Biomedical subjects

Larry A Sklar

Publications and source records attributed to Larry A Sklar.

At least 19 recordsLinked to original sources

PubMed2007

GPR30: A G protein-coupled receptor for estrogen.

Estrogen is a critical steroid in human physiology exerting its effect both at the transcriptional level as well as at the level of rapid intracellular signaling through second messengers. Many of estrogen's transcriptional effects have long been known to be mediated through classical nuclear steroid receptors but recent studies also demonstrate the existence of a 7-transmembrane G protein-coupled receptor, GPR30 that responds to estrogen with rapid cellular signaling. There is currently controversy over the ability of classical estrogen receptors to recapitulate GPR30-mediated signaling mechanisms and vice versa. This article will summarize recent literature and address the relationship between GPR30 and conventional estrogen receptor signaling.

Estrogens↗

PubMed2006

Linkage effects on binding affinity and activation of GPR30 and estrogen receptors ERalpha/beta with tridentate pyridin-2-yl hydrazine tricarbonyl-Re/(99m)Tc(I) chelates.

We describe a new structural class of neutral tridentate pyridin-2-yl hydrazine chelates for labeling with tricarbonyl Re/99mTc(I) under aqueous conditions and investigate the receptor binding of synthetic estradiol derivatives with the novel G-protein-coupled receptor GPR30 and estrogen receptors ERalpha/beta. The steroid linkage affected the affinity and selectivity of estrogen binding with these receptors. Fluorescence assays based on calcium signaling demonstrate that membrane-permeable chelates 2 and 3 interact with the receptors in whole cells. These results suggest that in vitro assays will facilitate the development of targeted imaging agents for intracellular receptors and the feasibility of targeting GPR30 and ERalpha/beta for diagnostic tumor imaging.

Chelating Agents↗

PubMed2006

Virtual and biomolecular screening converge on a selective agonist for GPR30.

Estrogen is a hormone critical in the development, normal physiology and pathophysiology of numerous human tissues. The effects of estrogen have traditionally been solely ascribed to estrogen receptor alpha (ERalpha) and more recently ERbeta, members of the soluble, nuclear ligand-activated family of transcription factors. We have recently shown that the seven-transmembrane G protein-coupled receptor GPR30 binds estrogen with high affinity and resides in the endoplasmic reticulum, where it activates multiple intracellular signaling pathways. To differentiate between the functions of ERalpha or ERbeta and GPR30, we used a combination of virtual and biomolecular screening to isolate compounds that selectively bind to GPR30. Here we describe the identification of the first GPR30-specific agonist, G-1 (1), capable of activating GPR30 in a complex environment of classical and new estrogen receptors. The development of compounds specific to estrogen receptor family members provides the opportunity to increase our understanding of these receptors and their contribution to estrogen biology.

Animals↗

PubMed2006

Superquenching as a detector for microsphere-based flow cytometric assays.

BACKGROUND: Fluorescent conjugated polymers display high fluorescence quantum yields and enhanced sensitivity to quenching (superquenching) by oppositely charged quenchers through energy or electron transfer. Fluorescent polymers and their quenchers are used in bead-based biosensor applications where the polymers are coated on particles. In this work, we investigate a detection method that utilizes superquenching on microspheres, which can be used for flow cytometric assays. METHODS: Microspheres were coated with the fluorescent cationic polyelectrolyte poly(p-phenylene-ethynylene) (PPE), and its superquenching by 9,10-anthraquinone-2,6-disulfonic acid (AQS) was examined by fluorometric methods in presence and in absence of a barrier to superquenching in the form of an anionic lipid bilayer. RESULTS: Flow cytometry detected superquenching of PPE on microspheres (MS-PPE) by AQS where high levels of reduction in fluorescence were observed. Adding different concentrations of AQS to MS-PPE yielded a Stern-Volmer quenching constant of 0.8x10(6) M-1. While forming an anionic lipid bilayer around the MS-PPE acted as a barrier to superquenching by AQS, disrupting the lipid bilayer allowed superquenching to take place. CONCLUSIONS: The sensitivity of flow cytometry in detecting fluorescence of microspheres and the amplified quenching sensitivity of fluorescent conjugated polymers both offer advantages over other fluorometric methods and conventional quenching detection. This study used superquenching of fluorescent polymers as a new tool in flow cytometry, thus combining the advantages offered by both method and detector. In addition, we employed the formation and the disruption of a supported lipid bilayer in mediating superquenching to offer new biosensing applications.

