烟曲霉pbs2基因在热应激与胞壁应激的双重应激中的作用

目的 通过研究烟曲霉对细胞壁合成干扰物质及棘白菌素类药物的敏感性、烟曲霉在受热应激后HOG通路成分的表达变化探讨pbs2基因在热应激和胞壁应激的双重应激中的作用.方法 烟曲霉野生株AF293和pbs2突变株在含钙荧光白(Calcofluor white,CFW),刚果红(Congo red,CR)和十二烷基硫酸钠(So...     

额外拷贝ERG6基因对烟曲霉的影响

通过构建烟曲霉ERG6基因额外拷贝株．研究该基因对烟曲霉生长速度、抗药物敏感性的影响。在烟曲霉基因组找出烟曲霉可能的ERG6基因的开放读码框(ORF),PCR扩增ERG6的ORF连同其上下游各约1 kb的DNA片段,利用DNA重组的方法将该片段克隆到载体pRG-AMA1-NotI。用重组后的质粒转化烟曲霉尿嘧啶营养缺陷株AF293．1。在MM和YAG培养基上观察转化子的生长速度。采用纸片扩散法和微量液基稀释法测定转化子对抗真菌药物敏感性。烟曲霉基因组中存在一个拷贝的ERG6基因,ORF大小为1,256 bp。其编码的蛋白与白念珠菌、酿酒酵母固醇甲基转移酶(Ers6p)的氨基酸相同率分别为57%和50%,相似率分别为70%和63%。烟曲霉中ERG6基因被成功克隆到了pRG-AMA1-Not I,产生了质粒pERG6。用pERG6和空载体pRG-AMA1-Not I转化AF293．1后,分别得到转化子AF-pERG6和AF-empty。AF-pERG6在MM和YAG培养基上的生长速度均比AF-empty慢。AF-pERG6和AF-empty对伊曲康唑、伏力康唑、特比萘芬、两性霉素B、卡泊芬净、灰黄霉素的敏感性没有差异。ERG6基因额外拷贝不影响烟曲霉对伊曲康唑、伏力康唑、特比萘芬、两性霉素B、卡泊芬净、灰黄霉素的敏感性,但是能使烟曲霉的生长速度减慢。     

压力应激影响烟曲霉致病力的研究进展

烟曲霉是环境中广泛存在的腐生真菌,是重要的机会致病菌。近年来随着免疫受损人群的增多,烟曲霉引起侵袭性曲霉病不断增加,在深部丝状真菌感染中居于首位。由于宿主体内环境与自然环境存在诸多差异,使得烟曲霉孢子适应体内环境条件是其能够生存并致病的前提。研究发现烟曲霉适应宿主环境的能力,如温度、氧化应激、渗透压、缺氧、营养缺乏等,与其致病力密切相关。对压力应激与致病力关系的研究有助于解析烟曲霉的致病机制,可为研发烟曲霉感染诊治的新途径提供理论基础。本文对烟曲霉的压力应激与其致病力的关系进行了综述。     

肉桂醛、柠檬醛对烟曲霉色素及关键基因alb1mRNA表达的影响

研究了肉桂醛、柠檬醛对烟曲霉色素及其关键基因alb1 mRNA表达的影响。结果表明随着肉桂醛、柠檬醛浓度的增加,菌苔逐渐变稀、薄且烟绿色色素逐渐变淡,甚至白化;肉桂醛、柠檬醛对alb1基因mRNA表达有明显抑制作用,随着药物浓度的降低这种抑制作用亦相应呈现不同程度的减弱,肉桂醛、柠檬醛可能通过抑制alb1 mRNA的表达导致烟曲霉缺失烟绿色色素。     

