蛇足石杉内生真菌Shiraia sp. Slf14中赖氨酸脱羧酶基因的克隆、表达及功能

【目的】阿尔茨海默症治疗药物石杉碱甲(Huperzine A,Hup A)的生物合成途径起始于赖氨酸脱羧酶(Lysine decarboxylase,LDC)。本研究克隆及表达了来源于产Hup A的植物内生真菌的LDC基因,并研究了其功能。【方法】采用RT-PCR扩增法,从一株产Hup A的蛇足石杉内生真菌Shiraia sp.Slf14获得LDC基因,构建表达质粒p ET-22b-LDC与p ET-32a-LDC,转化感受态细胞E.coli BL21,加入IPTG至终浓度为1×10~(–3) mol/L,于24°C、200 r/min培养8 h,诱导表达LDC蛋白质;通过Ni~(2+)金属亲和层析纯化重组LDC并建立酶促反应体系,利用TLC检测了LDC催化活性。利用生物信息学软件分析了LDC的理化性质及蛋白质的空间结构。【结果】成功克隆并异源表达出重组蛋白LDC与Trx-LDC,经SDS-PAGE电泳鉴定分子量分别为24.4 k Da和42.7 k Da,与预计大小相符。TLC结果表明LDC与Trx-LDC均具有赖氨酸脱羧酶活性。【结论】本研究从产Hup A的蛇足石杉内生真菌Shiraia sp.Slf14中成功克隆到LDC基因并进行了异源表达,检测到了其催化活性,为丰富LDC分子信息及阐明内生真菌中Hup A生物合成机制提供参考数据。     

Evaluation of Phytochemical Compositions and Biological Properties of Achillea gypsicola at Different Phenological Stages

Phytochemicals, which are commonly found at different levels in many medicinal plants, are natural strong antioxidants used in traditional medicine. In this research, determination of differences of phytochemical compositions and biological properties were aimed as periodically (pre‐, full and post flowering) and daily (6 am, 1 pm and 8 pm) in Achillea gypsicola Hub.‐Mor . The volatile oils belonging to A. gypsicola were obtained by hydrodistillation and analyzed by gas chromatography‐flame ionization detection (GC‐FID) and gas chromatography‐mass spectrometry (GC/MS). The antimicrobial activities of the volatile oils were determined with disc diffusion method. The microdilution method was used to determine minimum inhibitory concentration (MIC). Total phenolic and flavonoid contents were determined by spectrophotometric methods and antioxidant capacities were evaluated by 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) free radical, reducing power (RP) and metal chelating activity (MCA) assay. In addition, the phenolic acid and flavonoid compositions were evaluated by reversed phase‐high‐performance liquid chromatography (RP‐HPLC). This study presented a comprehensive report for the first time on evaluation of the phytochemical composition and the biological properties of A. gypsicola at different phenological stages. Thirty‐two compounds, containing the major component as camphor, 1,8‐cineole and borneol, were detected. Designated harvest time for the highest yield of volatile oils was found to be at full flowering stage‐1 pm. It has been observed that the volatile oil composition changes periodically and even daily. Also, in this research, menthol and menthone were found as the composition of volatile oil in Achillea species for the first time. Full flowering stage was found as the richest period in terms of phenolic acid and flavonoid compositions of A. gypsicola for the first time. The species examined in this research showed a high antioxidant and antimicrobial activity in comparison to other studies with Achillea species. The volatile oils exhibited high performances with range of inhibition zones (8.3–42.3 mm) and minimum inhibitory concentration values (2.25—144 μg/ml). Besides, a high correlation between antioxidant activity and phenolic content of A. gypsicola was found. These results suggest that A. gypsicola can be used as a safe source in the cosmetic, food and pharmaceutical industries.     

Pyrethroids as Promising Marine Antifoulants: Laboratory and Field Studies

Due to the regulations and bans regarding the use of traditional toxic chemicals against marine fouling organisms and the practical impediments to the commercialization of natural product antifoulants, there is an urgent need for compounds that are antifouling-active, environmentally friendly, and have a potential for commercial application. In this study, a series of common, commercially available pyrethroid products, which are generally used as environmentally safe insecticides, was evaluated for antifouling activity in the laboratory using an anti-settlement test with cyprids of the barnacle Balanus albicostatus and also in a field experiment. Laboratory assay showed that all eleven pyrethroids (namely, rich d-trans-allethrin, Es-biothrin, rich d-prallethrin, S-prallethrin, tetramethrin, rich d-tetramethrin, phenothrin, cyphenothrin, permethrin, cypermethrin, and high active cypermethrin) were able to inhibit barnacle settlement (EC₅₀ range of 0.0316 to 87.00 μg/ml) without significant toxicity. Analysis of structure–activity relationships suggested that the cyano group at the α-carbon position had a significant influence on the expression of antifouling activity in pyrethroids. In the field, the antifouling activity of pyrethroids was further confirmed, with the most potent pyrethroids being cypermethrin and high active cypermethrin, which displayed efficiency comparable with that of tributyltin. In summary, our investigation indicated that these pyrethroids have a great and practical commercial potential as antifouling agents.     

