不同培养基和三种金属离子对桦褐孔菌培养菌丝体的羊毛甾醇和麦角甾醇积累的影响

Lanosterol and ergosterol are the active principles with potential pharmacological activities in Inonotus obliquus. However, the two sterols are less accumulated in cultured mycelia of the fungus. In this study, different carbon and nitrogen sources and pH levels together with three metal ions were assayed for their effects on accumulation of the two sterols in the fungus. Among the tested media the growth medium consisting of glucose (1.5%), rice powder (0.5%), yeast extract (0.4%), wheat bran (0.1%), KH2PO4 (0.01%) and MgSO4·7H2O (0.05%) with pH level at 6.5 yielded a maximum production of the two sterols, which can further be increased following the treatment of Ag+, Cu2+ and Ca2+. Supplementing Ag+ at concentrations of 0.28 and 0.35μmol partially inhibited ergosterol biosynthesis, leading to an enhanced accumulation of lanosterol, the presence of intermediates of ergosterol biosynthetic pathway and a reduced accumulation of ergosterol in cultured mycelia of I.     

一氧化氮对桦褐孔菌抗氧化酚类化合物积累的影响

本研究探讨了NO对深层培养的桦褐孔菌积累抗氧化酚类化合物的影响。在桦褐孔菌的培养基中分别加入0.01mmol/L、0.1mmol/L以及1mmol/L的NO供体硝普钠,并测定总酚的含量及其抗氧化活性。发酵产物的抗氧化活性以清除DPPH和羟自由基活力表示。加入0.1mmol/L的硝普钠可使桦褐孔菌胞内外酚类化合物分别达到最高水平67mg/g和677mg/L。不同培养时期产生的胞内外酚类化合物都具有抗氧化活性,尤其是添加0.1mmol/L硝普钠的菌丝体。因此NO可用于上调深层发酵培养的桦褐孔菌酚类化合物的积累。     

真菌激发子对桦褐孔菌多酚积累的影响

作者旨在阐明真菌激发子对桦褐孔菌多酚积累的影响。以摇瓶法培养桦褐孔菌,在其培养液中加入真菌激发子和一氧化氮合酶抑制剂氨基胍,观察桦褐孔菌多酚和一氧化氮的积累并测定菌丝体内一氧化氮合酶和苯丙氨酸解氨酶的活性。多酚以Folin-Ciocalteu法测定,一氧化氮的积累量用硝酸还原酶法测定,一氧化氮合酶和苯丙氨酸解氨酶活性均以分光光度计法测定。结果表明,添加45μg/mL的激发子可使桦褐孔菌菌丝体多酚的积累量达到46.5mg/g,显著地高于正常培养的菌丝体多酚积累量34.6mg/g;同时加入45μg/mL的激发子和10mmol/L氨基胍则使菌丝体多酚积累的最高水平降为34.8mg/g。此外,激发子的加入显著促进了一氧化氮的产生并提高了苯丙氨酸解氨酶活性,而这种促进和提升作用为氨基胍所抑制。这表明真菌激发子能显著地提升桦褐孔菌多酚类化合物的积累,而一氧化氮可能是这种提升作用的信号传导分子。     

外源因子对桦褐孔菌发酵产桦褐孔菌醇的影响

食药用真菌因其丰富的天然活性物质成为具有开发潜力的药物来源。桦褐孔菌Inonotus obliquus作为一种珍稀的药用真菌,因其对糖尿病、消化系统疾病、心血管疾病、肝病和癌症等疾病有良好的治疗效果而受到广泛关注。桦褐孔菌醇（inotodiol）是桦褐孔菌特有的一种羊毛甾烷型三萜类化合物,具有多种抗癌活性。本文的主要目的是研究外源因子的添加对桦褐孔菌液态发酵产桦褐孔菌醇的影响,以及对桦褐孔菌醇合成途径中酶活的影响。结果表明：最佳外源因子是香叶醇,最佳添加浓度和添加时间分别为0.02%（V/V）和第144小时。发酵结束时（240h）桦褐孔菌醇的产量为27.89mg/L是对照组（9.23mg/L）的3.02倍。通过对比添加香叶醇后桦褐孔菌醇的产量变化以及合成途径中4种酶（法尼基焦磷酸合酶、角鲨烯合酶、角鲨烯环氧化酶和羊毛甾醇合酶）的活性变化,对香叶醇的作用机制进行了初步探究。研究结果表明添加香叶醇后,4种酶活性均较对照组有显著的提高,与此对应的桦褐孔菌醇产量也显著增加,说明这4种酶在桦褐孔菌醇合成途径中起到了积极的作用。     

