Youji Mitsui

Differences between early and late passage cell cultures on the organelle and macromolecular levels have been attributed to cellular "aging". However, concern has been expressed over whether changes in diploid cell populations after serial passage in vitro accurately reflect human cellular aging in vivo. Studies were therefore undertaken to determine if significant differences would be observed in the in vitro lifespans of skin fibroblast cultures from old and young normal, non-hospitalized volunteers and to examine if parameters that change with in vitro "aging" are altered as a function of age in vivo. Statistically signigificant (P less than 0.05) decreases were found in the rate of fibroblast migration, onset of cell culture senescence, in vitro lifespan, cell population replication rate, and cell number at confluency of fibroblast cultures derived from the old donor group when compared to parallel cultures from young donors. No significant differences were observed in modal cell volumes and cellular macromolecular contents. The differences observed in cell cultures from old and young donors were quantitatively and qualitatively distinct from those cellular alterations observed in early and late passage WI-38 cells (in vitro "aging"). Therefore, although early and late passage cultures of human diploid cells may provide an important cell system for examining loss of replicative potential, fibroblast cultures derived from old and young human donors may be a more appropriate model system for studying human cellular aging.

Endothelin-1 is a 21-amino acid potent vasoconstrictor peptide produced by vascular endothelial cells. We have cloned the whole length of the human preproendothelin-1 (PPET-1) gene and the corresponding cDNA and determined the complete nucleotide sequences. The 2026-nucleotide human mRNA for PPET-1 (excluding the polY(A) tail) is encoded in five exons distributed over 6836 base pairs of the genome. The 5'-flanking region of the gene contains (i) octanucleotide sequences for the phorbol ester-responsive elements, also known as the binding elements for FOS.JUN complex; (ii) consensus motifs for the binding site of nuclear factor 1, which may mediate the induction described previously of PPET-1 mRNA by transforming growth factor-beta; (iii) hexanucleotide sequences for the acute phase reactant regulatory elements that may be involved in the induction of endothelin-1 under acute physical stress in vivo. Further, the 3'-nontranslated sequence of human PPET-1 mRNA contains three AUUUA motifs, which may mediate selective translation-dependent destabilization of the mRNA. Northern blot analysis in cultured endothelial cells from human umbilical veins shows that PPET-1 mRNA is in fact rapidly induced by the active phorbol ester 12-O-tetradecanoylphorbol 13-acetate within 10 min. Analysis of mRNA life span by using actinomycin D demonstrates that PPET-1 mRNA has a short intracellular half-life of about 15 min and is superinduced by cycloheximide. This superinduction is found to be due to the stabilization of the mRNA by cycloheximide, as in the case of other known AUUUA-containing mRNAs. These findings suggest that the regulation of expression of PPET-1 mRNA may be mediated in part by these sequence elements.

A cDNA encoding a human endothelium-derived vasoconstrictor peptide, endothelin, was isolated from a human placenta cDNA library. The nucleotide sequence of this cDNA clone showed that the primary structure of the human preproendothelin has 212 amino acid residues and is highly homologous to porcine preproendothelin, and that human endothelin is identical with porcine endothelin.

A new peptide family (endothelin (ET] consisting of three members in mammals appears to be present in mice according to genomic Southern blot analysis. Two ET-related genes were identified by cloning and sequence analysis of a mouse genome. One encoded a peptide identical to porcine and human vasoconstrictor peptide ET, and the other encoded a novel peptide differing from ET in 3 amino acid residues, with 4 cysteines in the same positions as in ET. This novel peptide was synthesized and confirmed to have in vivo pressor activity similar to that of ET. Northern blot analysis, however, indicated the gene of this novel peptide to be expressed only in the intestine, and not in other tissues or cell lines, or endothelial cells. Furthermore, the peptide evoked a strong contractile response in the guinea pig ileum. This peptide may thus be reasonably classified as a gastrointestinal peptide, vasoactive intestinal contractor.