A multicenter study has been carried out to characterize 13 polymorphic short tandem repeat (STR) systems located on the male specific part of the human Y chromosome (DYS19, DYS288, DYS385, DYS388, DYS389I/II, DYS390, DYS391, DYS392, DYS393, YCAI, YCAII, YCAIII, DXYS156Y). Amplification parameters and electrophoresis protocols including multiplex approaches were compiled. The typing of non-recombining Y loci with uniparental inheritance requires special attention to population substructuring due to prevalent male lineages. To assess the extent of these subheterogeneities up to 3825 unrelated males were typed in up to 48 population samples for the respective loci. A consistent repeat based nomenclature for most of the loci has been introduced. Moreover we have estimated the average mutation rate for DYS19 in 626 confirmed fatherson pairs as 3.2 x 10(-3) (95% confidence interval limits of 0.00041-0.00677), a value which can also be expected for other Y-STR loci with similar repeat structure. Recommendations are given for the forensic application of a basic set of 7 STRs (DYS19, DYS3891, DYS389II, DYS390, DYS391, DYS392, DYS393) for standard Y-haplotyping in forensic and paternity casework. We recommend further the inclusion of the highly polymorphic bilocal Y-STRs DYS385, YCAII, YCAIII for a nearly complete individualisation of almost any given unrelated male individual. Together, these results suggest that Y-STR loci are useful markers to identify males and male lineages in forensic practice.

We report the molecular design and synthesis of EG00229, 2, the first small molecule ligand for the VEGF-A receptor neuropilin 1 (NRP1) and the structural characterization of NRP1−ligand complexes by NMR spectroscopy and X-ray crystallography. Mutagenesis studies localized VEGF-A binding in the NRP1 b1 domain and a peptide fragment of VEGF-A was shown to bind at the same site by NMR, providing the basis for small molecule design. Compound 2 demonstrated inhibition of VEGF-A binding to NRP1 and attenuated VEGFR2 phosphorylation in endothelial cells. Inhibition of migration of endothelial cells was also observed. The viability of A549 lung carcinoma cells was reduced by 2, and it increased the potency of the cytotoxic agents paclitaxel and 5-fluorouracil when given in combination. These studies provide the basis for design of specific small molecule inhibitors of ligand binding to NRP1.

We show for the first time, to our knowledge, that binding of vascular endothelial growth factor (VEGF) to the neuropilin-1 b1 domain is essential for VEGF complex formation with VEGFR2/KDR (kinase insert domain-containing receptor) and is important for endothelial cell migration and tubulogenesis

Although a number of target genes for the tumor suppressor p53 have been described, the mechanism of p53-dependent apoptosis is incompletely understood. Thus, it is essential to identify and characterize additional target genes that could mediate apoptosis. In the study reported here, we isolated a p53-regulated gene named NDRG1 (N-Myc down-regulated gene 1). Its expression is induced by DNA damage in a p53-dependent fashion. The promoter region of the NDRG1 gene contains a p53 binding site that confers p53-dependent transcriptional activation via a heterologous reporter. RNA interference and inducible gene expression approaches suggest that NDRG1 is necessary but not sufficient for p53-mediated caspase activation and apoptosis. This report further supports the notion that p53 controls a network of genes that are required for its apoptotic function.

