用核糖体工程技术二次开发海洋微生物菌株资源的研究

经对无活性的海洋来源放线菌B3054进行链霉素抗性筛选,获得了一株具有抗肿瘤活性的链霉素抗性突变株SY-1.从该突变株的发酵物中分离得到了4个活性化合物,其中1个为新天然产物.对该菌株突变前后的rpsL基因(编码核糖体蛋白S12)进行分析未发现发生突变,提示突变位点可能在核糖体的另外亚基上.实验结果表明,核糖体工程技术可用于无活性菌株资源的二次开发利用.     

海洋天然产物防污研究进展

多种海洋天然产物都具有防污活性 ,将其开发为高效无毒的防污涂料已成为防污技术发展方向之一。本文综述了海洋植物、海洋动物及海洋微生物中天然产物的防污活性 ,并对海洋天然产物防污研究进行了展望     

海洋链霉菌M518产的新星形孢菌素衍生物的生物活性

从海洋放线菌M518中分离到1个新结构化合物N-甲酰胺基-星形孢菌素(N-carboxamido-staurosporine) (1c),以及两个同类型已知化合物星形孢菌素(staurosporine)(1a)和N-甲酰基-星形孢菌素(N-formyl-staurosporine)(1b).为了解新化合物的生物活性,对37株人类肿瘤细胞和9种受试微生物细胞株进行了抑制活性筛选,发现化合物1c具有强烈的抗肿瘤活性,对37株肿瘤细胞的平均抑制浓度IC50,IC70和IC90分别为0.016、0.171和2.352μg·mL-1.其活性强于专利化合物1b.通过测定发现,分离到的三种孢菌素类化合物都具有较好的抗微藻活性.菌株M518经16S rRNA系统进化学分析,属于链霉菌属(Streptomyces sp.).     

一株产香兰素的海洋放线菌的筛选及发酵条件的研究

本文以漳州古雷半岛潮间带沉积物作为研究对象,筛选出能够发酵代谢产香兰素的高产菌株。为海洋放线菌发酵代谢进入工业化生产做出理论依据。     

微生物源食品保鲜剂的研究进展

目的 介绍近5年微生物源食品保鲜剂的研究进展,为研究高效、无毒、天然的食品保鲜剂提供理论和方法依据.方法 综述常见的微生物源食品保鲜剂,包括细菌源保鲜剂、真菌源保鲜剂和微生物代谢产物保鲜剂(乳酸链球菌素、￡-聚赖氨酸、溶菌酶和纳他霉素).简要说明其抑菌机理和存在的问题.结果 微生物源保鲜剂可以通过竞争营养,诱导系统抗性和产生活性代谢产物等方式抑制多种致病菌的生长繁殖,降低果蔬病害的发生率,保持食品良好的感官品质和理化特性,有效延长食品货架期.结论 微生物源保鲜剂为食品保鲜贮藏提供了新途径.其抑菌机理和潜在毒性需进一步明确,如何提高微生物源保鲜剂抗不良环境的稳定性还有待进一步研究.     

利用DNA重组技术构建产生多聚甘露糖醛酸的突变菌株

本文旨在以海洋细菌Pseudomonas sp.QDA褐藻多糖(alginate)生物合成操纵元中的algG基因为靶点,利用DNA重组技术改建褐藻多糖的生物合成途径,以期获得产生甘露糖醛酸的突变菌株.首先,利用PCR克隆algG基因两侧大小分别为1.7kb和1.8kb的DNA片段作为同源片段,用编码Gm抗性的aacC1基因替换algG基因,构建成打靶载体pEX100TUDG.将打靶载体转入QDA,利用Gm抗生素和蔗糖为选择压力进行培养,通过PCR和Southern杂交验证,获得了突变菌株.1H-NMR、PAGE和GPC分析发现,突变菌株胞外产物为多聚甘露糖醛酸.该突变菌株的获得丰富了具有较高药物开发价值的甘露糖醛酸的来源,为褐藻多糖途径工程的进一步研究提供了理论依据和技术支持.     

赖氨酸高产菌株的选育

赖氨酸作为一种重要的饲料用氨基酸,需求量一直在不断增长。传统的赖氯酸生产菌株都是多年来是经过多轮随机突变和筛选得到,而近年来随着基因重组技术的发展及对生物代谢过程的了解,人们已经能够通过基因重组技术,改变代谢途径,提高赖氨酸产量。目前有不少成功将野生菌株改造为高产菌株的案例,他们都可以作为合理设计代谢途径并结合各种组学进行微生物代谢途径改造的基础。本文主要描述通过代谢途径改造并结合高通量筛选技术,快速得到赖氨酸高产菌株的方法。     