热应激对大鼠丘脑、纹状体PLA2活性的影响

热应激作为一种外部应激信号 ,可以引起机体信号系统的反应。近年来 ,磷脂酰肌醇 (ＰＩ)信号转导系统受到人们的重视 ,它包括ＰＩ、磷脂酶Ｃ(ＰＬＣ)、磷脂酶Ａ2 (ＰＬＡ2 )、三磷酸肌醇 (ＩＰ3)、细胞内钙离子浓度 ([Ｃａ2 ]ｉ)等 ,参与许多重要的生理生化过程。其中 ,ＰＬＡ2 作为ＰＩ信号系统活化的启动因子 ,它在热应激时的变化颇受关注。离体实验表明 ,热可致ＰＬＡ2 的活性增强 ,在体实验中发现 ,热可使肝、肺组织中ＰＬＡ2 活性增强 ,膜磷脂 (ＭＰＬ)降解增强 ,用ＰＬＡ2 拮抗剂和膜稳定剂阿的平 ,可调节ＭＰＬ代谢 ,防止中暑发生 ,…     

碱性pH条件下白念珠菌钙离子通道CCH1和MID1基因对钙内流的影响及Crz1p转录因子对其的调控作用

白念珠菌Candida albicans对环境pH的适应能力与其致病性有密切关系,钙信号转导途径介导许多环境压力的应答并伴随胞内钙离子浓度的瞬间变化。通过构建钙通道基因CCH1和MID1的缺失突变株,在碱性pH条件下,研究其对胞内钙内流的影响以及转录因子Crz1p对CCH1和MID1基因的调控作用。使用二步法PCR介导的基因敲除技术构建cch1Δ/Δ和mid1Δ/Δ突变菌株,利用流式细胞术比较野生型和突变型菌株在碱性pH条件刺激下胞内钙的瞬间变化,进一步构建pPHO89-LacZ重组质粒并利用β-半乳糖苷     

不同生长阶段的烟曲霉对果蝇自噬水平的影响

目的探究不同生长阶段的烟曲霉对果蝇自噬水平的影响。方法用沙保弱平板培养烟曲霉,收获静息孢子;用沙保弱液体培养基震荡培养烟曲霉,在不同时间收获膨胀孢子、菌丝。以3种形态的烟曲霉分组处理转基因果蝇,组织切片观察烟曲霉形态,Rt-PCR(逆转录PCR)检测自噬相关蛋白LC3BⅡ、Beclin-1mRNA的转录水平,Western blot检测LC3BⅡ、Beclin-1的表达量。结果组织切片观察菌的形态与注入时的形态相符,Rt-PCR检测LC3BⅡ、Beclin-1mRNA的转录水平升高,同时,感染膨胀烟曲霉孢子及菌丝组的果蝇LC3BⅡ、Beclin-1蛋白的表达水平升高。结论烟曲霉膨胀孢子及菌丝能显著提高果蝇自噬水平,而静息孢子不能引起果蝇自噬应答。     

海洋红藻龙须菜对2种逆境温度胁迫的应激生理响应

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烟曲霉对哮喘大鼠糖皮质激素受体和β2-肾上腺素能受体表达的影响

为了揭示烟曲霉(Aspergillus fumigatus)对哮喘大鼠糖皮质激素受体(glucocorticoid receptor, GCR)和β2-肾上腺素能受体(β2-adrenergic receptor,β2AR)表达的影响,本研究利用卵白蛋白致敏和激发建立大鼠哮喘模型,并考察烟曲霉对大鼠肺组织GCR、β2AR、TLR2和TLR4表达的影响。研究显示,与对照组相比,烟曲霉组、卵白蛋白组和卵白蛋白+烟曲霉组的GCR m RNA和蛋白表达均显著下降。与卵白蛋白组相比,卵白蛋白+烟曲霉组的GCR mRNA和蛋白表达显著降低。烟曲霉对β2AR m RNA和蛋白的表达无明显影响。与对照组相比,暴露于烟曲霉后的大鼠肺组织TLR2 m RNA水平显著升高,而对照组和卵白蛋白组的TLR2mRNA水平无显著差异。各组TLR4 mRNA的水平无显著差异。卵白蛋白+烟曲霉组大鼠的肺组织样品通过培养呈阳性,并且可见烟曲霉菌落在马铃薯葡萄糖琼脂培养基上生长。PASM染色也显示该组大鼠肺部可见烟曲霉孢子定植,而其他组均为阴性且未发现肺部烟曲霉孢子。本研究表明,烟曲霉可下调哮喘患者肺组织中GCR mRNA和蛋白的表达。烟曲霉可能通过下调GCR的表达降低糖皮质激素的反应性,导致糖皮质激素抵抗性哮喘的发生,且具有正常免疫功能的大鼠能及时清除吸入的烟曲霉孢子,而哮喘或免疫抑制状态有助于烟曲霉在呼吸道定植。     