Anthraquinones↗

PubMed2006

Glutathione-S-transferase-green fluorescent protein fusion protein reveals slow dissociation from high site density beads and measures free GSH.

BACKGROUND: Glutathione, a ubiquitous tripeptide, is an important cellular constituent, and measurement of reduced and oxidized glutathione is a measure of the redox state of cells. Glutathione-S-transferase (GST) fusion proteins bind naturally to beads derivatized with glutathione, and elution of such bead-bound fusion proteins with buffer containing millimolar glutathione is a commonly used method of protein purification. Many protein-protein interactions have been established by using GST fusion proteins and measuring binding of fusion protein binding partners by GST pulldown assays, usually monitored by Western blot methodology. METHODS: Dextran beads suitable for flow cytometry were derivatized with glutathione. A fusion protein of GST and green fluorescent protein was used to define kinetic and equilibrium binding characteristics of GST fusion proteins to glutathione beads. Free glutathione competes with this binding, and this competition was used to measure free glutathione concentration. RESULTS: A 10 microl assay can measure 5 microl of 20 microM glutathione (100 pmol glutathione) in 2 h by flow cytometry. This concentration is two orders of magnitude lower than cellular glutathione concentrations, and three orders of magnitude lower than affinity chromatography eluates. One important result is that by generating high site density, the GST fusion proteins can be constrained to the surface of one bead without hopping to the next bead in multiplex assays. CONCLUSIONS: Glutathione in cellular lysates and GST-fusion protein affinity chromatography eluates can be measured by flow cytometry. Many interactions between GST fusion proteins and their fluorescent binding partners should be quantifiable by flow cytometry. Although a system may have the disadvantage that it has a low affinity and a correspondingly quick off-rate in solution, it may remain on beads if the site density can be increased to offer a slow apparent off rate.

Binding Sites↗

PubMed2006

Microsphere-based protease assays and screening application for lethal factor and factor Xa.

BACKGROUND: Proteases regulate many biological pathways in humans and are components of several bacterial toxins. Protease studies and development of protease inhibitors do not follow a single established methodology and are mostly protease specific. METHODS: We have created recombinant fusion proteins consisting of a biotinylated attachment sequence linked to a GFP via a protease cleavage site to develop a multiplexable microsphere-based protease assay system. Using the proteases lethal factor and factor Xa, we performed kinetic experiments to determine optimal conditions for inhibitor screens and detect known inhibitors using the HyperCyt flow cytometry system. RESULTS: We have demonstrated specific cleavage of lethal factor and factor Xa substrates, optimized screening conditions for these substrates, shown specific inhibition of the proteases, and demonstrated high throughput detection of these inhibitors. CONCLUSIONS: The assay developed here is adaptable to any site-specific protease, compatible with high throughput flow cytometry systems, and multiplexable. Coupled with flow cytometry, which provides continuous time resolution and intrinsic resolution of free vs. bound fluorophores, this assay will be useful for high throughput screening of protease inhibitors in general and could simplify assays designed to determine protease mechanism.

Antigens, Bacterial↗

PubMed2005

Integration of virtual screening with high-throughput flow cytometry to identify novel small molecule formylpeptide receptor antagonists.

