Magic roundabout is a new member of the roundabout receptor family that is endothelial specific and expressed at sites of active angiogenesis

We have used bioinformatic data mining to identify a novel, endothelial-specific gene encoding a protein with homology to the axon guidance protein roundabout (ROBO1). The new gene has been called magic roundabout (ROBO4; GenBank acc. no. AF361473) and is smaller than other members of the roundabout gene family. Thus, in the extracellular region, magic roundabout has only two of the five immunoglobulin and two of the three fibronectin domains present in other roundabout genes. Expression of magic roundabout in vitro was detected in only endothelial cells and was greater in cells exposed to hypoxia. In situ hybridization and immunohistochemistry validated the bioinformatic prediction that magic roundabout expression would be endothelial specific in vivo. Magic roundabout expression in the adult was restricted exclusively to sites of active angiogenesis, notably tumor vessels. The identification of magic roundabout shows that the roundabout gene family extends beyond neuronal tissue and that roundabout/slit interactions are likely to have a role in angiogenesis.     

Quantification of progeny classes in two facultatively apomictic accessions of Hieracium

Hieracium is an established model system for studying the cytological and genetic basis of gametophytic apomixis. In common with most known apomicts, the formation of 'maternal seed' is not exclusive in Hieracium, as apomixis operates in conjunction with a low level of sexuality. When this occurs the form of apomixis is described as 'facultative'. The formation of maternal seed in these plants is characterised by the avoidance of meiosis followed by the parthenogenetic development of an unreduced egg cell. In some ovules, however, meiosis does proceed, and sometimes the fertilisation of an egg cell presages embryogenesis. As a result, this mechanism of facultative apomixis leads to the formation of several different types of progeny, each representing a unique combination of meiosis/apomeiosis and fertilisation/parthenogenesis. Furthermore, fertilisation may involve either self or non-self pollen, leading to the recognition of six progeny classes from each individual plant. To facilitate an understanding of these processes we have developed a method for identifying individuals from different progeny classes based on the inheritance of introduced heterologous marker genes. This technique permits the screening of many thousands of seedlings at germination, and the consequent isolation of individuals associated with rare classes. Progeny profiles were determined for two apomictic accessions of Hieracium. Both were found to develop approximately 2.5% of their seed from meiotically derived eggs under the experimental conditions used and to have a rate of hybridity of approximately 2%. Evidence was also found for the action of a self-incompatibility mechanism operating in these plants despite the autonomous nature of apomixis in Hieracinum. As a demonstration of the utility of this approach, a study was conducted of polyembryony in one accession. The results indicate that there was a 7 fold greater likelihood that a meiotically derived seedling would arise in a polyembryonic seed than in a single-embryo seed. This indicates that facultative apomixis in Hieracium not only results from the simultaneous occurrence of sexual and asexual seed formation in the same capitulum as previously demonstrated, but most often as parallel processes within the same ovule.     

Purification and Functional Characterisation of Rhinocerase,a Novel Serine Protease from the Venom of Bitis gabonica rhinoceros

Background

Serine proteases are a major component of viper venoms and are thought to disrupt several distinct elements of the blood coagulation system of envenomed victims. A detailed understanding of the functions of these enzymes is important both for acquiring a fuller understanding of the pathology of envenoming and because these venom proteins have shown potential in treating blood coagulation disorders.

Methodology/Principal Findings

In this study a novel, highly abundant serine protease, which we have named rhinocerase, has been isolated and characterised from the venom of Bitis gabonica rhinoceros using liquid phase isoelectric focusing and gel filtration. Like many viper venom serine proteases, this enzyme is glycosylated; the estimated molecular mass of the native enzyme is approximately 36kDa, which reduces to 31kDa after deglycosylation. The partial amino acid sequence shows similarity to other viper venom serine proteases, but is clearly distinct from the sequence of the only other sequenced serine protease from Bitis gabonica. Other viper venom serine proteases have been shown to exert distinct biological effects, and our preliminary functional characterization of rhinocerase suggest it to be multifunctional. It is capable of degrading α and β chains of fibrinogen, dissolving plasma clots and of hydrolysing a kallikrein substrate.

