1.Preparation

 

Preparation of chitooligosaccharides from chitosan by a complex enzyme

 

Hu Zhang, Yuguang Du,  Xingju Yu, Masaru Mitsutomi, Sei-ichi Aiba

 

Carbohydrate Research 320 (1999) 257–260

 

Chitosan of 24% degree of acetylation was depolymerized by a mixture of cellulase, alpha amylase, and proteinase to give the title oligosaccharides. The removal of products by membrane separation permitted yield maximization of products having degree of polymerization in the 3–10 range.

 

2. Plant

 

Isolation of a novel Ser/Thr protein kinase gene from oligochitosan-induced tobacco and its role in resistance against tobacco mosaic viru

 

B. Feng, Y. Chen, C. Zhao, X. Zhao, X. Bai, Y. Du

 

Plant Physiology and Biochemistry 44 (2006) 596–603

 

Oligochitosan induces defense responses to pathogenic microbes in a wide variety of plants by acting as an elicitor. In the present study, mRNA differential display was used to investigate oligochitosan-induced transcriptional activation of defense-related genes. Accordingly, a novel Ser/Thr protein kinase gene was isolated and designated as oligochitosan-induced protein kinase (oipk). Molecular cloning showed that oipk contains six introns interrupted by seven exons. The open reading frame (ORF) of the gene is 1848 bp, which encodes a putative protein of 615 amino acids with the predicted molecular mass of 70.96 kDa and a pI of 6.32. A plant oipk antisense expression vector was constructed and transformed into tobacco by Agrobacterium tumefaciens. Decreased phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity and decreased resistance to tobacco mosaic virus (TMV) were observed in transgenic tobacco. RT-PCR analysis revealed that oipk was expressed at high levels after oligochitosan induction in wild-type tobacco, but not in transgenic tobacco. These results indicated that oipk is involved in the signal pathway of oligochitosan-induced resistance in tobacco.

 

 

cDNA microarray analysis of gene expression in Brassica napus treated with oligo- chitosan elicitor

 

H. Yin, S. Li, X. Zhao, Y. Du, X. Ma

 

Plant Physiology and Biochemistry 44 (2006) 910–916

 

An oilseed rape (Brassica napus H) cDNA microarray containing 8095 expressed sequence tags (ESTs) was used to analyze the B. napus gene expression changes elicited by oligochitosan. Transcript levels for 393 genes were altered twofold or more in oligochitosan-treated seedlings compared to control seedlings. Of the 393 genes, 257 were repressed and 136 were induced. Semi quantification RT-PCR of eight of these 393

 

genes confirmed the microarray results. These 393 genes were involved in different processes and had different functions including defense, primary metabolism, transcription, and signal transduction etc. Some of these genes were elicited by various pathogen-related or stress stimuli, and others were regulated by plant growth regulators like auxin and gibberellin. These manifested complicated interactions of oligochitosan and

 

phytohormones. An important jasmonic acid (JA) synthase (2-oxophytodienoate-10,11- reductase) gene, a JA-mediated defense required kinase ATMPK4-homolog gene, an ethylene receptor gene, and two ethylene responsive element binding protein (EREBP) genes were induced by oligochitosan, suggesting that oligochitosan activated the plant self-defense through JA/ET signaling pathway.

 

Induction of tobacco genes in response to oligochitosan

 

Fuyun Zhang, Bin Feng, Wei Li, Xuefang Bai, Yuguang Du, Yukui Zhang

 

Molecular Biology Reports, 2007 34(1), 35-40

 

Oligochitosan has a variety of biological activities. To understand its mechanism, DDRT-PCR, reverse Northern blot and quantitative relative RT-PCR were used to identify and isolate genes whose transcription were altered in cultured Nicotiana tabacum

 

(var. Samsun NN) plants that were treated with oligochitosan. Three genes whose mRNA levels significantly changed in response to oligochitosan wereisolated and identified. One gene is up-regulated, and two genes are down-regulated. These genes encode a DNAJ heat shock N-terminal domain-containing protein, a histone H1 gene and a hypothetical protein, whose function is unknown. The results suggest that the usefulness of mRNA differential display technique for the detection of plant metabolic pathways affected by oligochitosan.