酒精摄入量对小鼠肠道微生物、酶活性和血常规的影响

【背景】酒是影响机体健康的一把"双刃剑",饮酒对机体肠道微生态体系具有重要影响。【目的】研究不同酒精摄入量对小鼠肠道微生物、酶活性及血常规的影响,从肠道微生态和血常规角度探讨饮酒对身体健康的影响及作用机制。【方法】将SPF(Specific pathogen free)级实验小鼠随机分为对照组、低酒精量摄入组、中酒精量摄入组和高酒精量摄入组。对照组给予蒸馏水饮用,其余各组分别给予10%、20%和30%(体积比)的酒精水溶液作为小鼠的唯一饮用水,连续1个月后采集回肠内容物进行微生物和酶活性分析,采集眼球血进行血常规分析。【结果】与对照组相比,低酒精量摄入组小鼠肠道内乳酸菌数量显著增加(P0.05),大肠杆菌和细菌总数显著降低(P0.01或P0.05);高酒精量摄入组小鼠肠道乳酸菌、双歧杆菌数量显著降低(P0.01);与低酒精量摄入组和中酒精量摄入组相比,高酒精量摄入组小鼠肠道木聚糖酶、纤维素酶、蛋白酶和淀粉酶活性显著升高(P0.01);与对照组相比,低酒精量摄入组小鼠的红细胞比容显著降低(P0.05)。【结论】高酒精摄入量小鼠肠道有益菌群数量相对减少,肠道屏障功能受到影响;低酒精摄入量能调节小鼠肠道菌群结构和消化酶相对活性。     

Structural analysis of water-soluble polysaccharides in the fruiting body of Dictyophora indusiata and their in vivo antioxidant activities

The water-soluble Dictyophora indusiata polysaccharides (DIP) were extracted from the fruiting body of D. indusiata. The structural features of purified DIPs I and II were investigated. The results indicated that DIP I was composed of glucose (Glc) and mannose (Man) with molecular weight of 2100 kDa, while DIP II comprised of xylose (Xyl), galactose (Gal), glucose (Glc) and Man with molecular weight of 18.16 kDa. The glycosidic linkage of DIP I was composed of →1)-Glc-(6→: →1)-Man-(3,6→ with the ratio of 5.6:1.0, while DIP II was composed of →1)-Glc-(6→: →1)-Man-(3,6→: →1)-Xyl-(5→: →1)-Gal-(3→: →1)-Gal-(6→: with the ratio of 4.9: 15.5: 7.8: 1.0: 5.7. DIP significantly (P < 0.05) decreased the malondialdehyde (MDA), lipofuscin levels and increased the superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activities of mice. The strong in vivo antioxidant activity indicated DIP had great potential as functional food.     

Bioactivities of water-soluble polysaccharides from fruit shell of Camellia oleifera Abel: Antitumor and antioxidant activities

Polysaccharides were extracted from fruit shell of Camellia oleifera Abel. Fruit shell of Camellia oleifera Abel polysaccharide (WEP2) was a water-soluble compound. Its molecular weight was about 362 kDa. HPLC analysis showed that this polysaccharide was composed of rhamnose, fucose, arabinose, mannose, galactose and glucose in the molecular ratio of 4.05, 11.62, 1.78, 3.91, 8.76 and 27.06, respectively. The broad intense characteristic peak around 3463 cm⁻¹ due to the hydroxyl stretching vibration of the polysaccharide was observed in the polysaccharide. The characteristic absorption bands at 852 cm⁻¹ and 893 cm⁻¹ indicated that WEP2 contained both α-glycosidic and β-glycosidic linkages. WEP2 exhibited remarkable antitumor activity against Sarcoma180 cell compared to the negative control group. At the highest dose 40 mg/kg days, the tumor inhibition rate reached 65.2%. The scavenging effects of WEP2 to hydroxyl radical and superoxide radical anion were 72.5% and 86.3% at a concentration of 1.0 mg/ml, respectively.     

