Genetic engineering of complex feed enzymes into barley seed for direct utilization in animal feedstuff.

Currently, feed enzymes are primarily obtained through fermentation of fungi, bacteria, and other microorganisms. Although the manufacturing technology for feed enzymes has evolved rapidly, the activities of these enzymes decline during the granulating process and the cost of application has increased over time. An alternative approach is the use of genetically modified plants containing complex feed enzymes for direct utilization in animal feedstuff. We co-expressed three commonly used feed enzymes (phytase, ß-glucanase, and xylanase) in barley seeds using the Agrobacterium-mediated transformation method and generated a new barley germplasm. The results showed that these enzymes were stable and had no effect on the development of the seeds. Supplementation of the basal diet of laying hens with only 8% of enzyme-containing seeds decreased the quantities of indigestible carbohydrates, improved the availability of phosphorus, and reduced the impact of animal production on the environment to an extent similar to directly adding exogenous enzymes to the feed. Feeding enzyme-containing seeds to layers significantly increased the strength of the eggshell and the weight of the eggs by 10.0%-11.3% and 5.6%-7.7% respectively. The intestinal microbiota obtained from layers fed with enzyme-containing seeds was altered compared to controls and was dominated by Alispes and Rikenella. Therefore, the transgenic barley seeds produced in this study can be used as an ideal feedstuff for use in animal feed.

[Effects of Radix Angelicae Sinensis on airway mucus hypersecretion and the TNF-α/NF-κB signaling pathway in Yin-deficiency asthmatic mice].

Objective: To investigate the therapeutic effects of Radix Angelicae Sinensis (RADA) on airway mucus hypersecretion and the tumor necrosis factor-α/ nuclear factor- κB (TNF-α/NF-κB) signaling pathway in Yin-deficiency asthma mice. Methods: KM mice were randomly divided into control group, model group, ambroxol group and RADA low, medium and high dose (2, 4 and 8 g/kg) group(n=12). Ovalbumin and the thyroid gland were used to replicate the model of Yin-deficiency asthma. Asthma symptoms in mice , immune globulin E (IgE) , TNF-α , and the expressions of Mucin 5ac (Muc5ac) and NF- κB in lung tissue were observed under the intervention of RADA. Results: RADA at the doses of 2,4 and 8 g/kg could alleviate the asthma symptoms of Yin-deficiency asthma mice significantly, reduce the levels of IgE in serum and TNF-α in bronchoalveolar lavage fluid (BALF), and inhibite the overexpressions of Muc5ac and NF- κB in lung tissue. Conclusion: RADA has significant anti-asthmatic effect. One of its mechanisms is to inhibit TNF-α/NF- κB signaling pathway and to alleviate airway mucus hypersecretion.

Metabolic engineering of rice endosperm for betanin biosynthesis.

Betanin has been widely used as an additive for many centuries, and its use has increased because of its market application as an additive, high free radical scavenging activity, and safety, health-promoting properties. The main source of betanin is red beet, but many factors notably affect the yield of betanin from red beets. Betanin is not produced in cereal grains. Thus, developing biofortified crops with betanin is another alternative to health-promoting food additives. Here, rice endosperm was bioengineered for betanin biosynthesis by introducing three synthetic genes (meloS, BvDODA1S, and BvCYP76AD1S). The overexpression of these genes driven by rice endosperm-specific promoter established the betanin biosynthetic pathways in the endosperm, resulting in new types of germplasm - 'Betanin Rice' (BR). The BR grains were enriched with betanin and had relatively high antioxidant activity. Our results proved that betanin can be biosynthesized de novo in rice endosperm by introducing three genes in the committed betanin biosynthetic pathway. The betanin-fortified rice in this study can be used as a functional grain to promote health and as a raw material to process dietary supplements.

Induction of Apoptosis in Human Glioma Cells by Fucoxanthin via Triggering of ROS-Mediated Oxidative Damage and Regulation of MAPKs and PI3K-AKT Pathways.

