The NADPH oxidase 2 subunit p47phox binds to the WAVE regulatory complex and p22phox in a mutually exclusive manner.

The actin cytoskeleton and reactive oxygen species (ROS) both play crucial roles in various cellular processes. Previous research indicated a direct interaction between two key components of these systems: the WAVE1 subunit of the WAVE regulatory complex (WRC), which promotes actin polymerization and the p47phox subunit of the NADPH oxidase 2 complex (NOX2), which produces ROS. Here, using carefully characterized recombinant proteins, we find that activated p47phox uses its dual Src homology 3 domains to bind to multiple regions within the WAVE1 and Abi2 subunits of the WRC, without altering WRC's activity in promoting Arp2/3-mediated actin polymerization. Notably, contrary to previous findings, p47phox uses the same binding pocket to interact with both the WRC and the p22phox subunit of NOX2, albeit in a mutually exclusive manner. This observation suggests that when activated, p47phox may separately participate in two distinct processes: assembling into NOX2 to promote ROS production and engaging with WRC to regulate the actin cytoskeleton.

Identification of New Markers of Angiogenic Sprouting Using Transcriptomics: New Role for RND3.

BACKGROUND: New blood vessel formation requires endothelial cells to transition from a quiescent to an invasive phenotype. Transcriptional changes are vital for this switch, but a comprehensive genome-wide approach focused exclusively on endothelial cell sprout initiation has not been reported. METHODS: Using a model of human endothelial cell sprout initiation, we developed a protocol to physically separate cells that initiate the process of new blood vessel formation (invading cells) from noninvading cells. We used this model to perform multiple transcriptomics analyses from independent donors to monitor endothelial gene expression changes. RESULTS: Single-cell population analyses, single-cell cluster analyses, and bulk RNA sequencing revealed common transcriptomic changes associated with invading cells. We also found that collagenase digestion used to isolate single cells upregulated the Fos proto-oncogene transcription factor. Exclusion of Fos proto-oncogene expressing cells revealed a gene signature consistent with activation of signal transduction, morphogenesis, and immune responses. Many of the genes were previously shown to regulate angiogenesis and included multiple tip cell markers. Upregulation of SNAI1 (snail family transcriptional repressor 1), PTGS2 (prostaglandin synthase 2), and JUNB (JunB proto-oncogene) protein expression was confirmed in invading cells, and silencing JunB and SNAI1 significantly reduced invasion responses. Separate studies investigated rounding 3, also known as RhoE, which has not yet been implicated in angiogenesis. Silencing rounding 3 reduced endothelial invasion distance as well as filopodia length, fitting with a pathfinding role for rounding 3 via regulation of filopodial extensions. Analysis of in vivo retinal angiogenesis in Rnd3 heterozygous mice confirmed a decrease in filopodial length compared with wild-type littermates. CONCLUSIONS: Validation of multiple genes, including rounding 3, revealed a functional role for this gene signature early in the angiogenic process. This study expands the list of genes associated with the acquisition of a tip cell phenotype during endothelial cell sprout initiation.

Annexin A2 modulates phospholipid membrane composition upstream of Arp2 to control angiogenic sprout initiation.

The intersection of protein and lipid biology is of growing importance for understanding how cells address structural challenges during adhesion and migration. While protein complexes engaged with the cytoskeleton play a vital role, support from the phospholipid membrane is crucial for directing localization and assembly of key protein complexes. During angiogenesis, dramatic cellular remodeling is necessary for endothelial cells to shift from a stable monolayer to invasive structures. However, the molecular dynamics between lipids and proteins during endothelial invasion are not defined. Here, we utilized cell culture, immunofluorescence, and lipidomic analyses to identify a novel role for the membrane binding protein Annexin A2 (ANXA2) in modulating the composition of specific membrane lipids necessary for cortical F-actin organization and adherens junction stabilization. In the absence of ANXA2, there is disorganized cortical F-actin, reduced junctional Arp2, excess sprout initiation, and ultimately failed sprout maturation. Furthermore, we observed reduced filipin III labeling of membrane cholesterol in cells with reduced ANXA2, suggesting there is an alteration in phospholipid membrane dynamics. Lipidomic analyses revealed that 42 lipid species were altered with loss of ANXA2, including an accumulation of phosphatidylcholine (16:0_16:0). We found that supplementation of phosphatidylcholine (16:0_16:0) in wild-type endothelial cells mimicked the ANXA2 knock-down phenotype, indicating that ANXA2 regulated the phospholipid membrane upstream of Arp2 recruitment and organization of cortical F-actin. Altogether, these data indicate a novel role for ANXA2 in coordinating events at endothelial junctions needed to initiate sprouting and show that proper lipid modulation is a critical component of these events.

