Donald A. Fischman

Monoclonal antibodies (McAbs) against the myosin heavy chain (MHC) of adult chicken pectoralis muscle have been tested for reactivity with pectoralis myosin at selected stages of chick development in vivo and in vitro. Three such McAbs, MF 20 and MF 14, which bind to light meromyosin, and MF 30, which binds to myosin subfragment two (S2), were used to assay the appearance and accumulation of specific MHC epitopes with: (a) indirect, solid phase radioimmune assay (RIA), (b) immunoautoradiography, (c) immunofluorescence microscopy. McAb MF 20 bound strongly and equivalently to MHC at all stages of embryonic development in vivo. In contrast, the MF 30 epitope was barely detectable at 12 d of incubation but its concentration rose rapidly just before hatching. No detectable binding of MF 14 to pectoralis myosin could be measured during myogenesis in vivo until 1 wk after hatching. Immunofluorescence studies revealed that all three epitopes accumulate in the same myocytes of the developing pectoralis muscle. Since all three McAbs bound with high activity to native and denatured forms of myosin, it is unlikely that differential antibody reactivity can be explained by conformational changes in myosin during development in vivo. When myogenesis in vitro was monitored using the same McAbs, MF 20 bound to the MHC at all stages tested while reactivity of MF 30 and MF 14 with myosin from cultured muscle was never observed. Thus, this study demonstrates three different immunochemical states of the MHC during development in vivo of chick pectoralis muscle and the absence of later occurring immunochemical transitions in the MHC of cultured embryonic muscle.

Abstract Treatment of isolated human erythrocyte membranes with Triton X‐100 at ionic strength ⋍0.04 preferentially released all the glycerolipid and glycoprotein species. At low ionic strength, certain nonglycosylated polypeptides were also selectively solubilized. The liberated polypeptides were free of lipids, but some behaved as if associated into specific oligomeric complexes. Each detergent‐insoluble ghost residue appeared by electron microscopy to be a filamentous reticulum with adherent lipoid sheets and vesicles. The residues contained most of the membrane sphingolipids and the nonglycosylated proteins. The polypeptide elution profile obtained with nonionic detergents is therefore nearly reciprocal to that previously seen with a variety of agents which perturb proteins. These data afford further evidence that the externally‐oriented glycoproteins penetrate the membrane core where they are anchored hydrophobically, whereas the nonglycosylated polypeptides are, in general, bound by polar associations at the inner membrane surface. The filamentous meshwork of inner surface polypeptides may constitute a discrete, self‐associated continuum which provides rather than derives structural support from the membrance.

Cellular progenitors of the coronary vasculature are believed to enter the chicken heart during epicardial morphogenesis between stages 17 and 27 (days 3-5) of egg incubation. To trace cells which give rise to the coronary arteries in vivo, we applied retroviral cell tagging procedures and analyzed clonal populations of vascular smooth muscle, endothelium, and connective tissue in the hearts of post-hatch chickens. Our data provide direct proof that (i) vascular smooth muscle progenitors begin to enter the heart at stage 17, substantially after the heart begins propulsive contractions; (ii) cardiac myocytes, vascular smooth muscle, perivascular fibroblasts, and coronary endothelial cells all derive from independent precursors when these cells migrate into the heart; (iii) endothelial cells of the coronary vessels have a different clonal origin than endothelial cells of the endocardium; (iv) coronary arteries form by the coalescence of discontinuous colonies (i.e., in situ vasculogenesis), each derived from a founder cell tagged at the time of retroviral injection (stages 17-18); and (v) coronary arteries contain discrete segments composed of "polyclones." These studies indicate the feasibility of gene targeting to coronary progenitors through the use of recombinant retroviruses.

Intracellular targets of the ubiquitous second messenger cAMP are located at great distances from the most widely studied source of cAMP, the G protein responsive transmembrane adenylyl cyclases. We previously identified an alternative source of cAMP in mammalian cells lacking transmembrane spanning domains, the "soluble" adenylyl cyclase (sAC). We now demonstrate that sAC is distributed in specific subcellular compartments: mitochondria, centrioles, mitotic spindles, mid-bodies, and nuclei, all of which contain cAMP targets. Distribution at these intracellular sites proves that adenylyl cyclases are in close proximity to all cAMP effectors, suggesting a model in which local concentrations of cAMP are regulated by individual adenylyl cyclases targeted to specific microdomains throughout the cell.