Identification and characterization of a novel family of mammalian ependymin-related proteins (MERPs) in hematopoietic, nonhematopoietic, and malignant tissues.

Evidence is presented for a family of mammalian homologs of ependymin, which we have termed the mammalian ependymin-related proteins (MERPs). Ependymins are secreted glycoproteins that form the major component of the cerebrospinal fluid in many teleost fish. We have cloned the entire coding region of human MERP-1 and mapped the gene to chromosome 7p14.1 by fluorescence in situ hybridization. In addition, three human MERP pseudogenes were identified on chromosomes 8, 16, and X. We have also cloned the mouse MERP-1 homolog and an additional family member, mouse MERP-2. Then, using bioinformatics, the mouse MERP-2 gene was localized to chromosome 13, and we identified the monkey MERP-1 homolog and frog ependymin-related protein (ERP). Despite relatively low amino acid sequence conservation between piscine ependymins, toad ERP, and MERPs, several amino acids (including four key cysteine residues) are strictly conserved, and the hydropathy profiles are remarkably alike, suggesting the possibilities of similar protein conformation and function. As with fish ependymins, frog ERP and MERPs contain a signal peptide typical of secreted proteins. The MERPs were found to be expressed at high levels in several hematopoietic cell lines and in nonhematopoietic tissues such as brain, heart, and skeletal muscle, as well as several malignant tissues and malignant cell lines. These findings suggest that MERPs have several potential roles in a range of cells and tissues.

ORP-3, a human oxysterol-binding protein gene differentially expressed in hematopoietic cells.

Using differential display polymerase chain reaction, a gene was identified in CD34(+)-enriched populations that had with low or absent expression in CD34(-) populations. The full coding sequence of this transcript was obtained, and the predicted protein has a high degree of homology to oxysterol-binding protein. This gene has been designated OSBP-related protein 3 (ORP-3). Expression of ORP-3 was found to be 3- to 4-fold higher in CD34(+) cells than in CD34(-) cells. Additionally, expression of this gene was 2-fold higher in the more primitive subfraction of hematopoietic cells defined by the CD34(+)38(-) phenotype and was down-regulated with the proliferation and differentiation of CD34(+) cells. The ORP-3 predicted protein contains an oxysterol-binding domain. Well-characterized proteins expressing this domain bind oxysterols in a dose-dependent fashion. Biologic activities of oxysterols include inhibition of cholesterol biosynthesis and cell proliferation in a variety of cell types, among them hematopoietic cells. Characterization and differential expression of ORP-3 implicates a possible role in the mediation of oxysterol effects on hematopoiesis.

Atrial natriuretic peptide binding sites in the brain and pituitary gland of the toad, Bufo marinus: localisation and receptor characterisation.

The distribution and nature of 125I-atrial natriuretic peptide binding sites have been examined in the brain and pituitary gland of the toad, Bufo marinus, using tissue section autoradiography, affinity cross-linking and electrophoresis, guanylyl cyclase assays and molecular analysis of natriuretic peptide receptor C (NPR-C) and NPR-GC mRNA expression. The highest density of 125I-atrial natriuretic peptide binding sites occurred in the dorsal pallium, the habenular region, the torus semicircularis, the choroid plexus, and the pituitary gland. Less dense binding was observed in the medial pallium, the thalamic region, the hypothalamus, the optic tectum, and the interpeduncular nucleus. The natriuretic peptide receptor-C specific ligand, C-ANF, displaced the binding in all brain regions; however, some residual binding was observed in the habenular region, the hypothalamus, the choroid plexus, and the pituitary gland. In isolated brain membranes, 1 microM rat atrial natriuretic peptide increased cyclic guanosine monophosphate levels to 90% above basal. Affinity cross-linking followed by reducing electrophoresis showed that 125I-atrial natriuretic peptide bound to proteins of 65 kDa and 135 kDa respectively. Furthermore, molecular analysis demonstrated that natriuretic peptide receptor-C and guanylyl cyclase messenger ribonucleic acid are expressed in the brain. In combination with the autoradiography, the data indicated that atrial natriuretic peptide acting via specific receptors could be important in natriuretic peptide regulation of the brain.

Relationship between arginine vasotocin-like and natriuretic peptide-like immunoreactive structures in the brain of the toad Bufo marinus.

The distribution of natriuretic peptide-like immunoreactivity was investigated in the brain of Bufo marinus and compared with arginine vasotocin-like immunoreactivity using fluorescence immunohistochemistry. The antisera used were rabbit anti-porcine brain natriuretic peptide, which recognises the three main structural forms of natriuretic peptides, and guinea-pig antivasopressin, which recognises arginine vasotocin. Natriuretic peptide-like immunoreactive fibres were observed in many regions of the brain, being densest in the preoptic/hypothalamic region of the diencephalon and the interpeduncular nucleus of the mesencephalon. Natriuretic peptide-like immunoreactive cell bodies were observed in the dorsal and medial pallium, the medial amygdala, the preoptic nucleus, the ventral hypothalamus, the nucleus posterodorsalis tegmenti mesencephali, and the interpeduncular nucleus. No natriuretic peptide-like immunoreactivity was seen in the pituitary gland. The distribution of arginine vasotocin-like immunoreactivity was similar to that described previously for other amphibian species. Numerous immunoreactive cell bodies were present in the preoptic nucleus whilst immunoreactive fibres were observed in the preoptic/hypothalamic region as well as in extrahypothalamic regions such as the medial amygdala and the medial pallium. Double-labelling immunohistochemistry revealed no colocalisation of arginine vasotocin-like and natriuretic peptide-like immunoreactivities in the same neural elements. The results suggest that natriuretic peptides and arginine vasotocin have distinct distributions in the brain but that natriuretic peptide-like immunoreactive fibres in the hypothalamus could influence the activity of arginine vasotocin-like immunoreactive cell bodies.