Stage-specific formation of the equine blastocyst capsule is instrumental to hatching and to embryonic survival in vivo

Early embryonic development in the horse is characterized by the formation of an unusual acellular glycoprotein "capsule" between the trophectoderm and the overlying zona pellucida. This structure is first detected between days 6 and 7 after ovulation and completely envelops the spherical conceptus until as late as day 23 of gestation. In the present study, a micromanipulator was used to remove the capsule from 15 embryos on day 6-7 after ovulation. None of these denuded embryos developed into ultrasonographically detectable pregnancies after surgical transfer into recipient mares whereas four of six control embryos handled and transferred similarly but without capsule removal developed normally, thereby demonstrating clearly a role for the capsule in embryonic survival. In addition, observation of the embryonic investments after embryo collection and during micromanipulation led to the hypothesis that hatching of the horse embryo from its zona pellucida is assisted by the force of the expanding capsule, which causes the attenuating zona to literally burst open.     

Sodium channel beta1 subunit-mediated modulation of Nav1.2 currents and cell surface density is dependent on interactions with contactin and ankyrin

Voltage-gated sodium channels are composed of a pore-forming alpha subunit and at least one auxiliary beta subunit. Both beta1 and beta2 are cell adhesion molecules that interact homophilically, resulting in ankyrin recruitment. In contrast, beta1, but not beta2, interacts heterophilically with contactin, resulting in increased levels of cell surface sodium channels. We took advantage of these results to investigate the molecular basis of beta1-mediated enhancement of sodium channel cell surface density, including elucidating structure-function relationships for beta1 association with contactin, ankyrin, and Nav1.2. beta1/beta2 subunit chimeras were used to assign putative sites of contactin interaction to two regions of the beta1 Ig loop. Recent studies have shown that glutathione S-transferase fusion proteins containing portions of Nav1.2 intracellular domains interact directly with ankyrinG. We show that native Nav1.2 associates with ankyrinG in cells in the absence of beta subunits and that this interaction is enhanced in the presence of beta1 but not beta1Y181E, a mutant that does not interact with ankyrinG. beta1Y181E does not modulate Nav1.2 channel function despite efficient association with Nav1.2 and contactin. beta1Y181E increases Nav1.2 cell surface expression, but not as efficiently as wild type beta1. beta1/beta2 chimeras exchanging various regions of the beta1 Ig loop were all ineffective in increasing Nav1.2 cell surface density. Our results demonstrate that full-length beta1 is required for channel modulation and enhancement of sodium channel cell surface expression.     

TLRs mediate IFN-gamma production by human uterine NK cells in endometrium

The human endometrium (EM) contains macrophages, NK cells, T cells, B cells, and neutrophils in contact with a variety of stromal and epithelial cells. The interplay between these different cell types and their roles in defense against pathogen invasion in this specialized tissue are important for controlling infection and reproduction. TLRs are a family of receptors able to recognize conserved pathogen-associated molecular patterns. In this study, we determined the expression of TLRs on uterine NK (uNK) cells from the human EM and the extent to which uNK cells responded to TLR agonist stimulation. uNK cells expressed TLRs 2, 3, and 4, and produced IFN-gamma when total human endometrial cells were stimulated with agonists to TLR2 or TLR3 (peptidoglycan or poly(I:C), respectively). Activated uNK cell clones produced IFN-gamma upon stimulation with peptidoglycan or poly(I:C). However, purified uNK cells did not respond directly to TLR agonists, but IFN-gamma was produced by uNK cells in response to TLR stimulation when cocultured with APCs. These data indicate that uNK cells express TLRs and that they can respond to TLR agonists within EM by producing IFN-gamma. These data also indicate that the uNK cells do not respond directly to TLR stimulation, but rather their production of IFN-gamma is dependent upon interactions with other cells within EM.     

