粘细菌STXZ54的分离鉴定及其抗肿瘤活性

【目的】从广东省土样中分离并鉴定获得粘细菌,以丰富资源微生物库,并对其进行抗肿瘤活性的初步研究,为寻找新的抗肿瘤药物及其开发应用奠定基础。【方法】用灭活大肠杆菌诱导法,从广东土样中分离得到粘细菌,通过菌落形态观察、扫描电镜、生理生化特征、以及16s rRNA基因序列同源性分析,鉴定并归类细菌。测定不同生长时期代谢产物的抗肿瘤活性,分离纯化次级代谢产物得到抗肿瘤活性组分,测定抗肿瘤活性组分抗肿瘤谱及其对应的半致死浓度。利用激光共聚焦显微镜观察抗肿瘤活性组分作用B16后的亚细胞结构变化。【结果】分离并鉴定了粘细菌STXZ54新菌株,命名为Myxococcus macrosporus STXZ54,该粘细菌产生的抗肿瘤活性代谢产物能够较好地抑制B16、Hela、HCT-116、4T1、Hep-3B等多种肿瘤细胞,初步分离其代谢产物得到活性组分SGF5。经MTT实验算出SGF5对B16、Hela、Hep-3B、4T1细胞的IC50均在10μg/mL左右,对HCT-116细胞的IC50是70μg/mL。利用激光共聚焦显微镜观察到活性组分SGF5作用B16后亚细胞结构出现了明显的变化,结合细胞坏死与凋亡的检测初步判断SGF5能引起B16细胞的凋亡。【结论】从土样中分离得到粘细菌STXZ54,对分离到的活性组分进行了细胞毒性试验,证实其具有较好的抗肿瘤活性,有开发成抗肿瘤药物的潜在价值。     

土壤样品中粘细菌的分离鉴定及其生物活性检测

【目的】从土壤样品中分离粘细菌,对其进行纯化、归类与鉴定,以丰富粘细菌菌种资源;对纯化得到的粘细菌进行抑菌、杀虫及抗肿瘤生物活性的分析,为粘细菌次级代谢产物的开发奠定基础。【方法】采用灭活大肠杆菌和滤纸片诱导子实体法从土样中分离粘细菌,结合形态观察、生理生化特征和16S r DNA基因序列同源性分析,对分离到的各菌株进行鉴定并归类。通过平板扩散法、昆虫口服毒性测试、肿瘤细胞毒性实验等方法,检测粘细菌代谢产物生物活性。【结果】共分离得到35株粘细菌,初步分类为:粘球菌属(Myxococcus)9株;珊瑚球菌属(Corallococcus)9株;侏囊菌属(Nannocystis)11株;堆囊菌属(Sorangium)6株。从其中纯化得到8株粘细菌,对其进行了鉴定并命名。发现了具有高效且广谱的肿瘤细胞毒性效应菌株S22,S51和S55也具同样的细胞毒性;另外,还发现具有肿瘤细胞毒性的菌株S20和S22对枯草芽胞杆菌和白色念珠菌也具有较好的抑菌活性。【结论】土壤中存在丰富的粘细菌资源,发现了具有肿瘤细胞毒性的弱小珊瑚球菌与具有抑菌和强抗肿瘤活性的大孢珊瑚球菌,粘细菌是活性天然产物与新药开发的重要资源。     

