Synthesis and QSAR studies of novel triazole compounds containing thioamide as potential antifungal agents

Eighteen novel triazole compounds containing thioamide were designed and synthesized. Their structures were confirmed by elemental analysis, H-1 NMR, IR, and MS. The title compounds exhibited certain antifungal activity. And the geometry structures of the title compounds were optimized by means of the density functional theory (DFT) method at B3LYP/6-31G* level. The quantitative structure-activity relationship (QSAR) of the title compounds was systematically investigated. A correlative equation between FA and DELH, V was well established by using the multiple linear regression (MLR). (c) 2006 Elsevier Ltd. All rights reserved.

杜仲抗真菌蛋白晶体生长的原子力显微成像研究—快速生长与晶面生长速率

杜仲抗真菌蛋白(Eucommiaantifungalprotein,EAFP)的单晶体具有在几小时内就可长大的快速生长特性.用原子力显微成像(atomicforcemicroscope,AFM)技术,原位实时观测了EAFP单斜晶体生长过程中的｛10 0｝表面形貌动态变化,并分别在不同的过饱和度下测量了其生长速率.结果表明,EAFP晶体生长的速率与蛋白质溶液的过饱和度相关,在过饱和度高时(σ =1 78)晶面生长极快;在中等过饱和度(σ =1 5)下,其晶面台阶的生长速率沿b,c方向分别为 12nm/s和 2 4 2nm/s,比溶菌酶生长速率(6～ 7nm/s)快很多;在蛋白质浓度很低的情况下,其生长速率仍与其他蛋白质相当.EAFP晶体快速生长可能与该分子尺寸较小,内部结构紧凑,分子骨架呈刚性和分子表面性质等其固有特性密切相关.沉淀剂浓度对EAFP晶体生长也有影响.过饱和度很低时,提高沉淀剂浓度会干扰晶体生长.

杜仲抗真菌蛋白(EAFP)晶体动态生长研究

杜仲抗真菌蛋白(eucommia antifungal protein,EAFP),含有41个氨基酸残基,其中有10个半胱氨酸,半胱氨酸间形成的二硫桥键使分子构象很稳定.EAFP的晶体属单斜晶系,空间群为P21,晶胞参数为:a=1.9085nm,b=2.3225nm,c=3.0854nm10-6,β=98.64,分子量为4158.9.EAFP晶体生长速度快,对X射线的衍射能力强,很值得研究其生长的机理.利用原子力显微镜(AFM)对EAFP晶体的{100}面进行动态的生长研究,发现在中低过饱和度下主要以各向异性的单双链螺旋位错的生长模式进行生长,详细研究了这种螺旋的形成机制,并探讨了其结构基础.

用AFM研究杜仲抗真菌蛋白的晶体生长

随着后基因组时代的到来以及蛋白质组学研究的深入开展,研究蛋白质晶体生长成为生物化学和结构生物学领域一个广泛关注的课题。通过使用原子力显微镜(Atomic Force Microscope,简称AFM)对杜仲抗真菌蛋白(eucommia antifungal protein,简称EAFP)的晶体在有母液存在下原位实时动态地进行了晶面生长观察。研究结果表明:不同过饱和度对EAFP晶体生长形貌的影响较大,较高的过饱和度下生长很快,生长台阶密度高,较高的过饱和度下主要进行各向异性二维台阶的发生、发展,较低的过饱和度下主要采用螺旋位错的生长方式,当过饱和度极低时生长缓慢,且晶体表面有很多小孔存在,晶面生长很不完整;还对不同过饱和度下晶体生长速率进行了定量的测量,也反映了过饱和度对EAFP晶体生长的影响;同时对在AFM观察过程中由探针的扫描速度和方向对表面形貌的影响进行了讨论。

商陆抗真菌蛋白的基因克隆及其应用

商陆种子的7kD多肽被鉴定为一种抗真菌多肽，命名为PAFP（pokeweed antifungal protein）。它抑制Trichoderma viride, Fusatium及其它一些病原真菌的生长。本文构建了cDNA文库，而后从库中筛选和克隆PAFP基因。PAFP的编码序列－－201bp的DNA片段被扩增并插入pBluescript SK+载体。经酶切图谱分析和核苷酸顺序测定之后，这个片段与35S启动子连接并重组于双元载体pBin 19。此表达载体质粒转入农杆菌LBA 4404供转化植物之用。通过农杆菌介导的对西瓜的转化，所采用的基因还包括报告基因GUS和Bar，以及一种来自大麦的抗真菌蛋白的基因。以PCR扩增，GUS与NPT II活性检测，以及Southern杂交对转基因植物进行鉴定。

