Number Of Receptor Binding Sites Research Articles

Design Considerations of Liquid Gated High Field Modulated FET Biosensor for Enhanced Sensing Characteristics

Medical diagnostics are rapidly evolving to meet the requirements of personalized medicine and home-care based in-vitro diagnostics are in very high demand. Translating the clinical blood based assays into home-care tests pose several challenges. Primarily, we need to develop a compact, portable system that can enable the end user to perform the assay with minimal protocols that can be carried out without prior training, cost-effective and quick response time. More importantly, the home-care tests should retain the sensitivity and selectivity of the laboratory based assays. We previously developed high field modulated field effect transistor (FET) biosensors for the direct detection of protein biomarkers in whole blood. In this work, we investigate the sensor structure in detail to assess the parameters that govern the sensing characteristics. The basic sensor structure includes a functionalized reference electrode to which gate voltage is applied, placed at a narrow gap from the extended gate metal, which is connected to the FET’s gate terminal. The test solution containing the target analyte is placed across the gap such that it is sandwiched between the reference electrode and extended gate metal, together forming the sensing region. It was revealed from our previous studies that there are two ways in which the field generated across the test solution can be modulated: by applying higher amplitude of gate voltage and by decreasing the gap between the electrodes. It was found that at smaller gap distances, the potential drop in the solution is linearly dependent on the gap between the electrodes, and hence higher sensitivity can be observed for smaller gap configurations. In this work we initially explore the gap dependence of sensor response for micron scale and nano scale gap distances. The results indicate that noise characteristics and sensitivity are improved when the gap between the reference electrode and extended gate metal is decreased. The geometry of the sensing areas are also studied to evaluate the optimal electric field distribution across the test solution to generate enhanced sensing characteristics. Finally the surface functionalization is investigated to analyse the effect of surface coverage and number of receptor binding sites on the sensor performance. Through the integration of sensor design, geometry and optimal surface functionalization, we can improve the sensing characteristics, while satisfying the practical considerations of implementation of a home-care whole blood assay.

Design Considerations of Liquid Gated High Field Modulated FET Biosensor for Enhanced Sensing Characteristics

Medical diagnostics are rapidly evolving to meet the requirements of personalized medicine. We previously developed high field modulated field effect transistor (FET) biosensors for the direct detection of protein biomarkers in whole blood. In this work, we investigate the sensor structure in detail to assess the parameters that govern the sensing characteristics. The basic sensor structure includes a functionalized reference electrode to which gate voltage is applied, placed at a narrow gap from the extended gate metal, which is connected to the FET’s gate terminal. It was revealed from our previous studies that there are two ways in which the field generated across the test solution, placed in the gap, can be modulated: by applying higher amplitude of gate voltage and by decreasing the gap between the electrodes. It was found that at smaller gap distances, the potential drop in the solution is linearly dependent on the gap between the electrodes, and hence higher sensitivity can be observed for smaller gap configurations. In this work we initially explore the gap dependence of sensor response for micron scale gap distances. The results indicate that noise characteristics and sensitivity are improved when the gap between the reference electrode and extended gate metal is decreased. The geometry of the sensing areas are also studied to evaluate the optimal electric field distribution across the test solution to generate enhanced sensing characteristics. Finally the surface functionalization is investigated to analyse the effect of surface coverage and number of receptor binding sites on the sensor performance.

N-terminal SAP97 isoforms differentially regulate synaptic structure and postsynaptic surface pools of AMPA receptors.

