Loss of FGF receptor 1 signaling reduces skeletal muscle mass and disrupts myofiber organization in the developing limb.

The identities of extracellular growth factors that regulate skeletal muscle development in vivo are largely unknown. We asked if FGFs, which act as repressors of myogenesis in culture, play a similar role in vivo by ectopically expressing in the developing limb a truncated FGF receptor 1 (dnFGFR1) that acts as a dominant negative mutant. Hind limbs and the adjacent somites of Hamburger and Hamilton (HH) stage 17 chickens were infected with a replication-competent RCAS virus encoding dnFGFR1. By ED5, the virus had spread extensively within the limb and the adjacent somites with little rostral or caudal expansion of the infection along the axial midline. Viral infection and mutant receptor expression were coincident as revealed by the distribution of a viral coat protein and an HA epitope tag present on the carboxy terminus of dnFGFR1. Within 48 h following injection of dnFGFR1, we could detect no obvious changes in skeletal muscle precursor cell migration into the hind limb as compared to control limbs infected with an empty RCAN virus. However, by 3 days following infection of RCAS-dnFGFR1 virus, the level of skeletal muscle-specific myosin heavy chain was decreased and the expression pattern altered, suggesting disruption of skeletal muscle development. Two striking muscular phenotypes were observed in dnFGFR1-expressing limbs, including an average loss of 30% in skeletal muscle wet weight and a 50% decrease in myofiber density. At all ages examined the loss of skeletal muscle mass was accompanied by a loss of myoblasts and an unexpected concomitant loss of fibroblasts. Consistent with these observations, explants of infected cells revealed a reduction in the number of myonuclei in myotubes. Although the myofiber density per unit area was decreased over 50% compared to controls there were no detectable effects on myofiber diameter. The loss in myofiber density was, however, accompanied by an increase in the space surrounding individual myofibers and a generalized loss of myofiber integrity. It is noteworthy that long-bone development was unaffected by RCAS-dnFGFR1 infection, suggesting that FGFR2 and FGFR3 signaling was not disrupted. Our data provide conclusive evidence that FGFR1 signaling is necessary to maintain myoblast number and plays a role in myofiber organization.

Differentially expressed fibroblast growth factors regulate skeletal muscle development through autocrine and paracrine mechanisms.

Several FGF family members are expressed in skeletal muscle; however, the roles of these factors in skeletal muscle development are unclear. We examined the RNA expression, protein levels, and biological activities of the FGF family in the MM14 mouse skeletal muscle cell line. Proliferating skeletal muscle cells express FGF-1, FGF-2, FGF-6, and FGF-7 mRNA. Differentiated myofibers express FGF-5, FGF-7, and reduced levels of FGF-6 mRNA. FGF-3, FGF-4, and FGF-8 were not detectable by RT-PCR in either proliferating or differentiated skeletal muscle cells. FGF-I and FGF-2 proteins were present in proliferating skeletal muscle cells, but undetectable after terminal differentiation. We show that transfection of expression constructs encoding FGF-1 or FGF-2 mimics the effects of exogenously applied FGFs, inhibiting skeletal muscle cell differentiation and stimulating DNA synthesis. These effects require activation of an FGF tyrosine kinase receptor as they are blocked by transfection of a dominant negative mutant FGF receptor. Transient transfection of cells with FGF-1 or FGF-2 expression constructs exerted a global effect on myoblast DNA synthesis, as greater than 50% of the nontransfected cells responded by initiating DNA synthesis. The global effect of cultures transfected with FGF-2 expression vectors was blocked by an anti-FGF-2 monoclonal antibody, suggesting that FGF-2 was exported from the transfected cells. Despite the fact that both FGF-l and FGF-2 lack secretory signal sequences, when expressed intracellularly, they regulate skeletal muscle development. Thus, production of FGF-1 and FGF-2 by skeletal muscle cells may act as a paracrine and autocrine regulator of skeletal muscle development in vivo.

Increase in copy number of an integrated vector during continuous culture of Hansenula polymorpha expressing functional human haemoglobin.

Recombinant human haemoglobin A (rHbA) was produced by a leucine-requiring strain of Hansenula polymorpha which had been transformed with an integration vector containing the Saccharomyces cerevisiae LEU2 gene and cDNAs for the expression of alpha and beta globin each driven by the H. polymorpha MOX promoter. After 40 generations in a chemostat it was found that the integrated vector had become amplified in the host strain. In some cases this led to an increase in LEU2 gene dosage, but a loss of globin expression cassettes. In other cases the globin gene dosage also increased. These changes coincided with an increase in rHbA production in the culture, which was reversed when the dilution rate was increased. Isolates from a chemostat culture producing elevated levels of rHbA were grown in fed-batch fermentations, resulting in higher productivities than when inoculated with the parent strain. The rHbA produced was purified and characterized. Oxygen binding studies and electrospray mass spectrometry showed that the rHbA had been processed and assembled correctly, and behaved as a fully functional co-operative tetramer.

Isolation of mucoid strains of Pseudomonas aeruginosa from non-cystic-fibrosis patients and characterisation of the structure of their secreted alginate.

When the incubation period of primary isolation plates was extended to 48 h, mucoid strains of Pseudomonas aeruginosa were found in specimens from various infected sites in patients who did not have cystic fibrosis. The 17 mucoid isolates were characterised in terms of mucoid type, pyocin type, and their sensitivity or resistance to seven beta-lactam and two aminoglycoside antibiotics. The carbohydrate, uronic acid (alginate) and protein content of the water-soluble extracellular material of 15 strains was determined. This material was fractionated by ion-exchange chromatography, and the presence of alginate confirmed by the chemical assay of uronic acids and their quantitation by gas-liquid chromatography. Uronic acids were absent from a non-mucoid revertant of one strain. The strains produced alginate with a high content of mannuronic acid and substituted with O-acetyl groups. By proton nuclear magnetic resonance (1H-nmr) analysis the alginate from three strains was shown to lack polyguluronate blocks in its structure. These properties are also found in the alginate of mucoid P. aeruginosa strains from patients with cystic fibrosis.