Solution structure of delta-Am2766: a highly hydrophobic delta-conotoxin from Conus amadis that inhibits inactivation of neuronal voltage-gated sodium channels

The three-dimensional (3D) NMR solution structure (MeOH) of the highly hydrophobic delta-conotoxin delta-Am2766 from the molluscivorous snail Conus amadis has been determined. Fifteen converged structures were obtained on the basis of 262 distance constraints, 25 torsion-angle constraints, and ten constraints based on disulfide linkages and H-bonds. The root-mean-square deviations (rmsd) about the averaged coordinates of the backbone (N, C(alpha), C) and (all) heavy atoms were 0.62+/-0.20 and 1.12+/-0.23 A, respectively. The structures determined are of good stereochemical quality, as evidenced by the high percentage (100%) of backbone dihedral angles that occupy favorable and additionally allowed regions of the Ramachandran map. The structure of delta-Am2766 consists of a triple-stranded antiparallel beta-sheet, and of four turns. The three disulfides form the classical 'inhibitory cysteine knot' motif. So far, only one tertiary structure of a delta-conotoxin has been reported; thus, the tertiary structure of delta-Am2766 is the second such example. Another Conus peptide, Am2735 from C. amadis, has also been purified and sequenced. Am2735 shares 96% sequence identity with delta-Am2766. Unlike delta-Am2766, Am2735 does not inhibit the fast inactivation of Na+ currents in rat brain Na(v)1.2 Na+ channels at concentrations up to 200 nM.     

Cospeciation and horizontal transmission of avian sarcoma and leukosis virus gag genes in galliform birds

In a study of the evolution and distribution of avian retroviruses, we found avian sarcoma and leukosis virus (ASLV) gag genes in 26 species of galliform birds from North America, Central America, eastern Europe, Asia, and Africa. Nineteen of the 26 host species from whom ASLVs were sequenced were not previously known to contain ASLVs. We assessed congruence between ASLV phylogenies based on a total of 110 gag gene sequences and ASLV-host phylogenies based on mitochondrial 12S ribosomal DNA and ND2 sequences to infer coevolutionary history for ASLVs and their hosts. Widespread distribution of ASLVs among diverse, endemic galliform host species suggests an ancient association. Congruent ASLV and host phylogenies for two species of Perdix, two species of Gallus, and Lagopus lagopus and L. mutus also indicate an old association with vertical transmission and cospeciation for these ASLVs and hosts. An inference of horizontal transmission of ASLVs among some members of the Tetraoninae subfamily (grouse and ptarmigan) is supported by ASLV monophyletic groups reflecting geographic distribution and proximity of hosts rather than host species phylogeny. We provide a preliminary phylogenetic taxonomy for the new ASLVs, in which named taxa denote monophyletic groups.     

Differentiation of human retinal pigment epithelial cells into neuronal phenotype by N-(4-hydroxyphenyl)retinamide

ARPE-19, a human retinal pigment epithelial (RPE) cell line, has been widely used in studies of RPE function as well as gene expression. Here, we report the novel finding that N-(4-hydroxyphenyl)retinamide (fenretinide), a synthetic retinoic acid derivative and a potential chemopreventive agent against cancer, induced the differentiation of ARPE-19 cells into a neuronal phenotype. The treated cells lost their epithelial phenotype and exhibited a typical neuronal shape with long processes (four to five times longer than the cell body). The onset of fenretinide-induced neuronal differentiation was dose and time dependent, started within 1-2 days, and lasted at least 4 weeks. Immunohistochemical studies indicated that the expression of neurofilament proteins (NF160 and NF200), calretinin and neural cell adhesion molecule was increased in these differentiated cells. Western blot analysis indicated that cellular retinaldehyde-binding protein, which is normally expressed in RPE cells, was decreased in treated cells. Protein analysis on a two-dimensional gel followed by matrix-assisted laser desorption ionization-time of flight mass spectrometric analysis demonstrated that heat-shock protein 70 was increased after fenretinide treatment. Thus, fenretinide, a synthetic retinoid, is able to induce neuronal differentiation of human RPE cells in culture.     

Kaposi's sarcoma-associated herpesvirus induces the phosphatidylinositol 3-kinase-PKC-zeta-MEK-ERK signaling pathway in target cells early during infection: implications for infectivity

