Mouse ARF-related protein 1: genomic organization and analysis of its promoter.

ARF-related protein 1 (ARFRP1) is a membrane-associated GTPase which inhibits the ARF/Sec7-dependent activation of phospholipase D. We have recently shown that deletion of Arfrp1 in mice results in increased apoptosis of mesodermal cells during gastrulation, leading to early embryonic lethality. Here we describe the organization of the Arfrp1 gene and of its promoter region. The Arfrp1 gene spans approximately 7 kb and contains 8 exons. The proximal 5'-flanking regions of mouse and human ARFRP1 lack a TATA box and a CAAT box, are highly GC-rich and contain potential transcription factor binding sites. Interestingly, sequence analysis of human ARFRP1 showed its 5'-flanking region contains the first exon of another gene (DJ583P15.3 in the ensembl data base; www.ensembl.org) on the opposite strand. Promoter analysis revealed that the intergenic region between both genes (54 bp) exhibits bidirectional promoter activity. However, deletion analysis demonstrated that transcription of both genes is regulated by different cis-elements. Mutational analysis and electrophoretic mobility shift assays indicated that two short cRel- and cEts1-like elements in the 5'-flanking region of Arfrp1 (-76 to -53 and -45 to -23) are critical for the regulation of Arfrp1 expression.

Embryonic lethality caused by apoptosis during gastrulation in mice lacking the gene of the ADP-ribosylation factor-related protein 1.

ADP-ribosylation factor (ARF)-related protein 1 (ARFRP1) is a membrane-associated GTPase with significant similarity to the family of ARFs. We have recently shown that ARFRP1 interacts with the Sec7 domain of the ARF-specific guanine nucleotide exchange factor Sec7-1/cytohesin and inhibits the ARF/Sec7-dependent activation of phospholipase D in a GTP-dependent manner. In order to further analyze the function of ARFRP1, we cloned the mouse Arfrp1 gene and generated Arfrp1 null-mutant mice by gene targeting in embryonic stem cells. Heterozygous Arfrp1 mutants developed normally, whereas homozygosity for the mutant allele led to embryonic lethality. Cultured homozygous Arfrp1 null-mutant blastocysts were indistinguishable from wild-type blastocysts. In vivo, they implanted and formed egg cylinder stage embryos that appeared normal until day 5. Between embryonic days 6 and 7, however, apoptotic cell death of epiblast cells occurred in the embryonic ectoderm during gastrulation, as was shown by histological analysis combined with terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling. Epiblast cells that would normally differentiate to mesodermal cells detached from the ectodermal cell layer and were dispersed into the proamniotic cavity. In contrast, the development of extraembryonic structures appeared unaffected. Our results demonstrate that ARFRP1 is necessary for early embryonic development during gastrulation.

A motion sickness maximum around the 0.2 Hz frequency range of horizontal translational oscillation.

BACKGROUND: Low frequency translational oscillation can provoke motion sickness in land vehicles, ships and aircraft. Although controlled motion experiments indicate a progressive increase in nauseogenicity as frequency decreases toward 0.2 Hz, few data are available on the existence of a definite maximum nauseogenic potential of motion around 0.2 Hz, or decreased nauseogenicity below this frequency. HYPOTHESIS: Nauseogenicity should be maximal around 0.2 Hz. METHODS: We selected 12 subjects for high motion sickness susceptibility, and they were exposed to horizontal sinusoidal motion (1.0 m.s(-2) peak acceleration) at 3 different frequencies (0.1, 0.2 and 0.4 Hz), at 1-wk intervals at the same time of day, according to a factorial design. Subjects were seated comfortably in the upright position with head erect. Fore-aft motion was through the body and head X-axis. Motion was stopped (motion endpoint) at moderate nausea or after 30 min. RESULTS: The proportion of subjects experiencing moderate nausea was maximal at the intermediate frequency: 8/12 at 0.1 Hz, 12/12 at 0.2 Hz, 7/12 at 0.4 Hz. The mean time to motion endpoint was significantly (p < 0.01) shorter at the intermediate frequency: 18.0 min at 0.1 Hz; 11.2 min at 0.2 Hz; 20.2 min at 0.4 Hz. Similar frequency patterns emerged for other sickness ratings. The equivalent times to achieve moderate nausea using estimated values to correct for subjects who reached the 30 min time cut-off were: 22.7 min at 0.1 Hz; 11.2 min at 0.2 Hz; 28.1 min at 0.4 Hz. CONCLUSIONS: A maximum nauseogenic potential around 0.2 Hz was substantiated.