• 【黑曲霉和青霉培养物中6-和7-甲氧基黄酮的微生物转化。】 复制标题 收藏 收藏
    DOI:10.1515/znc-2012-7-809 复制DOI
    作者列表:Kostrzewa-Susłow E,Dmochowska-Gładysz J,Janeczko T,Sroda K,Michalak K,Palko A
    BACKGROUND & AIMS: :A detailed study of the biotransformation of 6- and 7-methoxyflavones by four fungal strains (Aspergillus niger strains MB, KB, and SBP; Penicillium chermesinum 113) was carried out. Products of demethylation and also demethylation combined with hydroxylation at C-4' were identified. The biotransformation products were stronger antioxidants than the substrates.
    背景与目标: : 进行了四种真菌菌株 (黑曲霉菌株MB,KB和SBP; 113青霉) 对6-和7-甲氧基黄酮的生物转化的详细研究。鉴定了去甲基化和在C-4 '处与羟基化结合的去甲基化产物。生物转化产物比底物更强的抗氧化剂。
  • 【通过生物累积和生物吸附去除染料的曲霉的比较性能评估。】 复制标题 收藏 收藏
    DOI:10.1007/s11356-012-1190-8 复制DOI
    作者列表:Kaushik P,Malik A
    BACKGROUND & AIMS: :Dyes used in various industries are discharged into the environment and pose major environmental concern. In the present study, fungal isolate Aspergillus lentulus was utilized for the treatment of various dyes, dye mixtures and dye containing effluent in dual modes, bioaccumulation (employing growing biomass) and biosorption (employing pre-cultivated biomass). The effect of dye toxicity on the growth of the fungal isolate was studied through phase contrast and scanning electron microscopy. Dye biosorption was studied using first and second-order kinetic models. Effects of factors influencing adsorption and isotherm studies were also conducted. During bioaccumulation, good removal was obtained for anionic dyes (100 mg/l), viz. Acid Navy Blue, Fast Red A and Orange-HF dye (99.4 %, 98.8 % and 98.7 %, respectively) in 48 h. Cationic dyes (10 mg/l), viz. Rhodamine B and Methylene Blue, had low removal efficiency (80.3 % [48 h] and 92.7 % [144 h], respectively) as compared to anionic dyes. In addition to this, fungal isolate showed toxicity response towards Methylene Blue by producing larger aggregates of fungal pellets. To overcome the limitations of bioaccumulation, dye removal in biosorption mode was studied. In this mode, significant removal was observed for anionic (96.7-94.3 %) and cationic (35.4-90.9 %) dyes in 24 h. The removal of three anionic dyes and Rhodamine B followed first-order kinetic model whereas removal of Methylene Blue followed second-order kinetic model. Overall, fungal isolate could remove more than 90 % dye from different dye mixtures in bioaccumulation mode and more than 70 % dye in biosorption mode. Moreover, significant color removal from handmade paper unit effluent in bioaccumulation mode (86.4 %) as well as in biosorption mode (77.1 %) was obtained within 24 h. This study validates the potential of fungal isolate, A. lentulus, to be used as the primary organism for treating dye containing wastewater.
    背景与目标: : 各种行业中使用的染料会排放到环境中,并引起重大环境问题。在本研究中,真菌分离物香菇曲霉被用于处理各种染料,染料混合物和含有废水的染料的双重模式,生物蓄积 (采用生长的生物质) 和生物吸附 (采用预培养的生物质)。通过相衬和扫描电子显微镜研究了染料毒性对真菌分离物生长的影响。使用一阶和二阶动力学模型研究了染料的生物吸附。还进行了影响吸附和等温线研究的因素的影响。在生物累积过程中,阴离子染料 (100 mg/l),即酸性海军蓝、快红A和橙-HF染料 (分别为99.4% 、98.8% 和98.7%) 在48小时内得到良好的去除。与阴离子染料相比,阳离子染料 (10 mg/l),即罗丹明b和亚甲基蓝具有低的去除效率 (分别为80.3% [48 h] 和92.7% [144 h])。除此之外,真菌分离株通过产生更大的真菌颗粒聚集体,显示出对亚甲基蓝的毒性反应。为了克服生物累积的局限性,研究了生物吸附模式下的染料去除。在这种模式下,在24小时内观察到阴离子 (96.7-94.3%) 和阳离子 (35.4-90.9%) 染料的显著去除。三种阴离子染料和罗丹明b的去除遵循一级动力学模型,而亚甲基蓝的去除遵循二级动力学模型。总的来说,真菌分离物可以在生物累积模式下从不同的染料混合物中去除90% 以上的染料,在生物吸附模式下去除70% 以上的染料。此外,在24小时内从生物累积模式 (86.4%) 和生物吸附模式 (77.1%) 的手工纸单元流出物中获得了显著的颜色去除。这项研究验证了真菌分离物A. lentulus作为处理含染料废水的主要生物的潜力。
  • 【影响丝状真菌构巢曲霉单倍体和二倍体有丝分裂重组频率的突变。】 复制标题 收藏 收藏
    DOI:10.1007/BF00341677 复制DOI
    作者列表:Parag Y,Parag G
    BACKGROUND & AIMS: :A haploid strain of Asp. nidulans with a chromosome segment in duplicate (one in normal position on chromosome I, one translocated to chromosome II) shows mitotic recombination, mostly by conversion, in adE in a frequency slightly higher than in the equivalent diploid. A method has been devised, using this duplication, for the selection of rec and uvs mutations. Six rec mutations have been found which decrease recombination frequency in the haploid. One mutation selected as UV sensitive showed a hundred fold increase in recombination frequency in the haploid (pop mutation) and probably the same in diploids. The increased frequency is both in gene conversion and in crossing over, and the exchanges appear in clusters of two or more. pop is allelic to uvsB (Jansen, 1970) which had been found to affect mitotic but not meiotic recombination. It is suggested that mutations of this type interfere with the control mechanism which determines that high recombination is confirmed to the meiotic nuclei and avoided in somatic nuclei.