The ß1-integrin plays a key role in LEC invasion in an optimized 3-D collagen matrix model.

Lymphangiogenesis, or formation of new lymphatic vessels, is a tightly regulated process that is controlled by growth factor signaling and biomechanical cues. Lymphatic endothelial cells (LECs) undergo remodeling, migration, and proliferation to invade the surrounding extracellular matrix (ECM) during both physiological and pathological lymphangiogenesis. This study optimized conditions for an in vitro three-dimensional (3-D) collagen-based model that induced LEC invasion and recapitulated physiological formation of lymphatic capillaries with lumens. Invasion of LECs was enhanced in the presence of sphingosine 1-phosphate (S1P). Effects of various known lymphangiogenic factors, vascular endothelial growth factor (VEGF)-A, basic fibroblast growth factor (bFGF), interleukin (IL)-8, and hepatocyte growth factor (HGF), were tested on LEC sprout formation synergistically with VEGF-C. Several of these growth factors significantly enhanced LEC invasion, and synergistic effects of some of these further enhanced the sprouting density and lumen volume. To determine the contribution of specific ECM components, we analyzed the expression of different integrin subunits. Basal expressions of the integrin α5- and integrin ß1-subunits were high in LECs. The addition of fibronectin, which mediates cellular responses through these integrins, enhanced LEC sprouting density and sprout length dose-dependently. siRNA-mediated knockdown of the integrin ß1-subunit suppressed LEC invasion and also inhibited VEGF receptor (VEGFR)3 and ERK activation. Furthermore, exposing LECs to the inflammatory mediator lipopolysaccharide (LPS) inhibited sprouting. This optimized model for LEC invasion includes S1P, VEGF-C, and fibronectin within a 3-D collagen matrix, along with VEGF-C, VEGF-A, bFGF, and HGF in the culture medium, and provides a useful tool to investigate the functional effect of various lymphangiogenic factors and inhibitors.

Hic-5 mediates the initiation of endothelial sprouting by regulating a key surface metalloproteinase.

During angiogenesis, endothelial cells must coordinate matrix proteolysis with migration. Here, we tested whether the focal adhesion scaffold protein Hic-5 (also known as TGFB1I1) regulated endothelial sprouting in three dimensions. Hic-5 silencing reduced endothelial sprouting and lumen formation, and sprouting defects were rescued by the return of Hic-5 expression. Pro-angiogenic factors enhanced colocalization and complex formation between membrane type-1 matrix metalloproteinase (MT1-MMP, also known as MMP14) and Hic-5, but not between paxillin and MT1-MMP. The LIM2 and LIM3 domains of Hic-5 were necessary and sufficient for Hic-5 to form a complex with MT1-MMP. The degree of interaction between MT1-MMP and Hic-5 and the localization of the complex within detergent-resistant membrane fractions were enhanced during endothelial sprouting, and Hic-5 depletion lowered the surface levels of MT1-MMP. In addition, we observed that loss of Hic-5 partially reduced complex formation between MT1-MMP and focal adhesion kinase (FAK, also known as PTK2), suggesting that Hic-5 bridges MT1-MMP and FAK. Finally, Hic-5 LIM2-LIM3 deletion mutants reduced sprout initiation. Hic-5, MT1-MMP and FAK colocalized in angiogenic vessels during porcine pregnancy, supporting that this complex assembles during angiogenesis in vivo. Collectively, Hic-5 appears to enhance complex formation between MT1-MMP and FAK in activated endothelial cells, which likely coordinates matrix proteolysis and cell motility.

