Identification of an osteocalcin isoform in fish with a large acidic prodomain

Osteocalcin is a small, secreted bone protein whose gene consists of four exons. In the course of analyzing the structure of fish osteocalcin genes, we recently found that the spotted green pufferfish has two possible exon 2 structures, one of 15 bp and the other of 324 bp. Subsequent analysis of the pufferfish cDNA showed that only the transcript with a large exon 2 exists. Exon 2 codes for the osteocalcin propeptide, and exon 2 of pufferfish osteocalcin is ∼3.4-fold larger than exon 2 previously found in other vertebrate species. We have termed this new pufferfish osteocalcin isoform OC2. Additional studies showed that the OC2 isoform is restricted to a unique fish taxonomic group, the Osteichthyes; OC2 is the only osteocalcin isoform found so far in six Osteichthyes species, whereas both OC1 and OC2 isoforms coexist in zebrafish and rainbow trout. The larger size of the OC2 propeptide is due to an acidic region that is likely to be highly phosphorylated and has no counterpart in the OC1 propeptide. We propose 1) that OC1 and OC2 are encoded by distinct genes that originated from a duplication event that probably occurred in the teleost fish lineage soon after divergence from tetrapods and 2) that the novel OC2 propeptide could be, if secreted, a phosphoprotein that participates in the regulation of biomineralization through its large acidic and phosphorylated propeptide

Accumulation of muscle ankyrin repeat protein transcript reveals local activation of primary myotube endcompartments during muscle morphogenesis

The characteristic shapes and positions of each individual body muscle are established during the process of muscle morphogenesis in response to patterning information from the surrounding mesenchyme. Throughout muscle morphogenesis, primary myotubes are arranged in small parallel bundles, each myotube spanning the forming muscles from end to end. This unique arrangement potentially assigns a crucial role to primary myotube end regions for muscle morphogenesis. We have cloned muscle ankyrin repeat protein (MARP) as a gene induced in adult rat skeletal muscle by denervation. MARP is the rodent homologue of human C-193 (Chu, W., D.K. Burns, R.A. Swerick, and D.H. Presky. 1995. J. Biol. Chem. 270:10236-10245) and is identical to rat cardiac ankyrin repeat protein. (Zou, Y., S. Evans, J. Chen, H.-C. Kuo, R.P. Harvey, and K.R. Chien. 1997. Development. 124:793-804). In denervated muscle fibers, MARP transcript accumulated in a unique perisynaptic pattern. MARP was also expressed in large blood vessels and in cardiac muscle, where it was further induced by cardiac hypertrophy. During embryonic development, MARP was expressed in forming skeletal muscle. In situ hybridization analysis in mouse embryos revealed that MARP transcript exclusively accumulates at the end regions of primary myotubes during muscle morphogenesis. This closely coincided with the expression of thrombospondin-4 in adjacent prospective tendon mesenchyme, suggesting that these two compartments may constitute a functional unit involved in muscle morphogenesis. Transfection experiments established that MARP protein accumulates in the nucleus and that the levels of both MARP mRNA and protein are controlled by rapid degradation mechanisms characteristic of regulatory early response genes. The results establish the existence of novel regulatory muscle fiber subcompartments associated with muscle morphogenesis and denervation and suggest that MARP may be a crucial nuclear cofactor in local signaling pathways from prospective tendon mesenchyme to forming muscle and from activated muscle interstitial cells to denervated muscle fibers

Recruitment, augmentation and apoptosis of rat osteoclasts in 1,25-(OH)2D3 response to short-term treatment with 1,25-dihydroxyvitamin D3in vivo

Background Although much is known about the regulation of osteoclast (OC) formation and activity, little is known about OC senescence. In particular, the fate of of OC seen after 1,25-(OH)2D3 administration in vivo is unclear. There is evidence that the normal fate of OC is to undergo apoptosis (programmed cell death). We have investigated the effect of short-term application of high dose 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on OC apoptosis in an experimental rat model. Methods OC recruitment, augmentation and apoptosis was visualised and quantitated by staining histochemically for tartrate resistant acid phosphatase (TRAP), double staining for TRAP/ED1 or TRAP/DAPI, in situ DNA fragmentation end labelling and histomorphometric analysis. Results Short-term treatment with high-dose 1,25-(OH)2D3 increased the recruitment of OC precursors in the bone marrow resulting in a short-lived increase in OC numbers. This was rapidly followed by an increase in the number of apoptotic OC and their subsequent removal. The response of OC to 1,25-(OH)2D3 treatment was dose and site dependent; higher doses producing stronger, more rapid responses and the response in the tibiae being consistently stronger and more rapid than in the vertebrae. Conclusions This study demonstrates that (1) after recruitment, OC are removed from the resorption site by apoptosis (2) the combined use of TRAP and ED1 can be used to identify OC and their precursors in vivo (3) double staining for TRAP and DAPI or in situ DNA fragmentation end labelling can be used to identify apoptotic OC in vivo

