The Prevalence of Pretransplant Frailty and Mental Distress in Hematopoietic Cell Transplantation and Association with Clinical Outcomes.

Frailty is a phenotype of decreased physiologic reserve associated with increased risk of toxicities and nonrelapse mortality (NRM) in hematopoietic cell transplant (HCT) recipients. The incidence, predictors, and adverse effects of pre-HCT frailty are not well known. We evaluated the association of pre-HCT frailty, defined using Fried's criteria, with age and baseline characteristics in patients ≥18 years undergoing autologous (auto) or allogeneic (allo) HCT for hematological malignancies. Assessments were performed as part of routine pre-HCT evaluations and then retrospectively analyzed. We additionally investigated the association of mental health distress indicators with frailty and the association between frailty and transplant outcomes including NRM and overall survival (OS) plus healthcare utilization. Patients undergoing HCT for hematological malignancies were analyzed (total n = 300; 162 auto, 138 allo). The overall prevalence of frailty was 18%, 21.7% among alloHCT, and 14.8% among autoHCT recipients, with similar distributions of frailty domains. Logistic regression analysis of the overall cohort revealed that older age was associated with an increased risk of frailty (odds ratio [OR] 1.37, 95% confidence interval [CI] [1.02-1.82]; P = 0.04). AlloHCT (OR 2.03, CI [1.07-3.84]; P = .03), and patient health questionnaire-9 (PHQ-9) (health depression) score ≥10 (OR 6.28, CI 1.93-20.43; P < .01) were each independently associated with pre-HCT frailty. In alloHCT patients, older age (OR 1.44, CI [1.00-2.06]; P = .05) was the only significant risk factor for pre-HCT frailty, while for autoHCT patients, only a higher PHQ-9 score was associated with frailty (OR 6.43, CI [1.34-30.82]; P = .02). For the whole cohort OS at 1 year was lower in frail recipients at 83% (95% CI, 70-91%) versus 92% (95% CI, 88-95%) in nonfrail (P = .04); with multivariate analysis showing higher risk of death in the frail group (hazard ratio [HR] 2.31, CI 0.97-5.46; P = .06). In the alloHCT cohort, multivariate analysis showed greater 1-year mortality in frail recipients (HR 2.55, CI [0.99-6.56]; P = .053). In the alloHCT recipients, we observed a 1-year NRM of 20% in frail patients versus 9% in nonfrail, and multivariate analysis showed a marginally higher risk of NRM in the frail group (HR 2.70, CI 0.90-8.10; P = .08). Frailty was not associated with higher risk of relapse in alloHCT or autoHCT recipients. Frail alloHCT patients experienced a longer initial hospital stay following HCT compared to nonfrail recipients (P < .01). We observed a high prevalence of pre-HCT frailty across all age groups, and identify older age is a risk factor for frailty, particularly in alloHCT recipients. Frailty is associated with a greater risk of NRM and lower survival which needs investigation in a larger cohort. Frailty associates with greater HCT complexity suggesting a need for early assessments and targeted interventions for this vulnerable population. Our findings suggest the utility of frailty and mental distress screening along with multidisciplinary interventions in pre-HCT to limit the morbidity of HCT.

Mammalian splicing factor SF1 interacts with SURP domains of U2 snRNP-associated proteins.

Splicing factor 1 (SF1) recognizes the branch point sequence (BPS) at the 3' splice site during the formation of early complex E, thereby pre-bulging the BPS adenosine, thought to facilitate subsequent base-pairing of the U2 snRNA with the BPS. The 65-kDa subunit of U2 snRNP auxiliary factor (U2AF65) interacts with SF1 and was shown to recruit the U2 snRNP to the spliceosome. Co-immunoprecipitation experiments of SF1-interacting proteins from HeLa cell extracts shown here are consistent with the presence of SF1 in early splicing complexes. Surprisingly almost all U2 snRNP proteins were found associated with SF1. Yeast two-hybrid screens identified two SURP domain-containing U2 snRNP proteins as partners of SF1. A short, evolutionarily conserved region of SF1 interacts with the SURP domains, stressing their role in protein-protein interactions. A reduction of A complex formation in SF1-depleted extracts could be rescued with recombinant SF1 containing the SURP-interaction domain, but only partial rescue was observed with SF1 lacking this sequence. Thus, SF1 can initially recruit the U2 snRNP to the spliceosome during E complex formation, whereas U2AF65 may stabilize the association of the U2 snRNP with the spliceosome at later times. In addition, these findings may have implications for alternative splicing decisions.

