Cross-Linked Pectin Nanofibers with Enhanced Cell Adhesion

Polysaccharides display poor cell adhesion due to the lack of cell binding domains. This severely limits their applications in regenerative medicine. This study reports novel cross-linked pectin nanofibers with dramatically enhanced cell adhesion. The nanofibers are prepared by at first oxidizing pectin with periodate to generate aldehyde groups and then cross-linking the nanofibers with adipic acid dihydrazide to covalently connect pectin macromolecular chains with adipic acid dihydrazone linkers. The linkers may act as cell binding domains. Compared with traditional Ca²⁺-cross-linked pectin nanofibers, the pectin nanofibers with high oxidation/cross-linking degree exhibit much enhanced cell adhesion capability. Moreover, the cross-linked pectin nanofibers exhibit excellent mechanical strength (with Young’s modulus ∼10 MPa) and much enhanced body degradability (degrade completely in 3 weeks or longer time). The combination of excellent cell adhesion capability, mechanical strength, and body degradability suggests that the cross-linked pectin nanofibers are promising candidates for in vivo applications such as tissue engineering and wound healing. This cross-linking strategy may also be used to improve the cell adhesion capability of other polysaccharide materials

Identification of natural products with neuronal and metabolic benefits through autophagy induction

<p>Autophagy is a housekeeping lysosomal degradation pathway important for cellular survival, homeostasis and function. Various disease models have shown that upregulation of autophagy may be beneficial to combat disease pathogenesis. However, despite several recently reported small-molecule screens for synthetic autophagy inducers, natural chemicals of diverse structures and functions have not been included in the synthetic libraries, and characterization of their roles in autophagy has been lacking. To discover novel autophagy-regulating compounds and study their therapeutic mechanisms, we used analytic chemistry approaches to isolate natural phytochemicals from a reservoir of medicinal plants used in traditional remedies. From this pilot plant metabolite library, we identified several novel autophagy-inducing phytochemicals, including Rg2. Rg2 is a steroid glycoside chemical that activates autophagy in an AMPK-ULK1-dependent and MTOR-independent manner. Induction of autophagy by Rg2 enhances the clearance of protein aggregates in a cell-based model, improves cognitive behaviors in a mouse model of Alzheimer disease, and prevents high-fat diet-induced insulin resistance. Thus, we discovered a series of autophagy-inducing phytochemicals from medicinal plants, and found that one of the compounds Rg2 mediates metabolic and neurotrophic effects dependent on activation of the autophagy pathway. These findings may help explain how medicinal plants exert the therapeutic functions against metabolic diseases.</p

The endocytosis of Gal-3.

<p>The HUVECs were incubated with biotinylated Gal-3 for 30 min and then either permeabilized or not before examination by IF analysis with Rhodamine- streptavidin. Images from a fluorescent microscope. Scale bar, 10 µm.</p

Response of carbohydrate concentrations.

<p>The differences between clipping without (real line) and with saliva (broken line) in carbohydrate concentrations, fructans (a₁–a₄), sucrose (b₁–b₄), glucose (c₁–c₄) and fructose (d₁–d₄) in component parts, leaf (a₁–d₁), stem (a₂–d₂), rhizome (a₃–d₃) and fibrous root (a₄–d₄), within 10 days after treatments in 2008. Bars represent standard errors. **, P<0.05; *, P<0.01.</p

The Two Endocytic Pathways Mediated by the Carbohydrate Recognition Domain and Regulated by the Collagen-like Domain of Galectin-3 in Vascular Endothelial Cells

<div><p>Galectin-3 plays an important role in endothelial morphogenesis and angiogenesis. We investigated the endocytosis of galectin-3 in human vascular endothelial cells and showed that galectin-3 could associate with and internalized into the cells in a carbohydrate-dependent manner. Our work also revealed that galectin-3 was transported to the early/recycling endosomes and then partitioned into two routes – recycling back to the plasma membrane or targeting to the late endosomes/lysosomes. Various N- and C-terminal truncated forms of galectin-3 were constructed and compared with the full-length protein. These comparisons showed that the carbohydrate-recognition domain of galectin-3 was required for galectin-3 binding and endocytosis. The N-terminal half of the protein, which comprises the N-terminal leader domain and the collagen-like internal repeating domain, could not mediate binding and endocytosis alone. The collagen-like domain, although it was largely irrelevant to galectin-3 trafficking to the early/recycling endosomes, was required for targeting galectin-3 to the late endosomes/lysosomes. In contrast, the leader domain was irrelevant to both binding and intracellular trafficking. The data presented in this study correlate well with different cellular behaviors induced by the full-length and the truncated galectin-3 and provide an alternative way of understanding its angiogenic mechanisms.</p> </div

The exocytosis of Gal-3.