The formylpeptide receptor (FPR) family of G-protein-coupled receptors contributes to the localization and activation of tissue-damaging leukocytes at sites of chronic inflammation. We developed a FPR homology model and pharmacophore (based on the bovine rhodopsin crystal structure and known FPR ligands, respectively) for in silico screening of approximately 480,000 drug-like small molecules. A subset of 4324 compounds that matched the pharmacophore was then physically screened with the HyperCyt flow cytometry platform in high-throughput, no-wash assays that directly measure human FPR binding, with samples (each approximately 2500 cells in 2 microl) analyzed at 40/min. From 52 confirmed hits (1.2% hit rate), we identified 30 potential lead compounds (inhibition constant, Ki= 1-32 microM) representing nine distinct chemical families. Four compounds in one family were weak partial agonists. All others were antagonists. This virtual screening approach improved the physical screening hit rate by 12-fold (versus 0.1% hit-rate in a random compound collection), providing an efficient process for identifying small molecule antagonists.

Amino Acid Sequence↗

PubMed2005

Dynamics of fluorescence dequenching of ostrich-quenched fluorescein biotin: a multifunctional quantitative assay for biotin.

We describe a simple and rapid quantitative assay for biotin and biotin conjugates. The assay is based on the kinetic analysis of the enhancement of fluorescence of streptavidin/fluorescein biotin complexes in the presence of biotin. The kinetic response of fluorescence enhancement is proportional to the concentration of biotin. Standard calibration curves based on the kinetic response are obtained and detection limits of approximately 10(-9)M are established. Because the assay is amenable for use in small volumes of 5-50 microL or bead-based assays, the detection limits can be extended to the femtomole range. Since the assay depends on kinetic analysis, routine quantitation can be achieved without reference to standard curves. The dynamic aspects allow the assay to be extended to a broader range of applications including its use as an indicator of reagent mixing in laminar-flow assays carried out in microfluidic devices.

Biotin↗

PubMed2005

ATP hydrolysis-dependent disassembly of the 26S proteasome is part of the catalytic cycle.

ATP hydrolysis is required for degradation of polyubiquitinated proteins by the 26S proteasome but is thought to play no role in proteasomal stability during the catalytic cycle. In contrast to this view, we report that ATP hydrolysis triggers rapid dissociation of the 19S regulatory particles from immunopurified 26S complexes in a manner coincident with release of the bulk of proteasome-interacting proteins. Strikingly, this mechanism leads to quantitative disassembly of the 19S into subcomplexes and free Rpn10, the polyubiquitin binding subunit. Biochemical reconstitution with purified Sic1, a prototype substrate of the Cdc34/SCF ubiquitin ligase, suggests that substrate degradation is essential for triggering the ATP hydrolysis-dependent dissociation and disassembly of the 19S and that this mechanism leads to release of degradation products. This is the first demonstration that a controlled dissociation of the 19S regulatory particles from the 26S proteasome is part of the mechanism of protein degradation.

Adenosine Triphosphatases↗

PubMed2005

Dissociation of I domain and global conformational changes in LFA-1: refinement of small molecule-I domain structure-activity relationships.

