解淀粉芽胞杆菌HZ-12中ysnE参与吲哚-3-乙酸合成的代谢途径研究

【目的】吲哚-3-乙酸是调控植物生长发育和生理活动的重要激素,吲哚-3-乙酸N-乙酰转移酶YsnE在吲哚-3-乙酸合成中发挥重要作用,本研究拟解析解淀粉芽胞杆菌中YsnE参与吲哚-3-乙酸合成的代谢途径。【方法】通过基因ysnE缺失和强化表达,分析ysnE对吲哚-3-乙酸合成影响,结合吲哚-3-乙酸合成中间物(吲哚丙酮酸、吲哚乙酰胺、色胺和吲哚乙腈)添加和体外酶转化实验,解析ysnE参与吲哚-3-乙酸合成的代谢途径。【结果】明确了YsnE在解淀粉芽胞杆菌HZ-12吲哚-3-乙酸合成中发挥重要作用。发现ysnE缺失菌株中的吲哚丙酮酸、吲哚乙酰胺和吲哚乙腈利用显著降低,揭示了YsnE主要发挥吲哚丙酮酸脱羧酶YclB和吲哚乙酰胺水解酶/腈水解酶/腈水合酶YhcX的功能,并通过参与吲哚丙酮酸、吲哚乙酰胺和吲哚乙腈途径来影响吲哚-3-乙酸合成。【结论】初步揭示了YsnE通过影响吲哚丙酮酸、吲哚乙酰胺和吲哚乙腈途径参与吲哚-3-乙酸合成的代谢机理,为吲哚-3-乙酸合成途径解析和代谢工程育种构建吲哚-3-乙酸高产菌株奠定了基础。     

抗油桐枯萎病木霉菌的分离鉴定及抑菌作用研究

油桐是我国重要的木本油料植物,其生产的桐油作为天然的优良干性油在工业上具有广泛用途。但由油桐专化型尖孢镰孢菌Fusarium oxysporum f. sp. fordiis（Fof-1）侵染引起的枯萎病给油桐产业造成了毁灭性灾害,目前尚无有效防治手段。众多实践证明,利用生防菌可以有效防治土传枯萎病。本研究发现,在抗病油桐根围土壤中木霉菌的相对丰度较高,并从中分离获得了16株木霉菌;通过形态学鉴定和ITS-TEF1双基因联合构建系统进化树,鉴定出4种木霉菌：拟康宁木霉Trichoderma koningiopsis（TkonT1）、螺旋木霉T. spirale（TspiT2）、深绿木霉T. atroviride（TatrT3）和哈茨木霉T. harzianum（TharT4）;通过对峙培养试验,发现木霉菌株TkonT1、TharT4和TspiT2具有较好的抑菌效果;进一步显微观察发现菌株TkonT1和TatrT3可缠绕在尖孢镰孢菌菌丝体上或穿入菌丝体内营寄生生长,吸收病菌菌丝体养分进而导致病菌菌丝体破裂和细胞原生质消解。结果表明,从抗病油桐根围土壤中获得的拮抗木霉菌株可用于油桐枯萎病的生物防治。     

紫陀螺菌化学成分及其抑制肿瘤细胞增殖活性的研究

以紫陀螺菌为对象,研究其子实体的化学成分及其抑制肿瘤细胞增殖活性。采用溶剂提取、柱层析和高效液相色谱等方法分离纯化化学成分,通过核磁共振和质谱技术鉴定单体化合物结构,运用结晶紫法评价单体化合物抑制肿瘤细胞增殖活性。从乙酸乙酯提取物中共分离鉴定6个单体化合物,分别为(22E,24R)-麦角甾-5,7,22-三烯-3β-醇(1)、3β,5α-二羟基-(22E,24R)-麦角甾-7,22-二烯-6-酮(2)、(22E,24R)-麦角甾-7,22-二烯-3β,5α,6β-三醇(3)、吲哚-3-甲酸甲酯(4)、4,4-二甲基-1,7-庚二酸(5)和(8E,10E)-12羰基十八碳-8,10-二烯酸(6),其中化合物1为主要成分,相对含量为23.8%。活性测试结果表明3对人乳腺癌细胞株MCF-7 细胞、人胰腺癌细胞株PANC-1细胞和人乳腺癌细胞株MDA-MB-231细胞具有微弱的细胞增殖抑制活性。本研究首次报道了紫陀螺菌化学成分,对深入挖掘其在健康领域中的开发价值具有重要意义。     