镉胁迫对家蚕脂肪体脂质过氧化物含量及抗氧化酶活性和mRNA表达的影响

【目的】探讨鳞翅目模式昆虫家蚕Bombyx mori作为重金属污染的监测指示生物在镉胁迫下的酶反应及相关的基因表达。【方法】给家蚕幼虫期全龄添食镉(Cd²⁺), 调查不同性别家蚕5龄幼虫脂肪体中脂质过氧化物丙二醛（MDA）的含量, 超氧化物歧化酶（SOD）、 过氧化氢酶（CAT）和谷胱甘肽过氧化物酶（GSH-Px）的活性及其基因表达水平的变化。【结果】Cd²⁺胁迫对雌雄家蚕MDA 含量均具有浓度效应关系, MDA含量随Cd²⁺胁迫浓度的升高而增加。Cd²⁺胁迫下, SOD和CAT活性表现为先升后降的变化趋势, Pearson相关性分析显示SOD和CAT活性变化有显著相关性(雄: R=0.770, P=0.001; 雌: R=0.854, P=0.000）。雌性家蚕脂肪体中CAT活性变化和Cat mRNA水平的表达具有正相关性（R=0.712, P=0.003）。雄性家蚕脂肪体中GSH-Px活性随Cd²⁺胁迫浓度的升高而增加, 显示浓度 效应关系, 12.5~50 mg/kg Cd²⁺胁迫组GSH-Px活性与对照相比有显著差异（P<0.05）, 其活性和GSH-Px mRNA水平的表达具有正相关性（R=0.834, P=0.000）; 雌性家蚕脂肪体中GSH-Px活性表现为先升后降的变化趋势, 12.5 mg/kg Cd²⁺胁迫组GSH-Px活性与对照相比有显著增加（P<0.01）。【结论】结果表明, 急性镉胁迫对家蚕脂肪体有明显的毒性作用, 其作用机制与脂质过氧化加剧和抗氧化酶活性变化有关。家蚕对重金属镉的解毒机制有性别相关性。     

Topoisomerase I‐Mediated Antiproliferative Activity of 10‐Substituted and 12‐Substituted Homocamptothecins

Homocamptothecin (hCPT) is an E‐ring modified camptothecin (CPT) analogue, which showed pronounced inhibitory activity of topoisomerase I. In search of novel hCPT‐type anticancer agents, two series of hCPT derivatives were synthesized and evaluated in vitro against three human tumor cell lines. The results indicated that the 10‐substituted hCPT derivatives had a considerably higher cytotoxic activity than the 12‐substituted ones. Among the 10‐substituted compounds, 8a, 8b, 9b , and 9i showed an equivalent or even more potent activity than the positive control drug topotecan against the lung cancer cell line A‐549. Moreover, the hCPT analogues 8a and 8b exhibited a higher topoisomerase I inhibitory activity than CPT at a concentration of 100 μM .     

Purification and Structural Characterization of Dendrobium officinale Polysaccharides and Its Activities

In this study, Dendrobium officinale polysaccharide (named DOPS-1) was isolated from the stems of Dendrobium officinale by hot-water extraction and purified by using Sephadex G-150 column chromatography. The structural characterization, antioxidant and cytotoxic activity were carried out. Based on the results of HPLC, GC, Congo red experiment, together with periodate oxidation, Smith degradation, SEM, FT-IR, and NMR spectral analysis, it expressed that DOPS-1 was largely composed of mannose, glucose and galacturonic acid in a molar ratio of 3.2 : 1.3 : 1. The molecular weight of DOPS-1 was 1530 kDa and the main chain was composed of (1→4)-β-D-Glcp, (1→4)-β-D-Manp and 2-O-acetyl-(1→4)-β-D-Manp. The measurement results of antioxidant activity showed that DOPS-1 had the strong scavenging activities on hydroxyl radicals, DPPH radicals and superoxide radicals and the high reducing ability in vitro. Moreover, DOPS-1 was cytotoxic to all three human cancer cells of MDA-MB-231, A549 and HepG2.     

Anti-cancer study and whey protein complexation of new lanthanum(III) complex with the aim of achieving bioactive anticancer metal-based drugs

In this study, a new lanthanum (III)-amino acid complex utilizing cysteine has been synthesized and characterized. The anticancer activities of the prepared La(III) complex against MCF-7 cell lines were studied. Results of MTT assay showed that at all three incubation times, the cytotoxic effect of prepared La(III) complex on MCF-7 breast cancer cell lines displays a time- and dose-dependent inhibitory effects. The interactions of the La(III) complex with two whey proteins (bovine serum albumin, BSA, and Bovine β-lactoglobulin, βLG) have been explored by using spectroscopic and molecular dicking methods. The obtained results indicated that La(III) complex strongly quenched the fluorescence of two carrier proteins in static quenching mode and also, BSA hah stronger binding affinity toward studied complex than βLG whit binding constant values of K_{BSA-La?Complex}?～?0.11?×?10⁴ M^?1 and K_{βLG-La?Complex}?～?0.63?×?10³ M^?1 at 300 K. The thermodynamic parameters revealed the contribution of hydrogen bond and Vander Waals interactions in both systems. The distances of the La(III) complex whit whey proteins were calculated using Förster energy transfer theory and proved existence of the energy transfer between two proteins and prepared La(III) complex with a high probability. FT-IR and UV–Vis absorption measurements indicated that the binding of the La(III) to BSA and βLG may induce conformational and micro-environmental changes of the proteins. The docking results indicate that the La(III) complex bind to residues located in the site II of BSA and second site of βLG.

Communicated by Ramaswamy H. Sarma