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A novel chitosan-based membrane that made of hydroxyethyl chitosan, gelatin and chondroitin sulfate was used as a carrier of corneal endothelial cells. The characteristics of the blend membrane including transparency, equilibrium water content, ion and glucose permeability were determined. The results showed that the optical transparency of the membrane was as good as the natural human cornea. The water content of this scaffold was 81.32% which was remarkably close to the native cornea. The membrane had a good ion permeability and its glucose permeability was even higher than natural human cornea. The cultured rabbit corneal endothelial cells formed a monolayer on the membrane. The results demonstrated that the membrane was suitable for corneal endothelial cells to attach and grow on it. In addition, the membranes in vivo could be degraded steadily with less inflammation and showed a good histocompatibility. These results demonstrated that the hydroxyethyl chitosan-chondroitin sulfate-gelatin blend membrane can potentially be used as a carrier for corneal endothelial cell transplantation.

INTRODUCTIONDiabetes mellitus (DM) is a highly prevalent disease all over the world. Its long-term tissue complications that affect small and large blood vessels are directly connected with the time of patients suffering from hyperglycemia. Chitosan is a polycationic copolymer consisting of β-1, 4-linked 2-acetamido-D-glucose and β-1, 4-linked 2-amino-D-glucose units. Crab and shrimp shell wastes are currently utilized as the major industrial source of biomass for large-scale production of chitosan. Chitosan which is biodegradable, non-toxic and biocompatible has been shown to be particularly useful in many fields [1] , including food, cosmetics, biomedicine, agriculture and environmental protection. Furthermore, it can be used as a bioactive material due to its biodegradable, non-toxic and non-allergenic natures. However, chitosan shows its biological activity only in acidic medium because of its poor solubility at pH above 6.5 and low absorbability of non-digestible and high molecular polysaccharides. Therefore, recent studies on chitosan have attracted interest in converting it to chitooligosaccharides(COS), because COS not only are water-soluble but also possess versatile functional properties such as antitumor enhancing properties [2,3] , immunostimulating effects [2,4] , antimicrobial activity [5] , free radical scavenging activity [6][7][8] , protective effects against infections [9] , arthritis controlling activity [10] , plant disease controlling activity [11,12] and angiotensin I converting enzyme inhibitory activity [13] . However, little attention has been paid to its activity in diabetes mellitus and related mode of action.In the present study, soluble chitooligosaccharides with low molecular weight were prepared by enzymatic hydrolysis of chitosan with chitosanase as previously described [14] . The purpose of this study was to examine the effect of chitooligosaccharides on proliferation of pancreatic islet cells and release of insulin in vitro. MATERIALS AND METHODS MaterialsChitosan (minimum 90% deacetylated, Mr: 500 000) was purchased from Jinan Haidebei Marine Bioengineering Abstract AIM: To investigate the effect of chitooligosaccharides on proliferation of pancreatic islet cells, release of insulin and 2 h plasma glucose in streptozotocin-induced diabetic rats. METHODS:In vitro , the effect of chitooligosaccharides on proliferation of pancreatic islet cells and release of insulin was detected with optical microscopy, colorimetric assay, and radioimmunoassay respectively. In vivo , the general clinical symptoms, 2 h plasma glucose, urine glucose, oral glucose tolerance were examined after sixty days of feeding study to determine the effect of chitooligosaccharides in streptozotocin-induced diabetic rats. RESULTS:Chitooligosaccharides could effectively a c c e l e ra t e t h e p r o l i f e ra t i o n o f p a n c r e a t i c i s l e t cells. Chitooligosaccharides (100 mg/L) had direct and prominent effect on pancreastic β cells and insulin release from islet cells. All concentrations of chitooli...

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Chitosanase is able to specifically cleave β-1,4-glycosidic bond linkages in chitosan to produce a chito-oligomer product, which has found a variety of applications in many areas, including functional food and cancer therapy. Although several structures for chitosanase have been determined, the substrate-binding mechanism for this enzyme has not been fully elucidated because of the lack of a high-resolution structure of the chitosanase-substrate complex. In the present study we show the crystal structure of a novel chitosanase OU01 from Microbacterium sp. in complex with its substrate hexa-glucosamine (GlcN)6, which belongs to the GH46 (glycoside hydrolyase 46) family in the Carbohydrate Active Enzymes database (http://www.cazy.org/). This structure allows precise determination of the substrate-binding mechanism for the first time. The chitosanase-(GlcN)6 complex structure demonstrates that, from the -2 to +1 position of the (GlcN)6 substrate, the pyranose rings form extensive interactions with the chitosanase-binding cleft. Several residues (Ser27, Tyr37, Arg45, Thr58, Asp60, His203 and Asp235) in the binding cleft are found to form important interactions required to bind the substrate. Site-directed mutagenesis of these residues showed that mutations of Y37F and H203A abolish catalytic activity. In contrast, the mutations T58A and D235A only lead to a moderate loss of catalytic activity, whereas the S27A mutation retains ~80% of the enzymatic activity. In combination with previous mutagenesis studies, these results suggest that the -2, -1 and +1 subsites play a dominant role in substrate binding and catalysis. DSF (differential scanning fluorimetry) assays confirmed that these mutations had no significant effect on protein stability. Taken together, we present the first mechanistic interpretation for the substrate (GlcN)6 binding to chitosanase, which is critical for the design of novel chitosanase used for biomass conversion.

Increasing evidence indicates that lymphocyte cytosolic protein 1 (LCP1) overexpression contributes to tumor progression; however, its role in osteosarcoma (OS) remains unclear. We aimed to investigate the potential effect of LCP1 in OS and the underlying mechanisms. We first demonstrated that LCP1 is upregulated in OS cell lines and tissues. Then, we found that aberrant expression of LCP1 could induce the proliferation and metastasis of OS cells in vitro and in vivo by destabilizing neuregulin receptor degradation protein-1 (Nrdp1) and subsequently activating the JAK2/STAT3 signaling pathway. When coculturing OS cells with bone marrow-derived mesenchymal stem cells (BMSCs) in vitro , we validated that oncogenic LCP1 in OS was transferred from BMSCs via exosomes. Moreover, microRNA (miR)-135a-5p, a tumor suppressor, was found to interact upstream of LCP1 to counteract the pro-tumorigenesis effects of LCP1 in OS. In conclusion, BMSC-derived exosomal LCP1 promotes OS proliferation and metastasis via the JAK2/STAT3 pathway. Targeting the miR-135a-5p/LCP1 axis may have potential in treating OS.