A tripeptide sequence within the nascent DaaP protein is required for mRNA processing of a fimbrial operon in Escherichia coli.

The biogenesis of F1845 fimbriae, a member of the Dr family of Escherichia coli adhesins, is regulated by endonucleolytic cleavage of the daaABCDPE primary transcript and differential stability of the resulting cleavage products. Processing of daa mRNA is dependent upon translation of a small open reading frame, designated daaP, which flanks the daa processing site. Here, we demonstrate that daa mRNA processing is directly coupled to daaP translation. Cleavage of the daaA-E mRNA was shown to require the tripeptide Gly-Pro-Pro (GPP), encoded by daaP codons 49-51 downstream of the processing site. Processing also required active translation through RNA located upstream of the processing site; however, processing did not depend on the amino acid sequence encoded by the region of daaP upstream of the processing site. Finally, determination of the processing site was shown to involve its location relative to the codons encoding the GPP tripeptide. These data show that translation of daaP is required in cis to promote RNA processing. These data suggest a model involving interaction of the nascent GPP tripeptide portion of the DaaP polypeptide with the ribosome, triggering cleavage of the associated mRNA at a fixed distance upstream. A model of active involvement of the ribosome in this process is proposed.

Effect of strain on human keratinocytes in vitro.

Tissue expansion, a technique to enlarge the skin surface area with an expandable balloon, has been widely used in reconstructive surgery. Although the effect of tissue expansion on in vivo skin physiology and histology has been well documented, it remains unclear whether keratinocytes or other cell types are responsible for these changes. Therefore, we investigated the in vitro effect of cyclic (10 cycles/min, 150 mmHg) or constant (continuous, 150 mmHg) strain on human keratinocyte phenotype and relevant mechanosignaling pathways. Our results demonstrate that keratinocytes subjected to cyclic strain exhibit a significant (P < 0.05) increase in cell proliferation (49.2+/-15.8%), DNA synthesis (37.7+/-4.5%), elongation (20.3+/-2.7%), and protein synthesis (17.9+/-6.6% increase) as compared with stationary controls. In contrast, keratinocytes subjected to constant strain were unaffected aside from a modest transitory increase in the proliferative rate. Keratinocytes subjected to cyclic strain aligned perpendicular to the force vector (24.2+/-1.6 degrees) as compared with stationary controls (40.4+/-2.2 degrees; the smaller degree indicates better alignment). We also report strain-induced reduction in the levels of cyclic adenosine mono phosphate (cAMP), protein kinase A (PKA), and prostaglandin E2 (PGE2) as compared with stationary controls (cAMP, 30+/-7.5%; PKA, 45+/-17%; PGE2, 58+/-4.3%; percent decrease vs. that of control). We conclude that direct application of cyclic strain on human keratinocytes modulates cell phenotype and cAMP-mediated signaling pathways in an inverse manner. Moreover, keratinocytes may play an important role in previously observed alterations in skin properties associated with tissue expansion and other strain-induced responses.

Transcriptional regulation of endothelin.

Three isoforms of endothelin (ET) exist, ET-l, ET-2,and ET-3.Nucleotide sequences for the three human ET genes are highly conserved. ET-l exactly matches the sequence ofET originally isolated from the condi- tioned medium of cultured bovine aortic endothelial cells (BAECs)."All three forms have been found in vascular, neural, adrenal, and kidney tissue, but are expressed in different proportions. Endothelial cells ex- clusively produce ETAll three isoforms have different vasoconstrictive potencies but are otherwise quali- tatively similar. ET-2 is the most potent vasoconstrictor, followed by ET-l and ET.