 

 

Induction of antiviral resistance and stimulary effect by oligochitosan in tobacco

 

Xiaoming Zhao, Xiaoping She, Yuguang Du, Xinmiao Liang

 

Pesticide Biochemistry and Physiology 87 (2007) 78–84

 

Oligochitosan was applied by spraying it on tobacco leaves for inhibition of tobacco mosaic virus (TMV). The maximum inhibition of TMV by oligochitosan was observed when inoculation occurred at 24 h after spraying 50 lg ml_1 oligochitosan. The production of H2O2 and NO in epidermal tobacco cells induced by oligochitosan was investigated by epidermal strip bioassay and LSCM, using cell permeable fluorophore diaminofluorescein diacetate (DAF-2D) and 20,70-dichlorofluorescin diacetate (H2DCF-DA), respectively. Epidermal tobacco cells treated with oligochitosan resulted in a strong increase of intracellular NO and H2O2. Oligochitosan and NO donor sodium nitroprusside (SNP) induced the defense reaction against tobacco mosaic virus (TMV), and increased phenylalanine ammonia-lyase (PAL) activity. Co-treatment of the tobacco cells with oligochitosan and NO scavenger CPTIO blocked the inducing resistance. The results indicated that the defense response induced by oligochitosan was connected with NO pathway.

 

 

Effects of oligochitosans on tobacco cells and role of endogenous nitric oxide burst in the resistance of tobacco to tobacco mosaic virus

 

X.M. Zhao, X.P. She, W. Yu, X.M. Liang, Y.G Du

 

Journal of Plant Pathology (2007), 89 (1), 69-79

 

Binding to tobacco cells of oliogochitosan labeled with the fluorophore 2-aminoacidone (2-AMAC) was investigated using laser scanning confocal microscopy (LSCM). Production of NO in epidermal tobacco cells treated with oligochitosan was investigated by epidermal strip bioassay and LSCM, using the cell-permeable fluorophore diamino- fluorescein diacetate (DAF-2D).Binding of the labeled oligochitosan to cell walls and membranes of tobacco cells was directly observed by LSCM. Binding antagonist assays showed that unlabeled oligsaccharides apart from oligochitosn did not inhibit the observed binding of labeled oligochitosan to cell walls and membranes. Treatment of epidermal tobacco cells with oligochitosan resulted in a strong increase of intracellular NO. Oligochitosan and the NO donor SNP induced a defense reaction against Tobacco mosaic virus (TMV), increased PAL activity and elevated PAL mRNA level. Co-treatment of oligochitosan and NO (scavenger CPTIO) blocked the inducing resistance indicating that the defense response induced by oligochitosan was connected with the NO pathway.

 

 

Oligochitosan induces cell death and hydrogen peroxide accumulation in tobacco suspension cells

 

Wenxia Wang, Shuguang Li, Xiaoming Zhao, Yuguang Du, Bincheng Lin

 

Pesticide Biochemistry and Physiology 90 (2008) 106–113

 

Oligochitosan has been shown to induce several plant defense responses. In the present work, the effect of oligochitosan on tobacco cell survival was investigated. The results showed that oligochitosan caused tobacco cell death in a dose-dependent manner. About 40.6% tobacco cells died when cultured for 72 h after 500 lg ml_1 oligochitosan treatment. Certain aspects of this cell death process appeared to be similar to apoptosis in animal cells. These included shrinkage of cytoplasm and condensation of chromatin. ] Oligochitosan also induced H2O2 accumulation in tobacco cell suspension culture. The role of H2O2 in the signal transduction that leads to cell death was investigated. Co-treatment of tobacco cells with oligochitosan and catalase inhibited H2O2 accumulation but did not inhibit the induction of cell death. The results suggested that apoptosis-like cell death of tobacco cells induced by oligochitosan is independent  of H2O2 signal pathway.

 

 

3. Antifungal

 

Antifungal activity of oligochitosan against Phytophthora capsici and other plant pathogenic fungi in vitro

 

Junguang Xu, Xiaoming Zhao, Xiuwen Han, Yuguang Du

 

Pesticide Biochemistry and Physiology 87 (2007) 220–228

 

Antifungal activity of oligochitosan against nine phytopathogens was investigated in vitro. Oligochitosan was more eVective than chitosan in inhibiting mycelial growth of Phytophthora capsici and its inhibition on diVerent stages in life cycle of P. capsici was observed. Rupture of released zoospores induced by oligochitosan was reduced by addition of 100mM glucose. The eVects of oligochitosan on mycelial growth and zoospore release, but not zoospore rupture, were reduced largely when pH value was above 7. The ultrastructural study showed that oligochitosan caused distortion and disruption of most vacuoles, thickening of plasmalemma, and appearance of unique tubular materials. Plasmalemmasomes in hyphal tip cells were not found in the presence of oligochitosan. These results suggest polycationic nature of oligochitosan contributes only partly to its antifungal activity and multiple modes of action of oligochitosan exist including the disruption of endomembrane system.