海南染木树茎的化学成分研究(Ⅰ)北大核心CSCD

研究海南染木树茎的化学成分,采用硅胶、Sehadex LH-20、ODS、HPLC等色谱技术对海南染木树茎乙醇提取物的乙酸乙酯部位进行分离和纯化,结合光谱数据和理化性质鉴定化合物的结构,分别为：4-甲基苯并喹啉-5,10-二酮（1）、白兰花碱（2）、鹅掌楸碱（3）、吲哚-3-甲醛（4）、羽扇豆醇（5）、霍烷-3,30-二醇（6）、熊果酸（7）、白桦酯酸（8）、30-羟基羽扇豆醇（9）、豆甾醇（10）、3β-乙酰基齐墩果醛（11）。其中,化合物4、7为首次从染木树属植物中分离得到,5、6、8、9-11为首次从该植物中分离得到。首次对化合物2的碳谱数据进行了归属。     

海洋链霉菌B170167发酵产物中含氮化合物及其细胞毒活性研究

利用色谱层析技术从海洋链霉菌B170167中分离得到11个含氮化合物,包括8个吲哚类和3个含氮杂环化合物。经波谱分析分别鉴定为Turbomycin A(1)、3,3-二-(3-吲哚)丙烷-1,2-二醇(2)、N-(2-(1H-Indol-3-yl)-2-oxoethyl)-acetamide(3)、N-乙酰基色氨酸(4)、9H-吡啶并[3,4-b]吲哚(5)、吲哚-3-甲醛(6)、3-吲哚丙酸(7)、3-吲哚甲酸(8)、光色素(9)、Ferrioxamine E(10)、3-羟基-2-甲基吡啶(11)。其中化合物11为首次从微生物中分离得到。化合物1、2、5、10、11显示出显著的细胞毒活性,化合物3、4、6、7、8显示中等程度的细胞毒活性。     

内生条纹拟盘多毛孢发酵液中细胞毒活性成分研究

从条纹拟盘多毛孢Pestalotopsis virgatula发酵液中分离得到8个化合物,其结构分别被鉴定为：2-(1-甲氧基-1-H-吲哚-3-基)乙醇 (1),2-(1-甲氧基-1-H-吲哚-3-基)乙酸 (2),3β-羟基-5α,8α-过氧化麦角甾-6,22-二烯 (3),麦角甾-4,6,8(14),22-四烯-3-酮 (4),对羟基苯乙醇 (5),邻苯二甲酸二异丁酯 (6),(E)-3-(4-羟基-3-甲氧苯基)败脂酸 (7) 和丁二酸 (8),化合物1–8均为首次从该菌种中分离得到。利用MTT法测试了化合物1 和2对5种人体肿瘤细胞的细胞毒活性,结果显示化合物1和2对5株肿瘤细胞株均具有一定选择性抑制活性。     

三株海洋木霉的应用潜力

【目的】探索3株海洋生境木霉的应用潜力。【方法】经过筛选和诱变,获得高抑菌活性及产孢量的木霉突变株;通过优化培养基、温度、初始p H考察其产孢量及最适培养条件;综合抑菌谱、重寄生及抑菌相关基因考察其抑菌活性;采用特殊培养基法考察其产纤维素酶、植酸酶、铁载体以及降解磷钾的能力,高效液相色谱法测定其产吲哚乙酸能力。【结果】3株木霉菌的产孢量分别为3.45×108、3.10×108和2.55×108 CFU/cm2,与野生型相比分别提高了88.52%、63.16%和180.22%;且均可产生厚垣孢子,其中XG20-1厚垣孢子产量最高,达到3.56×108 CFU/m L。3株木霉菌具有较广抑菌谱及对番茄早疫病菌的重寄生作用,同时扩增得到Tex1、Nag1、Eg1基因,生物学测试显示其均具有产纤维素酶、几丁质酶以及铁载体的能力,证明其抑菌活性是多种机制共同作用的结果;菌株可以降解磷钾,且吲哚乙酸产量分别为2.61、1.57和1.92 mg/L,具有促进植物生长的潜力。【结论】本文中3株木霉菌在开发为生防菌与生物肥料方面展现出良好的应用潜力。     

