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Pathogenic hantaviruses bind to the plexin-semaphorin-integrin (PSI) domain of inactive, β 3 integrins. Previous studies have implicated a cognate cis interaction between the bent conformation β 5 /β 3 integrins and an arginine-glycine-aspartic acid (RGD) sequence in the first extracellular loop of P2Y 2 R. With single-molecule atomic force microscopy, we show a specific interaction between an atomic force microscopy tip decorated with recombinant α IIb β 3 integrins and (RGD)P2Y 2 R expressed on cell membranes. Mutation of the RGD sequence to RGE in the P2Y 2 R removes this interaction. Binding of inactivated and fluorescently labeled Sin Nombre virus (SNV) to the integrin PSI domain stimulates higher affinity for (RGD)P2Y 2 R on cells, as measured by an increase in the unbinding force. In CHO cells, stably expressing α IIb β 3 integrins, virus engagement at the integrin PSI domain, recapitulates physiologic activation of the integrin as indicated by staining with the activation-specific mAB PAC1. The data also show that blocking of the Gα 13 protein from binding to the cytoplasmic domain of the β 3 integrin prevents outside-in signaling and infection. We propose that the cis interaction with P2Y 2 R provides allosteric resistance to the membrane-normal motion associated with the switchblade model of integrin activation, where the development of tensile force yields physiological integrin activation., (© 2017 Bondu et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Assays to identify small molecule inhibitors of cell transporters have long been used to develop potential therapies for reversing drug resistance in cancer cells. In flow cytometry, these approaches rely on the use of fluorescent substrates of transporters. Compounds which prevent the loss of cell fluorescence have typically been pursued as inhibitors of specific transporters, but further drug development has been largely unsuccessful. One possible reason for this low success rate could be a substantial overlap in substrate specificities and functions between transporters of different families. Additionally, the fluorescent substrates are often synthetic dyes that exhibit promiscuity among transporters as well. Here, we describe an assay in which a fluorescent analog of a natural metabolite, 3',5'-cyclic adenosine monophosphate (F-cAMP), is actively effluxed by malignant leukemia cells. The F-cAMP is loaded into the cell cytoplasm using a procedure based on the osmotic lysis of pinocytic vesicles. The flow cytometric analysis of the fluorescence retained in F-cAMP-loaded cells incubated with various compounds can subsequently identify inhibitors of cyclic AMP efflux (ICE).

Background: Tibial plateau fractures account for approximately 1% of all fractures. They usually occur after a direct high-energy trauma. Despite adequate treatment, these fractures can result in malalignment and secondary osteoarthritis (OA). Research concerning long-term functional outcome is limited. The primary aim of this study was to evaluate mid- to long-term functional outcome of surgically treated tibial plateau fractures. The secondary aim was to investigate whether radiological characteristics of OA one year after surgery are predictive of functional outcome at follow-up., Methods: All consecutive patients with fractures of the proximal tibia, which were surgically treated in our level-2 trauma centre between 2004 and 2010, were included in this study. Initial trauma radiographs were analysed for fracture classification, using both the Schatzker and AO/OTA classification systems, by three different raters. Immediate postoperative and 1-year postoperative radiographs were analysed for osteoarthritis by an experienced radiologist, using the Kellgren and Lawrence scale. Functional outcome of the included patients was measured using the Dutch version of the Knee injury and Osteoarthritis Outcome Score (KOOS) questionnaire., Results: Seventy one patients out of a group of 96 included patients completed the survey. Median KOOS scores are 89.8% for pain, 91.1% for 'other symptoms', 89.7% for daily function, 72.5% for sports and recreation and 75.0% for quality of life. Median KOOS overall score is 82.99%. We did not find a correlation between the KOOS scores and the absolute age for any of the subscales. There was no significant relationship between radiological characteristics of osteoarthritis and functional outcome., Conclusions: This is the first study to describe mid- to long-term functional outcome after ORIF for all types of tibial plateau fractures, with the use of the KOOS. Patients should be informed about the likelihood of lower functional outcome in the long-term. This study shows that radiological characteristics of osteoarthritis are not related with lower functional outcomes in the mid- to long-term., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Purpose: To retrospectively identify morphologic findings at computed tomographic (CT) colonography that are the most reliable in the differentiation of masslike chronic diverticular disease from sigmoid carcinoma in a large patient cohort., Materials and Methods: This study was approved by the institutional review boards. The need for signed consent was waived for this retrospective study. The cohort consisted of 212 patients (mean age, 68 years; 113 women, 99 men) with focal masslike findings in the sigmoid colon at CT colonography, representing chronic diverticular disease (n = 97) or sigmoid carcinoma (n = 115). CT colonography studies were scored according to presence or absence of potential discriminators by a panel of four readers in consensus. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy were calculated, and multivariate analysis was performed., Results: Absence of diverticula in the affected segment showed high NPV and PPV (0.95 and 0.93, respectively). Also, shoulder phenomenon showed a high NPV (0.92) and PPV (0.75). Segment length of 10 cm or less (NPV, 0.85; PPV, 0.61) and destroyed mucosal folds (NPV, 1.00; PPV, 0.62) had a high NPV but a low PPV. Although segments affected by carcinoma often showed straightened and eccentric growth patterns, no thick fascia sign, and more and larger local-regional lymph nodes (all P < .05), NPV was insufficient for discrimination (NPV ≤ 0.66). Combination of absence of diverticula and presence of shouldering showed a high diagnostic certainty (93%)., Conclusion: Carcinoma is best differentiated from masslike diverticular disease by the absence of diverticula in the affected segment and the presence of shoulder phenomenon., (© RSNA, 2014.)