LPS对冷应激大鼠Mφ、Sertoli细胞分泌IL-1的影响

目的 为探讨LPS对冷应激大鼠外周血巨噬细胞（Mφ）和睾丸支持细胞（Sertoli）分泌IL-1的影响,本文检测了大鼠Mφ细胞和Sertoli细胞IL-1的含量。方法体外分离培养大鼠Mφ细胞、大鼠睾丸Sertoli细胞和小鼠胸腺细胞,设37℃对照组与0℃、-4℃冷应激实验组,IL-1含量测定采用放免分析方法。结果外周血MφIL-1含量：0℃组高于37℃对照组,分别为20444.7±8936.3和10281.2±1435.8,差异有显著性（P〈0.05）;-4℃和37℃对照组分别为10307.2±2648.6和10281.2±1435.8,差异无显著性（P〉0.05）。睾丸Sertoli细胞IL-1含量：0℃组高于37℃对照组,LPS-和LPS＋分别为54.31±0.35和41.52±0.42与99.74±0.09和82.96±0.33,差异有显著性（P〈0.05）;-4℃和37℃对照组分别为38.11±0.14和41.52±0.42与45.84±0.11和82.96±0.33,差异有显著性（P〈0.05）。结论冷应激抑制LPS诱导Mφ和Sertoli细胞的IL-1含量分泌,但在早期Mφ和Sertoli细胞均出现免疫系统激活的过程,其作用机制有待深入研究。     

Ag/TiO2纳米材料对烟曲霉的抑制作用及机制

目的 合成Ag/TiO2纳米材料,对其进行表征测定,并探讨其对烟曲霉的抑制作用及具体机制。方法 采用光催化还原法制备Ag/TiO2纳米材料,紫外可见分析和扫描电镜对其进行表征测定;微量液基稀释法检测对烟曲霉的最低抑菌浓度（MIC）,以及生物量的抑制作用;ELISA试剂盒检测对真菌谷胱甘肽还原酶、总谷胱甘肽、线粒体膜电位的影响,荧光显微镜检测活性氧的产生。结果 成功制备Ag/TiO2纳米材料,分布均匀;对烟曲霉的MIC值为0.5 μg/mL,能完全抑制烟曲霉生物量,与单独纳米银相比,具有更好的抗菌活性。机制研究发现其主要通过降低烟曲霉体内谷胱甘肽及其还原酶的含量,诱导过量活性氧的产生,最终导致线粒体膜电位降低,使真菌细胞发生凋亡。结论 Ag/TiO2纳米材料可有效阻断烟曲霉等真菌在空气中的传播,具有广泛的应用前景。     

维生素D对烟曲霉感染的肺巨噬细胞 自噬机制的初步研究

目的探究维生素D在鼠肺细胞感染烟曲霉后对细胞自噬的影响。方法用一定量的烟曲霉活化孢子感染细胞后,一组细胞加入维生素D(维生素D组),一组细胞加入生理盐水,感染一定时间后用溶酶体探针检测自噬相关分子的表达;收集各组细胞并裂解细胞,离心取上清,用Western-blot法检测上清液中的LC3BII、Dectin-1及ROS的表达水平。结果活化的孢子感染肺巨噬细胞后,维生素D组自噬体与溶酶体共定位减少、吞噬孢子的速率在减少以及ROS水平降低,其对应的胞内Dectin-1、ROS、LC3BII减少且差异有统计学意义。结论烟曲霉感染肺巨噬细胞后,维生素D可通过减弱细胞自噬体与溶酶体的融合并下调自噬信号通路蛋白的表达以达到抵抗烟曲霉感染的作用。     

