor is a highly relevant drug target which plays an important role in the pathogenesis of inflammatory disease and also has been shown to be a co-receptor for entry of HIV into cells as well as to play a role in metastasis of certain cancer cells.
ACS nano, Jan 24, 2017
Remarkable progress has recently been made in the synthesis and characterization of engineered na... more Remarkable progress has recently been made in the synthesis and characterization of engineered nanoparticles for imaging and treatment of cancers, resulting in several promising candidates in clinical trials. Despite these advances, clinical applications of nanoparticle-based therapeutic/imaging agents remain limited by biological, immunological, and translational barriers. In order to overcome the existing status quo in drug delivery, there is a need for open and frank discussion in the nanomedicine community on what is needed to make qualitative leaps toward translation. In this Nano Focus, we present the main discussion topics and conclusions from a recent workshop: "Mechanisms and Barriers in Nanomedicine". The focus of this informal meeting was on biological, toxicological, immunological, and translational aspects of nanomedicine and approaches to move the field forward productively. We believe that these topics reflect the most important issues in cancer nanomedicine.
The Journal of Cell Biology, 1992
Approximately 23% of the glycolytic enzyme aldolase in the perinuclear region of Swiss 3T3 cells ... more Approximately 23% of the glycolytic enzyme aldolase in the perinuclear region of Swiss 3T3 cells is immobile as measured by FRAP. Previous studies suggest that the immobile fraction may be associated with the actin cytoskeleton (Pagliaro, L. and D. L. Taylor. 1988. J. Cell Biol. 107:981-991), and it has been proposed that the association of some glycolytic enzymes with the cytoskeleton could have functional significance, perhaps involving a fundamental relationship between glycolysis, cytoplasmic organization, and cell motility. We have tested the effect of a key glycolytic inhibitor and an actin cytoskeletal modulator on the mobility of aldolase in living cells directly, using fluorescent analog cytochemistry and FRAP. We report here that the competitive hexokinase inhibitor 2-deoxyglucose releases the bound fraction of aldolase in 3T3 cells within 10 min, and that this process is reversible upon washout of the inhibitor. A similar result is produced with the actin-binding agent, cytochalasin D. These results are consistent with models in which glycolytic enzymes are not exclusively diffusion-limited, soluble proteins, but may exist partially in the solid phase of cytoplasm. Such organization has significant implications for both the modulation of cytoplasmic structure and for cellular metabolism.
![Research paper thumbnail of Aldolase exists in both the fluid and solid phases of cytoplasm [published erratum appears in J Cell Biol 1988 Dec;107(6 Pt 1):following 2463]](https://attachments.academia-assets.com/53252083/thumbnails/1.jpg)
](The Journal of Cell Biology, 1988
We have prepared a functional fluorescent t. Abbreviations used in this paper: Daq, aqueous diffu... more We have prepared a functional fluorescent t. Abbreviations used in this paper: Daq, aqueous diffusion coefficient; Dcyto, apparent cytoplasmic diffusion coefficient; D/P, dye/protein ratio; F-actin, filamentous actin; FDP, fructose 1,6-diphosphate; FRAP, fluorescence redistribution after photobleaching; Rh, rhodamine.
Journal of Biochemical and Biophysical Methods, 1994
We have developed a system that greatly facilitates viscosity measurement under low-shear conditi... more We have developed a system that greatly facilitates viscosity measurement under low-shear conditions, based upon a falling ball viscometer interfaced to a personal computer. Three optical sensors indicate the rate of passage of a steel ball falling through a micro-capillary tube, and a potentiometer detects the angle of the tube. The resulting data are passed from a custom program with a graphical user interface to a spreadsheet. The spreadsheet then calculates and stores a value for the viscosity of the sample, using the data passed to it from the viscometer, and a look-up table of stored slope and intercept values calculated from precision viscosity standards. We used the viscometer to determine the kinetics of actin polymerization, and to measure the viscosity of F-actin-aldolase gels. This system provides significantly greater reproducibility and speed in data acquisition than does the traditional 'eyeball and stopwatch' method, and data reduction is virtually instantaneous.
