真菌纤溶化合物1的遗传毒性评价

目的:在已建立的良好实验室规范(GLP)平台,对真菌纤溶化合物1(FGFC1)进行系统的遗传毒性评估。方法:采用经典遗传毒性检测组合(Ames试验、体外培养CHO细胞染色体畸变试验和小鼠骨髓细胞微核试验)检测FGFC1的遗传毒性。结果:Ames试验结果提示,FGFC1在每皿0.5、5、50、500、1000 mg 5个剂量下,在加和不加代谢活化系统(S9)时,对鼠伤寒沙门氏菌均无致突变性。CHO细胞染色体畸变试验结果提示,在终浓度62.5、125.0和250.0 mg/mL 3个剂量组,在加和不加S9时,于作用24 h和48 h的条件下培养的CHO细胞,均未诱发染色体畸变。小鼠骨髓细胞微核试验在62.5、125.0和250.0 mg/kg 3个剂量下对骨髓细胞的微核诱发率,与溶剂对照组比较差异均无统计学意义(P>0.05)。结论:在本实验条件下,未检测到FGFC1的遗传毒性和潜在致癌性。

具有纤溶活性作用的海洋真菌代谢产物的分离与菌株鉴定

本文旨在分离具有纤溶活性作用的化合物和鉴定产生纤溶活性化合物的菌株FG216的种属分类。以马铃薯蔗糖培养基为种子培养基,改良查氏培养基为发酵培养基对菌株进行发酵培养,用甲醇作为提取溶剂,通过半制备型高效液相色谱从真菌FG216的1 L发酵液中分离和精制了12 mg纤溶活性化合物,该纤溶活性化合物在纤溶酶原和单链尿激酶性纤溶酶原激活剂相互活化反应体系中添加10μg/mL活性最高。对FG216菌株rDNA的ITS基因(ITS-5.8 S rDNA)进行PCR扩增、测序,从GenBank获取相似序列,通过序列比对分析和系统发育分析表明菌株FG216与Stachybotrys longispora同源性最高。从分离的海洋微生物葡萄穗霉属菌株FG216分离得到的纤溶活性化合物具体促进纤溶酶原和单链尿激酶性纤溶酶原激活剂相互活化的作用。

海洋微生物代谢产物FGFC1的纤溶促进作用

目的：研究海洋微生物产生的吡喃并异吲哚酮化合物FGFC1体外和实验动物体内促纤溶作用。方法：采用异硫氰酸荧光素（FITC）-纤维蛋白降解法和急性肺血栓大鼠模型法研究FGFC1体外和体内的纤溶促进作用。结果：在体外FITC-纤维蛋白降解实验中,FGFC1在0.5~25μmol/L浓度范围内,其纤溶促进作用随着浓度升高逐渐增强;当FGFC1浓度≥25μmol/L时,其纤溶促进作用维持在最高水平,EC50为5μmol/L。在大鼠急性肺血栓实验中,FGFC1的剂量为5或10mg/kg能溶解肺血栓,达到与5nmol/L剂量单链尿激酶型纤溶酶原激活剂相当的溶栓效果。结论：溶栓药物先导化合物FGFC1具有优良的纤溶促进作用和溶栓效果。

海洋长孢葡萄穗霉菌生物合成纤溶活性化合物的研究

目的基于FG216(Fungus stachytotrys longispora)产生纤溶化合物FGFC1(Fungi fibrinolitic compound 1)的可能生源途径,调控其次生代谢过程,获得新型纤溶活性或者其他活性化合物。方法 FG216经过种子培养和发酵培养,在发酵培养结束前24h,添加ADP、色氨酸、柠檬酸三钠、赖氨酸、苯丙氨酸、组氨酸、鸟氨酸、天冬酰胺等8种代谢产物调节剂(metabolite regulator,MTRL),添加量为发酵液的1%,用HPLC(High-pressure liquid chromatogram)检测FG216代谢产物产出情况。结果添加鸟氨酸为MTRL的FGFC1的产量增加至950mg/L,苯丙氨酸为MTRL的FG216代谢产物的HPLC图谱出现2个新的化合物峰。色氨酸为MTRL的FG216代谢产物的HPLC图谱出现3个新的化合物峰。ADP、柠檬酸三钠、赖氨酸、组氨酸、天冬酰胺为MTRL的FG216代谢产物的HPLC图谱未出现新的化合物峰。分离纯化5种目标化合物CA、CB、CC、CD、CE在纤溶活性评价反应体系(10μg/mL),其纤溶活性分别是FGFC1的30%、90%、50%、110%、17%。结论采用恰当的FG216发酵MTRL,能产生新型的纤溶活性化合物,推测通过调控代谢生源途径,能从FG216挖掘出活性更强或结构新颖的纤溶化合物。