粘细菌Myxococcus macrosporus STXZ54抗肿瘤活性物质的分离制备及其活性测定

粘细菌作为第三大类次级代谢产物产生菌,是挖掘抗肿瘤新药物的重要资源。将本实验室保藏的粘细菌菌株Myxococcus macrosporus STXZ54进行发酵培养后,通过对发酵液进行硫酸铵沉淀、丙酮沉淀等,分析了该菌株发酵液中抗肿瘤活性物质的性质,采用高效液相色谱技术对发酵液中的抗肿瘤活性物质进行了分离,并对该物质进行了肿瘤细胞毒性的测定,利用激光共聚焦显微镜观察该物质作用B16后的亚细胞结构变化。实验结果表明该物质可能为常温下性质较稳定的蛋白质类化合物,纯化到的单一组分WGF5对B16,Hela,MCF-7,Hep-3B肿瘤细胞均具有较强的抑制作用,其作用48 h的IC_(50)(最低半抑制浓度)分别为2.767μg/ml、2.204μg/ml、3.758μg/ml、3.073μg/ml。MTT实验以及激光共聚焦显微镜下观察分析发现,蛋白WGF5能够明显改变细胞形态并最终导致肿瘤细胞死亡。从粘细菌Myxococcus macrosporus STXZ54分离到的活性物质具有广谱高效的抗肿瘤效果,有开发成抗肿瘤新药物的潜在价值。     

粘细菌次生代谢产物中大环类化合物的研究进展

粘细菌可以产生多种次生代谢产物,是继放线菌、芽胞杆菌之后的第3大类次级代谢产物产生菌。本文综述了粘细菌次生代谢产物中大环类化合物的研究进展。首先介绍了粘细菌中分离的主要大环类化合物种类、结构特征及来源菌株;然后分别从抗肿瘤、抗菌、抗病毒等几方面对其生物活性进行概述;最后对粘细菌次生代谢产物中大环类化合物的相关研究工作进行了展望。     

叶柄粘球菌STXZ77的分离鉴定及抗肿瘤活性

目的：从土样中分离纯化粘细菌,对其进行鉴定与归类,以丰富粘细菌菌种资源,并对其进行抗肿瘤活性初步研究,为抗肿瘤药物开发奠定基础。方法：采用灭活大肠杆菌诱导法,从土样中分离纯化粘细菌,结合形态观察、生理生化特征及16S rRNA基因序列同源性分析进行菌株鉴定;向发酵液上清中加入XAD-16大孔吸附树脂提取发酵产物粗提物,CCK-8法进行体外抗肿瘤活性研究;RP-HPLC分离抗肿瘤活性组分,LC-MS/MS分析其分子质量。结果：分离并鉴定了STXZ77菌株,命名为Myxococcus stipitatus STXZ77。该菌株的发酵产物XAD-16树脂粗提物对小鼠黑色素瘤细胞B16、小鼠乳腺癌细胞4T1、人肝癌细胞SMMC-7721、人宫颈癌细胞HeLa、人结肠癌细胞SW480等多种肿瘤细胞具有较好的细胞毒性,作用24h的IC₅₀值分别为5.34μg/ml、13.50μg/ml、11.93μg/ml、28.70μg/ml、48.09μg/ml,而对正常细胞人脐静脉血管内皮细胞HUVEC的毒性较小,IC₅₀值为17.09μg/ml,小于B16、4T1及SMMC-7721的细胞毒性。RP-HPLC分离得到抗肿瘤活性组分AP-C,质谱分析其分子质量为422.99m/z。结论：从土样中分离得到粘细菌Myxococcus stipitatus STXZ77,从该菌中分离得到抗肿瘤活性组分AP-C,具有开发成抗肿瘤药物的潜在价值。     

粘细菌:一类重要的微生物资源

粘细菌 (Myxobacteria)是革兰氏阴性单细胞杆状细菌 ,具有有独特的生活史 ,在细胞分化发育和生物进化研究中占有重要地位。尤为重要的是粘细菌可产生丰富的次级代谢产物 ,是一类具有重要经济价值的微生物资源。本文简单地介绍了粘细菌生物学特性、分离纯化、系统分类、活性物质的研究 ,以推动粘细菌资源的全面研究、开发和利用。     

