ABSTRACT
Sphingosine 1-phosphate receptor 1 (S1P(1)) is critical for lymphocyte recirculation and is a clinical target for treatment of multiple sclerosis. By generating a short-duration S1P(1) agonist and mice in which fluorescently tagged S1P(1) replaces wild-type receptor, we elucidate physiological and agonist-perturbed changes in expression of S1P(1) at a subcellular level in vivo. We demonstrate differential downregulation of S1P(1) on lymphocytes and endothelia after agonist treatment.
Subject(s)
Gene Knock-In Techniques , Green Fluorescent Proteins/chemistry , Multiple Sclerosis/drug therapy , Receptors, Lysosphingolipid/agonists , Receptors, Lysosphingolipid/therapeutic use , Animals , Down-Regulation/drug effects , Endothelium/drug effects , Endothelium/metabolism , Flow Cytometry , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Green Fluorescent Proteins/metabolism , Lymphocytes/drug effects , Lymphocytes/metabolism , Mice , Multiple Sclerosis/metabolism , Multiple Sclerosis/pathology , Receptors, Lysosphingolipid/metabolism , Time FactorsABSTRACT
Asymmetric scandium(III)-catalyzed rearrangement of 3-allyloxyflavones was utilized to prepare chiral, nonracemic 3,4-chromanediones in high yields and enantioselectivities. These synthetic intermediates have been further elaborated to novel heterocyclic frameworks including angular pyrazines and dihydropyrazines. The absolute configuration of rearrangement products was initially determined by a nonempirical analysis of circular dichroism (CD) using time-dependent density functional theory (TDDFT) calculations and verified by X-ray crystallography of a hydrazone derivative. Initial studies of the mechanism support an intramolecular rearrangement pathway that may proceed through a benzopyrylium intermediate.
Subject(s)
Chromans/chemical synthesis , Flavones/chemistry , Scandium/chemistry , Catalysis , Chromans/chemistry , Circular Dichroism , Crystallography, X-Ray , Models, Molecular , Molecular Structure , StereoisomerismABSTRACT
Poplars (Populus spp.) comprise an important component of circumpolar boreal forest ecosystems and are the model species for tree genomics. In this study, we surveyed genetic variation and population differentiation in three nuclear genes among populations of balsam poplar (Populus balsamifera) in North America. We examined nucleotide sequence variation in alcohol dehydrogenase 1 (Adh1) and glyceraldehyde 3-phosphate dehydrogenase (G3pdh), two well-studied nuclear loci in plants, and abscisic acid insensitivity 1B (ABI1B), a locus coincident with timing of seasonal dormancy in quantitative trait locus (QTL) studies of hybrid poplars. We compared estimates of baseline population genetic parameters for these loci with those obtained in studies of other poplar species, particularly European aspen (Populus tremula). Average pairwise nucleotide diversity (pi(tot) = 0.00216-0.00353) was equivalent to that in Populus trichocarpa, but markedly less than that in P. tremula. Elevated levels of population structure were observed in ABI1B between the northern and southern regions (F(CT) = 0.184, P < 0.001) and among populations (F(ST) = 0.256, P < 0.001). These results suggest that geographic or taxonomic factors are important for understanding patterns of variation throughout the genus Populus. Our findings have the potential to aid in the design of sampling regimes for conservation and breeding stock and contribute to historical inferences regarding the factors that shaped the genetic diversity of boreal plant species.
Subject(s)
Genetic Variation , Alcohol Dehydrogenase/genetics , Base Sequence , Geography , Glyceraldehyde 3-Phosphate/genetics , Haplotypes , North America , Population Dynamics , Populus/enzymology , Populus/genetics , Sample SizeABSTRACT
Aryl ether C-glycoside scaffolds have been prepared from tri-O-acetyl-D-glucal by C-glycosylation followed by allylic substitution with phenols mediated by Pd(0). The aryl ethers were subjected to either [3,3]-sigmatropic rearrangement to produce 3-pyranyl-phenols or Au(III)-mediated ring contraction to create highly substituted tetrahydrofurans. [structure: see text]
Subject(s)
Furans/chemical synthesis , Glycosides/chemistry , Glycosides/chemical synthesis , Phenols/chemical synthesis , Furans/chemistry , Glycosylation , Gold/chemistry , Molecular Structure , Phenols/chemistryABSTRACT
[reaction: see text] The synthesis of a complex hybrid oxime library is reported utilizing convergent ligation of alkoxyamine and carbonyl monomers via "chemical domain shuffling". Initial biological screening of the library against human small cell lung carcinoma (A549) cells led to the identification of a novel hybrid dimer in contrast to the corresponding monomeric compounds which were found to be inactive.