枯草芽孢杆菌生物学特性及其与稻瘟病菌互作的蛋白质组学研究

枯草芽孢杆菌（Bacillus subtilis）是革兰氏阳性细菌研究的模式菌株，为重要的生防菌剂。本论文以枯草芽孢杆菌ATCC6051作对照，首次对新型菌株KB-1111和KB-1122进行了生物学特性、生防潜力以及与水稻稻瘟病致病菌互作的蛋白质组学研究。 形态学观察表明，菌株KB-1111和KB-1122在细胞形态、芽孢的大小、运动性、菌落褶皱和色素的生成等方面与菌株ATCC6051相似，具有枯草芽孢杆菌的典型特征。生理生化测定以及对多种碳源的利用结果显示，三个菌株大部分指标检测结果相同，只在几个方面等存在差异。 体外平板对峙抑菌试验说明，枯草芽孢杆菌ATCC6051、KB-1111和KB-1122对8种作物、果蔬代表性病害致病真菌具有明显的拮抗效果。其中，菌株KB-1122的广谱抗真菌活性优于KB-1111，而KB-1111又强于对照菌株ATCC6051，尤其是对稻瘟病（M. grisea P131）和蔬菜菌核病（S. sclerotiorum）显现出强烈的抑制作用，具备生防拮抗菌的优秀性能。 比较蛋白质组学分析结果表明，液体悬浮培养枯草芽孢杆菌KB-1111、KB-1122二维蛋白质组表达谱至少有11个胞内蛋白和10个胞外蛋白出现丰度差异。其中，菌株KB-1122中胁迫或逆境反应相关ATP酶、顺乌头酸水合酶和alpha-淀粉酶前体在细胞内蛋白质组，以及分泌型蛋白―内切葡聚糖酶在胞外蛋白质组中的高丰度表达可能与菌株KB-1122的优势拮抗能力相关。 将对数生长期的枯草芽孢杆菌KB-1122与菌丝丰富期的稻瘟病菌P131悬浮混合共培养发现，在24小时的共培养过程中，稻瘟病菌P131菌丝体及芽管经历了致变、破裂、细胞质溢出直至菌丝体崩溃等一系列变化，枯草芽孢杆菌KB-1122表现出强烈的拮抗效应。差异显示蛋白质组学研究表明，共培养菌体蛋白质组至少有39个蛋白点丰度发生显著变化，其中33个蛋白点得到成功鉴定，包括12个上调蛋白和21个下调蛋白。根据鉴定结果分析，这些上调的蛋白质全部来源于枯草芽孢杆菌，而下调的蛋白全部属于稻瘟病菌。共培养过程中的培养液蛋白质组至少有20个蛋白点丰度发生显著变化，其中18个蛋白点得到成功鉴定。根据以上分析结果初步认为，3-磷酸甘油醛脱氢酶、丝氨酸蛋白激酶和内切葡聚糖酶在枯草芽孢杆菌KB-1122与稻瘟病菌P131相互作用的过程中可能是B. subtilis KB-1122发挥抗真菌活性的关键性蛋白。