Establishment of a three-dimensional model to study human uterine angiogenesis.

STUDY QUESTION: Can primary human uterine microvascular endothelial cells (UtMVECs) be used as a model to study uterine angiogenic responses in vitro that are relevant in pregnancy? SUMMARY ANSWER: UtMVECs demonstrated angiogenic responses when stimulated with proangiogenic factors, including sphingosine 1-phosphate (S1P), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), physiological levels of wall shear stress (WSS), human chorionic gonadotropin (hCG) and various combinations of estrogen and progesterone. WHAT IS KNOWN ALREADY: During sprouting angiogenesis, signaling from growth factors and cytokines induces a monolayer of quiescent endothelial cells (ECs) lining the vasculature to degrade the extracellular matrix and invade the surrounding tissue to form new capillaries. During pregnancy and the female reproductive cycle, the uterine endothelium becomes activated and undergoes sprouting angiogenesis to increase the size and number of blood vessels in the endometrium. STUDY DESIGN, SIZE, DURATION: The study was designed to examine the angiogenic potential of primary human UtMVECs using the well-characterized human umbilical vein EC (HUVEC) line as a control to compare angiogenic potential. ECs were seeded onto three-dimensional (3D) collagen matrices, supplemented with known proangiogenic stimuli relevant to pregnancy and allowed to invade for 24 h. Sprouting responses were analyzed using manual and automated methods for quantification. PARTICIPANTS/MATERIALS, SETTING, METHODS: RT-PCR, Western blot analysis and immunostaining were used to characterize UtMVECs. Angiogenic responses were examined using 3D invasion assays. Western blotting was used to confirm signaling responses after proangiogenic lipid, pharmacological inhibitor, and recombinant lentiviral treatments. All experiments were repeated at least three times. MAIN RESULTS AND THE ROLE OF CHANCE: After ensuring that UtMVECs expressed the proper endothelial markers, we found that UtMVECs invade 3D collagen matrices dose-dependently in response to known proangiogenic stimuli (e.g. S1P, VEGF, bFGF, hCG, estrogen, progesterone and WSS) present during early pregnancy. Invasion responses were positively correlated with phosphorylation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) and p42/p44 mitogen-activated protein kinase (ERK). Inhibition of these second messengers significantly impaired sprouting (P < 0.01). Gene silencing of membrane type 1-matrix metalloproteinase using multiple approaches completely abrogated sprouting (P < 0.001). Finally, UtMVECs displayed a unique ability to undergo sprouting in response to hCG, and combined estrogen and progesterone treatment. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: The study of uterine angiogenesis in vitro has limitations and any findings many not fully represent the in vivo state. However, these experiments do provide evidence for the ability of UtMVECs to be used in functional sprouting assays in a 3D environment, stimulated by physiological factors that are produced locally within the uterus during early pregnancy. WIDER IMPLICATIONS OF THE FINDINGS: We show that UtMVECs can be used reliably to investigate how growth factors, hormones, lipids and other factors, such as flow, affect angiogenesis in the uterus. STUDY FUNDING/COMPETING INTERESTS: This work was supported by NIH award HL095786 to K.J.B. The authors have no conflicts of interest.

Fluid shear stress promotes proprotein convertase-dependent activation of MT1-MMP.

During angiogenesis, endothelial cells (ECs(1)) initiate new blood vessel growth and invade into the extracellular matrix (ECM). Membrane type-1 matrix metalloproteinase (MT1-MMP) facilitates this process and translocates to the plasma membrane following activation to promote ECM cleavage. The N-terminal pro-domain within MT1-MMP must be processed for complete activity of the proteinase. This study investigated whether MT1-MMP activation was altered by sphingosine 1-phosphate (S1P) and wall shear stress (WSS), which combine to stimulate EC invasion in three dimensional (3D) collagen matrices. MT1-MMP was activated rapidly and completely by WSS but not S1P. Proprotein convertases (PCs) promoted MT1-MMP processing, prompting us to test whether WSS or S1P treatments increased PC activity. Like MT1-MMP, PC activity increased with WSS, while S1P had no effect. A pharmacological PC inhibitor completely blocked S1P- and WSS-induced EC invasion and MT1-MMP translocation to the plasma membrane. Further, a recombinant PC inhibitor reduced MT1-MMP activation and decreased lumen formation in invading ECs, a process known to be controlled by MT1-MMP. Thus, we conclude that PC and MT1-MMP activation are mechanosensitive events that are required for EC invasion into 3D collagen matrices.

