Effect of Active Oxygen Radicals on Protein and Carbohydrate Moieties of Recombinant Human Erythropoietin

Our previous study showed that active oxygen radicals generated from a Fenton system and a xanthine plus xanthine oxidase system caused serious loss of in vivo bioactivity of recombinant human erythropoietin (EPO), a highly glycosylated protein. In the present study, we characterized the oxidative modifications to the protein and carbohydrate moiety of EPO, which lead to a reduction of its bioactivity. In vitro bioactivity was reduced when EPO was treated with oxygen radi cals generated from a Fenton system in the presence of 0.016 mM H₂0₂, and the reduction was directly proportional to the loss of in vivo bioactivity. SDS-PAGE analysis showed that dimer formation and degradation was observed under more severe conditions (Fenton reaction with 0.16 mM H₂0₂). The tryptophan destruction was detected at 0.016 mM H₂O₂ and well correlated with the loss of in vitro bioactivity, whereas loss of other amino acids were occurred under more severe conditions. Treatment with the Fenton system did not result in any specific damage on the carbohydrate moiety of EPO, except a reduction of sialic acid content under severe condition. These results suggest that active oxygen radicals mainly react with the protein moiety rather than the carbohydrate moiety of EPO. Destruction of tryptophan residues is the most sensitive marker of oxidative damage to EPO, suggesting the importance of tryptophan in the active EPO structure. Deglycosylation of EPO caused an increase of susceptibility to oxygen radicals compared to intact EPO. The role of oligosaccharides in EPO may be to protect the protein structure from active oxygen radicals.     

A heliangolide from Chromolaena glaberrima

The investigation of Chromolaena glaberrima afforded, besides the known heliangolide chromolaenide, a new 4-dehydrochromolaenide. The structure of the new compound was elucidated by spectroscopic methods.     

ABUNDANCE OF IRRAWADDY DOLPHINS (ORCAELLA BREVIROSTRIS) AND GANGES RIVER DOLPHINS (PLATANISTA GANGETICA GANGETICA) ESTIMATED USING CONCURRENT COUNTS MADE BY INDEPENDENT TEAMS IN WATERWAYS OF THE SUNDARBANS MANGROVE FOREST IN BANGLADESH

Independent observer teams made concurrent counts of Irrawaddy dolphins Orcaella brevirostris and Ganges River dolphins Platanista gangetica gangetica in mangrove channels of the Sundarbans Delta in Bangladesh. These counts were corrected for missed groups using mark-recapture models. For Irrawaddy dolphins, a stratified Lincoln-Petersen model, which incorporated group size and sighting conditions as covariates, and a Huggins conditional likelihood model, which averaged models that individually incorporated group size, sighting conditions, and channel width as covariates, generated abundance estimates of 397 individuals (CV = 10.2%) and 451 individuals (CV = 9.6%), respectively. For Ganges River dolphins, a stratified Lincoln-Petersen model, which incorporated group size as a covariate, and a Huggins conditional likelihood model, which averaged the same models described above, generated abundance estimates of 196 individuals (CV = 12.7%) and 225 individuals (CV = 12.6%), respectively. Although the estimates for both models were relatively close, the analytical advantages of the Huggins models probably outweigh those of the Lincoln-Petersen models. However, the latter should be considered appropriate when simplicity is a priority. This study found that waterways of the Sundarbans support significant numbers of Irrawaddy and Ganges River dolphins, especially compared to other areas where the species have been surveyed.     

Soluble signalling factors derived from differentiated cartilage tissue affect chondrogenic differentiation of rat adult marrow stromal cells.

BACKGROUND: Chondral defects show lack of proper regeneration whereas osteochondral lesions display limited regeneration capacity. Latter is probably due to immigration of chondroprogenitor cells from the subchondral bone. Known chondroprogenitor cells for cartilage tissues are multi-potent adult marrow stromal or mesenchymal stem cells (MSCs). In vitro chondrogenic differentiation of these precursor cells usually require cues from growth and signalling factors provided in vivo by surrounding tissues and cells. We hypothesise that signalling factors secreted by differentiated cartilage tissue can initiate and maintain chondrogenic differentiation status of MSCs. METHODS: To study such paracrine communication between allogenic rat articular cartilage and rat MSCs embedded in alginate beads a novel coculture system without addition of external growth factors has been established. RESULTS: Impact of cartilage on differentiating MSCs was observed at two different time points. Firstly, sustained expression of Sox9 was observed at an early stage which indicated induction of chondrogenic differentiation. Secondly, late stage repression of collagen X indicated pre-hypertrophic arrest of differentiation. In the culture supernatant we have identified vascular endothelial growth factor alpha (VEGF-164 alpha), matrix metalloproteinase (MMP) -13 and tissue inhibitors of MMPs (TIMP-1 and TIMP-2) which could be traced back either to the cartilage explant or to the MSCs under the influence of cartilage. CONCLUSION: The identified factors might be involved in regulation of collagen X gene and protein expression and therefore, may have an impact on the control and regulation of MSCs differentiation.