Isolation, characterization, and genome sequencing of a novel chitin deacetylase producing Bacillus aryabhattai TCI-16.

Chitin deacetylase (CDA) is a chitin degradation enzyme that catalyzes the conversion of chitin to chitosan by the deacetylation of N-acetyl-D-glucosamine residues, playing an important role in the high-value utilization of waste chitin. The shells of shrimp and crab are rich in chitin, and mangroves are usually recognized as an active habitat to shrimp and crab. In the present study, a CDA-producing bacterium, strain TCI-16, was isolated and screened from the mangrove soil. Strain TCI-16 was identified and named as Bacillus aryabhattai TCI-16, and the maximum CDA activity in fermentation broth reached 120.35 ± 2.40 U/mL at 36 h of cultivation. Furthermore, the complete genome analysis of B. aryabhattai TCI-16 revealed the chitin-degrading enzyme system at genetic level, in which a total of 13 putative genes were associated with carbohydrate esterase 4 (CE4) family enzymes, including one gene coding CDA, seven genes encoding polysaccharide deacetylases, and five genes encoding peptidoglycan-N-acetyl glucosamine deacetylases. Amino acid sequence analysis showed that the predicted CDA of B. aryabhattai TCI-16 was composed of 236 amino acid residues with a molecular weight of 27.3 kDa, which possessed a conserved CDA active like the known CDAs. However, the CDA of B. aryabhattai TCI-16 showed low homology (approximately 30%) with other microbial CDAs, and its phylogenetic tree belonged to a separate clade in bacteria, suggesting a high probability in structural novelty. In conclusion, the present study indicated that the novel CDA produced by B. aryabhattai TCI-16 might be a promising option for bioconversion of chitin to the value-added chitosan.

Arecoline inhibits epithelial cell viability by upregulating the apoptosis pathway: implication for oral submucous fibrosis.

Oral submucous fibrosis (OSF) is a chronic inflammatory disease characterized by the accumulation of excess collagen, and areca nut chewing has been proposed as a significant etiological factor for disease manifestation. However, the underlying molecular mechanisms regarding areca nut chewing-induced OSF are only partially understood. Herein, we reported that arecoline markedly induced morphologic change in HaCaT epithelial cells, but had no obvious effect on Hel fibroblast cells. MTS assay revealed that arecoline significantly suppressed HaCaT cell viability. Moreover, flow cytometric analysis indicated that arecoline substantially promoted HaCaT cell, but not Hel cell apoptosis in a dose-dependent manner. Furthermore, arecoline-induced HaCaT cell apoptosis was found to be associated with increased expression and activation of cleaved-Bid, cleaved-PARA and cleaved-caspase-3. Collectively, our results suggest that HaCaT epithelial cells are more sensitive than Hel fibroblast cells to arecoline-induced cytotoxicity, which may be involved in the pathogenesis of OSF.

Arecoline suppresses HaCaT cell proliferation through cell cycle regulatory molecules.

Betel nut chewing is the most common cause of oral submucous fibrosis (OSF). Arecoline is the main component of the betel nut, and is associated with the occurrence and development of OSF through cytotoxicity, genotoxicity and DNA damage. Similar types of stimuli elicit differential responses in different cells. In the present study, we investigated the effects of arecoline on the HaCaT epithelial and Hel fibroblast cell lines. The data showed that arecoline affected HaCaT cell morphology. MTT assay revealed that arecoline suppressed HaCaT cell proliferation. Furthermore, we found that arecoline induced the cell cycle arrest of HaCaT cells. In comparison with the untreated control cells, following treatment with ≥75 µg/ml arecoline an increased percentage of HaCaT cells remained at the G0/G1 phase of the cell cycle, accompanied by a reduced percentage of cells in the S phase. However, arecoline treatment did not significantly alter Hel cell cycle distribution. In the HaCaT epithelial cells, arecoline downregulated expression of the G1/S phase regulatory proteins cyclin D1, CDK4, CDK2, E2F1 as determined by reverse transcription-PCR analysis and western blotting. In summary, arecoline inhibits HaCaT epithelial cell proliferation and survival, in a dose-dependent manner, and cell cycle arrest in the G1/S phase, while this is not obvious in the Hel fibroblast cells. Potentially, our findings may aid in the prevention of arecoline-associated human OSF.