    背景与目标: : 具有一式两份染色体片段的Asp.Nidulan的单倍体菌株 (一个在I号染色体上处于正常位置,一个易位为II号染色体) 在adE中显示有丝分裂重组,主要是通过转化,其频率略高于等效的二倍体。已经设计出一种使用这种重复的方法来选择rec和uvs突变。已发现六个rec突变降低了单倍体的重组频率。一个被选为对紫外线敏感的突变显示单倍体 (pop突变) 的重组频率增加了一百倍,而二倍体的重组频率可能相同。增加的频率既包括基因转化,也包括杂交,交换出现在两个或更多的簇中。pop与已发现影响有丝分裂但不影响减数分裂重组的uvsB (Jansen,1970) 等位。建议这种类型的突变会干扰控制机制,该机制决定了减数分裂核得到证实并避免了体细胞核中的高重组。
  • 【在自给自足的照明微生物燃料电池中使用产生漆酶的曲霉NYKA 510作为阴极生物催化剂。】 复制标题 收藏 收藏
    DOI:10.4014/jmb.1907.07031 复制DOI
    作者列表:Abdallah YK,Estevez AT,Tantawy DEDM,Ibraheem AM,Khalil NM
    BACKGROUND & AIMS: :In the present work, we isolated and identified Aspergillus sydowii NYKA 510 as the most potent laccase producer. Its medium constituents were optimized to produce the highest possible amount of laccase, which was after 7 days at 31°C and pH 5.2. Banana peel and peptone excelled in inducing laccase production at concentrations of 15.1 and 2.60 g/l, respectively. Addition of copper sulfate elevated enzyme yield to 145%. The fungus was employed in a microbial fuel cell (MFC). The best performance was obtained at 2000 Ω achieving 0.76 V, 380 mAm-2, 160 mWm-2, and 0.4 W. A project to design a self-sufficient lighting unit was implemented by employing a system of 2 sets of 4 MFCs each, connected in series, for electricity generation. A scanning electron microscopy image of A. sydowii NYKA 510 was utilized in algorithmic form generation equations for the design. The mixed patterning and patterned customized mass approach were developed by the authors and chosen for application in the design.
    背景与目标: : 在目前的工作中,我们分离并鉴定了奈卡曲霉510是最有效的漆酶生产者。优化其培养基成分以产生最高可能量的漆酶,这是在31 °C和pH 5.2下7天后。香蕉皮和蛋白ept分别在15.1和2.60g/l的浓度下诱导漆酶产生。硫酸铜的添加提高了145% 的酶产量。真菌用于微生物燃料电池 (MFC)。在2000 Ω 处获得最佳性能,达到0.76 V、380 mAm-2、160 mWm-2和0.4 W。通过采用2套串联连接的4个mfc的系统来实现设计自给自足的照明单元的项目。将A. sydowii NYKA 510的扫描电子显微镜图像用于设计的算法形式生成方程。作者开发了混合图案和图案定制的质量方法,并选择在设计中应用。
  • 【构巢曲霉鸟氨酸代谢的一些遗传方面。】 复制标题 收藏 收藏
    DOI:10.1007/BF00446919 复制DOI
    作者列表:Arst HN Jr
    BACKGROUND & AIMS: :A possible minor route of ornithine catabolism in Aspergillus nidulans might begin with the ornithine decarboxylase reaction and end with the succinic semialdehyde dehydrogenase reaction. It is therefore of interest that the putative structural genes for these two enzymes, puA and ssuA, respectively, are tightly linked group II. However, this linkage is unlikely to have regulatory significance because ileA, the structural gene for threonine dehydratase, separates them. The gene order in this region is ssuA-ileA-puA-mauB-anB. (mauB- mutations result in loss of monoamine oxidase whilst anB- mutations lead to aneurin auxotrophy.) 2. An auxotrophy for ornithine or putrescine in A. nidulans occurs in double mutants lacking arginase and blocked before ornithine in the arginine biosynthetic pathway. Some residual ornithine synthesis in such double mutants can be catalysed by ornithine delta-transaminase, especially if it is synthesised constitutively.
