用于丝状真菌的苯菌灵标记基因benA表达载体的构建

为了构建适用于丝状真菌遗传转化的表达载体,在质粒pAN52-1的高效组成型三磷酸甘油醛脱氢酶基因启动子PgpdA和色氨酸C基因终止子TtrpC之间加入内切酶NotⅠ和ClaⅠ两个位点,构成质粒pAN52-2;把质粒pAN52-2中从启动子PgpdA到终止子TtrpC的基因片段通过PstⅠ和XbaⅠ酶切位点导入到pUC19载体上,并且应用PCR的方法在启动子Pg-pdA的前端引入HindⅢ酶切位点,构成质粒pUCPT-2;通过PCR,在苯菌灵抗性基因benA两端分别加入内切酶NotⅠ和ClaⅠ酶切位点,并克隆到pGEM-T载体上,得到质粒pGEM-Ben;用NotⅠ分别酶切质粒pUCPT-2与pGEM-Ben,连接苯菌灵抗性基因benA到pUCPT-2上,得到了pUCPT-Ben载体。经PCR、酶切和核苷酸序列检测,证实了PgpdA-benA-TtrpC表达元件连接成功,即得到了苯菌灵抗性基因高效表达载体。

马尔尼菲青霉菌内源启动子驱动的苯菌灵抗性基因盒构建及其应用

马尔尼菲青霉菌是一种区域性流行于东南亚及中国南部地区的条件致病性温度依赖性双相型病原真菌,它能对免疫功能低下者造成致命的全身性感染,25℃时呈现菌丝状生长,而在37℃或在宿主体内为酵母相生长。为深入研究马尔尼菲青霉菌致病性和双相转化的分子机制,原有的遗传转化筛选标记基因已不能满足研究需要。为此,我们通过用马尔尼菲青霉菌微管蛋白β亚基基因启动子替换粗脉孢菌苯菌灵抗性基因的启动子,构建了一个苯菌灵抗性基因盒,并成功地将其运用于马尔尼菲青霉菌的遗传转化研究中。

Novel Stilbene Glycosides from Polygonum multiflorum

Two new stilbene glycosides (1 and 2), together with nine known compounds (3-11), were isolated from the water extract of Polygonum multiflorum Thunb. The structures of the new compounds were elucidated by their chemical properties and spectroscopic analyses, including extensive 2D NMR experiments. Compound 2 showed strong DNA cleavage activity, and compounds 1, 2 and 10 (2, 3, 4′, 5_tetrahydroxy_ trans _stilbene_2_O_β_ D _glucopyranoside) exhibited significant inhibition of lipid peroxidation.

Structure, properties and application to water-soluble coatings of complex antimicrobial agent Ag-carboxymethyl chitosan-thiabendazole

The structure, properties and application to water-soluble coatings of a new complex antimicrobial agent Ag-carboxylmethyl citosan-thiabendazole （Ag-CMCTS-TBZ） prepared from different materiel ratios were reported. The silver ions were preferably coordinated with the free-NH2 groups and the -OH groups of secondary alcohol and carboxyl in CMCTS. TBZ preferably bonded to carboxyl group in CMCTS by electrostatic force and hydrogen bonding. Increase in silver ions content in the complex agent improved to some limited extent the antibacterial activity, but enhanced coloring and cost of the complex agent. Increase in TBZ content resulted in increase of antifungal activity, but decrease of water solubility of the complex agent. The antimicrobial MICs of the complex agent to Esherichia coil, Staphylococcus aureus, Candida albicans, Aspergillus niger, Mucor sp. were 20 -80, 15 -60, 20 - 55, 40 - 250, and 400 - 1700 mg/kg, respectively. Addition of 0.1% of this complex agent to acrylic emulsion paint made the paint without substantial change in color, luster, viscosity, odor or pH value, but with an excellent and chronically persisting broad-spectra antimicrobial activity.

Synthesis and antitumor activities of 2-(E)-(4-cyclopentyloxy-3-methoxylbenzylidene)cyclopentanone derivatives

Aim To design and synthesize a series of 2-（E）-（4-cyclopentyloxy-3-methoxylbenzylidene）cyclopentanone derivatives, and to determine their antitumor activities in vitro. Method The target compounds were synthesized. Their antitumor activities were assayed using human hepatic carcinoma ceil line （Bel-7402） and human oral cavity epidermis squamoceilular carcinoma cell line （KB）. Results Five compounds were obtained. Three of them were not reported in the literature and their chemical structures were confirmed by IR, ^1H NMR, MS and elemental analysis. Preliminary screening results showed that compound 5 possessed better biological activity with IC50 1.62 μmol·L^-1 against Bel-7402 and 8.04 μmol·L^-1 against KB, but much weaker than 5- Fluorouracil. Conclusion Mannich base derivatives of 2-（E）-（4-cyclopentyloxy-3-methoxylbenzylidene）cyclopentanone exhibited some antitumor activities.

