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The phosphoprotein plastin was originally identified as an abundant transformation-induced polypeptide of chemically transformed neoplastic human fibroblasts. This abundant protein is normally expressed only in leukocytes, suggesting that it may play a role in hemopoietic cell differentiation. Protein microsequencing of plastin purified from leukemic T lymphocytes by high-resolution two-dimensional gel electrophoresis produced eight internal oligopeptide sequences. An oligodeoxynucleotide probe corresponding to one of the oligopeptides was used to clone cDNAs from transformed human fibroblasts that encoded the seven other oligopeptides predicted for human plastin. Sequencing and characterization of two cloned cDNAs revealed the existence of two distinct, but closely related, isoforms of plastin--l-plastin, which is expressed in leukocytes and transformed fibroblasts, and t-plastin, which is expressed in normal cells of solid tissues and transformed fibroblasts. The leukocyte isoform l-plastin is expressed in a diverse variety of human tumor cell lines, suggesting that it may be involved in the neoplastic process of some solid human tumors.

Endothelin, a newly discovered endothelium-derived peptide, has potent vasoactive properties in vivo and in vitro. The actions of endothelin in clinical conditions of hypertension have not yet been defined. This study examined the possible role of endothelin in the vasospasm and hypertension associated with a well-defined syndrome of gestational hypertension, preeclampsia. Our results indicate that the concentration of immunoreactive endothelin is elevated significantly in plasma obtained from women with preeclampsia and rapidly returns to a normal pregnancy value within 48 hours of delivery, as predicted by the prompt clinical resolution of this disorder. The findings suggest that endothelin may contribute to the vasospasm associated with this syndrome and lend further support to the involvement of endothelial cells in the pathophysiology of preeclampsia.

The hypothesis that activation of apoptosis and DNA fragmentation is involved in TNF-mediated cytolysis of U937 tumor cells was investigated. Morphological, biochemical, and kinetic criteria established that TNF activates apoptosis as opposed to necrosis. Within 2-3 h of exposure to TNF, U937 underwent the morphological alterations characteristic of apoptosis. This was accompanied by cleavage of DNA into multiples of nucleosome size fragments. Both of these events occurred 1-2 h prior to cell death as defined by trypan blue exclusion or 51Cr release. DNA fragmentation was not a non-specific result of cell death since U937 cells lysed under hypotonic conditions did not release DNA fragments. The percentage of cells undergoing apoptosis depended on the concentration of TNF and was augmented by the addition of cycloheximide. A TNF-resistant variant derived from U937 did not undergo apoptosis in response to TNF, even in the presence of cycloheximide. Furthermore, TNF could still activate NFkB in this variant, suggesting that this pathway is not involved in TNF-mediated cytotoxicity. Two agents known to inhibit TNF-mediated cytotoxicity, ZnSO4 and 3-aminobenzamide, were shown to inhibit TNF-induced apoptosis. Taken altogether, these data support the hypothesis that activation of apoptosis is at least one essential step in the TNF lytic pathway in the U937 model system.

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Two different mutant human j-actin genes have been introduced into normal diploid human (KD) fibroblasts and their immortalized derivative cell line, HuT-12, to assess the impact of an abnormal cytoskeletal protein on cellular phenotypes such as morphology, growth characteristics, and properties relating to the neoplastic phenotype. A mutant ,-actin containing a single mutation (Gly-244 --Asp-244) was stable and was incorporated into cytoskeletal stress fibers. Transfected KD cells which expressed the stable mutant I(-actin in excess of normal ,-actin were morphologically altered. In contrast, a second mutant ,-actin gene containing two additional mutations (Gly-36 -* Glu-36 and Glu-83 -* Asp-83, as well as Gly-244 -* Asp-244) did not alter cell morphology when expressed at high levels in transfected cells, but the protein was labile and did not accumulate in stress fibers. In both KD and HuT-12 cells, endogenous ,-and y-actin decreased in response to high-level expression of the stable mutant I-actin, in a manner consistent with autoregulatory feedback of actin concentrations. Since the percent decreases in the endogenous I8-and y-actins were equal, the ratio of net 0-actin (mutant plus normal) to y-actin was significantly increased in the transfected cells. Antisera capable of distinguishing the mutant from the normal epitope revealed that the mutant j-actin accumulated in stress fibers but did not participate in the formation of the actin filament-rich perinuclear network. These observations suggest that different intracellular locations differentially incorporate actin into cytoskeletal microfilaments. The dramatic impact on cell morphology and on P-actin/y-actin ratios in the transfected diploid KD cells may be related to the acquisition of some of the characteristics of cells that underwent the neoplastic transformation event that originally led to the appearance of the j-actin mutations.Actin is a ubiquitous, highly abundant protein that is responsible for a variety of cellular activities, including motility and the structural properties of the cytoplasm. This protein, highly conserved in evolution, forms complex structures in all eucaryotic cells by a combination of selfpolymerization and interactions with a host of binding proteins (reviewed in reference 37). These complex interactions have been the subject of intense biochemical and structural study but have not yet been amenable to genetic analysis. Few structural mutations in actin are known, as might be expected for a protein whose actions are so central to normal cell morphology and function. The recessive flightless mutants of Drosophila melanogaster show a disordered structure of the sarcomeres (18, 31) and induction of stress proteins (15,18,31) in the indirect flight muscles of homozygotes in which mutant alleles of a tissue-specific actin are expressed (18,31).Human fibroblasts which express mutant P-actins have been generated by the neoplastic transformation of diploid KD fibroblasts in vitro and the isolation of stable focusderived, neoplast...

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