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 ($2), 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.Isoforms of myosin have been demonstrated in adult (1,3,10,19,28,37) and embryonic (11,12,20,27,30,35,36) striated muscles of both birds and mammals. Three experimental formats have been used to substantiate these myosin variants within embryonic muscles. First, heterogeneity of myosin can be detected by electrophoresis on native pyrophosphate gels (14). Secondly, differences exist in the peptide maps obtained by limited proteolysis of adult and embryonic myosins (27,35,36). Finally, immunological dissimilarities exist between myosins isolated from homologous adult and embryonic muscles (11,12,20,26,35,36). Masaki and Yoshizaki (20) have demonstrated that myofibrils from embryonic chick pectoralis muscle bind antibodies specific for myosin of adult cardiac muscle, whereas similar myofibrils from adult pectoralis muscle do not. It was later proven that these cross-reactive determinants reside in the myosin heavy chain (MHC) subunit. More recently, Gauthier et al. (11,12) and Rubinstein and Kelley (26) have shown that developing rat skeletal myofibers contain MHCs which share antigenic homology with myosin isolated from both adult fast-and slow-twitch muscles. However, the adult myofibers bound specifically to either the "anti-slow" myosin or "anti-fast" myosin antiserum preparations. Immunochemical differences between myosin preparations purified from homologous em...

Treatment of isolated human erythrocyte membranes with Triton X-100 at ionic strength 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 t o 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, selfassociated continuum which provides rather than derives structural support from the membrance. 0.04 preferentially released all the glycerolipid and glycoprotein species.

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.

Cellular progenitors of the coronary vasculature are believed to enter the chicken heart during epicardlal 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 (t) 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; (ON) 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.The embryonic chicken heart begins rhythmic contractions at Hamburger-Hamilton stage 10 ("'28 hr of incubation) (1, 2), but for the first 6 days of chicken embryogenesis the myocardial wall is avascular and nourished by diffusion through the endocardium (3)(4)(5). Such diffusion is facilitated by extensive trabecular channels which markedly increase endocardial surface area (6). Overt coronary vasculogenesis begins on day 6 of incubation (stage 29), first as venous sinusoids in communication with the trabecular channels, and secondarily as coronary arterial vessels which anastomose with the sinusoids (7,8). A closed coronary vasculature is completed after day 14 ofembryogenesis (3-5). Up to stage 15, the heart is composed of only two cell types: myocytes and endothelial cells; neither connective tissue, coronary vessels, neural elements, nor the conduction system is evident histologically (9, 10). Progenitor cells of the coronary vessels along with connective-tissue precursors are believed to enter the heart as the epicardial mantle envelopes the myocardium at stages 17-27 (10-12). Except for one study using chicken-quail chimeras to trace endothelial cells in the developing avian vasculature (13), no lineage analyses of coronary progenitors have been published. In this report we present an application of retroviral cell tagging (14,15) procedures for tracking smooth muscle, endothelial, and connective-tissue progenitors during coronary morphogenesis. We demonstrate the independence offibroblast, smooth muscle, and endothelial lineages and prove that coronary vessels form by in situ vasculogenesis rather than by angiogenic outgrowth from the root of the aorta. 106 active virions per ml, were concentrated by ultracentrifugation (...

Bicarbonate-responsive “soluble” adenylyl cyclase resides, in part, inside the mammalian cell nucleus where it stimulates the activity of nuclear protein kinase A to phosphorylate the cAMP response element binding protein (CREB). The existence of this complete and functional, nuclear-localized cAMP pathway establishes that cAMP signals in intracellular microdomains and identifies an alternate pathway leading to CREB activation.