鹅膏毒肽与RNA聚合酶II相互作用的分子机制研究

研究鹅膏毒肽与RNA聚合酶II相互作用的分子机制,利用分子对接方法获得了9种鹅膏毒肽与RNA聚合酶II相互作用的结合模式、结合位点、对接能和抑制常数等信息,并对鹅膏毒肽的毒性与结构间的构效关系进行了考察。结果表明：利用分子对接方法获得的鹅膏毒肽与RNA聚合酶II相互作用的信息与实验结果相一致;不同R2取代基引起毒素与聚合酶II结合能力强弱不同,从而导致鹅膏毒肽分子间的毒性差异。结果证实了运用分子对接方法探索多肽分子与蛋白质相互作用的可行性,为在分子水平上研究多肽与蛋白质的相互作用开拓了新的思路。     

几种不同鹅膏菌毒素对真核生物RNA聚合酶Ⅱ抑制作用的比较研究

用鹅膏菌属（Amanita）含α-毒伞肽的毒素粗提液培养新鲜绿豆,结果在36小时后,各种不同毒苗的毒素粗提液培养的绿豆生长情况有明显不同,用紫外吸收法测定蛋白质含量,发现毒素粗提液培养的绿豆细胞中蛋白质含量比蒸馏水培养的有明显下降,这表明α-毒伞肽的作用机理的确是通过抑制RNA聚合酶Ⅱ而寻致蛋白质合成减少。     

几种不同鹅膏菌毒素对真核生物RNA聚合酶Ⅱ抑制作用的比较研究

用鹅膏菌属含α－毒伞肽的毒素粗提液培养新鲜绿豆,结果在３６小时后,各种不同毒 的毒素粗提液培养的绿豆生长情况有明显不同,用紫外吸收法测定蛋白质含量,发现毒素粗提液培养的绿豆细胞中蛋白质含量比蒸馏水培养的有明显下降,这表明α－毒伞肽的作用机理确是通过抑制ＲＮＡ聚合酶Ⅱ而导致蛋白质合成减少。     

灰花纹鹅膏中三个鹅膏毒肽的分离和结构研究

从灰花纹鹅膏(Amanita fuliginea)中分离得到3个已知鹅膏毒肽类成分,利用波谱方法(^1H-NMR、^13C-NMR、2D-NMR和FAB-MS等)对这些结构较为复杂的环肽类成分进行了鉴定,结构分别为α-Amanitin、β-Amanitin和Amanin。     

长白山鹅膏菌肽类毒素的HPLC分析

采用HPLC法对长白山地区分布的10种鹅膏菌中的-鹅膏毒肽(-amanitin)、鹅膏毒肽(-amanitin)和鬼笔毒肽(phalloidin)3种毒素的含量进行了测定。结果表明:白鹅膏(A.verna)和鳞柄白鹅膏(A.virsa)中含有-amanitin和-amanitin两种毒素,二者-amanitin的含量分别为 1861.85g/g和2477.02g/g,均高于欧洲产毒鹅膏(A.phalloides)中的含量(1607g/g)而接近灰花纹鹅膏Amanita fuliginea中的量(2633.80g/g)。毒鹅膏A.phalloides中含有3种毒素,并且菌蕾中的含量高于成熟子实体,尤其菌蕾中Phalloidin的含量(1113.35g/g)是灰花纹鹅膏成熟子实体中(432.5g/g)的3倍。     

我国鹅膏菌新发现种--致命鹅膏(Amanita exitialis)的肽类毒素分析

采用高效液相色谱(HPLC)技术对在广州发现的鹅膏菌新种——致命鹅膏(Amanita exitialis)不同组织部位的肽类毒素(鹅膏毒肽和鬼笔毒肽)的含量进行了分析,结果表明,致命鹅膏是一种剧毒蘑菇,其毒素含量相当高,子实体中组织部位不同,毒素含量以及鹅膏毒肽和鬼笔毒肽在其中的分布也不一样,菌盖中的毒素含量最高,达8152．6μg／g干重,菌柄的毒素含量次之,为3742．3μg／g干重,菌托中的毒素含量最低,只有1142．5μg／g干重;在菌盖、菌柄和菌托中都以鹅膏毒肽为主,尤其以α-amanitin的相对含量最高,但从菌盖至菌柄到菌托,鬼笔毒肽尤其是Phallacidin的相对含量依次增加。     

我国鹅膏菌新发现种——致命鹅膏(Amanita exitialis)的肽类毒素分析

采用高效液相色谱（HPLC）技术对在广州发现的鹅膏菌新种——致命鹅膏(Amanita exitialis)不同组织部位的肽类毒素(鹅膏毒肽和鬼笔毒肽)的含量进行了分析,结果表明,致命鹅膏是一种剧毒蘑菇,其毒素含量相当高,子实体中组织部位不同,毒素含量以及鹅膏毒肽和鬼笔毒肽在其中的分布也不一样,菌盖中的毒素含量最高,达8152.6μg/g干重,菌柄的毒素含量次之,为3742.3μg/g干重,菌托中的毒素含量最低,只有1142.5μg/g干重;在菌盖、菌柄和菌托中都以鹅膏毒肽为主,尤其以αamanitin的相对含量最高,但从菌盖至菌柄到菌托,鬼笔毒肽尤其是Phallacidin的相对含量依次增加。     