Journal of Biomolecular Screening, 2003
Several factors are known to increase the noise and variability of cell-based assays used for hig... more Several factors are known to increase the noise and variability of cell-based assays used for high-throughput screening. In particular, edge effects can result in an unacceptably high plate rejection rate in screening runs. In an effort to minimize these variations, the authors analyzed a number of factors that could contribute to edge effects in cell-based assays. They found that pre-incubation of newly seeded plates in ambient conditions (air at room temperature) resulted in even distribution of the cells in each well. In contrast, when newly seeded plates were placed directly in the CO(2) incubator, an uneven distribution of cells occurred in wells around the plate periphery, resulting in increased edge effect. Here, the authors show that the simple, inexpensive approach of incubating newly seeded plates at room temperature before placing them in a 37 degrees C CO(2) incubator yields a significant reduction in edge effect.
Current Opinion in Chemical Biology, 2004
Protein-protein interactions play a key role in the signal transduction pathways that regulate ce... more Protein-protein interactions play a key role in the signal transduction pathways that regulate cellular function. Three years ago, few descriptions of small molecule protein-protein interaction inhibitors (SMPPIIs) existed in the literature. Today, the number of examples of both the biology and chemistry of such interaction inhibitors is growing rapidly. This growth occurs at the convergence of medicinal chemistry, signaling biology and novel assay technology for profiling emerging compound classes and modes of action. Protein translocation assays provide a unique new tool for identifying, profiling, and optimizing SMPPIIs. This review summarizes recent work in the field, and outlines future developments we can anticipate. Abbreviations iNOS inducible nitric oxide synthase MoA mode of action PI3K phosphatidylinositol 3 kinase PCA principal component analysis PIP3 phosphatidylinositol 3,4,5-triphosphate SMPPII small molecule protein-protein interaction inhibitor Emerging classes of protein-protein interaction inhibitors Pagliaro et al. 443 www.sciencedirect.com
ASSAY and Drug Development Technologies, 2004
Redistribution (BioImage) A/S, Søborg, Denmark) is a novel high-throughput screening technology t... more Redistribution (BioImage) A/S, Søborg, Denmark) is a novel high-throughput screening technology that monitors translocation of specific protein components of intracellular signaling pathways within intact mammalian cells, using green fluorescent protein as a tag. A single Redistribution assay can be used to identify multiple classes of compounds that act at, or upstream of, the level of the protein target used in the primary screening assay. Such compounds may include both conventional and allosteric enzyme inhibitors, as well as protein-protein interaction modulators. We have developed a series of Redistribution assays to discover and characterize compounds that inhibit tumor necrosis factor-alpha biosynthesis via modulation of the p38 mitogen-activated protein kinase (MAPK) pathway. A primary assay was designed to identify low-molecular-weight compounds that inhibit the activation-dependent nuclear export of the p38 kinase substrate MAPK-activated protein kinase 2 (MK2). Hits from the primary screen were categorized, using secondary assays, either as direct inhibitors of MK2 nuclear export, or as inhibitors of the upstream p38 MAPK pathway. Activity profiles are presented for a nuclear export inhibitor, and a compound that structurally and functionally resembles a known p38 kinase inhibitor. These results demonstrate the utility of Redistribution technology as a pathway screening method for the identification of diverse and novel compounds that are active within therapeutically important signaling pathways.
Methods in molecular biology (Clifton, N.J.), 2007
This chapter describes the design and development of cell-based assays, in which quantitation of ... more This chapter describes the design and development of cell-based assays, in which quantitation of the intracellular translocation of a target protein--rather than binding or catalytic activity--provides the primary assay readout. These are inherently high content assays, and they provide feedback on cellular response at the systems level, rather than data on activities of individual, purified molecules. Multiple protein translocation assays can be used to profile cellular signaling pathways and they can play a key role in determination of mechanism of action for novel classes of compounds with therapeutic potential. This assay technology has developed from laboratory curiosity into main stream industrial research over the past decade, and its promise is beginning to be realized as data acquisition and analysis technology evolve to take advantage of the rich window into systems biology provided by translocation assays.