青蒿琥酯抗蜡螟烟曲霉感染自噬机制初步研究

目的 探究青蒿琥酯在蜡螟感染烟曲霉后,对蜡螟的自噬相关蛋白的表达影响。方法 用一定量的烟曲霉的活化孢子感染蜡螟,经过1 h,用青蒿琥酯注射一组蜡螟,两性霉素B注射一组蜡螟,剩余的作为感染组。12 h后,取各组蜡螟进行病理切片染色,观察各组的病理情况;取各组的蜡螟的淋巴液,收集各组的孢子,用真菌活性检测试剂盒检测孢子活性并将淋巴细胞分离、裂解,离心取上清,用Western-blot法检测上清液中的Dectin-1、ROS、LC3Ⅱ的表达水平。结果 青蒿琥酯注射组的蜡螟病理切片中的孢子比感染组数量少且聚集在一起,未长出菌丝,而体外分离的真菌孢子,青蒿琥酯组的活性明显受到抑制。经Western-blot显示青蒿琥酯能增强淋巴细胞Dectin-1、ROS、LC3Ⅱ的表达。结论 青蒿琥酯可通过抑制烟曲霉的活性和增强蜡螟淋巴细胞的自噬水平,对抗蜡螟烟曲霉感染。     

Aspergillus fumigatus exoβ(1‐3)glucanases family GH55 are essential for conidial cell wall morphogenesis

The cell wall of Aspergillus fumigatus is predominantly composed of polysaccharides. The central fibrillar core of the cell wall is composed of a branched β(1‐3)glucan, to which the chitin and the galactomannan are covalently bound. Softening of the cell wall is an essential event during fungal morphogenesis, wherein rigid cell wall structures are cleaved by glycosyl hydrolases. In this study, we characterised the role of the glycosyl hydrolase GH55 members in A. fumigatus fungal morphogenesis. We showed that deletion of the six genes of the GH55 family stopped conidial cell wall maturation at the beginning of the development process, leading to abrogation of conidial separation: the shape of conidia became ovoid, and germination was delayed. In conclusion, the reorganisation and structuring of the conidial cell wall mediated by members of the GH55 family is essential for their maturation, normal dissemination, and germination.     

The effect of gene tubC on the vegetative growth of benomyl-resistant strains of Aspergillus nidulans

Abstract Two benomyl-resistant mutants, benD3 tubC41 and benD4 tubC42 , of Aspergillus nidulans were isolated after UV treatment. The tubC mutations permitted good conidiation of these strains in culture media containing benomyl and were responsible for increasing their benomyl resistance levels. This implies that β3-tubulin, a product of the tubC gene, in addition to being involved in fungal conidiation, participates in the vegetative growth of the fungus. The tubC gene was located in linkage group I.     

Neuroprotective effect of PEP-1-peroxiredoxin2 on CA1 regions in the hippocampus against ischemic insult

Background

Oxidative stress is a leading cause of various diseases, including ischemia and inflammation. Peroxiredoxin2 (PRX2) is one of six mammalian isoenzymes (PRX1–6) that can reduce hydrogen peroxide (H₂O₂) and organic hydroperoxides to water and alcohols.

Methods

We produced PEP-1-PRX2 transduction domain (PTD)-fused protein and investigated the effect of PEP-1-PRX2 on oxidative stress-induced neuronal cell death by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Western blot, immunofluorescence microscopy, and immunohistochemical analysis.

Results

Our data showed that PEP-1-PRX2, which can effectively transduce into various types of cells and brain tissues, could be implicated in suppressing generation of reactive oxygen species, preventing depolarization of the mitochondrial membrane, and inhibiting the apoptosis pathway in H₂O₂-stimulated HT22, murine hippocampal neuronal cells, likely resulting in protection of HT22 cells against H₂O₂-induced toxicity. In addition, we found that in a transient forebrain ischemia model, PEP-1-PRX2 inhibited the activation of astrocytes and microglia in the CA1 region of the hippocampus and lipid peroxidation and also prevented neuronal cell death against ischemic damage.