木霉属3个中国新记录种

报道了采自我国黑龙江、吉林及西藏的木霉属Trichoderma 3个中国新记录种：内生木霉Trichoderma endophyticum、意大利木霉T. italicum和酒色木霉T. vinosum。首次发现近深绿木霉T. paratroviride的有性阶段,提供了上述种的详细描述及宏观和微观特征的图示,并探讨了其分类地位。     

多粘类芽孢杆菌HY96—2发酵液化学成分研究

从多粘类芽孢杆菌(Paenibacillus polymyxa HY96-2)的发酵液中分离得到了12个化合物,其结构通过波谱分析分别鉴定为苯甲酸(1)、对羟基苯甲酸(2)、对羟基苯丙酸(3)、2-苯基乳酸(4)、3-苯基乳酸(5)、琥珀酸(6)、棕榈酸甲酯(7)、大豆甙元(8)、环(甘氨酸-L-丙氨酸)二肽(9)、吲哚-3-乙酸(10)、L-苯丙氨酸(11)和2R,3R-丁二醇(12),其中化合物1～9均为首次从该菌中分离得到,化合物1、2、5和6对青枯劳尔氏菌的最小抑菌浓度(MIC)分别为1、3、2和3 mg/mL.     

α-溴-2、3、4、5、6五氟甲苯衍生气相色谱同时测定吲-3-乙酸和脱落酸

用α-溴-2、3、4、5、6五氟甲苯作衍生剂,在55℃反应90分钟可与吲哚-3-乙酸和脱落酸生成酯。这两种酯可用气相色谱分离,电子捕获检测器测定。本方法操作简单、灵敏度高。最小检测量:吲哚-3-乙酸和脱落酸分别为10~(-14)g 和10~(-13)g。     

Anti-proliferative lichen compounds with inhibitory activity on 12(S)-HETE production in human platelets

Several lichen compounds, i.e. lobaric acid (1), a β-orcinol depsidone from Stereocaulon alpinum L., (+)-protolichesterinic acid (2), an aliphatic -methylene-γ-lactone from Cetraria islandica Laur. (Parmeliaceae), (+)-usnic acid (3), a dibenzofuran from Cladonia arbuscula (Wallr.) Rabenh. (Cladoniaceae), parietin (4), an anthraquinone from Xanthoria elegans (Link) Th. Fr. (Calaplacaceae) and baeomycesic acid (5), a β-orcinol depside isolated from Thamnolia vermicularis (Sw.) Schaer. var. subuliformis (Ehrh.) Schaer. were tested for inhibitory activity on platelet-type 12(S)-lipoxygenase using a cell-based in vitro system in human platelets. Lobaric acid (1) and (+)-protolichesterinic acid (2) proved to be pronounced inhibitors of platelet-type 12(S)-lipoxygenase, whereas baeomycesic acid (5) showed only weak activity (inhibitory activity at a concentration of 100 μg/ml: 1 93.4±6.62%, 2 98,5±1.19%, 5 14.7±2.76%). Usnic acid (3) and parietin (4) were not active at this concentration. 1 and 2 showed a clear dose–response relationship in the range of 3.33–100 μg/ml. According to the calculated IC₅₀ values the highest inhibitory activity was observed for the depsidone 1 (IC₅₀=28.5 μM) followed by 2 (IC₅₀=77.0 μM). The activity of 1 was comparable to that of the flavone baicalein, which is known as a selective 12(S)-lipoxygenase inhibitor (IC₅₀=24.6 μM).     

拟南芥IAA酰胺合成酶GH3-6负调控干旱和盐胁迫的反应

生长素是一种重要的植物激素, 几乎参与了植物所有的生命活动过程。GH3-6具有IAA酰胺合成酶活性, 催化氨基酸与IAA形成IAA的氨基轭合物, 发挥暂时或永久灭活IAA的作用。该文探讨了GH3-6基因在拟南芥(Arabidopsis thaliana)逆境适应过程中的功能。结果显示GH3-6基因受干旱、ABA和高盐的诱导表达。与野生型相比, GH3-6基因过表达突变体dfl1-D对干旱的抗性明显减弱, 叶片失水速率更快。在抗盐方面, dfl1-D也显著弱于野生型。在3种逆境(干旱、ABA和高盐)胁迫下, GH3-6基因的高表达抑制了逆境响应基因RD22、KIN1、RD29A和DREB1A的表达。而且在干旱胁迫下, dfl1-D中ABA的含量明显低于野生型。研究结果证明, 高表达GH3-6基因负调控拟南芥对逆境的抗性。     

