Optimum krill phospholipids content in microdiets for gilthead seabream (Sparus aurata) larvae

The aim of the present study was to determine the optimum dietary levels of krill phospholipids (KPL) for sea bream (Sparus aurata) larvae, and its influence on larval development and digestive enzymes activity. Larvae were fed five formulated microdiets with five different levels of KPL. Complete replacement of live preys with the experimental microdiets for seabream larvae produced high survival and growth rates, particularly in fish fed the highest levels of KPL. In the present study, increase in dietary KPL up to 120 g kg^?1 (100 g kg^?1 total PL) significantly improved larval survival and growth, whereas further increase did not improve those parameters. An increase in alkaline phosphatase, trypsin and lipase activity with the elevation of KPL up to 120 g kg^?1 was also found denoting a better functioning of digestive system. Besides, there was a linear substrate stimulatory effect of dietary KPL on phospholipase A2 activity. Finally, increasing dietary KPL lead to better assimilation of n‐3 HUFA especially eicosapentaenoic acid, reflected in the higher content of these fatty acids in both neutral and polar lipids of the larvae. In summary, KPL were found to be an excellent source of lipids for seabream larvae. Optimum inclusion levels of this ingredient in microdiets to completely substitute live preys at this larval age were found to be 120 g kg^?1 KPL.     

磷脂酶C参与ParA1诱导的烟草悬浮细胞死亡和防卫反应

【目的】研究寄生疫霉Phytophthora parasitica分泌的蛋白激发子ParA1诱导烟草悬浮细胞后,磷脂酶C(phospholipase C,PLC)参与ParA1诱导烟草过敏细胞死亡(hypersensitive cell death,HCD)和防卫反应。【方法】采用药理学方法,在烟草悬浮细胞中添加PLC抑制剂,比较烟草悬浮细胞受ParA1诱导后细胞死亡和多种防卫反应的差别。【结果】PLC特异性抑制剂U73122能完全抑制ParA1引起的烟草悬浮细胞死亡,氧迸发、胞外基质碱化和植保素scopoletin(7-羟基-6-甲氧基香豆素)积累都能显著地被抑制,5种防卫基因hin1、hsr203J、PR(pathogenesis-related)-1a、PR-1b和PAL等的转录表达也都受到U73122不同程度的抑制。【结论】ParA1处理烟草悬浮细胞后,PLC参与了ParA1诱导细胞死亡和防卫反应的信号传导过程。     

肠道微生物宏基因组文库构建及酯解酶基因筛选

利用宏基因组学方法,以人肠道微生物样品为原材料,构建了1个约30 000个克隆的fosmid文库.以三丁酸甘油酯为底物,通过功能筛选,获得1个酯解酶阳性克隆.对该阳性克隆构建亚克隆,挑选具有酯解酶活性的阳性亚克隆进行测序分析,最终获得1个肠道微生物来源的酯解酶基因(GenBank登录号:JQ972699).结果表明,文库克隆的平均插入片段约为40 kb,没有重复插入片段克隆.获得的酯解酶基因推演蛋白与Pyramidobacter piscolens W5455的patatin样磷脂酶同源性最高,氨基酸一致性为95％.生物信息学分析结果表明该基因可能通过Vd型分泌方式进行分泌并发挥功能.本研究是通过构建人肠道微生物宏基因组大片段文库并结合重组子功能筛选获得酯解酶的首次报道,可为食品工业提供新的酯解酶来源和筛选方法.     

意大利蜜蜂蜂毒磷脂酶A2基因在大肠杆菌中的表达

将意大利蜜蜂蜂毒磷脂酶A2(AmPLA2)成熟肽编码区基因(405 bp) 插入表达载体pETBlue-1,在大肠杆菌Tuner(DE3)placⅠ中诱导表达,经SDS-PAGE电泳检测,表达产物分子量为15 kD,约占细菌总蛋白的4.3%;用兔抗AmPLA2多克隆血清为第一抗体作Western blot检测,表达产物显示与天然AmPLA2同样的特异性印迹,证实AmPLA2 基因已在大肠杆菌中得到表达.     