7种冬青对苯气体胁迫的生理响应

为揭示7个冬青品种对苯气体胁迫的生理响应,筛选出抗苯气体污染的种质资源,采用人工模拟苯气体胁迫的方法进行试验。研究表明:随着苯气体胁迫时间的延长,7个冬青品种的质膜透性、叶绿素a/b、MDA含量和脯氨酸含量均呈上升趋势,可溶性蛋白含量呈下降趋势,SOD活性和POD活性基本呈先上升后下降的趋势。7个冬青品种扦插苗中‘兰少女’冬青和大别山冬青耐苯性强;钝齿冬青、刺叶冬青和全缘冬青次之;‘金宝石’冬青和冬青抗苯性差。

Relationship between Ethanol and 2-phenylethanol Stress Tolerance and Fatty Acid Compositions of Saccharomyces cerevisiae

In the present study, we investigated of the ethanol （Eth）, 2-phenylethanol （2-PE） and ethanol/2-phenylethanol （Eth/2-PE） stress tolerance and established relationship between stress tolerance and fatty acid compositions of wine yeast strain S. cerevisiae UCM Y-524 in relation to different cultivation factors. Changes in cell membrane fatty acid profiles studied under different temperature and media. The active oxygenation, semi-aerobic cultivation supplemented Tween 80 conditions were used in different combinations. The concentrations of 2-PE and Eth, the fatty acids methyl esters （FAMES） in the media were measured by GC-MS analyses. 2-PE, Eth, FAMES were identified by NIST02 MS database at the 2-PE and Eth standard solution （Merck, Germany）, bacterial FAMES standard （Supelco）. Palmitoleic and oleic acid were dominated for S. cerevisiae UCM Y-524. The unsaturated/saturated fatty acid （UFA/SFA） ratio was higher than five for S. cerevisiae UCM Y-524 at 14 ~C in simple medium and about more than three for other conditions. It has been found that yeast cells grown in the presence of the 2-PE, Eth and Eth/2-PE appear to increase the amount of unsaturated fatty acids, especially oleic acid, in cellular lipids especially under active oxygenation supplemented with Tween 80. S. cerevisiae UCM Y-524 has a higher tolerance under 2-PE, Eth and Eth/2-PE stress at 28 ~C in a simple medium under aerobic conditions. A higher concentration of unsaturated fatty acids in the membrane S. cerevisiae UCM Y-524 and a lower temperature under aerobic conditions, has been considered to have a positive influence on the excretion of 2-phenylethanol.

Improvement in Oxidative Stability of Soybean Oil by Mango Kernel Extracts

This work was undertaken to explore the potential of fruit waste materials as sources of natural antioxidants. Kernels of four varieties of mangoes of Indian origin were studied. Kernels were extracted with five different solvents, tested for extraction efficiency and total phenolic content （TPC）. As the methanol proved to be the best solvent, it was used for further analysis. The methanolic extracts of kernels had reducing power （RP） and scavenged 2, 2 diphenyl-l-picryl hydrazyl （DPPH） radical, which is related to their antioxidant activity （AA）. When analyzed in HPLC, the extracts showed the presence of phenolic compounds. The extracts had the higher capacity to reduce the formation of peroxides and slow down the rate of oxidation than butylated hydroxyl toluene （BHT） in refined, bleached deodorized soybean oil （RBD SBO）. Based on the results obtained mango kernels are potential source of natural antioxidants owing to their antioxidant activity.

Synthesis and antimicrobial activity of 2-（3＇, 5＇-disubstituted-indoly-2＇-yl）-4H-3, 1-benzoxazin-4-ones and their derivatives

As benzoxazin-4-ones and quinazolines linked to indole nucleus acting as a good pharmacophore, the synthesis of these compounds has significant meaning. The key intermediates 2-（2＇, 5＇-disubstituted-indol-2＇-yl）-4H-3, 1-benzoxazin-4-ones （3） were synthesized from cyclocondensation of indole-2-carbonyl chlorides （2） and anthranilic acid. Compound （3） on reaction with thiosemicarbazide and o-phenylene diamine afforded the compound （4） and （6） respectively. Compound （4） subjected to intramolecular cyclization under thermal conditions above its melting point afforded the compound （5）. Similarly compound （3） on fusion with o- phenylene diamine gave compound （7）. Structures of these compounds were confirmed by their spectral studies. The compounds were screened for their antimicrobial activity and the results were reported.