粘细菌基因组学研究进展

粘细菌(Myxobacteria)隶属于δ变形菌纲(Deltaproteobacteria)的粘球菌目(Myxococcales),是一类革兰氏阴性杆状细菌。它是继放线菌和真菌之后又一重要的活性次级代谢产物产生菌,尽管如此,由于分离纯化困难,粘细菌的研究进展一直较为缓慢。随着测序技术的进步和生物信息学的应用,大量粘细菌基因组被完成测序和报道。本文对粘细菌研究意义及该类资源开发价值、分离培养存在的困难进行了阐述,对粘细菌基因组注释及目前已测菌株的全基因组进行了归纳总结,同时介绍了基因组学在粘细菌生态、捕食机制、子实体形成以及次级代谢产物合成方面的研究进展。本文有助于了解基因组学在粘细菌研究中的重要价值,为联合应用多组学技术深入研究粘细菌代谢机制和社会性行为提供了参考,对粘细菌基础研究、资源发掘和开发利用具有重要意义。     

链霉菌Streptomyces.sp NJ0510的分离、鉴定及其抑菌活性的研究

目的:从湖边树木下的土壤中筛选分离链霉菌并研究其抑菌活性。方法:通过对菌的形态学和生理生化特征分析确定其种属。通过滤纸片法研究其次级代谢物的抑菌谱。结果:分离得到的一株链霉菌Streptomyces.sp NJ0510,次级代谢产物能抑制G 细菌,抑菌圈可达10mm以上,而对大肠杆菌等没有抑制活性。结论:该菌代谢产物能够抗耐药的金色葡萄球菌,具有潜在的应用前景。     

一株新粘细菌生物学性质的研究

对粘细菌纤维堆囊菌So ce cpu-1生物学特性进行了研究.结果表明,该粘细菌能利用如滤纸或玉米秸杆等纤维素.So ce cpu-1次级代谢产物的抑茵实验表明,该粘细菌具有广谱抗菌活性:对人白血病细胞K562有较好的抑制效果.     

海滨土壤粘细菌次级代谢产物的性质及产量调控的研究*

黄海边土壤中筛选到具有广谱抑菌活性白色粘细菌So ce cpu-1,次级代谢物活性组分在210nm紫外吸收值最大。研究了不同培养条件对次级代谢产物的产量影响。结果表明,So ce cpu-1在含10％w／v D312中性吸附树脂的M₇培养基中,通氧量为总体积的70％,添加该粘细菌次级代谢产物的粗提物5μL作为诱导物,30℃,200r／min,培养6d,次级代谢产物产量可达到最大值。     

Investigation of cytochromes P450 in myxobacteria: excavation of cytochromes P450 from the genome of Sorangium cellulosum So ce56

The exploitation of cytochromes P450 for novel biotechnological application and for the investigation of their physiological function is of great scientific interest in this post genomic era, where an extraordinary biodiversity of P450 genes has been derived from all forms of life. The study of P450s in the myxobacterium Sorangium cellulosum strain So ce56, the producer of novel secondary metabolites of pharmaceutical interest is the research topic, in which we were engaged since the beginning of its genome sequencing project. We herein disclosed the cytochrome P450 complements (CYPomes) of spore-forming myxobacterial species, Stigmatella aurantiaca DW4/3-1, Haliangium ochraceum DSM 14365 and Myxococcus xanthus DK1622, and their potential pharmaceutical significance has been discussed.     

Distribution of Sorangium cellulosum

The myxobacterium Sorangium cellulosum exists and plays an important role in soil ecology by its ability to degrade cellulosic materials; it was found to be widely distributed in North American soils.     

Unusual outer membrane lipid composition of the gram-negative, lipopolysaccharide-lacking myxobacterium Sorangium cellulosum So ce56

The gram-negative myxobacterium Sorangium cellulosum So ce56 bears the largest bacterial genome published so far, coding for nearly 10,000 genes. Careful analysis of this genome data revealed that part of the genes coding for the very well conserved biosynthesis of lipopolysaccharides (LPS) are missing in this microbe. Biochemical analysis gave no evidence for the presence of LPS in the membranes of So ce56. By analyzing the lipid composition of its outer membrane sphingolipids were identified as the major lipid class, together with ornithine-containing lipids (OL) and ether lipids. A detailed analysis of these lipids resulted in the identification of more than 50 structural variants within these three classes, which possessed several interesting properties regarding to LPS replacement, mediators in myxobacterial differentiation, as well as potential bioactive properties. The sphingolipids with the basic structure C9-methyl-C(20)-sphingosine possessed as an unusual trait C9-methylation, which is common to fungi but highly uncommon to bacteria. Such sphingolipids have not been found in bacteria before, and they may have a function in myxobacterial development. The OL, also identified in myxobacteria for the first time, contained acyloxyacyl groups, which are also characteristic for LPS and might replace those in certain functions. Finally, the ether lipids may serve as biomarkers in myxobacterial development.     

