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Early skeletal muscle development is accompanied by changes in the composition of the cytoskeleton. In this report we analyze the distribution of the intermediate filament nestin in the developing mouse limb buds in vivo and in mesenchymal cells isolated from limb buds in vitro. The subcellular distribution of nestin mRNA and protein in muscle cells was also analyzed. We find a shift in nestin expression during early limb bud development. At embryonic day 11 (E11), low levels of nestin (protein) were expressed in the mesenchymal cells of the developing limb bud. Later, nestin mRNA and protein were down-regulated in the mesenchymal condensations undergoing chondrogenesis (E12 and E13), but remained expressed predominantly in the ectodermal cells and in the differentiating myoblasts. At E18, only muscle fibres, endothelial cells and nerves were nestin positive. This shift in expression was reproduced in vitro, in micro-mass cultures of mesenchymal cells. In E11 cultures, nestin protein was initially expressed in all cells. Upon formation of cartilage foci (after 2-3 days in culture), nestin immunoreactivity was not observed in cartilage, and low levels were detected in the cells located between the foci. A subpopulation of mono- and multinucleated cells, peripheral to the cartilage nodules, expressed the muscle-specific intermediate filament desmin protein together with high levels of nestin protein. The proportion of nestin-expressing cells could be changed by addition of specific signalling molecules. Insulin-like growth factors I and II (IGF I and II) increased the number of nestin-positive cells, while basic fibroblast growth factor (FGF) reduced the number of nestin-expressing cells. Finally, we present evidence for a different subcellular localization of nestin protein and mRNA: the mRNA is predominantly located in the ends of the muscle cell, whereas the protein is found in the central region. Intracellular localization of nestin mRNA may constitute an additional level of regulation of the cytoskeleton during muscle development.

Background: The toxic effects of acute citalopram overdose are reported by the Swedish Poisons Information Centre., Design: Case reports received from Swedish hospitals during 1995 have been analyzed. Forty-four cases of pure citalopram intoxication have been studied in detail., Results: At doses below 600 mg, mild symptoms were observed. Doses above 600 mg caused ECG abnormalities and convulsions in some patients, while doses greater than 1900 mg caused such symptoms in all patients., Conclusions: The findings are consistent with previous reports claiming that selective serotonin reuptake inhibitors are less toxic compared to tricyclic antidepressants. However, there is a risk of developing serious symptoms when large doses have been ingested.

Tenascin-C (TN-C) is an extracellular matrix protein expressed during development in several tissues, but restricted to only a few areas in normal adult tissues. By immunizing mice with human fetal myoblasts we generated a monoclonal antibody to TN-C and mapped the epitope to the aminoterminal end containing EGF-like repeats. Using this antibody we detected by immunohistochemistry TN-C in the epimysium and perimysium of human fetal muscles, as well as in nonfibrillar deposits in myoblast cultures. In situ hybridization did not reveal any signal within human fetal muscle groups, suggesting that non-muscle cells synthesize the majority of the tenascin that localizes in and around human fetal muscle. Immunohistochemical analysis of muscle biopsies from Duchenne/Becker muscular dystrophy and myositis patients revealed that TN-C is expressed in skeletal muscle. Although the patterns of TN-C immunoreactivity were quite different in the two disease entities, the endomysial TN-C reactivity in both DMD/BMD and in myositis invariably correlated with the presence of macrophages.

