X-ray structure analysis of a unique D-amino-acid oxidase from the thermophilic fungus Rasamsonia emersonii strain YA.

D-Amino-acid oxidases (DAAOs) catalyze the oxidative deamination of neutral and basic D-amino acids. The DAAO from the thermophilic fungus Rasamsonia emersonii strain YA (ReDAAO) has a high thermal stability and a unique broad substrate specificity that includes the acidic D-amino acid D-Glu as well as various neutral and basic D-amino acids. In this study, ReDAAO was crystallized by the hanging-drop vapor-diffusion method and its crystal structure was determined at a resolution of 2.

A novel thermostable D-amino acid oxidase of the thermophilic fungus Rasamsonia emersonii strain YA.

D-Amino acid oxidase (DAAO) is a valuable flavoenzyme capable of being used in various practical applications, such as in determining D-amino acids and producing a material for semisynthetic cephalosporins, requiring higher thermal stability, higher catalytic activity, and broad substrate specificity. In this study, we isolated the thermophilic fungus Rasamsonia emersonii strain YA, which can grow on several D-amino acids as the sole nitrogen source, from a compost and characterized DAAO (ReDAAO) of the fungus. ReDAAO expressed in Escherichia coli exhibited significant oxidase activity against various neutral and basic D-amino acids, in particular hydrophobic D-amino acids.

Characterization and improvement of substrate-binding affinity of D-aspartate oxidase of the thermophilic fungus Thermomyces dupontii.

D-Aspartate oxidase (DDO) is a valuable enzyme that can be utilized in the determination of acidic D-amino acids and the optical resolution of a racemic mixture of acidic amino acids, which require its higher stability, higher catalytic activity, and higher substrate-binding affinity. In the present study, we identified DDO gene (TdDDO) of a thermophilic fungus, Thermomyces dupontii, and characterized the recombinant enzyme expressed in Escherichia coli. In addition, we generated a variant that has a higher substrate-binding affinity.

Visualization of the calcitonin receptor-like receptor and its receptor activity-modifying proteins during internalization and recycling.

Expression of the calcitonin receptor-like receptor (CRLR) and its receptor activity modifying proteins (RAMPs) can produce calcitonin gene-related peptide (CGRP) receptors (CRLR/RAMP1) and adrenomedullin (AM) receptors (CRLR/RAMP2 or -3). A chimera of the CRLR and green fluorescent protein (CRLR-GFP) was used to study receptor localization and trafficking in stably transduced HEK 293 cells, with or without co-transfection of RAMPs. CRLR-GFP failed to generate responses to CGRP or AM without RAMPs.

The RAMP2/CRLR complex is a functional adrenomedullin receptor in human endothelial and vascular smooth muscle cells.

Adrenomedullin, a potently hypotensive peptide isolated from human pheochromocytoma, is known to elicit a rise in cAMP levels within mammalian endothelial and smooth muscle cells. Until now, however, little has been known about the adrenomedullin receptor. Recently, a group called receptor activity-modifying proteins that complex with the calcitonin receptor-like receptor, and thereby regulate its transport and ligand specificity, were identified.

Molecular cloning and characterization of a transcription factor for the C-type natriuretic peptide gene promoter.

Our previous studies on the promoter function of the human C-type natriuretic peptide (CNP) gene revealed the existence of two GC-rich cis elements essential for gene transcription in rat pituitary-derived GH3 cells. To isolate transcription factors that bind to those GC-rich elements, we screened a lambda ZAP cDNA library derived from GH3 cells by Southwestern screening. Several positive clones with specific binding abilities were obtained, and one was identical as TSC-22, a speculated transcriptional modulator stimulated by transforming growth factor beta (TGF-beta) of unknown function.

Adrenomedullin stimulates two signal transduction pathways, cAMP accumulation and Ca2+ mobilization, in bovine aortic endothelial cells.

The biological action of adrenomedullin, a novel hypotensive peptide, on bovine aortic endothelial cells, was examined. The specific binding of adrenomedullin to these cells was observed, and adrenomedullin was found to induce intracellular cAMP accumulation in a dose-dependent manner. EC50 for the cAMP accumulation was about 100 times lower than the apparent IC50 for the binding assay.

C-type natriuretic peptide stimulates secretion of growth hormone from rat-pituitary-derived GH3 cells via a cyclic-GMP-mediated pathway.

Although C-type natriuretic peptide (CNP) has been shown to exist at the highest concentration in the anterior pituitary in rat tissues, its physiological role(s) there is (are) not clear. In this study, we report a novel function of CNP examined with anterior pituitary-derived cell lines, GH3 and AtT20/D16v-F2. Both CNP and atrial natriuretic peptide (ANP) increased cellular cGMP levels in both cell lines in dose-dependent manners.

Cell-type-specific function of the C-type natriuretic peptide gene promoter in rat anterior pituitary-derived cultured cell lines.

The promoter function of the human C-type natriuretic peptide (CNP) gene in various cultured cells was examined by transient transfection assays. The CNP promoter functioned very effectively in GH3 cells, which originated from the growth hormone-producing tumor of the rat anterior pituitary and somatomammotroph phenotype, but functioned much less effectively in GH1 cells, another type of rat pituitary-derived cell with a somatotroph phenotype, and rat primary cardiocytes. The CNP promoter did not function at all in other cells, including AtT20 cells of murine pituitary corticotroph origin.

The role of the C-terminal region of rat brain natriuretic peptide in receptor selectivity.

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) have different C-terminal tail structures compared with the rather conservative ring structures which consist of 17 amino acid residues. To examine the different effects of the tail structures of ANP and BNP on their interaction with receptors, we synthesized several peptide analogs and measured their biological actions in three different assay systems. Deletion of the C-terminal tail from rat BNP did not effect the vasorelaxation activity against rat aorta, but it promoted cGMP production in cultured rat aortic smooth muscle cells (RASMC).