氨基酸和霉菌水提物对深层发酵桦褐孔菌菌丝体黄酮积累及其抗氧化活性的影响

黄酮类化合物是桦褐孔菌菌丝体中多酚类化合物的重要组成部分,也是该菌治疗众多疾病的有效成分之一。然而人工培养桦褐孔菌黄酮等酚类化合物积累甚少,导致药理活性的明显下降。为此,我们研究了3种氨基酸和4种霉菌水提物对深层发酵桦褐孔菌黄酮的积累及其抗氧化能力的影响。在所试验的3种氨基酸和4种霉菌水提物中,L-酪氨酸,黄曲霉和毛霉水提物能有效地增加该菌黄酮的积累。人工培养菌体中的黄酮至少由4种黄酮苷组成,苷元分别是槲皮素、柚皮素、山奈酚和异鼠李素。深层发酵菌丝体具有一定的抗氧化能力,并与总黄酮的含量呈正相关。由L-酪氨酸,黄曲霉和毛霉水提物调控生长的桦褐孔菌菌丝体,能有效地清除超氧阴离子、羟基自由基和DPPH自由基。     

桦褐孔菌菌丝体及甾类化合物的发酵条件优化

对桦褐孔菌深层发酵培养基进行了筛选,以菌丝体及甾类化合物产量为目标对发酵条件进行了优化,确定最佳发酵条件为：30g/L葡萄糖,2.5g/L黄豆粉,2.5g/L蛋白胨,3g/L KH2PO4,0.8g/L MgSO4,0.8g/L CaSO4,初始pH4.0,接种量15%,装液量100mL/500mL,转速150r/min,28℃恒温培养。此条件下培养11d,菌丝体干重达12.52g/L,甾体类化合物的产量达112.44mg/L。     

桦褐孔菌发酵液提取物对不同细胞株的药理作用

本文采用桦褐孔菌发酵液提取物(IOFE)对人肝癌细胞株HepG_2、人胃癌细胞株SGC7901、正常组织来源的人肝细胞HL-7702,进行体外细胞试验,结果表明,IOFE在低浓度处理条件下对HepG_2和SGC7901均有抑制作用,且对SGC7901抑制效果最好;在高浓度条件下对HepG_2和SGC7901的生长具有一定的促进作用;IOFE对氟尿嘧啶损伤后的HL-7702具有非常高的修复作用,随浓度的升高,修复作用逐渐增强,在高浓度3 000μg/ml处理条件下,修复率为303.01%。因此,IOFE对肿瘤细胞的作用表现可知提取物含有复杂的成分,这些成分具有抑制或促进细胞增殖的作用,随着浓度的升高促进作用的成分占优势,但对正常细胞,甚至化疗药物损伤后的细胞却有更好的促进作用。     

5-氮杂胞苷对桦褐孔菌多酚积累的影响

本文旨在研究DNA甲基转移酶抑制剂5-氮杂胞苷对桦褐孔菌多酚合成的调控。以液体摇瓶法培养桦褐孔菌,并在培养液中添加5-氮杂胞苷。采用荧光定量PCR测定与多酚合成相关的编码苯丙氨酸解氨酶（pal）、4-香豆酸辅酶A连接酶（4cl）和硬毛素合成酶（sps）基因表达水平,染色质免疫沉淀技术对编码硬毛素合成酶基因启动子区的组蛋白甲基化修饰进行检测,Folin-Ciocalteu法测定桦褐孔菌细胞内和发酵液中多酚的含量。结果显示,5-氮杂胞苷的添加提高了桦褐孔菌体内pal、4cl和sps基因的表达水平,改变了sps启动子区的组蛋白甲基化修饰,即降低了H3K9三甲基化修饰水平,提高了H3K4和H3K36三甲基化修饰水平,显著提高了桦褐孔菌细胞内和发酵液中多酚的积累量。5-氮杂胞苷诱导下桦褐孔菌细胞内多酚积累量达(46.6±2.8)mg/g,明显高于对照组多酚积累量(28.7±1.0)mg/g,并且胞外多酚的含量由对照组的(66.9±1.3)mg/L提高至(92.3±2.3)mg/L。此外,经5-氮杂胞苷处理后胞内多酚清除DPPH自由基、超氧阴离子和羟自由基的能力显著提高。可见,5-氮杂胞苷可以作为调节因子激发桦褐孔菌液体培养条件下多酚的合成,可以成为进一步提高桦褐孔菌液体发酵产物中多酚产量的技术手段之一。     