Structure, phosphorylation and U2AF65 binding of the N-terminal domain of splicing factor 1 during 3'-splice site recognition.

Recognition of the 3'-splice site is a key step in pre-mRNA splicing and accomplished by a dynamic complex comprising splicing factor 1 (SF1) and the U2 snRNP auxiliary factor 65-kDa subunit (U2AF65). Both proteins mediate protein-protein and protein-RNA interactions for cooperative RNA-binding during spliceosome assembly. Here, we report the solution structure of a novel helix-hairpin domain in the N-terminal region of SF1 (SF1(NTD)). The nuclear magnetic resonance- and small-angle X-ray scattering-derived structure of a complex of the SF1(NTD) with the C-terminal U2AF homology motif domain of U2AF65 (U2AF65(UHM)) reveals that, in addition to the known U2AF65(UHM)-SF1 interaction, the helix-hairpin domain forms a secondary, hydrophobic interface with U2AF65(UHM), which locks the orientation of the two subunits. Mutational analysis shows that the helix hairpin is essential for cooperative formation of the ternary SF1-U2AF65-RNA complex. We further show that tandem serine phosphorylation of a conserved Ser80-Pro81-Ser82-Pro83 motif rigidifies a long unstructured linker in the SF1 helix hairpin. Phosphorylation does not significantly alter the overall conformations of SF1, SF1-U2AF65 or the SF1-U2AF65-RNA complexes, but slightly enhances RNA binding. Our results indicate that the helix-hairpin domain of SF1 is required for cooperative 3'-splice site recognition presumably by stabilizing a unique quaternary arrangement of the SF1-U2AF65-RNA complex.

Diversity and coexistence of tick-borne pathogens in central Germany.

In total, 1000 Ixodes ricinus L. ticks were collected from a small recreational forest area in central Germany (Thuringia) and investigated for the presence of Borrelia spp., Babesia spp., Anaplasma spp., Rickettsia spp., Coxiella burnetii, and Francisella tularensis. Overall, 43.6% of the ticks were infected with at least one pathogen. In 8.4% of ticks double infections were detected, and 1.6% harbored more than two pathogens. In this study, we present data on the coexistence of established and emerging pathogens in questing nymphs and adult ticks in a recreational area in central Germany, indicating the need for further studies for a reliable risk assessment.

The metabotropic glutamate receptor 7 allosteric modulator AMN082: a monoaminergic agent in disguise?

Metabotropic glutamate receptor 7 (mGluR7) remains the most elusive of the eight known mGluRs primarily because of the limited availability of tool compounds to interrogate its potential therapeutic utility. The discovery of N,N'-dibenzhydrylethane-1,2-diamine dihydrochloride (AMN082) as the first orally active, brain-penetrable, mGluR7-selective allosteric agonist by Mitsukawa and colleagues (Proc Natl Acad Sci USA 102:18712-18717, 2005) provides a means to investigate this receptor system directly. AMN082 demonstrates mGluR7 agonist activity in vitro and interestingly has a behavioral profile that supports utility across a broad spectrum of psychiatric disorders including anxiety and depression. The present studies were conducted to extend the in vitro and in vivo characterization of AMN082 by evaluating its pharmacokinetic and metabolite profile. Profiling of AMN082 in rat liver microsomes revealed rapid metabolism (t(1/2) < 1 min) to a major metabolite, N-benzhydrylethane-1,2-diamine (Met-1). In vitro selectivity profiling of Met-1 demonstrated physiologically relevant transporter binding affinity at serotonin transporter (SERT), dopamine transporter (DAT), and norepinephrine transporter (NET) (323, 3020, and 3410 nM, respectively); whereas the parent compound AMN082 had appreciable affinity at NET (1385 nM). AMN082 produced antidepressant-like activity and receptor occupancy at SERT up to 4 h postdose, a time point at which AMN082 is significantly reduced in brain and plasma while the concentration of Met-1 continues to increase in brain. Acute Met-1 administration produced antidepressant-like activity as would be expected from its in vitro profile as a mixed SERT, NET, DAT inhibitor. Taken together, these data suggest that the reported in vivo actions of AMN082 should be interpreted with caution, because they may involve other mechanisms in addition to mGluR7.