Experimental Cell Research, Apr 30, 1989
The lectin concanavalin A (Con A) inhibits adhesion of cells of complementary mating types into p... more The lectin concanavalin A (Con A) inhibits adhesion of cells of complementary mating types into pairs during conjugation in the ciliate Tetrahymena thermophila.
J Biol Chem, 1996
We used site-directed mutagenesis of rabbit muscle aldolase, falling ball viscometry, co-sediment... more We used site-directed mutagenesis of rabbit muscle aldolase, falling ball viscometry, co-sedimentation binding assays, and negative stain electron microscopy, to identify specific residues involved in the aldolase-actin interaction. Three mutants, R42A (Arg --> Ala), K107A (Lys --> Ala), and R148A (Arg --> Ala), had minimal actin binding activity relative to wild type (wt) aldolase, and one mutant, K229A (Lys --> Ala), had intermediate actin binding activity. A mutant with approximately 4,000-fold reduced catalytic activity, D33S (Asp --> Ser), had normal actin binding activity. The aldolase substrates and product, fructose 1,6-bisphosphate, fructose 1-phosphate, and dihydroxyacetone phosphate, reversed the gelling of wt aldolase and F-actin, consistent with at least partial overlap of catalytic and actin-binding sites on aldolase. Molecular modeling reveals that the actin-binding residues we have identified are clustered in or around the catalytic pocket of the molecule. These data confirm that the aldolase-actin interaction is due to specific binding, and they suggest that electrostatic interactions between specific residues, rather than net charge, mediate this interaction. Low concentration of wt and D33S aldolase caused formation of high viscosity actin gel networks, while high concentrations of wt and D33S aldolase resulted in solation of the gel by bundling actin filaments, consistent with a potential role for this enzyme in the regulation of cytoplasmic structure.
Undersea Hyperbaric Medicine Journal of the Undersea and Hyperbaric Medical Society Inc, Jul 1, 1995
We have developed an optical pressure chamber designed for use with high numerical aperture oil i... more We have developed an optical pressure chamber designed for use with high numerical aperture oil immersion microscope objectives at working pressures up to 1,000 psi (67 atm abs). The chamber is optimized for studies of living, adherent, cultured mammalian cells using high resolution epifluorescence and phase contrast microscopy, and biophysical techniques such as fluorescence redistribution after photobleaching and optical trapping. The primary optical window assembly of the chamber can be removed and placed into a standard 35-mm tissue culture dish, allowing for culture, microinjection, and micromanipulation of adherent cells before they are loaded into the chamber. The chamber is designed to fit into a commercially available stage heater for temperature control, and we used a computer-controlled high pressure liquid chromatography pump for pressure control. A graphic software interface allows the user to program "dive" profiles and to link temperature and pressure data with digital image files of specimens under study. A minor modification of the present design will allow perfusion at high pressure.
Journal of Nanomaterials, 2014
Photothermal therapy (PTT) treatments have shown strong potential in treating tumors through thei... more Photothermal therapy (PTT) treatments have shown strong potential in treating tumors through their ability to target destructive heat preferentially to tumor regions. In this paper we demonstrate that PTT in a murine melanoma model using gold nanorods (GNRs) and near-infrared (NIR) light decreases tumor volume and increases animal survival to an extent that is comparable to the current generation of melanoma drugs. GNRs, in particular, have shown a strong ability to reach ablative temperatures quickly in tumors when exposed to NIR light. The current research tests the efficacy of GNRs PTT in a difficult and fast growing murine melanoma model using a NIR light-emitting diode (LED) light source. LED light sources in the NIR spectrum could provide a safer and more practical approach to photothermal therapy than lasers. We also show that the LED light source can effectively and quickly heat in vitro and in vivo models to ablative temperatures when combined with GNRs. We anticipate that this approach could have significant implications for human cancer therapy.
