Miniaturization of fluorescence polarization receptor-binding assays using CyDye-labeled ligands

Fluorescence polarization (FP) is an established technique for the study of biological interactions and is frequently used in the high-throughput screening (HTS) of potential new drug targets. This work describes the miniaturization of FP receptor assays to 1536-well formats for use in HTS. The FP assays were initially developed in 384-well microplates using CyDye-labeled nonpeptide and peptide ligands. Receptor expression levels varied from approximately 1 to 10 pmols receptor per mg protein, and ligand concentrations were in the 0.5- to 1.0-nM range. The FP assays were successfully miniaturized to 1536-well formats using Cy3B-labeled ligands, significantly reducing reagent consumption, particularly the receptor source, without compromising assay reliability. Z' factor values determined for the FP receptor assays in both 384- and 1536-well formats were found to be > 0.5, indicating the assays to be robust, reliable, and suitable for HTS purposes.     

From molecules to behavior: new clues for RGS function in the striatum

RGS proteins act as negative regulators of G protein signaling, and there is growing evidence that the RGS family is important for regulating signaling in neurons. Two articles in this issue of Neuron (Martemyanov et al. and Rahman et al.) shed light on the function of one family member, RGS9-2, in behavioral responses to dopamine signaling in the striatum and on the relationship between its structure and its function.     

The nucleoid-associated protein StpA binds curved DNA, has a greater DNA-binding affinity than H-NS and is present in significant levels in hns mutants

The StpA protein is closely related to H-NS, the well-characterised global regulator of gene expression which is a major component of eubacterial chromatin. Despite sharing a very high degree of sequence identify and having biochemical properties in common with H-NS, the physiological function of StpA remains unknown. We show that StpA exhibits similar DNA-binding activities to H-NS. Although both display a strong preference for binding to curved DNA, StpA binds DNA with a four-fold higher affinity than H-NS, with K(d)s of 0.7 microM and 2.8 microM, respectively. It has previously been reported that expression of stpA is derepressed in an hns mutant. We have quantified the amount of StpA protein produced under this condition and find it to be only one-tenth the level of H-NS protein in wild-type cells. Our findings explain why the presence of StpA does not compensate for the lack of H-NS in an hns mutant, and why the characteristic pleiotropic hns mutant phenotype is observed.     

Genomewide search for type 2 diabetes susceptibility genes in four American populations

Type 2 diabetes is a serious, genetically influenced disease for which no fully effective treatments are available. Identification of biochemical or regulatory pathways involved in the disease syndrome could lead to innovative therapeutic interventions. One way to identify such pathways is the genetic analysis of families with multiple affected members where disease predisposing genes are likely to be segregating. We undertook a genomewide screen (389-395 microsatellite markers) in samples of 835 white, 591 Mexican American, 229 black, and 128 Japanese American individuals collected as part of the American Diabetes Association's GENNID study. Multipoint nonparametric linkage analyses were performed with diabetes, and diabetes or impaired glucose homeostasis (IH). Linkage to diabetes or IH was detected near markers D5S1404 (map position 77 cM, LOD = 2.80), D12S853 (map position 82 cM, LOD = 2.81) and GATA172D05 (X-chromosome map position 130 cM, LOD = 2.99) in whites, near marker D3S2432 (map position 51 cM, LOD = 3.91) in Mexican Americans, and near marker D10S1412 (map position 14 cM, LOD = 2.39) in African Americans mainly collected in phase 1 of the study. Further analyses showed evidence for interactions between the chromosome 5 locus and region on chromosome 12 containing the MODY 3 gene (map position 132 cM) and between the X-chromosome locus and region near D12S853 (map position 82 cM) in whites. Although these results were not replicated in samples collected in phase 2 of the GENNID study, the region on chromosome 12 was replicated in samples from whites described by Bektas et al. (1999).     

