Hic-5, a paxillin homologue, binds to the protein-tyrosine phosphatase PEST (PTP-PEST) through its LIM 3 domain

The Hic-5 protein is encoded by a transforming growth factor-beta1- and hydrogen peroxide-inducible gene, hic-5, and has striking similarity to paxillin, especially in their C-terminal LIM domains. Like paxillin, Hic-5 is localized in focal adhesion plaques in association with focal adhesion kinase in cultured fibroblasts. We carried out yeast two-hybrid screening to identify cellular factors that form a complex with Hic-5 using its LIM domains as a bait, and we identified a cytoplasmic tyrosine phosphatase (PTP-PEST) as one of the partners of Hic-5. These two proteins are associated in mammalian cells. From in vitro binding experiments using deletion and point mutations, it was demonstrated that the essential domain in Hic-5 for the binding was LIM 3. As for PTP-PEST, one of the five proline-rich sequences found on PTP-PEST, Pro-2, was identified as the binding site for Hic-5 in in vitro binding assays. Paxillin also binds to the Pro-2 domain of PTP-PEST. In conclusion, Hic-5 may participate in the regulation of signaling cascade through its interaction with distinct tyrosine kinases and phosphatases.     

Vascular endothelial growth factor accelerates compensatory lung growth after unilateral pneumonectomy

We hypothesize that compensatory lung growth after unilateral pneumonectomy in a murine model is, in part, angiogenesis dependent and can be altered using angiogenic agents, possibly through regulation of endothelial cell proliferation and apoptosis. Left pneumonectomy was performed in mice. Mice were then treated with proangiogenic factors [vascular endothelial growth factor (VEGF); basic fibroblast growth factor (bFGF)], VEGF receptor antibodies (MF-1, DC101), and VEGF receptor small molecule chemical inhibitors. Lung volume and mass were measured. The lungs were analyzed using immunohistochemistry by CD31 staining, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling, type II pneumocytes staining, and proliferating cell nuclear antigen. Compensatory lung growth was complete by postoperative day 10 and was associated with diffuse apoptosis of endothelial cells and pneumocytes. This process was accelerated by VEGF, such that growth was complete by postoperative day 4 with similar associated apoptosis. bFGF had no effect on lung growth. MF-1 and DC101 had no effect. The VEGF receptor small molecule chemical inhibitors also had no effect. VEGF, but not bFGF, accelerates growth. VEGF receptor inhibitors do not block growth, suggesting that other proangiogenic factors play a role or can compensate for VEGF receptor blockade. Diffuse apoptosis, endothelial cell and pneumocyte, occurs at cessation of both normal compensatory and VEGF-accelerated growth. Angiogenesis modulators may control growth via regulation of endothelial cell proliferation and apoptosis, although the exact relationship between endothelial cells and pneumocytes has yet to be determined. The fact that bFGF did not accelerate growth in our model when it did accelerate regeneration in the liver model suggests that angiogenesis during organ regeneration is regulated in an organ-specific manner.     

N-linked oligosaccharides are not involved in the function of a cell-cell binding glycoprotein E-cadherin

E-cadherin is a Ca2+-dependent cell-cell adhesion molecule identified as a glycoprotein with a molecular weight (MW) of 124,000. To study the role of the sugar moieties of this adhesion molecule, we tested the effect of tunicamycin on aggregation mediated by E-cadherin of teratocarcinoma cells. Immunoblot analysis using a monoclonal antibody to E-cadherin showed that in cells treated with tunicamycin this adhesion molecule is converted into two forms with MW of 118,000 and 131,000. The smaller one was exposed on the cell surface and showed a trypsin sensitivity characteristic to E-cadherin, suggesting that this is the peptide moiety of E-cadherin whose glycosylation with N-linked oligosaccharides was blocked by tunicamycin. The larger one was not removed by trypsin treatment of cells, suggesting an intracellular location. These tunicamycin-treated cells aggregated in a Ca2+-dependent manner, and the aggregation was inhibited by a monoclonal antibody to E-cadherin. These results suggested that N-linked oligosaccharides are not involved in the functional sites of this adhesion molecule.     

Highly potent nociceptin analog containing the Arg-Lys triple repeat

One of the structural characteristics of a neuropeptide nociceptin is the existence of Arg-Lys (RK) residues at positions 8-9 and 12-13; both RKs have been suggested to bind to the acidic amino acid cluster in the second extracellular loop of the seven transmembrane domain receptor ORL1. With a design strategy of attempting to obtain an analog that binds more strongly to the receptor's acidic cluster, we synthesized a series of nociceptin analogs in which the RK dipeptide unit was placed at positions 6-7, 10-11, or 14-15 adjacent to the parent RKs. Among these nociceptin analogs containing the RK triple repeat, [Arg-Lys(6-7)]- and [Arg-Lys(10-11)]nociceptins exhibited weak activities (6-9 and 60-90% of nociceptin, respectively) both in the receptor binding assay and in the [(35)S]GTPgammaS binding functional assay. In contrast, [Arg-Lys(14-15)]nociceptin was found to be very potent in both assays (3-fold in binding and 17-fold in GTPgammaS functional assay). [Arg-Lys(14-15)]nociceptin was the first peptide analog found to be stronger than the parent nociceptin, and structure-activity studies have suggested that the incorporated Arg-Lys(14-15) interacts with either the receptor acidic amino acid cluster or the receptor aromatic amino acid residues.     