Journal of Microscopy, 1994
Optical fibres bent in two mutually perpendicular planes have proven useful for randomizing illum... more Optical fibres bent in two mutually perpendicular planes have proven useful for randomizing illumination in light microscopes. These optical scramblers can increase the resolution and/or contrast obtained with several modes of light microscopy. Here, computer simulations are used to investigate several parameters affecting light randomization in curved optical fibres in order to further the theoretical basis for scrambler design. Light passing through 90" bends of optical fibre of varying radii of curvature was modelled by ray tracing in two dimensions, and scrambling mechanisms were observed. The effects of varying the position and angle of entry of light on the phase and direction of propagation of the emergent light were determined. It was found that (a) thorough scrambling does not necessarily require high numerical aperture (NA) entry of light into the fibre, (b) considerable order persists after a single 90" bend of an idealized fibre and (c) a higher degree of scrambling (at the cost of transmission efficiency) is achieved in more tightly curved fibres. The pathlength variations introduced by scrambling proved smaller than typical laser coherence lengths, requiring temporal scrambling (vibrating the fibre).
Hormone and Metabolic Research, 1997
Experimental Cell Research, 1997
plasm has been accumulating for many years, but mod-The glycolytic enzyme aldolase is concentrate... more plasm has been accumulating for many years, but mod-The glycolytic enzyme aldolase is concentrated in a els for metabolic organization have been difficult to test domain around stress fibers in living Swiss 3T3 cells, directly, due to the complex nature of cytoplasm. Glybut the mechanism by which aldolase is localized has colysis has been studied extensively in vitro using solunot been revealed. We have recently identified a molection biochemistry, but there is growing evidence that ular binding site for F-actin on aldolase, and we hyglycolysis in vivo can not be explained by simple extrappothesized that this specific binding interaction, olation of in vitro data.
Experimental Cell Research, 1987
The lectin concanavalin A (conA; 25 ug/ml) inhibits conjugation in the ciliate Tetrahymenu, and b... more The lectin concanavalin A (conA; 25 ug/ml) inhibits conjugation in the ciliate Tetrahymenu, and binds to receptors localized at the junction between conjugating cells. We report here that succinyl+onA (30 &ml) has similar activity, but that two other mannosespecific lectins, lentil and pea lectins, have inhibitory activities more than tenfold lower in this system, indicating that factors other than mannose specificity are essential for biological activity. By using fluorescein-isothiocyanate (FITC)-conA, we have found that extraction of cells with the detergent Triton X-100 removes conA receptors from the extractionresistant cytoskeleton, but that the binding of conA to its receptor before extraction associates the ligand-receptor complex with the cytoskeleton. Under the hypothesis that the conA receptor may be a mating type receptor, we have used this ligand-induced differential cytoskeletal association, in conjunction with electrophoresis and Western blotting, to identify a glycoprotein with an apparent molecular weight (MW) of 23000 D which may be a mating type receptor. Our data are consistent with a model in which a direct interaction between the conA receptor and the cytoskeleton, rather than receptor cross-linking, is the biologically significant activity of ligand binding. 0 1987 Acade.nic PES.
Experimental Cell Research, 1989
The lectin concanavalin A (Con A) inhibits adhesion of cells of complementary mating types into p... more The lectin concanavalin A (Con A) inhibits adhesion of cells of complementary mating types into pairs during conjugation in the ciliate Tetrahymena thermophila.
Combinatorial Chemistry & High Throughput Screening, 2005
G protein-coupled receptors (GPCRs) have been one of the most productive classes of drug targets ... more G protein-coupled receptors (GPCRs) have been one of the most productive classes of drug targets for several decades, and new technologies for GPCR-based discovery promise to keep this field active for years to come. While molecular screens for GPCR receptor agonist- and antagonist-based drugs will continue to be valuable discovery tools, the most exciting developments in the field involve cell-based assays for GPCR function. Some cell-based discovery strategies, such as the use of beta-arrestin as a surrogate marker for GPCR function, have already been reduced to practice, and have been used as valuable discovery tools for several years. The application of high content cell-based screening to GPCR discovery has opened up additional possibilities, such as direct tracking of GPCRs, G proteins and other signaling pathway components using intracellular translocation assays. These assays provide the capability to probe GPCR function at the cellular level with better resolution than has previously been possible, and offer practical strategies for more definitive selectivity evaluation and counter-screening in the early stages of drug discovery. The potential of cell-based translocation assays for GPCR discovery is described, and proof-of-concept data from a pilot screen with a CXCR4 assay are presented. This chemokine receptor is a highly relevant drug target which plays an important role in the pathogenesis of inflammatory disease and also has been shown to be a co-receptor for entry of HIV into cells as well as to play a role in metastasis of certain cancer cells.
