An endothelium-derived 21-residue vasoconstrictor peptide, endothelin, has been isolated, and shown to be one of the most potent vasoconstrictors known. Cloning and sequencing of preproendothelin complementary DNA shows that mature endothelin is generated through an unusual proteolytic processing, and regional homologies to a group of neurotoxins suggest that endothelin is an endogenous modulator of voltage-dependent ion channels. Expression of the endothelin gene is regulated by several vasoactive agents, indicating the existence of a novel cardiovascular control system.

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, nonhospitalized volunteers and to examine if parameters that change with in vitro "aging" are altered as a function of age in The limited lifespan of cultured human diploid fibroblasts in vitro has led to their extensive utilization as a model system for studying human cellular aging (1-3). These human fetal lung fibroblasts in their last passages are often referred to as representing "aged" cell populations (4-6). Differences between early and late passage cell cultures on the organelle and macromolecular levels have been attributed to cellular "aging" (4-6). However, concern has been expressed over whether changes in diploid cell populations after serial passage in vitro accurately reflects human cellular aging in vivo (7-9). One of the most frequently cited justifications for the use of cultured fibroblasts for studying human cellular aging is the reported decline in the cumulative number of cell population replications in vitro and earlier onset of cell culture senescence with the increasing age of the cell culture donor (10, 11). Recently, however, the statistical analysis and population selection used in these studies have been questioned (7,8).The studies to be described in this report had two major goals: (i) to determine if statistically significant differences would be observed in the onset of cell culture senescence and the cumulative replication capacity of fibroblast cultures derived from old and young normal, nonhospitalized human volunteers; and (ii) to examine if parameters that change with increased in vitro "aging" are altered as a function of in vivo age. This latter asAbbreviation: CPD, cell population doublings. * Present address: Tokyo Metropolitan Institute of Gerontology, Sakaecho, Itabashiku, Tokyo-173, Japan. pect was approached by comparing cell population replication rate, percent replicating cells, cell number at confluency, cell volume, and cellular macromolecular contents in cell cultures from old and young human donors (aging in vivo) as well as in early and late passage cell cultures ("aging" in vitro). MATERIALS AND METHODSCell Culture. After informed consent had been obtained, skin punch biopsies, 2 mm in diameter, were performed on the inner aspect of the upper arm of nonhospitalized male volunteers. The subjects were interviewed prior to biopsy, and those individuals with a history of diabetes or who were receiving steroid medication were not included in this study. The skin specimen was divided into four equal explants and two each were placed between coverglass sandwiches in Leighton tubes. ...

The mortalin genes, mot-1 and mot-2, are hsp70 family members that were originally cloned from normal and immortal murine cells, respectively. Their proteins differ by only two amino acid residues but exhibit different subcellular localizations, arise from two distinct genes, and have contrasting biological activities. We report here that the two proteins also differ in their interactions with the tumor suppressor protein p53. The pancytosolic mot-1 protein in normal cells did not show colocalization with p53; in contrast, nonpancytosolic mot-2 and p53 overlapped significantly in immortal cells. Transfection of mot-2 but not mot-1 resulted in the repression of p53-mediated transactivation in p53-responsive reporter assays. Inactivation of p53 by mot-2 was supported by the down-regulation of p53-responsive genes p21 WAF-1 and mdm-2 in mot-2-transfected cells only. Furthermore, NIH 3T3 cells transfected with expression plasmid encoding green fluorescent proteintagged mot-2 but not mot-1 showed an abrogation of nuclear translocation of wild-type p53. These results demonstrate a novel mechanism of p53 inactivation by mot-2 protein.Evidence has been accumulating that inactivation of p53, a tumor suppressor and cellular transcription factor (1), is involved in cellular transformation and immortalization (2-5). Extensive analyses of p53 have defined at least four functional domains, including an amino terminus transactivation domain (amino acids 1-44), a sequence-specific DNA-binding domain (amino acids 100 -300), a carboxyl terminus oligomerization domain, and a regulatory domain (amino acids Ref. 6), and shown that the conformation of p53 and its interactions with other proteins have key roles in its various cellular activities (7,8). Several cellular proteins, including some of the hsp70 family members, have been shown to interact with p53 (9 -12). Although mutational or mdm-2-mediated inactivation of p53 is a common event involved in cellular transformation (1), p53 is inactivated in a considerable number of tumors and transformed cells by an unknown mechanism(s).We initially cloned mortalins mot-1 and mot-2, which code for pancytosolically and perinuclearly distributed members of the hsp70 family of proteins, from normal and immortal murine cells, respectively (13,14). The open reading frames of the two types of murine mortalins differ in two nucleotides, encode proteins differing in two amino acids, arise from distinct genes, and have contrasting biological activities (13-16). RNA in situ hybridization and immunohistochemical studies on mortalin in normal murine tissues showed a higher level of expression in nondividing cell populations than in dividing cells. However, tumor tissues were seen to have a high intensity of mortalin staining by an antibody that reacts with both the mot-1 and mot-2 proteins (17, 18). Mortalin was also identified as PBP-74, mtHSP70, and Grp75 and has been assigned roles in antigen processing, in vivo nephrotoxicity, and radioresistance in independent studies from other groups (19,20)...

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.

The Caenorhabditis elegans homolog of mortalin/mthsp70/Grp75 (called mot-2 hereafter) was isolated by screening of a nematode cDNA library with mouse mot-2 cDNA. The isolated clone matched to hsp70F of C. elegans. Analysis with two of the antibodies raised against hsp70F revealed that unlike mammalian mot-2, it is heat inducible. Transient induction of hsp70F by heat shock led to a slight (<13%) extension in the C. elegans life span. The transgenic worms that constitutively over-expressed hsp70F predominantly in muscle showed life span extension (approximately 43% for mean and approximately 45% for maximum life span) as compared to the wild-type and green fluorescent protein-transgenic worms. Life span extension of human cells was obtained by over-expression of mot-2 [Kaul et al. (2000) FEBS Lett. 474, 159-164]. Our results show, for the first time, that this member of the hsp70 family governs the longevity of worms and thus there are common pathways that determine mammalian and worm longevity.

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The murine mortalin genes, mot-1 and mot-2, are members of the hsp70 family of proteins and di er from each other by only two amino acid residues. Mot-1 is expressed in normal cells and has pancytosolic cellular distribution whereas mot-2 is found in the perinuclear region of immortal cells. We report here that a high level of expression of mot-2 protein resulted in malignant transformation of cells as analysed by anchorage independent growth and nude mice assays. A high level of protein expression is attributed to the 900 bp 3' untranslated region of the cDNA which does not have any transforming activity per se. Mortalin cDNA clones isolated from human transformed cells were also found to have transforming activity in similar assays and a high level of expression was apparent in some of the human immortalized cells that showed non-pancytosolic mortalin immuno¯uorescence. Taken together, the data suggest that nonpancytosolic mortalin may have a role in tumorigenesis.