Conclusions

These findings suggest that the transduced PEP-1-PRX2 has neuroprotective functions against oxidative stress-induced cell death in vitro and in vivo.

General significance

PEP-1-PRX2 could be a potential therapeutic agent for oxidative stress-induced brain diseases such as ischemia.     

The role of cytochrome P450 2E1 on ethanol-mediated oxidative stress and HIV replication in human monocyte-derived macrophages

BackgroundAlcohol consumption is considered to be a major health problem among people living with HIV/AIDS. Our previous reports have shown that ethanol reduced intracellular concentrations of antiretroviral drugs elvitegravir and darunavir in the HIV-1-infected U1 cell line. Ethanol also increased HIV-1 replication despite the presence of elvitegravir. Our previous finding has also shown that the levels of cytochrome P450 enzyme 2E1 (CYP2E1) and oxidative stress in blood monocytes were induced, while the concentration of alcohol in the plasma was reduced in HIV-1-infected alcohol users compared to uninfected alcohol users. However, the role of CYP2E1 in ethanol-enhanced oxidative stress and HIV-1 replication is still unclear.MethodsThis study examined the chronic effects (14 days) of ethanol on HIV viral load, oxidative DNA damage, expression of CYP2E1, expression of antioxidant enzymes (AOEs), expression of reactive oxygen species (ROS) in human monocyte-derived macrophages (MDM). Further, to evaluate the role of CYP2E1 in mediating ethanol-induced viral replication, CYP2E1 siRNA and CYP2E1 selective inhibitor were used in the HIV-1-infected U1 cell line following ethanol treatment.ResultsChronic ethanol exposure demonstrated an increase in oxidative DNA damage and CYP2E1 expression in both non-infected and HIV-1-infected MDM. Our results showed that ethanol chronic exposure increased HIV-1 replication by ~3-fold in HIV-1-infected MDM. This ethanol-enhanced HIV-1 replication was associated with an increased oxidative DNA damage, an increased expression of CYP2E1, and a decreased expression of antioxidant enzyme PRDX6. In HIV-1-infected U1 cell line, we observed a decreased viral replication (~30%) and a decreased DNA damage (~100%) after repression of CYP2E1 by siRNA, upon ethanol exposure. We also observed a decreased viral replication (~25%) after inhibition of CYP2E1 by using selective CYP2E1 inhibitor.ConclusionsThe data suggest that chronic ethanol exposure increases HIV-1 replication in MDM, at least in part, through CYP2E1-mediated oxidative stress. These results are clinically relevant to potentially find effective treatment strategies for HIV-1-infected alcohol users.     

The Keap1 signaling in the regulation of HSP90 pathway

The Keap1 protein is the master modulator of Nrf2 pathway; moreover, it is the hub of such important processes as cancer, cell stress, inflammation, and chemio- and radio-resistance. That is why Keap1 has become an intriguing pharmacological target. Many recent data show that Keap1 interacts with HSP90 protein. In this study, we use ferulic acid (FA) as antioxidant and anti-inflammatory agent, able to relieve inflammatory response. It is known that treatment with 100 μg of FA can significantly decrease the oxidative stress, so it turns to be useful to study the antioxidant regulation. The RAW 264.7 cells transfected with si-Keap1 and LPS treated are the in vitro model used to study the effects of Keap1 silencing on HSP90 activities and the FA antioxidant modulation. Immunoblot data and qPCR analysis show that Keap1 is involved in HSP90 modulation and on anti-oxidative response. Keap1 silencing affects negatively COX2 activation; in fact western blot and qPCR analysis conducted on RAW 264.7 cells Keap1silenced highlight that LPS treatment does not induce COX2 activation. In addition, the FA anti-oxidative and modulatory effect is abolished in COX2 pathway. The same results are point out using human A549 cell line with an allelic mutation on Keap1 gene, and the protein results are partially inactive. This preliminary study points out that Keap1protein is involved in HSP90 and anti-oxidative pathway regulation.