烟曲霉硫氧还蛋白还原酶双抗体夹心ELISA法的建立及初步应用

目的建立检测烟曲霉硫氧还蛋白还原酶Gli T(thioredoxin reductase Gli T,TR)的双抗体夹心ELISA法,并用于几种真菌培养上清液中TR蛋白的检测。方法纯化鼠抗人TR单克隆抗体并进行辣根过氧化物酶(HRP)标记。以纯化羊抗TR多克隆抗体为包被抗体,HRP标记鼠抗人TR单克隆抗体为检测抗体,建立双抗体夹心ELISA法。用方阵滴定法确定包被抗体和检测抗体的最适浓度;对方法的精密度、特异性和最低检测限进行评价;比较分析其检测3种常见曲霉(烟曲霉、黄曲霉、黑曲霉)培养上清中天然TR蛋白的能力,并与3种常见假丝酵母菌(白假丝酵母菌、热带假丝酵母菌、光滑假丝酵母菌)作比较。结果方阵滴定的结果显示最适包被抗体浓度为1μg/m L,最适检测抗体的稀释度为1∶3 000。抗原最低检测限达2.25ng/m L。重组TR浓度为5 ng/m L和20 ng/m L时,批内和批间变异系数分别为8.57%、12.69%和6.73%、10.45%。阻断实验显示该法有较好的特异性,阻断率达84.7%。该法可以检出烟曲霉24 h培养上清中的天然TR,TR含量与烟曲霉菌丝含量呈正相关。本法与另2种曲霉和3种假丝酵母菌培养上清无交叉反应。结论成功建立并优化了检测烟曲霉TR抗原的ELISA法。该法具有良好的精密度及特异性,对烟曲霉感染有潜在的诊断价值。     

Design,Synthesis and Evaluation of Rhodanine Derivatives as Aldose Reductase Inhibitors

Aldose reductase (ALR) enzyme plays a significant role in conversion of excess amount of glucose into sorbitol in diabetic condition, inhibitors of which decrease the secondary complication of diabetes mellitus. To understand the structural interaction of inhibitors with ALR enzyme and develop more effective ALR inhibitors, a series of substituted 5-phenylbenzoate containing N-substituted rhodanine derivatives were synthesized and evaluated for their in vitro ALR inhibitory activity. Docking studies of these compounds were carried out, which revealed that the 5-phenylbenzoate moiety deeply influenced the key π-π stacking while 4-oxo-2-thioxothiazolidines contributed in hydrogen bond interactions. The phenyl ring of benzylidene system occupied in specific pocket constituted from Phe115, Phe122, Leu300 and Cys303 while the rhodanine ring forms a tight net of hydrogen bond with Val47 at anionic binding site of the enzyme. The structural insights obtained from the docking study gave better understanding of rhodanine and macromolecular interaction and will help us in further designing and improving of ALR inhibitory activity of rhodanine analogs.     

珍珠菜中黄酮苷类成分研究

研究珍珠菜Lysimachia clethroides的黄酮苷类成分。珍珠菜乙醇提取物经硅胶、Sephadex LH-20和Pre-HPLC等色谱技术分离纯化,通过质谱和核磁共振等波谱学方法鉴定化合物的结构。从珍珠菜中分离得到11个黄酮苷类化合物,分别鉴定为紫云英苷(1),异槲皮苷(2),异鼠李素-3-O-β-D-吡喃葡萄糖苷(3),槲皮素-3-O-β-D-6"-乙酰基吡喃葡萄糖苷(4),槲皮素-7-O-β-D-吡喃葡萄糖苷(5),江户樱花苷(6),2-羟基-柚皮素-5-O-β-D-吡喃葡萄糖苷(7),山柰酚-3-O-芸香糖苷(8),山柰酚-3-O-洋槐糖苷(9),芦丁(10),山柰酚-3,7-二-O-β-D-吡喃葡萄糖苷(11)。化合物4, 7和11为首次从该属植物中分离得到,化合物3 、 5和9为首次从该种植物中分离得到。体外活性测试结果显示化合物2, 6, 8具有一定的醛糖还原酶抑制活性,IC₅₀分别为2.69,1.00,1.80 μmol·L^-1;化合物1 ~ 11对5种人肿瘤细胞均没有明显的细胞毒活性(IC₅₀ > 10 μmol·L^-1)。     

药用植物功能基因的研究思路与展望——以甘草为例

近些年来有关功能基因的研究已成为药用植物研究领域的热点,研究内容涉及基因克隆、功能鉴定、多态性分析、表达模式分析以及功能基因与代谢途径的相关性分析等诸多方面。甘草是我国最常用的大宗药材之一,其主要活性成分为甘草酸,具有多种药理功能,在国内外市场上应用广泛。本文以甘草为例,对甘草酸代谢途径中已报道的功能基因进行调查研究,在基因克隆、基因功能、基因多态性等方面进行分析,以期为揭示甘草酸合成代谢的分子机制奠定基础,并为其他药用植物功能基因的研究提供思路。     