SimArray: a user-friendly and user-configurable microarray design tool

Background  

Microarrays were first developed to assess gene expression but are now also used to map protein-binding sites and to assess allelic variation between individuals. Regardless of the intended application, efficient production and appropriate array design are key determinants of experimental success. Inefficient production can make larger-scale studies prohibitively expensive, whereas poor array design makes normalisation and data analysis problematic.     

The same ELA class II risk factors confer equine insect bite hypersensitivity in two distinct populations

Insect bite hypersensitivity (IBH) is a chronic allergic dermatitis common in horses. Affected horses mainly react against antigens present in the saliva from the biting midges, Culicoides ssp, and occasionally black flies, Simulium ssp. Because of this insect dependency, the disease is clearly seasonal and prevalence varies between geographical locations. For two distinct horse breeds, we genotyped four microsatellite markers positioned within the MHC class II region and sequenced the highly polymorphic exons two from DRA and DRB3, respectively. Initially, 94 IBH-affected and 93 unaffected Swedish born Icelandic horses were tested for genetic association. These horses had previously been genotyped on the Illumina Equine SNP50 BeadChip, which made it possible to ensure that our study did not suffer from the effects of stratification. The second population consisted of 106 unaffected and 80 IBH-affected Exmoor ponies. We show that variants in the MHC class II region are associated with disease susceptibility (p (raw)?=?2.34?×?10(-5)), with the same allele (COR112:274) associated in two separate populations. In addition, we combined microsatellite and sequencing data in order to investigate the pattern of homozygosity and show that homozygosity across the entire MHC class II region is associated with a higher risk of developing IBH (p?=?0.0013). To our knowledge this is the first time in any atopic dermatitis suffering species, including man, where the same risk allele has been identified in two distinct populations.     

A set of polymorphic nuclear intron markers for conservation genetics and phylogeography of <Emphasis Type="Italic">Euphorbia</Emphasis> species (<Emphasis Type="Italic">Pedilanthus</Emphasis> clade)

We developed seven nuclear intron markers for Euphorbia lomelii. New exon-primed intron-crossing (EPIC) oligonucleotides were used for initial amplification and sequencing, then locus-specific primers and restriction-fragment-length polymorphism genotyping assays were designed. Loci showed no significant deviation from Hardy–Weinberg and linkage equilibrium, and they cross-amplify in at least three congeneric species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Metabolic and morphological differences between rapidly proliferating cancerous and normal breast epithelial cells

The metabolic and morphological characteristics of two human epithelial breast cell populations--MCF7 cells, a cancerous cell line, and 48R human mammary epithelial cells (48R HMECs), a noncancerous, finite lifespan cell strain--were compared at identical growth rates. Both cell types were induced to grow rapidly in nutrient-rich media containing 13C-labeled glucose, and the isotopic enrichment of cellular metabolites was quantified to calculate metabolic fluxes in key pathways. Despite their similar growth rates, the cells exhibited distinctly different metabolic and morphological profiles. MCF7 cells have an 80% smaller exposed surface area and contain 26% less protein per cell than the 48R cells. Surprisingly, rapidly proliferating 48R cells exhibited a 225% higher per-cell glucose consumption rate, a 250% higher per-cell lactate production rate, and a nearly identical per-cell glutamine consumption rate relative to the cancer cell line. However, when fluxes were considered on the basis of exposed area, the cancer cells were observed to have higher glucose, lactate, and glutamine fluxes, demonstrating superior transport capabilities per unit area of cell membrane. MCF7 cells also consumed amino acids at rates much higher than are generally required for protein synthesis, whereas 48R cells generally did not. Pentose phosphate pathway activity was higher in MCF7 cells, and the flux of glutamine to glutamate was less reversible. Energy efficiency was significantly higher in MCF7 cells, as a result of a combination of their smaller size and greater reliance on the TCA cycle than the 48R cells. These observations support evolutionary models of cancer cell metabolism and suggest targets for metabolic drugs in metastatic breast cancers.