    背景与目标: : 构巢曲霉鸟氨酸分解代谢的一种可能的次要途径可能始于鸟氨酸脱羧酶反应,并以琥珀酸半醛脱氢酶反应结束。因此,令人感兴趣的是,这两种酶的推定结构基因分别是puA和ssuA,是紧密相连的II组。然而,这种连接不太可能具有调节意义,因为苏氨酸脱水酶的结构基因ileA将它们分离。该区域的基因顺序为ssuA-ileA-puA-mauB-anB。(mauB突变会导致单胺氧化酶的丢失,而anB突变会导致神经营养缺陷。) 2。A.Niddulans中鸟氨酸或腐胺的营养缺陷型发生在缺乏精氨酸酶的双突变体中,并且在精氨酸生物合成途径中鸟氨酸之前被阻断。此类双突变体中的某些残留鸟氨酸合成可以由鸟氨酸 δ-转氨酶催化,特别是如果它是组成型合成的。
  • 【构巢曲霉的胞质 (细胞色素P450补体)。】 复制标题 收藏 收藏
    DOI:10.1016/j.fgb.2008.08.010 复制DOI
    作者列表:Kelly DE,Krasevec N,Mullins J,Nelson DR
    BACKGROUND & AIMS: :The cytochromes P450 (CYPs) are found in all biological kingdoms and genome sequencing projects continue to reveal an ever increasing number. The principle aim of this paper is to identify the complete CYPome of Aspergillus nidulans from the genome sequence version AN.3 deposited at the Broad institute, assign the appropriate CYP nomenclature and define function where possible. The completed analysis revealed a total of 111 CYP genes, 3 of which were previously unknown and 8 pseudogenes, representing 89CYP families, 21 of which are unique. We have identified 28 potential gene clusters associated with one or more CYP genes and discussed those with putative PKS and NRPS associated function. The chromosomal location of the genes, predicted cellular location of the proteins and possible function(s) are discussed.
    背景与目标: : 细胞色素P450 (CYPs) 在所有生物界都有发现,基因组测序项目继续显示出越来越多的数量。本文的主要目的是从广泛研究所存放的基因组序列版本AN.3中鉴定构巢曲霉的完整cypme,分配适当的CYP命名法并在可能的情况下定义功能。完成的分析揭示了总共111个CYP基因,其中3个以前是未知的,8个假基因代表89CYP家族,其中21个是独特的。我们已经确定了28个与一个或多个CYP基因相关的潜在基因簇,并讨论了具有假定的PKS和NRPS相关功能的基因簇。讨论了基因的染色体位置,蛋白质的预测细胞位置以及可能的功能。
  • 【烟曲霉诱导的肺微血管内皮细胞早期炎症反应: p38 MAPK的作用和水飞蓟宾的抑制。】 复制标题 收藏 收藏
    DOI:10.1016/j.intimp.2017.05.038 复制DOI
    作者列表:Song J,Pan W,Sun Y,Han J,Shi W,Liao W
    BACKGROUND & AIMS: :Human invasive pulmonary aspergillosis (IPA) is a serious infectious disease mainly caused by Aspergillus fumigatus (A. fumigatus). Pulmonary microvascular endothelial cells (PMVECs) are important ones in the human lung tissue. However, it remains unclear about the role of PMVECs in IPA. In the present study, we cocultured PMVECs with A. fumigatus. We observed that A. fumigatus induced dose- and time-dependent increases of interleukin 6 (IL-6), interleukin 1β (IL-1β) and intercellular adhesion molecule 1 (ICAM-1) concentration in the cultures. Significant increases in IL-6, IL-1β, E-selectin, and ICAM-1 mRNA expression were also observed in the cultures treated with A. fumigatus. While preincubation with SB203580 (10μM) did not cause significant changes in IL-6, IL-1β and ICAM-1 concentration in the cocultures, significant IL-6, IL-1β and ICAM-1 concentration decreases were observed in the cocultures preincubated with SB203580 (20μM). Neither SP600125 (10-20μM) nor PD98059 (10-20μM) caused significant changes in IL-6, IL-1β and ICAM-1 concentration in the cocultures. PCR results also showed that SB203580 (20μM) (neither SP600125 (20μM) nor PD98059 (20μM)) preincubation significantly decreased IL-6, IL-1β, E-selectin and ICAM-1 mRNA expression in the cocultures. In addition, significant p38 MAPK phosphorylation increase was observed in the PMVECs cultures treated with A. fumigatus. Furthermore, silibinin pre-treatment and post-treatment were observed to significantly down-regulate mRNA and protein expression of proinflammatory factors and adhesion molecules in the cocultures. Finally, we observed that silibinin significantly inhibited A. fumigatus-induced p38 MAPK activation in PMVECs. Our results indicated that PMVECs might participate in IPA early inflammation which is mediated by p38 MAPK. Silibinin may inhibit A. fumigatus-induced inflammation in PMVECs through p38 MAPK.