[Protective effect of allicin on human periodontal ligament cells with nicotine-induced oxidative damage].

OBJECTIVE: To explore the protective effect of allicin on nicotine-induced oxidative damage to human periodontal ligament cells (HPDLCs). METHODS: (1) Establish nicotine-induced oxidative damage model on HPDLCs. Use water-soluble tetrazolium (WST) colorimetric method to find out the nicotine concentration (X) that could inhibit HPDLCs' growth for the following experiments. (2) HPDLCs of the fifth passage were divided into 5 groups: The control group, the nicotine group and the nicotine+allicin groups(the concentration of allicin was 15, 30, and 60 microg x mL(-1) respectively). Different kinds of culture media were added. Similarly, use WST colorimetric method to choose the allicin concentration (Y) that could significantly improve the survival rate of HPDLCs. (3) HPDLCs were divided into 3 groups: The control group, the nicotine group, the nicotine+allicin group and different media were added. The glutathion (GSH) concentrations in HPDLCs were determined in 1, 4, 8, 12 and 24h respectively. RESULTS: 0.8 mg x mL(-1) nicotine could inhibit the HPDLCs survival rate significantly (77% of the control, P < 0.05). But 60 microg x mL(-1) allicin could prevent the inhibition effects evidently, improving the survival rate to 112% of that of the nicotine group (P < 0.05) and reaching the survival rate level of control group (P > 0.05). The GSH concentrations of nicotine+allicin group were higher than that of the nicotine group always (P < 0.05) and by 82% at 8 h after culture, but had no difference with that of the control group (P > 0.05). CONCLUSION: 60 microg x mL(-1) allicin can protect the HPDLCs against oxidative damage induced by nicotine.

[Application of bioinformatics tools in analysis of differentially expressed genes in oral submucosal fibrosis].

OBJECTIVE: To apply the bioinformatics tools for analyzing the differentially expressed genes in oral submucous fibrosis (OSF) to obtain the implied biological significance. METHODS: By using DAVID and Onto-express bioinformatic tools, 865 differentially expressed genes in OSF were analyzed and the analysis of chromosome location, gene ontology (GO) and genetic-association diseases were performed. RESULTS: A majority of the differentially expressed genes were located on chromosome 1,2,5,6,7,11,12 (P < 0.01). GO classification of the differentially expressed genes identified the biological process subgroups, including genes involved in immune response, defense response and so on. The cellular component subgroups were associated with extracellular matrix, cytoskeleton and membrane, molecular function subgroups related to protein binding, extracellular matrix structural constituent and signal transducer activity. The diseases genetically associated with these genes included infection, immune and cardiovascular diseases. CONCLUSIONS: Bioinformatics can provide the quick and parallel analysis of massive data got from gene microarrays and enable the function classification of the differentially expressed genes, which provides new ideas on the research of pathogenesis and epidemiology of OSF.

[Effect of astragalus polysaccharides on the proliferation and ultrastructure of dog bone marrow stem cells induced into osteoblasts in vitro].

OBJECTIVE: To observe the growth and osteogenic property of cultured dog bone marrow stem cells (BMSCs) by investigating the effects of astragalus polysaccharides (APS) on the proliferation and ultrastructure of BMSCs into osteoblasts in vitro. METHODS: BMSCs osteogenic property was detected by improved Wright-Giemsa, Gomori and alizarin dyeing method. The proliferation and differentiation of the induced BMSCs with APS in different concentration and time were detected by MTT assay and the morphologic change of the induced BMSCs was observed by transmission electron microscope (TEM). RESULTS: BMSCs osteogenic property was detected with Wright-Giemsa deep-bluing, Gomori method blacking and with more mineral nodules alizarin dyeing method carmining. APS with concentration of 0.005 mg/mL can promote the proliferation of the induced BMSCs in short-term culture (1th, 3th day) and 50 mg/mL can decrease the effect through long-term culture (5th day). Observed by TEM (5th day), the number of mitochondria, rough endoplasmic reticulum increased and the extracellular matrix was excreted more in the induced BMSCs by APS with concentration of 0.005 mg/mL. However, not only the number of mitochondria, rough endoplasmic reticulum reduced but also the structure was swollen, degenerative, membrance damaged in the induced BMSCs by APS with concentration of 50 mg/mL. CONCLUSION: APS with lower concentration in short-term culture may promote BMSCs proliferation and differentiation.

