Netrin-4 acts as a pro-angiogenic factor during zebrafish development

Netrins form a heterogeneous family of laminin-related molecules with multifunctional activities. Netrin-4, the most distant member of this family, is related to the laminin β chain and has recently been proposed to play an important role in embryonic and pathological angiogenesis. However, the data reported so far lead to the apparently contradictory conclusions supporting Netrin-4 as either a pro- or an anti-angiogenic factor. To elucidate this controversy, Netrin-4 was analyzed for a vascular activity in both cell-based models (human umbilical vein endothelial cells and human umbilical artery endothelial cells) and two zebrafish models: the wild-type AB/Tü strain and the transgenic Tg(fli1a:EGFP)(y1) strain. We show that Netrin-4 is expressed in endothelial cells and in the zebrafish vascular system. We also show evidence that Netrin-4 activates various kinases and induces various biological effects directly linked to angiogenesis in vitro. Using a morpholinos strategy, we demonstrate that Netrin-4 expression is crucial for zebrafish vessel formation and that a blood vessel formation defect induced by netrin-4 morpholinos can be partially rescued through drug delivery leading to protein kinase activation. Together these data underscore the crucial role of Netrin-4 in blood vessel formation and the involvement of protein kinases activation in Netrin-4-induced biological effects related to vascular development.     

Regulation of temporal identities during Drosophila neuroblast lineage development

One of the major goals of neurobiology is to describe, in molecular terms, how a neural progenitor cell can generate an ordered series of uniquely fated neurons and glia. It has become clear that many, or all, neural-subtype identities can be linked to sequentially changing regulatory programs within neural precursors. Recent studies shed light on regulatory inputs and timing mechanisms that generate temporally defined cell identities, and new contributions are beginning to establish a link between the temporal network and cell function.     

Survivin acts as anti-apoptotic factor during the development of bovine pre-implantation embryos

Survivin, an inhibitor of apoptotic protein containing a single baculoviral inhibit apoptotic protein repeat domain, is a bifunctional protein that suppresses apoptosis and regulates cell division. Thus, we used double stranded RNA (dsRNA) interference to manipulate survivin expression in bovine embryos and analyze its role in blocking apoptosis and facilitating development of pre-implantation embryos. In vitro fertilized embryos (1-cell) were injected with survivin dsRNA, and expression of survivin mRNA was evaluated by real-time quantitative RT-PCR. To analyze survivin protein expression, we performed immunocytochemistry using a rabbit anti-bovine suvivin antibody. Expression levels of survivin mRNA and protein were decreased in the dsRNA group compared to the sham group. Rates of in vitro blastocyst development were lower in the survivin dsRNA-injected group than in the sham-injected group. Also, the total cell number seen in blastocysts was decreased in the dsRNA group. TUNEL assays of DNA fragmentation indicated an increased apoptotic index in the dsRNA group compared to the sham group. These results indicate that survivin is important for optimal development of bovine blastocysts and confirm that survivin expression suppresses apoptosis of pre-implantation embryos.     

Erk5 is activated and acts as a survival factor in mitosis

Erk5 is a recently discovered MAPK claimed to be responsible for some of the roles attributed to Erk1/2; here we report that it is activated in mitosis in comparison to G1/S. When Erk5 is inactivated pharmacologically or largely ablated by RNAi, cell survival in mitosis is diminished. We have previously shown Bim, a BH3-only protein of the Bcl-2 family, to be phosphorylated in mitosis, in a MEK-dependent manner (M. Grãos, A. D. Almeida, S. Chatterjee, Biochem. J. 388 (2005) 185). Inactivation of Erk5 in mitosis causes dephosphorylation of Bim. Bim is in the mitochondria in mitosis and when dephosphorylated interacts with Bax, inducing caspase activation. We also show that in mitosis Bim co-immunoprecipitates with Erk5 and Erk5 phosphorylates GST-Bim in in vitro kinase reaction. Taken together, our results identify a new target of the still largely mysterious Erk5 and suggest that Erk5 in mitosis may be a decisive step for the survival of proliferating cells.     

GABA as a trophic factor during development

The process of synaptogenesis has been studied by many investigators to determine the factors which regulate synapse formation. We have used neonatal rabbit retina to investigate the role of the gamma-aminobutyric acid (GABA) neurotransmitter system during development. By utilizing an in vitro incubation treatment of isolated eyecups we found that treatment with nipecotic acid, a GABA uptake blocker, resulted in a 4-fold increase in the amount of specific 3H-muscimol binding. In addition, incubation of the tissue in the presence of the GABA agonists muscimol, 4,5,6,7-tetrahydroisoxazolo [5,4-c]pyridine-3-ol (THIP), or GABA itself led to similar increases in specific 3H-muscimol binding. The findings support the conclusion from previous studies that the induction of GABA receptors observed after in vivo treatment of 1-day-old rabbits with nipecotic acid resulted from an increase in the extracellular concentration of GABA. A possible role for GABA in the regulation of GABAergic synapse formation is presented in this report.     