Fucoxanthin, a natural carotenoid derived from algae, exhibits novel anticancer potential. However, fucoxanthin with high purity is hard to prepare, and the anticancer mechanism remains elusive. In the present study, fucoxanthin with high purity was prepared and purified from the marine microalgae Nitzschia sp. by silica-gel column chromatography (SGCC), and the underlying mechanism against human glioma cells was evaluated. The results showed that fucoxanthin time- and dose-dependently inhibited U251-human-glioma-cell growth by induction of apoptosis (64.4 ± 4.8, P < 0.01) accompanied by PARP cleavage and caspase activation (244 ± 14.2, P < 0.01). Mechanically, fucoxanthin time-dependently triggered reactive-oxygen-species (ROS)-mediated DNA damage (100 ± 7.38, P < 0.01), as evidenced by the phosphorylation activation of Ser1981-ATM, Ser428-ATR, Ser15-p53, and Ser139-histone. Moreover, fucoxanthin treatment also time-dependently caused dysfunction of MAPKs and PI3K-AKT pathways, as demonstrated by the phosphorylation activation of Thr183-JNK, Thr180-p38, and Thr202-ERK and the phosphorylation inactivation of Ser473-AKT. The addition of kinase inhibitors further confirmed the importance of MAPKs and PI3K-AKT pathways in fucoxanthin-induced cell-growth inhibition (32.5 ± 3.6, P < 0.01). However, ROS inhibition by the antioxidant glutathione (GSH) effectively inhibited fucoxanthin-induced DNA damage, attenuated the dysfunction of MAPKs and PI3K-AKT pathways, and eventually blocked fucoxanthin-induced cytotoxicity (54.3 ± 5.6, P < 0.05) and cell apoptosis (32.7 ± 2.5, P < 0.05), indicating that ROS production, an early apoptotic event, is involved in the fucoxanthin-mediated anticancer mechanism. Taken together, these results suggested that fucoxanthin induced U251-human-glioma-cell apoptosis by triggering ROS-mediated oxidative damage and dysfunction of MAPKs and PI3K-AKT pathways, which validated that fucoxanthin may be a candidate for potential applications in cancer chemotherapy and chemoprevention.

[Effect of Yuyin Ruangan Granule on TGF-ß1 expression in hepatic fibrosis rats].

OBJECTIVES: To observe the preventive and therapeutic action of Yuyin Ruangan Granule (YRG, Traditional Chinese Medicine) in hepatic fibrosis rats model and its effect on transforming growth factor-ß1 (TGF-ß1) expression. METHODS: The Wistar rats were randomly divided into 6 group (n=10), and the model of hepatic fibrosis rats was established by subcutaneous injected with carbon tetrachloride (CCL4), fed on high-fat diet and 20% ethanol for 6 weeks, to survey the effect and mechanism of YRG preventing hypatic fibrosis by detecting liver function (the activity of alanine aminotransferase(ALT), aspartate aminotransferase(AST), etc.) of liver fibrosis rats, liver fibrosis indicators (hyaluronic acid, Ⅲ procollagen, type IV collagen, laminin and hepatic pathology, etc.), and TGF-ß1 expression in liver tissue after 6 weeks treated with YRG through intragastric administration (q. d.). RESULTS: At the 7th week, fibrotic lesions appears distinctly in liver tissue of model group compared with control group (P<0.01), YRG of 6.2~28.8 g/kg could significantly decrease hepatic index, ALT and AST activities, content of hyaluronic acid(HA), Ⅲ procollagen (PCⅢ), type Ⅳ collagen(C-Ⅳ), laminin (LN) in serum, relieve liver fibrosis pathological changes and inhibit TGF-ß1 expression in fibrotic liver tissue (P<0.05, P<0.01). CONCLUSIONS: YRG has significantly preventive effects on liver fibrosis rats model, and it may be one of its mechanisms to inhibit expression of TGF-ß1.

Enhanced Therapeutic Potential of Nano-Curcumin Against Subarachnoid Hemorrhage-Induced Blood-Brain Barrier Disruption Through Inhibition of Inflammatory Response and Oxidative Stress.

Curcumin and nano-curcumin both exhibit neuroprotective effects in early brain injury (EBI) after experimental subarachnoid hemorrhage (SAH). However, the mechanism that whether curcumin and its nanoparticles affect the blood-brain barrier (BBB) following SAH remains unclear. This study investigated the effect of curcumin and the poly(lactide-co-glycolide) (PLGA)-encapsulated curcumin nanoparticles (Cur-NPs) on BBB disruption and evaluated the possible mechanism underlying BBB dysfunction in EBI using the endovascular perforation rat SAH model. The results indicated that Cur-NPs showed enhanced therapeutic effects than that of curcumin in improving neurological function, reducing brain water content, and Evans blue dye extravasation after SAH. Mechanically, Cur-NPs attenuated BBB dysfunction after SAH by preventing the disruption of tight junction protein (ZO-1, occludin, and claudin-5). Cur-NPs also up-regulated glutamate transporter-1 and attenuated glutamate concentration of cerebrospinal fluid following SAH. Moreover, inhibition of inflammatory response and microglia activation both contributed to Cur-NPs' protective effects. Additionally, Cur-NPs markedly suppressed SAH-mediated oxidative stress and eventually reversed SAH-induced cell apoptosis in rats. Our findings revealed that the strategy of using Cur-NPs could be a promising way in improving neurological function in EBI after experimental rat SAH.

Induction of S-Phase Arrest in Human Glioma Cells by Selenocysteine, a Natural Selenium-Containing Agent Via Triggering Reactive Oxygen Species-Mediated DNA Damage and Modulating MAPKs and AKT Pathways.

