The organization of actin and myosin in vascular endothelial cells in situ was studied by immunofluorescence microscopy. Examination of perfusion-fixed, whole mounts of normal mouse and rat descending thoracic aorta revealed the presence of axially oriented stress fibers containing both actin and myosin within the endothelial cells. In both species, the proportion of cells containing stress fibers varied from region to region within the same vessel. Some endothelial cells in mouse mesenteric vein and in rat inferior vena cava also contained stress fibers. Quantitative studies of the proportion of endothelial cells containing stress fibers in the descending thoracic aorta of age-matched normotensive and spontaneously hypertensive rats revealed significant differences. When animals of the same sex of the two strains were compared, the proportion was approximately two times greater in the spontaneously hypertensive rats. The proportion of endothelial cells containing stress fibers was about two times greater in males than in females of both strains. These observations suggest that multiple factors, including anatomical, sex, and hemodynamic differences, influence the organization of the endothelial cell cytoskeleton in situ.Stress fibers in tissue-cultured cells are linear, phase-dense, cytoplasmic fibrils that are demonstrable by light microscopy and consist of bundles of microfilaments (3). Immunofluorescence techniques have revealed actin and myosin, as well as accessory contractile proteins, within these structures (15,24,25,40), but their functional significance in vitro has remained unclear (4). Furthermore, on only a few occasions have similar structures been observed in situ (6,32,(42)(43)(44).Using immunofluorescence microscopy, work in this laboratory (6) demonstrated stress fibers in the scleroblasts located near the edge or over the radial ridge of the fish scale. Inasmuch as these regions are likely to experience great shearing forces, this suggested that in situ stress fibers play a role in cellular adhesion. Stress fiber-like structures were also observed within other cells but their function was interpreted as being different from that of the fibers found in the scleroblasts. As early as 1953, Palade (29) observed filaments within the basal cytoplasm of capillary endothelial cells. Subsequently, many investigators, using transmission electron microscopy, reported the presence of micro filaments (presumably containing actin; see reference 11) in the endothelial cells of various blood vessels in normotensive animals (20). In certain endothelial cells, these microfilaments were organized into bundles (7). Cross-striated microfilament bundles (simi-416 lar in appearance to the stress fibers of certain tissue-cultured cells) have been observed in the arterial endothelium (aorta and cerebral arteries) of hypertensive rats (17,19). However, similar structures were not found in normotensive rats; thus, it was postulated that cross-striated microfllament bundles are an adaptive response of the endot...

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To identify the DNA sequences that regulate the expression of the sarcomeric myosin heavy-chain (MHC) genes in muscle cells, a series of deletion constructs of the rat embryonic MHC gene was assayed for transient expression after introduction into myogenic and nonmyogenic cells. The sequences in 1.4 kilobases of 5'-flanking DNA were found to be sufficient to direct expression of the MHC gene constructs in a tissue-specific manner (i.e., in differentiated muscle cells but not in undifferentiated muscle and nonmuscle cells Sarcomeric myosin heavy chain, like other contractile proteins, exists as a family of distinct isoforms that are predominantly expressed during a given stage of muscle development or in a specific muscle tissue or both (11,12,36,39,69,76,77). In vertebrates, the different myosin heavy-chain (MHC) isoforms are encoded by a multigene family of distinct but closely related members (38,39,53,57,78). Structural studies on a variety of MHC genes from a number of different organisms have shown that these genes, at least in higher vertebrates, are comprised of approximately 25 kilobases (kb) of DNA and display a highly complex exon-intron organization (38, 51, 68). The rat embryonic skeletal muscle MHC gene (43,44,57,66,68,78) has recently been sequenced in its entirety and was found to be split into 41 exons distributed over 24 kb of DNA (68). In addition, most sarcomeric MHC genes are clustered in the genome (13,35,38,75), and several of them are tightly linked with only a few kb of intergenic sequence separating one gene from the next (38,39). However, the relevance of these structural features for the tightly controlled mode of expression of the MHC multigene family remains unclear.Differential MHC gene expression seems to be largely regulated at the level of transcription (3,36,43,51,72,74) In the present work, we have begun to define the cisregulatory elements involved in the cell-type-specific expression of the rat embryonic MHC gene. To this end, different MHC minigenes and CAT reporter genes were constructed, and their transcriptional activity was tested in transient expression assays upon transfection into nonmyogenic and myogenic cells. The results show that 1.4 kb of 5'-flanking sequence is sufficient to direct the tissue-specific expression of this gene, whereas intragenic sequences do not appear to be of major importance. In addition to the promoter, which in itself is not muscle specific, a minimum of two functionally distinct 5' sequence elements controls the expression of the rat embryonic MHC gene. The sequences from -173 to -142 base pairs (bp) confer tissue specificity to the gene by inhibiting its expression in nonmuscle cells. This musclespecific negative regulatory sequence needs upstream elements located between -1413 and -174 bp for full activity. The upstream elements can be functionally replaced by the simian virus 40 (SV40) enhancer but do not act like typical enhancers (31). These results demonstrate that cell-typespecific MHC gene expression is controlled by the concerted a...

Historically, sodium azide has been used to anesthetize the nematode Caenorhabditis elegans; however, the mechanism by which it survives this exposure is not understood. In this study, we report that exposure of wild-type C elegans to 10 mM sodium azide for up to 90 minutes confers thermotolerance (defined as significantly increased survival probability [SP] at 37 degrees C) on the animal. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed enhanced Hsp70 expression, whereas Western blot analysis revealed the induction of Hsp16. We also tested the only known C elegans Hsp mutant def-21 (codes for Hsp90), which constitutively enters the stress-resistant state known as the dauer larvae. Daf-21 mutants also acquire sodium azide-induced thermotolerance, whereas 3 non-Hsp, constitutive dauer-forming mutants exhibited a variable response to azide exposure. We conclude that the ability of C elegans to survive exposure to azide is associated with the induction of at least 2 stress proteins.