Subject(s)
Combinatorial Chemistry Techniques , Oximes/chemistry , Oximes/chemical synthesis , Indicators and Reagents , Molecular StructureABSTRACT
[structure: see text] Chitin synthase (CS) polymerizes UDP-GlcNAc to form chitin (poly-beta(1,4)-GlcNAc), a key component of fungal cell wall biosynthesis. Little is known about the substrate specificity of chitin synthase or the scope of substrate modification the enzyme will tolerate. Following a previous report suggesting that 6-O-dansyl GlcNAc is biosynthetically incorporated into chitin, we became interested in developing an assay for CS activity based on incorporation of a fluorescent substrate. We describe the synthesis of two fluorescent UDP-GlcNAc analogues and their evaluation as chitin synthase substrates.
Subject(s)
Chitin Synthase/metabolism , Uridine Diphosphate N-Acetylglucosamine/analogs & derivatives , Uridine Diphosphate N-Acetylglucosamine/metabolism , Chitin/biosynthesis , Chitin/chemistry , Chitin/metabolism , Fluorescent Dyes/chemistry , Molecular Structure , Substrate Specificity , Uridine Diphosphate N-Acetylglucosamine/chemical synthesis , Uridine Diphosphate N-Acetylglucosamine/chemistryABSTRACT
Chitin synthase (CS) is essential for fungal cell wall biosynthesis and is an attractive medicinal target. Expanded results from our efforts to develop mechanism based inhibitors of CS are presented here. Specifically, we describe uridine dimers linked by tartrate amides as potential pyrophosphate mimics.
Subject(s)
Antifungal Agents/chemical synthesis , Chitin Synthase/antagonists & inhibitors , Antifungal Agents/chemistry , Antifungal Agents/pharmacology , Cross-Linking Reagents , Dimerization , Enzyme Inhibitors/chemistry , Structure-Activity Relationship , Tartrates , UridineABSTRACT
Chitin synthase polymerizes UDP-GlcNAc to form chitin (poly-beta(1,4)-GlcNAc) and is essential for fungal cell wall biosynthesis. The alternating orientation of the GlcNAc residues within the chitin chain has led to the proposal that chitin synthase possesses two active sites. We report the results of the first direct test of this possibility. Two simple uridine-derived dimeric inhibitors are shown to exhibit 10-fold greater inhibition than a monomeric control, consistent with the presence of two active sites. This observation has important implications for the development of antifungal agents, as well as the understanding of polymerizing glycosyltransferases.
Subject(s)
Chitin Synthase/chemistry , Chitin Synthase/metabolism , Binding Sites , Carbohydrate Sequence , Chitin/biosynthesis , Chitin Synthase/antagonists & inhibitors , Deoxyuridine/analogs & derivatives , Deoxyuridine/chemical synthesis , Deoxyuridine/pharmacology , Dimerization , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Molecular Sequence Data , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae Proteins/antagonists & inhibitors , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Uridine Diphosphate/analogs & derivatives , Uridine Diphosphate/metabolismABSTRACT
Chitin synthase is responsible for the biosynthesis of chitin, an essential component of the fungal cell wall. There is a long-standing question as to whether "processive" transferases such as chitin synthase operate in the same manner as non-processive transferases. The question arises from analysis of the polysaccharide structure--in chitin, for instance, each sugar residue is rotated approximately 180 degrees relative to the preceding sugar in the chain. This requires that the enzyme account for the alternating "up/down" configuration during biosynthesis. An enzyme with a single active site, analogous to the non-processive transferases--would have to accommodate a distorted glycosidic linkage at every other synthetic step. An alternative proposal is that the enzyme might assemble the disaccharide donor, addressing the "up/down" conformational problem prior to polymer synthesis. We present compelling evidence that this latter hypothesis is incorrect.
