ABSTRACT
Interleukin (IL)-18 is the interferon-gamma-inducing factor and has other proinflammatory properties. The precise role of IL-18 in immunoinflammatory diseases remains poorly understood. In this study, we show that in vivo electrotransfer of an expression-plasmid DNA encoding for murine IL-18 binding protein (BP) (the endogenous inhibitor of IL-18) prevents fatty streak development in the thoracic aorta of apoE knockout mice and slows progression of advanced atherosclerotic plaques in the aortic sinus. More importantly, transfection with the IL-18BP plasmid induces profound changes in plaque composition (decrease in macrophage, T cell, cell death, and lipid content and increase in smooth muscle cell and collagen content) leading to a stable plaque phenotype. These results identify for the first time a critical role for IL-18/IL-18BP regulation in atherosclerosis and suggest a potential role for IL-18 inhibitors in reduction of plaque development/progression and promotion of plaque stability. The full text of this article is available at http://www.circresaha.org.
Subject(s)
Arteriosclerosis/prevention & control , DNA, Complementary/administration & dosage , Glycoproteins/administration & dosage , Interleukin-18/antagonists & inhibitors , Signal Transduction/drug effects , Animals , Aorta, Thoracic/drug effects , Aorta, Thoracic/pathology , Apolipoproteins E/deficiency , Apolipoproteins E/genetics , Arteriosclerosis/genetics , Arteriosclerosis/pathology , DNA, Complementary/genetics , Disease Models, Animal , Disease Progression , Electroporation , Genetic Therapy/methods , Glycoproteins/genetics , Injections, Intramuscular , Intercellular Signaling Peptides and Proteins , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Phenotype , Plasmids/administration & dosage , Plasmids/genetics , Signal Transduction/genetics , Sinus of Valsalva/pathology , Treatment OutcomeABSTRACT
The public availability of numerous microbial genomes is enabling the analysis of bacterial biology in great detail and with an unprecedented, organism-wide and taxon-wide, broad scope. Streptococcus pneumoniae is one of the most important bacterial pathogens throughout the world. We present here sequences and functional annotations for 2.1-Mbp of pneumococcal DNA, covering more than 90% of the total estimated size of the genome. The sequenced strain is a clinical isolate resistant to macrolides and tetracycline. It carries a type 19F capsular locus, but multilocus sequence typing for several conserved genetic loci suggests that the strain sequenced belongs to a pneumococcal lineage that most often expresses a serotype 15 capsular polysaccharide. A total of 2,046 putative open reading frames (ORFs) longer than 100 amino acids were identified (average of 1,009 bp per ORF), including all described two-component systems and aminoacyl tRNA synthetases. Comparisons to other complete, or nearly complete, bacterial genomes were made and are presented in a graphical form for all the predicted proteins.
Subject(s)
Genome, Bacterial , Pneumococcal Infections/microbiology , Streptococcus pneumoniae/genetics , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Genes, Bacterial/genetics , Molecular Sequence Data , Prokaryotic Cells , RNA, Transfer/metabolismABSTRACT
Xenopus oocytes were injected with RNAs for the two inward-rectifier potassium channel subunits Kir3.1 (GIRK1) and Kir3.4 (rcKATP or CIR) in addition to RNA from the neuroblastoma cell line KAN-TS. Potassium currents were evoked by neuropeptide Y in oocytes injected with polyadenylated RNA or with cRNA from pools of a neuroblastoma (KAN-TS) cDNA library, and progressive subdivision of responding pools yielded a single cDNA. The encoded protein contains 381 amino acids, has the seven hydrophobic domains characteristic of G protein-coupled receptors, and is 31% identical to the Y1 receptor: potassium currents were induced by neuropeptide Y (EC50=60pm) and Y2-selective analogues. Coexpression with potassium channel subunits will be a generally useful method for the cloning of G protein-coupled receptors.
Subject(s)
Neuropeptide Y/metabolism , Potassium Channels/genetics , Receptors, Neuropeptide Y/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Humans , Molecular Sequence Data , Neuroblastoma , Neuropeptide Y/pharmacology , Oocytes/physiology , Oocytes/ultrastructure , Potassium Channels/metabolism , Potassium Channels/physiology , RNA/genetics , RNA, Messenger/genetics , Receptors, Neuropeptide Y/metabolism , Sequence Homology, Amino Acid , Tumor Cells, Cultured , XenopusABSTRACT
The structure of the mouse 5-HT3 receptor gene, 5-HT3R-A, is most similar to nicotinic acetylcholine receptor (nAChR) genes, in particular to the gene encoding the neuronal nAChR subunit alpha 7. These genes share among other things the location of three adjacent introns, suggesting that 5-HT3R-A and nAChR genes arose from a common precursor gene. The alternative use of two adjacent splice acceptor sites in intron 8 creates, in addition to the original 5-HT3R-A cDNA (5-HT3R-AL), a shorter isoform (5-HT3R-AS) which lacks six codons in the segment that translates into the major intracellular domain. This splice consensus sequence is not found in human genomic DNA. In mouse, we demonstrate by RNAse protection assay that 5-HT3R-AS mRNA is approximately 5 times more abundant than 5-HT3R-AL mRNA in both neuroblastoma cell lines and neuronal tissues. We used the Semliki Forest virus expression system for electrophysiological characterization of 5-HT3R-AS and 5-HT3R-AL in mammalian cells. No differences in electrophysiological characteristics, such as voltage dependence, desensitization kinetics, or unitary conductance were found between homomeric 5-HT3R-AS and 5-HT3R-AL receptors. Their properties are very similar to those of 5-HT3 receptors in mouse neuroblastoma cell lines.
Subject(s)
Alternative Splicing , Gene Expression , Genetic Variation , Mice/genetics , Receptors, Serotonin/biosynthesis , Receptors, Serotonin/genetics , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Cloning, Molecular , Codon/genetics , Consensus Sequence , DNA/chemistry , DNA/genetics , DNA Primers , Humans , Introns , Kinetics , Molecular Sequence Data , Muscles/metabolism , Neurons/metabolism , Polymerase Chain Reaction , Protein Biosynthesis , Receptors, Serotonin/physiology , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Sequence Homology, Nucleic Acid , Serotonin/pharmacology , Transfection , VertebratesABSTRACT
Immunoglobulin E (IgE) mediates many allergic responses. CD23 is a 45-kilodalton type II transmembrane glycoprotein expressed in many cell types. It is a low-affinity IgE receptor and interacts specifically with CD21, thereby modulating IgE production by B lymphocytes in vitro. In an in vivo model of an allergen-specific IgE response, administration of a rabbit polyclonal antibody to recombinant human truncated CD23 resulted in up to 90 percent inhibition of ovalbumin-specific IgE synthesis. Both Fabs and intact IgG inhibited IgE production in vitro and in vivo. Thus, CD23 participates in the regulation of IgE synthesis in vivo and so could be important in allergic disease.
