P2X4 receptors interact with both P2X2 and P2X7 receptors in the form of homotrimers.

BACKGROUND AND PURPOSE: The P2X receptor family consists of seven subunit types - P2X1-P2X7. All but P2X6 are able to assemble as homotrimers. In addition, various subunit permutations have been reported to form heterotrimers. Evidence for heterotrimer formation includes co-localization, co-immunoprecipitation and the generation of receptors with novel functional properties; however, direct structural evidence for heteromer formation, such as chemical cross-linking and single-molecule imaging, is available in only a few cases. Here we examined the nature of the interaction between two pairs of subunits - P2X2 and P2X4, and P2X4 and P2X7. EXPERIMENTAL APPROACH: We used several experimental approaches, including in situ proximity ligation, co-immunoprecipitation, co-isolation on affinity beads, chemical cross-linking and atomic force microscopy (AFM) imaging. KEY RESULTS: Both pairs of subunits co-localize upon co-transfection, interact intimately within cells, and can be co-immunoprecipitated and co-isolated from cell extracts. Despite this, chemical cross-linking failed to show evidence for heteromer formation. AFM imaging of isolated receptors showed that all three subunits had the propensity to form receptor dimers. This self-association is likely to account for the observed close interaction between the subunit pairs, in the absence of true heteromer formation. CONCLUSIONS AND IMPLICATIONS: We conclude that both pairs of receptors interact in the form of distinct homomers. We urge caution in the interpretation of biochemical evidence indicating heteromer formation in other cases.

High-throughput mapping of protein occupancy identifies functional elements without the restriction of a candidate factor approach.

There are a variety of in vivo and in vitro methods to determine the genome-wide specificity of a particular trans-acting factor. However there is an inherent limitation to these candidate approaches. Most biological studies focus on the regulation of particular genes, which are bound by numerous unknown trans-acting factors. Therefore, most biological inquiries would be better addressed by a method that maps all trans-acting factors that bind particular regions rather than identifying all regions bound by a particular trans-acting factor. Here, we present a high-throughput binding assay that returns thousands of unbiased measurements of complex formation on nucleic acid. We applied this method to identify transcriptional complexes that form on DNA regions upstream of genes involved in pluripotency in embryonic stem cells (ES cells) before and after differentiation. The raw binding scores, motif analysis and expression data are used to computationally reconstruct remodeling events returning the identity of the transcription factor(s) most likely to comprise the complex. The most significant remodeling event during ES cell differentiation occurred upstream of the REST gene, a transcriptional repressor that blocks neurogenesis. We also demonstrate how this method can be used to discover RNA elements and discuss applications of screening polymorphisms for allelic differences in binding.

Relationship of growth and serum growth hormone concentration in the prepubertal labrador bitch.

Body weights of purebred Labrador Retriever bitches were recorded from birth through 40 weeks of age. Blood samples were collected twice weekly between 6 and 26 weeks of age. Samples were analyzed for serum growth hormone concentrations using a validated growth hormone radioimmunoassay. From a mean birth weight of 0.46 kg, the animals reached their mean adult body weight (24.8 kg) by 40 weeks of age. Growth rate showed a sigmoid response curve with age. Growth rate was linear between 4 and 26 weeks having a correlation coefficient of 0.96, a slope of 0.98, and an intercept of -2.28. Concentrations of growth hormone during the same period exhibited a bimodal distribution. From 8.3 +/- 0.6 ng/ml at 7 weeks, mean growth hormone concentration steadily declined reaching 1.8 +/- 0.4 ng/ml at 12 weeks. There was a secondary increase to 9.7 +/- 0.6 ng/ml at 20 weeks and a subsequent decline to 6.4 +/- 0.9 ng/ml at 26 weeks. The mean percent growth increment (defined as percent weight increase per week over previous body weight) was calculated to study actual growth. The growth increment was 34% at 6 weeks, 14.8% at 12 weeks, 15.1% at 16 weeks, and 3.5% at 26 weeks of age. Once hourly blood samples for 6 hours were collected at 10 and 24 weeks of age from six animals to determine the episodic release pattern of growth hormone and evaluate possible stress from venipuncture. Growth hormone concentrations ranged from 2.0 to 23.0 ng/ml in individual animals, with at least one episodic release of growth hormone detected within this time. Venipuncture did not stimulate any rise in growth hormone.

Influence of inbreeding on reproductive performance, ejaculate quality and testicular volume in the dog.

An established foxhound colony employing an outbreeding and inbreeding program was evaluated to assess the influence of increased homozygosity on overall reproductive performance and ejaculate quality. A total of 14 outbred and four inbred male dogs were naturally bred to 544 outbred and 51 inbred bitches, respectively. Mean conception rate and total number of puppies whelped and number born alive were greater (P<0.01) in outbred compared to inbred lines. Duration of gestation and number of puppies surviving from birth to weaning were not different between outbred and inbred groups. Ejaculate quality of studs also appeared affected by genotype with outbred males producing a greater (P<0.025) average sperm count/ejaculate and sperm count/ml of ejaculate. Ejaculate volume, % motility of spermatozoa and testes volume, although not significantly different, also favored the outbred compared to inbred sires. These results: 1) indicate that a breeding program utilizing dogs with inbreeding coefficients ranging from .125-.558 can experience reduced reproductive performance; 2) suggest that a portion of the infertility associated with conception may be attributed to the male, since inbred studs produced lower quality ejaculates compared to outbred counterparts.