Proteomic profiling of endothelial invasion revealed receptor for activated C kinase 1 (RACK1) complexed with vimentin to regulate focal adhesion kinase (FAK).

Angiogenesis is critical for many physiological and pathological processes. To identify molecules relevant to angiogenesis, we performed a proteomic screen comparing invading versus non-invading endothelial cells in three-dimensional collagen matrices. We found up-regulated levels of receptor for activated C kinase 1 (RACK1) and the intermediate filament protein vimentin that correlated with increased endothelial cell invasion. Because both RACK1 and vimentin have been linked to focal adhesion kinase (FAK), we investigated whether this pathway regulated invasion. RACK1 depletion reduced invasion responses, and this was associated with attenuated activation of FAK. Knockdown of vimentin significantly decreased levels of phosphorylated and total FAK. Treatment with a pharmacological inhibitor of FAK dose-dependently reduced invasion, indicating a crucial role for FAK activity during invasion. Because RACK1 and vimentin were both up-regulated with sphingosine 1-phosphate treatment, required for invasion, and regulated FAK, we tested whether they complexed together. RACK1 complexed with vimentin, and growth factors enhanced this interaction. In addition, RACK1, vimentin, and FAK formed an intermolecular complex in invading endothelial cultures in three dimensions in response to stimulation by sphingosine 1-phosphate and growth factors. Moreover, depletion of RACK1 decreased the association of vimentin and FAK, suggesting that RACK1 was required for stabilizing vimentin-FAK interactions during sprouting. Silencing of vimentin and RACK1 decreased cell adhesion and focal contact formation. Taken together, these results demonstrate that proangiogenic signals converge to enhance expression and association of RACK1 and vimentin, which regulated FAK, resulting in successful endothelial sprout formation in three-dimensional collagen matrices.

Identification of a major locus interacting with MC1R and modifying black coat color in an F2 Nellore-Angus population.

BACKGROUND: In cattle, base color is assumed to depend on the enzymatic activity specified by the MC1R locus, i.e. the extension locus, with alleles coding for black (E(D)), red (e), and wild-type (E+). In most mammals, these alleles are presumed to follow the dominance model of E(D) > E+ > e, although exceptions are found. In Bos indicus x Bos taurus F2 cattle, some E(D)E+ heterozygotes are discordant with the dominance series for MC1R and display various degrees of red pigmentation on an otherwise predicted black background. The objective of this study was to identify loci that modify black coat color in these individuals. RESULTS: Reddening was classified with a subjective scoring system. Interval analyses identified chromosome-wide suggestive (P < 0.05) and significant (P < 0.01) QTL on bovine chromosomes (BTA) 4 and 5, although these were not confirmed using single-marker association or Bayesian methods. Evidence of a major locus (F = 114.61) that affects reddening was detected between 60 and 73 Mb on BTA 6 (Btau4.0 build), and at 72 Mb by single-marker association and Bayesian methods. The posterior mean of the genetic variance for this region accounted for 43.75% of the genetic variation in reddening. This region coincided with a cluster of tyrosine kinase receptor genes (PDGFRA, KIT and KDR). Fitting SNP haplotypes for a 1 Mb interval that contained all three genes and centered on KIT accounted for the majority of the variation attributed to this major locus, which suggests that one of these genes or associated regulatory elements, is responsible for the majority of variation in degree of reddening. CONCLUSIONS: Recombinants in a 5 Mb region surrounding the cluster of tyrosine kinase receptor genes implicated PDGFRA as the strongest positional candidate gene. A higher density marker panel and functional analyses will be required to validate the role of PDGFRA or other regulatory variants and their interaction with MC1R for the modification of black coat color in Bos indicus influenced cattle.