DFT Study of the Solvent Effects on the Structure, UV-Vis Spectra and the Antioxidant Activity of Caffeic Acid Phenethyl Ester and Some of Its Derivatives

In this study, the antioxidative （3-methyl-2-butenyl caffeate）, BC efficiency of CAPE （caffeic acid phenethyl ester） and four of its derivatives （MBC （benzoic caffeate）, P3HC （phenethyl-3-hydroxy-cinnamate） and P4HC （phenethyl-4-hydroxy-cinnamate）） are compared in vacuum and in seven solvents. It turned out that the AA （antioxidant activity） in increasing order was P3HC 〈 P4HC 〈 CAPE 〈 MBC. Effects of solvents on the structure and the antioxidant activity of P3HC, P4HC, BC, MBC and CAPE, were studied at 133LYP/6-31G （d, p） then B3LYP/6-3 I＋G （d, p） level of theory using the conductor polarized continuum model methods. Thermodynamically, the authors showed that solvent effects on bond dissociation enthalpy are very weak （within 25 kJ/mol）, but sufficient to influence hydrogen bonds, O-H bond lengths and showed the preferential sites of hydrogen atom cleavage. In addition, solvent notably influences and changes the nature of the scavenging process of ROS （reactive oxygen species）, favouring by this way the HHAT （homolytic hydrogen atom transfer） in non polar solvents, the SPLET （sequential proton loss electron transfer） in polar solvents. Moreover, in chloroform and for the five molecules studied the SET-PT （sequential electron transfer proton transfer） mechanism is preferred compared to the HHAT, because in this solvent the IP is lower than the BDE. TD-DFT calculations revealed that solvent induce a bathochromic effect （red-shift of the wavelengths） coupled to hyperchromic or hypochromic effects.

Phenol Composition and Susceptibility to Fusarium oxysporum Dianthi in Carnation

Phenol composition of two carnation （Dianthus caryophyllus） cvs ＂Gloriana＂ and ＂Roland＂, partially and highly resistant to F. oxysporum f. sp. dianthi, respectively, has been investigated with the aim of assessing the possible role of phenolic constituents both as biochemical markers of resistance and antifungal molecules. Analyses were performed through chromatography, NMR and MS methods. The results indicate that protocatechuic and vanillic acids are present in healthy and infected tissues of both cvs; the flavonol glycoside peltatoside. 2,6-Dimethoxybenzoic acid was detected in small amounts only in infected ＂Gloriana＂ cv while the highly resistant ＂Roland＂ cv contained the flavone datiscetin. This latter compound, due to its noticeable antifungal activity evidenced in in vitro trials, could be conceived both as a biochemical marker of resistance towards this pathogen and as a phytoanticipin.

In vivo Anticancer Activities of Benzophenone Semicarbazone against Ehrlich Ascites Carcinoma Cells in Swiss Albino Mice

Objective Benzophenone semicarbazone(BSC) was synthesized and characterized to identify compounds with anticancer activities. Methods Anticancer activities were studied against Ehrlich Ascites Carcinoma(EAC) cells in Swiss albino mice by monitoring parameters such as tumor weight measurement,survival time of tumor bearing mice,tumor cell growth inhibition,and so on.Some hematological parameters,such as red blood cells,white blood cells,and hemoglobin content,were also measured. Results The results showed that BSC has a positive effect against EAC cells.An assessment was conducted by comparing these results with those obtained using the standard drug bleomycin. Conclusions The BSC compound can be considered as a potent anticancer agent.

Synthesis of Novel Series of Benzothieno [2,3-d] Pyrimidine Derivatives, Promising Anticancer Agents

An extension of the authors＇ previous discovery of in vitro antitumor activity of substituted thino [2,3-d] prymidine derivatives is reported. The synthesis of some new spirothino [2,3-d] prymidine （4a-f）, imidazolidin, substituted prymidinyl and substituted thiazolidine thino [2,3-d] prymidine derivatives have been described. Thirteen of the obtained compounds were selected by the NCI and evaluated for their in vitro anticancer activity. Seven of the investigated compounds, 4a, 8a, 9a, （12a, b）, 14a and 15a, displayed high anticancer activity in the primary assay. These compounds have been selected for a full anticancer screening against a 60-cell panel assay where they showed non-selective broad spectrum and promising activity against all cancer cell lines. Compounds 12a and 12b proved to be the active members in this study compared to 5-fluorouracil and cyclophosphamide as reference drugs, respectively. Compounds 12a and 12b were identified as promising lead compounds, evaluated for their in-vitro antitumor activity.

