15 research outputs found
A novel a-L-Arabinofuranosidase of Family 43 Glycoside Hydrolase (Ct43Araf ) from Clostridium thermocellum
Articles in International JournalsThe study describes a comparative analysis of biochemical, structural and functional properties of two recombinant
derivatives from Clostridium thermocellum ATCC 27405 belonging to family 43 glycoside hydrolase. The family 43 glycoside
hydrolase encoding a-L-arabinofuranosidase (Ct43Araf) displayed an N-terminal catalytic module CtGH43 (903 bp) followed
by two carbohydrate binding modules CtCBM6A (405 bp) and CtCBM6B (402 bp) towards the C-terminal. Ct43Araf and its
truncated derivative CtGH43 were cloned in pET-vectors, expressed in Escherichia coli and functionally characterized. The
recombinant proteins displayed molecular sizes of 63 kDa (Ct43Araf) and 34 kDa (CtGH43) on SDS-PAGE analysis. Ct43Araf
and CtGH43 showed optimal enzyme activities at pH 5.7 and 5.4 and the optimal temperature for both was 50uC. Ct43Araf
and CtGH43 showed maximum activity with rye arabinoxylan 4.7 Umg21 and 5.0 Umg21, respectively, which increased by
more than 2-fold in presence of Ca2+ and Mg2+ salts. This indicated that the presence of CBMs (CtCBM6A and CtCBM6B) did
not have any effect on the enzyme activity. The thin layer chromatography and high pressure anion exchange
chromatography analysis of Ct43Araf hydrolysed arabinoxylans (rye and wheat) and oat spelt xylan confirmed the release of
L-arabinose. This is the first report of a-L-arabinofuranosidase from C. thermocellum having the capacity to degrade both pnitrophenol-
a-L-arabinofuranoside and p-nitrophenol-a-L-arabinopyranoside. The protein melting curves of Ct43Araf and
CtGH43 demonstrated that CtGH43 and CBMs melt independently. The presence of Ca2+ ions imparted thermal stability to
both the enzymes. The circular dichroism analysis of CtGH43 showed 48% b-sheets, 49% random coils but only 3% a-helices
SWI/SNF Chromatin-Remodeling Factor Smarcd3/Baf60c Controls Epithelial-Mesenchymal Transition by Inducing Wnt5a Signaling
We previously identified a gene signature predicted to regulate the epithelial-mesenchymal transition (EMT) in both epithelial tissue stem cells and breast cancer cells. A phenotypic RNA interference (RNAi) screen identified the genes within this 140-gene signature that promoted the conversion of mesenchymal epithelial cell adhesion molecule-negative (EpCAM(−)) breast cancer cells to an epithelial EpCAM(+/high) phenotype. The screen identified 10 of the 140 genes whose individual knockdown was sufficient to promote EpCAM and E-cadherin expression. Among these 10 genes, RNAi silencing of the SWI/SNF chromatin-remodeling factor Smarcd3/Baf60c in EpCAM(−) breast cancer cells gave the most robust transition from the mesenchymal to epithelial phenotype. Conversely, expression of Smarcd3/Baf60c in immortalized human mammary epithelial cells induced an EMT. The mesenchymal-like phenotype promoted by Smarcd3/Baf60c expression resulted in gene expression changes in human mammary epithelial cells similar to that of claudin-low triple-negative breast cancer cells. These mammary epithelial cells expressing Smarcd3/Baf60c had upregulated Wnt5a expression. Inhibition of Wnt5a by either RNAi knockdown or blocking antibody reversed Smarcd3/Baf60c-induced EMT. Thus, Smarcd3/Baf60c epigenetically regulates EMT by activating WNT signaling pathways
Placental miR-340 mediates vulnerability to activity based anorexia in mice
Anorexia nervosa is characterised by self-starvation but its etiology is not completely understood. Here the authors describe how prenatal stress can induce activity-based anorexia in the offspring during early adulthood by upregulating miR-340 expression in the placenta that affects expression of nutrient transporters