Zhihong Liang

Mycotoxin Ochratoxin A-induced cell death and changes in oxidative metabolism of Arabidopsis thaliana

We evaluated the phytotoxicity of mycotoxin ochratoxin A (OTA) from Aspergillus and Penicillium strains on Arabidopsis thaliana. The results demonstrate that the growth of Arabidopsis thaliana on media containing OTA was inhibited significantly. Moreover, OTA induced necrotic lesions in detached leaves, which are reminiscent of hypersensitive response lesions that are activated during plant–pathogen interactions and other abiotic stress factors. From our study, we can see that OTA exposure stimulated a biphasic oxidative burst in the leaves, resulting in the generation of hydrogen peroxide (H2O2) and superoxide anion radicals (O2·−) and in the concomitant down-regulation of antioxidant enzyme defense responses and up-regulation of lipid peroxidation. These results suggested that OTA damage might result from reactive oxygen species pathways. Our experiments provide a useful model plant system for research on OTA-induced plant cell death.

The effect of various environmental factors on the ethidium monazite and quantitative PCR method to detect viable bacteria

Aims:  Ethidium monoazide in combination with quantitative PCR (EMA–qPCR) has been considered as a promising method to enumerate viable cells; however, its efficacy can be significantly affected by disinfection conditions and various environments. In this study, thermal disinfection, osmotic pressure and acids with different pH values were systematically investigated to achieve the optimum conditions.

Transcript and protein profiling analysis of OTA-induced cell death reveals the regulation of the toxicity response process in Arabidopsis thaliana

Ochratoxin A (OTA) is a toxic isocoumarin derivative produced by various species of mould which mainly grow on grain, coffee, and nuts. Recent studies have suggested that OTA induces cell death in plants. To investigate possible mechanisms of OTA phytotoxicity, both digital gene expression (DGE) transcriptomic and two-dimensional electrophoresis proteomic analyses were used, through which 3118 genes and 23 proteins were identified as being up- or down-regulated at least 2-fold in Arabidopsis leaf in response to OTA treatment. First, exposure of excised Arabidopsis thaliana leaves to OTA rapidly causes the hypersensitive reponse, significantly accelerates the increase of reactive oxygen species and malondialdehyde, and enhances antioxidant enzyme defence responses and xenobiotic detoxification. Secondly, OTA stimulation causes dynamic changes in transcription factors and activates the membrane transport system dramatically. Thirdly, a concomitant persistence of compromised photosynthesis and photorespiration is indicative of a metabolic shift from a highly active to a weak state. Finally, the data revealed that ethylene, salicylic acid, jasmonic acid, and mitogen-activated protein kinase signalling molecules mediate the process of toxicity caused by OTA. Profiling analyses on Arabidopsis in response to OTA will provide new insights into signalling transduction that modulates the OTA phytotoxicity mechanism, facilitate mapping of regulatory networks, and extend the ability to improve OTA tolerance in Arabidopsis.

Red Ginseng and Semen Coicis can improve the structure of gut microbiota and relieve the symptoms of ulcerative colitis.

ETHNOPHARMACOLOGICAL RELEVANCE Many Chinese herbs are traditionally used as medicine to improve the functions of gastrointestinal tract. Some of these herbs are also promising agents for the improvement of the gut microbiota and the treatment of ulcerative colitis. MATERIALS AND METHODS By screening seven traditional Chinese herbs, we found that Red Ginseng and Semen Coicis were the most effective in promoting the growth of probiotics including Lactobacillus and Bifidobacterium in vitro. We then evaluated the effects of Red Ginseng and Semen Coicis on the growth of the bacterial pathogens (Escherichia coli, Staphylococcus aureus, and Salmonella spp.) in vitro. In in vivo experiment, we gavage administrated trinitro-benzene-sulfonic acid induced ulcerative colitis (UC) rats with Red Ginseng and Semen Coicis extracts. After two weeks treatment, we analyzed the structure of the gut microbiota and examined the UC symptoms by employing qPCR and animal pathology detection techniques. RESULTS Both Red Ginseng and Semen Coicis promoted the growth of probiotics - Bifidobacterium and Lactobacillus in vitro. Red Ginseng also inhibited the growth of some pathogen strains. In vivo, Red Ginseng and Semen Coicis improved the structure of gut microbiota and relieved the symptoms of ulcerative colitis in vivo. Compared with Semen Coicis, Red Ginseng was more effective in relieving the symptoms of ulcerative colitis. CONCLUSIONS Red Ginseng could promote the growth of probiotic bacteria in vitro. Red Ginseng and, to a lesser extent Semen Coicis, gave positive results in an experimental in vivo model for ulcerative colitis.