Integrin expression and distribution was studied in cloned human fetal G6 myoblasts and myotubes. Immunoprecipitation of beta 1 integrins from surface iodinated and metabolically labeled G6 cells typically showed a five-fold induction of a beta 1 integrin associated protein upon differentiation. Under non-reducing conditions this beta 1 associated protein migrated as 145 kD. No such beta 1 associated protein was observed in the myogenic L8 rat cell line, before or after differentiation. The beta 1 integrin associated cell surface protein present in G6 myotubes remained associated with the beta 1 subunit in the presence of 1% Triton X-100 and 0.5 M NaCl. Like integrin alpha-chains, the protein dissociated from the beta 1 integrin subunit at low pH. Immunoprecipitation of G6 myotubes further indicated the presence of alpha 1, alpha 3, alpha 5, and alpha v integrins, and small amounts of alpha 4 and alpha 6 integrins. Immunodepletion with integrin alpha-chain antibodies to alpha 1, alpha 3, alpha 4, alpha 5, alpha 6, and alpha v integrin chains could not deplete the beta 1 integrin associated protein, indicating that it did not interact with any of these known integrin heterodimers. Upon treatment with reducing agents, the beta 1 integrin associated protein migrated in SDS-PAGE as a 155 kD protein. The decreased mobility in SDS-PAGE upon reduction is a feature shared with alpha 1, alpha 2, and alpha 9 integrin alpha-chains. Antibodies to alpha 1 immunoprecipitated an integrin heterodimer distinct from the 155 kD protein.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of fibronectin (FN) and vitronectin (VN) receptors for cell adhesion and matrix assembly was analyzed during human fetal myogenesis in vivo and in vitro. In human fetal muscle at 10 weeks gestational age FN and laminin are present in the extracellular matrix. Analysis of the integrin repertoire at this developmental stage reveals that the differentiated muscle cells in vivo express alpha 5 and alpha 6 integrins, but not alpha v, alpha 1, and alpha 3 integrins. However, in vitro cultured myoblasts (G6) isolated from the same gestational age express alpha v, alpha 1, and alpha 3 integrins in addition to alpha 5 and alpha 6 integrins. A more detailed analysis of FN and VN receptors in vitro shows that the localization of different alpha v heterodimers into focal contacts is differently regulated. Alpha v beta 1, and alpha v beta 3, are present at focal contacts throughout in vitro myogenesis whereas alpha v beta 5 appears to depend on an endogenously produced factor to localize to focal contacts. The alpha v beta 1, alpha v beta 5, and alpha 3 beta 1 heterodimers, often reported not to focalize, did form focal contacts in G6 cells, indicating that these myoblasts possess components facilitating the formation of cytoskeletal linkages containing these integrins. Alpha 5 beta 1 colocalized with FN in myoblast cultures, whereas myotubes lacked both FN and alpha 5 beta 1 on the cell surface. In summary, we show that concomitant with in vitro differentiation of G6 cells, FN matrix contacts are abolished, but vitronectin receptors continue to fulfill an anchoring function during the differentiation process in vitro. Further studies are needed to assess the relative importance of the FN and VN binding integrins for the differentiation process in comparison with the laminin-binding integrins alpha 6 and alpha 7, also present on these cells.

During skeletal muscle development three intermediate filament proteins are expressed: nestin, vimentin, and desmin. Vimentin and desmin belong to the class III intermediate filaments and are closely related to each other, whereas nestin is a more distantly related, class VI, intermediate filament. It was previously observed by conventional immunocytochemistry that the intracellular patterns of nestin, desmin, and vimentin appeared indistinguishable, despite nestin's more distant evolutionary relationship. We here extend this analysis by applying three-dimensional fluorescence digital imaging microscopy to compare the intracellular distribution of nestin with that of desmin, vimentin, actin, and tubulin in G6 human fetal skeletal muscle cells. We show that in vitro differentiation of G6 cells can produce an intermediate filament expression pattern similar to that observed during myogenesis in vivo, i.e., downregulation of vimentin but not of nestin and desmin during myotube maturation. The image analysis demonstrated that the degree of overlap between nestin and desmin/vimentin was very extensive in myoblasts and in multinucleate myotubes in all regions of the cells. In contrast, nestin did not colocalize with tubulin or actin in G6 myoblasts. In particular, nestin immunoreactivity was not detected at the microtubule-organizing center, and it was only sparsely observed at the cell periphery where actin stress fibers were seen. Our data lend further support to the notion that nestin interacts very closely with the two more distantly related class III intermediate filament proteins desmin and vimentin in the entire muscle cell, before and after myotube formation. A comparison of conserved amino acid residues in the different IFs suggest that charged amino acid residues in the alpha-helical rod domain may play a role in the interaction.

The intermediate filament nestin is transiently expressed in developing skeletal muscle. In the present investigation, we analyzed by immunohistochemistry the presence of nestin, as well as vimentin and desmin, in skeletal muscle affected by two diseases characterized by various degrees of necrosis and muscle regeneration: Duchenne/Becker muscular dystrophy and myositis. Nestin-positive areas were found in all analyzed muscle biopsies of both diseases. The same areas were, in most cases, also positive for vimentin and stained more intensely for desmin than surrounding myofibers. Only nestin was found specifically in myopathic muscle fibers; vimentin was in addition present in muscle fibroblasts and desmin in all myofibers. The areas staining positive for nestin were typically basophilic, small-diameter myofibers, often with centrally located nuclei. With the interesting exception of a 73-year-old healthy control with abundant ring fibers, nestin was not detected in the muscle of healthy controls. The intracellular distribution of nestin in the myopathic muscle fibers, as well as in the ring fibers, was confined to the vicinity of Z-bands. The presence of nestin protein in myopathic regenerating areas and in ring fibers correlated more closely to the presence of desmin than to vimentin immunoreactivity. Our results suggest that nestin is specifically expressed in newly formed muscle fibers also during regeneration, and that nestin may serve as a useful marker of regenerating muscle fibers in pathological conditions.