桦褐孔菌不同提取成分对小鼠免疫功能比较研究

采用正交水提醇沉淀法提取桦褐孔菌成分,得出其最佳提取方法。采用腹腔巨噬细胞吞噬法、测量小鼠免疫器官质量法和测定老龄大鼠SOD酶的活性,研究其对正常小鼠免疫功能的影响及对老龄大鼠抗氧化能力。与乙醇回流提取法和索氏提取法生理盐水组进行比较,得出桦褐孔菌成分可显著提高正常小鼠腹腔巨噬细胞的吞噬功能。通过比较,水提法得到的桦褐孔菌有效成分提高免疫力的作用最强,也证明了桦褐孔菌多糖能通过提高超氧化物歧化酶活性增强老龄大鼠机体的抗氧化能力。     

Amo 1618 effects on incorporation of 14C-MVA and 14C-acetate into sterols in Nicotiana and Digitalis seedlings and cell-free preparations from Nicotiana

Incorporation of radioactivity from acetate-[¹⁴C] and MVA-[¹⁴C] into sterols and sterol precursors in tobacco was inhibited by Amo 1618; differing patterns of accumulation were obtained with the two precursors, suggesting more than one point of inhibition. This was borne out with cell-free preparations with which it was demonstrated that both HMG-CoA reductase and squalene-2,3-epoxide cyclase were inhibited, the latter more strongly than the former. GLC analysis of gross sterol and hydrocarbon fractions confirmed previous indications that incorporation of radioactivity into individual sterols was inhibited by Amo 1618. Finally, incorporation of MVA-[¹⁴C] into sterols and sterol precursors of Digitalis was significantly altered by the retardant, thus expanding the generality of the relationship between sterol (particularly 4-desmethylsterol) biosynthesis inhibition and retardant effect.     

Comparison of the cytotoxic,pro-oxidant and pro-inflammatory characteristics of different oxysterols

Oxidized low-density lipoproteins play important roles in the development of atherosclerosis and contain several lipid-derived, bioactive molecules which are believed to contribute to atherogenesis. Of these, some cholesterol oxidation products, refered to as oxysterols, are suspected to favor the formation of atherosclerotic plaques involving cytotoxic, pro-oxidant and pro-inflammatory processes. Ten commonly occurring oxysterols (7α-, 7β-hydroxycholesterol, 7-ketocholesterol, 19-hydroxycholesterol, cholesterol-5α,6α-epoxide, cholesterol-5β,6β-epoxide, 22R-, 22S-, 25-, and 27-hydroxycholesterol) were studied for both their cytotoxicity and their ability to induce superoxide anion production (O₂^{⋅ −}) and IL-8 secretion in U937 human promonocytic leukemia cells. Cytotoxic effects (phosphatidylserine externalization, loss of mitochondrial potential, increased permeability to propidium iodide, and occurrence of cells with swollen, fragmented and/or condensed nuclei) were only identified with 7β-hydroxycholesterol, 7-ketocholesterol and cholesterol-5β,6β-epoxide, which also induce lysosomal destabilization associated or not associated with the formation of monodansylcadaverine-positive cytoplasmic structures. No relationship between oxysterol-induced cytotoxicity and HMG-CoA reductase activity was found. In addition, the highest O₂^{⋅ −} overproduction quantified with hydroethidine was identified with 7β-hydroxycholesterol, 7-ketocholesterol and cholesterol-5β,6β-epoxide, with cholesterol-5α, 6α-epoxide and 25-hydroxycholesterol. The highest capacity to simultaneously stimulate IL-8 secretion (quantified by ELISA and by using a multiplexed, particle-based flow cytometric assay) and enhance IL-8 mRNA levels (determined by RT-PCR) was observed with 7β-hydroxycholesterol and 25-hydroxycholesterol. None of the effects observed for the oxysterols were detected for cholesterol. Therefore, oxysterols may have cytotoxic, oxidative, and/or inflammatory effects, or none whatsoever.     