Hydroxylated jasmonic acid and related compounds from Botryodiplodia theobromae

From the culture filtrate of the fungus Botryodiplodia theobromae five hydroxylated cyclopentane fatty acids of the jasmonic acid type were isolated and identified as (11 S -(-)-hydroxyjasmonic acid; (11R)-(-)-hydroxyjasmonic acid; (-)-12-hydroxyjasmonic acid; (-)-8ξ-hydroxyjasmonic acid; (-)-3-oxo-2-(1ξ-hydroxy-2Z-pentenyl)cyclopent-1-yl-butyric acid; (-)-3-oxo-2(4ξ-hydroxy-2Z-pentenyl)cyclopent-1-yl-butyric acid. In addition, the corresponding hydroxylated iso-jasmonic acid analogues were found as minor constituents. During silica gel chromatography 11,12-didehydrojasmonic acid, 11ξ-acetoxyjasmonic acid, 3-oxo-2-(4ξ-acetoxy-2Z-pentenyl)cyclopent-1-yl-butyric acid 3-oxo-2-(2Z,4-pentadienyl)cyclopent-1-yl-butyric acid were formed as artefacts.     

Biocatalytic production of 2(E)-hexenal from hydrolysed linseed oil

Natural 2(E)-hexenal was produced in two steps from hydrolysed linseed oil, which contains the most linolenic acid among the available natural sources. In the first step 13-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid (13-HPOT) was formed from linolenic acid (100 mM) by soybean lipoxygenase-1 (Lox-1) isoenzyme with oxygen as co-substrate. The reaction resulted in 57 mM 13-HPOT with a yield of 62%. In the second step 13-HPOT (20 mM) was cleaved by green bell pepper hydroperoxide lyase resulting in 1.6 mM 2(E)-hexenal and 5.9 mM 3(Z)-hexenal (37% yield for the hexenal isomers together). Hexenals were isolated from the reaction mixture by repeated steam distillations. During distillations the 2(E)-hexenal:3(Z)-hexenal isomer ratio was changed from 0.27 to 7.86 as a consequence of heat.     

Synergistic anti-proliferative effects of vitamin D derivatives and 9-cis retinoic acid in SH-SY5Y human neuroblastoma cells.

This study examines the effect of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃], two vitamin D analogues (KH 1060 and EB 1089, which are 20-epi-22-oxa and 22,24-diene-analogues, respectively), 9-cis retinoic acid and all-trans retinoic acid on proliferation of SH-SY5Y human neuroblastoma cells, after treatment for 7 days. Cell number did not change when the cells were incubated with 1, 10 or 100 nM 1,25(OH)₂D₃ or its derivatives, but significantly decreased in the presence of the two retinoids (0.001–10 μM final concentration). A synergistic inhibition was observed, when SH-SY5Y cells were treated combining 0.1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or 10 nM KH 1060, and 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or 10 nM EB 1089. Acetylcholinesterase activity showed a significant increase, in comparison with controls, after treatment of the cells for 7 days with 0.1 or 1 μM 9-cis retinoic acid, alone or combined with 10 nM 1,25(OH)₂D₃ or 10 nM KH 1060 or 10 nM EB 1089. This increase was synergistic, combining 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or EB 1089. The levels of the c-myc encoded protein remarkably decreased after treatment of SH-SY5Y cells for 1, 3, 7 days with 0.1 and 1 μM 9-cis retinoic acid, alone or combined with 10 nM 1,25(OH)₂D₃ or 10 nM KH 1060 or 10 nM EB 1089. In particular, the association of 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or 10 nM EB 1089 resulted in a synergistic c-myc inhibition, in comparison with that obtained in the presence of the retinoid alone. These findings may have therapeutic implications in human neuroblastoma.     

