cGMP-mediated signaling via cGKIalpha is required for the guidance and connectivity of sensory axons

Previous in vitro studies using cGMP or cAMP revealed a cross-talk between signaling mechanisms activated by axonal guidance receptors. However, the molecular elements modulated by cyclic nucleotides in growth cones are not well understood. cGMP is a second messenger with several distinct targets including cGMP-dependent protein kinase I (cGKI). Our studies indicated that the alpha isoform of cGKI is predominantly expressed by sensory axons during developmental stages, whereas most spinal cord neurons are negative for cGKI. Analysis of the trajectories of axons within the spinal cord showed a longitudinal guidance defect of sensory axons within the developing dorsal root entry zone in the absence of cGKI. Consequently, in cGKI-deficient mice, fewer axons grow within the dorsal funiculus of the spinal cord, and lamina-specific innervation, especially by nociceptive sensory neurons, is strongly reduced as deduced from anti-trkA staining. These axon guidance defects in cGKI-deficient mice lead to a substantial impairment in nociceptive flexion reflexes, shown using electrophysiology. In vitro studies revealed that activation of cGKI in embryonic dorsal root ganglia counteracts semaphorin 3A-induced growth cone collapse. Our studies therefore reveal that cGMP signaling is important for axonal growth in vivo and in vitro.     

Beta3-adrenergic eNOS stimulation in left ventricular murine myocardium

This study investigates mechanisms underlying beta3-adrenergic activation of the endothelial nitric oxide synthase (eNOS) in myocardial tissue of wild-type (WT) and beta3-adrenoceptor knockout (beta3-KNO) mice, in the absence and presence of BRL 37344 (BRL), the preferential beta3-adrenoceptor selective agonist. Nitric oxide (NO)-liberation was measured after the application of BRL (10 micromol/L), using fluorescence dye diaminofluorescein (DAF), in left ventricular cardiac preparations. Phosphorylation of eNOSSer1177, eNOSThr495, eNOSSer114, and eNOS translocation, and alterations of 8-isoprostaglandin F2alpha (a parameter for reactive oxygen radical generation), after application of BRL (10 micromol/L), were studied using immunohistochemical stainings in isolated, electrically stimulated (1 Hz) right atrial (RA) and left ventricular (LV) myocardium. An increased NO release after BRL application (10 micromol/L) was observed in the RA and LV myocardial tissue of WT mice, but not in beta3-KNO mice. This NO liberation in WT mice was paralleled by an increased eNOSSer1177, but not eNOSThr495, phosphorylation. A cytosolic eNOS translocation was observed after the application of BRL (10 micromol/L) only in the RA myocardial tissue of WT mice. A BRL (10 micromol/L)-dependent increase in eNOSSer114 phosphorylation was observed only in the LV myocardial tissue of WT mice; this was paralleled by an increase in 8-isoprostaglandin F2alpha. In murine myocardium, 3 beta3-adrenoceptor-dependent activation pathways for eNOS exist (i.e., a translocation and phosphorylation of eNOSSer1177 and eNOSSer114). These pathways are used in a regional-dependent manner. beta3-adrenergic oxygen-derived free radical production might be important in situations of enhanced beta3-adrenoceptor activation, as has been described in human heart failure.     

Fesselin is a natively unfolded protein

Fesselin is a heat stable proline-rich actin binding protein. The stability, amino acid composition, and ability to bind to several proteins suggested that fesselin may be unfolded under native conditions. While the complete sequence of fesselin is unknown an analysis of a closely related protein, synaptopodin 2 from Gallus gallus, indicates that fesselin consists of a series of unstructured regions interspersed between short folded regions. To determine if fesselin is natively unfolded, we compared fesselin to a known globular protein (myosin S1) and a known unfolded protein Cad22 (the COOH terminal 22 kDa fragment of caldesmon). Fesselin, and Cad22, had larger Stokes radii than globular proteins of equivalent mass. The environments of tryptophan residues of fesselin and Cad22 were the same in the presence and absence of 6 M guanidine hydrochloride. Fesselin had a circular dichroism spectrum that was primarily random coil. Changes in pH over the range of 1.5-11.5 did not alter that spectrum. Increasing the temperature to 85 degrees C caused an increase in the degree of secondary structure. Calmodulin binding to fesselin altered the environment of the tryptophan residues so that they became less sensitive to the quencher acrylamide. These results show that fesselin is a natively unfolded protein.     