Grafted Deproteinized Natural Rubber as an Impact Modifier in Styrene-Methyl Methacrylate Copolymer Sheet

This research was studied to prepare styrene-methyl methacrylate copolymer sheet （S-co-MMA sheet） by using DPNR （deproteinized natural rubber） as an impact modifier. The DPNR was prepared by adding SDS （sodium dodecyl sulfate） into the HANR （high-ammoniated natural rubber latex） and followed by centrifugation. It was found that DPNR was successfully prepared with the lowest nitrogeneous content about 0.034±0.01% w/w by adding SDS for 1.00 phr and centrifugation at 12,000 rpm. Then DPNR was grafted with styrene and methyl methacrylate （DPNR-g-S/MMA） obtained by emulsion polymerization. The effects of DPNR-g-S/MMA were studied by varied the amount of SDS and redox initiator. From this result the grafting efficiency DPNR-g-S/MMA was higher than that of NR-g-S/MMA. The DPNR-g-S/MMA was used as an impact modifier in S-co-MMA sheet. Furthermore, the appropriate quantities of styrene monomer and DPNR-g-S/MMA were also investigated. While DPNR-g-S/MMA was used as impact modifier in S-co-MMA sheet, the result was shown DPNR-g-S/MMA improve impact strength of S-co-MMA sheet. Scanning electron micrographs of S-co-MMA sheet with DPNR-g-S/MMA was found the smooth fracture surface. Thus impact strength of S-co-MMA/gDPNR sheet was high and physical properties of S-co-MMA/gDPNR sheet could be able to be accepted in industry. Concisely, DPNR-g-S/MMA can be used as an impact modifier in S-co-MMA sheet.

A Study on Antitoxic Role of Vesicular Monoamine Transporter 2 in Transgenic Chinese Hamster Overy Cells

Objective : To study the antitoxic role of vesicular monoamine transporter 2 (VMAT2) in transgenic Chinese Hamster ovary (CHO) cell. Methods :With the technology of trans-gene from PC 12 to CHO, MTT reduction assay was used to detect MPP+ toxic effect on wild type CHO (wtCHO) and transgenic CHO. Meanwhile, the role of reserpine was also observed in MPP+ toxic effects. Results :The sensitivity of transgenic CHO to MPP+ was much less than that of wtCHO with 0. 5 mmol/L MPP+. Transgenic CHO had the same sensitivity as wtCHO if rotenone was given. WtCHO, by given reserpine alone, didn't change its sensitivity to MPP+. Conclusions :VMAT2 has protective effect on transgenic CHO by transporting MPP+ to vesicles.

Anti-inflammatory Activity and Antioxidant Potential of Aqueous Extracts from Stem Bark of Geoffroea decorticans

In the present work we investigated, for the first time, the anti-inflammatory activity and the antioxidant properties of aqueous and ethanolic extracts, obtained from stem bark of Geoffroea decorticans （Gill. ex Hook. et Am.） Burk. （Fabaceae）. G. decorticans, commonly known as ＂chafiar＂ or ＂chafiarcillo＂, is a traditional argentinean plant used as emollient, balsamic, antitussive, expectorant and anti-inflammatory. The stem bark was collected from San Francisco del Monte de Oro, San Luis, Argentina. Anti-inflammatory activity was evaluated by carrageenan-induced paw edema in rats. Antioxidant activity was tested using 1,1-Diphenyl-2-picrylhydrazyl radical-scavenging activity （DPPH）, 2,2＂-Azino-bis（3-ethylbenzthiazoline-6-sulphonic acid） radical scavenging activity （ABTS） and ferric ion-reducing power （RP-Fe） assays. Aqueous extract 10% p/v showed anti-inflammatory activity （3h, 48% inhibition, 5h 37% inhibition and 7h 17% inhibition） and antioxidant activity （DPPH, ICs0 （mg/mL） = 0.098 =1： 0.032; ABTS, ICs0 （mg/mL） = 0.022 ~ 0.343, RP-Fe IC50 （mg/mL） = 1.124 ~ 0.146）. In the other hand, the ethanolic extract 5% p/v, presented anti-inflammatory activity （3h, 34% inhibition, 5h 38% inhibition and 7h 35% inhibition） and antioxidant activity （DPPH, IC50 （mg/mL） = 0.133 q- 0.027; ABTS, IC50 （mg/mL） = 0.086 ＋ 0.262, RP-Fe IC50 （mg/mL） = 7.089 ± 0.104）. These results suggest that, also fruits, the aqueous and ethanolic extracts from the stem bark of G. decorticans present significant anti-inflammatory activity and antioxidant properties.