    背景与目标: : 人侵袭性肺曲霉病 (IPA) 是一种主要由烟曲霉 (a.fumigatus) 引起的严重传染病。肺微血管内皮细胞 (pmvec) 是人肺组织中的重要细胞。然而,目前尚不清楚PMVECs在IPA中的作用。在本研究中,我们将PMVECs与烟曲霉共培养。我们观察到烟曲霉诱导培养物中白介素6 (IL-6),白介素1β (IL-1β) 和细胞间粘附分子1 (ICAM-1) 浓度的剂量和时间依赖性增加。在用烟曲霉处理的培养物中也观察到IL-6,IL-1β,E-选择素和ICAM-1 mRNA表达的显着增加。虽然与SB203580 (10μm) 的预孵育不会引起共培养物中IL-6,IL-1β 和ICAM-1浓度的显着变化,但在与SB203580 (20μm) 预孵育的共培养物中观察到显着的IL-6,IL-1β 和ICAM-1浓度降低。SP600125 (10-20μM) 和PD98059 (10-20μM) 均未引起共培养物中IL-6,IL-1β 和ICAM-1浓度的显着变化。PCR结果还表明,SB203580 (20μm) (SP600125 (20μm) 和PD98059 (20μm)) 的预孵育显着降低了共培养物中IL-6,IL-1β,E-选择素和ICAM-1 mRNA的表达。此外,在用烟曲霉处理的PMVECs培养物中观察到显着的p38 MAPK磷酸化增加。此外,观察到水飞蓟宾治疗前和治疗后可显着下调共培养物中促炎因子和粘附分子的mRNA和蛋白质表达。最后,我们观察到水飞蓟宾显着抑制了烟曲霉诱导的pmvec中的p38 MAPK激活。我们的结果表明,PMVECs可能参与了由p38 MAPK介导的IPA早期炎症。水飞蓟宾可通过p38 MAPK抑制烟曲霉诱导的PMVECs炎症。
  • 【通过分生孢子途径的靶向失调降低烟曲霉的毒力。】 复制标题 收藏 收藏
    影响因子 :
    发表时间:2020-02-04
    来源期刊:mBio
    DOI:10.1128/mBio.03202-19 复制DOI
    作者列表:Stewart JIP,Fava VM,Kerkaert JD,Subramanian AS,Gravelat FN,Lehoux M,Howell PL,Cramer RA,Sheppard DC
    BACKGROUND & AIMS: :Inhalation of conidia of the opportunistic mold Aspergillus fumigatus by immunocompromised hosts can lead to invasive pulmonary disease. Inhaled conidia that escape immune defenses germinate to form filamentous hyphae that invade lung tissues. Conidiation rarely occurs during invasive infection of the human host, allowing the bulk of fungal energy to be directed toward vegetative growth. We hypothesized that forced induction of conidiation during infection can suppress A. fumigatus vegetative growth, impairing the ability of this organism to cause disease. To study the effects of conidiation pathway dysregulation on A. fumigatus virulence, a key transcriptional regulator of conidiation (brlA) was expressed under the control of a doxycycline-inducible promoter. Time- and dose-dependent brlA overexpression was observed in response to doxycycline both in vitro and in vivo. Exposure of the inducible brlA overexpression strain to low doses of doxycycline under vegetative growth conditions in vitro induced conidiation, whereas high doses arrested growth. Overexpression of brlA attenuated A. fumigatus virulence in both an invertebrate and mouse model of invasive aspergillosis. RNA sequencing studies and phenotypic analysis revealed that brlA overexpression results in altered cell signaling, amino acid, and carbohydrate metabolism, including a marked upregulation of trehalose biosynthesis and a downregulation in the biosynthesis of the polysaccharide virulence factor galactosaminogalactan. This proof of concept study demonstrates that activation of the conidiation pathway in A. fumigatus can reduce virulence and suggests that brlA-inducing small molecules may hold promise as a new class of therapeutics for A. fumigatus infection.IMPORTANCE The mold Aspergillus fumigatus reproduces by the production of airborne spores (conidia), a process termed conidiation. In immunocompromised individuals, inhaled A. fumigatus conidia can germinate and form filaments that penetrate and damage lung tissues; however, conidiation does not occur during invasive infection. In this study, we demonstrate that forced activation of conidiation in filaments of A. fumigatus can arrest their growth and impair the ability of this fungus to cause disease in both an insect and a mouse model of invasive infection. Activation of conidiation was linked to profound changes in A. fumigatus metabolism, including a shift away from the synthesis of polysaccharides required for cell wall structure and virulence in favor of carbohydrates used for energy storage and stress resistance. Collectively, these findings suggest that activation of the conidiation pathway may be a promising approach for the development of new agents to prevent or treat A. fumigatus infection.