Conclusions/Significance

A novel multifunctional viper venom serine protease has been isolated and characterised. The activities of the enzyme are consistent with the known in vivo effects of Bitis gabonica envenoming, including bleeding disorders, clotting disorders and hypotension. This study will form the basis for future research to understand the mechanisms of serine protease action, and examine the potential for rhinocerase to be used clinically to reduce the risk of human haemostatic disorders such as heart attacks and strokes.     

Effect of progesterone on the activation of neurones of the supraoptic nucleus during parturition

Parturition is driven by a pulsatile pattern of oxytocin secretion, resulting from burst firing activity of supraoptic oxytocin neurones and reflected by induction of Fos expression. Rats were injected with progesterone on day 20 of pregnancy to investigate the role of the decreasing progesterone:ratio oestrogen ratio, which precedes delivery, in the activation of supraoptic neurones. Progesterone delayed the onset of birth by 28 h compared with vehicle (control) and prolonged the duration of delivery, which was overcome by pulsatile injections of oxytocin, indicating that the slow delivery may reflect impaired oxytocin secretion. Parturient rats pretreated with progesterone had fewer Fos immunoreactive nuclei in the supraoptic nucleus than did parturient rats pretreated with vehicle. The number of Fos immunoreactive nuclei was not restored after oxytocin injection, indicating that appropriate activation of oxytocin neurones is impaired by progesterone and also that there is a lack of stimulatory afferent drive. Fos expression increased in the nucleus of the tractus solitarius during parturition in rats pretreated with either vehicle or progesterone, but not in rats that had been pretreated with progesterone and induced with oxytocin, indicating that this input was inhibited. Endogenous opioids inhibit oxytocin neurones in late pregnancy and the opioid antagonist, naloxone, increases Fos expression in supraoptic nuclei by preventing inhibition. However, progesterone attenuated naloxone-induced Fos expression in the supraoptic nucleus in late pregnancy and naloxone administered during parturition did not accelerate the duration of births delayed by progesterone administration, indicating that progesterone does not act by hyperactivation of endogenous opioid tone. RU486, a progesterone receptor antagonist, enhanced supraoptic neurone Fos expression in late pregnancy, indicating progesterone receptor-mediated actions. Thus, progesterone withdrawal is necessary for appropriate activation of supraoptic and tractus solitarius neurones during parturition.     

Vascular endothelial growth factor (VEGF)-D and VEGF-A differentially regulate KDR-mediated signaling and biological function in vascular endothelial cells

Vascular endothelial growth factor (VEGF)-D binds to VEGF receptors (VEGFR) VEGFR2/KDR and VEGFR3/Flt4, but the signaling mechanisms mediating its biological activities in endothelial cells are poorly understood. Here we investigated the mechanism of action of VEGF-D, and we compared the signaling pathways and biological responses induced by VEGF-D and VEGF-A in endothelial cells. VEGF-D induced KDR and phospholipase C-gamma tyrosine phosphorylation more slowly and less effectively than VEGF-A at early times but had a more sustained effect and was as effective as VEGF-A after 60 min. VEGF-D activated extracellular signal-regulated protein kinases 1 and 2 with similar efficacy but slower kinetics compared with VEGF-A, and this effect was blocked by inhibitors of protein kinase C and mitogen-activated protein kinase kinase. In contrast to VEGF-A, VEGF-D weakly stimulated prostacyclin production and gene expression, had little effect on cell proliferation, and stimulated a smaller and more transient increase in intracellular [Ca(2+)]. VEGF-D induced strong but more transient phosphatidylinositol 3-kinase (PI3K)-mediated Akt activation and increased PI3K-dependent endothelial nitric-oxide synthase phosphorylation and cell survival more weakly. VEGF-D stimulated chemotaxis via a PI3K/Akt- and endothelial nitric-oxide synthase-dependent pathway, enhanced protein kinase C- and PI3K-dependent endothelial tubulogenesis, and stimulated angiogenesis in a mouse sponge implant model less effectively than VEGF-A. VEGF-D-induced signaling and biological effects were blocked by the KDR inhibitor SU5614. The finding that differential KDR activation by VEGF-A and VEGF-D has distinct consequences for endothelial signaling and function has important implications for understanding how multiple ligands for the same VEGF receptors can generate ligand-specific biological responses.     