壳聚糖衍生物抑菌性能及机理的研究

壳聚糖是一种天然的聚阳离子氨基多糖，由甲壳素经脱乙酰反应得到。作为天然可再生资源，壳聚糖以其特有的安全无毒、可生物降解、生物相容等特性，在农业、生物工程、制药、环保等领域引起广泛关注。随着对壳聚糖研究的不断深入，壳聚糖的抑菌活性及机理成为研究热点之一，但是，大多集中于壳聚糖分子量和脱乙酰度对其抑菌活性的影响，而对壳聚糖衍生物的抑菌性能及机理研究较少。本文合成了N–季铵盐，N,O–季铵盐，羧甲基壳聚糖希夫碱及其铜配合物，壳聚糖有机酸盐，并对它们的抑菌活性进行了研究，同时探讨了引入基团与抑菌活性之间的关系。 测定了壳聚糖及其季铵盐对黄瓜枯萎病菌、黄瓜炭疽病菌和桃褐腐病菌的抑制活性，结果表明壳聚糖季铵盐的抑制活性高于壳聚糖，其中，抑制活性最强的是N–异丁基–N,N–二甲基壳聚糖，当样品浓度为500μg/mL时，对三种病原菌的抑制率分别为：67.52%、70.42%和76.25%。壳聚糖分子带正电荷的氨基可以与菌体表面带负电的物质作用，导致菌体死亡，而壳聚糖季铵盐分子中的正电性可以加强这种作用，从而增强抑菌活性，而且随着正电性的增强，抑菌活性增强。 为了进一步研究壳聚糖季铵盐的正电性与其抑菌活性的关系，合成了含有氟、溴、氯吸电子基团苯环取代的壳聚糖季铵盐，通过计算分别得到取代基团的电负性以及氨基正电性。结果表明，随着取代基团电负性的增强，季铵盐氨基正电性增强，抑菌活性增强。其中，抑菌活性最强的是N–(2–氟)苄基–N,N–二甲基壳聚糖，当样品浓度为500μg/mL时，对三种病原菌的抑制率分别为：80.85%、100%和100%。 将壳聚糖的–NH2与有机酸的–H通过离子键结合制备得到了系列的固态壳聚糖有机酸盐：壳聚糖甲酸盐、壳聚糖乙酸盐、壳聚糖丙酸盐、壳聚糖丁酸盐和壳聚糖戊酸盐，抑菌活性结果表明，随着衍生物链长的增加，抑菌活性增强，而且衍生物抑菌活性与取代基团的电负性成正相关。 以羧甲基壳聚糖为原料，合成了羧甲基壳聚糖希夫碱及其铜配合物，各类衍生物的抑菌活性强弱顺序如下：羧甲基壳聚糖希夫碱铜配合物>羧甲基壳聚糖希夫碱>羧甲基壳聚糖，从而说明了基团的相互增效作用能增强衍生物的抑菌活性。 以N–季铵盐为原料，以环氧丙基三甲基氯化铵为改性剂合成了具有–N+(CH3)2R和–N+(CH3)3两种基团的N,O–季铵盐，抑菌活性结果表明，N,O–季铵盐的抑菌活性

Maximins S, a novel group of antimicrobial peptides from toad Bombina maxima

Amphibian skin secretions are rich in antimicrobial peptides acting as important components of innate defense system against invading microorganisms. A novel type of peptide, designated as maximin S, was deduced by random sequencing of 793 clones from a constructed Bombina maxima skin cDNA library. The putative primary structures of maximin S peptides can be grouped into five species, in which maximin S I has 14 amino acid residues and the rest of maximin S peptides (S2-S5) all have 18 amino acid residues. Unlike most of the amphibian antimicrobial peptides so far identified, the newly characterized four maximin S precursors are composed of maximin S I and different combinations of tandem repeated maximin S2-S5 linked by internal peptides. Except maximin S I, the predicted secondary structures of maximin S2-S5 show a similar amphipathic alpha-helical structure. MALDI-TOF mass spectrometry analysis of partially isolated skin secretions of the toad indicates that most of the deduced maximin S peptides are expressed. Two deduced maximin S peptides (S1, S4) were synthesized and their antimicrobial activities were tested. Maximin S4 only had an antibiotic activity against mycoplasma and had no antibacterial or antifungal activity toward tested strains. Maximin S1 had no activity under the same conditions. (C) 2004 Elsevier Inc. All rights reserved.

Novel cathelicidin-derived antimicrobial peptides from Equus asinus

In the present study, EA-CATH1 and EA-CATH2 were identified from a constructed lung cDNA library of donkey (Equus asinus) as members of cathelicidin-derived antimicrobial peptides, using a nested PCR-based cloning strategy. Composed of 25 and 26 residues, respectively, EA-CATH1 and EA-CATH2 are smaller than most other cathelicidins and have no sequence homology to other cathelicidins identified to date. Chemically synthesized EA-CATH1 exerted potent antimicrobial activity against most of the 32 strains of bacteria and fungi tested, especially the clinically isolated drug-resistant strains, and minimal inhibitory concentration values against Gram-positive bacteria were mostly in the range of 0.3-2.4 mu g center dot mL-1. EA-CATH1 showed an extraordinary serum stability and no haemolytic activity against human erythrocytes in a dose up to 20 mu g center dot mL-1. CD spectra showed that EA-CATH1 mainly adopts an alpha-helical conformation in a 50% trifluoroethanol/water solution, but a random coil in aqueous solution. Scanning electron microscope observations of Staphylococcus aureus (ATCC2592) treated with EA-CATH1 demonstrated that EA-CATH could cause rapid disruption of the bacterial membrane, and in turn lead to cell lysis. This might explain the much faster killing kinetics of EA-CATH1 than conventional antibiotics revealed by killing kinetics data. In the presence of CaCl2, EA-CATH1 exerted haemagglutination activity, which might potentiate an inhibition against the bacterial polyprotein interaction with the host erythrocyte surface, thereby possibly restricting bacterial colonization and spread.

