ABSTRACT
The A1-adenosine receptor (A1-AR) is a G protein-coupled receptor that mediates many of the physiological effects of adenosine in the brain, heart, kidney, and adipocytes. Currently, ligand interactions with the A1-AR can be quantified on large cell populations only by using radioligand binding. To increase the resolution of these measurements, we have designed and characterized a previously undescribed fluorescent antagonist for the A1-AR, XAC-BY630, based on xanthine amine congener (XAC). This compound has been used to quantify ligand-receptor binding at a single cell level using fluorescence correlation spectroscopy (FCS). XAC-BY630 was a competitive antagonist of A1-AR-mediated inhibition of cAMP accumulation [log10 of the affinity constant (pKb) = 6.7)] and stimulation of inositol phosphate accumulation (pKb = 6.5). Specific binding of XAC-BY630 to cell surface A1-AR could also be visualized in living Chinese hamster ovary (CHO)-A1 cells by using confocal microscopy. FCS analysis of XAC-BY630 binding to the membrane of CHO-A1 cells revealed three components with diffusion times (tauD) of 62 micros (tauD1, free ligand), 17 ms (tauD2, A1-AR-ligand), and 320 ms (tauD3). Confirmation that tauD2 resulted from diffusion of ligand-receptor complexes came from the similar diffusion time observed for the fluorescent A1-AR-Topaz fusion protein (15 ms). Quantification of tauD2 showed that the number of receptor-ligand complexes increased with increasing free ligand concentration and was decreased by the selective A1-AR antagonist, 8-cyclopentyl-1,3-dipropylxanthine. The combination of FCS with XAC-BY630 will be a powerful tool for the characterization of ligand-A1-AR interactions in single living cells in health and disease.
Subject(s)
Adenosine A1 Receptor Antagonists , Receptor, Adenosine A1/physiology , Xanthines/pharmacology , Animals , Base Sequence , Binding Sites , CHO Cells , Cell Membrane/physiology , Cricetinae , DNA Primers , Microscopy, Confocal , Polymerase Chain Reaction , Xanthines/pharmacokineticsABSTRACT
The proteasome, an essential component of the ATP-dependent proteolytic pathway in eukaryotic cells, is responsible for the degradation of most cellular proteins and is believed to be the main source of MHC class I-restricted antigenic peptides for presentation to CTL. Inhibition of the proteasome by lactacystin or various peptide aldehydes can result in defective Ag presentation, and the pivotal role of the proteasome in Ag processing has become generally accepted. However, recent reports have challenged this observation. Here we examine the processing requirements of two HLA A*0201-restricted epitopes from HIV-1 reverse transcriptase and find that they are produced by different degradation pathways. Presentation of the C-terminal ILKEPVHGV epitope is impaired in ME275 melanoma cells by treatment with lactacystin, and is independent of expression of the IFN-gamma-inducible proteasome beta subunits LMP2 and LMP7. In contrast, both lactacystin treatment and expression of LMP7 induce the presentation of the N-terminal VIYQYMDDL epitope. Consistent with these observations we show that up-regulation of LMP7 by IFN-gamma enhances presentation of the VIYQYMDDL epitope. Hence interplay between constitutive and IFN-gamma-inducible beta-subunits of the proteasome can qualitatively influence Ag presentation. These observations may have relevance to the patterns of immunodominance during the natural course of viral infection.
Subject(s)
Epitopes, T-Lymphocyte/metabolism , HIV Reverse Transcriptase/immunology , HIV Reverse Transcriptase/metabolism , HIV-1/enzymology , Interferon-gamma/pharmacology , T-Lymphocytes, Cytotoxic/immunology , ATP Binding Cassette Transporter, Subfamily B, Member 2 , ATP-Binding Cassette Transporters/genetics , Acetylcysteine/analogs & derivatives , Acetylcysteine/pharmacology , Antigen Presentation/drug effects , Antigen Presentation/genetics , Cell Line , Cysteine Endopeptidases/metabolism , Cysteine Endopeptidases/physiology , Enzyme Inhibitors/pharmacology , Gene Deletion , HIV-1/immunology , Histocompatibility Antigens Class II/genetics , Humans , Multienzyme Complexes/metabolism , Multienzyme Complexes/physiology , Proteasome Endopeptidase Complex , Proteins/genetics , Proteins/physiology , T-Lymphocytes, Cytotoxic/virology , Tumor Cells, Cultured , Viral Matrix Proteins/deficiency , Viral Matrix Proteins/geneticsABSTRACT
We describe genetic variation at three microsatellite single loci and six allozyme loci of seven natural Atlantic salmon populations from Ireland and Spain. A comparison of genetic variability detected at both types of loci is performed. Also, the relative value of microsatellite single locus variability with regard to the identification of individual salmon populations is assessed. Microsatellite loci are shown to display higher levels of variation than allozyme loci. Mean number of alleles (6 +/- 1.53) and heterozygosity (0.46 +/- 0.04) at microsatellite loci are greater than those found for allozymes (1.85 +/- 0.05 and 0.21 +/- 0.03, respectively), and some microsatellite alleles appear to be specific for a location or geographical area. Allozyme and microsatellite variation show the same pattern of differentiation between populations with Irish and Spanish populations grouped into different clusters. However, greater values of genetic distance were found among microsatellite (D = 0.0747 +/- 0.011) rather than among allozyme loci (D = 0.0449 +/- 0.008). These results provide evidence that microsatellite-based analysis of genetic variation will be useful in the identification of individual populations of Atlantic salmon.
Subject(s)
Genetic Variation , Microsatellite Repeats , Proteins/genetics , Salmon/genetics , Alleles , Animals , Base Sequence , DNA Primers/genetics , Genetics, Population , Heterozygote , Ireland , SpainABSTRACT
A repetitive element was isolated from the genome of Atlantic salmon. Nucleotide sequence analysis revealed the existence of variant monomers that range in length from approximately 200 to 230 bp. Repeat monomers contain regions of cryptic simplicity, internal repetition, and long direct repeats with deletions and insertions between individual units. The repetitive element was shown to have a tandem unit arrangement and was estimated to occupy between two and three percent of the Atlantic salmon genome. Southern blot analysis revealed the repetitive element to be unique to Atlantic salmon and brown trout species. In situ hybridization analysis showed this element to be localized at the main nucleolar organizer region bearing chromosomes of Atlantic salmon (Salmo salar), AS cell line (derived from S. salar), and brown trout (Salmo trutta).
Subject(s)
Nucleolus Organizer Region/genetics , Repetitive Sequences, Nucleic Acid , Salmon/genetics , Trout/genetics , Animals , Base Sequence , Blotting, Southern , Cell Line , Chromosome Mapping , Cloning, Molecular , Conserved Sequence , Fishes/genetics , In Situ Hybridization, Fluorescence , Molecular Sequence Data , Sequence Analysis, DNAABSTRACT
We report the cloning and characterization of a high-copy-number, tandem-repeat satellite DNA sequence from the genome of the Pacific oyster, Crassostrea gigas (Cg). The monomeric unit was found to be 166 (+/- 2) bp in length with 79-94% homology between monomers of the array. The sequence is A+T-rich (60%) and lacks internal repetition and substructural features. The repeat was estimated to account for 1-4% of the Cg genome. Fluorescence in situ hybridization (FISH) studies mapped the repeat to two distinct heterochromatic regions of two pairs of homologous chromosomes on Cg embryonic metaphases. Also, the number of metaphase chromosomes containing this repeat varied with the ploidy of the cell.