 

Oligochitosan inhibits Phytophthora capsici by penetrating the cell membrane and putative binding to intracellular targets

Junguang Xu, Xiaoming Zhao, Xiuli Wang, Zongbao Zhao, Yuguang Du

 

Pesticide Biochemistry and Physiology 88 (2007) 167–175

 

The mechanism of action of oligochitosan, which has shown great antifungal activity against Phytophthora capsici in vitro, was studied using 2-aminoacridone-labeled oligochitosan (2-AMAC-oligochitosan) and a gel-retardation experiment. Internalization of 2-AMAColigochitosan in cysts, germtubes and sporangia of P. capsici was conWrmed by confocal laser scan microscopy (CLSM), and the degree of uptake depended on the incubation concentration. 2-AMAC-oligochitosan localized mainly in the cytoplasm and showed no binding to both cell wall and cell membrane. Mannose, an inhibitor for oligochitosan uptake by macrophages, could not inhibit the internalization of oligochitosan in P. capsici. The gel-retardation experiment showed that oligochitosan bound strongly to DNA and RNA of P. capsici. These results indicate that oligochitosan exerts its antifungal activity by penetrating the cell membrane and putative binding to intracellular targets such as DNA and RNA.

 

 

4. Human health（experiments on animal）

 

Anti-angiogenic activities of chitooligosaccharides

 

Haige Wu, Ziang Yao, Xuefang Bai, Yuguang Du, Bingcheng Lin

 

Carbohydrate Polymers 73 (2008) 105–110

 

Chitooligosaccharides (COS) was obtained from chitosan by depolymerization with enzyme and analyzed by HPLC and TOF-MS, and the results indicated that the polymerization degree of COS was 2–18. In order to explore the anti-angiogenic activities of COS, the effect of COS on chicken chorioallantoic membrane (CAM) angiogenesis and on proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) induced by human hepatoma carcinoma cells (HCC) culture fluid was measured. The results showed COS had no toxicity to normal HUVECs, but could inhibit the CAM angiogenesis and the proliferation, migration and tube formation of induced HUVECs. All of these results indicate that COS have potential anti-angiogenic activities and can counteract the stimulation of HCC-culture-fluid on endothelial cells at a certain level.

 

Chitooligosaccharides induce apoptosis of human hepatocellular carcinoma cells via up-regulation of Bax

 

Qingsong Xu, Jiangli Dou, Peng Wei, Chengyu Tan, Xiaojing Yun, Yihong Wu, Xuefang Bai, Xiaojun Ma, Yuguang Du

 

Carbohydrate Polymers 71 (2008) 509–514

 

Chitooligosaccharides (COS) have been shown to regulate various cellular and biological functions. However, the effect of COS on apoptosis of hepatocellular carcinoma cells remains unclear. In this study, the activity and mechanism of COS against human hepatocellular carcinoma cells (SMMC-7721 cells) were investigated in vitro. The experiments showed that COS notably induced the apoptosis of SMMC-7721 cells and increased the cleavage of poly(ADP-ribose) polymerase. It presented a dose-dependent manner, and the apoptotic rate amounted to about 38% after treatment with 0.8 mg/ml COS for 72 h. The mRNA and protein levels of Bax were up-regulated by COS. These results demonstrated that COS induced apoptosis of SMMC-7721 cells. The possible mechanism is that COS up-regulate pro-apoptotic protein Bax, and trigger the cells a start-up of the apoptosis program.

 

 

Effects of chitooligosaccharides on rabbit neutrophils in vitro

 

Jiangli Dou, Chengyu Tan, Yuguang Du, Xuefang Bai, Keyi Wang, Xiaojun Ma

 

Carbohydrate Polymers 69 (2007) 209–213

 

The effects of chitooligosaccharides(COS) on resting and PMA-activated neutrophils were estimated. MTT assays, NO estimation and superoxide detection revealed that chito- oligosaccharides at concentrations of 25, 50, 75, and 100 lg/ml increased the viability, ability to produce reactive oxygen intermediates and nitrogen intermediates of resting neutrophils. Superoxide detection, degranulation assay and adhesion assay suggested that chitooligosaccharides at concentrations from 50 to 150 lg/ml reduced PMA-induced activation of neutrophils.