Interaction domains and nuclear targeting signals in subunits of the U2 small nuclear ribonucleoprotein particle-associated splicing factor SF3a.

Human splicing factor SF3a is a component of the mature U2 small nuclear ribonucleoprotein particle (snRNP) and its three subunits of 60, 66, and 120 kDa are essential for splicing in vitro and in vivo. The SF3a heterotrimer forms in the cytoplasm and enters the nucleus independently of the U2 snRNP. Here, we have analyzed domains required for in vitro interactions between the SF3a subunits. Our results indicate that the SF3a66-SF3a120 interaction is mediated by a 27-amino acid region in SF3a120 C-terminal to the second suppressor-of-white-apricot and prp21/spp91 domain and amino acids 108-210 of SF3a66. Neither of these sequences contains known structural motifs, suggesting that the interaction domains are novel. Moreover, an ∼100-amino acid region, including the SURP2 domain of SF3a120 but extending into neighboring regions, is sufficient for binding to SF3a60. Analysis of determinants for nuclear import of SF3a demonstrates that SF3a120 provides the major nuclear localization signal and SF3a60 contributes to nuclear import.

Analysis of in situ pre-mRNA targets of human splicing factor SF1 reveals a function in alternative splicing.

The conserved pre-mRNA splicing factor SF1 is implicated in 3' splice site recognition by binding directly to the intron branch site. However, because SF1 is not essential for constitutive splicing, its role in pre-mRNA processing has remained mysterious. Here, we used crosslinking and immunoprecipitation (CLIP) to analyze short RNAs directly bound by human SF1 in vivo. SF1 bound mainly pre-mRNAs, with 77% of target sites in introns. Binding to target RNAs in vitro was dependent on the newly defined SF1 binding motif ACUNAC, strongly resembling human branch sites. Surprisingly, the majority of SF1 binding sites did not map to the expected position near 3' splice sites. Instead, target sites were distributed throughout introns, and a smaller but significant fraction occurred in exons within coding and untranslated regions. These data suggest a more complex role for SF1 in splicing regulation. Indeed, SF1 silencing affected alternative splicing of endogenous transcripts, establishing a previously unexpected role for SF1 and branch site-like sequences in splice site selection.

Novel alpha-7 nicotinic acetylcholine receptor agonists containing a urea moiety: identification and characterization of the potent, selective, and orally efficacious agonist 1-[6-(4-fluorophenyl)pyridin-3-yl]-3-(4-piperidin-1-ylbutyl) urea (SEN34625/WYE-103914).

Alpha-7 nicotinic acetylcholine receptor (alpha7 nAChR) agonists are promising therapeutic candidates for the treatment of cognitive impairment. We report a series of novel, potent small molecule agonists (4-18) of the alpha7 nAChR deriving from our continuing efforts in the areas of Alzheimer's disease and schizophrenia. One of the compounds of the series containing a urea moiety (16) was further shown to be a selective agonist of the alpha7 nAChR with excellent in vitro and in vivo profiles, brain penetration, and oral bioavailability and demonstrated in vivo efficacy in multiple behavioral cognition models. Structural modifications leading to the improved selectivity profile and the biological evaluation of this series of compounds are discussed.

5-Cyclic amine-3-arylsulfonylindazoles as novel 5-HT6 receptor antagonists.

Novel 5-cyclic amine-3-arylsulfonylindazoles were prepared, and several analogues within this class have been identified as high-affinity 5-HT(6) receptor ligands with improved pharmacokinetic and pharmacological properties. One selected example, 18b, showed good brain penetrability and a generally favorable pharmacokinetic profile with procognitive efficacy in the rat novel object recognition assay. The synthesis and structure-activity relationship of this potent class are discussed.

Detection of Rickettsia spp. and Anaplasma phagocytophilum in Ixodes ricinus ticks in a region of Middle Germany (Thuringia).