Asymmetric catalysis 87. Enantioselective allylation of 1,5-dimethylbarbituric acid with Pd catalysts and new optically active PN ligands

The new PN ligands 5, 6 and 7 were prepared by Schiff base condensation of 2-formylphenyl(diphenyl)phosphine (1) with the optically active amines (R)-(−)-2-aminobutanol (2), (S)-(+)-2-aminobutanol (3) and (1S,2S)-2-amino- 1-phenyl-1,3-propanediol (4). These new ligands were used in the Pd catalysed allylation of 1,5-dimethylbarbituric acid with allylacetate. 5-Allyl-1,5-dimethylbarbituric acid was obtained with an optical induction of up to 12.7% ee.     

Regioselective acylation of several polyhydroxylated natural compounds by Candida antarctica lipase B

Regioselective acylation of four polyhydroxylated natural compounds, deacetyl asperulosidic acid (1), asperulosidic acid (2), puerarin (3) and resveratrol (4) by Candida antarctica Lipase B in the presence of various acyl donors (vinyl acetate, vinyl decanoate or vinyl cinnamoate) was studied. Compounds 1, 2 and 4 were regioselectively acetylated with vinyl acetate to afford products, 3'-O-acetyl-10-O-deacetylasperulosidic acid (1a), 3',6'-O-diacetyl-10-O-deacetylasperulosidic acid (1b), 3'-O-acetylasperulosidic acid (2a), 3',6'-O-diacetylasperulosidic acid (2b), 4'-O-acetylresveratrol (4a), respectively, with yields of 22 to 50%, while reactions with vinyl decanoate and vinyl cinnamoate were slow with lower yields. Compound 3 was readily acylated with all three acyl donors and quantitatively converted to products 6'-O-acetylpuerarin (3a), 6'-O-decanoylpuerarin (3b), 6'-O-cinnamoylpuerarin (3c), respectively. The structures of these acylated products were determined by spectroscopic methods (MS and NMR).     

Photochemical reactions of transition metal organyl complexes with olefins Part 10. Light-induced formal [5+2] cycloadditions at manganese

Tricarbonyl-η⁵-2,4-dimethyl-2,4-pentadien-1-yl-manganese (1) forms upon UV irradiation in THF at 208 K solvent stabilized dicarbonyl-η⁵-2,4-dimethyl-2,4-pentadien-1-yl-tetrahydrofurane-manganese (2). With butynedioic acid dimethyl ester (3) and diphenylacetylene (5) complex 2 yields tricarbonyl-η⁵-1,2-dimethoxycarbonyl-4,6-dimethyl- cyclohepta-2,4-dien-1-yl-manganese (4) and tricarbonyl-η-4,6-dimethyl-1,2-diphenyl-cyclohepta-2,4-dien-1-yl- manganese (6) in a formal [5+2] cycloaddition. Addition of carbon monoxide and a 1,4-H shift completes the reaction. Propynoic acid methyl ester (7) forms the 2:1 adduct dicarbonyl-η^5:2-1,3-dimethyl-6-methoxycarbonyl-6- (E-2′-methoxycarbonylvinyl)-cyclohepta-2,4-dien-1-yl-manganese (8). The crystal and molecular structure of 8 was determined by X-ray structure analysis. The molecular structures of the complexes 4 and 6 were established by IR and NMR spectroscopy. Formation mechanisms of 4, 6 and 8 are discussed. Crystal data for 8: monoclinic space group P2₁/c, a=802.6(3), b=1136.6(1), c=8872.3(3) pm, β=93.14(2)°, V=1.705 nm³, Z=4.     

灵芝菌丝体中一个三萜类化合物的核磁归属及活性初探

通过液体振荡-静置两阶段发酵获得灵芝菌丝体,并采用硅胶柱色谱层析、反相柱层析和甲醇重结晶的方法,从中分离得到4个三萜类化合物。根据NMR、MS等波谱数据分析,化合物分别被鉴定为lanosta-7,9(11),24-trien-3α-acetoxy-26-oic acid（1）、灵芝酸R（2）、灵芝酸T（3）和灵芝酸S（4）,其中化合物1的核磁信号全归属为首次报道。4个三萜类化合物均具有较好的抑制肿瘤细胞L1210及K562增殖的活性,且化合物1的体外抗肿瘤活性为首次证实,其对肿瘤细胞L1210及K562增殖的半数抑制浓度IC₅₀分别为22.17μmol/L和54.79μmol/L。