Population genetic structure and long-distance dispersal among seabird populations: implications for colony persistence

Dramatic local population decline brought about by anthropogenic-driven change is an increasingly common threat to biodiversity. Seabird life history traits make them particularly vulnerable to such change; therefore, understanding population connectivity and dispersal dynamics is vital for successful management. Our study used a 357-base pair mitochondrial control region locus sequenced for 103 individuals and 18 nuclear microsatellite loci genotyped for 245 individuals to investigate population structure in the Atlantic and Pacific populations of the pelagic seabird, Leach's storm-petrel Oceanodroma leucorhoa leucorhoa. This species is under intense predation pressure at one regionally important colony on St Kilda, Scotland, where a disparity between population decline and predation rates hints at immigration from other large colonies. AMOVA, F(ST), Φ(ST) and Bayesian cluster analyses revealed no genetic structure among Atlantic colonies (Global Φ(ST) = -0.02 P > 0.05, Global F(ST) = 0.003, P > 0.05, STRUCTURE K = 1), consistent with either contemporary gene flow or strong historical association within the ocean basin. The Pacific and Atlantic populations are genetically distinct (Global Φ(ST) = 0.32 P < 0.0001, Global F(ST) = 0.04, P < 0.0001, STRUCTURE K = 2), but evidence for interocean exchange was found with individual exclusion/assignment and population coalescent analyses. These findings highlight the importance of conserving multiple colonies at a number of different sites and suggest that management of this seabird may be best viewed at an oceanic scale. Moreover, our study provides an illustration of how long-distance movement may ameliorate the potentially deleterious impacts of localized environmental change, although direct measures of dispersal are still required to better understand this process.     

Hybrid 80S monomers formed from subunits of ribosomes from protozoa,fungi, plants,and mammals

Free 80S ribosomes of eukaryotic organisms are dissociated by KCl (0.8–1.0 m) in the presence of 2-mercaptoethanol and magnesium ions (10–15mm); the large and small subunits so formed can be recombined to yield 80S monomers. We have now studied the ability of ribosomal subunits from protozoa (Tetrahymena pyriformis), fungi (Allomyces arbuscula, Saccharomyces cerevisiae), plants (pea, wheat), and mammals (rat, mouse, rabbit) to combine to form hybrid ribosomes. In general, both subunits of the species studied participate in the formation of hybrid particles, with the exception of the 60S subunit of Tetrahymena, which does not combine with the small subunit of fungal, plant, or mammalian ribosomes. The interaction of subunits from rat and Tetrahymena ribosomes has been visualized by an electron microscope study of negatively stained preparations. The base sequences of the ribosomal RNAs of these organisms have been compared to those of Saccharomyces by nucleic acid hybridization-competition.This work was supported by a fellowship #PF-529 from the American Cancer Society and by United States Public Health Service, National Institutes of Health grant GM 12449.     

Identification of Tspan9 as a novel platelet tetraspanin and the collagen receptor GPVI as a component of tetraspanin microdomains

Platelets are essential for wound healing and inflammatory processes, but can also play a deleterious role by causing heart attack and stroke. Normal platelet activation is dependent on tetraspanins, a superfamily of glycoproteins that function as 'organisers' of cell membranes by recruiting other receptors and signalling proteins into tetraspanin-enriched microdomains. However, our understanding of how tetraspanin microdomains regulate platelets is hindered by the fact that only four of the 33 mammalian tetraspanins have been identified in platelets. This is because of a lack of antibodies to most tetraspanins and difficulties in measuring mRNA, due to low levels in this anucleate cell. To identify potentially platelet-expressed tetraspanins, mRNA was measured in their nucleated progenitor cell, the megakaryocyte, using serial analysis of gene expression and DNA microarrays. Amongst 19 tetraspanins identified in megakaryocytes, Tspan9, a previously uncharacterized tetraspanin, was relatively specific to these cells. Through generating the first Tspan9 antibodies, Tspan9 expression was found to be tightly regulated in platelets. The relative levels of CD9, CD151, Tspan9 and CD63 were 100, 14, 6 and 2 respectively. Since CD9 was expressed at 49000 cell surface copies per platelet, this suggested a copy number of 2800 Tspan9 molecules. Finally, Tspan9 was shown to be a component of tetraspanin microdomains that included the collagen receptor GPVI (glycoprotein VI) and integrin alpha6beta1, but not the von Willebrand receptor GPIbalpha or the integrins alphaIIbbeta3 or alpha2beta1. These findings suggest a role for Tspan9 in regulating platelet function in concert with other platelet tetraspanins and their associated proteins.