Menaquinone-7 production from maize meal hydrolysate by Bacillus isolates with diphenylamine and analogue resistance

A menaquinone-7（MK-7） high-producing strain needs to be isolated to increase MK-7 production, in order to meet a requirement of MK-7 given the low MK-7 content in food products. This article focuses on developing MK-7 high-producing strains via screening and mutagenesis by an atmospheric and room temperature plasma（ARTP） mutation breeding system. We isolated an MK-7-producing strain Y-2 and identified it as Bacillus amyloliquefaciens, which produced（7.1±0.5） mg/L of MK-7 with maize meal hydrolysate as carbon source. Then, an MK-7 highproducing strain B. amyloliquefaciens H.β.D.R.-5 with resistance to 1-hydroxy-2-naphthoic acid, β-2-thienylalanine, and diphenylamine was obtained from the mutation of the strain Y-2 using an ARTP mutation breeding system. Using strain H.β.D.R.-5, efficient production of MK-7 was achieved（（30.2±2.7） mg/L）. In addition, the effects of nitrogen sources, prenyl alcohols, and MgSO_4 on MK-7 production were investigated, suggesting that soymeal extract combined with yeast extract, isopentenol, and MgSO4 was beneficial. Under the optimized condition, the MK-7 production and biomass-specific yield reached（61.3±5.2） mg/L and 2.59 mg/L per OD600 unit respectively in a 7-L fermenter. These results demonstrated that strain H.β.D.R.-5 has the capacity to produce MK-7 from maize meal hydrolysate, which could reduce the substrate cost.

Novel benzimidazole derived naphthalimide triazoles: synthesis, antimicrobial activity and interactions with calf thymus DNA

A novel series of benzimidazole derived naphthalimide triazoles and some corresponding triazoliums have been successfully synthesized and characterized by 1H NMR, 13 C NMR, 1H-1H COSY, IR and HRMS spectra. All the new compounds were screened for their antimicrobial activities in vitro by two-fold serial dilution. 2-Chlorobenzyl triazolium 8g and compound 9b with octyl group exhibited the best antibacterial activities among all the tested compounds, especially against S. aureus with inhibitory concentration of 2 μg/mL which was equipotent potency to Norfloxacin(MIC=2 μg/mL) and more active than Chloromycin(MIC=7 μg/mL). Triazoliums 8g and 8f bearing 3-fluorobenzyl moiety displayed the best antifungal activities(MIC=2-19 μg/mL) against all the tested fungal strains without being toxic to PC12 cell line within concentration of 128 μg/m L. Further investigations by fluorescence and UV-Vis spectroscopic methods revealed that the compound 8g could effectively intercalate into calf thymus DNA to form the 8g-DNA complex which could block DNA replication, exerting powerful antimicrobial activities.

Antimicrobial, anticancer and DNA binding studies of transition metal(Ⅱ)complexes containing 2-phenyl-4-selenazole carboxylic acid

Three novel transition metal complexes [ML2（phen）]＇H20 （M= Mn, Co, Zn; HL= C10H702NSe, 2-phenyl-4-selenazole carboxylic acid, phen= 1,10-phenanthroline） 1-3 were synthesized and characterized by elemental analysis, IR spectra. Their crystal structures were deterimined by single-crystal X-ray diffraction method. The antibacterial activities of the complexes against five species of bacteria, Escherichia coli （E. coli）, Staphylococcus epidermidis （S. epidermidis）, Staphylococcus aureus （S. au- reus）, Acinetobacter baumanii （A. baumanii） and Streptococcus viridans （S. viridans）, were tested respectively. The anticancer activities of the complexes against human pancreatic cancer line PANC-28 and human hepatocarcinoma line HuH7 were also studied. The interactions between the complexes and DNA were studied by ethidium bromide （EB） fluorescent probe.

Modification of the alkylating agent chlorambucil with spiropiperazinium salts

In order to improve the antitumor activity and reduce the toxicity of the alkylating agent chlorambucil, a series of spiropiperazinium salts of chlorambucil （8） were designed and synthesized. It was found that compound 8f exhibited potential in vivo activity against H22.