ACS nano, Jan 24, 2017
Remarkable progress has recently been made in the synthesis and characterization of engineered na... more Remarkable progress has recently been made in the synthesis and characterization of engineered nanoparticles for imaging and treatment of cancers, resulting in several promising candidates in clinical trials. Despite these advances, clinical applications of nanoparticle-based therapeutic/imaging agents remain limited by biological, immunological, and translational barriers. In order to overcome the existing status quo in drug delivery, there is a need for open and frank discussion in the nanomedicine community on what is needed to make qualitative leaps toward translation. In this Nano Focus, we present the main discussion topics and conclusions from a recent workshop: "Mechanisms and Barriers in Nanomedicine". The focus of this informal meeting was on biological, toxicological, immunological, and translational aspects of nanomedicine and approaches to move the field forward productively. We believe that these topics reflect the most important issues in cancer nanomedicine.
The Journal of Cell Biology, 1992
Approximately 23% of the glycolytic enzyme aldolase in the perinuclear region of Swiss 3T3 cells ... more Approximately 23% of the glycolytic enzyme aldolase in the perinuclear region of Swiss 3T3 cells is immobile as measured by FRAP. Previous studies suggest that the immobile fraction may be associated with the actin cytoskeleton (Pagliaro, L. and D. L. Taylor. 1988. J. Cell Biol. 107:981-991), and it has been proposed that the association of some glycolytic enzymes with the cytoskeleton could have functional significance, perhaps involving a fundamental relationship between glycolysis, cytoplasmic organization, and cell motility. We have tested the effect of a key glycolytic inhibitor and an actin cytoskeletal modulator on the mobility of aldolase in living cells directly, using fluorescent analog cytochemistry and FRAP. We report here that the competitive hexokinase inhibitor 2-deoxyglucose releases the bound fraction of aldolase in 3T3 cells within 10 min, and that this process is reversible upon washout of the inhibitor. A similar result is produced with the actin-binding agent, cytochalasin D. These results are consistent with models in which glycolytic enzymes are not exclusively diffusion-limited, soluble proteins, but may exist partially in the solid phase of cytoplasm. Such organization has significant implications for both the modulation of cytoplasmic structure and for cellular metabolism.
The Journal of Cell Biology, 1988
We have prepared a functional fluorescent t. Abbreviations used in this paper: Daq, aqueous diffu... more We have prepared a functional fluorescent t. Abbreviations used in this paper: Daq, aqueous diffusion coefficient; Dcyto, apparent cytoplasmic diffusion coefficient; D/P, dye/protein ratio; F-actin, filamentous actin; FDP, fructose 1,6-diphosphate; FRAP, fluorescence redistribution after photobleaching; Rh, rhodamine.
Journal of Biochemical and Biophysical Methods, 1994
We have developed a system that greatly facilitates viscosity measurement under low-shear conditi... more We have developed a system that greatly facilitates viscosity measurement under low-shear conditions, based upon a falling ball viscometer interfaced to a personal computer. Three optical sensors indicate the rate of passage of a steel ball falling through a micro-capillary tube, and a potentiometer detects the angle of the tube. The resulting data are passed from a custom program with a graphical user interface to a spreadsheet. The spreadsheet then calculates and stores a value for the viscosity of the sample, using the data passed to it from the viscometer, and a look-up table of stored slope and intercept values calculated from precision viscosity standards. We used the viscometer to determine the kinetics of actin polymerization, and to measure the viscosity of F-actin-aldolase gels. This system provides significantly greater reproducibility and speed in data acquisition than does the traditional 'eyeball and stopwatch' method, and data reduction is virtually instantaneous.
