Zebrafish ProVEGF-C Expression, Proteolytic Processing and Inhibitory Effect of Unprocessed ProVEGF-C during Fin Regeneration

BACKGROUND: In zebrafish, vascular endothelial growth factor-C precursor (proVEGF-C) processing occurs within the dibasic motif HSIIRR(214) suggesting the involvement of one or more basic amino acid-specific proprotein convertases (PCs) in this process. In the present study, we examined zebrafish proVEGF-C expression and processing and the effect of unprocessed proVEGF-C on caudal fin regeneration. METHODOLOGY/PRINCIPAL FINDINGS: Cell transfection assays revealed that the cleavage of proVEGF-C, mainly mediated by the proprotein convertases Furin and PC5 and to a less degree by PACE4 and PC7, is abolished by PCs inhibitors or by mutation of its cleavage site (HSIIRR(214) into HSIISS(214)). In vitro, unprocessed proVEGF-C failed to activate its signaling proteins Akt and ERK and to induce cell proliferation. In vivo, following caudal fin amputation, the induction of VEGF-C, Furin and PC5 expression occurs as early as 2 days post-amputation (dpa) with a maximum levels at 4-7 dpa. Using immunofluorescence staining we localized high expression of VEGF-C and the convertases Furin and PC5 surrounding the apical growth zone of the regenerating fin. While expression of wild-type proVEGF-C in this area had no effect, unprocessed proVEGF-C inhibited fin regeneration. CONCLUSIONS/SIGNIFICANCES: Taken together, these data indicate that zebrafish fin regeneration is associated with up-regulation of VEGF-C and the convertases Furin and PC5 and highlight the inhibitory effect of unprocessed proVEGF-C on fin regeneration

A new bioassay-guided fractionation from root of Bryonia dioica: A promising strategy for drug discovery

International audienc

Screening of plant extracts for antimicrobial and anticancer activities

International audienceThe screening of several crude plant extracts from Gabon for their antimicrobial and antitumoral activities showed us interesting results. After chromatography separation and purification of the alkaloid extracts from Isolona hexaloba (Annonaceae) roots, followed by spectral data analysis, we characterized two bisbenzylisoquinoline: curine and guattegaumerine [1,2,3].We then, examined whether these compounds can regulate bacterial stress and growth. Interestingly, both molecules activate the formation of bacterial biofilm. However, none of the two, at concentrations ranging from 10-17 to 10-6 M, showed an effect on Escherichia coli, Bacillus subtilis, or a mucoid strain of Pseudomonas aeruginosa growth.Using the rat hepatocarcinoma cells (HTC-R) that are resistant to anticancer drugs, we tested the effects of curine and guattegaumerine on the efflux of Rhodamine123 from HTC-R, a substrate for P-glycoprotein encoded by mdr1 gene. Curine and guattegaumerine at 10µM were able to reduce Rhodamine123 efflux by 50% and were not verapamil (Rhodamine efflux inhibitor) competitors.The cytotoxic effect of curine and guattegaumerine was also evaluated on HTC-R cell growth. No effect was found for concentrations ranging from 10-8 to 10-5 M. In contrast, at 10-4 M, curine and guattegaumerine inhibited cell proliferation by 90 and 70% respectively. Thus, we concluded that due to their antiproliferative effect, these two compounds are not specific substrates for P-glycoprotein but may present potential inhibitors for cell growth in cancer treatment

Increased calvaria cell differentiation and bone matrix formation induced by fibroblast growth factor receptor 2 mutations in Apert syndrome.

Apert syndrome, associated with fibroblast growth factor receptor (FGFR) 2 mutations, is characterized by premature fusion of cranial sutures. We analyzed proliferation and differentiation of calvaria cells derived from Apert infants and fetuses with FGFR-2 mutations. Histological analysis revealed premature ossification, increased extent of subperiosteal bone formation, and alkaline phosphatase- positive preosteoblastic cells in Apert fetal calvaria compared with age-matched controls. Preosteoblastic calvaria cells isolated from Apert infants and fetuses showed normal cell growth in basal conditions or in response to exogenous FGF-2. In contrast, the number of alkaline phosphatase- positive calvaria cells was fourfold higher than normal in mutant fetal calvaria cells with the most frequent Apert FGFR-2 mutation (Ser252Trp), suggesting increased maturation rate of cells in the osteoblastic lineage. Biochemical and Northern blot analyses also showed that the expression of alkaline phosphatase and type 1 collagen were 2-10-fold greater than normal in mutant fetal calvaria cells. The in vitro production of mineralized matrix formed by immortalized mutant fetal calvaria cells cultured in aggregates was also increased markedly compared with control immortalized fetal calvaria cells. The results show that Apert FGFR-2 mutations lead to an increase in the number of precursor cells that enter the osteogenic pathway, leading ultimately to increased subperiosteal bone matrix formation and premature calvaria ossification during fetal development, which establishes a connection between the altered genotype and cellular phenotype in Apert syndromic craniosynostosis