Abstract. Cultured cardiac myocytes were stained with antibodies to sarcomeric ot-actinin, troponin-I, tx,-actin, myosin heavy chain (MHC), titin, myomesin, C-protein, and vinculin. Attention was focused on the distribution of these proteins with respect to nonstriated myofibrils (NSMFs) and striated myofibrils (SMFs). In NSMFs, ot-actinin is found as longitudinally aligned, irregular ,,o0.3-#m aggregates. Such aggregates are associated with ot-actin, troponin

Advanced glycation end products (AGEs) are linked with the development of diabetic retinopathy; however, the pathogenic mechanisms are poorly defined. Vascular endothelial growth factor (VEGF) levels are increased in ischemic and nonischemic diabetic retina, and VEGF is required for the development of retinal and iris neovascularization. Moreover, VEGF alone can induce much of the concomitant pathology of diabetic retinopathy. In this study, we found that AGEs increased VEGF mRNA levels in the ganglion, inner nuclear, and retinal pigment epithelial (RPE) cell layers of the rat retina. In vitro, AGEs increased VEGF mRNA and secreted protein in human RPE and bovine vascular smooth muscle cells. The AGE-induced increases in VEGF expression were dose-and time-dependent, inhibited by antioxidants, and additive with hypoxia. Use of an anti-VEGF antibody blocked the capillary endothelial cell proliferation induced by the conditioned media of AGE-treated cells. AGEs may participate in the pathogenesis of diabetic retinopathy through their ability to increase retinal VEGF gene expression.

Abstract. Experiments are described supporting the proposition that the assembly of stress fibers in nonmuscle cells and the assembly of myofibrils in cardiac cells share conserved mechanisms. Double staining with a battery of labeled antibodies against membraneassociated proteins, myofibrillar proteins, and stress fiber proteins reveals the following: (a) dissociated, cultured cardiac myocytes reconstitute intercalated discs consisting of adherens junctions (AJs) and desmosomes at sites of cell-cell contact and sub-sarcolemmal adhesion plaques (SAPs) at sites of cellsubstrate contact; (b) each AJ or SAP associates proximally with a striated myofibril, and conversely every striated myofibril is capped at either end by an AJ or a SAP; (C) the invariant association between a given myofibril and its SAP is especially prominent at the earliest stages of myofibrillogenesis; nascent myofibrils are capped by oppositely oriented SAPs; (d) the insertion of nascent myofibrils into AJs or into SAPs invariably involves vinculin, a-actin, and sarcomeric ot-actinin (s-ot-actinin); (e) AJs are positive for A-CAM but negative for talin and integrin; SAPs lack A-CAM but are positive for talin and integrin; (f) in cardiac cells all ot-actinin-containing structures invariably are positive for the sarcomeric isoform, o~-actin and related sarcomeric proteins; they lack non-sot-actinin, -y-actin, and caldesmon; (g) in fibroblasts all ot-actinin-containing structures are positive for the non-sarcomeric isoform, -y-actin, and related nonsarcomeric proteins, including caldesmon; and (h) myocytes differ from all other types of adherent cultured cells in that they do not assemble authentic stress fibers; instead they assemble stress fiber-like structures of linearly aligned I-Z-I-like complexes consisting exclusively of sarcomeric proteins. CONSIDERABLE information has accumulated regarding the molecular composition and possible functions of the submembranous complexes into which stress fibers insert (4,5,11,26,34,46). In cell-cell junctions of the adherens type the distal tips of the microfilament bundles insert into submembranous F-actin/o~-actinin/vinculin complexes that often involve additional membrane-associated molecules such as A-CAM (31, 60), radixin (58) etc. A somewhat different class of submembranous complexes characterizes the termini of stress fibers where they insert into the cell-substrate junctions of spread, nontranslocating cultured ceils. These F-actin/ot-actinin/vinculin complexes, known as adhesion plaques (APs),t are also positive for talin (11), paxillin (59), integrin (17), and other proteins. APs are also sites for a Ca2 § protease (5) and a1. Abbreviations used in this paper: AJ, adherens junction; AP, adhesion plaque; MHC, myosin heavy chain; s, sarcomeric; SAP, subsarcolemmal adhesion plaque.protein kinase C (35) suggesting they may be involved in transmembrane signaling. In both the adherens junction (AJs) and the APs, ct-actinin appears to play a bifunctional role. Distally, facing the inner surface of the c...

Based on the assumption that a conserved differentiation program governs the assembly of sarcomeres in skeletal muscle in a manner analogous to programs for viral capsid assembly, we have defined the temporal and spatial distribution of 10 muscle-specific proteins in mononucleated myoblasts as a function of the time after terminal cell division. Single cells in mitosis were identified in monolayer cultures of embryonic chicken pectoralis, followed for selected time points (0-24 h postmitosis) by video time-lapse microscopy, and then fixed for immunofluorescence staining. For convenience, the myoblasts were termed x-h-old to define their age relative to their mitotic "birthdate." All 6 h myoblasts that emerged in a mitogen-rich medium were desmin+ but only 50% were positive for a alpha-actin, troponin-I, alpha-actinin, MyHC, zeugmatin, titin, or nebulin. By 15 h postmitosis, approximately 80% were positive for all of the above proteins. The up-regulation of these 7 myofibrillar proteins appears to be stochastic, in that many myoblasts were alpha-actinin+ or zeugmatin+ but MyHC- or titin- whereas others were troponin-I+ or MyHC+ but alpha-actinin- or alpha-actin-. In 15-h-old myoblasts, these contractile proteins were organized into nonstriated myofibrils (NSMFs). In contrast to striated myofibrils (SMFs), the NSMFs exhibited variable stoichiometries of the sarcomeric proteins and these were not organized into any consistent pattern. In this phase of maturation, two other changes occurred: (1) the microtubule network was reorganized into parallel bundles, driving the myoblasts into polarized, needle-shaped cells; and (2) the sarcolemma became fusion-competent. A transition from NSMFs to SMFs took place between 15 and 24 h (or later) postmitosis and was correlated with the late appearance of myomesin, and particularly, MyBP-C (C protein). The emergence of one, or a string of approximately 2 mu long sarcomeres, was invariably characterized by the localization of myomesin and MyBP-C to their mature positions in the developing A-bands. The latter group of A-band proteins may be rate-limiting in the assembly program. The great majority of myoblasts stained positively for desmin and myofibrillar proteins prior to, rather than after, fusing to form myotubes. This sequential appearance of muscle-specific proteins in vitro fully recapitulates myofibrillar assembly steps in myoblasts of the myotome and limb bud in vivo, as well as in nonmuscle cells converted to myoblasts by MyoD. We suggest that this cell-autonomous myoblast differentiation program may be blocked at different control points in immortalized myogenic cell lines.

RNase J is a conserved ribonuclease that belongs to the β-CASP family of nucleases. It possesses both endo- and exo-ribonuclease activities, which play a key role in pre-rRNA maturation and mRNA decay. Here we report high-resolution crystal structures of Deinococcus radiodurans RNase J complexed with RNA or uridine 5′-monophosphate in the presence of manganese ions. Biochemical and structural studies revealed that RNase J uses zinc ions for two-metal-ion catalysis. One residue conserved among RNase J orthologues (motif B) forms specific electrostatic interactions with the scissile phosphate of the RNA that is critical for the catalysis and product stabilization. The additional manganese ion, which is coordinated by conserved residues at the dimer interface, is critical for RNase J dimerization and exonuclease activity. The structures may also shed light on the mechanism of RNase J exo- and endonucleolytic activity switch.