Eckol isolated from Ecklonia cava attenuates oxidative stress induced cell damage in lung fibroblast cells

We have investigated the cytoprotective effect of eckol, which was isolated from Ecklonia cava, against oxidative stress induced cell damage in Chinese hamster lung fibroblast (V79-4) cells. Eckol was found to scavenge 1,1-diphenyl-2-picrylhydrazyl radical, hydrogen peroxide (H(2)O(2)), hydroxy radical, intracellular reactive oxygen species (ROS), and thus prevented lipid peroxidation. As a result, eckol reduced H(2)O(2) induced cell death in V79-4 cells. In addition, eckol inhibited cell damage induced by serum starvation and radiation by scavenging ROS. Eckol was found to increase the activity of catalase and its protein expression. Further, molecular mechanistic study revealed that eckol increased phosphorylation of extracellular signal-regulated kinase and activity of nuclear factor kappa B. Taken together, the results suggest that eckol protects V79-4 cells against oxidative damage by enhancing the cellular antioxidant activity and modulating cellular signal pathway.     

Phylogenetic relationships among genotypes of worldwide collection of spring and winter ryes (Secale cereale L.) determined by RAPD-PCR markers

Genetic similarities among 20 spring and 22 winter accessions of agronomically different ryes from fourteen countries were estimated by employing random amplified polymorphic DNA (RAPD) techniques. Cluster analysis of genetic distance data showed that 42 genotypes were readily classifiable into two main groups: spring and winter groups. Within the spring group, cultivars fell into a North European and an American-Chinese group. Cultivars of winter rye fell into four groups: Northern European, Russian, American and Chinese lines. A UPGMA-dendrogram based on genetic distances of cultivars of rye within the winter and spring groups showed that the clusters corresponded well to their geographical locations. The results indicated that isolation has played an important role in the evolution of rye, and that temporal isolation has influenced the genetic diversity of rye more than geographical isolation. In this experiment, RAPD proved to be a rapid, reliable and practicable method of revealing polymorphisms in rye populations.     

Saturation Mutagenesis of Toluene ortho-Monooxygenase of Burkholderia cepacia G4 for Enhanced 1-Naphthol Synthesis and Chloroform Degradation

Directed evolution of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 previously created the hydroxylase α-subunit (TomA3) V106A variant (TOM-Green) with increased activity for both trichloroethylene degradation (twofold enhancement) and naphthalene oxidation (six-times-higher activity). In the present study, saturation mutagenesis was performed at position A106 with Escherichia coli TG1/pBS(Kan)TOMV106A to improve TOM activity for both chloroform degradation and naphthalene oxidation. Whole cells expressing the A106E variant had two times better naphthalene-to-1-naphthol activity than the wild-type cells (V_max of 9.3 versus 4.5 nmol·min⁻¹·mg of protein⁻¹ and unchanged K_m), and the regiospecificity of the A106E variant was unchanged, with 98% 1-naphthol formed, as was confirmed with high-pressure liquid chromatography. The A106E variant degrades its natural substrate toluene 63% faster than wild-type TOM does (2.12 ± 0.07 versus 1.30 ± 0.06 nmol·min⁻¹·mg of protein⁻¹ [mean ± standard deviation]) at 91 μM and has a substantial decrease in regiospecificity, since o-cresol (50%), m-cresol (25%), and p-cresol (25%) are formed, in contrast to the 98% o-cresol formed by wild-type TOM. The A106E variant also has an elevated expression level compared to that of wild-type TOM, as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Another variant, the A106F variant, has 2.8-times-better chloroform degradation activity based on gas chromatography (V_max of 2.61 versus 0.95 nmol·min⁻¹·mg of protein⁻¹ and unchanged K_m) and chloride release (0.034 ± 0.002 versus 0.012 ± 0.001 nmol·min⁻¹·mg of protein⁻¹). The A106F variant also was expressed at levels similar to those of wild-type TOM and 62%-better toluene oxidation activity than wild-type TOM (2.11 ± 0.3 versus 1.30 ± 0.06 nmol·min⁻¹·mg of protein⁻¹). A shift in regiospecificity of toluene hydroxylation was also observed for the A106F variant, with o-cresol (28%), m-cresol (18%), and p-cresol (54%) being formed. Statistical analysis was used to estimate that 292 colonies must be screened for a 99% probability that all 64 codons were sampled during saturation mutagenesis.     

枯草芽孢杆菌ccpA基因敲除及对其核黄素产量的影响

CcpA蛋白是介导枯草芽孢杆菌碳分解代谢物阻遏(CCR)的全局调控因子,由ccpA基因编码。CCR效应的存在影响B.subtilis对葡萄糖的利用,降低B.subtilis生产发酵产品的效率。采用基因重组技术敲除了核黄素发酵菌株B.subtilis24/pMX45的ccpA基因,构建了CcpA缺陷株B.subtilis24A1/pMX45。发酵结果显示:B.subtilis24A1/pMX45能够在70h内基本耗尽10%的葡萄糖,生物量达到1.5×109个细胞/mL,溢流代谢产物积累量减少,在8%和10%葡萄糖浓度下,B.subtilis24A1/pMX45核黄素产量分别比B.subtilis24/pMX45提高了62%和95%。CcpA的缺陷,可以缓解葡萄糖引起的CCR效应,显著提高菌株的核黄素产量。     

TUNEL法检测乳酸杆菌对小鼠宫颈鳞癌的凋亡诱导作用

目的应用脱氧核苷酸转移酶介导的dUTP缺口末端标记法———TUNEL法,检测乳酸杆菌(Lactoba-cillus,LB)对小鼠宫颈鳞癌的凋亡诱导作用。方法建立615近交系小鼠U14移植瘤动物模型,并随机分组,TUNEL法检测各组细胞凋亡指数(apoptosis index,AI)。结果乳酸杆菌实验组的AI为(11.25±4.06)%,与对照组的差异有非常显著性(P<0.01),与抗癌药顺铂组的差异有显著性(P<0.05)。结论乳酸杆菌可诱导U14移植瘤细胞凋亡,这可能是其抑癌机制之一。