Corticosterone analgesia is mediated by the spinal production of neuroactive metabolites that enhance GABAergic inhibitory transmission on dorsal horn rat neurons

Corticosterone (CORT) is a glucocorticoid produced by adrenal glands under the control of the hypothalamic–pituitary–adrenal axis. Circulating CORT can enter the central nervous system and be reduced to neuroactive 3α5α‐reduced steroids, which modulate GABA_A receptors. In the dorsal spinal cord, GABAergic transmission modulates integration of nociceptive information. It has been shown that enhancing spinal inhibitory transmission alleviates hyperalgesia and allodynia. Therefore, the spinal neuronal network is a pivotal target to counteract pain symptoms. Thus, any increase in spinal 3α5α‐reduced steroid production enhancing GABAergic inhibition should reduce nociceptive message integration and the pain response. Previously, it has been shown that high levels of plasma glucocorticoids give rise to analgesia. However, to our knowledge, nothing has been reported regarding direct non‐genomic modulation of neuronal spinal activity by peripheral CORT. In the present study, we used combined in vivo and in vitro electrophysiology approaches, associated with measurement of nociceptive mechanical sensitivity and plasma CORT level measurement, to assess the impact of circulating CORT on rat nociception. We showed that CORT plasma level elevation produced analgesia via a reduction in C‐fiber‐mediated spinal responses. In the spine, CORT is reduced to the neuroactive metabolite allotetrahydrodeoxycorticosterone, which specifically enhances lamina II GABAergic synaptic transmission. The main consequence is a reduction in lamina II network excitability, reflecting a selective decrease in the processing of nociceptive inputs. The depressed neuronal activity at the spinal level then, in turn, leads to weaker nociceptive message transmission to supraspinal structures and hence to alleviation of pain.     

Effect of two monoterpene phenols on antioxidant defense system in Candida albicans

Thymol and carvacrol from the class of monoterpene phenols are one of the most potent plant essential oil components possessing antimicrobial effects. Known for their wide bioactive spectrum, these positional isomers of isopropyl cresol deplete ergosterol content, compromise membrane permeability, block efflux pumps and restore antifungal susceptibility to fluconazole in resistant Candida strains. Exposure to these natural compounds induces a cascade of stress responses, which are important to comprehend their microbicidal mechanisms. This study evaluates the antioxidant defense response to lower concentrations of thymol and carvacrol in Candida albicans. The antioxidant defense responses in C. albicans are important for developmental mechanisms pertaining to resistance against the immune system, infection establishment and drug resistance. In this view, primary and secondary antioxidant defense enzymes, and oxidative stress markers including glutathione and lipid peroxidation were determined in C. albicans cells exposed to lower concentrations of thymol and carvacrol. These compounds were found to induce oxidative stress and compromised the antioxidant defense system in C. albicans at lower concentrations. This study helps in understanding the ‘in cell’ antifungal mechanisms of natural monoterpene phenols originating from oxidative stress. Thymol and carvacrol induced membrane deterioration reported earlier, is further explained as a result of a toxic radical cascade mediated by lipid peroxidation. Findings reinforce the observed toxic oxidizing effects of these compounds as a consequence of direct damage to antioxidant components and not to their genetic manipulations.     

糖尿病并发症相关基因醛糖还原酶酵母细胞模型的建立

目的 构建由绿色荧光蛋白（GFP）标记的糖尿病慢性并发症相关基因醛糖还原酶（AR）表达载体，并在酵母细胞中表达，建立AR抑制剂筛选模型。方法 构建AR与GFP的嵌合基因，将此AR：：GFP嵌合基因克隆到酵母表达载体pYEX-BX中，再将重组载体pYEX-BX-AR：：GFP转化至酵母宿主菌INVSC1，在SC-UD选择性培养基中表达AR：：GFP。结果 PCR扩增出约l106bp大小片段，为AR基因片段；RT-PCR在酵母细胞中检测到ARmRNA的表达；经CuS04诱导后可见AR：：GFP融合蛋白的绿色荧光；诱导剂CuSO4终浓度在100～200μmol／L范围内相对荧光强度较高，细胞密度适中；培养基SC-UD的pH值为6左右最适合细胞生长和荧光表达。结论 AR：：GFP在酿酒酵母中成功地表达，酵母细胞是研究目的基因功能与表达的良好模式菌。     

平糖舒目散治疗糖尿病的作用机制初探

目的:通过醛糖还原酶高通量模型对中药方剂平糖舒目散的筛选,探寻其治疗糖尿病的作用机制。方法:用高通量醛糖还原酶筛模型来筛选平糖舒目散,研究方剂平糖舒目散对醛糖还原酶的抑制作用。结果:平糖舒目散对醛糖还原酶有较强的抑制作用,其半数抑制浓度是0.8 g/L。结论:中药方剂平糖舒目散治疗糖尿病慢性并发症的作用机制之一是抑制醛糖还原酶活性。