Ochratoxin A biocontrol and biodegradation by Bacillus subtilis CW 14.

BACKGROUND Ochratoxin A (OTA) is a mycotoxin produced by some Aspergillus and Penicillium species. In this study a strain of Bacillus subtilis was tested for its effects on OTA-producing Aspergillus and OTA degradation. The mechanisms of the effects were also investigated. RESULTS A strain of Bacillus spp. isolated from fresh elk droppings was screened out using the methods described by Guan et al. (Int J Mol Sci 9:1489-1503 (2008)). The 16S rRNA gene sequence suggested that it was B. subtilis CW 14. It could inhibit the growth of the OTA-producing species Aspergillus ochraceus 3.4412 and Aspergillus carbonarius, with inhibition rates of 33.0 and 33.3% respectively. At 6 µg mL(-1) OTA, both viable and autoclaved (121 °C, 20 min) cells of CW 14 bound more than 60% of OTA. In addition, OTA was degraded by the cell-free supernatant of CW 14. By high-performance liquid chromatography, the cell-free supernatant degraded 97.6% of OTA after 24 h of incubation at 30 °C, and no degradation products were produced. The fastest degradation occurred during the first 2 h. In 3 g samples of contaminated maize, 47.1% of OTA was degraded by 50 mL inocula of overnight cultures of CW 14. CONCLUSION These findings indicated that B. subtilis CW 14 could both prevent OTA contamination and degrade OTA in crops.

Cloning, expression and characterization of recombinant elastase from Pseudomonas aeruginosa in Picha pastoris

The gene lasB from Pseudomonas aeruginosa, which encoded elastase, was cloned and firstly successfully expressed in Pichia pastoris stain KM71 under the control of AOX promoter. The effects on the recombinant elastase activities of different pH, different temperatures and different metal ions were assayed. The full-length gene (1497 bp) encodes a preproenzyme including an N-terminal signal peptide (23 aa), a propeptide (197 aa) and mature elastase (301 aa). The recombinant elastase was secreted into culture supernatants using signal sequence from lasB and showed a single band at about 34 kDa by SDS-PAGE. The recombinant elastase expression hit the highest level of approximately 450 mg/L and the specific elastolytic activity of the recombinant elastase was 130 U/ml, which was approximately 26-fold higher than that of elastase obtained from P. aeruginosa. The optimal temperature and pH of the recombinant elastase was 28 degrees C and 7.4, respectively. The enzyme possessed high resistance to heat, and can be activated by Ca(2+). These enzyme properties suggested that it could be produced in an industrial scale and has the potential to be a commercial enzyme.

A novel antifungal peptide from foxtail millet seeds

BACKGROUND Antifungal proteins (AFP) help plants to combat phytopathogenic fungi and thus protect plants from the devastating damage caused by fungal infections and prevent massive economic losses. To date, several proteins with antibacterial and/or antifungal properties have been isolated and characterized from different plant species and tissues; however, there are no reports concerning the antifungal peptide from foxtail millet seeds. RESULTS An antifungal peptide with a molecular mass of 26.9 kDa was isolated from dry seeds of the foxtail millet (Setaria italica (L.) Beauv.), using a procedure that involved four chromatographic steps. The antifungal peptide was adsorbed on CM-Sepharose, Affi-gel blue gel and Superdex 75. It was further purified by C(18) reverse-phase high-performance liquid chromatography and submitted for analysis of peptide mass fingerprint. The Mascot peptide mass fingerprint of the isolated protein hit no existing protein (score >60), and it was proved to be a novel antifungal peptide. It inhibited mycelial growth in Alternaria alternate with an IC(50) of 1.3 µmol L(-1) , and it also exhibited antifungal activity against Trichoderma viride, Botrytis cinerea and Fusarium oxysporum. Transmission electron microscopy of mold forms of Alternaria alternate after incubation with 20 µg mL(-1) of the antifungal protein for 48 h revealed marked ultrastructural changes in the fungus. CONCLUSION A novel antifungal peptide with high potency was isolated from foxtail millet seeds.