The location and density of postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors is controlled by scaffolding proteins within the postsynaptic density (PSD). SAP97 is a PSD protein with two N-terminal isoforms, α and β, that have opposing effects on synaptic strength thought to result from differential targeting of AMPA receptors into distinct synaptic versus extrasynaptic locations, respectively. In this study, we have applied dSTORM super resolution imaging in order to localize the synaptic and extrasynaptic pools of AMPA receptors in neurons expressing α or βSAP97. Unexpectedly, we observed that both α and βSAP97 enhanced the localization of AMPA receptors at synapses. However, this occurred via different mechanisms: αSAP97 increased PSD size and consequently the number of receptor binding sites, whilst βSAP97 increased synaptic receptor cluster size and surface AMPA receptor density at the PSD edge and surrounding perisynaptic sites without changing PSD size. αSAP97 also strongly enlarged presynaptic active zone protein clusters, consistent with both presynaptic and postsynaptic enhancement underlying the previously observed αSAP97-induced increase in AMPA receptor-mediated currents. In contrast, βSAP97-expressing neurons increased the proportion of immature filopodia that express higher levels of AMPA receptors, decreased the number of functional presynaptic terminals, and also reduced the size of the dendritic tree and delayed the maturation of mushroom spines. Our data reveal that SAP97 isoforms can specifically regulate surface AMPA receptor nanodomain clusters, with βSAP97 increasing extrasynaptic receptor domains at peri-synaptic and filopodial sites. Moreover, βSAP97 negatively regulates synaptic maturation both structurally and functionally. These data support diverging presynaptic and postsynaptic roles of SAP97 N-terminal isoforms in synapse maturation and plasticity. As numerous splice isoforms exist in other major PSD proteins (e.g., Shank, PSD95, and SAP102), this alternative splicing may result in individual PSD proteins having divergent functional and structural roles in both physiological and pathophysiological synaptic states.

Synthesis and Preclinical Evaluation of 11C-UCB-J as a PET Tracer for Imaging the Synaptic Vesicle Glycoprotein 2A in the Brain.

The synaptic vesicle glycoprotein 2A (SV2A) is found in secretory vesicles in neurons and endocrine cells. PET with a selective SV2A radiotracer will allow characterization of drugs that modulate SV2A (e.g., antiepileptic drugs) and potentially could be a biomarker of synaptic density (e.g., in neurodegenerative disorders). Here we describe the synthesis and characterization of the SV2A PET radiotracer (11)C-UCB-J ((R)-1-((3-((11)C-methyl-(11)C)pyridin-4-yl)methyl)-4-(3,4,5-trifluorophenyl)pyrrolidin-2-one) in nonhuman primates, including whole-body biodistribution. (11)C-UCB-J was prepared by C-(11)C-methylation of the 3-pyridyl trifluoroborate precursor with (11)C-methyl iodide via the Suzuki-Miyaura cross-coupling method. Rhesus macaques underwent multiple scans including coinjection with unlabeled UCB-J (17, 50, and 150 μg/kg) or preblocking with the antiepileptic drug levetiracetam at 10 and 30 mg/kg. Scans were acquired for 2 h with arterial sampling and metabolite analysis to measure the input function. Regional volume of distribution (VT) was estimated using the 1-tissue-compartment model. Target occupancy was assessed using the occupancy plot; the dissociation constant (Kd) was determined by fitting self-blocking occupancies to a 1-site model, and the maximum number of receptor binding sites (Bmax) values were derived from baseline VT and from the estimated Kd and the nondisplaceable distribution volume (VND). (11)C-UCB-J was synthesized with greater than 98% purity. (11)C-UCB-J exhibited high free fraction (0.46 ± 0.02) and metabolized at a moderate rate (39% ± 5% and 24% ± 3% parent remaining at 30 and 90 min) in plasma. In the monkey brain, (11)C-UCB-J displayed high uptake and fast kinetics. VT was high (∼25-55 mL/cm(3)) in all gray matter regions, consistent with the ubiquitous expression of SV2A. Preblocking with 10 and 30 mg/kg of levetiracetam resulted in approximately 60% and 90% occupancy, respectively. Analysis of the self-blocking scans yielded a Kd estimate of 3.4 nM and Bmax of 125-350 nM, in good agreement with the in vitro inhibition constant (Ki) of 6.3 nM and regional Bmax in humans. Whole-body biodistribution revealed that the liver and the brain are the dose-limiting organs for males and females, respectively. (11)C-UCB-J exhibited excellent characteristics as an SV2A PET radiotracer in nonhuman primates. The radiotracer is currently undergoing first-in-human evaluation.

Up-regulation of MUC2 and IL-1β expression in human colonic epithelial cells by Shigella and its interaction with mucins.

