Post-translational and transcriptional regulation of phenylpropanoid biosynthesis pathway by Kelch repeat F-box protein SAGL1

Plant Molecular Biology - A Kelch repeat F-box containing protein, SMALL AND GLOSSY LEAVES1 (SAGL1) regulates phenylpropanoid biosynthesis as a post-translational regulator for PAL1 (phenylalanine...     

Generation of a Persistently Infected MDBK Cell Line with Natural Bovine Spongiform Encephalopathy (BSE)

Bovine spongiform encephalopathy (BSE) is a zoonotic transmissible spongiform encephalopathy (TSE) thought to be caused by the same prion strain as variant Creutzfeldt-Jakob disease (vCJD). Unlike scrapie and chronic wasting disease there is no cell culture model allowing the replication of proteinase K resistant BSE (PrP^BSE) and the further in vitro study of this disease. We have generated a cell line based on the Madin-Darby Bovine Kidney (MDBK) cell line over-expressing the bovine prion protein. After exposure to naturally BSE-infected bovine brain homogenate this cell line has shown to replicate and accumulate PrP^BSE and maintain infection up to passage 83 after initial challenge. Collectively, we demonstrate, for the first time, that the BSE agent can infect cell lines over-expressing the bovine prion protein similar to other prion diseases. These BSE infected cells will provide a useful tool to facilitate the study of potential therapeutic agents and the diagnosis of BSE.     

A newly synthesized,potent tyrosinase inhibitor: 5-(6-Hydroxy-2-naphthyl)-1,2,3-benzenetriol

In searching for new agents with a depigmenting effect, we synthesized a derivative of resveratrol, 5-(6-hydroxy-2-naphthyl)-1,2,3-benzenetriol (5HNB) with a potent tyrosinase inhibitory activity. 5HNB inhibited mushroom tyrosinase with an IC₅₀ value of 2.95 μM, which is more potent than the well-known anti-tyrosinase activity of kojic acid (IC₅₀ = 38.24). The results of the enzymatic inhibition kinetics by Lineweaver–Burk analysis indicated 5HNB inhibits tyrosinase non-competitively when l-tyrosine was used as the substrate. Based on the strong inhibitory action of 5HNB, it is expected that 5HNB can suppress melanin production in which tyrosinase plays the essential role. Our expectation was confirmed by the experimentations with B16 melanoma cells in which 5HNB inhibited melanin production. We propose that 5HNB might have skin-whitening effects as well as therapeutic potential for treating skin pigmentation disorders.     

AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation

The phosphoinositide 3-kinase–Akt signaling pathway is essential to many biological processes, including cell proliferation, survival, metabolism, and angiogenesis, under pathophysiological conditions. Although 3-phosphoinositide–dependent kinase 1 (PDK1) is a primary activator of Akt at the plasma membrane, the optimal activation mechanism remains unclear. We report that adhesion molecule with IgG-like domain 2 (AMIGO2) is a novel scaffold protein that regulates PDK1 membrane localization and Akt activation. Loss of AMIGO2 in endothelial cells (ECs) led to apoptosis and inhibition of angiogenesis with Akt inactivation. Amino acid residues 465–474 in AMIGO2 directly bind to the PDK1 pleckstrin homology domain. A synthetic peptide containing the AMIGO2 465–474 residues abrogated the AMIGO2–PDK1 interaction and Akt activation. Moreover, it effectively suppressed pathological angiogenesis in murine tumor and oxygen-induced retinopathy models. These results demonstrate that AMIGO2 is an important regulator of the PDK1–Akt pathway in ECs and suggest that interference of the PDK1–AMIGO2 interaction might be a novel pharmaceutical target for designing an Akt pathway inhibitor.     

Surrogate reporters for enrichment of cells with nuclease-induced mutations

Zinc-finger nucleases (ZFNs) and TAL-effector nucleases (TALENs) are powerful tools for creating genetic modifications in eukaryotic cells and organisms. But wild-type and mutant cells that contain genetic modifications induced by these programmable nucleases are often phenotypically indistinguishable, hampering isolation of mutant cells. Here we show that transiently transfected episomal reporters encoding fluorescent proteins can be used as surrogate genes for the efficient enrichment of endogenous gene-modified cells by flow cytometry.     

Development of selective agonists and antagonists of P2Y receptors

Although elucidation of the medicinal chemistry of agonists and antagonists of the P2Y receptors has lagged behind that of many other members of group A G protein-coupled receptors, detailed qualitative and quantitative structure–activity relationships (SARs) were recently constructed for several of the subtypes. Agonists selective for P2Y₁, P2Y₂, and P2Y₆ receptors and nucleotide antagonists selective for P2Y₁ and P2Y₁₂ receptors are now known. Selective nonnucleotide antagonists were reported for P2Y₁, P2Y₂, P2Y₆, P2Y₁₁, P2Y₁₂, and P2Y₁₃ receptors. At the P2Y₁ and P2Y₁₂ receptors, nucleotide agonists (5′-diphosphate derivatives) were converted into antagonists of nanomolar affinity by altering the phosphate moieties, with a focus particularly on the ribose conformation and substitution pattern. Nucleotide analogues with conformationally constrained ribose-like rings were introduced as selective receptor probes for P2Y₁ and P2Y₆ receptors. Screening chemically diverse compound libraries has begun to yield new lead compounds for the development of P2Y receptor antagonists, such as competitive P2Y₁₂ receptor antagonists with antithrombotic activity. Selective agonists for the P2Y₄, P2Y₁₁, and P2Y₁₃ receptors and selective antagonists for P2Y₄ and P2Y₁₄ receptors have not yet been identified. The P2Y₁₄ receptor appears to be the most restrictive of the class with respect to modification of the nucleobase, ribose, and phosphate moieties. The continuing process of ligand design for the P2Y receptors will aid in the identification of new clinical targets.     

Clec14a is specifically expressed in endothelial cells and mediates cell to cell adhesion

Clec14a is a member of the thrombomodulin (TM) family, but its function has not yet been determined. Here, we report that Clec14a is a plasma membrane protein of endothelial cells (ECs) expressed specifically in the vasculature of mice. Deletion mutant analysis revealed that Clec14a mediates cell–cell adhesion through its C-type lectin-like domain. Knockdown of Clec14a in ECs suppressed cell migratory activity and filopodial protrusion, and delayed formation of tube-like structures. These findings demonstrate that Clec14a is a novel EC-specific protein that appears to play a role in cell–cell adhesion and angiogenesis.