Sturgeon osteocalcin shares structural features with matrix gla protein evolutionary relationship and functional implications

Osteocalcin (OC) and matrix Gla protein (MGP) are considered evolutionarily related because they share key structural features, although they have been described to exert different functions. In this work, we report the identification and characterization of both OC and MGP from the Adriatic sturgeon, a ray-finned fish characterized by a slow evolution and the retention of many ancestral features. Sturgeon MGP shows a primary structure, post-translation modifications, and patterns of mRNA/protein distribution and accumulation typical of known MGPs, and it contains seven possible Gla residues that would make the sturgeon protein the most gamma-carboxylated among known MGPs. In contrast, sturgeon OC was found to present a hybrid structure. Indeed, although exhibiting protein domains typical of known OCs, it also contains structural features usually found in MGPs (e. g. a putative phosphorylated propeptide). Moreover, patterns of OC gene expression and protein accumulation overlap with those reported for MGP; OC was detected in bone cells and mineralized structures but also in soft and cartilaginous tissues. We propose that, in a context of a reduced rate of evolution, sturgeon OC has retained structural features of the ancestral protein that emerged millions of years ago from the duplication of an ancient MGP gene and may exhibit intermediate functional features.Portuguese Science and Technology Foundation [POCTI/MAR/57921/2004, SFRH/BD/9077/2002]; Fundo Europeu De Desenvolvimento Regional (FEDER) (Portugal); National Funding; Center of Marine Sciences (CCMAR

The Rachel Carson Letters and the Making of Silent Spring

Environment, conservation, green, and kindred movements look back to Rachel Carson’s 1962 book Silent Spring as a milestone. The impact of the book, including on government, industry, and civil society, was immediate and substantial, and has been extensively described; however, the provenance of the book has been less thoroughly examined. Using Carson’s personal correspondence, this paper reveals that the primary source for Carson’s book was the extensive evidence and contacts compiled by two biodynamic farmers, Marjorie Spock and Mary T. Richards, of Long Island, New York. Their evidence was compiled for a suite of legal actions (1957-1960) against the U.S. Government and that contested the aerial spraying of dichlorodiphenyltrichloroethane (DDT). During Rudolf Steiner’s lifetime, Spock and Richards both studied at Steiner’s Goetheanum, the headquarters of Anthroposophy, located in Dornach, Switzerland. Spock and Richards were prominent U.S. anthroposophists, and established a biodynamic farm under the tutelage of the leading biodynamics exponent of the time, Dr. Ehrenfried Pfeiffer. When their property was under threat from a government program of DDT spraying, they brought their case, eventually lost it, in the process spent US$100,000, and compiled the evidence that they then shared with Carson, who used it, and their extensive contacts and the trial transcripts, as the primary input for Silent Spring. Carson attributed to Spock, Richards, and Pfeiffer, no credit whatsoever in her book. As a consequence, the organics movement has not received the recognition, that is its due, as the primary impulse for Silent Spring, and it is, itself, unaware of this provenance

Injectable Peptide Decorated Functional Nanofibrous Hollow Microspheres to Direct Stem Cell Differentiation and Tissue Regeneration

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/110559/1/adfm201402618-sup-0001-S1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/110559/2/adfm201402618.pd

Electromechanical Coupling between Skeletal and Cardiac Muscle: Implications for Infarct Repair

Skeletal myoblasts form grafts of mature muscle in injured hearts, and these grafts contract when exogenously stimulated. It is not known, however, whether cardiac muscle can form electromechanical junctions with skeletal muscle and induce its synchronous contraction. Here, we report that undifferentiated rat skeletal myoblasts expressed N-cadherin and connexin43, major adhesion and gap junction proteins of the intercalated disk, yet both proteins were markedly downregulated after differentiation into myo-tubes. Similarly, differentiated skeletal muscle grafts in injured hearts had no detectable N-cadherin or connexin43; hence, electromechanical coupling did not occur after in vivo grafting. In contrast, when neonatal or adult cardiomyocytes were cocultured with skeletal muscle, ∼10% of the skeletal myotubes contracted in synchrony with adjacent cardiomyocytes. Isoproterenol increased myotube contraction rates by 25% in coculture without affecting myotubes in monoculture, indicating the cardiomyocytes were the pacemakers. The gap junction inhibitor heptanol aborted myotube contractions but left spontaneous contractions of individual cardiomyocytes intact, suggesting myotubes were activated via gap junctions. Confocal microscopy revealed the expression of cadherin and connexin43 at junctions between myotubes and neonatal or adult cardiomyocytes in vitro. After microinjection, myotubes transferred dye to neonatal cardiomyocytes via gap junctions. Calcium imaging revealed synchronous calcium transients in cardiomyocytes and myotubes. Thus, cardiomyocytes can form electromechanical junctions with some skeletal myotubes in coculture and induce their synchronous contraction via gap junctions. Although the mechanism remains to be determined, if similar junctions could be induced in vivo, they might be sufficient to make skeletal muscle grafts beat synchronously with host myocardium