海洋产蛋白酶细菌发酵液防污活性研究

传统的防污剂对海洋环境造成严重污染,随着环保意识的增强以及相关规定的制订,各国竞相开展新型无毒防污剂的研究。本文以海洋产蛋白酶菌株发酵产物为活性物质,研究蛋白酶粗提物对污损生物硅藻(navicula sp.)和贻贝(mytilus edulis)附着行为的影响。结果表明,所研究菌株的发酵液对硅藻(navicula sp.)和贻贝(mytilus edulis)的附着有明显抑制作用。贻贝(mytilus edulis)毒性实验显示,细菌发酵液对贻贝无毒。因此,海洋微生物蛋白酶产生菌粗酶提取物可以作为环保型防污功能添加剂。     

膜电位调控棕色固氮菌表达新生理特性的研究

膜电位对棕色固氮菌整体细胞固氮活性有调控作用。外加扫描电位能调控缺固氮酶活性中心、无固氮活性的棕色固氮菌突变菌株 CA30表达略高于天然棕色固氮菌的固氮活性。     

维胺酸的癌化学预防作用研究

维按酸（RⅡ）是中国医学科学院药物研究所研制的Ⅰ类新药。多中心临床观察证明,其对口腔白斑,胃和宫颈上皮不典型增生,外阴白色病损等癌前疾病具较好治疗作用。临床前研究证实维胺酸能明显抑制多种致癌物诱发的突变和减轻染色体损伤,可以阻遏TPA类物质对鸟氨酸脱羧酶（ODC）活性的诱导,降低蛋白激酶C（PKC）活性,具有明显抗致突变、抗促癌作用。在一组由亚硝胺、DMBA诱发的动物口腔、胃、皮肤及膀胱癌及癌前病变的实验中,显示出它有明显抗化学致癌作用。RⅡ可以影响多种肿瘤细胞增殖,显示分化诱导作用。电镜组化、细胞周期分     

Scattering properties of microalgae: the effect of cell size and cell wall

The main objective of this work was to investigate how the cell size and the presence of a cell wall influence the scattering properties of the green microalgae Chlamydomonas reinhardtii. The growth cycle of two strains, one with a cell wall and one without, was synchronized to be in the same growth phase. Measurements were conducted at two different phases of the growth cycle on both strains of the algae. It was found that the shape of the scattering phase function was very similar for both strains at both growth phases, but the regular strain with a cell wall scatters more strongly than the wall-less mutant. It was also found that the mutant strain has a stronger increase in scattering than the regular strain, as the algae grow, and that the scattering from the regular strain is more wavelength dependent than from the mutant strain.     

海洋有益菌的筛选与鉴定

从健康的斑节对虾（Penaeus monodon）幼体及其养殖环境中分离到213株海洋细菌,采用十字叉划线法和琼脂扩散法进行体外拮抗试验,从中筛选到5株对水产养殖动物病原哈维氏弧菌（Vibro harueyi）和鳗弧菌（Vibrio anguillarum）等菌株生长有抑制作用的海洋细菌。根据其形态特征以及生理生化反应特征,初步鉴定为交替单胞菌属（Alteromonas）,其中菌株A18鉴定为橙色交替单胞菌（Alteromonas aurantia）。     

Antimicrobial Activity of Cinnamaldehyde and Eugenol and Their Activity after Incorporation into Cellulose‐based Packaging Films

Cinnamaldehyde and eugenol were investigated for their antimicrobial activity against 10 pathogenic and spoilage bacteria and three strains of yeast, using an agar‐well diffusion assay. The minimum inhibitory concentrations (MICs) of these compounds were determined using an agar dilution method. Finally, cinnamaldehyde‐incorporated and eugenol‐incorporated methyl cellulose films were prepared to obtain active antimicrobial packaging materials. These antimicrobial cellulose‐based packaging films were investigated for antimicrobial activity against target microorganisms using both an agar‐disc diffusion technique and a vapour diffusion technique. At a concentration of 50 µl/ml, cinnamaldehyde and eugenol revealed antimicrobial activity against all test strains. They showed zones of inhibition, ranging from 8.7 to 30.1 mm in diameter. Eugenol and cinnamaldehyde possessed ‘moderate?strong inhibitory’ and ‘strong?highly strong inhibitory’ characteristics, respectively. With MICs of 0.78?50 µl/ml, cinnamaldehyde and eugenol also inhibited the growth of all test microorganisms. Among the test microorganisms, Aeromonas hydrophila and Enterococcus faecalis were the most sensitive to cinnamaldehyde and eugenol. Cinnamaldehyde showed lower MICs against all test strains than those of eugenol. In an agar‐disc diffusion assay, cellulose‐based film containing cinnamaldehyde or eugenol totally failed to exhibit a clear inhibitory zone. However, it showed positive activity against all selected test strains in terms of size and enumeration of microbial colonies in a vapour diffusion assay. This study shows the potential use of cinnamaldehyde and eugenol for application in antimicrobial packaging film or coating. Copyright © 2011 John Wiley & Sons, Ltd.     