    背景与目标: : 免疫功能低下的宿主吸入机会性霉菌烟曲霉的分生孢子可导致侵袭性肺部疾病。逃避免疫防御的吸入分生孢子萌发,形成侵入肺组织的丝状菌丝。在人类宿主的侵袭性感染期间,很少发生分生孢子,从而使大量的真菌能量直接用于营养生长。我们假设在感染过程中强制诱导分生孢子可以抑制烟曲霉的营养生长,从而损害该生物引起疾病的能力。为了研究分生孢子调节途径失调对烟曲霉毒力的影响,在强力霉素诱导的启动子的控制下表达了分生孢子的关键转录调节因子 (brlA)。在体外和体内均观察到对强力霉素的反应,时间和剂量依赖性brlA过表达。在体外诱导的分生孢子的营养生长条件下,诱导型brlA过表达菌株暴露于低剂量的强力霉素中,而高剂量则阻止了生长。在无脊椎动物和侵袭性曲霉病小鼠模型中,brlA的过表达减弱了烟曲霉的毒力。RNA测序研究和表型分析表明,brlA过表达导致细胞信号传导,氨基酸和碳水化合物代谢的改变,包括海藻糖生物合成的显着上调和多糖毒力因子半乳糖半乳聚糖生物合成的下调。这一概念验证研究表明,烟曲霉分生孢子途径的激活可以降低毒力,并表明brlA诱导的小分子可能有望成为烟曲霉感染的一类新疗法。重要性烟曲霉通过产生空气中的孢子 (分生孢子) 繁殖,称为分生孢子的过程。在免疫功能低下的个体中,吸入的烟曲霉分生孢子可以发芽并形成细丝,穿透并损伤肺组织; 但是,在侵入性感染期间不会发生分生孢子。在这项研究中,我们证明了烟曲霉细丝中分生孢子的强制激活可以阻止其生长并损害这种真菌在昆虫和小鼠侵袭性感染模型中引起疾病的能力。分生孢子的激活与烟曲霉代谢的深刻变化有关,包括从细胞壁结构和毒力所需的多糖合成转向用于能量存储和抗逆性的碳水化合物。总的来说,这些发现表明,分生孢子途径的激活可能是开发预防或治疗烟曲霉感染的新药物的有希望的方法。
  • 【构巢曲霉实验种群补偿适应的基因组学。】 复制标题 收藏 收藏
    DOI:10.1534/g3.116.036152 复制DOI
    作者列表:Dettman JR,Rodrigue N,Schoustra SE,Kassen R
    BACKGROUND & AIMS: :Knowledge of the number and nature of genetic changes responsible for adaptation is essential for understanding and predicting evolutionary trajectories. Here, we study the genomic basis of compensatory adaptation to the fitness cost of fungicide resistance in experimentally evolved strains of the filamentous fungus Aspergillus nidulans The original selection experiment tracked the fitness recovery of lines founded by an ancestral strain that was resistant to fludioxonil, but paid a fitness cost in the absence of the fungicide. We obtained whole-genome sequence data for the ancestral A. nidulans strain and eight experimentally evolved strains. We find that fludioxonil resistance in the ancestor was likely conferred by a mutation in histidine kinase nikA, part of the two-component signal transduction system of the high-osmolarity glycerol (HOG) stress response pathway. To compensate for the pleiotropic negative effects of the resistance mutation, the subsequent fitness gains observed in the evolved lines were likely caused by secondary modification of HOG pathway activity. Candidate genes for the compensatory fitness increases were significantly overrepresented by stress response functions, and some were specifically associated with the HOG pathway itself. Parallel evolution at the gene level was rare among evolved lines. There was a positive relationship between the predicted number of adaptive steps, estimated from fitness data, and the number of genomic mutations, determined by whole-genome sequencing. However, the number of genomic mutations was, on average, 8.45 times greater than the number of adaptive steps inferred from fitness data. This research expands our understanding of the genetic basis of adaptation in multicellular eukaryotes and lays out a framework for future work on the genomics of compensatory adaptation in A. nidulans.
    背景与目标: : 了解负责适应的遗传变化的数量和性质对于理解和预测进化轨迹至关重要。在这里,我们研究了在实验进化的丝状真菌构巢曲霉菌株中,对杀真菌剂抗性的适应性成本进行补偿性适应的基因组基础。最初的选择实验跟踪了由祖先菌株建立的品系的适应性恢复,该品系对氟二氧嘧啶具有抗性,但在没有杀真菌剂的情况下支付了健身费用。我们获得了祖先的A.Niddulans菌株和八个实验进化的菌株的全基因组序列数据。我们发现,祖先的氟二氧杂环素抗性可能是由组氨酸激酶nikA的突变赋予的,该突变是高渗透压甘油 (HOG) 应激反应途径的两组分信号转导系统的一部分。为了补偿抗性突变的多效性负面影响,在进化系中观察到的随后的适应性增益可能是由HOG途径活性的二次修饰引起的。代偿性适应性增加的候选基因显着被应激反应功能所代表,其中一些与HOG途径本身特别相关。在进化品系中,基因水平的平行进化很少见。根据适应性数据估算的自适应步骤的预测数量与通过全基因组测序确定的基因组突变数量之间存在正相关关系。然而,基因组突变的数量平均比从适应性数据推断的适应性步骤的数量大8.45倍。这项研究扩大了我们对多细胞真核生物适应的遗传基础的理解,并为未来在a.Niddulans的补偿性适应基因组学方面的工作奠定了框架。
  • 【铁饥饿导致曲霉中Cu/Zn-超氧化物歧化酶的表达增加。】 复制标题 收藏 收藏
    DOI:10.1016/s0014-5793(00)02206-7 复制DOI
    作者列表:Oberegger H,Zadra I,Schoeser M,Haas H
    BACKGROUND & AIMS: :In a search for iron-regulated proteins of Aspergillus nidulans and Aspergillus fumigatus a 16-kDa protein was identified which is about 5-fold upregulated during iron starvation in both species and which can be approximately 500-fold enriched by simple one-step chromatography on Amberlite XAD-16 resin. N-terminal protein sequence analysis and cloning of the respective A. nidulans cDNA identified this protein as a Cu/Zn-superoxide dismutase (SODA). Northern analysis revealed that upregulation of sodA expression occurs at the level of transcript accumulation. This seems to be a specific low iron response and not a general starvation answer since sodA transcript levels do not respond to carbon or nitrogen starvation. In contrast, copper depletion leads to transcriptional downregulation of sodA. Furthermore, sodA expression was found still to be subject to iron regulation in an A. nidulans mutant lacking SREA, a regulator of iron homeostasis, indicating that sodA expression is regulated by an SREA-independent mechanism. The data presented suggest that SODA plays a protective role under iron deplete conditions.