Rab7 facilitates vesicular transport and delivery from early endosomes to late endosomes as well as from late endosomes to lysosomes. The role of Rab7 in vesicular transport is dependent on its interactions with effector proteins, among them Rab-interacting lysosomal protein (RILP), which aids in the recruitment of active Rab7 (GTP-bound) onto dynein-dynactin motor complexes to facilitate late endosomal transport on the cytoskeleton. Here we detail a novel bead-based flow cytometry assay to measure Rab7 interaction with the Rab-interacting lysosomal protein (RILP) effector protein and demonstrate its utility for quantitative assessment and studying drug-target interactions. The specific binding of GTP-bound Rab7 to RILP is readily demonstrated and shown to be dose-dependent and saturable enabling K d and B max determinations. Furthermore, binding is nearly instantaneous and temperature-dependent. In a novel application of the assay method, a competitive small molecule inhibitor of Rab7 nucleotide binding (CID 1067700 or ML282) is shown to inhibit the Rab7-RILP interaction. Thus, the assay is able to distinguish that the small molecule, rather than incurring the active conformation, instead 'locks' the GTPase in the inactive conformation. Together, this work demonstrates the utility of using a flow cytometry assay to quantitatively characterize protein-protein interactions involving small GTPases and which has been adapted to high-throughput screening. Further, the method provides a platform for testing small molecule effects on protein-protein interactions, which can be relevant to drug discovery and development.

Background: Annually 16,000 appendectomies are performed in the Netherlands, of which 15-20% are negative. In 2010, to reduce this unacceptable percentage of superfluous appendectomies, the Dutch Association for Surgery introduced the 'Appendicitis Guideline'. This guideline recommends the use of imaging. In this observational prospective study the added value of imaging in everyday clinical practice is evaluated., Methods: All patients with suspected appendicitis were included at the emergency department of a Dutch teaching hospital during the period from September 2011 to May 2012 (n = 350; 237 adults and 113 children under 18 years). Adherence to the guideline was evaluated., Results: 75 Patients (21%) were not referred for imaging because of a low suspicion or alternative diagnosis. In 16 patients (5%) the guideline was not followed. Of the 259 patients (74%) who underwent ultrasonography, 105 (30%) also underwent computed tomography (CT). 127 appendectomies were performed, showing appendicitis in 112 patients (88%); 15 appendectomies (12%) were negative. In the latter group, 12 were performed after false positive imaging results, and 3 following inconclusive imaging results., Conclusion: When using imaging in the diagnosis of appendicitis, the percentage of negative appendectomies remains close to the percentage declared as unacceptable by the publishers of the guideline., (© 2015 S. Karger AG, Basel.)

Objectives: Computed tomographic colonography (CTC) is a less burdensome alternative to colonoscopy in excluding colorectal cancer (CRC) in symptomatic patients. We evaluated the proportion of patients who underwent CTC in whom CRC was missed., Methods: Patients who had undergone CTC in the period 1 January 2007 to 1 January 2011 were merged with all cases of CRC recorded in the Cancer Registry between 1 January 2007 and 1 July 2011 to identify all patients who had undergone CTC less than 2 years before CRC had been diagnosed., Results: In 53 out of 1,855 patients who had undergone CTC, CRC was diagnosed. Of these, 40 patients had suspected CRC and 5 had large polyps at CTC. In five patients with an indeterminate mass, further investigation confirmed malignancy. One cancer in the caecum was missed because of poor distension. Two cancers were missed: one in the distal rectum and one in the ascending colon. Sensitivity of CTC for CRC was 94.3 % (95 % CI 88-100 %). The true miss rate, excluding the inadequate distended study, was 2 out of 53 (3.8 %)., Conclusion: This study shows that the miss rate for CTC is low, which means that CTC is accurate in excluding CRC in symptomatic patients at a relatively low risk of CRC.

We developed a platform combining fluorogen-activating protein (FAP) technology with high-throughput flow cytometry to detect real-time protein trafficking to and from the plasma membrane in living cells. The hybrid platform facilitates drug discovery for trafficking receptors such as G protein-coupled receptors and was validated with the β₂-adrenergic receptor (β₂AR) system. When a chemical library containing ∼1200 off-patent drugs was screened against cells expressing FAP-tagged β₂ARs, all 33 known β₂AR-active ligands in the library were successfully identified, together with a number of compounds that might regulate receptor internalization in a nontraditional manner. Results indicated that the platform identified ligands of target proteins regardless of the associated signaling pathway; therefore, this approach presents opportunities to search for biased receptor modulators and is suitable for screening of multiplexed targets for improved efficiency. The results revealed that ligands may be biased with respect to the rate or duration of receptor internalization and that receptor internalization may be independent of activation of the mitogen-activated protein kinase pathway.

Academia and small business research units are poised to play an increasing role in drug discovery, with drug repurposing as one of the major areas of activity. Here we summarize project status for a number of drugs or classes of drugs: raltegravir, cyclobenzaprine, benzbromarone, mometasone furoate, astemizole, R-naproxen, ketorolac, tolfenamic acid, phenothiazines, methylergonovine maleate and beta-adrenergic receptor drugs, respectively. Based on this multi-year, multi-project experience we discuss strengths and weaknesses of academic-based drug repurposing research. Translational, target and disease foci are strategic advantages fostered by close proximity and frequent interactions between basic and clinical scientists, which often result in discovering new modes of action for approved drugs. On the other hand, lack of integration with pharmaceutical sciences and toxicology, lack of appropriate intellectual coverage and issues related to dosing and safety may lead to significant drawbacks. The development of a more streamlined regulatory process world-wide, and the development of pre-competitive knowledge transfer systems such as a global healthcare database focused on regulatory and scientific information for drugs world-wide, are among the ideas proposed to improve the process of academic drug discovery and repurposing, and to overcome the "valley of death" by bridging basic to clinical sciences.