胞质型磷脂酶A2基因克隆及其表达量随大黄鱼仔稚鱼生长发育的变化

利用同源克隆技术和cDNA末端快速扩增(RACE)技术克隆大黄鱼胞质型磷脂酶A2(cPLA2)基因的cDNA全长.此外,应用实时定量PCR法检测不同日龄大黄鱼仔稚鱼cPLA2基因的表达量.序列分析结果表明:cPLA2基因全长2 550 bp(GenBank登录号:KF006240.1),其中5'端非编码区176 bp,开放阅读框2 169 bp,3'端非编码区205 bp,共编码723个氨基酸.系统进化树分析结果表明:克隆所得大黄鱼cPLA2基因与其他鱼类的cPLA2基因亲缘关系较近,与哺乳动物的亲缘关系较远.定量检测结果表明:大黄鱼仔稚鱼cPLA2基因的表达量随日龄的增加为先显著升高(1、3、7日龄vs.15日龄,P＜0.05),在15日龄时达到最高值,随后显著下降(15日龄vs.19日龄,P＜0.05),之后趋于平稳.由此可知,大黄鱼从仔稚鱼到幼鱼早期的变形过程中,磷脂分解代谢的关键酶cPLA2基因的表达量呈现有规律的变化,这可能对机体保持体内磷脂的动态平衡、维护细胞膜的流动性具有重要的意义.大黄鱼仔稚鱼cPLA2基因表达量的变化趋势在一定程度上可以衡量大黄鱼消化系统的发育情况.     

Pharmacological mechanisms of black cohosh in Sprague-Dawley rats

Background

Studies indicate that extracts and purified components from black cohosh inhibit the growth of human breast cancer cells, but the molecular targets and signaling pathways have not yet been defined.

Purpose

This study examines the pharmacological mechanisms and toxicological effects in the short term of the herb black cohosh on female Sprague–Dawley rats.

Materials and methods

To assess effects on gene activity and lipid content, we treated female Sprague–Dawley rats with an extract of black cohosh enriched in triterpene glycosides (27%) at 35.7 or 0 mg/kg. Four animals for each group were sacrificed at 1, 6 and 24 h after treatment; liver tissue and serum samples were obtained for gene expression and lipid analysis.

Results

Microarray analysis of rat liver tissue indicated that black cohosh markedly downregulated mitochondrial oxidative phosphorylation genes. Phospholipid biosynthesis and remodeling, PI3-Kinase and sphingosine signaling were upregulated, driven largely by an upregulation of several isoforms of phospholipase C. Hierarchical clustering indicated that black cohosh clustered with antiproliferative compounds, specifically tubulin binding vinca alkaloids and DNA alkylators. In support of this, black cohosh repressed the expression of cyclin D1 and ID3, and inhibited the proliferation of HepG2, p53 positive, liver cancer cells. Black cohosh reduced the level of free fatty acids at 6 and 24 h and triglycerides at 6 h in the serum, but increased the free fatty acid and triglyceride content of the treated livers at 24 h.

Conclusion

Our results suggest that black cohosh warrants further study for breast cancer prevention and therapy.     

不同种类食用菌对黑粪蚊生长发育、繁殖及酯酶活性的影响

【目的】研究黑粪蚊（Scatopse sp.）取食不同食用菌后的生长发育、存活情况及体内磷酸酯酶和乙酰胆碱酯酶活力,为生产上利用不同种类食用菌的合理布局防止和减轻黑粪蚊危害提供科学依据。【方法】在人工控制温度（25±0.5） ℃、黑暗、相对湿度（80±10）%条件下,人工接虫,系统饲养观察平菇、木耳、银耳3种食用菌对黑粪蚊生长发育、繁殖和磷酸酯酶、乙酰胆碱酯酶活力的影响。【结果】取食平菇的黑粪蚊全幼虫期的发育历期为10.4 d,显著短于取食木耳（14.8 d）和银耳（18.9 d）的幼虫;黑粪蚊取食平菇时,幼虫存活率、蛹质量和雌虫产卵量均最大,分别为61.15%,0.74 mg和145.15粒,其次为取食木耳,取食银耳的最低,分别为50.79%,0.48 mg和85.79粒。取食平菇的黑粪蚊成虫寿命（3.0 d）显著长于取食木耳（2.4 d）,取食银耳的成虫寿命居中。取食不同种类食用菌,对黑粪蚊蛹发育历期、羽化率和雌性比率影响不显著。取食平菇的黑粪蚊种群趋势指数达31.37,明显高于取食木耳（16.62）和银耳（13.49）。幼虫取食不同种类食用菌,其酸性磷酸酯酶和碱性磷酸酯酶的活力由高到低依次均为银耳>木耳>平菇,乙酰胆碱酯酶的活力由高到低依次为平菇>木耳>银耳。【结论】不同种类食用菌对黑粪蚊的生长发育、存活繁殖、磷酸酯酶和乙酰胆碱酯酶活力均有显著影响,其中平菇对黑粪蚊生长发育和繁殖最有利。     

