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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. Searches for ligands for orphan G-protein-coupled receptors (GPCRs)1 have discovered many novel peptides and have identified the previously unknown receptors of bioactive substances (1-9). Studies on the newly identified ligands and their receptors have given us a more precise understanding of the physiological processes involved in the endocrine, cardiovascular, reproductive, immune, inflammatory, digestive, metabolic, and central nervous systems (1-11). In addition, these studies have provided opportunities to discover innovative drugs that exert their pharmacological effects by interacting with an identified receptor as an agonist or antagonist (12). GPR7 and GPR8, for which the ligands have not been identified, are structurally related orphan GPCRs. Two genes for GPR7 and GPR8 were originally isolated from human genomic DNA by O'Dowd et al. (13). Human GPR7 highly resembles human GPR8, with an amino acid identity of 64%. Among various families of GPCRs, GPR7 and GPR8 share high similarity to the opioid and somatostatin receptor families. In mammalian brain, gene expression of GPR7 and GPR8 was detected by Northern blot and in situ hybridization analyses (13). Especially in rat brain, GPR7 mRNA was detected in regions of the cortex, hippocampus, and hypothalamus (14). Profiles of GPR7 and GPR8 expressed mainly in brain suggest that the endogenous ligands for the two receptors have several functions in the central nervous system.In this study, we report the purification, cloning, and characterization of neuropeptide W (NPW). We attempted to purify the agonist peptide for GPR8. The cDNA encoding the agonist peptide for GPR8 demonstrates the existence of neuropeptide W-23 (NPW23) and neuropeptide W-30 (NPW30), which exhibit no meaningful similarity to any known peptides. With the functional and binding characterization of NPW for GPR7 and GPR8, we show that NPW is the endogenous ligand for both of these recept...

Abstract-Endothelin-1 (ET-1) is a powerful vasoconstrictor peptide produced by endothelial and smooth muscle cells.Many lines of biological evidence suggest that the ET-1 gene is a candidate gene for hypertension. Moreover, recent association studies suggested that a G/T polymorphism with an amino acid substitution (Lys/Asn) at codon 198 in exon 5 of the ET-1 gene interacts with body mass index (BMI) in association with blood pressure. They suggested that T carriers are more sensitive to weight gain than GG homozygotes in association with blood pressure. However, association studies are often irreproducible, and the first study often suggests a stronger genetic effect than is found by subsequent studies. We therefore assessed the interaction in 2 large Japanese populations. The present study showed a nonsignificant but similar trend to the results of previous reports. Moreover, in line with previous reports, this study revealed a significant interaction between the ET-1 K198N (G/T) polymorphism and BMI in association with hypertension in our populations (Pϭ0.027). The interaction was significant, even after adjustment for gender and age (Pϭ0.045) and for all confounding factors (Pϭ0.044). T carriers were more sensitive to weight gain than GG homozygotes in association with hypertension. Considering the combined impact of obesity and hypertension on the development of cardiovascular and cerebrovascular disorders, T allele carriers might represent elective targets for therapy to lower their body weight. Key Words: endothelin Ⅲ hypertension, essential Ⅲ genetics Ⅲ polymorphism Ⅲ body mass index E ndothelin-1 (ET-1) is a powerful vasoconstrictor peptide produced by vascular endothelial cells. 1 Some patients with moderate-to-severe essential hypertension, similar to some experimental rat models with severe blood pressure elevation, exhibit enhanced endothelial expression of the ET-1 gene. 2 Plasma ET-1 concentration is elevated in hypertensive patients. 3-6 An endothelin-receptor antagonist significantly lowered blood pressure in patients with essential hypertension. 7 Given these lines of biological evidence, the ET-1 gene is a candidate responsible for hypertension.Hypertension is a common, complex phenotype and has been intensively studied to identify susceptibility loci in humans. Nonetheless, there is no known genotypic polymorphism consistently associated with hypertension in humans, thus far. Moreover, albeit that the development of hypertension is considered to be due at least partly to gene-gene and gene-environmental interactions, fewer interaction analyses have been conducted than simple association analyses. In this regard, the ET-1 gene is an attractive candidate because, in addition to its biological function, this gene has been shown to interact with body mass index (BMI) in association with blood pressure in 3 large populations. 8,9 However, association studies are often irreproducible, and the first study often suggests a stronger genetic effect than is found by subsequent studies. 10 We therefore a...

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Forty-six strains of Malassezia spp. with atypical biochemical features were isolated from 366 fresh clinical isolates from human subjects and dogs. Isolates obtained in this study included 2 (4.7%) lipiddependent M. pachydermatis isolates; 1 (2.4%) precipitate-producing and 6 (14.6%) non-polyethoxylated castor oil (Cremophor EL)-assimilating M. furfur isolates; and 37 (34.3%) M. slooffiae isolates that were esculin hydrolyzing, 17 (15.7%) that were non-tolerant of growth at 40°C, and 2 (1.9%) that assimilated polyethoxylated castor oil. Although their colony morphologies and sizes were characteristic on CHROMagar Malassezia medium (CHROM), all strains of M. furfur developed large pale pink and wrinkled colonies, and all strains of M. slooffiae developed small (<1 mm) pale pink colonies on CHROM. These atypical strains were distinguishable by the appearance of their colonies grown on CHROM. Three clinically important Malassezia species, M. globosa, M. restricta, and M. furfur, were correctly identified by their biochemical characteristics and colony morphologies. The results presented here indicate that our proposed identification system will be useful as a routine tool for the identification of clinically important Malassezia species in clinical laboratories.Members of the genus Malassezia are among the microbiological flora of the skin of homoiothermic animals. Most species of this genus are lipid-dependent yeasts which colonize the seborrheic part of the skin, and they have been reported to be associated with pityriasis versicolor, seborrheic dermatitis, Malassezia folliculitis, and atopic dermatitis (1,6,19,20,24,29). Although M. furfur was previously thought to be the causative agent or trigger factor in all of these skin disorders, Guého et al. (9) reclassified this genus into five species in 1996. Malassezia has since been reclassified into seven species based on molecular biological analysis of nuclear ribosomal DNA/ RNA (9, 10), and the results agreed with those of mitochondrial ribosomal DNA analyses (30). As members of the genus Malassezia share similar morphological and biochemical characteristics, it was thought that differentiating between them based on phenotypic features would be difficult. While molecular biological techniques are the most reliable for the identification of Malassezia, they are not available in most clinical laboratories. Therefore, culture methods for the identification of Malassezia species are required. Some of these identification or differentiation methods have been reported previously. Guillot et al. reported a method of identification based on lipid usage pattern, catalase reaction, growth temperature, and cell shape (11). Hammer and Riley reported the production of a precipitate by some Malassezia strains on Dixon's agar (12); for example, M. furfur, M. obtusa, and M. slooffiae were precipitate-negative strains, while M. sympodialis and M. globosa were precipitate-positive strains. Mayser et al. reported that some Malassezia species hydrolyzed esculin and assimilated pol...

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