Rickettsia spp. and Anaplasma spp. are regarded as potentially emerging tick-borne pathogens, but so far data on prevalence rates in questing ticks and reports on human diseases in several parts of Europe are rarely available. In this study, 430 nymphs and 570 adult Ixodes (I.) ricinus ticks were collected from a frequently visited forest region of Thuringia (Zeitzgrund, near Stadtroda) in 2006 (n=506) and 2007 (n=494). Individual ticks were investigated for a part of the citrate synthase gene (gltA) of Rickettsia spp. and the 16S rRNA gene of Anaplasma phagocytophilum. Positive amplicons were identified with restriction fragment length polymorphism (RFLP) and/or sequencing. Overall, 14.7% (147/1000) of investigated ticks were infected with Rickettsia spp. After sequencing of 64/147 positive amplicons R. helvetica (29/64) was detected predominantly. Prevalence varied in different developmental stages between 9.3% (40/430) in nymphs and 18.8% (107/570) in adults. A. phagocytophilum-specific DNA was detected in 5.4% (54/1000) of ticks with an infection rate of 4.7% (20/430) in nymphs and 6.0% (34/570) in adults. In 1% (10/1000) of ticks coinfections with Rickettsia spp. and A. phagocytophilum were found. Our study provides interesting insights into the circulation and cocirculation of different rickettsial species and A. phagocytophilum in the same biotope.

Pharmacological comparison of muscarinic ligands: historical versus more recent muscarinic M1-preferring receptor agonists.

In functional assay assessments using the five muscarinic receptor subtypes, a second generation of muscarinic M(1)-preferring receptor agonists [AC-42 (1), AC-260584 (2), 77-LH-28-1 (3) and LY-593039 (4)] was shown to have higher selectivity for muscarinic M(1) over M(3) receptor as compared to historical agonists [talsaclidine (8), sabcomeline (10), xanomeline (11), WAY-132983 (12), cevimeline (9) and NGX-267 (6)]. Another striking difference of these more recent compounds is their affinities for the dopamine D(2) and 5-HT(2B) receptors. Taken together, these results suggest that the newer compounds may have a greater clinical safety profile, especially with regard to muscarinic M(3) receptor-mediated events, than the historical agonists, but their affinities for other receptors may still compromise their use to validate the therapeutic potential of muscarinic M(1) receptor agonists.

Old and new pharmacology: positive allosteric modulation of the alpha7 nicotinic acetylcholine receptor by the 5-hydroxytryptamine(2B/C) receptor antagonist SB-206553 (3,5-dihydro-5-methyl-N-3-pyridinylbenzo[1,2-b:4,5-b']di pyrrole-1(2H)-carboxamide).

The alpha7 nicotinic acetylcholine receptor (nAChR) has been implicated in Alzheimer's disease and schizophrenia, leading to efforts targeted toward discovering agonists and positive allosteric modulators (PAMs) of this receptor. In a Ca2+ flux fluorometric imaging plate reader assay, SB-206553 (3,5-dihydro-5-methyl -N-3-pyridinylbenzo [1, 2-b:4,5 -b']-di pyrrole-1(2H)-carboxamide), a compound known as a 5-hydroxytryptamine(2B/2C) receptor antagonist, produced an 8-fold potentiation of the evoked calcium signal in the presence of an EC(20) concentration of nicotine and a corresponding EC(50) of 1.5 muM for potentiation of EC(20) nicotine responses in GH4C1 cells expressing the alpha7 receptor. SB-206553 was devoid of direct alpha7 receptor agonist activity and selective against other nicotinic receptors. Confirmation of the PAM activity of SB-206553 on the alpha7 nAChR was obtained in patch-clamp electrophysiological experiments in GH4C1 cells, where it failed to evoke any detectable currents when applied alone, yet dramatically potentiated the currents evoked by an EC(20) (17 microM) and EC(100) (124 microM) of acetylcholine (ACh). Native nicotinic receptors in CA1 stratum radiatum interneurons of rat hippocampal slices could also be activated by ACh (200 microM), an effect that was entirely blocked by the alpha7-selective antagonist methyllycaconitine (MLA). These ACh currents were potentiated by SB-206553, which increased the area of the current response significantly, resulting in a 40-fold enhancement at 100 microM. In behavioral experiments in rats, SB-206553 reversed an MK-801 (dizocilpine maleate)-induced deficit in the prepulse inhibition of acoustic startle response, an effect attenuated in the presence of MLA. This latter observation provides further evidence in support of the potential therapeutic utility of alpha7 nAChR PAMs in schizophrenia.

Functional properties of alpha7 nicotinic acetylcholine receptors co-expressed with RIC-3 in a stable recombinant CHO-K1 cell line.