BackgroundThe entire gastrointestinal tract is protected by a mucous layer, which contains complex glycoproteins called mucins. MUC2 is one such mucin that protects the colonic mucosa from invading microbes. The initial interaction between microbes and mucins is an important step for microbial pathogenesis. Hence, it was of interest to investigate the relationship between host (mucin) and pathogen interaction, including Shigella induced expression of MUC2 and IL-1β during shigellosis.MethodsThe mucin-Shigella interaction was revealed by an in vitro mucin-binding assay. Invasion of Shigella dysenteriae into HT-29 cells was analyzed by Transmission electron microscopy. Shigella induced mucin and IL-1β expression were analyzed by RT-PCR and Immunofluorescence.ResultsThe clinical isolates of Shigella were found to be virulent by a congo-red binding assay. The in vitro mucin-binding assay revealed both Shigella dysenteriae and Shigella flexneri have binding affinity in the increasing order of: guinea pig small intestinal mucin<guinea pig colonic mucin< Human colonic mucin. Invasion of Shigella dysenteriae into HT-29 cells occurs within 2 hours. Interestingly, in Shigella dysenteriae infected conditions, significant increases in mRNA expression of MUC2 and IL-1β were observed in a time dependent manner. Further, immunofluorescence analysis of MUC2 shows more positive cells in Shigella dysenteriae treated cells than untreated cells.ConclusionsOur study concludes that the Shigella species specifically binds to guinea pig colonic mucin, but not to guinea pig small intestinal mucin. The guinea pig colonic mucin showed a greater binding parameter (R), and more saturable binding, suggesting the presence of a finite number of receptor binding sites in the colonic mucin of the host. In addition, modification of mucins with TFMS and sodium metaperiodate significantly reduced mucin-bacterial binding; suggesting that the mucin-Shigella interaction occurs through carbohydrate epitopes on the mucin backbones. Overproduction of MUC2 may alter adherence and invasion of Shigella dysenteriae into human colonic epithelial cells.

Synthesis and in vitro characterization of radioiodinatable benzodiazepines selective for type 1 and type 2 cholecystokinin receptors.

Radiolabeled antagonists of specific peptide receptors identify a higher number of receptor binding sites than agonists and may thus be preferable for in vivo tumor targeting. In this study, two novel radioiodinated 1,4-benzodiazepines, (S)-1-(3-iodophenyl)-3-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)urea (9) and (R)-1-(3-iodophenyl)-3-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)urea (7), were developed. They were characterized in vitro as high affinity selective antagonists at cholecystokinin types 1 and 2 (CCK(1) and CCK(2)) receptors using receptor binding, calcium mobilization, and internalization studies. Their binding to human tumor tissues was assessed with in vitro receptor autoradiography and compared with an established peptidic CCK agonist radioligand. The (125)I-labeled CCK(1) receptor-selective compound 9 often revealed a substantially higher amount of CCK(1) receptor binding sites in tumors than the agonist (125)I-CCK. Conversely, the radioiodinated CCK(2) receptor-selective compound 7 showed generally weaker tumor binding than (125)I-CCK. In conclusion, compound 9 is an excellent radioiodinated nonpeptidic antagonist ligand for direct and selective labeling of CCK(1) receptors in vitro. Moreover, it represents a suitable candidate to test antagonist binding to CCK(1) receptor-expressing tumors in vivo.

Post-phosphorylation prolyl isomerisation of gephyrin represents a mechanism to modulate glycine receptors function

The microtubule binding protein gephyrin plays a prominent role in establishing and maintaining a high concentration of inhibitory glycine receptors juxtaposed to presynaptic releasing sites. Here, we show that endogenous gephyrin undergoes proline-directed phosphorylation, which is followed by the recruitment of the peptidyl-prolyl isomerase Pin1. The interaction between gephyrin and Pin1 is strictly dependent on gephyrin phosphorylation and requires serine-proline consensus sites encompassing the gephyrin proline-rich domain. Upon binding, Pin1 triggers conformational changes in the gephyrin molecule, thus enhancing its ability to bind the beta subunit of GlyRs. Consistently, a downregulation of GlyR clusters was detected in hippocampal neurons derived from Pin1 knockout mice, which was paralleled by a reduction in the amplitude of glycine-evoked currents. Our results suggest that phosphorylation-dependent prolyl isomerisation of gephyrin represents a mechanism for regulating GlyRs function.