The human Bcl-2 family includes six antiapoptotic members (Bcl-2, Bcl-B, Bcl-W, Bcl-X(L), Bfl-1, and Mcl-1) and many proapoptotic members, wherein a balance between the two determines cell life or death in many physiological and disease contexts. Elevated expression of various antiapoptotic Bcl-2 members is commonly observed in cancers, and chemical inhibitors of these proteins have been shown to promote apoptosis of malignant cells in culture, in animal models, and in human clinical trials. All six antiapoptotic members bind a helix from the proapoptotic family member Bim, thus quenching Bim's apoptotic signal. Here, we describe the use of a multiplex, high-throughput flow cytometry assay for the discovery of small molecule modulators that disrupt the interaction between the antiapoptotic members of the Bcl-2 family and Bim. The six antiapoptotic Bcl-2 family members were expressed as glutathione-S-transferase fusion proteins and bound individually to six glutathione bead sets, with each set having a different intensity of red fluorescence. A fluorescein-conjugated Bcl-2 homology region 3 (BH3) peptide from Bim was employed as a universal ligand. Flow cytometry measured the amount of green peptide bound to each bead set in a given well, with inhibitory compounds resulting in a decrease of green fluorescence on one or more bead set(s). Hits and cheminformatically selected analogs were retested in a dose-response series, resulting in three "active" compounds for Bcl-B. These three compounds were validated by fluorescence polarization and isothermal titration calorimetry. We discuss some of the lessons learned about screening a chemical library provided by the National Institutes of Health Small Molecule Repository (∼195,000 compounds) using high-throughput flow cytometry.

The B-cell lymphoma-2 (Bcl-2) family contains six antiapoptotic members, each with a hydrophobic pocket in which Bcl-2 homology region 3 (BH3) helices bind. This binding quenches apoptotic signals from activated BH3 family members. Many tumor cells either have increased expression of one of these six proteins or become overexpressed under treatment. Six fusion proteins made up of glutathione-S-transferase and each of the Bcl-2 members are bound individually to six glutathione bead sets, each set being easily distinguished by its different intensity of red fluorescence. The coated bead sets are washed, combined and incubated with green fluorescent Bim-BH3 peptide and a small molecule in 10-μl wells for 1 h. The green fluorescence signal for each bead set is resolved, and selective inhibitors are expected to reduce the signal for individual bead sets. Each 384-well plate is analyzed in 12 min, measuring 200 of 2,000 beads (∼10%) of each type per well.

Objective: To investigate in how many patients with bowel or abdominal complaints, referred by the primary care physician (PCP) for exclusion of colorectal carcinoma (CRC), the more invasive colonoscopy could be avoided on the basis of the findings of CT colonography., Design: Retrospective, descriptive., Methods: All consecutive patients who underwent CT colonography in our centre on the request of their PCP from December 2006 to June 2009 were included. Demographic and referral data were collected. CT colonography results were described according to the 'CT Colonography Reporting and Data System'. We also investigated how many patients had to undergo colonoscopy in the 6 months following CT colonography., Results: 398 patients (154 men and 244 women) with a median age of 61 years (range: 22-91) were included. Follow-up colonoscopy was indicated by CT colonography in 30 patients (7.5%) for suspected colorectal carcinoma, polyps > 10 mm, or 3 or more polyps 6-9 mm in size. In 33 patients (8.3%) follow-up colonoscopy or CT colonography was indicated for 1 or 2 polyps 6-9 mm in size, or suspicious lesions. 11 of these patients (2.8%) underwent colonoscopy. In 335 patients (84.2%) polyps > 6 mm or malignancies could be excluded. 18 of these patients (4.5%) still had a colonoscopy. In total, colonoscopy was spared in 341 patients (85.7%). Significant or potentially significant extra-colonic pathological abnormalities were found in 63 patients (15.8%)., Conclusion: Our results support the theory that in the vast majority of patients with low or moderate suspicion of CRC referred by their PCP, invasive colonoscopy could be avoided, because CRC and polyps could be excluded by CT colonography. CT colonography could be a valuable additional diagnostic tool in primary care.

Ras-like small GTPases cycle between GTP-bound active and GDP-bound inactive conformational states to regulate diverse cellular processes. Despite their importance, detailed kinetic or comparative studies of family members are rarely undertaken due to the lack of real-time assays measuring nucleotide binding or exchange. Here we report a bead-based flow cytometric assay that quantitatively measures the nucleotide binding properties of glutathione-S-transferase (GST) chimeras for prototypical Ras family members Rab7 and Rho. Measurements are possible in the presence or absence of Mg(2+), with magnesium cations principally increasing affinity and slowing nucleotide dissociation rates 8- to 10-fold. GST-Rab7 exhibited a 3-fold higher affinity for guanosine diphosphate (GDP) relative to guanosine triphosphate (GTP) that is consistent with a 3-fold slower dissociation rate of GDP. Strikingly, GST-Rab7 had a marked preference for GTP with ribose ring-conjugated BODIPY FL. The more commonly used gamma-NH-conjugated BODIPY FL GTP analogue failed to bind to GST-Rab7. In contrast, both BODIPY analogues bound equally well to GST-RhoA and GST-RhoC. Comparisons of the GST-Rab7 and GST-RhoA GTP binding pockets provide a structural basis for the observed binding differences. In sum, the flow cytometric assay can be used to measure nucleotide binding properties of GTPases in real time and to quantitatively assess differences between GTPases.

Bone marrow thymocytes in part mediate the bone-preserving effects of estrogen by decreasing their production of osteoclast growth factors such as interleukin-1 and -6 and tumor necrosis factor alpha in the presence of physiological amounts of estradiol. Although several in vitro studies implicate the T-lymphocyte as a candidate mediator of estrogen signaling in the skeleton, whether these cells or any lymphocytes ordinarily express one or both nuclear estrogen receptors was previously unresolved. The purpose of our investigation was therefore to ascertain, by using real-time PCR, immmunoblotting, and cytometric techniques, if any of the nuclear estrogen receptors could be detected in normal peripheral blood mononuclear cells (PBMNC) collected from healthy volunteers. The results of immunoblotting experiments revealed that both estrogen receptor alpha (ESR1) and beta (ESR2) proteins are expressed in nuclei, but not in the cytoplasm of PBMNC harvested from all of the 15 healthy male and female volunteers (aged 23-50 years) we tested. PBMNCs contained mRNA coding for the two major full-length isoforms of ESR2 and the expression of ESR2 protein was localized within a lymphocyte subpopulation by cytometric analysis. Our data provide further evidence that lymphocytes and monocytes are responsive to estrogen and underscore its importance in modulating the immune response, as well as the vascular and skeletal health of men and women.

