SummaryWe demonstrate here that CD59, an inhibitor of the membrane attack complex (MAC) of the complement system, is present in cell-free seminal plasma (SP) at a concentration of at least 20 #g/ml. Analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and Edman degradation indicated that this protein, SP CD59, was similar, if not identical, to CD59 isolated from erythrocyte (E) membranes (E CD59). Like purified E CD59, SP CD59 also possesses a glycosyl phosphatidyl inositol (GPI) anchor and incorporates into the membranes of heterologous cells where it inhibits lysis by the human MAC. This phenomenon could be demonstrated not only if cells were incubated with purified SP CD59 but also if unfractionated SP were used. Further, CD59 in unfractionated SP bound to washed spermatozoa, increasing their membrane content of the protein. The mechanism by which this protein retains its GPI anchor while apparently present in the fluid phase is of interest and was further investigated. Using the techniques of high-speed centrifugation, fast performance liquid chromatography fractionation, and electron microscopy, we found that all detectable SP CD59 was associated with vesicular extracellular organelles. These organelles, named "prostasomes" were previously known to be present in SP and to interact with spermatozoa, although their function was uncertain. Interaction of heterologous E with prostasomes rendered the cells more resistant to lysis by human MACs. We propose that these organelles represent a pool of CD59 from which protein lost from spermatozoa, perhaps as a result of low level complement attack or of normal membrane turnover, can be replenished.

We have used quantitative electron microscope autoradiography to study uptake and distribution of arachidonate in HSDMIC1 murine fibrosarcoma cells and in EPU-1B, a mutant HSDMtC1 line defective in high affinity arachidonate uptake. Cells were labeled with [3H]arachidonate for 15 min, 40 min, 2 h, or 24 h. Label was found almost exclusively in cellular phospholipids; 92-96% of incorporated radioactivity was retained in cells during fixation and tissue processing. All incorporated radioactivity was found to be associated with cellular membranes. Endoplasmic reticulum (ER) contained the bulk of [3H]arachidonate at all time points in both cell types, while mitochondria, which contain a large portion of cellular membrane, were labeled slowly and to substantially lower specific activity. Plasma membrane (PM) also labeled slowly, achieving a specific activity only one-sixth that of ER at 15 rain in HSDM~Ct cells (6% of total label) and one-third of ER in EPU-1B (10% of total label). Nuclear membrane (NM) exhibited the highest specific activity of labeling at 15 min in HSDM~C1 cells (twice that of ER) but was not preferentially labeled in the mutant. Over 24 h, PM label intensity increased to that of ER in both cell lines. However, NM activity diminished in HSDM~C1 cells by 24 h to a small fraction of that in ER. In response to agonists, HSDM~C~ cells release labeled arachidonate for icosanoid synthesis most readily when they have been labeled for short times. Our results therefore suggest that NM and ER, sites of cyclooxygenase in murine fibroblasts, are probably sources for release of [3H]arachidonate, whereas PM and mitochondria are unlikely to be major sources of icosanoid precursors.Arachidonic acid (C20:4~5.8.1 u4) is the major polyunsaturated fatty acid of most mammalian cells and occurs primarily esterified in the sn-2 position of cellular phospholipids (11). In addition to serving as a structural membrane component, arachidonate is the primary substrate for the synthesis of icosanoids. These oxygenated metabolites, including prostaglandins, leukotrienes, thromboxane, and related compounds, modulate important biological processes including inflammation, allergy, and hemostasis (30). Icosanoid precursor fatty acids (chiefly arachidonate) are used by specific metabolic pathways, distinct from those used by other fatty acids, in cells that make these mediators. These cell types avidly accumulate arachidonate from their surroundings and esterify it into cellular lipids by virtue of an acyl-CoA synthetase specific for icosanoid precursor fatty acids (20, 34). A mutant cell line that lacks this enzyme is defective both in high affinity arachidonate uptake and its subsequent release in response to the agonist bradykinin (19).Upon appropriate stimulation, icosanoid synthesis is initiated by the selective release of arachidonate from phospholipids, chiefly phosphatidylinositol and phosphatidylcholine. In every case studied thus far, the availability of unesterified arachidonate, and hence this specific release ...

Fibronectin, a glycoprotein that binds to collagen and modifies the adhesion properties and motility of cells in culture, is present in the interstitium of rat hearts. To localize fibronectin more precisely and to assess its relationship to the myocyte and to connective tissue elements, we employed a double antibody technique to label myocardial fibronectin with electron-dense ferritin to permit an ultrastructural analysis. Fibronectin was found to be associated with collagen, and in some cases appeared to link collagen fibers. Fibronectin was also found inserted along the surfaces of cardiac myocytes, connecting these cells to perimyocytic collagen. These ultrastructural relationships imply that fibronectin is a major component of the myocardial interstitium, and may affect myocardial compliance and control the motion of myocytes during the contraction and relaxation of the heart.

The manner in which growth factors acting at the cell surface regulate activity of myogenic basic-helix-loop-helix proteins in the nucleus and thus control the fate of committed skeletal myoblasts remains poorly understood. In this study, we report that immunoreactive G s protein ␣ -subunits (

Transcription factor GATA-4 belongs to a family of zinc finger proteins involved in lineage determination. GATA-4 is first expressed in yolk sac endoderm of the developing mouse and later in cardiac tissue, gut epithelium and gonads. To delineate the role of this transcription factor in differentiation and early development, we studied embryoid bodies derived from mouse embryonic stem (ES) cells in which both copies of the Gata-4 gene were disrupted. Light and electron microscopy demonstrated that embryoid bodies formed from wild-type and heterozygous deficient ES cells were covered with a layer of visceral yolk sac endoderm, whereas no yolk sac endoderm was evident on the surface of the homozygous deficient embryoid bodies. Independently selected homozygous deficient cell lines displayed this distinctive phenotype, suggesting that it was not an artifact of clonal variation. Biochemical markers of visceral endoderm formation, such as alpha-feto-protein, hepatocyte nuclear factor-4 and binding sites for Dolichos biflorus agglutinin, were absent from the homozygous deficient embryoid bodies. Examination of other differentiation markers in the mutant embryoid bodies, studies of ES cell-derived teratocarcinomas and chimeric mouse analysis demonstrated that GATA-4-deficient ES cells have the capacity to differentiate along other lineages. We conclude that, under in vitro conditions, disruption of the Gata-4 gene results in a specific block in visceral endoderm formation. These homozygous deficient cells should yield insights into the regulation of yolk sac endoderm development and the factors expressed by visceral endoderm that influence differentiation of adjoining ectoderm/mesoderm.