The involvement of sequence variation and expression of CX3CR1 in the pathogenesis of age-related macular degeneration.

This study examined the association between the sequence variation/expression of CX3CR1, a chemokine receptor, and age-related macular degeneration (AMD). Peripheral blood from 85 AMD patients and 105 subjects without AMD (controls), as well as ocular tissue from 40 pathological sections with AMD and two normal eye sections, were screened for V249I and T280M, two single nucleotide polymorphisms (SNPs) in CX3CR1. An increased prevalence, with the highest odds ratio of 3.57, of the I249 and M280 carriers was found among the AMD cases as compared with the controls. When comparing CX3CR1 expression in the archived eye sections, CX3CR1 transcripts were not detectable in the maculae of AMD eyes bearing T/M280; however, transcripts were detected in the maculae of normal eyes bearing T/T280 or T/M280 as well as in the AMD maculae bearing T/T280. Furthermore, lower CX3CR1 protein expression was observed in the maculae of AMD eyes bearing T/M280 compared with the controls bearing T/T280. The I249 and M280 alleles result in a lowered number of receptor binding sites and a decreased ligand affinity. Our data suggest that a decrease, caused by sequence variation and/or lower CX3CR1 expression, in CX3CR1-induced cellular activities could contribute to AMD development.

Muscarinic receptor protein expression in the ocular tissues of the chick during normal and myopic eye development

Muscarinic receptor signalling has been implicated in both the embryonic and postnatal development of ocular structures as well as in myopic eye growth. A radioligand binding assay was used to determine whether changes in muscarinic receptor density and/or affinity occurred in the chick retina, choroid and sclera during early post-hatching development or with the induction of myopia. Specific receptor binding sites were saturable with increasing concentrations of the muscarinic receptor ligand [ 3H] N-methyl-scopolamine in the retina and choroid but not in the sclera. In normal eyes, binding density in the retina was not altered from age P5 to P10 (447±14 vs. 411±13 fmol/mg of protein, P=0.07). However, in the choroid, the number of receptor binding sites significantly increased between P5 and P10 (637±39 vs. 1125±121 fmol/mg of protein, P<0.01). Binding affinity ( K D) was not altered with age in either the retina or choroid. Myopia was induced in chicks by deprivation of form vision, using translucent diffusers, from age P3. Despite the induction of significant degrees of ocular elongation and myopia at P5 (−8.7±0.3 D, P<0.01) and P10 (−22.5±1.3 D, P<0.01), neither muscarinic receptor density nor affinity were altered in the retina or choroid of myopic eyes. These findings indicate that regulation of muscarinic receptor numbers in the chick choroid is occurring in normal post-hatching development of this tissue. However, myopic eye enlargement was not associated with changes in muscarinic receptor protein expression in the chick retina and choroid.

Effect of atrazine and zearalenone on the numer of receptor binding sites for 3H-estradiol in the rat uterus cytosol

The effect of a daily dose of 14 mg atrazine and/or 2.5 mg zearalenone that were administered to rats during 5 days of estrous cycle on the number of receptor binding sites for 3H-estradiol in uterine cytosol was investigated. Comparison with a control group of animals showed no effect of atrazine influence on the number of receptor binding sites for 3H-estradiol in the rat uterine cytosol until day 6 of the experiment, when a significantly lower value was recorded (P &amp;lt; 0.05). In the group of animals administered zearalenone, a decline in the number of receptor binding sites for 3H-estradiol in uterine cytosol was observed. On day 4 of the experiment, the number of binding sites was significantly greater (P &amp;lt; 0.05) in this group than in the control group of animals, whereafter it gradually decreased to a significantly lower value (P &amp;lt; 0.05) as compared to the control group on day 6. In the group of animals administered a mixture of atrazine and zearalenone, the number of receptor binding sites for 3H-estradiol in uterine cytosol did not decrease but showed a similar pattern as in the control group of animals, however, at a statistically significantly (P &amp;lt; 0.05) higher level.