Newly Identified Peptide, Peptide Lv, Promotes Pathological Angiogenesis.

Background We recently discovered a small endogenous peptide, peptide Lv, with the ability to activate vascular endothelial growth factor receptor 2 and its downstream signaling. As vascular endothelial growth factor through vascular endothelial growth factor receptor 2 contributes to normal development, vasodilation, angiogenesis, and pathogenesis of various diseases, we investigated the role of peptide Lv in vasodilation and developmental and pathological angiogenesis in this study. Methods and Results The endothelial cell proliferation, migration, and 3-dimensional sprouting assays were used to test the abilities of peptide Lv in angiogenesis in vitro. The chick chorioallantoic membranes and early postnatal mice were used to examine its impact on developmental angiogenesis. The oxygen-induced retinopathy and laser-induced choroidal neovascularization mouse models were used for in vivo pathological angiogenesis. The isolated porcine retinal and coronary arterioles were used for vasodilation assays. Peptide Lv elicited angiogenesis in vitro and in vivo. Peptide Lv and vascular endothelial growth factor acted synergistically in promoting endothelial cell proliferation. Peptide Lv-elicited vasodilation was not completely dependent on nitric oxide, indicating that peptide Lv had vascular endothelial growth factor receptor 2/nitric oxide-independent targets. An antibody against peptide Lv, anti-Lv, dampened vascular endothelial growth factor-elicited endothelial proliferation and laser-induced vascular leakage and choroidal neovascularization. While the pathological angiogenesis in mouse eyes with oxygen-induced retinopathy was enhanced by exogenous peptide Lv, anti-Lv dampened this process. Furthermore, deletion of peptide Lv in mice significantly decreased pathological neovascularization compared with their wild-type littermates. Conclusions These results demonstrate that peptide Lv plays a significant role in pathological angiogenesis but may be less critical during development. Peptide Lv is involved in pathological angiogenesis through vascular endothelial growth factor receptor 2-dependent and -independent pathways. As anti-Lv dampened the pathological angiogenesis in the eye, anti-Lv may have a therapeutic potential to treat pathological angiogenesis.

Matrix density alters zyxin phosphorylation, which limits peripheral process formation and extension in endothelial cells invading 3D collagen matrices.

This study was designed to determine the optimal conditions required for known pro-angiogenic stimuli to elicit successful endothelial sprouting responses. We used an established, quantifiable model of endothelial cell (EC) sprout initiation where ECs were tested for invasion in low (1 mg/mL) and high density (5 mg/mL) 3D collagen matrices. Sphingosine 1-phosphate (S1P) alone, or S1P combined with stromal derived factor-1α (SDF) and phorbol ester (TPA), elicited robust sprouting responses. The ability of these factors to stimulate sprouting was more effective in higher density collagen matrices. S1P stimulation resulted in a significant increase in invasion distance, and with the exception of treatment groups containing phorbol ester, invasion distance was longer in 1mg/mL compared to 5mg/mL collagen matrices. Closer examination of cell morphology revealed that increasing matrix density and supplementing with SDF and TPA enhanced the formation of multicellular structures more closely resembling capillaries. TPA enhanced the frequency and size of lumen formation and correlated with a robust increase in phosphorylation of p42/p44 Erk kinase, while S1P and SDF did not. Also, a higher number of significantly longer extended processes formed in 5mg/mL compared to 1mg/mL collagen matrices. Because collagen matrices at higher density have been reported to be stiffer, we tested for changes in the mechanosensitive protein, zyxin. Interestingly, zyxin phosphorylation levels inversely correlated with matrix density, while levels of total zyxin did not change significantly. Immunofluorescence and localization studies revealed that total zyxin was distributed evenly throughout invading structures, while phosphorylated zyxin was slightly more intense in extended peripheral processes. Silencing zyxin expression increased extended process length and number of processes, while increasing zyxin levels decreased extended process length. Altogether these data indicate that ECs integrate signals from multiple exogenous factors, including changes in matrix density, to accomplish successful sprouting responses. We show here for the first time that zyxin limited the formation and extension of fine peripheral processes used by ECs for matrix interrogation, providing a molecular explanation for altered EC responses to high and low density collagen matrices.