Eukaryotic translation initiation factor 5 (eIF5) acts as a classical GTPase-activator protein

GTP hydrolysis occurs at several specific stages during the initiation, elongation, and termination stages of mRNA translation. However, it is unclear how GTP hydrolysis occurs; it has previously been suggested to involve a GTPase active center in the ribosome, although proof for this is lacking. Alternatively, it could involve the translation factors themselves, e.g., be similar to the situation for small G in which the GTPase active site involves arginine residues contributed by a further protein termed a GTPase-activator protein (GAP). During translation initiation in eukaryotes, initiation factor eIF5 is required for hydrolysis of GTP bound to eIF2 (the protein which brings the initiator Met-tRNA(i) to the 40S subunit). Here we show that eIF5 displays the hallmarks of a classical GAP (e.g., RasGAP). Firstly, its interaction with eIF2 is enhanced by AlF(4)(-). Secondly, eIF5 possesses a conserved arginine (Arg15) which, like the "arginine fingers" of classical GAPs, is flanked by hydrophobic residues. Mutation of Arg15 to methionine abolishes the ability of eIF5 either to stimulate GTP hydrolysis or to support mRNA translation in vitro. Mutation studies suggest that a second conserved arginine (Arg48) also contributes to the GTPase active site of the eIF2.eIF5 complex. Our data thus show that eIF5 behaves as a classical GAP and that GTP hydrolysis during translation involves proteins extrinsic to the ribosome. Indeed, inspection of their sequences suggests that other translation factors may also act as GAPs.     

Growth hormone releasing peptide-6 acts as a survival factor in glutamate-induced excitotoxicity

Chronic systemic treatment given to adult male rats with growth hormone releasing peptide-6, an agonist of the ghrelin receptor, increases insulin-like growth factor I levels in various brain regions, including the hypothalamus and cerebellum. Furthermore, intracellular signalling cascades normally associated with anti-apoptotic actions are activated in the same areas and are coincident with decreased basal cell death. Because abnormally high concentrations of glutamate can lead to overexcitation of neurones leading to cell damage and/or death, we investigated whether administration of growth hormone releasing peptide-6 attenuates monosodium glutamate-induced apoptosis in the rat hypothalamus and cerebellum. Glutamate increased activation of caspase 9 followed by cleavage of caspase 7, which in turn fragmented poly(ADP-ribose) polymerase, terminating in cell death in both the hypothalamus and cerebellum. Growth hormone releasing peptide-6 reversed glutamate-induced cell death by decreasing activation of caspases 9 and 7 and poly(ADP-ribose) polymerase fragmentation. These results provide a better understanding of the neuroprotective role of growth hormone secretagogues and the mechanisms involved.     

The basement membrane protein laminin-5 acts as a soluble cell motility factor

One of the basement membrane (BM) proteins, laminin-5 (LN5), is known to support efficient cell adhesion and migration through interaction with integrins on the basal plasma membrane. Here, we show that a soluble form of LN5 induced migration of human epithelial cells and carcinoma cells by interacting with integrins on the apical cell surface. Although both LN5 and laminin-10/11 (LN10/11) promoted cell migration when coated onto a plastic surface as insoluble substrata, only LN5 stimulated cell migration in its soluble form on other substrata such as fibronectin (FN), vitronectin (VN) and collagen. Soluble LN5 interacted with integrins alpha3beta1 and alpha6beta1 on the apical cell surface and stimulated cell migration, while the cell morphology was largely dependent on the underlying substratum. Thus, integrin signals from the apical surface and the basal surface synergistically regulated cytoskeletal organization and cell motility. Soluble and insoluble LN5 induced cell motility by activating signal pathways via protein kinase C (PKC), phosphoinositide 3-OH kinase (PI3-K) and MAP kinase. The PKC dependency was more prominent for soluble LN5 than insoluble LN5, and was absent in the stimulation by insoluble LN10/11. In vitro scratch assays with keratinocytes, self-produced soluble LN5 bound to the apical cell surface of migrating cells at the scratched edges, suggesting that soluble LN5 may contribute to cell migration in pathological conditions such as wound healing and tumor invasion.     

Amylases during different stages of seed development in wheat

The β-Amylase activity was high in seeds of pre-Milk and milk stages but low in seeds of half seed and dough stages and very low at fully ripe stage of seed development. Zymogen (latent β-Amylase) was absent in half seed stage but appeared at pre-Milk stage. The content of zymogen increased progressively with advance in ripening time. No α-Amylase activity was detected at any stages of seed development in wheat.     

Induction of helmet development by a Chaoborus factor in Daphnia ambigua during juvenile stages

Daphnia ambigua juveniles developed spike-like helmets inducedby a water-soluble factor from Chaoborus, when reared in Chaoborusflavicans-conditioned medium. A study was made of the time atwhich the Chaoborus factor affects helmet development. Juvenilesdeveloped the helmets when individuals were exposed to the Chaoborusfactor after being born, but did not develop the helmets whenthe individuals were exposed to the factor only in the egg orembryo stages. These results indicate that D.ambigua respondsto the Chaoborus factor during the juvenile stages.     

Netrin-1 acts as a survival factor via its receptors UNC5H and DCC

The membrane receptors DCC and UNC5H have been shown to be crucial for axon guidance and neuronal migration by acting as receptors for netrin-1. DCC has also been proposed as a dependence receptor inducing apoptosis in cells that are beyond netrin-1 availability. Here we show that the netrin-1 receptors UNC5H (UNC5H1, UNC5H2, UNC5H3) also act as dependence receptors. UNC5H receptors induce apoptosis, but this effect is blocked in the presence of netrin-1. Moreover, we demonstrate that UNC5H receptors are cleaved in vitro by caspase in their intracellular domains. This cleavage may lead to the exposure of a fragment encompassing a death domain required for cell death induction in vivo. Finally, we present evidence that during development of the nervous system, the presence of netrin-1 is crucial to maintain survival of UNC5H- and DCC-expressing neurons, especially in the ventricular zone of the brainstem. Altogether, these results argue for a role of netrin-1 during the development of the nervous system, not only as a guidance cue but as a survival factor via its receptors DCC and UNC5H.