α—鹅膏毒肽和二羟鬼笔毒肽的制备和鉴定

α-鹅膏毒（环）肽和二羟鬼笔毒（环）肽是剧毒的鹅膏菌和其它几种致死毒菌中由一些修饰氨基酸组成的环肽毒素,由于α-鹅膏毒肽对真核生物的mRNA合成的专一性抑制和二羟鬼笔毒肽对肌动蛋白的专一性束缚,因而它们在分子生物学和细胞学研究中具有重要应用,对其需求逐渐增加,为此,作者使用了一种改良的毒素提取方法,以制备高效液相色谱从灰花纹鹅膏菌中分离制备α-鹅膏毒肽和二羟鬼笔毒肽,并通过紫外吸收光谱和质谱进行鉴定,表明α-鹅膏毒肽和二羟鬼笔毒肽的分离效果好,纯度高,本方法对其它毒菌中的α-鹅膏毒肽和二羟鬼笔毒肽的分离制备具有同样的应用价值。     

云南楚雄致命鹅膏中环肽毒素的检测与分析

采用反相高效液相色谱法对采自云南楚雄双柏县的致命鹅膏在3个不同生长期中不同部位的6种环肽毒素含量进行了检测和分析。结果表明,致命鹅膏含有α-, β-鹅膏毒肽、羧基三羟鬼笔毒肽和羧基二羟鬼笔毒肽,未检出γ-鹅膏毒肽和二羟鬼笔毒肽。生长期毒素总量最高（9.3mg/g）、从成熟期（7.5mg/g）到衰老期（6.5mg/g）逐渐降低,但鬼笔毒肽的相对含量随着年龄增长而逐渐增加,鹅膏毒肽与鬼笔毒肽比值从生长期、成熟期到衰老期分别为2.6、1.4和0.9。在3个不同发育阶段中,4种毒素含量从菌盖、菌柄到菌托逐渐降低,而鬼笔毒肽的相对含量逐渐增加。α-鹅膏毒肽和β-鹅膏毒肽在生长期菌盖中含量最高,分别为7.4mg/g和3.1mg/g,而羧基三羟鬼笔毒肽和羧基二羟鬼笔毒肽在衰老期的菌盖中含量最高,分别为2.8mg/g和2.1mg/g。     

β—鹅膏毒肽的反相高效液相色谱分离纯化

用反相高效液相色谱,以0．02mol/L醋酸铵—乙腈为流动相的梯度洗脱模式,在295nm吸收值的条件下,灰花纹鹅膏菌Amanita fuliginea的肽类毒素可以被成功的分离和纯化。单个肽类毒素的鉴定是用反相高效液相色谱和质谱同时进行。用这一方法可从灰花纹鹅膏菌中分离纯化出β-鹅膏毒肽(β-amanitin),产量可达到：1158μg／g(干重),产品纯度达98％以上,回收率为95．3％。β-鹅膏毒肽的分子量为919．3Da。这个方法可用于其它鹅膏菌肽类毒素的分离纯化。     

Cross Talk between β1 and αV Integrins: β1 Affects β3 mRNA Stability

There is increasing evidence that a fine-tuned integrin cross talk can generate a high degree of specificity in cell adhesion, suggesting that spatially and temporally coordinated expression and activation of integrins are more important for regulated cell adhesive functions than the intrinsic specificity of individual receptors. However, little is known concerning the molecular mechanisms of integrin cross talk. With the use of beta(1)-null GD25 cells ectopically expressing the beta(1)A integrin subunit, we provide evidence for the existence of a cross talk between beta(1) and alpha(V) integrins that affects the ratio of alpha(V)beta(3) and alpha(V)beta(5) integrin cell surface levels. In particular, we demonstrate that a down-regulation of alpha(V)beta(3) and an up-regulation of alpha(V)beta(5) occur as a consequence of beta(1)A expression. Moreover, with the use of GD25 cells expressing the integrin isoforms beta(1)B and beta(1)D, as well as two beta(1) cytoplasmic domain deletion mutants lacking either the entire cytoplasmic domain (beta(1)TR) or only its "variable" region (beta(1)COM), we show that the effects of beta(1) over alpha(V) integrins take place irrespective of the type of beta(1) isoform, but require the presence of the "common" region of the beta(1) cytoplasmic domain. In an attempt to establish the regulatory mechanism(s) whereby beta(1) integrins exert their trans-acting functions, we have found that the down-regulation of alpha(V)beta(3) is due to a decreased beta(3) subunit mRNA stability, whereas the up-regulation of alpha(V)beta(5) is mainly due to translational or posttranslational events. These findings provide the first evidence for an integrin cross talk based on the regulation of mRNA stability.     