Effects of metal ions on activity of plasmin

The effects of some metal ions on amidolytic and fibrinogenolytic activities of highly purified human plasmin were investigated in vitro. In the presence of Zn²⁺, Cu²⁺, Cd²⁺, and Au⁺ in the incubation mixture at the concentrations of 1×10⁻⁵−1×10⁻³ M, the anidolytic plasmin activity was strongly inhibited, whereas Ca²⁺ and Mg²⁺ at the same concentrations were not effective. The analysis of the kinetic study has shown that Zn²⁺ or Cu²⁺ acts as mixed-type inhibitors of plasmin activity. The inhibition of amidolytic plasmin activity by Zn²⁺ and Cu²⁺ was reduced in the presence of EDTA, histidine, or albumin. Incubation of plasmin with Zn²⁺ or Cu²⁺ (at the concentration of 5×10⁻⁴ M) resulted in complete loss of its proteolytic action on fibrinogen, whereas Cd²⁺ and Au⁺ under the same conditions only partially inhibited this process.     

Inhibition of purified bovine liver glutathione reductase with some metal ions

Glutathione reductase (GR; E.C. 1.6.4.2) is a flavoprotein that catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG). In this study we tested the effects of Al³⁺, Ba²⁺, Ca²⁺, Li⁺, Mn²⁺, Mo⁶⁺, Cd²⁺, Ni²⁺, and Zn²⁺ on purified bovine liver GR. In a range of 10?μM–10?mM concentrations, Al³⁺, Ba²⁺, Li⁺, Mn²⁺, and Mo⁶⁺, and Ca²⁺ at 5?μM–1.25?mM, had no effect on bovine liver GR. Cadmium (Cd²⁺), nickel (Ni²⁺), and zinc (Zn²⁺) showed inhibitory effects on this enzyme. The obtained IC₅₀ values of Cd²⁺, Ni²⁺, and Zn²⁺ were 0.08, 0.8, and 1?mM, respectively. Cd²⁺ inhibition was non-competitive with respect to both GSSG (Ki_GSSG 0.221?±?0.02?mM) and NADPH (Ki_NADPH 0.113?±?0.008?mM). Ni²⁺ inhibition was non-competitive with respect to GSSG (Ki_GSSG 0.313?±?0.01?mM) and uncompetitive with respect to NADPH (Ki_NADPH 0.932?±?0.03?mM). The effect of Zn²⁺ on GR activity was consistent with a non-competitive inhibition pattern when the varied substrates were GSSG (Ki_GSSG 0.320?±?0.018?mM) and NADPH (Ki_NADPH 0.761?±?0.04?mM), respectively.     

荷叶离褶伞生物学特性研究

荷叶离褶伞菌丝生长的温度范围是5~35℃,最适温度25℃;子实体分化的温度范围13~22℃,最适温度19℃;0~3℃的温差有利于子实体的分化,温差大于9℃子实体不能形成;孢子萌发的温度范围是10~25℃,最适温度20℃;菌丝能在pH4~11范围内生长,最适pH4~5;菌丝和孢子的致死温度分别是45℃30min和50℃15min。能利用淀粉、蔗糖、葡萄糖、果糖、玉米粉、乳糖和甘露糖作碳源,但蔗糖和葡萄糖为碳源生长势强,果糖生长势弱;甘氨酸、蛋白胨、硫酸铵、黄豆粉、硝酸铵、谷氨酸和麦麸均可作为氮源,在麦麸上生长速度快,长势旺盛;硫酸铵、硝酸铵和谷氨酸虽能作为氮源,但长势很弱;能在C/N15/1~50/1的范围内生长,C/N为50/1时生长速度最快。在完全黑暗的条件下不能形成子实体,连续光照和12h光暗交替两种处理都可形成子实体,且两种处理间差异不显著。     

Effect of the nitrogen source on caffeine degradation by Aspergillus tamarii

AIMS: To evaluate caffeine degradation and nitrogen requirements during Aspergillus tamarii growth in submerged culture. METHODS AND RESULTS: Aspergillus tamarii spores produced on a coffee infusion agar medium added with sucrose were used. Several caffeine and ammonium sulphate concentrations (0-1 and 0-1.36 g l-1, respectively) were tested simultaneously on fungal biomass production and caffeine degradation. An additional caffeine pulse (4 g l-1) was added for all experiments after 48 h of fermentation. Results revealed that when using 0.90 g l-1 of caffeine and 0.14 g l-1 of ammonium sulphate, biomass production and caffeine degradation were enhanced. Highest biomass production (Xmax = 9.87 g l-1) with a specific growth rate (micro) of 0.073 h-1 and caffeine degradation rate of 0.033 g l-1 h-1, was observed under these conditions. CONCLUSIONS: Caffeine degradation as well as biomass production were characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: These studies set the stage for future characterization studies of intracellular enzymes involved in caffeine degradation. Moreover, results observed may help in the biotreatment of residues from the coffee agroindustry.