Variant alleles of the D2 dopamine receptor gene and obesity

The mesocorticolimbic dopaminergic reward pathways have a role in the neuromodulation of appetite. There are data supporting a role of allelic variants of the D2 dopamine receptor ( DRD2) gene and the number of receptor binding sites in vulnerability to substance abuse and obesity. We recently demonstrated that the A1 and B1 genotypes are in linkage disequilibrium and predictive of smoking status. Previous studies have evaluated the relationship between obesity and A1 but not B1 genotypes. Our objective was to assess the relationships between obesity and DRD2 TaqI A and TaqI B genotypes in healthy individuals. Subjects were 139 Caucasian average weight and 37 obese individuals (body mass index ⩾30) identified as comparison subjects for ongoing case-control studies. Among the obese group, only 41.7% exhibited the A2 genotypes and 58.3% the A1 genotypes compared with 68.8% and 31.2% respectively for the average weight subjects (P=0.002). There was a similar pattern for B2 genotypes (51.4% compared with 78.9% respectively, P=0.003). The risk of obesity associated with the DRD2 A1 genotypes was 3.48 (95% confidence intervals = 1.55, 7.80), compared with 4.55 (1.94, 10.69) for the DRD2 B1 genotypes. Individuals who had the A1 or B1 genotypes had higher BMI than those with the wildtype genotypes (P=0.086 and 0.05 respectively). The prevalence of the A1 genotypes was 2 of 5 (40%) obese individuals who never smoked, 12 of 22 (54.6%) for obese former smokers, and 7 of 9 (77.9%) for obese current smokers. The comparable percentages for the B1 genotypes were 0%, 56.5% and 55.6%. Further emphasis needs to be placed on identifying the genetic basis for obesity in order to develop targeted weight reduction interventions, that may improve potential for success.

Beta(2)-adrenoceptor agonist-induced upregulation of tachykinin NK(2) receptor expression and function in airway smooth muscle.

Neurokinin A (NKA) induces bronchoconstriction mediated by tachykinin NK(2) receptors in animals and humans, and may be increased in asthma. Because beta(2)-adrenoceptor agonists are the most widely used bronchodilators in asthma, we investigated the effects of the beta(2)-adrenoceptor agonist fenoterol on NK(2) receptor messenger RNA (mRNA) and receptor density as well as the functional responses of bovine tracheal smooth muscle to the NK(2) receptor agonist [beta-Ala(8)]-NKA(4-10) in vitro, using Northern blot analysis, receptor binding, and organ bath studies. Incubation with fenoterol induced a time- and concentration-dependent upregulation of NK(2) receptor mRNA (71% increase after 12 h at 10(-7) M fenoterol), which was abolished by propranolol (a nonselective beta-adrenoceptor agonist) and ICI118551 (a selective beta(2)-adrenoceptor antagonist), but not by CGP20712A (a selective beta(1)-adrenoceptor antagonist), indicating that fenoterol acts via beta(2)-adrenoceptors. These effects were mimicked by forskolin and prostaglandin E(2) (PGE(2)), both agents that increase cyclic adenosine monophosphate (cAMP), and by the cAMP analogue 8-bromo-cAMP. The upregulation was blocked by cycloheximide, indicating that it requires new protein synthesis, and was accompanied by an increase in both the stability of NK(2) receptor mRNA and the rate of NK(2) receptor gene transcription. Radioligand binding assay using the selective NK(2) receptor antagonist [(3)H]SR48968 showed a significant increase in the number of receptor binding sites after 12 h and 18 h, which was accompanied by an increased contractile responsiveness to the NK(2) receptor agonist [beta-Ala(8)]-NKA(4-10). Dexamethasone completely prevented the fenoterol-induced increase in NK(2) receptor mRNA and in the contractile response. We conclude that beta(2)-adrenoceptor agonists induce upregulation of functional NK(2) receptors in airway smooth muscle by increasing cAMP, and that this can be prevented by a corticosteroid. The increased responsiveness could be relevant to asthma control and mortality.

Glucocorticoids reduce tachykinin NK2 receptor expression in bovine tracheal smooth muscle.