Materials composed of the Drosophila melanogaster protein ultrabithorax are cytocompatible.

The Drosophila melanogaster Hox protein ultrabithorax (Ubx) has the interesting ability to hierarchically self-assemble in vitro into materials that have mechanical properties comparable to natural elastin. Ubx materials can be easily functionalized by gene fusion, generating potentially useful scaffolds for cell and tissue engineering. Here, we tested the cytocompatibility of fibers composed of Ubx or an mCherry-Ubx fusion protein. Fibers were cultured with three primary human cell lines derived from vasculature at low passage: umbilical vein endothelial cells, brain vascular pericytes, or aortic smooth muscle cells. No direct or indirect toxicity was observed for any cell line, in response to fibers composed of either plain Ubx or mCherry-Ubx. Cells readily adhered to Ubx fibers, and cells attached to fibers could be transferred between tissue cultures without loss of viability for at least 96 h. When attached to fibers, the morphology of the three cell lines differed somewhat, but all cells in contact with Ubx fibers exhibited a microtubular network aligned with the long axis of Ubx fibers. Thus, Ubx fibers are cytocompatible with cultured primary human vascular cells.

Genome-wide survey of SNP variation uncovers the genetic structure of cattle breeds.

The imprints of domestication and breed development on the genomes of livestock likely differ from those of companion animals. A deep draft sequence assembly of shotgun reads from a single Hereford female and comparative sequences sampled from six additional breeds were used to develop probes to interrogate 37,470 single-nucleotide polymorphisms (SNPs) in 497 cattle from 19 geographically and biologically diverse breeds. These data show that cattle have undergone a rapid recent decrease in effective population size from a very large ancestral population, possibly due to bottlenecks associated with domestication, selection, and breed formation. Domestication and artificial selection appear to have left detectable signatures of selection within the cattle genome, yet the current levels of diversity within breeds are at least as great as exists within humans.

A physical map of the bovine genome.

BACKGROUND: Cattle are important agriculturally and relevant as a model organism. Previously described genetic and radiation hybrid (RH) maps of the bovine genome have been used to identify genomic regions and genes affecting specific traits. Application of these maps to identify influential genetic polymorphisms will be enhanced by integration with each other and with bacterial artificial chromosome (BAC) libraries. The BAC libraries and clone maps are essential for the hybrid clone-by-clone/whole-genome shotgun sequencing approach taken by the bovine genome sequencing project. RESULTS: A bovine BAC map was constructed with HindIII restriction digest fragments of 290,797 BAC clones from animals of three different breeds. Comparative mapping of 422,522 BAC end sequences assisted with BAC map ordering and assembly. Genotypes and pedigree from two genetic maps and marker scores from three whole-genome RH panels were consolidated on a 17,254-marker composite map. Sequence similarity allowed integrating the BAC and composite maps with the bovine draft assembly (Btau3.1), establishing a comprehensive resource describing the bovine genome. Agreement between the marker and BAC maps and the draft assembly is high, although discrepancies exist. The composite and BAC maps are more similar than either is to the draft assembly. CONCLUSION: Further refinement of the maps and greater integration into the genome assembly process may contribute to a high quality assembly. The maps provide resources to associate phenotypic variation with underlying genomic variation, and are crucial resources for understanding the biology underpinning this important ruminant species so closely associated with humans.

Identification of endometrial genes regulated by early pregnancy, progesterone, and interferon tau in the ovine uterus.