Heterologous functional expression of alpha7 nicotinic acetylcholine receptors (nAChRs) is difficult to achieve in mammalian cell lines, and the reasons have been associated with a lack of expression of the putative chaperone factor RIC-3. Here, we describe the generation and functional and pharmacological characterization of a Chinese hamster ovary (CHO)-K1 cell line co-expressing the human alpha7 nAChR and RIC-3. Stable recombinant cells expressing alpha7 nAChR on the plasma membrane were selected by binding of fluorochrome-conjugated alpha-bungarotoxin and fluorescence-activated cell sorting. The presence of functional alpha7 channels was demonstrated by whole cell patch clamp recordings. Nicotine and acetylcholine induced rapid desensitizing currents with 50% effective concentration values of 14 and 37 microM, respectively, with agonist-evoked currents detected in approximately 75% of the cell population. Surprisingly, when tested in a FLIPR (Molecular Devices, Sunnyvale, CA) Ca(2+) assay, activation of alpha7 nAChRs was measured only when nicotinic agonists were applied either in the presence of the positive allosteric modulator (PAM) PNU-120596 or after pretreatment of cells with the tyrosine kinase inhibitor genistein. No Ca(2+) influx was measured upon addition of agonists alone or together with allosteric potentiators such as 5-hydroxyindole that predominantly increase the apparent peak amplitude without robustly affecting the current desensitization rate, as exemplified by PNU-120596. These results show that functional alpha7 nAChRs can stably be expressed in the non-neuronal CHO-K1 cell line. This recombinant cell system is useful for characterization of alpha7 nAChRs and to study the mechanism of action of chemical modulators, in particular the detection of PAMs capable of slowing receptor desensitization kinetics.

Characterization of Gpr101 expression and G-protein coupling selectivity.

This report describes the identification and characterization of the murine orphan GPCR, Gpr101. Both human and murine genes were localized to chromosome X. Similar to its human ortholog, murine Gpr101 mRNA was detected predominantly in the brain within discrete nuclei. A knowledge-restricted hidden Markov model-based algorithm, capable of accurately predicting G-protein coupling selectivity, indicated that both human and murine GPR101 were likely coupled to Gs. This prediction was supported by the elevation of cyclic AMP levels and the activation of a cyclic AMP response element-luciferase reporter gene in HEK293 cells over-expressing human GPR101. Consistent with this, over-expression of human GPR101 in a yeast-based system yielded an elevated, agonist-independent reporter gene response in the presence of a yeast chimeric Galphas protein. These results indicate that GPR101 participates in a potentially wide range of activities in the CNS via modulation of cAMP levels.

Validation of DRG-like F11 cells for evaluation of KCNQ/M-channel modulators.

F11 cells are derived from a fusion between mouse embryonic neuroblastoma and rat dorsal root ganglion (DRG) neurons. These cells have been shown to retain many features of native DRG neurons, including synthesis of neurotransmitters, expression of neuropeptide receptors, and voltage-gated calcium channels. In this study, we describe the presence of KCNQ2/3 channels in F11 cells as determined by both reverse transcription-polymerase chain reaction and functional assessment. Electrophysiological recordings in whole-cell configuration performed in F11 cells revealed the functional expression of a KCNQ/M-current with characteristic slow deactivation kinetics, similar to the KCNQ/M-current recorded from dissociated DRG neurons. Deactivation tail currents elicited by conventional M-current protocols were enhanced by a specific KCNQ/M-channel opener, WAY-1, and inhibited by the specific blocker XE991 [10,10-bis(4-pyridinylmethyl)-9(10H)- anthracenone]. Using a non-radioactive atomic absorption Rb+ efflux assay, we further validated that Rb+ efflux can be induced in differentiated F11 cells by activation of KCNQ/M-channels. These findings have led us to conclude that F11 cells can be used as a DRG cell model to evaluate effects of KCNQ/M-channel modulators.

Human splicing factor SF3a, but not SF1, is essential for pre-mRNA splicing in vivo.

The three subunits of human splicing factor SF3a are essential for the formation of the functional 17S U2 snRNP and prespliceosome assembly in vitro. RNAi-mediated depletion indicates that each subunit is essential for viability of human cells. Knockdown of single subunits results in a general block in splicing strongly suggesting that SF3a is a constitutive splicing factor in vivo. In contrast, splicing of several endogenous and reporter pre-mRNAs is not affected after knockdown of SF1, which functions at the onset of spliceosome assembly in vitro and is essential for cell viability. Thus, SF1 may only be required for the splicing of a subset of pre-mRNAs. We also observe a reorganization of U2 snRNP components in SF3a-depleted cells, where U2 snRNA and U2-B'' are significantly reduced in nuclear speckles and the nucleoplasm, but still present in Cajal bodies. Together with the observation that the 17S U2 snRNP cannot be detected in extracts from SF3a-depleted cells, our results provide further evidence for a function of Cajal bodies in U2 snRNP biogenesis.