转反义PLDγ基因小麦的获得及其抗白粉病的初步鉴定

利用穗茎注射法将反义PLDγ基因导入烟农15和农大1521两个小麦品种中,旨在获得抗病性增强的转基因植株。结果表明:T3代经PCR及PCR-Southern杂交鉴定,目的基因已经整合到小麦的基因组中,并能在小麦基因组内稳定遗传;烟农15和农大1521的6个转基因株系的抗白粉病的能力明显增强。     

不同种类食用菌对黑粪蚊生长发育、繁殖及酯酶活性的影响

【目的】研究黑粪蚊(Scatopse sp.)取食不同食用菌后的生长发育、存活情况及体内磷酸酯酶和乙酰胆碱酯酶活力,为生产上利用不同种类食用菌的合理布局防止和减轻黑粪蚊危害提供科学依据。【方法】在人工控制温度(25±0.5)℃、黑暗、相对湿度(80±10)%条件下,人工接虫,系统饲养观察平菇、木耳、银耳3种食用菌对黑粪蚊生长发育、繁殖和磷酸酯酶、乙酰胆碱酯酶活力的影响。【结果】取食平菇的黑粪蚊全幼虫期的发育历期为10.4d,显著短于取食木耳(14.8d)和银耳(18.9d)的幼虫;黑粪蚊取食平菇时,幼虫存活率、蛹质量和雌虫产卵量均最大,分别为61.15%,0.74mg和145.15粒,其次为取食木耳,取食银耳的最低,分别为50.79%,0.48mg和85.79粒。取食平菇的黑粪蚊成虫寿命(3.0d)显著长于取食木耳(2.4d),取食银耳的成虫寿命居中。取食不同种类食用菌,对黑粪蚊蛹发育历期、羽化率和雌性比率影响不显著。取食平菇的黑粪蚊种群趋势指数达31.37,明显高于取食木耳(16.62)和银耳(13.49)。幼虫取食不同种类食用菌,其酸性磷酸酯酶和碱性磷酸酯酶的活力由高到低依次均为银耳>木耳>平菇,乙酰胆碱酯酶的活力由高到低依次为平菇>木耳>银耳。【结论】不同种类食用菌对黑粪蚊的生长发育、存活繁殖、磷酸酯酶和乙酰胆碱酯酶活力均有显著影响,其中平菇对黑粪蚊生长发育和繁殖最有利。     

Purification and characterization of phospholipase A2 from the pyloric caeca of red sea bream, Pagrus major

A phospholipase A₂ was purified 55,000-fold in a yield of 10% from the lipid-free extract of powder of the pyloric caeca of red sea bream to near homogeneity by sequential column chromatography on S-sepharose fast flow, butyl-cellulofine, Asahipak ES-502C cation-exchange HPLC, TSK gel G3000SW gel-filtration HPLC, and Asahipak ODP-50 reversed-phase HPLC. The final preparation showed a single band with the apparent molecular mass of 14 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and an estimated specific activity was 717 µmol min^-1 mg^-1 protein. The purified enzyme had a pH optimum in the range of pH 8.0–9.0 and required the presence of both 8 mM of Ca²⁺ and from 2 to 10 mM of sodium deoxycholate for its maximal activity, using 2 mM of phosphatidylcholine as a substrate. The purified enzyme preferentially hydrolyzed the 2-acyl ester bonds of both phosphatidylglycerol and phosphatidylcholine in the presence of sodium deoxycholate, followed in order by phosphatidylethanolamine and phosphatidyl-serine. In contrast to porcine pancreatic PLA₂, pyloric caeca PLA₂ hydrolyzed mixed-micellar phosphatidylcholine substrate effectively, regardless of the kinds of bile salts used. These results indicate that Ca²⁺-dependent low molecular mass PLA₂, so called secretory PLA₂, occurs in the pyloric caeca of red sea beam.