Identification of the Cu2+ binding sites in the N-terminal domain of the prion protein by EPR and CD spectroscopy

Recent evidence indicates that the prion protein (PrP) plays a role in copper metabolism in the central nervous system. The N-terminal region of human PrP contains four sequential copies of the highly conserved octarepeat sequence PHGGGWGQ spanning residues 60-91. This region selectively binds divalent copper ions (Cu(2+)) in vivo. To elucidate the specific mode and site of binding, we have studied a series of Cu(2+)-peptide complexes composed of 1-, 2-, and 4-octarepeats and several sub-octarepeat peptides, by electron paramagnetic resonance (EPR, conventional X-band and low-frequency S-band) and circular dichroism (CD) spectroscopy. At pH 7.45, two EPR active binding modes are observed where the dominant mode appears to involve coordination of three nitrogens and one oxygen to the copper ion, while in the minor mode two nitrogens and two oxygens coordinate. ESEEM spectra demonstrate that the histidine imidazole contributes one of these nitrogens. The truncated sequence HGGGW gives EPR and CD that are indistinguishable from the dominant binding mode observed for the multi-octarepeat sequences and may therefore comprise the fundamental Cu(2+) binding unit. Both EPR and CD titration experiments demonstrate rigorously a 1:1 Cu(2+)/octarepeat binding stoichiometry regardless of the number of octarepeats in a given peptide sequence. Detailed spin integration of the EPR signals demonstrates that all of the bound Cu(2+) is detected thereby ruling out strong exchange coupling that is often found when there is imidazolate bridging between paramagnetic metal centers. A model consistent with these data is proposed in which Cu(2+) is bound to the nitrogen of the histidine imidazole side chain and to two nitrogens from sequential glycine backbone amides.     

Sacral neural crest cells colonise aganglionic hindgut in vivo but fail to compensate for lack of enteric ganglia

The vagal neural crest is the origin of majority of neurons and glia that constitute the enteric nervous system, the intrinsic innervation of the gut. We have recently confirmed that a second region of the neuraxis, the sacral neural crest, also contributes to the enteric neuronal and glial populations of both the myenteric and the submucosal plexuses in the chick, caudal to the level of the umbilicus. Results from this previous study showed that sacral neural crest-derived precursors colonised the gut in significant numbers only 4 days after vagal-derived cells had completed their migration along the entire length of the gut. This observation suggested that in order to migrate into the hindgut and differentiate into enteric neurons and glia, sacral neural crest cells may require an interaction with vagal-derived cells or with factors or signalling molecules released by them or their progeny. This interdependence may also explain the inability of sacral neural crest cells to compensate for the lack of ganglia in the terminal hindgut of Hirschsprung's disease in humans or aganglionic megacolon in animals. To investigate the possible interrelationship between sacral and vagal-derived neural crest cells within the hindgut, we mapped the contribution of various vagal neural crest regions to the gut and then ablated appropriate sections of chick vagal neural crest to interrupt the migration of enteric nervous system precursor cells and thus create an aganglionic hindgut model in vivo. In these same ablated animals, the sacral level neural axis was removed and replaced with the equivalent tissue from quail embryos, thus enabling us to document, using cell-specific antibodies, the migration and differentiation of sacral crest-derived cells. Results showed that the vagal neural crest contributed precursors to the enteric nervous system in a regionalised manner. When quail-chick grafts of the neural tube adjacent to somites 1-2 were performed, neural crest cells were found in enteric ganglia throughout the preumbilical gut. These cells were most numerous in the esophagus, sparse in the preumbilical intestine, and absent in the postumbilical gut. When similar grafts adjacent to somites 3-5 or 3-6 were carried out, crest cells were found within enteric ganglia along the entire gut, from the proximal esophagus to the distal colon. Vagal neural crest grafts adjacent to somites 6-7 showed that crest cells from this region were distributed along a caudal-rostral gradient, being most numerous in the hindgut, less so in the intestine, and absent in the proximal foregut. In order to generate aneural hindgut in vivo, it was necessary to ablate the vagal neural crest adjacent to somites 3-6, prior to the 13-somite stage of development. When such ablations were performed, the hindgut, and in some cases also the cecal region, lacked enteric ganglionated plexuses. Sacral neural crest grafting in these vagal neural crest ablated chicks showed that sacral cells migrated along normal, previously described hindgut pathways and formed isolated ganglia containing neurons and glia at the levels of the presumptive myenteric and submucosal plexuses. Comparison between vagal neural crest-ablated and nonablated control animals demonstrated that sacral-derived cells migrated into the gut and differentiated into neurons in higher numbers in the ablated animals than in controls. However, the increase in numbers of sacral neural crest-derived neurons within the hindgut did not appear to be sufficiently high to compensate for the lack of vagal-derived enteric plexuses, as ganglia containing sacral neural crest-derived neurons and glia were small and infrequent. Our findings suggest that the neuronal fate of a relatively fixed subpopulation of sacral neural crest cells may be predetermined as these cells neither require the presence of vagal-derived enteric precursors in order to colonise the hindgut, nor are capable of dramatically altering their proliferation or d