Changes in biosynthesis and metabolism of glutathione upon ochratoxin A stress in Arabidopsis thaliana

Ochratoxin A (OTA) is one of the most toxic mycotoxins, which is toxic to plants and simulates oxidative stress. Glutathione is an important antioxidant in plants and is closely associated with detoxification in cells. We have previously shown that OTA exposure induces obvious expression differences in genes associated with glutathione metabolism. To characterize glutathione metabolism and understand its role in OTA phytotoxicity, we observed the accumulation of GSH in the detached leaves of Arabidopsis thaliana under OTA treatment. OTA stimulated a defense response through enhancing glutathione-S-transferase, glutathione peroxidase, glutathione reductase activities, and the transcript levels of these enzymes were increased to maintain the total glutathione content. Moreover, the level of oxidized glutathione (GSSG) was increased and the ascorbate-glutathione cycle fluctuated in response to OTA. The depletion of glutathione using buthionine sulfoximine (BSO, inhibitor of glutamate-cysteine ligase) had no profound effect on OTA toxicity, as glutathione was regenerated through the ascorbate-glutathione cycle to maintain the total glutathione content. The ROS, MDA and GSH accumulation was significantly affected in the mutant gsh1, gr1 and gpx2 after treatment with OTA, which indicated that glutathione metabolism is directly involved in the oxidative stress response of Arabidopsis thaliana subjected to OTA. In conclusion, date demonstrate that glutathione-associated metabolism is closely related with OTA stress and glutathione play a role in resistance of Arabidopsis subjected to OTA.

Comparative Physicochemical Properties and Structure of Rice Containing the sck+cryIAc Genes and Its Nontransgenic Counterpart

The physicochemical properties and structure of an insect-resistant rice, Liangyou Kefeng Nr. 6 (IRR), containing the sck and cryIAc genes were compared with those of its nontransgenic counterpart designated as Liangyou 2186 (control), considering their key role in determining commercial value. Basically the appearance of IRR was not affected in terms of size and shape after foreign gene transformation but improved with lower chalkiness. The milling yield of IRR with lower chalkiness was higher measured by head rice yield compared with its parental control. The differences in appearance and milling quality were confirmed by the structure of raw rice grain by scanning electron microscopy (SEM). Slight differences were observed in pasting properties and textural quality determined by rapid viscosity analyzer and texture analyzer which was in agreement with the result of the structure of cooked rice grain by SEM. The above differences might be as a result of a positional effect of T-DNA insertion. On the whole, the appearance, milling quality, and eating quality of IRR were not adversely affected by transgenes, which will facilitate its acceptance by the consumer after commercialization.

A subchronic feeding study of dicamba-tolerant soybean with the dmo gene in Sprague-Dawley rats.

The dicamba-tolerant soybean MON87708 expresses the dicamba mono-oxygenase (DMO) enzyme that is encoded by the dmo gene. In order to evaluate the safety of this soybean, a 90-day subchronic feeding toxicity study (13 weeks) was conducted on Sprague-Dawley rats. A total of 140 rats were divided into 7 groups (10/sex/group), including a standard commercial diet control group. The genetically modified (GM) soybean MON87708 and the near isogenic non-GM soybean A3525 were respectively processed to unhulled, full-fat, and heat-treated powder, then mixed into the diet at levels of 7.5%, 15%, and 30% (wt/wt) with the main nutrients of the various diets balanced and then fed to 6 groups. The remaining group of rats fed with a commercial rat diet served as blank control. Some isolated parameters indicated statistically significant differences in body weight, feed consumption/utilization, hematology, serum biochemistry, and relative organ weights. These differences were not consistent across gender or test-diet dose, which were attributed to incidental and biological variability. In conclusion, the results demonstrated that the transgenic soybean MON87708 containing DMO was as safe as non-transgenic isogenic counterpart with historical safe use.