Neurokinin A is not only a potent bronchoconstrictor, but also has immuno-modulatory effects in animals and man, mediated via tachykinin NK2 receptors. We have examined the effect of the glucocorticoid, dexamethasone, on tachykinin NK2 receptor mRNA and the number of tachykinin NK2 receptors in bovine tracheal smooth muscle in vitro by Northern blot analysis using a human tachykinin NK2 receptor cDNA probe and receptor binding assay using [3H]SR48968 [(S)-N-methyl-N[4-acetylamino-4-phenylpiperidino-2(3,4-dichlorophenyl) butyl]benzamide]. Tachykinin NK2 receptor mRNA showed a time-dependent suppression (62% reduction after 6 h at 10(-7) M of dexamethasone), as well as a concentration-dependent suppression after the incubation with dexamethasone (IC50 = 1.3 x 10(-8) M). This suppression was abolished by the glucocorticoid receptor antagonist, mifepristone (RU38486), indicating that dexamethasone acts via the glucocorticoid receptor. It was also abolished by the protein synthesis inhibitor, cycloheximide (10 microg/ml), indicating that new protein synthesis is required on this suppression. Using the RNA polymerase inhibitor actinomycin D (5 microg/ml), we showed that the stability of tachykinin NK2 receptor mRNA was not affected by dexamethasone (t1/2 = 5 h). Nuclear run-on assays revealed a 51% reduction in the rate of tachykinin NK2 receptor gene transcription after treatment with dexamethasone for 6 h. Radioligand binding assay using an selective tachykinin NK2 receptor antagonist, [3H]SR48968 showed a significant decrease in the number of receptor binding sites after 16 h (Bmax = 262 +/- 23 versus 213 +/- 13 fmol/mg protein for vehicle and dexamethasone treatment respectively, P < 0.05), with no significant change at the earlier time points. These results suggest that glucocorticoids act on glucocorticoid receptors to decrease tachykinin NK2 receptor expression by decreasing the rate of tachykinin NK2 receptor gene transcription.

Differential regulation of natriuretic peptide receptors on ciliary body epithelial cells.

Atrionatriuretic peptide (ANP) lowers intraocular pressure in the eyes of humans and rabbits. We examined the effects of natriuretic peptides on cGMP formation and 125I-labelled-ANP binding to cultured cells derived from ciliary body epithelium, the site of aqueous humour formation in the eye. ANP, brain natriuretic peptide (BNP) and C-natriuretic peptide (CNP) at 1 microM stimulated cGMP formation 8.2(+/-1.2)-fold, 4.8(+/-0.6)-fold and 87.3(+/-12.1)-fold respectively. 125I-ANP bound to intact cells at a single site, with a dissociation constant KD=0.30+/-0.01 nM. BNP was as effective as ANP in displacing 125I-ANP, whereas CNP displaced label with a slightly higher IC50. 125I-ANP binding was displaced >95% by c-ANP, a specific ligand for natriuretic peptide C receptors (NPR-C). Cross-linking of 125I-ANP to cells labelled predominantly a protein of Mr 62000. These data suggest that 125I-ANP binding was primarily to NPR-C, whereas cGMP stimulation occurred primarily via natriuretic peptide B receptors (NPR-B). Vasopressin and histamine, both activators of the inositol phosphate/diacylglycerol phosphate pathway in non-pigmented ciliary epithelial cells, inhibited CNP stimulation of guanylate cyclase (NPR-B) and 125I-ANP binding (NPR-C) by 30-38%. Inhibition was mimicked by PMA, dioctanoylglycerol and phorbol didecanoate, whereas 4alpha phorbol didecanoate had no effect. Staurosporine and bisindolylmaleimide both blocked inhibition of 125I-ANP binding and cGMP formation by PMA. These results suggest that protein kinase C (PKC) down-regulates both NPR-B and NPR-C. PKC down-regulation of NPR-B varied inversely with CNP concentration. Inhibition by 1 microM PMA was 30.6(+/-4.0)% with 500 nM CNP, but 83.4(+/-8.8)% with 10 nM CNP, indicating that increasing CNP could partially overcome inhibition by PMA. Since extracellular CNP levels were not affected by PKC activation, the effect of PKC on NPR-B is best explained as a reduction in NPR-B affinity for CNP. NPR-C measured as 125I-ANP binding was likewise reduced 36.4(+/-5.1)% by exposure to PMA. In contrast with NPR-B inhibition, however, inhibition of NPR-C was due largely to a reduction in the number of receptor binding sites per cell rather than a reduction in receptor affinity for ligand. The data therefore suggest that both NPR-B and NPR-C are down-regulated by PKC, but that the mechanisms of down-regulation of the two receptors are different.