The role of strand 1 of the C β-sheet in the structure and function of α1-antitrypsin

Serpins inhibit cognate serine proteases involved in a number of important processes including blood coagulation and inflammation. Consequently, loss of serpin function or stability results in a number of disease states. Many of the naturally occurring mutations leading to disease are located within strand 1 of the C beta-sheet of the serpin. To ascertain the structural and functional importance of each residue in this strand, which constitutes the so-called distal hinge of the reactive center loop of the serpin, an alanine scanning study was carried out on recombinant alpha(1)-antitrypsin Pittsburgh mutant (P1 = Arg). Mutation of the P10' position had no effect on its inhibitory properties towards thrombin. Mutations to residues P7' and P9' caused these serpins to have an increased tendency to act as substrates rather than inhibitors, while mutations at P6' and P8' positions caused the serpin to behave almost entirely as a substrate. Mutations at the P6' and P8' residues of the C beta-sheet, which are buried in the hydrophobic core in the native structure, caused the serpin to become highly unstable and polymerize much more readily. Thus, P6' and P8' mutants of alpha(1)-antitrypsin had melting temperatures 14 degrees lower than wild-type alpha(1)-antitrypsin. These results indicate the importance of maintaining the anchoring of the distal hinge to both the inhibitory mechanism and stability of serpins, the inhibitory mechanism being particularly sensitive to any perturbations in this region. The results of this study allow more informed analysis of the effects of mutations found at these positions in disease-associated serpin variants.     

Inhibition of α-, β- and γ-carbonic anhydrases from the pathogenic bacterium Vibrio cholerae with aromatic sulphonamides and clinically licenced drugs – a joint docking/molecular dynamics study

The binding mode of aromatic sulphonamides and clinically licenced drugs to the three carbonic anhydrase (CA, EC 4.2.1.1) isoforms from the human pathogen V. cholerae was here thouroghly characterised by a joint docking and molecular dynamics in silico protocol. In fact, VchCA, VchCAβ, and VchCAγ are crucial in the pathogen life cycle and growth and represent innovative targets to fight V. cholerae proliferation overcoming the spreading chemoresistance to the available drugs. A set of 40 sulphonamides/sulfamates VchCAs inhibitors was studied using the proteins homology built 3 D models unveiling the key and stable interactions responsible for a potent CA inhibition. This study has the aim to offer insights and guidelines for the future rational design of potent and selective inhibitors targeting CA isoforms from V. cholerae or other human pathogens.     

Washingtonia filifera seed extracts inhibit the islet amyloid polypeptide fibrils formations and α-amylase and α-glucosidase activity

Washingtonia filifera seeds have revealed to possess antioxidant properties, butyrylcholinesterase and xanthine oxidase inhibition activities. The literature has indicated a relationship between Alzheimer’s disease (AD) and type-2 diabetes (T2D). Keeping this in mind, we have now evaluated the inhibitory properties of W. filifera seed extracts on α-amylase, α-glucosidase enzyme activity and the Islet Amyloid Polypeptide (IAPP) fibrils formation.Three extracts from seeds of W. filifera were evaluated for their enzyme inhibitory effect and IC₅₀ values were calculated for all the extracts. The inhibition mode was investigated by Lineweaver-Burk plot analysis and the inhibition of IAPP aggregate formation was monitored.W. filifera methanol seed extract appears as the most potent inhibitor of α-amylase, α-glucosidase, and for the IAPP fibril formation.Current findings indicate new potential of this extract that could be used for the identification or development of novel potential agents for T2D and AD.     

Synthesis and bioactivities evaluation of oleanolic acid oxime ester derivatives as α-glucosidase and α-amylase inhibitors

Different oleanolic acid (OA) oxime ester derivatives (3a-3t) were designed and synthesised to develop inhibitors against α-glucosidase and α-amylase. All the synthesised OA derivatives were evaluated against α-glucosidase and α-amylase in vitro. Among them, compound 3a showed the highest α-glucosidase inhibition with an IC₅₀ of 0.35 µM, which was ∼1900 times stronger than that of acarbose, meanwhile compound 3f exhibited the highest α-amylase inhibitory with an IC₅₀ of 3.80 µM that was ∼26 times higher than that of acarbose. The inhibition kinetic studies showed that the inhibitory mechanism of compounds 3a and 3f were reversible and mixed types towards α-glucosidase and α-amylase, respectively. Molecular docking studies analysed the interaction between compound and two enzymes, respectively. Furthermore, cytotoxicity evaluation assay demonstrated a high level of safety profile of compounds 3a and 3f against 3T3-L1 and HepG2 cells.

Highlights

1. Oleanolic acid oxime ester derivatives (3a–3t) were synthesised and screened against α-glucosidase and α-amylase.
2. Compound 3a showed the highest α-glucosidase inhibitory with IC50 of 0.35 µM.
3. Compound 3f presented the highest α-amylase inhibitory with IC50 of 3.80 µM.
4. Kinetic studies and in silico studies analysed the binding between compounds and α-glucosidase or α-amylase.