Water and electrolyte balance in the vascular space during graded exercise in humans

We analyzed the changes in water content and electrolyte concentrations in the vascular space during graded exercise of short duration. Six male volunteers exercised on a cycle ergometer at 20 degrees C (relative humidity = 30%) as exercise intensity was increased stepwise until voluntary exhaustion. Blood samples were collected at exercise intensities of 29, 56, 70, and 95% of maximum aerobic power (VO2max). A curvilinear relationship between exercise intensity and Na+ concentration in plasma ([Na+]p) was observed. [Na+]p significantly increased at 70% VO2max and at 95% VO2max was approximately 8 meq/kgH2O higher than control. The change in lactate concentration in plasma ([Lac-]p) was closely correlated with the change in [Na+]p (delta[Na+]p = 0.687 delta[Lac-]p + 1.79, r = 0.99). The change in [Lac-]p was also inversely correlated with the change in HCO3- concentration in plasma (delta[HCO3-]p = -0.761 delta[Lac-]p + 0.22, r = -1.00). At an exercise intensity of 95% VO2max, 60% of the increase in plasma osmolality (Posmol) was accounted for by an increase in [Na+]p. These results suggest that lactic acid released into the vascular space from active skeletal muscles reacts with [HCO3-]p to produce CO2 gas and Lac-. The data raise the intriguing notion that increase in [Na+]p during exercise may be caused by elevated Lac-.     

Development of larval motor circuits in Drosophila

How are functional neural circuits formed during development? Despite recent advances in our understanding of the development of individual neurons, little is known about how complex circuits are assembled to generate specific behaviors. Here, we describe the ways in which Drosophila motor circuits serve as an excellent model system to tackle this problem. We first summarize what has been learned during the past decades on the connectivity and development of component neurons, in particular motor neurons and sensory feedback neurons. We then review recent progress in our understanding of the development of the circuits as well as studies that apply optogenetics and other innovative techniques to dissect the circuit diagram. New approaches using Drosophila as a model system are now making it possible to search for developmental rules that regulate the construction of neural circuits.     

Structure of the Ctr1 copper trans'PORE'ter reveals novel architecture

Copper is essential for biological processes such as free radical detoxification, mitochondrial respiration and iron metabolism. A central player in copper homeostasis is the high-affinity integral plasma membrane copper transporter Ctr1. However, the precise mechanisms by which Ctr1 functions are not known. Here, we highlight an important breakthrough in our understanding of how Ctr1 facilitates Cu(I) movement across membranes: the publication of structural details for human Ctr1 obtained from 2D crystallography and electron microscopy.     

A novel role for copper in Ras/mitogen-activated protein kinase signaling

Copper (Cu) is essential for development and proliferation, yet the cellular requirements for Cu in these processes are not well defined. We report that Cu plays an unanticipated role in the mitogen-activated protein (MAP) kinase pathway. Ablation of the Ctr1 high-affinity Cu transporter in flies and mouse cells, mutation of Ctr1, and Cu chelators all reduce the ability of the MAP kinase kinase Mek1 to phosphorylate the MAP kinase Erk. Moreover, mice bearing a cardiac-tissue-specific knockout of Ctr1 are deficient in Erk phosphorylation in cardiac tissue. in vitro investigations reveal that recombinant Mek1 binds two Cu atoms with high affinity and that Cu enhances Mek1 phosphorylation of Erk in a dose-dependent fashion. Coimmunoprecipitation experiments suggest that Cu is important for promoting the Mek1-Erk physical interaction that precedes the phosphorylation of Erk by Mek1. These results demonstrate a role for Ctr1 and Cu in activating a pathway well known to play a key role in normal physiology and in cancer.     

cDNA cloning of the housefly pigment-dispersing factor (PDF) precursor protein and its peptide comparison among the insect circadian neuropeptides.

Pigment-dispersing factor (PDF), an 18-amino acid neuropeptide, is a principal circadian neurotransmitter for the circadian rhythms of the locomotor activity in flies. Recently, two completely different types of PDF precursor were clarified; that of the cricket Gryllus bimaculatus and that of the last-summer cicada Meimuna opalifera. The G. bimaculatus PDF precursor is extraordinarily short and comprises a nuclear localization signal (NLS), while the M. opalifera PDF precursor is of ordinary length, comparable to that seen for the precursors of crustacean beta-PDH homologues. Although their PDF peptide regions were exactly the same, the regions containing a signal peptide combined with a PDF-associated peptide (PAP) were remarkably different from each other. Such a grouping suggested a fundamental role for the PAP peptide in the circadian clock, perhaps associated with PDF function. In the present study, the cDNA cloning of PDF from the adult brains of the housefly Musca domestica was carried out and it was found that an isolated clone (527 bp) encodes a PDF precursor protein of ordinary length. The PDF peptide shows a high sequence identity (78%-94%) and similarity (89%-100%) to insect PDFs and also to the crustacean beta-PDH peptides. In particular, there is only a single amino acid difference between the PDFs of Musca and Drosophila; at position 14 Ser for Musca PDF and Asn for Drosophila PDF. A characteristic Ser10 in Drosophila was retained in Musca, indicating the presence of a structural profile unique to these PDFs. The results of sequence analyses suggest that Musca and Drosophila PDFs are to be considered members of a single group that has evolved structurally. When the primary structure of the PAP regions was compared, the Musca PDF precursor also belonged to the same group as that to which the Drosophila PDF precursor belongs.