    背景与目标: : 在寻找构巢曲霉和烟曲霉的铁调节蛋白时,鉴定出一种16 kDa蛋白,该蛋白在两种物种的铁饥饿期间上调约5倍,并且可以通过简单的一步色谱法富集约500倍在Amberlite XAD-16树脂上。N末端蛋白序列分析和相应的A.Niddulans cDNA的克隆将该蛋白鉴定为Cu/Zn-超氧化物歧化酶 (SODA)。Northern分析表明,苏打表达的上调发生在转录本积累的水平上。这似乎是一种特定的低铁反应,而不是一般的饥饿答案,因为苏打水转录水平对碳或氮的饥饿没有反应。相反,铜的耗竭导致苏打的转录下调。此外,在缺乏铁稳态调节剂SREA的A.Niddulans突变体中,发现苏打表达仍受铁调节,这表明苏打表达受SREA独立机制的调节。提供的数据表明,苏打在铁耗尽条件下起着保护作用。
  • 【构巢曲霉黄嘌呤羟化酶活性位点变体的表征。】 复制标题 收藏 收藏
    DOI:10.1016/j.abb.2007.11.002 复制DOI
    作者列表:Li M,Müller TA,Fraser BA,Hausinger RP
    BACKGROUND & AIMS: :Xanthine/alpha-ketoglutarate (alphaKG) dioxygenase (XanA) is a non-heme mononuclear Fe(II) enzyme that decarboxylates alphaKG to succinate and CO(2) while hydroxylating xanthine to generate uric acid. In the absence of a XanA crystal structure, a homology model was used to target several putative active site residues for mutagenesis. Wild-type XanA and ten enzyme variants were purified from recombinant Escherichia coli cells and characterized. The H149A and D151A variants were inactive and the H340A variant exhibited only 0.17% of the wild-type enzyme activity, consistent with the proposed role of His149, Asp151, and His340 as Fe ligands. The K122A variant led to a 2-fold increase in the K(d) of alphaKG as measured by fluorescence quenching analysis, in agreement with Lys122 acting to stabilize the binding of alphaKG. The N358A variant exhibited a 23-fold decrease in k(cat)/K(m) compared to wild-type XanA, pointing to a key role of Asn358 in catalysis. 9-Methylxanthine was exploited as an alternate substrate, and the C357A, E137A, and D138A variants were found to exhibit relatively enhanced activity consistent with Cys357, Glu137, and Asp138 being proximal to N-9 or involved in its proper positioning. 6,8-Dihydroxypurine was identified as a slow-binding competitive inhibitor of XanA, and significant decreases (E137A and D138A) or increases (Q356A and N358A) in K(i)(app) of the variants were interpreted in terms of distinct interactions between this compound and the corresponding active site side chains. Further support for Cys357 residing at the active site was obtained using thiol-specific reagents that inactivated wild-type enzyme (with partial protection by substrate), whereas the C357A variant was resistant to these reagents. The Q101A, Q356A, and C357A variants showed elevated ferroxidase activity in the absence of substrates, pointing to the presence of the corresponding side chains at the active site. These results confirm most aspects of the homology model and provide additional insight into the enzyme reactivity.