2] Generating and characterizing retinoid receptors from Escherichia coli and insect cell expression systems

Publisher Summary This chapter focuses on the production and characterization of recombinant retinoid receptors in bacteria and insect cells, analysis of receptors using equilibrium saturation and competition binding analyses, and the use of sucrose density gradients (SDGs) to examine how DNA influences ligand binding to retinoid receptors (RAR and RXR) proteins. RAR/pATH fusion proteins containing the intact ligand-binding domain can also be detected by specific radioligand binding. However, this requires denaturation and refolding of the expressed protein prior to assay. The chapter describes the expression system for the generation of recombinant retinoid receptors that is based on the lytic virus, Autographa californica nuclear polyhedrosis virus (AcNPV). Saturation equilibrium binding and Scatchard analysis is used to determine the affinity of a receptor for radiolabeled ligand (Kd) and the number of receptor-binding sites in a given sample (Bmax). A variation on this method is to label the receptor at a single concentration of ligand. This is useful to screen samples rapidly for ligand-binding activity, or to check a novel compound for its ability to compete with radiolabeled ligand for binding to receptor.

Interaction of a cholera toxin derivative containing a reduced number of receptor binding sites with intact cells in culture

Hybrid CTB (hCTB), having only one or two functional binding sites, has been constructed from two chemically inactivated derivatives of CTB. One inactive derivative consisted of CTB formylated in the lone Trp-88 of each β-chain (fCTB), whereas the other inactive derivative consisted of CTB specifically succinylated in three amino groups located in or near the receptor binding site (sssCTB). hCTB, fCTB and sssCTB were able to reassociate with CTA and form the corresponding holotoxins hCT, fCT and sssCT as measured by gel filtration chromatography. In contrast to fCT and sssCT, hCT could increase the cAMP content of intact Vero cells in a time- and dose-dependent way: concentrations as low as a few nanograms of hCT per milliliter caused a significant increase in the intracellular cAMP level. The maximal cAMP level induced by hCT (1 μg/ml) was, however, more than 2-fold lower than that elicited by its native counterpart. At saturating ligand concentrations and at 37°C, the lag periods and rates of CT and hCT induced cAMP accumulation were essentially the same. Treatment of Vero and HeLa cells with GM 1 did not affect their difference in response to CT and hCT. When Vero cells treated with hCT were incubated for longer periods of time, a further slow accumulation of cAMP occurred until after about 20 h cAMP levels of cells exposed to CT or hCT were essentially the same. In contrast to Vero and HeLa cells, human skin fibroblasts exhibited an almost identical response to CT as well as to hCT. Acidotropic agents such as chloroquine and monensin affected the CT and hCT induced increase in cAMP content of Vero cells, fibroblasts and GM 1 treated Hela cells in a similar way. The results are consistent with the view that CT receptor recognition domains are shared between adjacent β-chains, that pentavalent binding appears not to be essential for cytotoxicity and that in the cell types studied intracellular processing of CT, hCT is involved.

Luminescent oxygen channeling immunoassay: measurement of particle binding kinetics by chemiluminescence.

A method for monitoring formation of latex particle pairs by chemiluminescence is described. Molecular oxygen is excited by a photosensitizer and an antenna dye that are dissolved in one of the particles. 1 delta gO2 diffuses to the second particle and initiates a high quantum yield chemiluminescent reaction of an olefin that is dissolved in it. The efficiency of 1 delta gO2 transfer between particles is approximately 3.5%. The technique permits real-time measurement of particle binding kinetics. Second-order rate constants increase with the number of receptor binding sites on the particles and approach diffusion control. By using antibody-coated particles, a homogeneous immunoassay capable of detecting approximately 4 amol of thyroid-stimulating hormone in 12 min was demonstrated. Single molecules of analyte produce particle heterodimers that are detected even when no larger aggregates are formed.