Identification of a novel cathelicidin gene in the rainbow trout, Oncorhynchus mykiss

We report the cloning of a novel antimicrobial peptide gene, termed rtCATH_1, found in the rainbow trout, Oncorhynchus mykiss. The predicted 216-residue rtCATH_1 prepropeptide consists of three domains: a 22-residue signal peptide, a 128-residue cathelin-like region containing two identifiable cathelicidin family signatures, and a predicted 66-residue C-terminal cationic antimicrobial peptide. This predicted mature peptide was unique in possessing features of different known (mammalian) cathelicidin subgroups, such as the cysteine-bridged family and the specific amino-acid-rich family. The rtCATH_1 gene comprises four exons, as seen in all known mammalian cathelicidin genes, and several transcription factor binding sites known to be of relevance to host defenses were identified in the 5' flanking region. By Northern blot analysis, the expression of rtCATH_1 was detected in gill, head kidney, and spleen of bacterially challenged fish. Primary cultures of head kidney leukocytes from rainbow trout stimulated with lipopolysaccharide or poly(I (.) C) also expressed riCATH_1. A 36-residue peptide corresponding to the core part of the fish cathelicidin was chemically synthesized and shown to exhibit potent antimicrobial activity and a low hemolytic effect. Thus, rtCATH_1 represents a novel antimicrobial peptide gene belonging to the cathelicidin family and may play an important role in the innate immunity of rainbow trout.

Agrobacterium-mediated transformation of Kentucky bluegrass

Embryogenic calli of Kentucky bluegrass, named Md, were induced from mature seeds and embryos, and proliferated on medium K3 containing 2,4-dichlorophenoxyacetic acid (2,4-D, 10.0 mumol/L), 6-benzylaminopurine (BAR, 0.5 mumol/L) and K5 which was the K3 medium supplemented with cupric sulfa (0.5 mumol/L) under dim-light condition (20-30 mumol.m(-2).s-1, 16 h light) at 24 degreesC. Embryogenic calli were transformed with plasmids pDM805 Carring bar and gus genes, Which was mediated by an Agrobacterium strain AGL1, four transgenic lines were obtained. The important factors that affect the transformation efficiency and obtain desirable number of transgenic plants included: (1) the quality of embryogenic calli; (2) light condition and time of co-cultivation; (3) concentration of antibiotics used for suppressing the overgrowth of Agrobacterium in the course of transformed plant regeneration; (4) selection pressure, etc. The micro nutrient of cupric had significant influence on the quality of embryogenic calli. This presentation is the first successful protocol of Kentucky bluegrass transformation mediated by Agrobacterium.

海洋枯草芽孢杆菌3512A抗真菌脂肽的研究

芽孢杆菌 (Bacillus) 被认为是一种具有潜在的能有效抑制植物病原真菌并促进植物生长作用的有益菌种，它能产生许多种不同结构的抗菌物质，其中脂肽类抗菌物质是其中重要的一类。这些抗菌脂肽具有对动植物无害、对环境友好、不易产生交叉抗性等特点，因而在生物防治由真菌引起的病害中起着十分重要的作用。海洋中也存在着大量芽孢杆菌，并且由于海洋的特殊生存环境有可能开发出与陆生芽孢杆菌性质或功能不同的代谢产物。 本论文从108株海洋芽孢菌中筛选到一株抗真菌活性显著、脂肽产量高的海洋枯草芽孢杆菌3512A（Bacillus subtilis 3512A）。对该菌株产生的脂肽进行分离纯化，结合酸沉淀、Flash Chromatography、HPLC等现代色谱手段，从其发酵液中分离得到了 4 个纯化合物，分别是流分 4 中分离到的Comd.1 和Comd.2，以及流分 8 中分离到的ALP1 和 ALP3。流分 4 中的 Comd.1 和Comd.2 分子量分别为1036D和1050D，结合1D, 2D-NMR, MS 等结构分析后确定分子式分别为 C53H93N7O13 和 C54H95N7O13，Comd.2为Comd.1的甲基化产物，均属于surfactin 的同系物，其它组分还有待进一步测定分析。而流分 8 中得到的三个化合物 ALP1、ALP2 和ALP3，对ALP1 和ALP3进行鉴定，通过 TLC 原位酸水解实验、1H-NMR 图谱分析等推断 ALP1 和 ALP3也属于环状脂肽类化合物。对这两种脂肽化合物的氨基酸组成进行分析，发现这两种化合物的氨基酸组成相同，推测可能是一组同系物，且这两种化合物的氨基酸组成不同于目前已报道的脂肽类化合物的氨基酸组成，可能是一类新的脂肽类物质，其具体化学结构还有待于进一步分析鉴定。 考察3512A所产粗脂肽的热稳定性和pH稳定性，结果显示粗脂肽的热稳定性很好，即使在115 ℃、30 min, 活性只有部分损失；粗脂肽对酸的耐受性比较好，对碱有部分耐受性，pH10后活性降低显著。通过单因素法考查培养基组成和培养条件对3512A产脂肽能力的影响，研究结果表明，以蔗糖或葡萄糖作碳源，以硝酸铵为氮源，pH为8.0，每250 ml三角瓶装液量60 ml，以5% 的接种量28℃，180 r/min培养48 h，脂肽产量最高，达到1003 mg/L。