1,3-dimethyl-6-nitroacridine derivatives induce apoptosis in human breast cancer cells by targeting DNA

The acridine derivatives can interact with the double-stranded DNA, which is regarded as the biological target of the anticancer drugs in cancer treatment. We designed and synthesized a new series of 1,3-dimethyl-6-nitroacridine derivatives as potential DNA-targeted anticancer agents. These compounds could partially intercalate into the calf thymus DNA, differing from the parent acridine. The results showed that the substitutions of the acridine ring had great effect on DNA binding affinity. The binding constants determined by UV-vis spectroscopy were found to be 10⁵?M^?1 grade. Anticancer activity of these compounds was screened using MTT assay. Most compounds inhibited 50% cancer cell growth at concentration below 30?μM, the results were consistent with the DNA binding ability. Compounds 1 and 6 were found to have more effective cytotoxicity, especially in human breast cancer cell lines. To investigate the action mechanism, we studied cell apoptosis, morphological changes, and cell cycle distribution in MCF-7 and MDA-MB-231 cells. Compounds 1 and 6 caused MCF-7 and MDA-MB-231 cells death due to apoptosis, and induced cell apoptosis in a dose-dependent manner. They also had significant effect on cell cycle progression and arrested cell cycle at G2/M phase. The results demonstrated that compounds 1 and 6 are promising candidates for cancer treatment.     

Anti Cervix Cancer Activity of Co-immobilized Tumor Necrosis Factor-α and Interferon-γ

Tumor necrosis factor α （TNF-α） and interferon-γ （IFN-γ） are cytokines with strong antitumor activities. They were reacted with a photoactive arylazide-4-azidobenzoic acid, resulting in photoactive TNF-α and IFN-γ. The infrared （IR） spectra of these products showed the characteristic absorption of an azido group at 2127 cm^-1. By photo-immobilization, this modified TNF-α and IFN-γ were immobilized on polystyrene membranes for cell culture to prepare biomaterials. The micro-morphology of photoactive cytokines was observed with a scanning electron microscope （SEM）. The inhibitory effect on growth of Hela cells and inducing apoptosis activity of these two cytokines were analyzed by growth curve, transmission electron microscope （TEM） and fluorescence active cell sorter （FACS）. The results showed that co-immobilization of IFN-γ and TNF-α had significant inhibitory effect on growth of Hela cells, inhibitory rate up to 82%, and IFN-γ had obviously synergistic action.     

FLT3-specific curcumin micelles enhance activity of curcumin on FLT3-ITD overexpressing MV4-11 leukemic cells

Curcumin, a major active compound in the turmeric rhizome, has many biological properties, especially anti-leukemia activity. The overexpression of FMS-like tyrosine kinase 3 protein with internal tandem duplication (FLT3-ITD) mutation protein was related to the poor prognosis and disease progression of leukemia. In this study, the cytotoxicity and inhibitory effect of curcumin on cell cycle of FLT3-ITD overexpressing MV4-11 leukemic cells were evaluated. Moreover, curcumin polymeric micelles conjugated with FLT3-specific peptide (FLT3-Cur-micelles) were prepared using a film hydration method to increase curcumin solubility and the inhibitory effect on MV4-11 cells was evaluated. Cytotoxicity and cell cycle analysis were performed using an MTT assay and flow cytometry, respectively. Physical properties of FLT3-Cur-micelles, including particle size, size distribution, morphology, and entrapment efficiency (EE), were evaluated. Cellular uptake of the micelles on MV4-11 cells was determined by flow cytometry and fluorescence microscopy. FLT3-Cur-micelles were observed with size less than 50?nm and high EE of >75%. In addition, FLT3-Cur-micelles demonstrated excellent internalization and increased curcumin accumulation in leukemic cells when compared to free curcumin. Furthermore, FLT3-Cur-micelles exhibited a strong cytotoxic effect on MV4-11 cells with IC₅₀ value of 1.1?µM, whereas the blank micelles showed no effect. Furthermore, FLT3-Cur-micelles showed no significant effect on normal human PBMCs with IC₅₀ value >25?µM. In summary, FLT3-Cur-micelles are a promising nanocarrier system for enhancing anti-leukemic activity of curcumin and suitable for further preclinical studies.     