Neuropilin 1 (NRP1) is a receptor for class 3 semaphorins and vascular endothelial growth factor (VEGF) A and is essential for cardiovascular development. Biochemical evidence supports a model for NRP1 function in which VEGF binding induces complex formation between NRP1 and VEGFR2 to enhance endothelial VEGF signalling. However, the relevance of VEGF binding to NRP1 for angiogenesis in vivo has not yet been examined. We therefore generated knock-in mice expressing Nrp1 with a mutation of tyrosine (Y) 297 in the VEGF binding pocket of the NRP1 b1 domain, as this residue was previously shown to be important for high affinity VEGF binding and NRP1-VEGFR2 complex formation. Unexpectedly, this targeting strategy also severely reduced NRP1 expression and therefore generated a NRP1 hypomorph. Despite the loss of VEGF binding and attenuated NRP1 expression, homozygous Nrp1 Y297A/Y297A mice were born at normal Mendelian ratios, arguing against NRP1 functioning exclusively as a VEGF 164 receptor in embryonic angiogenesis. By overcoming the mid-gestation lethality of full Nrp1-null mice, homozygous Nrp1 Y297A/Y297A mice revealed essential roles for NRP1 in postnatal angiogenesis and arteriogenesis in the heart and retina, pathological neovascularisation of the retina and angiogenesis-dependent tumour growth. KEY WORDS: NRP1, VEGF, Angiogenesis, Arteriogenesis, Retina, Hindbrain INTRODUCTIONNRP1 is a transmembrane receptor for the VEGF 165 isoform (VEGF 164 in mice) and the neuronal guidance cue SEMA3A, with essential roles in both vascular and neuronal development (reviewed by Pellet-Many et al., 2008;Raimondi and Ruhrberg, 2013). Accordingly, Nrp1-null mice die before birth with severe cardiovascular and neuronal defects (Kitsukawa et al., 1997;Kawasaki et al., 1999 Centre for Cardiovascular Biology and Medicine, BHF Laboratories, Division of Medicine, University College London, 5 University Street, London WC1E 6JJ, UK. *These authors contributed equally to this work ‡ These authors contributed equally to this work § Authors for correspondence (I.Zachary@ucl.ac.uk; c.ruhrberg@ucl.ac.uk) This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.Received 25 August 2013; Accepted 3 November 2013 whereas mice carrying a mutated extracellular domain that abolishes SEMA3A, not VEGF 164 , binding show defective nerve, but not blood vessel, patterning (Gu et al., 2003;Vieira et al., 2007). These and other genetic, biochemical and cell biological data support a model in which VEGF 165 binding induces complex formation between NRP1 and VEGFR2 (KDR -Mouse Genome Informatics) to enhance VEGFR2 signalling during EC migration in vitro (e.g. Soker et al., 2002;Wang et al., 2003;Evans et al., 2011) and arteriogenesis in vivo (Lanahan et al., 2013).The extracellular NRP1 a1/a2 and b1/b2 domains are crucial f...

NRP1 (neuropilin-1) is a co-receptor for members of the VEGF (vascular endothelial growth factor) family in endothelial cells, but is increasingly implicated in signalling induced by other growth factors. NRP1 is expressed in VSMCs (vascular smooth muscle cells), but its function and the mechanisms involved are poorly understood. The present study aimed to determine the role of NRP1 in the migratory response of HCASMCs (human coronary artery smooth muscle cells) to PDGF (platelet-derived growth factor), and to identify the signalling mechanisms involved. NRP1 is highly expressed in HAoSMCs (human aortic smooth muscle cells) and HCASMCs, and modified in VSMCs by CS (chondroitin sulfate)-rich O-linked glycosylation at Ser612. HCASMC migration induced by PDGF-BB and PDGF-AA was inhibited by NRP1 siRNA (small interfering RNA), and by adenoviral overexpression of an NRP1 mutant lacking the intracellular domain (Ad.NRP1ΔC). NRP1 co-immunoprecipitated with PDGFRα (PDGF receptor α), and immunofluorescent staining indicated that NRP1 and PDGFRα co-localized in VSMCs. NRP1 siRNA also inhibited PDGF-induced PDGFRα activation. NRP1-specific siRNA, Ad.NRP1ΔC and removal of CS glycans using chondroitinase all inhibited PDGF-BB and -AA stimulation of tyrosine phosphorylation of the adapter protein, p130Cas (Cas is Crk-associated substrate), with little effect on other major signalling pathways, and p130Cas knockdown inhibited HCASMC migration. Chemotaxis and p130Cas phosphorylation induced by PDGF were inhibited by chondroitinase, and, additionally, adenoviral expression of a non-glycosylatable NRP1S612A mutant inhibited chemotaxis, but not p130Cas phosphorylation. These results indicate a role for NRP1 and NRP1 glycosylation in mediating PDGF-induced VSMC migration, possibly by acting as a co-receptor for PDGFRα and via selective mobilization of a novel p130Cas tyrosine phosphorylation pathway.