We have used rapid-mix flow cytometry to analyze the early subsecond dynamics of the disassembly of ternary complexes of G protein-coupled receptors (GPCRs) immobilized on beads to examine individual steps associated with guanine nucleotide activation. Our earlier studies suggested that the slow dissociation of Galpha and Gbetagamma subunits was unlikely to be an essential component of cell activation. However, these studies did not have adequate time resolution to define precisely the disassembly kinetics. Ternary complexes were assembled using three formyl peptide receptor constructs (wild type, formyl peptide receptor-Galpha(i2) fusion, and formyl peptide receptor-green fluorescent protein fusion) and two isotypes of the alpha subunit (alpha(i2) and alpha(i3)) and betagamma dimer (beta(1)gamma(2) and beta(4)gamma(2)). At saturating nucleotide levels, the disassembly of a significant fraction of ternary complexes occurred on a subsecond time frame for alpha(i2) complexes and tau(1/2)< or =4s for alpha(i3) complexes, time scales that are compatible with cell activation. beta(1)gamma(2) isotype complexes were generally more stable than beta(4)gamma(2)-associated complexes. The comparison of the three constructs, however, proved that the fast step was associated with the separation of receptor and G protein and that the dissociation of the ligand or of the alpha and betagamma subunits was slower. These results are compatible with a cell activation model involving G protein conformational changes rather than disassembly of Galphabetagamma heterotrimer.

Background: Many cellular signal transduction cascades have protein kinases as critical components. Small molecule protein kinase inhibitors can be effective as laboratory probes and drugs. Methods that allow two or more kinases to be evaluated simultaneously for inhibition by a small molecule would allow unequivocal tests of specificity and selectivity of action of the small molecule., Methods: Two hexahistidine-tagged activin receptor-like kinases were expressed in E. coli, purified, and bound to nickel beads. A fluorescent kinase ligand (F-KL) that binds to the ATP-binding site of these kinases with nanomolar affinity was developed. Binding of F-KL with kinase on the bead made the beads bright, and inhibitors decreased the brightness., Results: A test panel of 17 nonfluorescent kinase inhibitors, spanning two orders of magnitude affinity for the kinases, gave K(d) values for the kinases that correlated well with a fluorescence polarization assay. Results were obtained for the kinases in duplex, using an autosampler to send beads from a 96-well plate to a flow cytometer in a format suitable for high throughput screening., Conclusions: Inhibitors of kinases can be measured in duplex in a high throughput format by flow cytometry, if a suitable fluorescent ligand is available.

The binding of full and partial agonist ligands (L) to G protein-coupled receptors (GPCRs) initiates the formation of ternary complexes with G proteins [ligand-receptor-G protein (LRG) complexes]. Cyclic ternary complex models are required to account for the thermodynamically plausible complexes. It has recently become possible to assemble solubilized formyl peptide receptor (FPR) and beta(2)-adrenergic receptor (beta(2)AR) ternary complexes for flow cytometric bead-based assays. In these systems, soluble ternary complex formation of the receptors with G proteins allows direct quantitative measurements which can be analyzed in terms of three-dimensional concentrations (molarity). In contrast to the difficulty of analyzing comparable measurements in two-dimensional membrane systems, the output of these flow cytometric experiments can be analyzed via ternary complex simulations in which all of the parameters can be estimated. An outcome from such analysis yielded lower affinity for soluble ternary complex assembly by partial agonists compared with full agonists for the beta(2)AR. In the four-sided ternary complex model, this behavior is consistent with distinct ligand-induced conformational states for full and partial agonists. Rapid mix flow cytometry is used to analyze the subsecond dynamics of guanine nucleotide-mediated ternary complex disassembly. The modular breakup of ternary complex components is highlighted by the finding that the fastest step involves the departure of the ligand-activated GPCR from the intact G protein heterotrimer. The data also show that, under these experimental conditions, G protein subunit dissociation does not occur within the time frame relevant to signaling. The data and concepts are discussed in the context of a review of current literature on signaling mechanism based on structural and spectroscopic (FRET) studies of ternary complex components.

Objective: To determine the safety and the long-term results of primary stent placement for localized distal aortic occlusive disease., Design: Retrospective observational study., Patients and Methods: From July 1998 to July 2005 17 patients (14 female and 3 men, mean age 57 years (39-80)) were treated for intermittent claudication. Five of these patients underwent additional endovascular treatment of focal iliac lesions., Results: Technical success defined as residual stenosis of less than 50% or a trans-stenotic systolic pressure gradient <10% was achieved in 14 of 17 (82%) patients. Major complications included dissection at the puncture site in one patient and thrombosis of additional iliac stents in another patient. Both of these complications were successfully treated. During a mean follow-up of 27 months (range 1-86), four patients had recurrence of symptoms due to in-stent restenoses (n=2), femoral (n=1) or iliac occlusion (n=1), respectively. By Kaplan-Meier analysis, primary aortic hemodynamic patency was 83% at 3 years. Secondary aortic hemodynamic patency was 100%. The primary clinical patency was 68% at 3 years., Conclusion: Primary stent placement for distal aortic stenoses is an alternative to surgical treatment because of its high patency and relatively low complication rates.

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., (Copyright (c) 2006 International Society for Analytical Cytology.)

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.