Improvement of pea biomass and seed productivity by simultaneous increase of phloem and embryo loading with amino acids

The development of sink organs such as fruits and seeds strongly depends on the amount of nitrogen that is moved within the phloem from photosynthetic‐active source leaves to the reproductive sinks. In many plant species nitrogen is transported as amino acids. In pea (Pisum sativum L.), source to sink partitioning of amino acids requires at least two active transport events mediated by plasma membrane‐localized proteins, and these are: (i) amino acid phloem loading; and (ii) import of amino acids into the seed cotyledons via epidermal transfer cells. As each of these transport steps might potentially be limiting to efficient nitrogen delivery to the pea embryo, we manipulated both simultaneously. Additional copies of the pea amino acid permease PsAAP1 were introduced into the pea genome and expression of the transporter was targeted to the sieve element‐companion cell complexes of the leaf phloem and to the epidermis of the seed cotyledons. The transgenic pea plants showed increased phloem loading and embryo loading of amino acids resulting in improved long distance transport of nitrogen, sink development and seed protein accumulation. Analyses of root and leaf tissues further revealed that genetic manipulation positively affected root nitrogen uptake, as well as primary source and sink metabolism. Overall, the results suggest that amino acid phloem loading exerts regulatory control over pea biomass production and seed yield, and that import of amino acids into the cotyledons limits seed protein levels.     

GDF-15 is an inhibitor of leukocyte integrin activation required for survival after myocardial infarction in mice

Inflammatory cell recruitment after myocardial infarction needs to be tightly controlled to permit infarct healing while avoiding fatal complications such as cardiac rupture. Growth differentiation factor-15 (GDF-15), a transforming growth factor-β (TGF-β)-related cytokine, is induced in the infarcted heart of mice and humans. We show that coronary artery ligation in Gdf15-deficient mice led to enhanced recruitment of polymorphonuclear leukocytes (PMNs) into the infarcted myocardium and an increased incidence of cardiac rupture. Conversely, infusion of recombinant GDF-15 repressed PMN recruitment after myocardial infarction. In vitro, GDF-15 inhibited PMN adhesion, arrest under flow and transendothelial migration. Mechanistically, GDF-15 counteracted chemokine-triggered conformational activation and clustering of β(2) integrins on PMNs by activating the small GTPase Cdc42 and inhibiting activation of the small GTPase Rap1. Intravital microscopy in vivo in Gdf15-deficient mice showed that Gdf-15 is required to prevent excessive chemokine-activated leukocyte arrest on the endothelium. Genetic ablation of β(2) integrins in myeloid cells rescued the mortality of Gdf15-deficient mice after myocardial infarction. To our knowledge, GDF-15 is the first cytokine identified as an inhibitor of PMN recruitment by direct interference with chemokine signaling and integrin activation. Loss of this anti-inflammatory mechanism leads to fatal cardiac rupture after myocardial infarction.     