<p>A: HUVECs were incubated in SFM with 15 µg/ml Gal-3 for 30 min at 37°C and then washed five times at 4°C. The cells were placed in fresh SFM and then incubated at 37°C or 4°C for a total of 120 min with fresh media changes every 30 min. The media collected from each time point were precipitated by TCA and analyzed by western blotting. B: The HUVECs were incubated with 15 µg/ml Gal-3 for 30 min at 4°C and then washed five times at 4°C. The cells were then placed in fresh SFM and incubated at 37°C for 0, 20, 40, 60, or 120 min. Both the cells and the media were analyzed with western blotting.</p

Results of two ways ANOVA for the effects of saliva, clipping and their interaction on height, aboveground biomass (AGB), belowground biomass (BGB), tillers and buds both in 2006 and 2007.

<p>**, <i>P</i><0.05;</p><p>*, <i>P</i><0.01.</p

Cloning, expression and biochemical characterization of a GH1 β-glucosidase from <i>Cellulosimicrobium cellulans</i>

<p>β-Glucosidase plays an important role in the degradation of cellulose. In this study, a novel β-glucosidase <i>ccbgl1b</i> gene for a glycosyl hydrolase (GH) family 1 enzyme was cloned from the genome of <i>Cellulosimicrobium cellulans</i> and expressed in <i>Escherichia coli</i> BL21 cells. The sequence contained an open reading frame of 1494 bp, encoded a polypeptide of 497 amino acid residues. The recombinant protein CcBgl1B was purified by Ni sepharose fastflow affinity chromatography and had a molecular weight of 57 kDa, as judged by SDS-PAGE. The optimum β-glucosidase activity was observed at 55 °C and pH 6.0. Recombinant CcBgl1B was found to be most active against aryl-glycosides <i>p</i>-nitrophenyl-β-D-glucopyranoside (<i>p</i>NPβGlc), followed by <i>p</i>-nitrophenyl-β-D-galactopyranoside (<i>p</i>NPβGal). Using disaccharides as substrates, the enzyme efficiently cleaved β-linked glucosyl-disaccharides, including sophorose (β-1,2-), laminaribiose (β-1,3-) and cellobiose (β-1,4-). In addition, a range of cello-oligosaccharides including cellotriose, cellotetraose and cellopentaose were hydrolysed by CcBgl1B to produce glucose. The interaction mode between the enzyme and the substrates driving the reaction was modelled using a molecular docking approach. Understanding how the GH1 enzyme CcBgl1B from <i>C. cellulans</i> works, particularly its activity against cello-oligosaccharides, would be potentially useful for biotechnological applications of cellulose degradation.</p

The endocytic pathways of Gal-3 in HUVEC.

<p>A: The cells were incubated with DTAF-Gal-3 and Cy3-transferrin (only at 30 min) at 37°C for 10, 30, or 120 min and were then processed for IF analysis with antibodies against EEA1, Lamp-1 and Golgin-97. B: The cells were incubated with DTAF-Gal-3 at 4°C for 60 min. After washing, the cells were either fixed immediately (0 min) or transferred to 37°C for 5 min or 10 min and then fixed. C: The cells were incubated with DTAF-Gal-3 and Cy3-transferrin at 20°C for 60 min. The images were obtained with a confocal microscope (A and B) or a fluorescence microscope (C). Scale bar, 10 µm.</p

The association of Gal-3 with HUVECs.

<p>A: Cell association is concentration dependent. The cells were incubated for 30 min in SFM containing 0, 0.1, 0.2, 0.5, 1, 2, 5 or 15 µg/ml Gal-3. B: Cell association is time dependent. The cells were incubated in SFM containing either 2 or 15 µg/ml Gal-3 for 5, 30, 60, 120, or 240 min. C: Cell association is lectin specific. The cells were incubated for 30 min in SFM containing 15 µg/ml Gal-3 in the presence or absence of 50 mM lactose or sucrose. After incubation, the cells were extensively washed with cold SFM. Western blotting analysis was performed with anti-Gal-3 and anti-actin antibodies. Arrowhead, degraded form of Gal-3.</p