LFA-1 (alphalbeta2) is constitutively expressed on leukocytes, but its activity is rapidly regulated. This rapid activation has been proposed to be associated with conformation changes in the inserted ("I") domain within the headpiece of LFA-1 as well as conversion of the molecules from bent to extended forms. To study these molecular changes as they relate to affinity regulation of LFA-1, we developed and synthesized a fluorescent derivative of BIRT-377 [Kelly et al. (2001) J. Immunol.] to examine changes in LFA-1 affinity in a flow cytometer with live cells. BIRT-377 binds to the ligand-binding or "I" domain of LFA-1. Structure-activity relationships studies indicated that an aminoalkyl group could be added to the central hydantoin group without significantly affecting binding. Using this modified derivative [1-(N-fluoresceinylthioureidobutyl)-[5R]-(4-bromobenzyl)-3-(3,5-dichlorophenyl)-5-methyl-imidazolidine-2,4-dione (FBABIRT)], we analyzed the affinity of FBABIRT binding to LFA-1 on live cells. The binding affinity increases, and the dissociation rate decreases with divalent cation (Mn(2+)) stimulation. We then used FBABIRT with fluorescent resonance energy transfer (FRET) to show that LFA-1 changes its height relative to the cell surface when cells were treated with dithiothreitol (DTT) but not Mn(2+). Competition assays among FBABIRT and BIRT derivatives defined structure-affinity relationships that refine the current model of BIRT-377 binding to the I domain. Our data supports the model in which BIRT-377 binds to the I domain and stabilizes the bent structure of LFA-1, while divalent cation activation results in a small conformational change in the I domain without significant extension of LFA-1. DTT, in contrast, induces a conversion to the extended form of LFA-1 in the presence of BIRT-377 on live cells. The structure-activity studies suggest that BIRT-377 is a fully optimized inhibitor.

Antibodies, Blocking↗

PubMed2005

A transmembrane intracellular estrogen receptor mediates rapid cell signaling.

The steroid hormone estrogen regulates many functionally unrelated processes in numerous tissues. Although it is traditionally thought to control transcriptional activation through the classical nuclear estrogen receptors, it also initiates many rapid nongenomic signaling events. We found that of all G protein-coupled receptors characterized to date, GPR30 is uniquely localized to the endoplasmic reticulum, where it specifically binds estrogen and fluorescent estrogen derivatives. Activating GPR30 by estrogen resulted in intracellular calcium mobilization and synthesis of phosphatidylinositol 3,4,5-trisphosphate in the nucleus. Thus, GPR30 represents an intracellular transmembrane estrogen receptor that may contribute to normal estrogen physiology as well as pathophysiology.

Animals↗

PubMed2005

Small-volume rapid-mix device for subsecond kinetic analysis in flow cytometry.

BACKGROUND: Rapid-mix flow cytometry has emerged as a powerful tool for mechanistic analysis of ligand binding, cell response, and molecular assembly. Although progress has come from improving sample delivery capabilities, little attention has been paid to the volumetric requirements associated with precious biological reagents. METHODS: By using programmable syringes, valves, and other fluidic components, we created a modular, precisely regulated rapid-mix device for the delivery of small-volume samples to the flow cytometer. The device was tested using a bead-based assay in which the binding kinetics between native biotin and fluorescein biotin-bearing beads were characterized. RESULTS: Bead suspensions and reagents paired in 35- to 45-microl aliquots were efficiently mixed by the device and delivered to the flow cytometer. Kinetic data associated with the fluorescein biotin beads were analyzed and used to calibrate the performance characteristics of the device in terms of sample delivery and mixing efficiency. CONCLUSION: The rapid-mix device is capable of detecting subsecond kinetics of biological reactions using microliter volume of samples. Dimensions of the device have been minimized, and the quantitative aspects of sample delivery and analysis have been optimized. Further, the modular design has been optimized for adaptation to a variety of experimental protocols.

Biotin↗

PubMed2005

Inhibition of chemoattractant N-formyl peptide receptor trafficking by active arrestins.