During early pregnancy in ruminants, progesterone (P4) from the corpus luteum and interferon tau (IFNT) from the conceptus act on the endometrium to regulate genes important for uterine receptivity and conceptus growth. The use of the uterine gland knockout (UGKO) ewe has demonstrated the critical role of epithelial secretions in regulation of conceptus survival and growth. A custom ovine cDNA array was used to identify alterations in gene expression of endometria from Day 14 cyclic, pregnant, and UGKO ewes (study 1) and from cyclic ewes treated with P4 or P4 with ZK 136,317 antiprogestin and control proteins or IFNT (study 2). In study 1, expression of 47 genes was more than 2-fold different between Day 14 pregnant and cyclic endometria, whereas 23 genes was different between Day 14 cyclic and UGKO endometria. In study 2, 70 genes were different due to P4 alone, 74 genes were affected by IFNT in a P4-dependent manner, and 180 genes were regulated by IFNT in a P4-independent manner. In each study, an approximately equal number of genes were found to be activated or repressed in each group. Endometrial genes increased by pregnancy and P4 and/or IFNT include B2M, CTSL, CXCL10, G1P3, GRP, IFI27, IFIT1, IFITM3, LGALS15, MX1, POSTN, RSAD2, and STAT5A. Transcripts decreased by pregnancy and P4 and/or IFNT include COL3A1, LUM, PTMA, PUM1, RPL9, SPARC, and VIM. Identification and analysis of these hormonally responsive genes will help define endometrial pathways critical for uterine support of peri-implantation conceptus survival, growth, and implantation.

Glycogen branching enzyme (GBE1) mutation causing equine glycogen storage disease IV.

Comparative biochemical and histopathological evidence suggests that a deficiency in the glycogen branching enzyme, encoded by the GBE1 gene, is responsible for a recently identified recessive fatal fetal and neonatal glycogen storage disease (GSD) in American Quarter Horses termed GSD IV. We have now derived the complete GBE1 cDNA sequences for control horses and affected foals, and identified a C to A substitution at base 102 that results in a tyrosine (Y) to stop (X) mutation in codon 34 of exon 1. All 11 affected foals were homozygous for the X34 allele, their 11 available dams and sires were heterozygous, and all 16 control horses were homozygous for the Y34 allele. The previous findings of poorly branched glycogen, abnormal polysaccharide accumulation, lack of measurable GBE1 enzyme activity and immunodetectable GBE1 protein, coupled with the present observation of abundant GBE1 mRNA in affected foals, are all consistent with the nonsense mutation in the 699 amino acid GBE1 protein. The affected foal pedigrees have a common ancestor and contain prolific stallions that are likely carriers of the recessive X34 allele. Defining the molecular basis of equine GSD IV will allow for accurate DNA testing and the ability to prevent occurrence of this devastating disease affecting American Quarter Horses and related breeds.

Haplotype variation in bovine Toll-like receptor 4 and computational prediction of a positively selected ligand-binding domain.

Toll-like receptor 4 (TLR4) is a cell-surface receptor that activates innate and adaptive immune responses. Because it recognizes a broad class of pathogen-associated molecular patterns presented by lipopolysaccharides and lipoteichoic acid, TLR4 is a candidate gene for resistance to a large number of diseases. In particular, mouse models suggest TLR4 as a candidate gene for resistance to major agents in bovine respiratory disease and Johne's disease. The coding sequence of bovine TLR4 is divided into three exons, with intron/exon boundaries and intron sizes similar to those of human TLR4 transcript variant 1. We amplified each exon in 40 individuals from 11 breeds and screened the sequence for single-nucleotide polymorphisms (SNPs). We identified 32 SNPs, 28 of which are in the coding sequence, for an average of one SNP per 90 bp of coding sequence. Eight SNPs were nonsynonymous and potentially alter specificity of pathogen recognition or efficiency of signaling. To evaluate the functional importance of these SNPs, we used codon-substitution models to detect diversifying selection in an extracellular region that may physically interact with ligands. One nonsynonymous SNP is located within this region, and other substitutions are in adjacent regions that may interact with coreceptor molecules. The 32 SNPs were found in 20 haplotypes that can be assigned to geographic ranges of origin. Haplotype-tagging SNP analysis indicated that 12 SNPs need to be genotyped to distinguish these 20 haplotypes. These data provide a basic understanding of bovine TLR4 sequence variation and supply haplotype markers for disease association studies.