产纤维素酶黏细菌在生物转化的作用

目的:预处理对木质纤维素降解的影响.方法:从土壤中分离筛选到高纤维素酶活的黏细菌菌株So ce sh1008.该菌具有CMC酶活(CMCase)及微晶纤维素酶活性.研究NaOH联合黏细菌降解盐蒿、稻草、棉花秸秆和甘蔗渣四种木质纤维素的情况.结果:碱(2％ NaOH) -黏细菌处理的方法优于黏细菌-碱的方法,其中降解棉花秸秆降解效果最明显,以5.0g木质纤维素为原料,其最终干重损失达2.1g,溶液中总糖含量和还原糖含量均值分别为12.8 mg/mL和0.93 mg/mL.酵母菌发酵产乙醇的研究结果表明,最佳发酵时间为47h,碱-黏细菌甘蔗渣降解液发酵效果最好,乙醇产出达6.0％.结论:黏细菌联合2％ NaOH能有效降解甘蔗渣,提高乙醇产量.     

Characterisation,genome size and genetic manipulation of the myxobacterium <Emphasis Type="Italic">Sorangium cellulosum</Emphasis> So ce56

In this study, Sorangium cellulosum So ce56 was phenotypically and genotypically analysed in order to evaluate whether this strain can be used in a comprehensive genome project as a representative of the secondary metabolite-producing myxobacteria. In contrast to many other strains of S. cellulosum, strain So ce56 was found to have various advantageous features, including fast and homogeneous growth in submerged cultures and the ability to complete its morphological differentiation cycle on agar, even when the inoculant originates from a liquid culture. Two groups of secondary metabolites isolated from the culture broth were identified, the polyketides etnangien and chivosazole. The presence of polyketide synthase-encoding genes in the genome of strain So ce56 was demonstrated via PCR. The phenotypic classification was confirmed by comparison of 16S rDNA sequences which showed that S. cellulosum So ce56 clusters within a separate lineage together with S. cellulosum ATCC 25531 and the epothilone producer S. cellulosum So ce90. The genome of S. cellulosum So ce56 belongs to the largest bacterial genomes described so far. It is estimated to be 12.2 Mb in size, by pulsed-field gel electrophoresis. In order to demonstrate that S. cellulosum So ce56 is a convenient strain for molecular biological studies, a genetic manipulation system was developed. Using triparental mating, polyketide synthase-encoding genes were inactivated, leading to chivosazole-negative mutants.     

CD1A-positive cells and HSP60 (HSPD1) levels in keratoacanthoma and squamous cell carcinoma

CD1a is involved in presentation to the immune system of lipid antigen derived from tumor cells with subsequent T cell activation. Hsp60 is a molecular chaperone implicated in carcinogenesis by, for instance, modulating the immune reaction against the tumor. We have previously postulated a synergism between CD1a and Hsp60 as a key factor in the activation of an effective antitumor immune response in squamous epithelia. Keratoacantomas (KAs) are benign tumors that however can transform into squamous cell carcinomas (SCCs), but the reasons for this malignization are unknown. In a previous study, we found that CD1a-positive cells are significantly more numerous in KA than in SCC. In this study, we analyzed a series of KAs and SCCs by immunohistochemistry for CD1a and Hsp60. Our results show that the levels of both are significantly lower in KA than in SCC and support the hypothesis that KA may evolve towards SCC if there is a failure of the local modulation of the antitumor immune response. The data also show that immunohistochemistry for CD1a and Hsp60 can be of help in differential diagnosis between KAs and well-differentiated forms of SCC.