Human herpesvirus 8 (HHV-8) is implicated in the pathogenesis of Kaposi's sarcoma. HHV-8 envelope glycoprotein B (gB) possesses the RGD motif known to interact with integrin molecules, and HHV-8 infectivity was inhibited by RGD peptides, by antibodies against alpha3 and beta1 integrins, and by soluble alpha3beta1 integrin (S. M. Akula, N. P. Pramod, F.-Z. Wang, and B. Chandran, Cell 108:407-419, 2002). Anti-gB antibodies immunoprecipitated the virus alpha3 and beta1 complexes, and virus-binding studies suggest a role for alpha3beta1 in HHV-8 entry. HHV-8 infection induced the integrin-mediated activation of focal adhesion kinase (FAK), implicating a role for integrin and the associated signaling pathways in HHV-8 entry into the target cells. Immediately after infection, target cells exhibited morphological changes and cytoskeletal rearrangements, suggesting the induction of signal pathways. As early as 5 min postinfection, HHV-8 activated the MEK-ERK1/2 pathway. The focal adhesion components phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase C-zeta (PKC-zeta) were recruited as upstream mediators of the HHV-8-induced ERK pathway. Anti-HHV-8 gB-neutralizing antibodies and soluble alpha3beta1 integrin inhibited the virus-induced signaling pathways. Early kinetics of the cellular signaling pathway and its activation by UV-inactivated HHV-8 suggest a role for virus binding and/or entry but not viral gene expression in this induction. Studies with human alpha3 integrin-transfected Chinese hamster ovary cells and FAK-negative mouse DU3 cells suggest that the alpha3beta1 integrin and FAK play roles in the HHV-8 mediated signal induction. Inhibitors specific for PI 3-kinase, PKC-zeta, MEK, and ERK significantly reduced the virus infectivity without affecting virus binding to the target cells. Examination of viral DNA entry suggests a role for PI 3-kinase in HHV-8 entry into the target cells and a role for PKC-zeta, MEK, and ERK at a post-viral entry stage of infection. These findings implicate a critical role for integrin-associated mitogenic signaling in HHV-8's infection of target cells and suggest that, by orchestrating the signal cascade, HHV-8 may create an appropriate intracellular environment to facilitate the infection.     

Phosphorylation of pyrimidine L-deoxynucleoside analog diphosphates. Kinetics of phosphorylation and dephosphorylation of nucleoside analog diphosphates and triphosphates by 3-phosphoglycerate kinase

Anticancer and antiviral D- and L-nucleoside analogs are phosphorylated stepwise in the cells to the pharmacologically active triphosphate metabolites. We recently reported that in the last step, L-deoxynucleoside analog diphosphates are phosphorylated by 3-phosphoglycerate kinase (PGK). To explain the preference of PGK for L- over D-deoxynucleoside analog diphosphates, the kinetics of their phosphorylation were compared with the dephosphorylation of the respective triphosphates using recombinant human PGK. The results attributed favorable phosphorylation of L-deoxynucleoside analog diphosphates by PGK to differences in k(cat), which were consequences of varied orientations of the sugar and diphosphates in the catalytic site of PGK. The amino acids involved in the catalytic reaction of PGK (including Glu(344), Lys(220), and Asn(337)) were therefore mutated. The impact of mutations on the phosphorylation of L- and D-deoxynucleoside analog diphosphates was different from those on dephosphorylation of the respective triphosphates. This suggested that the interactions of the nucleoside analogs with amino acids during the transition state are different in the phosphorylation and dephosphorylation reactions. Thus, reversible action of the enzyme may not involve the same configuration of the active site. Furthermore, the amino acid determinants of the action of PGK for L-deoxynucleotides were not the same as for the D-deoxynucleotides. This study also suggests the potential impact of nucleoside analog diphosphates and triphosphates on the multiple cellular functions of PGK, which may contribute to the action of the analogs.     

CRISPR Screens Uncover Genes that Regulate Target Cell Sensitivity to the Morphogen Sonic Hedgehog

1. Download high-res image (232KB)
2. Download full-size image

     

Anti-inflammatory activity of dimethyl octenol and oleanene tetrol isolated from <Emphasis Type="Italic">Trianthema decandra</Emphasis> L.

Dimethyl octenol from chloroform extract and oleanene tetrol from water extract of Trianthema decandra (TD) were isolated and characterized by using HPLC, UV, FT-IR, NMR, LC–MS and CHNS, their structure were elucidated from their respective spectral data. The anti-inflammatory activity of chloroform extract, water extract, dimethyl octenol and oleanene tetrol of T. decandra were studied and underlying cellular and molecular mechanisms of action were investigated in vitro and in vivo using macrophage-like cell line (RAW264.7 cells) and type II collagen induced arthritis mice models. Nitric oxide production was inhibited and TNF-α secretion was supressed in stimulated RAW cells treated with the chloroform extract and dimethyl octenol of T. decandra. Further, the chloroform and water extract, dimethyl octenol and oleanene tetrol inhibited protein denaturation and stabilized HRBC membranes in vitro. Reduction in inflammation as a measure of paw diameter was recorded in all the treated animals when compared to control animals. Catalase, peroxidase and glutathione peroxidase levels significantly increased in the joint tissue of treated groups. The possible mechanism of action of these compounds was studied using in silico molecular docking methods with phospholipase A₂ (PLA₂), cycloxygenase-1 (COX-1) and cycloxygenase-2 (COX-2) as targets. Among the three target proteins, the inhibition of the inflammatory protein PLA₂ and COX-2 towards dimethyl octenol and oleanene tetrol respectively. Our results contribute towards confirmation of the traditional use of TD and its compounds for the therapy of rheumatoid arthritis and other inflammatory joint disorders.     

Water-mediated interactions enable smooth substrate transport in a bacterial efflux pump

Background

Efflux pumps of the Resistance-Nodulation-cell Division superfamily confer multi-drug resistance to Gram-negative bacteria. The most-studied polyspecific transporter belonging to this class is the inner-membrane trimeric antiporter AcrB of Escherichia coli. In previous studies, a functional rotation mechanism was proposed for its functioning, according to which the three monomers undergo concerted conformational changes facilitating the extrusion of substrates. However, the molecular determinants and the energetics of this mechanism still remain unknown, so its feasibility must be proven mechanistically.