海洋纤溶活性化合物FGFC1和FGFC2影响血小板聚集特性的研究

目的研究新型海洋双吲哚纤溶活性化合物FGFC1对血小板聚集的影响,发现纤溶活性化合物FGFC1与血小板相关因子变化的关系,同时比较双吲哚化合物FGFC1和单吲哚化合物FGFC2作用于血小板的异同。方法从新鲜兔血收集血小板,调整反应体系血小板密度,以乙酰水杨酸为阳性对照,采用微量比浊法分析FGFC1和FGFC2对血小板聚集率的影响,用酶联免疫法测定FGFC1和FGFC2对血小板生理因子含量的变化。结果 FGFC1对ADP和AA诱导的血小板聚集具有抑制作用,最大抑制率分别达到42.58%±1.7%和47.82%±2.18%;FGFC2对ADP、AA和胶原诱导的血小板聚集均具有抑制作用,最大抑制率分别为50.12%±1.02%、45.28%±1.09%和50.28%±2.12%。FGFC1与FGFC2均能提升血小板内环磷酸腺苷含量,且FGFC2能明显降低血小板内血栓素A2含量。结论海洋双吲哚纤溶活性化合物显著抑制血小板聚集,FGFC1通过影响血小板环磷酸腺苷的含量实现抑制血小板聚集,FGFC2可能通过不止一种途径抑制血小板聚集。吲哚化合物抑制血小板聚集与分子结构密切相关。

促纤溶活性化合物FGFC1的分离纯化

建立从发酵液中分离纯化高纯度促纤溶活性化合物FGFC1的方法。长孢葡萄穗霉Stachybotrys longispora FG216发酵液经固液分离得到菌丝体,菌丝体经乙醇抽提得FGFC1抽提液,采用AmberliteTM XAD-1600型树脂吸附抽提液中的部分色素与杂质,使用Uni PS30-300型色谱柱进一步去除色素与同系物杂质,得到FGFC1纯化液,调至pH 3沉淀并真空干燥,即得FGFC1固态粉末,其结构经质谱(MS)和核磁(NMR)初步验证。该法制备的FGFC1纯度为97%,总收率为48%。

Soluble Carbohydrates Repress the Cellulolytic Activity of Clostridium cellulovorans and Eubacterium cellulosolvens

Studies have provided indirect evidence that cellulolytic activity of some anaerobic bacteria is repressed by carbohydrates, such as glucose. This effect is known as carbon catabolite repression （CCR）. Previous work has found that cellulolytic activity of Clostridium cellulovorans and Eubacterium cellulosolvens are regulated. Many cellulolytic systems of these organisms are expressed only in the presence of cellulose or cellobiose （the disaccharide of cellulose）. Some of these cellulose-induced systems also appear subject to CCR when more soluble substrates, such as glucose, are also available. To determine if such repression directly effects cellulolytic activity of C. cellulovorans and E. cellulosolvens, these organisms were cultivated in media containing a glucose analog. We then measured the ability of low levels of analog to inhibit growth of the organisms when cellobiose or cellulose were the energy substrates. Our results found that growth of both C. cellulovorans and E. cellulosolvens in cellobiose-containing medium are strongly inhibited by glucose analogs. In addition, both organisms exhibited delayed and slower growth in cellulose-containing medium when a glucose analog was added. These results provide direct demonstration that these cellulolytic bacteria are subject to CCR. This repression of cellulolysis may affect both of these organisms＇ ability to serve as industrial platforms for biomass degradation, and may interfere with the contribution of E. cellulosolvens toward animal digestion of cellulose. These results were also in sharp contrast to what has been reported regarding CCR activity in Clostridium cellulolyticum, which actively expresses cellulases in the presence of low levels of glucose.

Effect of Long-term Experimental Warming on the Nutritional Quality of Alpine Meadows in the Northern Tibet

The nutritional quality of grasslands is closely related to recruitment of young and population dynamics of livestock and wild herbivores. However, the response of nutritional quality to climate warming has not been fully understood in the alpine meadow on the Tibetan Plateau, especially in the Northern Tibet. Here, we investigated the effect of experimental warming(beginning in 2008) on nutritional quality in three alpine meadows(site A: 4313 m, B: 4513 m and C: 4693 m) in the Northern Tibet. Crude protein(CP), neutral detergent fiber(NDF), acid detergent fiber(ADF), crude ash(Ash), ether extract(EE) and water-soluble carbohydrate(WSC) were examined in 2018–2019. Experimental warming only increased the content of CP by 27.25%, ADF by 89.93% and NDF by 41.20%, but it decreased the content of Ash by 57.76% in 2019 at site B. The contents of CP and WSC both increased with soil moisture(SM). The content of CP decreased with vapor pressure deficit(VPD). The combined effect of SM and VPD was greater than air temperature(Ta) in controlling the variations of the CP content, ADF content and nutritional quality. Compared to Ta, VPD explained more of the variation in NDF and Ash content. All of these findings suggest that warming effects on nutritional quality may vary with site and year, and water availability may have a stronger effect on the nutritional quality than temperature in the alpine meadow of the Northern Tibet.