Flow cytometers are well known for their ability to analyze and sort cells at high rates based on physiological responses and expression of protein markers. The potential for flow cytometry in G-protein-coupled receptor (GPCR) research, however, is less well appreciated. Potential applications include: (i) the homogenous discrimination of free and bound ligands or proteins in both cellular and microsphere-based assays; and (ii) multiplexed ('suspension array') analysis of cell responses and protein-protein interactions. Innovative sample-handling systems also provide sub-second resolution of interaction kinetics and 1 second per well throughput of microliter-sized samples from multiwell plates. Flow cytometric methods using microspheres for analysis of GPCRs that interact with intracellular and extracellular binding partners such as ligands, G proteins and kinases have been established. These analyses can produce quantitative pharmacological data analogous to radioligand assays, and, in some cases, the probes can be integrated into the assembly as fluorescent fusion proteins.

We developed a novel and generalized approach to investigate G protein-coupled receptor molecular assemblies. We solubilized a fusion protein consisting of the beta(2)-adrenergic receptor and green fluorescent protein (GFP) for bead-based flow cytometric analysis. beta(2)-Adrenergic receptor GFP bound to dihydroalprenolol-conjugated beads, providing a K(d) for the fusion protein and, in competition with beta(2)-adrenergic receptor ligands, K(d) values for agonists and antagonists. Beads displaying chelated nickel bound purified hexahistidine-tagged G protein heterotrimers and, subsequently, the binary complex of agonist with beta(2)-adrenergic receptor GFP. The dose-response curves of ternary complex formation revealed maximal assembly for ligands previously classified as full agonists and reduced assembly for ligands previously classified as partial agonists. Guanosine 5'-3-O-(thio)triphosphate-induced dissociation rates of the ternary complex were the same for full and partial agonists. Soluble G protein, competing with ternary complexes on beads provided an affinity estimate of agonist-receptor complexes to G protein. When performed simultaneously, the two assemblies discriminated between agonist, antagonist or inactive molecule in a manner appropriate for high throughput, small volume drug discovery. The assemblies can be further generalized to other G protein coupled receptor protein-protein interactions.

G protein-coupled receptors form a ternary complex of ligand, receptor, and G protein heterotrimer (LRG) during signal transduction from the outside to the inside of a cell. Our goal was to develop a homogeneous, small-volume, bead-based approach compatible with high-throughput flow cytometry that would allow evaluation of G protein coupled receptor molecular assemblies. Dextran beads were derivatized to carry chelated nickel to bind hexahistidine-tagged green fluorescent protein (GFP) and hexahistidine-tagged G proteins. Ternary complexes were assembled on these beads using fluorescent ligand with wild-type receptor or a receptor-Gialpha2 fusion protein, and with a nonfluorescent ligand and receptor-GFP fusion protein. Streptavidin-coated polystyrene beads used biotinylated anti-FLAG antibodies to bind FLAG-tagged G proteins for ternary complex assembly. Validation was achieved by showing time and concentration dependence of ternary complex formation. Affinity measurements of ligand for receptor on particles, of the ligand-receptor complex for G protein on the particles, and receptor-Gialpha2 fusion protein for Gbetagamma, were consistent with comparable assemblies in detergent suspension. Performance was assessed in applications representing the potential of these assemblies for ternary complex mechanisms. We showed the relationship for a family of ligands between LR and LRG affinity and characterized the affinity of both the wild-type and GFP fusion receptors with G protein. We also showed the potential of kinetic measurements to allow observation of individual steps of GTP-induced ternary complex disassembly and discriminated a fast step caused by RG disassembly compared with the slower step of Galphabetagamma disassembly.

The effects of ambient temperature (T; 15 C vs. 30 C from 6 wk of age onwards), dietary Arg:Lys ratio (Arg:Lys ratio; 1.00 vs. 1.25), dietary electrolyte balance (DEB: 164 vs. 254 meq/kg), and their interactions on growth performance and carcass yields of male turkeys were studied. The experiment was designed as a split plot, including T x DEB as the main plot and Arg:Lys ratio as the sub-plot, with 24 pens containing 35 male turkeys each. Feed consumption, BW gain, mortality, and processing yields were measured. Temperature had a clear effect on performance during all age periods. Feed intake was significantly lower for the high T group compared with the low T group (322.7 vs. 432.3 g/bird per day; P < 0.001). Consequently, BW gain during the experimental period (28 to 140 d of age) was significantly lower for the high T group compared with the low T group (14.54 vs. 18.74 kg; P < 0.001). Feed:gain during the period of 28 to 140 d of age was significantly lower for the high T group compared with the low T group (2.51 vs. 2.61; P < 0.001). The high dietary Arg:Lys ratio increased feed intake significantly until 56 d of age (200.6 vs. 197.6; P < or = 0.034). A high Arg:Lys ratio resulted in significantly higher BW gain until 98 d of age (10.03 vs. 9.84 kg; P < or = 0.024). The Arg:Lys ratio did not affect feed:gain throughout the experiment. Dietary electrolyte balance did not affect performance parameters. No consistent two- or three-way interactions were observed. Processing yields were only affected significantly by T, and not by Arg:Lys ratio or DEB main effects. High T resulted in lower cold carcass (73.2 vs. 74.9%) and breast meat yields (33.5 vs. 36.0%), and higher thigh (18.9 vs. 18.1%), drumstick (14.5 vs. 13.2%), and wing yields (11.7 vs. 10.6%) compared with low T. We concluded that growth performance is compromised by higher T, and altering the Arg:Lys ratio or DEB does not alleviate this impaired performance. Dietary Arg levels seem to be important when dietary Lys is marginal relative to the requirement.