[Effects of astragalus polysaccharides-chitosan/polylactic acid scaffolds and bone marrow stem cells on repairing supra-alveolar periodontal defects in dogs].

OBJECTIVE: To explore the effect of astragalus polysaccharides-chitosan/polylactic acid (AP-C/PLA) scaffolds and bone marrow stem cells (BMSCs) on periodontal regeneration of experimentally horizontal periodontal defects in dogs. METHODS: Dog BMSCs were isolated from the bone marrow and then cultured in a conditioned medium to be induced for osteogenesis. The expressions of Type I collagen and alkaline phosphatase (ALP) were examined by immunohistochemistry and histochemistry in the induced BMSCs, respectively. The BMSCs were harvested and implanted with astragalus polysaccharides-chitosan/polylactic acid (AP-C/PLA) and chitosan/polylactic acid (C/PLA) scaffolds. Horizontal alveolar bone defects (5 mm depth, 2 mm width) were produced surgically in the buccal side of the mandibular premolar 3 and 4 of the 10 dogs. The defects were randomly repaired with a cell-scaffold construction (10 teeth per group): root planning only (surgical control), AP-C/PLA with a conditioned medium (medium control), C/PLA with BMSCs (scaffolds control), and AP-C/PLA with BMSCs (experimental group) . The dogs were killed at 4 weeks and 8 weeks after the surgery, and block sections of the defects were collected for the histologic and histometric analysis. RESULTS: BMSCs induced in vitro exhibited an osteogenic phenotype with expressing Type I collagen and ALP histologically. The bone nodule structure was observed in the experimental group 4 weeks postsurgically. The engineered bone became more mature,similar to the native bone 8 weeks postsurgically. The amount of new bone regeneration and the rate of new bone filling to the defect height of the experimental group were significantly different from those of the surgical control, medium control, and scaffolds control [(2.90+/-0.41) mm vs (0.83+/-0.30) mm, (1.46+/-0.55) mm, (2.67+/-0.26) mm; 57.46% vs 15.68 %, 30.13%, 51.87%)] (P<0.01, P<0.01, P<0.05). CONCLUSION: Astragalus polysaccharides can promote the new bone formation on the periodontal defects. The technology of tissue engineering with AP-C/PLA scaffolds and induced BMSCs may contribute to the periodontal regeneration.

[Effects of astragalus polysaccharides-chitosan/polylactic acid composite material on biological behavior of canine bone marrow stromal cells cultured in vitro].

OBJECTIVE: To observe the biological behavior of canine bone marrow stromal cells (BMSCs) cultured in vitro with the astragalus polysaccharides-chitosan/polylactic acid (AP-C/PLA) and with the chitosan/polylactic acid (C/PLA) and to find a suitable compound material for periodontal tissue engineering. METHODS: BMSCs (induced 14 days by 50 mg/L vitamine C, 10(-8) mol/L dexamethasone, 10 mmol/L beta-sodium glycerylphosphate) were cultured on AP-C/PLA or C/PLA for 5 days respectively. The BMSCs attachment and the morphology were observed with scanning electronic microscope and the combining rates were counted. Type I collagen synthesis was examined with immunohistochemistry staining and the content of osteocalin was determined with radio-immunological method. RESULTS: Combining rates, type I collagen synthesis, and the content of osteocalin of BMSCs on AP-C/PLA were significantly higher than those on C/PLA. CONCLUSION: AP-C/PLA may promote the BMSC proliferation, differentiation and extracellular matrix synthesis, and it can be used as a good scaffold material for bone tissue engineering.

[Clinical study on the relationship between tooth abrasion and the habits of chewing betel nut].

OBJECTIVE: To investigate the relationship between tooth abrasion and long-term chewing betel nut. METHODS: The time, frequency, clinical features of occlusal abrasion, and results of pantomography were studied in 64 patients with the habits of chewing betel nut. RESULTS: The occlusal surfaces of all patients had abrasion from mild to severe. The longer the chewing time, the more severe the occlusal abrasion (P < 0.01). The occlusal abrasion became more severe with the increase in chewing frequency (P < 0.01). The severe abrasion was accompanied with periapical periodontitis and the resorption of alveolar bone. CONCLUSION: Long-term chewing betel nut can result in the abrasion of occlusal surface.