Generation of stably-transfected Caco2-BBE cell expressing pEGFPN1-PHB A single PHB PCR product corresponding to the entire coding region of PHB

es several ethical questions when used in humans and are therefore not applicable. Another potential explanation for the unaltered activation of the analysed signalling pathways is that the biopsies were not taken at the appropriate time-points after rHuEpo exposure. From cell cultures it is known that Epo stimulation leads to phosphorylation of STAT5, Akt, and ERK, which peaks after 15 min and remains detectable after 60 min. Probably the time for activation of 8 Epo Receptor Expression in Skeletal Muscle these pathways in vivo will be slightly prolonged, due to transportation of the rHuEpo to the tissue of interest. It is possible that the first biopsies in study A were taken after maximal activation. Therefore, a biopsy was taken 1 h post rHuEpo administration in study B, which however did not reveal detectable phosphorylation of the pertinent signalling molecules. Considering that Epo has a half-life between 213 h, an upregulation would have been expected within the time-points analysed in the present studies. To date, no studies have been able to document robust effects of acute rHuEpo treatment in skeletal muscle. Thus, it remains a question whether the Epo-R is biologically active in skeletal muscle tissue. The results from the present study fits well with the mRNA measurements performed by Lundby et al. on the same biopsies as analysed in study A. They were not able to find a systematic regulation of a number of analysed mRNA molecules in relation to rHuEpo treatment. Epo does however seem to affect progenitor cells of the muscle tissue . Ogilvie et al. found the Epo-R to be present on these cell types and that stimulation by Epo mediates phosphorylation and PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22187127 thereby activation of JAK2 and to a minor extent also STAT5. Stimulation led to increased cell proliferation and decreased differentiation, which was accompanied by increased mRNA levels of MyoD and Myf5 and decreased mRNA levels of myogenin. Furthermore, it was Epo Receptor Expression in Skeletal Muscle Spot # Protein Uniprot # Matched fragments MS results Sequence coverage 27 17 29 38 41 49 Score 72 37 73 121 156 182 Matched fragments 4/49 2/49 3/34 7/29 8/48 7/52 1/36 2/16 – MS/MS results Sequence coverage 14 7 14 28 33 33 4 8 Score 256 159 240 338 649 618 59 129 – A Desmin Actin P17661 9/36 5/36 B C D E F G H Myosin light chain 1 V/sB Creatine kinase M-type Creatine kinase M-type Creatine kinase M-type RU 58841 web Glyceraldehyd-3-phosphate dehydrogenase Glyceraldehyd-3-phosphate dehydrogenase P05976 P06732 P06732 P06732 P04406 P04406 – 5/22 9/18 12/35 15/39 – Matched fragments; the number of peptides matched with the protein/the total amount of peptides in the sample. Sequence coverage; the percentage of the total protein that the matched peptides cover. Score; the score given by MASCOT, for MS results a score above 66 was considered significant, for MS/ MS the significance level was a score of 3437. Mascot: http://www.matrixscience.com. Uniprot: http://www.uniprot.org. doi:10.1371/journal.pone.0031857.t002 shown that Epo increased the proliferation of satellite cells significantly more than placebo during the first 14 days upon muscle trauma. This suggests a role for Epo in maintaining or expanding the pool of proliferating muscle progenitor cells during differentiation. This theory was partly supported by previously published results from study A, where MRF4 mRNA was transiently up-regulated 6 h after rHuEpo administration, whereas other markers of satellite cell differentiation w

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