Objective: To determine the prevalence of hypercholesterolaemia and the use of lipid-lowering medication in patients with a manifest vascular disease., Design: Prospective, cross-sectional., Methods: In patients who visited the University Medical Centre Utrecht, the Netherlands, for the first time with a manifestation of atherosclerosis in the period 1 September 1996-15 November 1998, we determined by a single measurement of the cholesterol if they were eligible for lipid-lowering medication according to the cholesterol cut-off value mentioned in the new Dutch cholesterol guidelines (1998) of the Dutch Institute for Health Care Improvement., Results: The study group comprised 737 patients: 539 (73%) males and 198 (27%) females, with a mean age of 62 year. 500 (68%) were eligible for lipid-lowering treatment. 66 patients (9%) were being treated according to the guidelines and in 106 (14%) the aim of a cholesterol lower than 5.0 mmol/1 was not reached despite cholesterol lowering medication. In 328 patients (45%) hyperlipidaemia was not treated pharmacologically., Conclusion: Two-thirds of the patients with manifest vascular disease had hypercholesterolaemia. Many of these patients were not yet being treated.

Evidence is emerging that the contribution of atherosclerosis to the development of abdominal aortic aneurysm may differ from that of other manifestations of arterial disease. B-mode ultrasound may be helpful in understanding the characteristics and factors that contribute to the development of different manifestations of arterial disease. We examined whether there is a difference in common carotid intima-media thickness (IMT), an indicator of generalized atherosclerosis, in patients with peripheral arterial disease (PAD) and abdominal aortic aneurysm (AAA). IMT of the left and right common carotid artery was measured in the first 172 patients (123 PAD and 49 AAA) enrolled in the Second Manifestations of ARTerial disease (SMART) study, a cohort study among patients with a manifestation of atherosclerotic vascular disease or risk factors for atherosclerosis. Mean IMT was 0.98 +/- 0.34 mm in patients with PAD and 0.91 +/- 0.20 mm in patients with AAA, with an age and sex adjusted mean difference of 0.18 mm (95% CI 0.08; 0.28). After additional adjustments for cardiovascular risk factors, the difference remained 0.11 mm (95% Cl 0.01; 0.21). Common carotid IMT in patients with AAA is on average smaller than in patients with PAD, independent of other determinants of IMT. These findings support the view that the development of AAA cannot completely be explained by atherosclerosis and is in part due to other pathophysiological mechanisms.

The Second Manifestations of ARTerial disease (SMART) study is a single-centre prospective cohort study among patients, newly referred to the hospital with (1) clinically manifest atherosclerotic vessel disease, or (2) marked risk factors for atherosclerosis. The first objectives of the SMART study are to determine the prevalence of concomitant arterial disease at other sites, and risk factors in patients presenting with a manifestation of arterial disease or vascular risk factor and to study the incidence of future cardiovascular events and its predictors in these high-risk patients. At least 1000 patients, aged 18 to 80 years, will undergo baseline examinations, including a questionnaire on cardiovascular disease, height, weight and blood pressure measurements, blood tests for glucose, lipids, creatinine and homocysteine, urinary tests for microproteinuria, resting twelve-lead electrocardiogram, ultrasound scanning of the abdominal aorta, kidneys and the carotid arteries, measurements of common carotid intima-media thickness and arterial stiffness, and a treadmill test to assess atherosclerosis of the leg arteries. Abnormal findings are reported to the treating specialist and general practitioner with a treatment suggestion according to current practice guidelines. Recruitment and baseline examinations began in September 1996. All cohort members will be followed for clinical cardiovascular events for a minimum of three years. In the scope of secondary prevention, the study is expected to support the design of solid based screening and treatment programmes and evidence-based cardiovascular medicine to reduce morbidity and mortality, and improve quality of life, in high-risk patients.

Purpose: The prevalence of asymptomatic internal carotid artery stenosis (ICAS) in patients with peripheral arterial disease (PAD) and characteristics that are associated with ICAS were studied., Methods: We used data from the first 600 patients enrolled in the Second Manifestations of ARTerial disease (SMART) study, a single-center, prospective cohort study among patients referred with a manifestation of cardiovascular disease, diabetes mellitus, hypertension, or hyperlipidemia. Included in the analysis were 162 patients with PAD or a history of PAD, who were not known to have ICAS at the time of referral and who had no history of cerebrovascular symptoms or previous carotid endarterectomy. ICAS was detected with duplex scanning and defined as a peak systolic velocity more than 150 cm/s (diameter reduction 50% or higher) on at least one side. Cardiovascular risk factors were measured. Logistic regression analysis was performed to investigate associations between these characteristics and ICAS., Results: The prevalence of previously unknown ICAS was 14%. A patient age of 67 years or older, body weight of 68 kg or less, and diastolic blood pressure of 75 mm Hg or lower were independently associated with ICAS. The Prevalence Of Icas In Patients With One Of These Characteristics (38% Of The Patients) Was 8%, In Those With Two Characteristics (21% Of The Patients) Was 32%, And In Those With Three Characteristics (6% Of The Patients) Was 50%., Conclusions: The prevalence of ICAS increases as much as 50% in patients who have PAD and the risk indicators of an age of 67 years or older, a body weight of 68 kg or less, and a diastolic blood pressure of 75 mm Hg or lower, and, therefore, these characteristics may be used as a means of increasing the likelihood of detecting ICAS.

Background: Common carotid intima-media thickness (IMT) and distensibility are markers of structural and functional vessel wall properties. Both parameters have been found in population-based studies to be associated with cardiovascular risk factors and prevalent cardiovascular disease. We investigated cross-sectionally whether IMT and distensibility are associated with cardiovascular risk in patients who already have vascular disease or atherosclerotic risk factors and evaluated the diagnostic ability of IMT and distensibility to discriminate between low- and high-risk patients., Methods and Results: IMT and distensibility (change of diameter) of the left and right common carotid arteries were measured in the first 570 patients (537 for distensibility) enrolled in the Second Manifestations of ARTerial disease (SMART) study, a cohort study among patients with a manifestation of vascular disease or cardiovascular risk factors. Three risk scores were used to classify each patient's vascular risk. Areas under the curve (AUCs) of receiver-operating characteristic curves were calculated for IMT and distensibility after the patients were dichotomized on the median of the risk scores as the outcome. Risk scores increased nearly linearly with increasing IMT and decreasing distensibility. The AUCs for IMT predicting high-risk patients were 0.77, 0.73, and 0.77 based on the 3 risk scores. The AUCs for distensibility were 0. 65, 0.62, and 0.66., Conclusions: Common carotid IMT and distensibility are clear markers of cardiovascular risk in patients who already have vascular disease or atherosclerotic risk factors. IMT appears to discriminate between low- and high-risk patients better than distensibility.