By means of a multicenter study, a large number of males have been characterized for Y-chromosome specific short tandem repeats (STRs) or microsatellites. A complete summary of the allele frequency distributions for these Y-STRs is presented in the Appendix. This manuscript describes in more detail some of the population genetic and evolutionary aspects for a restricted set of seven chromosome Y STRs in a selected number of population samples. For all the chromosome Y STRs markedly different region-specific allele frequency distributions were observed, also when closely related populations were compared. Haplotype analyses using AMOVA showed that when four different European male groups (Germans, Dutch, Swiss, Italians) were compared, less than 10% of the total genetic variability was due to differences between these populations. Nevertheless, these pairwise comparisons revealed significant differences between most population pairs. Assuming a step-wise mutation model and a mutation frequency of 0.21%, it was estimated that chromosome Y STR-based evolutionary lines of descent can be reliably inferred over a time-span of only 1950 generations (or about 49,000 years). This reduces the reliability of the inference of population affinities to a historical, rather than evolutionary time scale. This is best illustrated by the construction of a human evolutionary tree based on chromosome Y STRs in which most of the branches connect in a markedly different way compared with trees based on classical protein polymorphisms and/or mtDNA sequence variation. Thus, the chromosome Y STRs seem to be very useful in comparing closely related populations which cannot probably be separated by e.g. autosomal STRs. However, in order to be used in an evolutionary context they need to be combined with more stable Y-polymorphisms e.g. base-substitutions.

The orphan nuclear receptor estrogen-related receptor (ERR) ␣ is a downstream effector of the transcriptional coactivator PGC-1␣ in the regulation of genes important for mitochondrial oxidative capacity. PGC-1␣ is also a potent activator of the transcriptional program required for hepatic gluconeogenesis, and in particular of the key gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK). We report here that the regulatory sequences of the PEPCK gene harbor a functional ERR␣ binding site. However, in contrast to the co-stimulating effects of ERR␣ and PGC-1␣ on mitochondrial gene expression, ERR␣ acts as a transcriptional repressor of the PEPCK gene. Suppression of ERR␣ expression by small interfering RNA leads to reduced binding of ERR␣ to the endogenous PEPCK gene, and an increase in promoter occupancy by PGC-1␣, suggesting that part of the ERR␣ function at this gene is to antagonize the action of PGC-1␣. In agreement with the in vitro studies, animals that lack ERR␣ show increased expression of gluconeogenic genes, including PEPCK and glycerol kinase, but decreased expression of mitochondrial genes, such as ATP synthase subunit ␤ and cytochrome c-1. Our findings suggest that ERR␣ has opposing effects on genes important for mitochondrial oxidative capacity and gluconeogenesis. The different functions of ERR␣ in the regulation of these pathways suggest that enhancing ERR␣ activity could have beneficial effects on glucose metabolism in diabetic subjects by two distinct mechanisms: increasing mitochondrial oxidative capacity in peripheral tissues and liver, and suppressing hepatic glucose production.Nuclear receptors mediate the effects of many hormonal and dietary signals. These receptors bind to specific genomic sequences, recruit coactivators or corepressors of transcription, and regulate accordingly the expression of genes important for a wide range of biological processes, including development, reproduction, and metabolism (for reviews, see Refs. 1 and 2 and references therein). The estrogen-related receptor (ERR) 2 ␣ is a nuclear receptor with high sequence similarity to the estrogen receptors, and the founding member of a small family of orphan receptors that also includes ERR␤ and ERR␥ (3, 4). Despite their similarity to estrogen receptors, ERRs are not activated by estrogens or other known natural agonists (reviewed in Refs. 5 and 6). Structural studies of ERR␣ and ERR␥ indicate that these receptors can achieve a transcriptionally active conformation in the absence of a ligand, and suggest that ERR activity may not be subject to regulation by small lipophilic molecules (7,8). As an alternative mechanism of regulation, ERR␣ activity is controlled by the availability of specific coactivators that act as protein ligands (9 -11). Notably, the transactivation function of ERR␣ is weak in many cells where other nuclear receptors are active, and is greatly enhanced by expression of the transcriptional coactivators PGC-1␣ or PGC-1␤ (9 -11). PGC-1␣ not only activates the transcriptional function of ERR␣ bu...