吸水链霉菌、放线菌Sp.253和金色毛壳菌的次生代谢产物研究

链霉菌是十分重要的一类放线菌，绝大多数的抗生素都由该类细菌产生。毛壳属真菌是一类重要的丝状真菌，从中也发现有很多结构新颖、活性独特的活性物质。因此本论文对两株链霉菌的活性成分及一株金毛壳菌的次生代谢产物进行了研究。 1．从吸水链霉菌（Streptomyces hygroscopicus 1.358）液态发酵产物（乙酸乙酯提取物）中分离得到3个化合物，通过波谱方法鉴定为RK955A (1)、Nigericin（2）、Elaiophylin（3）。以青霉素耐药-金黄色葡萄球菌作为指示菌的抗菌活性测定表明，三者均具有较强抗菌活性。 2．通过抗肿瘤体外活性筛选模型筛选得到得到一株链霉属土壤放线菌，从中分离得到六个化合物：苯乙酰胺（4）、苯丙酰胺（5）、肉桂酰胺（6）、3-（N-（甲酰胺基）乙酰基）吲哚（7）、鸟苷磷酸（8）、鸟苷（9）。 3．从金色毛壳菌（Chaetomium aureus）的固态培养物中分离得到13个化合物，利用波谱方法将其鉴定为：金毛壳菌素A（10）、金毛壳菌素B（11）、Eugenetin（12）、Eugenitol（13）、Chaetoquadrin A（14）、Chaetoquadrin B（15）、Chaetoquadrin G（16）、Chaetoquadrin H（17）、Chaetochromin A（18）、Sterigmatocystin（19）、O-methylsterigmatocystin（20）、3β-羟基-麦角甾-5，7，22-三烯（21）和过氧麦角甾醇（22）。 4．综述了聚醚类抗生素的结构、生物合成、生物活性及作用机理。 The genus Streptomyces (Actinomycetes) is an important group of microbe. Most antibiotics known nowdays are discovered from species of Streptomyces. The fungi of the genus Chaetomium have attracted much attention because various kinds of secondary metabolites with diverse bioactivities have been found from them. Thus, the bioactive compounds from two strains of Streptomyces and the secondary metabolites of Chaetomium aureus were investigated. 1. Three compounds were isolated from the ethyl acetate extract of the fermentation broth of Streptomyces hygroscopicus. They are identified to be elaiophylin (1), nigericin (2), and antibiotic RK955A (3) on the basis of their spectroscopic data. Compounds 1-3 possess antibacterial activities against Staphyloccocus aureus. 2. It was found that the extract of the fermented broth of a strain of Actinomycetes could inhibit some tumor cel lines. Separation of the bioactive fraction led to the isolation of six compounds. They were characterized to be phenylacetamide (4), phenylpropylamide (5), trans-cinnamamide (6), 3- (N- (formylmethyl) acetamide) indole (7), guanylicacid (8), and guanosine (9). 3. From the fermented broth of Chaetomium aureus, 13 compounds were isolated for the first time. They were determined to be chaetomiumycin A (10), chaetomiumycin B (11), eugenetin (12), eugenitol (13), chaetoquadrin A (14), chaetoquadrin B (15), chaetoquadrin G (16), chaetoquadrin H (17), chaetochromin A (18), sterigmatocystin (19), O-methylsterigmatocystin (20), 3β-hydroxyergosta-5, 7, 22-triene (21) and peroxy-ergosterol (22). Compounds 10 and 11 are new ones. 4. Structure, biosynthesis, biological activity, and mechanisms of polyether antibiotics were reviewed.