    背景与目标: : 黄嘌呤/α-酮戊二酸 (αkg) 双加氧酶 (XanA) 是一种非血红素单核Fe(II) 酶,可将 αkg脱羧为琥珀酸和CO(2),同时将黄嘌呤羟基化以生成尿酸。在没有XanA晶体结构的情况下,使用同源性模型来靶向几个推定的活性位点残基进行诱变。从重组大肠杆菌细胞中纯化野生型XanA和十种酶变体并进行了表征。H149A和D151A变体是无活性的,H340A变体仅表现出0.17% 的野生型酶活性,这与His149,Asp151和His340作为Fe配体的拟议作用一致。通过荧光猝灭分析测得,K122A变体导致 αkg的K(d) 增加了2倍,这与Lys122起稳定 αkg结合的作用一致。与野生型XanA相比,N358A变体的k(cat)/K(m) 降低了23倍,表明Asn358在催化中的关键作用。利用9-甲基黄嘌呤作为替代底物,发现C357A,E137A和D138A变体表现出相对增强的活性,与Cys357,Glu137和Asp138接近N-9或参与其正确定位一致。6,8-二羟基嘌呤被鉴定为XanA的慢结合竞争性抑制剂,并且变体的K(i)(app) 中的显着降低 (E137A和D138A) 或增加 (Q356A和N358A) 解释为该化合物与相应活性位点侧链之间的不同相互作用。使用使野生型酶失活的硫醇特异性试剂 (通过底物部分保护) 获得了驻留在活性位点的Cys357的进一步支持,而C357A变体对这些试剂具有抗性。Q101A,Q356A和C357A变体在不存在底物的情况下显示出更高的铁氧化酶活性,表明在活性位点存在相应的侧链。这些结果证实了同源模型的大多数方面,并提供了对酶反应性的更多了解。
  • 【丝状真菌构巢曲霉基因高效置换的快速方法。】 复制标题 收藏 收藏
    DOI:10.1093/nar/28.22.e97 复制DOI
    作者列表:Chaveroche MK,Ghigo JM,d'Enfert C
    BACKGROUND & AIMS: :The construction of mutant fungal strains is often limited by the poor efficiency of homologous recombination in these organisms. Higher recombination efficiencies can be obtained by increasing the length of homologous DNA flanking the transformation marker, although this is a tedious process when standard molecular biology techniques are used for the construction of gene replacement cassettes. Here, we present a two-step technology which takes advantage of an Escherichia coli strain expressing the phage lambda Red(gam, bet, exo) functions and involves (i) the construction in this strain of a recombinant cosmid by in vivo recombination between a cosmid carrying a genomic region of interest and a PCR-generated transformation marker flanked by 50 bp regions of homology with the target DNA and (ii) genetic exchange in the fungus itself between the chromosomal locus and the circular or linearized recombinant cosmid. This strategy enables the rapid establishment of mutant strains carrying gene knock-outs with efficiencies >50%. It should also be appropriate for the construction of fungal strains with gene fusions or promoter replacements.
    背景与目标: : 突变真菌菌株的构建通常受到这些生物中同源重组效率低下的限制。通过增加位于转化标记物两侧的同源DNA的长度,可以获得更高的重组效率,尽管当使用标准分子生物学技术构建基因替换盒时,这是一个繁琐的过程。在这里,我们提出了一种两步技术,该技术利用了表达噬菌体 λ 红 (gam,bet,exo) 的功能,并涉及 (i) 通过携带目标基因组区域的粘粒与PCR生成的转化标记之间的体内重组,在该菌株中构建重组粘粒,其两侧是与目标DNA同源性的50 bp区域,并且 (ii) 真菌本身之间的遗传交换染色体基因座和环状或线性化的重组粘粒。该策略能够以> 50% 的效率快速建立携带基因敲除的突变株。它也应适用于具有基因融合或启动子替换的真菌菌株的构建。
  • 【与肺包虫病相关的曲霉定植的外科治疗。】 复制标题 收藏 收藏
    DOI: 复制DOI
    作者列表:Vasquez JC,Montesinos E,Rojas L,Peralta J,Delarosa J,Leon JJ
    BACKGROUND & AIMS: :Colonization with Aspergillus sp. usually occurs in previously formed lung cavities. Cystectomy is a widely used surgical technique for hydatid lung disease that can also leave residual cavities and potentially result in aspergilloma. We present two cases of this rare entity and a case with Aspergillus sp. colonization of an existing ruptured hydatid cyst.
