Versatile roles of R-Ras GAP in neurite formation of PC12 cells and embryonic vascular development.

Ras GTPase-activating proteins (GAP) are negative regulators of Ras that convert active Ras-GTP to inactive Ras-GDP. R-Ras GAP is a membrane-associated molecule with stronger GAP activity for R-Ras, an activator of integrin, than H-Ras. We found that R-Ras GAP is down-regulated during neurite formation in rat pheochromocytoma PC12 cells by nerve growth factor (NGF), which is blocked by the transient expression of R-Ras gap or dominant negative R-ras cDNA. By establishing a PC12 subclone that stably expresses exogenous R-Ras GAP, it was found that NGF reduced endogenous R-Ras GAP but not exogenous R-Ras GAP, suggesting that down-regulation of R-Ras GAP occurs at the transcription level. To clarify the physiological role of R-Ras GAP, we generated mice that express mutant Ras GAP with knocked down activity. While heterozygotes are normal, homozygous mice die at E12.5-13.5 of massive subcutaneous and intraparenchymal bleeding, probably due to underdeveloped adherens junctions between capillary endothelial cells. These results show essential roles of R-Ras GAP in development and differentiation: its expression is needed for embryonic development of blood vessel barriers, whereas its down-regulation facilitates NGF-induced neurite formation of PC12 cells via maintaining activated R-Ras.

A transposable element-mediated gene divergence that directly produces a novel type bovine Bcnt protein including the endonuclease domain of RTE-1.

Ruminant Bcnt protein with a molecular mass of 97 kDa (designated p97Bcnt) includes a region derived from the endonuclease domain of a retrotransposable element RTE-1. Human and mouse Bcnt proteins lack the corresponding region but have a highly conserved 82-amino acid region at the C-terminus that is not present in p97Bcnt. By screening a bovine BAC library, we found two more bcnt-related genes: human-type bcnt (h-type bcnt) and its processed pseudogene. Whereas the pseudogene is localized on chromosome 26, both bcntp97 and the h-type bcnt genes are found on bovine chromosome 18, a synteny region of human chromosome 16 on which human BCNT is localized. Complete nucleotide sequencing of the BAC clone reveals that the bcntp97 and h-type bcnt genes are located just 6 kb apart in a tandem manner. The two h-type bcnt and bcntp97genes are active at both the transcriptional level and the protein level. H-type bovine Bcnt is more like human BCNT than p97Bcnt, when compared at their N-terminal regions. However, phylogenetic analysis using the N-terminal region of the bcnt gene family revealed that the duplication of bovine genes occurred within the bovine lineage with significantly accelerated substitution in bcntp97. This acceleration was not ascribed definitely to positive selection. After duplication, one of the bovine bcnt genes recruited the endonuclease domain of an intronic RTE-1 repeat accompanied by the accelerated substitution at the 5'-ORF, resulting in creation of a novel type of Bcnt protein in bovine.

Analysis of Embryonic Chick Periderm by Monoclonal Antibody Specific against Periderm1 : (periderm/ectoderm/monoclonal antibody/cytokeratin/chick embryo).

The monoclonal antibody, 6H11, specific against embryonic chick periderm, was produced and found capable of recognizing polypeptides with 45 Kd in cytoskeletal fractions of skin and limb buds. These polypeptides were separated into two and one isoelectric variant, respectively, by two dimensional gel electrophoresis, and assigned to the acidic subfamily of cytokeratin. The ectoderm of the limb buds at stage 24 stained with 6H11. At stages 25 to 26, the experession of 6H11 in the ectoderm could no longer be detected except in a limited number of 6H11-positive cells present in the ectoderm. These positive cells were subsequently noted on the periphery of the ectoderm. They increased in number to finally form the periderm. Periderm in the dorsolateral trunk was also formed similarly by 6H11-positive cells but the timing of formation differed in the dorsal and lateral trunk. In the formation of skin integuments, such as feathers and scales, no regionality of 6H11-expression was found. The expression of 6H11 eventually decreased and ceased to be evident at about the time of hatching. Its expression in the basal epidermis during embryogenesis could not be detected. In the following, a possible mechanism of the periderm formation is discussed.