Characterization of the solubilized mosquito vitellogenin receptor

In this study we solubilized and characterized the receptor for the major egg yolk protein precursor, vitellogenin (Vg), from the yellow fever mosquito, Aedes aegypti. The receptor was solubilized from vitellogenic ovary membranes using octyl-β- d-glucoside (OG). Under equilibrium binding conditions, [ 35S]Vg bound with high affinity ( K d = 2.8 × 10 −8 M) to a single class of binding sites in solubilized ovary extracts. The solubilized receptor was present in ovarian extracts and bound selectively A. aegypti Vg and its storage form, vitellin (Vn). The receptor preparation was heat and trypsin sensitive. Binding of Vg to its receptor could be inhibited as well dissociated with suramin. The receptor was visualized by ligand-blotting as a 205 kDa protein under non-reducing conditions. It did not share immunological cross-reactivity with antibodies to chicken and locust Vg receptors. Vitellogenin, Vn and its purified subunits competed for binding to the receptor in the order, Vg ≈ Vn > Vn large subunit > Vn small subunit. Binding of dephosphorylated Vg was significantly reduced. Deglycosylated Vg, on the other hand, formed high molecular weight aggregates resulting in artifactually high binding which indicates importance of glycosylation for the stability of Vg molecule. During egg maturation, the number of receptor binding sites in ovaries correlated with the rate of Vg uptake and peaked between 24–30 h after which it reduced to no binding by 48 h post blood meal.

Receptor binding of epidermal growth factor in cultured human choriocarcinoma cell lines: effects of actinomycin-D and methotrexate.

Binding of epidermal growth factor (EGF) to its receptor was evaluated in the four cultured choriocarcinoma cell lines BeWo, NaUCC-1, NaUCC-2, and NaUCC-3. Also, the effect of the anti-tumor drugs actinomycin-D (Act-D) and methotrexate (MTX) on the EGF receptor binding was investigated in these cell lines. Incubation of these cells with [125I]EGF at 37 degrees C resulted in a higher binding than that at 22 degrees C or at 4 degrees C. These bindings were saturable during 30- to 60-min incubation, and were specific and reversible. Scatchard analysis showed that the maximal number of receptor binding sites was 2.89 X 10(3)/cell in BeWo cells, 2.04 X 10(3)/cell in NaUCC-1 cells, 1.84 X 10(3)/cell in NaUCC-2 cells, and 1.01 X 10(3)/cell in NaUCC-3 cells. Preincubation with Act-D or MTX for 24 hr decreased the number of receptor binding sites (26%-53%) and slightly increased the receptor binding affinities. Combination of the two drugs resulted in a further diminution of EGF receptor binding sites in BeWo, NaUCC-1, and NaUCC-3 cells, respectively, but reversed the Act-D effect in NaUCC-2 cells. These results indicated that choriocarcinoma tissue is rich in EGF receptors, that the anti-tumor drugs Act-D and MTX diminish the receptor binding sites in the tissue, and suggest that MTX might induce a drug resistance to Act-D in some choriocarcinoma tissue.

A kinetic study of human IgG binding to placental Fcγ-receptor

Various methods for studying the IgG interaction with its placental receptor have been examined to establish kinetic parameters. IgG binding has an average rate constant k 1, of (2.57 ± 0.94) × 10 7/ M per min. However, high displacement doses (1000 μ/g) of unlabelled IgG resulted in a curvilinear dissociation curve with two binding sites: one with a fast dissociation rate constant, k 2 of 0.100/min and a slow one with k 2 of 0.010/min. IgG binding was associated with a high average affinity of (3.70 ± 1.65) × 10 8/ M and a total number of receptor binding sites of (2.07 ± 0.93) × 10 14 sites/mg of membrane protein, in close agreement with previous results. The stoichiometry of IgG to Fcγ-receptor was established as a 4:1 binding ratio from log dose-response curves as opposed to a 1:1 binding ratio from previous reports.