The plasma membrane consists of a lipid bilayer with integral membrane proteins stabilized by regulated linkages to the cortical actin cytoskeleton. The regulation is necessary for cells to change shape ormigrate. The ERM (ezrin-radixin-moesin) proteins are believed to provide such links, with the N-terminal halves associating with integral membrane proteins, either directly or indirectly through adapter molecules like EBP50 (ERM binding phosphoprotein, 50 kDa), and their C-terminal halves associating with F-actin. However, isolated ERM proteins largely exist in a dormant state by virtue of an intramolecular interaction between amino- and carboxyl-terminal domains, thereby masking membrane and cytoskeletal association sites. C-terminal threonine phosphorylation of a fragment of radixin has been found to destroy its ability to bind the amino-terminal domain without affecting the C-terminal F-actin binding site. Here we show that C-terminal phosphorylation of full-length, dormant ezrin and moesin by protein kinase C-theta simultaneously unmasks both the F-actin and EBP50 binding sites. Increased phosphorylation of moesin in cells correlated with increased association of moesin with the cortical actin cytoskeleton. These results show that activation of ERM proteins can be accomplished by phosphorylation of a single C-terminal threonine residue.

In the assessment of arterial stiffness, pulse pressure is measured. Presently, there is no consensus on how pulse pressure should be measured. Distensibility of the left and right common carotid arteries was measured noninvasively in 224 patients participating in the Second Manifestations of ARTerial disease (SMART) study. Blood pressure was recorded every 4 min, using a semiautomatic oscillometric device. Distensibility coefficients (DC) were calculated with pulse pressure obtained as an average of (A) all measurements during the session; (B) the second, third, and fourth measurement; (C) measurements before and after distensibility assessment; and (D) three measurements nearest to distensibility assessment. Associations of cardiovascular risk factors with the four calculated DCs were evaluated with linear regression analysis. DC estimates were slightly more precise with methods A and B than with C or D. The magnitude of the associations showed a slight trend to higher precision for methods A and B. Pulse pressures obtained as an average of all or the second, third, and fourth blood pressure measurements during an arterial stiffness measurement session yield slightly more precise estimates of DC. However, the differences between the methods are small; therefore, we suggest that pragmatic arguments dominate the choice between the methods.

Objective: To assess current practice in screening for asymptomatic vascular disease and risk factors in patients referred with vascular disease or cardiovascular risk factors., Design: Descriptive, retrospective., Setting: University Hospital Utrecht, Utrecht, the Netherlands., Method: By means of the computerized hospital registration system all patients who were referred to the outpatient clinic with carotid stenosis, peripheral artery disease, abdominal aortic aneurysm, diabetes mellitus, hyperlipidaemia or hypertension during one year were identified. By means of the same hospital registration system the frequency of diagnostic tests performed to detect atherosclerosis or risk factors in these patients within a period of 5 months round the first attendance was determined., Results: 372 Patients with a vascular disease and 317 patients with a risk factor were identified. Tests to detect carotid stenosis, peripheral artery disease or an abdominal aortic aneurysm were each performed in less then 6% of all patients except the test for abdominal aortic aneurysm. Tests to detect coronary artery disease were performed in about 50% of all patients. Tests to detect diabetes mellitus were performed in 35% of the patients with vascular disease and in 81% of the patients presenting with hyperlipidaemia or hypertension. Tests to detect hyperlipidaemia were performed in 18% of the patients with a vascular disease and in 76% of the patients with diabetes or hypertension., Conclusion: The results of this study suggest that in current practice patients referred for vascular disease or cardiovascular risk factors are infrequently screened for asymptomatic macrovascular disease and risk factors.

Moesin, a member of the ezrin-radixin-moesin (ERM) family of membrane/cytoskeletal linkage proteins, is known to be threonine-phosphorylated at Thr558 in activated platelets within its conserved putative actin-binding domain. The pathway leading to this phosphorylation step and its control have not been previously elucidated. We have detected and characterized reactions leading to moesin phosphorylation in human leukocyte extracts. In vitro phosphorylation of endogenous moesin, which was identified by peptide microsequencing, was dependent on phosphatidylglycerol (PG) or to a lesser extent, phosphatidylinositol (PI), but not phosphatidylserine (PS) and diacylglycerol (DAG). Analysis of charge shifts, phosphoamino acid analysis, and stoichiometry was consistent with a single phosphorylation site. By using mass spectroscopy and direct microsequencing of CNBr fragments of phospho-moesin, the phosphorylation site was identified as KYKT*LRQIR (where * indicates the phosphorylation site) (Thr558), which is conserved in the ERM family. Recombinant moesin demonstrated similar in vitro phospholipid-dependent phosphorylation compared with the endogenous protein. The phosphorylation site sequence of moesin displays a high degree of conservation with the pseudosubstrate sequences of the protein kinase C (PKC) family. We identified the kinase activity as PKC-theta on the basis of immunodepletion of the moesin kinase activity and copurification of PKC-theta with the enzymic activity. We further demonstrate that PKC-theta displays a preference for PG vesicles over PI or PS/DAG, with minimal activation by DAG, as well as specificity for moesin compared with myelin basic protein, histone H1, or other cellular proteins. Expression of a human His6-tagged PKC-theta in Jurkat cells and purification by Ni2+ chelate chromatography yield an active enzyme that phosphorylates moesin. PG vesicle binding experiments with expressed PKC-theta and moesin demonstrate that both bind to vesicles independently of one another. Thus, PKC-theta is identified as a major kinase within cells with specificity for moesin and with activation under non-classical PKC conditions. It appears likely that this activity corresponds to a specific intracellular pathway controlling the function of moesin as well as other ERM proteins.