Selenocysteine (SeC) a natural available selenoamino acid exhibits novel anticancer activities against human cancer cell lines. However, the growth inhibitory effect and mechanism of SeC in human glioma cells remain unclear. The present study reveals that SeC time- and dose-dependently inhibited U251 and U87 human glioma cells growth by induction of S-phase cell cycle arrest, followed by the marked decrease of cyclin A. SeC-induced S-phase arrest was achieved by inducing DNA damage through triggering generation of reactive oxygen species (ROS) and superoxide anion, with concomitant increase of TUNEL-positive cells and induction of p21waf1/Cip1 and p53. SeC treatment also caused the activation of p38MAPK, JNK and ERK, and inactivation of AKT. Four inhibitors of MAPKs and AKT pathways further confirmed their roles in SeC-induced S-phase arrest in human glioma cells. Our findings advance the understanding on the molecular mechanisms of SeC in human glioma management.

Protective effect of apigenin on ischemia/reperfusion injury of the isolated rat heart.

Apigenin (Api), a mainly bioactive component of Apium graveolens L. var. dulce DC. (a traditional Chinese medicinal herb), possesses a wide range of biological activities, including antioxidant effects. It also has been shown to associate with lower prevalence of cardiovascular diseases, but its mechanisms of action remain unclear. The aim of the present study is to investigate the role of Api in isolated rat heart model of ischemia/reperfusion (I/R). Langendorff-perfused isolated rat hearts were used in our study. Api was added to the perfusate before ischemia and during reperfusion in the isolated pulsed rat heart exposed to 30-min ischemia followed by 50-min reperfusion. The treatment with Api conferred a cardioprotective effect, and the treated hearts demonstrated an improved ischemic cardiac functional recovery, a decreased myocardial infarct size, a reduced activities of creatine kinase isoenzyme and lactate dehydrogenase in the coronary flow, a reduced number of apoptotic cardiomyocytes, a reduced activity of caspase-3, up-regulation of the anti-apoptotic protein Bcl-2 and down-regulation of the pro-apoptotic protein Bax. In addition, Api inhibited the phosphorylation of p38 MAPKS during I/R. In conclusion, these observations provide preliminary evidence that Api can protect cardiomyocytes from I-/R-induced injury, at least partially, through the inhibition of p38 MAPKS signaling pathway.

Mutation by DNA shuffling of 5-enolpyruvylshikimate-3-phosphate synthase from Malus domestica for improved glyphosate resistance.

A new 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Malus domestica (MdEPSPS) was cloned and characterized by rapid amplification of cDNA ends to identify an EPSPS gene appropriate for the development of transgenic glyphosate-tolerant plants. However, wild-type MdEPSPS is not suitable for the development of transgenic glyphosate-tolerant plants because of its poor glyphosate resistance. Thus, we performed DNA shuffling on MdEPSPS, and one highly glyphosate-resistant mutant with mutations in eight amino acids (N63D, N86S, T101A, A187T, D230G, H317R, Y399R and C413A.) was identified after five rounds of DNA shuffling and screening. Among the eight amino acid substitutions on this mutant, only two residue changes (T101A and A187T) were identified by site-directed mutagenesis as essential and additive in altering glyphosate resistance, which was further confirmed by kinetic analyses. The single-site A187T mutation has also never been previously reported as an important residue for glyphosate resistance. Furthermore, transgenic rice was used to confirm the potential of MdEPSPS mutant in developing glyphosate-resistant crops.

Complementary screening, identification and application of a novel class II 5-enopyruvylshikimate-3-phosphate synthase from Bacillus cereus.

A novel aroA gene encoding 5-enolpyruvylshikimate-3-phosphate synthase from Bacillus cereus was identified and overexpressed by genomic library construction and complementary screening. The enzyme was then purified to homogeneity. We also transformed the aroA ( B. cereus ) gene into Arabidopsis thaliana by a floral dip method, and demonstrated that transgenic A. thaliana plants exhibited significant glyphosate resistance compared with the wild type. These results strongly suggested that the strategy was highly efficient and advantageous for rapidly cloning aroA genes from microorganisms in natural environments.

[Influence of H2O2 on degradation of residual pesticides and constituents in Radix Sophorae Flavescentis].

OBJECTIVE: To investigate the characteristic and influential factors of the degradation of residual pesticides and alkaloids in Radix Sophorae Flavescentis by H2O2. METHOD: The spiked samples were treated in H2O2 in different reaction time, concentration and pH value. The pesticide residuals were determined by GC-MS, and the contents of alkaloids were determined by HPLC. RESULT: H2O2 had highly activity in degrading organophosphorus and pyrethroid, but had less activity to organochlorines. The degradation processes of organophosphorus and pyrethroid followed first-order kinetics equations, and were influenced by the pH value, the concentration of H2O2 and reaction time. The contents of alkaloids in Radix Sophorae Flavescentis changed not obviously after treatment with 3 mL x L(-1) H2O2 less than 6 hours under neutral condition. CONCLUSION: H2O2 is a useful reagent for the degradation of organophosphorus and pyrethroid in crude drug.