AAP1 regulates import of amino acids into developing Arabidopsis embryos

The embryo of Arabidopsis seeds is symplasmically isolated from the surrounding seed coat and endosperm, and uptake of nutrients from the seed apoplast is required for embryo growth and storage reserve accumulation. With the aim of understanding the importance of nitrogen (N) uptake into developing embryos, we analysed two mutants of AAP1 (At1g58360), an amino acid transporter that was localized to Arabidopsis embryos. In mature and desiccated aap1 seeds the total N and carbon content was reduced while the total free amino acid levels were strongly increased. Separately analysed embryos and seed coats/endosperm of mature seeds showed that the elevated amounts in amino acids were caused by an accumulation in the seed coat/endosperm, demonstrating that a decrease in uptake of amino acids by the aap1 embryo affects the N pool in the seed coat/endosperm. Also, the number of protein bodies was increased in the aap1 endosperm, suggesting that the accumulation of free amino acids triggered protein synthesis. Analysis of seed storage compounds revealed that the total fatty acid content was unchanged in aap1 seeds, but storage protein levels were decreased. Expression analysis of genes of seed N transport, metabolism and storage was in agreement with the biochemical data. In addition, seed weight, as well as total silique and seed number, was reduced in the mutants. Together, these results demonstrate that seed protein synthesis and seed weight is dependent on N availability and that AAP1-mediated uptake of amino acids by the embryo is important for storage protein synthesis and seed yield.     

The p120 family of cell adhesion molecules

p120 is the prototypic member of the p120 subfamily of armadillo-related proteins that includes p0071, delta-catenin/NPRAP, ARVCF and the more distantly related plakophilins 1-3. Like armadillo, beta-catenin and plakoglobin these proteins are involved in mediating cell-cell adhesion. Besides their junctional localization they also reveal a cytoplasmic and nuclear localization. Non-cadherin-associated, cytoplasmic p120 functions in Rho signaling and regulation of cytoskeletal organization and actin dynamics. The nuclear function remains largely unsolved. Some characteristics seem to be shared by the various members of the family but it seems unlikely that p120-related proteins have solely redundant functions and compete for interactions with identical binding partners. Stabilization of cadherins at the membrane seems a common function of p120, p0071, delta-catenin and ARVCF but it is not yet known if and how these proteins confer distinct properties to cellular junctions. Moreover, p0071, NPRAP and ARVCF have a C-terminal PDZ-binding motif that is lacking in p120 pointing to distinct roles of these proteins. PDZ domains are found in a series of proteins involved in establishing cell polarity in epithelial cells. Thus, p120 proteins may not only be master regulators of cadherin abundance and activity but play additional roles in regulating cell polarity. This review focuses on the putative roles of p120 proteins in cell polarity.     

Protein transport in Archaea: Sec and twin arginine translocation pathways

The transport of proteins into and across hydrophobic membranes is an essential cellular process. The majority of proteins that are translocated in an unfolded conformation traverse the membrane by way of the universally conserved Sec pathway, whereas the twin arginine translocation pathway is responsible for the transport of folded proteins across the membrane. Structural, biochemical and genetic analyses of these processes in Archaea have revealed unique archaeal features, and have also provided a better understanding of these pathways in organisms of all domains. Further study of these pathways in Archaea might elucidate fundamental principles involved in each type of transport and could help determine their relative costs and benefits as well as evolutionary adaptations in protein secretion strategies.     

Archaeal and bacterial SecD and SecF homologs exhibit striking structural and functional conservation

The majority of secretory proteins are translocated into and across hydrophobic membranes via the universally conserved Sec pore. Accessory proteins, including the SecDF-YajC Escherichia coli membrane complex, are required for efficient protein secretion. E. coli SecDF-YajC has been proposed to be involved in the membrane cycling of SecA, the cytoplasmic bacterial translocation ATPase, and in the stabilizing of SecG, a subunit of the Sec pore. While there are no identified archaeal homologs of either SecA or SecG, many archaea possess homologs of SecD and SecF. Here, we present the first study that addresses the function of archaeal SecD and SecF homologs. We show that the SecD and SecF components in the model archaeon Haloferax volcanii form a cytoplasmic membrane complex in the native host. Furthermore, as in E. coli, an H. volcanii deltasecFD mutant strain exhibits both severe cold sensitivity and a Sec-specific protein translocation defect. Taken together, these results demonstrate significant functional conservation among the prokaryotic SecD and SecF homologs despite the distinct composition of their translocation machineries.