Techniques have been developed to produce microbial phytase for addition to diets for simple-stomached animals, with the aim to improve phosphorus availability from phytate-P in plant sources. The activity of the crude microbial phytase showed pH optima at pH 5.5 and 2.5. The enzyme was able to degrade phytate in vitro in soya-bean meal, maize and a liquid compound feed for pigs. When microbial phytase was added to low-P diets for broilers the availability of P increased to over 60% and the amount of P in the droppings decreased by 50%. The growth rate and feed conversion ratio on the low-P diets containing microbial phytase were comparable to or even better than those obtained on control diets. Addition of microbial phytase to diets for growing pigs increased the apparent absorbability of P by 24%. The amount of P in the faeces was 35% lower.

The effect of various factors on the incidence of leg abnormalities, with particular reference to twisted leg, in broilers was studied. The incidence of twisted legs was influenced by strain and for males was twice that for females. There was also a higher incidence in cages than on litter with the type of cage floor having an effect: broilers reared on floors of metal wire and perforated sheets had more leg problems than those reared on plastic mats and plastic-covered wire. Although vitamin and mineral supplementation had no effect on caged broilers, a restriction of metabolisable energy (ME) intake during the first 14 d after hatching halved the frequency of leg abnormalities compared with those fed ad libitum. Studies of cage size and location of water suggested that a lack of exercise increases the incidence of leg abnormalities.

The number of binding sites and the dissociation constants were determined for the binding of bilirubin to human liver ligandin and to human serum albumin. Albumin has a primary bilirubin binding site (KD = 0.03 microM), measured by the peroxidase procedure, and two apparently equivalent secondary binding sites (KD = 2 microM), determined by fluorescence quenching experiments. By contrast, ligandin does not have a corresponding high affinity site. The absence of this high affinity site was shown both by the peroxidase procedure and by direct competition between albumin and ligandin for bilirubin. Bilirubin binding to ligandin, measured by fluorescence quenching, is complex. At both pH 6.5 and 7.4, two interacting sites were observed with a Hill coefficient of 1.5, K' approximately 5 microM. Bilirubin binding to ligandin is not independent of glutathione S-transferase activity. Depending upon pH and upon the order with which the reactants are added, bilirubin can markedly alter the transferase activity. The results are interpreted in terms of kinetically stable conformational isomers of ligandin induced by bilirubin or by glutathione.

A new molded polypropylene, diamond-like carbon (DLC)-coated mixing cell has been developed for use in the batch microcalorimeter. Reagent volume can be varied from 25 microliters to 100 microliters. A 10 microcalorie reaction heat can be measured to 5%. Repeat reactions can be done as often as every 10 min for a fast reaction. Reactions can be started within 1 h or less after loading. A pre-equilibrator and a temperature-controlled syringe drive unit permit solutions to be stored at 4 degrees C while being run at any temperature from -20 degrees C to 40 degrees C. The kinetics and enthalpy of reaction of NAD-NADase have been measured. delta H is about 21 kcal/mol endothermic.

The long-distance electron transfer observed in the complex formed between ferrocytochrome c and compound I, the peroxide-oxidized form of cytochrome c peroxidase (CCP), has been proposed to occur through the participation of His 181 of CCP and Phe 87 of yeast iso-1 cytochrome c [Poulos, T. L., & Kraut, J. (1980) J. Biol. Chem. 255, 10322-10330]. We have examined the role of His 181 of CCP in this process through characterization of a mutant CCP in which His 181 has been replaced by glycine through site-directed mutagenesis. Data from single-crystal X-ray diffraction studies, as well as the visible spectra of the mutant CCP and its 2-equiv oxidation product, compound I, show that at pH 6.0 the protein is not dramatically altered by the His 181----Gly mutation. The rate of peroxide-dependent oxidation of ferrocytochrome c by the mutant CCP is reduced only 2-fold relative to that of the parental CCP, under steady-state conditions. Transient kinetic measurements of the intracomplex electron transfer rate from ferrous cytochrome c to compound I indicate that the rate of electron transfer within the transiently formed complex at high ionic strength (mu = 114 mM, pH = 6) is also reduced by approximately 2-fold in the mutant CCP protein. The relatively minor effect of the loss of the imidazole side chain at position 181 on the kinetics of electron transfer in the CCP-cytochrome c complex precludes an obligatory participation of His 181 in electron transfer from ferrous cytochrome c to compound I.(ABSTRACT TRUNCATED AT 250 WORDS)

The three major monomer hemoglobins from Glycera dibranchiata erythrocytes isolated in this laboratory were sequenced from their N-termini. A stretch of amino acid sequence identity was used to determine the sequence of a mixed oligodeoxynucleotide that would be complementary to all 12 possible mRNA sequences coding for the amino acids. A cDNA library was constructed by using poly(A+) RNA from G. dibranchiata erythrocytes, the library was probed with the oligonucleotide, and the longest positive inserts found were subcloned into a sequencing plasmid and then sequenced. The first one was 745 bases long, containing 85 bases of 5'-untranslated RNA, an open reading frame of 444 bases coding for 148 amino acids, and a 3'-untranslated region of 216 bases. The predicted amino acid sequence matches the first 25 amino acids of G. dibranchiata monomer globin component IV. The sequence contains an N-terminal methionine plus 18 other mostly conservative sequence changes compared to the published sequence of Imamura et al. (1972), which appears from our partial sequencing to be monomer globin component II. We confirm the presence of leucine in the E7 position, which is histidine in most myoglobins and hemoglobins.