Journal of Biomolecular Screening, 2003
Several factors are known to increase the noise and variability of cell-based assays used for hig... more Several factors are known to increase the noise and variability of cell-based assays used for high-throughput screening. In particular, edge effects can result in an unacceptably high plate rejection rate in screening runs. In an effort to minimize these variations, the authors analyzed a number of factors that could contribute to edge effects in cell-based assays. They found that pre-incubation of newly seeded plates in ambient conditions (air at room temperature) resulted in even distribution of the cells in each well. In contrast, when newly seeded plates were placed directly in the CO(2) incubator, an uneven distribution of cells occurred in wells around the plate periphery, resulting in increased edge effect. Here, the authors show that the simple, inexpensive approach of incubating newly seeded plates at room temperature before placing them in a 37 degrees C CO(2) incubator yields a significant reduction in edge effect.
Current Opinion in Chemical Biology, 2004
Protein-protein interactions play a key role in the signal transduction pathways that regulate ce... more Protein-protein interactions play a key role in the signal transduction pathways that regulate cellular function. Three years ago, few descriptions of small molecule protein-protein interaction inhibitors (SMPPIIs) existed in the literature. Today, the number of examples of both the biology and chemistry of such interaction inhibitors is growing rapidly. This growth occurs at the convergence of medicinal chemistry, signaling biology and novel assay technology for profiling emerging compound classes and modes of action. Protein translocation assays provide a unique new tool for identifying, profiling, and optimizing SMPPIIs. This review summarizes recent work in the field, and outlines future developments we can anticipate. Abbreviations iNOS inducible nitric oxide synthase MoA mode of action PI3K phosphatidylinositol 3 kinase PCA principal component analysis PIP3 phosphatidylinositol 3,4,5-triphosphate SMPPII small molecule protein-protein interaction inhibitor Emerging classes of protein-protein interaction inhibitors Pagliaro et al. 443 www.sciencedirect.com
ASSAY and Drug Development Technologies, 2004
Redistribution (BioImage) A/S, Søborg, Denmark) is a novel high-throughput screening technology t... more Redistribution (BioImage) A/S, Søborg, Denmark) is a novel high-throughput screening technology that monitors translocation of specific protein components of intracellular signaling pathways within intact mammalian cells, using green fluorescent protein as a tag. A single Redistribution assay can be used to identify multiple classes of compounds that act at, or upstream of, the level of the protein target used in the primary screening assay. Such compounds may include both conventional and allosteric enzyme inhibitors, as well as protein-protein interaction modulators. We have developed a series of Redistribution assays to discover and characterize compounds that inhibit tumor necrosis factor-alpha biosynthesis via modulation of the p38 mitogen-activated protein kinase (MAPK) pathway. A primary assay was designed to identify low-molecular-weight compounds that inhibit the activation-dependent nuclear export of the p38 kinase substrate MAPK-activated protein kinase 2 (MK2). Hits from the primary screen were categorized, using secondary assays, either as direct inhibitors of MK2 nuclear export, or as inhibitors of the upstream p38 MAPK pathway. Activity profiles are presented for a nuclear export inhibitor, and a compound that structurally and functionally resembles a known p38 kinase inhibitor. These results demonstrate the utility of Redistribution technology as a pathway screening method for the identification of diverse and novel compounds that are active within therapeutically important signaling pathways.
Methods in molecular biology (Clifton, N.J.), 2007
This chapter describes the design and development of cell-based assays, in which quantitation of ... more This chapter describes the design and development of cell-based assays, in which quantitation of the intracellular translocation of a target protein--rather than binding or catalytic activity--provides the primary assay readout. These are inherently high content assays, and they provide feedback on cellular response at the systems level, rather than data on activities of individual, purified molecules. Multiple protein translocation assays can be used to profile cellular signaling pathways and they can play a key role in determination of mechanism of action for novel classes of compounds with therapeutic potential. This assay technology has developed from laboratory curiosity into main stream industrial research over the past decade, and its promise is beginning to be realized as data acquisition and analysis technology evolve to take advantage of the rich window into systems biology provided by translocation assays.