Recent studies have highlighted the emergence of a class of G protein-coupled receptors that are internalized in an arrestin-independent manner. In addition to demonstrating that the N-formyl peptide receptor belongs in this family, we have recently shown that recycling of the receptor requires the presence of arrestins. To further elucidate mechanisms of arrestin-dependent regulation of G protein-coupled receptor processing, we examined the effects of altering the receptor-arrestin complex on ternary complex formation and cellular trafficking of the N-formyl peptide receptor by studying two active arrestin-2 mutants (truncated arrestin-2 [1-382], and arrestin-2 I386A, V387A, F388A). Complexes between the N-formyl peptide receptor and active arrestins exhibited higher affinity in vitro than the complex between the N-formyl peptide receptor and wild-type arrestin and furthermore were observed in vivo by colocalization studies using confocal microscopy. To assess the effects of these altered interactions on receptor trafficking, we demonstrated that active, but not wild-type, arrestin expression retards N-formyl peptide receptor internalization. Furthermore, expression of arrestin-2 I386A/V387A/F388A but not arrestin-2 [1-382] inhibited recycling of the N-formyl peptide receptor, reflecting an expanded role for arrestins in G protein-coupled receptor processing and trafficking. Whereas the extent of N-formyl peptide receptor phosphorylation had no effect on the inhibition of internalization, N-formyl peptide receptor recycling was restored when the receptor was only partially phosphorylated. These results indicate not only that a functional interaction between receptor and arrestin is required for recycling of certain G protein-coupled receptors, such as the N-formyl peptide receptor, but that the pattern of receptor phosphorylation further regulates this process.

Amino Acid Sequence↗

PubMed2005

Post-high-throughput screening analysis: an empirical compound prioritization scheme.

An empirical scheme to evaluate and prioritize screening hits from high-throughput screening (HTS) is proposed. Negative scores are given when chemotypes found in the HTS hits are present in annotated databases such as MDDR and WOMBAT or for testing positive in toxicity-related experiments reported in TOXNET. Positive scores were given for higher measured biological activities, for testing negative in toxicity-related literature, and for good overlap when profiled against drug-related properties. Particular emphasis is placed on estimating aqueous solubility to prioritize in vivo experiments. This empirical scheme is given as an illustration to assist the decision-making process in selecting chemotypes and individual compounds for further experimentation, when confronted with multiple hits from high-throughput experiments. The decision-making process is discussed for a set of G-protein coupled receptor antagonists and validated on a literature example for dihydrofolate reductase inhibition.

Automation↗

PubMed2005

High-throughput screening with HyperCyt flow cytometry to detect small molecule formylpeptide receptor ligands.

High-throughput flow cytometry (HTFC), enabled by faster automated sample processing, represents a promising high- content approach for compound library screening. HyperCyt is a recently developed automated HTFC analysis system by which cell samples are rapidly aspirated from microplate wells and delivered to the flow cytometer. The formylpeptide receptor (FPR) family of G protein-coupled receptors contributes to the localization and activation of tissue-damaging leukocytes at sites of chronic inflammation. Here, the authors describe development and application of an HTFC screening approach to detect potential anti-inflammatory compounds that block ligand binding to FPR. Using a homogeneous no-wash assay, samples were routinely processed at 1.5 s/well (approximately 2500 cells analyzed/sample), allowing a 96-well plate to be processed in less than 2.5 min. Assay sensitivity and accuracy were validated by detection of a previously documented active compound with relatively low FPR affinity (sulfinpyrazone, inhibition constant [K(i)]=14 microM) from among a collection of 880 compounds in the Prestwick Chemical Library. The HyperCyt system was therefore demonstrated to be a robust, sensitive, and highly quantitative method with which to screen lead compound libraries in a 96-well format.

Flow Cytometry↗

PubMed2004

Near-simultaneous and real-time detection of multiple analytes in affinity microcolumns.