    背景与目标: : 曲霉属的定植。通常发生在先前形成的肺腔中。膀胱切除术是一种广泛使用的用于包虫病肺疾病的外科手术技术,该技术也可能留下残留的腔并可能导致曲霉菌。我们介绍了这种罕见实体的两例病例和曲霉属的病例。现有破裂的包虫囊肿定植。
  • 【兔实验性曲霉菌心内膜炎。】 复制标题 收藏 收藏
    DOI: 复制DOI
    作者列表:Carrizosa J,Kohn C,Levison ME
    BACKGROUND & AIMS: :Aspergillus endocarditis in man usually occurs on prosthetic cardiac valves and gives rise to large vegetations which embolize easily producing peripheral organ infarction and infection. Blood cultures are usually sterile and the disease is difficult to cure with antimicrobial agents. Aspergillus endocarditis was studied in rabbits to determine the course, degree of fungemia, and response to treatment with amphotericin B (A), 5 flucytosine (5 FC) or A + 5 FC. Polyethylene tubing was introduced into the left ventricle through the carotid artery and 24 hours later animals were inoculated with 10(4) to 10(7) spores of a strain of Aspergillus fumigatus. Large occlusive vegetations developed on the aortic valves. Spontaneous mortality reached 67 per cent after 3 days. Despite large aggregates of mycelia seen beneath a layer of amorphous material on microscopic sections, vegetations contained only 10(3) to 10(5) colony forming units (CFU) of aspergilli per gram, suggesting the aspergilli in tissues were clumped. Disseminated infection involving kidney, lung, liver, spleen, and brain occurred. Animals without intracardiac tubing which received the same inoculum of spores did not develop endocarditis, but showed evidence of disseminated infection. Blood after 24 hours of infection grew aspergilli only when large volumes were cultured and then only a small fraction of the total volume of blood obtained for culture yielded aspergilli, suggesting that the aspergilli in blood were clumped. Sterile vegetations in the absence of an intracardiac catheter were resistant to infection with aspergilli, but once established, infection with aspergilli persisted on vegetations despite removal of the catheter. Treatment of infected animals with A (1 mg. per kilogram), 5 FC (25 or 50 mg. per kilogram) or A + 5 FC daily intraperitoneally, significantly lowered the number of CFU per gram of vegetation.
    背景与目标: : 人类的曲霉菌心内膜炎通常发生在人工心脏瓣膜上,并引起大的赘生物,这些赘生物容易栓塞,从而产生外周器官梗塞和感染。血液培养物通常是无菌的,这种疾病很难用抗菌剂治愈。在兔子中研究了曲霉菌心内膜炎,以确定其病程,菌血症程度以及对两性霉素b (A),5氟胞嘧啶 (5 FC) 或A 5 FC治疗的反应。将聚乙烯管通过颈动脉引入左心室,并在24小时后给动物接种烟曲霉菌株的10(4) 至10(7) 个孢子。主动脉瓣上形成了大的闭塞性植被。3天后自发死亡率达到67%。尽管在微观切片上的无定形材料层下看到了大量的菌丝体,但植被每克仅包含10(3) 至10(5) 个菌落形成单位 (CFU) 的曲霉,这表明组织中的曲霉结块了。发生累及肾、肺、肝、脾和脑的播散性感染。没有接受相同孢子接种的心内管的动物不会发生心内膜炎,但显示出弥漫性感染的证据。感染24小时后,血液仅在大量培养时才生长曲霉,然后在用于培养的血液总体积中只有一小部分产生曲霉,这表明血液中的曲霉结块了。在没有心内导管的情况下,无菌植物对曲霉感染具有抵抗力,但是一旦确定,尽管移除了导管,曲霉感染仍持续存在。每天腹膜内用A (1 mg。每公斤),5 FC (25或50 mg。每公斤) 或5 FC处理受感染的动物,可显着降低每克植被的CFU数量。
  • 【通过响应面方法 (RSM) 优化培养基参数,以增强曲霉属的角质酶的生产。RL2Ct。】 复制标题 收藏 收藏
    DOI:10.1007/s13205-016-0460-4 复制DOI
    作者列表:Kumari V,Kumar V,Chauhan R,Asif M,Bhalla TC
    BACKGROUND & AIMS: :Cutinases are hydrolytic enzymes which catalyzes esterification and trans-esterification reactions that make them highly potential industrial biocatalyst. In the present investigation microorganisms showing cutinase activity were isolated from plant samples. The strain showing maximum cutinase activity was identified by 18S rDNA sequencing as Aspergillus sp. RL2Ct and was selected for further studies. To achieve maximum enzyme production, the medium components affecting cutinase production were screened by Plackett-Burman followed by central composite design. The results obtained suggested that cutin, temperature and CaCl2 have influenced the cutinase production significantly with very high confidence levels. Cutinase production was maximum (663 U/mg protein) when using cutin prepared from orange peel as sole source of carbon. An overall 4.33-fold increase in the production of cutinase was observed after optimization of culture conditions (including 2.5-fold increase using RSM) during 24 h of incubation. The production time of Aspergillus sp. RL2Ct cutinase is significantly lower than the most of the earlier reported cutinase-producing fungus.
    背景与目标: : 角质酶是一种水解酶,可催化酯化和反式酯化反应,使其具有很高的工业生物催化剂潜力。在本研究中,从植物样品中分离出具有角质酶活性的微生物。通过18S rDNA测序将显示最大角质酶活性的菌株鉴定为曲霉属。RL2Ct并选择用于进一步研究。为了实现最大的酶产量,通过Plackett-Burman筛选了影响角质酶产量的培养基成分,然后进行了中央复合设计。获得的结果表明,角质,温度和CaCl2以很高的置信度显着影响了角质酶的产生。当使用从橙皮制备的角质作为唯一碳源时,角质酶产量最大 (663 U/mg蛋白质)。在培养24小时期间,在优化培养条件 (包括使用RSM增加2.5倍) 之后,观察到角质酶的产生总体增加了4.33倍。曲霉属 (Aspergillus sp. RL2Ct) 角质酶的生产时间明显低于早期报道的大多数角质酶生产真菌。

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