ABSTRACT
We have developed a real-time PCR array method to comprehensively detect genetically modified (GM) organisms. In the method, genomic DNA extracted from an agricultural product is analyzed using various qualitative real-time PCR assays on a 96-well PCR plate, targeting for individual GM events, recombinant DNA (r-DNA) segments, taxon-specific DNAs, and donor organisms of the respective r-DNAs. In this article, we report the single-laboratory validation of both DNA extraction methods and component PCR assays constituting the real-time PCR array. We selected some DNA extraction methods for specified plant matrixes, i.e., maize flour, soybean flour, and ground canola seeds, then evaluated the DNA quantity, DNA fragmentation, and PCR inhibition of the resultant DNA extracts. For the component PCR assays, we evaluated the specificity and LOD. All DNA extraction methods and component PCR assays satisfied the criteria set on the basis of previous reports.
Subject(s)
Food Analysis/methods , Organisms, Genetically Modified/genetics , Real-Time Polymerase Chain Reaction/methods , Zea mays/genetics , Base Sequence , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Lysophosphatidyl-L-serine (lysoPS) is thought to be an immunological regulator because it dramatically augments the degranulation of rat peritoneal mast cells (RPMCs). This stimulatory effect may be mediated by a lysoPS receptor, but its molecule has not been identified yet. During a ligand fishing study for the orphan G-protein-coupled receptor 34 (GPR34), we found that lysoPS caused a dose-dependent inhibition of forskolin-stimulated cAMP accumulation in human GPR34-expressing Chinese hamster ovary (CHO/hGPR34) cells. The CHO/hGPR34 cells were unresponsive to other structurally related phospholipids examined. Quantitative real-time-PCR demonstrated that mRNAs of GPR34 are particularly abundant in mast cells. The effective lysoPS concentration for RPMC degranulation was similar to that required for GPR34 activation, and the structural requirement of lysoPS for RPMC degranulation was in good agreement with that observed in CHO/hGPR34 cells. These results suggest that GPR34 is the functional mast cell lysoPS receptor.
Subject(s)
Lysophospholipids/metabolism , Mast Cells/chemistry , Receptors, Lysophospholipid/metabolism , Amino Acid Sequence , Animals , CHO Cells , Cell Degranulation/drug effects , Cloning, Molecular , Cricetinae , Cricetulus , Enzyme Activation , Extracellular Signal-Regulated MAP Kinases/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Lysophospholipids/pharmacology , Male , Mast Cells/drug effects , Mice , Molecular Sequence Data , Rats , Rats, Wistar , Sequence AlignmentABSTRACT
Urotensin II (UII) has been reported as the most potent known vasoconstrictor. While rat and mouse orthologs of UII precursor protein have been reported, only the tentative structures of UII peptides of these animals have been demonstrated, since prepro-UII proteins lack typical processing sites for their mature peptides. In the present study, we isolated a novel peptide, UII-related peptide (URP), from the extract of the rat brain as the sole immunoreactive substance to anti-UII antibody; the amino acid sequence of the peptide was determined as ACFWKYCV. cDNAs encoding rat, mouse, and human precursor proteins for URP were cloned and revealed that the sequences of mouse and human URP peptides are the same as that for rat URP. Prepro-URP gene is expressed in several rat tissues such as those of the thymus, spleen, testis, and spinal cord, although with lower levels than the prepro-UII gene. In the human, the prepro-URP gene is expressed comparably to prepro-UII in several tissues except the spinal cord. URP was found to bind and activate the human or rat UII receptors (GPR14) and showed a hypotensive effect when administered to anesthetized rats. These results suggest that URP is the endogenous and functional ligand for UII receptor in the rat and mouse, and possibly in the human. We also describe the preparation of specific monoclonal antibodies raised against UII peptide and the establishment of a highly sensitive enzyme immunoassay system for UII peptides.
Subject(s)
Brain/metabolism , Peptide Hormones/chemistry , Peptide Hormones/physiology , Urotensins/metabolism , Amino Acid Sequence , Animals , Blood Pressure , CHO Cells , Calcium/metabolism , Cloning, Molecular , Cricetinae , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Humans , Immunoenzyme Techniques , Intracellular Signaling Peptides and Proteins , Ligands , Male , Mice , Molecular Sequence Data , Peptides/chemistry , Polymerase Chain Reaction , Protein Binding , Rats , Rats, Wistar , Sequence Homology, Amino Acid , Time Factors , Tissue DistributionABSTRACT
The structurally related orphan G-protein-coupled receptors GPR7 and GPR8 are expressed in the central nervous system, and their ligands have not been identified. Here, we report the identification of the endogenous ligand for both of these receptors. We purified the peptide ligand from porcine hypothalamus using stable Chinese hamster ovary cell lines expressing human GPR8 and cloned the cDNA encoding its precursor protein. The cDNA encodes two forms of the peptide ligand with lengths of 23 and 30 amino acid residues as mature peptides. We designated the two ligands neuropeptide W-23 (NPW23) and neuropeptide W-30 (NPW30). The amino acid sequence of NPW23 is completely identical to that of the N-terminal 23 residues of NPW30. Synthetic NPW23 and NPW30 activated and bound to both GPR7 and GPR8 at similar effective doses. Intracerebroventricular administration of NPW23 in rats increased food intake and stimulated prolactin release. These findings indicate that neuropeptide W is the endogenous ligand for both GPR7 and GPR8 and acts as a mediator of the central control of feeding and the neuroendocrine system.