A miniaturized immunoassay system based on beads in poly(dimethylsiloxane) microchannels for analyzing multiple analytes has been developed. The method involves real-time detection of soluble molecules binding to receptor-bearing microspheres, sequestered in affinity column format inside a microfluidic channel. Identification and quantitation of analytes occurs via direct fluorescence measurements or fluorescence resonance energy transfer. A preliminary account of this work based on single-analyte format has been published in this journal (Buranda, T.; Huang, J.; Perez-Luna, V. H.; Schreyer, B.; Sklar, L. A.; Lopez, G. P. Anal. Chem. 2002, 74, 1149-1156). We have extended the work to a multianalyte model system composed of discrete segments of beads that bear distinct receptors. Near-simultaneous and real-time detection of diverse analytes is demonstrated. The importance of this work is established in the exploration of important factors related to the design, assessment, and utility of affinity microcolumn sensors. First, beads derivatized with surface chemistry suitable for the attachment of fluorescently labeled biomolecules of interest are prepared and characterized in terms of functionality and receptor site densities by flow cytometry. Second, calibrated beads are incorporated in microfluidic channels. The analytical device that emerges replicates the basic elements of affinity chromatography with the advantages of microscale and real-time direct measurement of bound analyte on beads rather than the indirect determination from eluted sample typical of affinity chromatography. In addition, the two-compartment analysis of the assay data as demonstrated in single-analyte columns provides a template upon which the dynamics of multiple-analyte assays can be characterized using existing theoretical models and be tested experimentally. The assay can potentially detect subfemtomole quantities of protein with high signal-to-noise ratio and a large dynamic range spanning nearly 4 orders of magnitude in analyte concentration in microliter to submicroliter volumes of analyte fluid. The approach has the potential to be generalized to a host of bioaffinity assay methods including analysis of protein complexes (e.g., biomolecular indicators of diseases). Proof-of-principle analytes include FLAG peptide and carcinoembryonic antigen detected at physiologically relevant concentration levels.

Carcinoembryonic Antigen↗

PubMed2004

An investigation of liquid carryover and sample residual for a high-throughput flow cytometer sample delivery system.

The phenomenon of intersample contamination in air-segmented continuous-flow assays has been studied for many years, and new uses are being found for these sampling techniques every day. One application that has been developed recently employs a flow cytometer to conduct high-throughput screening assays of biological compounds. We have explored the sources of intersample contamination in the system and shown how methods developed previously can be applied to describe these phenomena. Using a simple model, we were able to accurately measure liquid film thickness in the sample tubing and demonstrate the effects of intersample contamination in a flow cytometer assay. Also, measures have been taken to reduce the level of intersample contamination in cytometric screening assays, helping to make the system a more viable tool for drug screening applications.

Capillaries↗

PubMed2004

Dysregulated FcepsilonRI signaling and altered Fyn and SHIP activities in Lyn-deficient mast cells.

Studies in B cells from Lyn-deficient mice have identified Lyn as both a kinetic accelerator and negative regulator of signaling through the BCR. The signaling properties of bone marrow-derived mast cells from Lyn(-/-) mice (Lyn(-/-) BMMCs) have also been explored, but their signaling phenotype remains controversial. We confirm that Lyn(-/-) BMMCs release more beta-hexosaminidase than wild-type BMMCs following FcepsilonRI cross-linking and show that multiple mast cell responses to FcepsilonRI cross-linking (the phosphorylation of receptor subunits and other proteins, the activation of phospholipase Cgamma isoforms, the mobilization of Ca(2+), the synthesis of phosphatidylinositol 3,4,5-trisphosphate, the activation of the alpha(4)beta(1) integrin, VLA-4) are slow to initiate in Lyn(-/-) BMMCs, but persist far longer than in wild-type cells. Mechanistic studies revealed increased basal as well as stimulated phosphorylation of the Src kinase, Fyn, in Lyn(-/-) BMMCs. Conversely, there was very little basal or stimulated tyrosine phosphorylation or activity of the inositol phosphatase, SHIP, in Lyn(-/-) BMMCs. We speculate that Fyn may substitute (inefficiently) for Lyn in signal initiation in Lyn(-/-) BMMCs. The loss of SHIP phosphorylation and activity very likely contributes to the increased levels of phosphatidylinositol 3,4,5-trisphosphate and the excess FcepsilonRI signaling in Lyn(-/-) BMMCs. The unexpected absence of the transient receptor potential channel, Trpc4, from Lyn(-/-) BMMCs may additionally contribute to their altered signaling properties.

Adaptor Proteins, Signal Transducing↗