Branch site haplotypes that control alternative splicing.

We show that the allele-dependent expression of transcripts encoding soluble HLA-DQbeta chains is determined by branchpoint sequence (BPS) haplotypes in DQB1 intron 3. BPS RNAs associated with low inclusion of the transmembrane exon in mature transcripts showed impaired binding to splicing factor 1 (SF1), indicating that alternative splicing of DQB1 is controlled by differential BPS recognition early during spliceosome assembly. We also demonstrate that naturally occurring human BPS point mutations that alter splicing and lead to recognizable phenotypes cluster in BP and in position -2 relative to BP, implicating impaired SF1-BPS interactions in disease-associated BPS substitutions. Coding DNA variants produced smaller fluctuations of exon inclusion levels than random exonic substitutions, consistent with a selection against coding mutations that alter their own exonization. Finally, proximal splicing in this multi-allelic reporter system was promoted by at least seven SR proteins and repressed by hnRNPs F, H and I, supporting an extensive antagonism of factors balancing the splice site selection. These results provide the molecular basis for the haplotype-specific expression of soluble DQbeta, improve prediction of intronic point mutations and indicate how extraordinary, selection-driven DNA variability in HLA affects pre-mRNA splicing.

A role for Cajal bodies in the final steps of U2 snRNP biogenesis.

The biogenesis of Sm-type small nuclear ribonucleoproteins (snRNPs) involves the export of newly transcribed small nuclear RNAs (snRNAs) to the cytoplasm, assembly with seven common proteins and modification at the 5' and 3' termini. Binding of snRNP-specific proteins and snRNA modification complete the maturation process. This is thought to occur after reimport of the core snRNPs into the nucleus. The heterotrimeric splicing factor SF3a converts a pre-mature 15S U2 snRNP into the functional 17S particle. To analyze cellular aspects of this process, we studied domains in SF3a60 and SF3a66 that are required for their localization to nuclear speckles. Regions in SF3a60 and SF3a66 that mediate the binding to SF3a120 are necessary for nuclear import of the proteins, suggesting that the SF3a heterotrimer forms in the cytoplasm. SF3a60 and SF3a66 deleted for zinc finger domains required for the incorporation of SF3a into the U2 snRNP are nuclear, indicating that the 17S U2 snRNP is assembled in the nucleus. However, these proteins show an aberrant nuclear distribution. Endogenous SF3a subunits colocalize with U2 snRNP in nuclear speckles, but cannot be detected in Cajal bodies, unlike core U2 snRNP components. By contrast, SF3a60 and SF3a66 lacking the zinc finger domains accumulate in Cajal bodies and are diffusely distributed in the cytoplasm, suggesting a function for Cajal bodies in the final maturation of the U2 snRNP.

Evaluation of FLIPR Calcium 3 Assay Kit--a new no-wash fluorescence calcium indicator reagent.

We have evaluated the FLIPR Calcium 3 Assay Kit (Calcium 3), a new no-wash fluorescence calcium indicator dye reagent, for the measurement of agonist-stimulated calcium signaling in cells expressing the serotonin 2C (5-HT(2C)), metabotropic glutamate receptor 5 (mGluR5) and the vasopressin 2 (V2) G-protein-coupled receptors. Calcium 3 yielded equivalent (5-HT(2C)) or superior (mGluR5 and V2) sensitivity to FLUO-4 as indexed by the change in fluorescence counts following agonist application. Assay variability, indexed by CV, using Calcium 3 or FLUO-4 was equivalent with 5-HT(2C) receptor responses although CVs were reduced using Calcium 3 in the examples of the mGluR5 and V2 receptors. Receptor pharmacologies based on agonist EC(50) values were identical when either Calcium 3 or FLUO-4 were utilized. Our results validate Calcium 3 as a compelling alternative to FLUO-4 in the choice of fluorescent dye reagent for studying G-protein-coupled receptors, providing the advantage of a homogenous, no-wash assay format.