Comparison in purity and antitumor effect of brand and generic paclitaxel against human ovarian cancer cells by an in vitro experimental model

Context: The purity and the therapeutic effectiveness of the generic paclitaxel have not yet been examined and compared to the original brand form. Objective: This study aimed to compare the in vitro purity and biological effects of original brand form (Taxol) and a generic drug of paclitaxel. Materials and Methods: Purity was determined by high-performance liquid chromatography analysis, cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay, cell proliferation by clonogenic assay, morphology by Liu's staining, and cell cycle distribution by DNA histogram. Results: Taxol and generic paclitaxel shared similar high-performance liquid chromatography profiles with a major peak at the same retention time and ultraviolet spectrum. Generic paclitaxel inhibited the cell viability to an extent greater than Taxol. By assessing the IC₅₀, generic paclitaxel also exhibited a greater inhibitory activity on clonogenicity of human ovarian adenocarcinoma SKOV-3 cells. Although both generic paclitaxel and Taxol arrested SKOV-3 and ES-2 cells at G2/M phase with concurrent development of hypoploid and polyploid cells, Taxol treatment exhibited markedly less extent of these changes. Observation of cellular morphology revealed a greater amount of mitotic catastrophe-like and apoptotic cells in generic paclitaxel-treated cells than Taxol-treated cells. Discussion and Conclusion: The results suggest that generic paclitaxel may possess a greater cell death inducing capacity and clonogenicity inhibitory activity against ovarian cancer cells than the original brand Taxol of the same purity. We conclude that this experimental model for assessing the difference between generic and brand name drugs might be considered as a reference while determining their interchangeability and could be easily established in a hospital-based laboratory.     

鳗弧菌溶血素基因的克隆及突变株的构建

PCR扩增出鳗弧菌M3溶血素基因的保守区序列,根据保守区序列设计引物,利用反向PCR和巢式PCR扩增出溶血素基因的部分序列,结合构建的鳗弧菌M3的基因组文库,克隆出溶血素基因的全长序列。根据基因推测的氨基酸编码序列与已发表的血清型为A的鳗弧菌PT84057有86％的相似性。用自杀质粒对鳗弧菌M3染色体上的溶血素基因进行了插入突变,将突变株对金鱼进行肌肉注射,结果发现,突变株的毒力没有发生显著的变化,证明所克隆的溶血素基因对鳗弧菌M3的毒力没有直接的贡献。     

Analytical profiling of biosynthetic intermediates involved in the gentamicin pathway of Micromonospora echinospora by high-performance liquid chromatography using electrospray ionization mass spectrometric detection

In the present study, we developed a sensitive and highly selective method of detecting the biosynthetic intermediates involved in the gentamicin pathway from a cell culture of Micromonospora echinospora. A novel extraction method utilizing a dual solid-phase extraction (SPE) technique was employed to purify and recover all of the gentamicin-related components from the cell culture broth, and high-performance liquid chromatography (HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS/MS) was used to analyze the extractant for gentamicin intermediates. The pH of the culture broth was adjusted to an acidic condition of pH 2 prior to the extraction. The samples were first cleaned with a reversed-phase AccuBOND C(18) cartridge, and then the aminoglycosidic components were purified using a cationic exchanger OASIS MCX cartridge. The detection limit of a gentamicin standard spiked in blank medium processed by this method was found to be approximately 5 ng for each component of the gentamicin C complex, and the mean recovery for each component of standard gentamicin was above 91% when analyzed by HPLC-ESI-MS/MS. We further demonstrated that this method enables the analytical profiling of the gentamicin-related compounds produced by wild-type M. echinospora ATCC 15835, which mainly produces the gentamicin C complex, and the UV-induced mutant strain KCTC 10506BP, which produces gentamicin B as the major product. Seven intermediates (paromamine, gentamicin A2, B, X2, A, JI-20A, and JI-20B) besides the gentamicin C complex were detected in the culture broth of both M. echinospora strains when analyzed by MS/MS for the distinct fragmentation patterns of each gentamicin component. This report displays the first example of the HPLC profiling in a wide range of structurally related biosynthetic intermediates involved in the gentamicin pathway.     

Steady ratcheting strains accumulation in varying temperature fatigue tests of PMMA

The evolutions of ratcheting strains of polymethyl methacrylate (PMMA) at different temperatures and stress levels were experimentally investigated. A steady ratcheting strain growth region with a constant rate was observed in all specimens, which occupied significant part of total fatigue failure life. Experimental results also showed that the steady ratcheting growth rate varied with applied temperatures and loading. In this paper, theory of thermally activated process for glassy polymers was used to describe the plastic deformations during the cycle. Based on the correlations between ratcheting strains per cycle and hysteresis loop energy, a new ratcheting strains accumulative model for polymer materials was developed, which quantificationally elucidated the effects of temperature, loading frequency, mean stress and stress amplitude on the accumulative rate of ratcheting strains. Comparing the predications from the proposed model with experimental ratcheting strain data of PMMA, it was found that the model could describe the steady ratcheting strain accumulative behaviors under arbitrary temperatures and loading conditions exactly.