Cytochalasins from Xylaria sp. CFL5, an Endophytic Fungus of Cephalotaxus fortunei

Three previously undescribed cytochalasins,named xylariasins A‒C(1‒3),together with six known ones(4‒9)were iso-lated from Xylaria sp.CFL5,an endophytic fungus of Cephalotaxus fortunei.The chemical structures of all new compounds were elucidated on the basis of extensive spectroscopic data analyses and electronic circular dichroism calculation,as well as optical rotation calculation.Biological activities of compounds 1,4‒9 were evaluated,including cytotoxic,LAG3/MHC II binding inhibition and LAG3/FGL1 binding inhibition activities.Compounds 6 and 9 possessed cytotoxicity against AGS cells at 5μM,with inhibition rates of 94%and 64%,respectively.In addition,all tested isolates,except compound 6,exhibited obvious inhibitory activity against the interaction of both LAG3/MHC II and LAG3/FGL1.Compounds 1,5,7,and 8 inhibited LAG3/MHC II with IC50 values ranging from 2.37 to 4.74μM.Meanwhile,the IC50 values of compounds 1,7,and 8 against LAG3/FGL1 were 11.78,4.39,and 7.45μM,respectively.

Vercytochalasins A and B: Two unprecedented biosynthetically related cytochalasins from the deep-sea-sourced endozoic fungus Curvularia verruculosa

Vercytochalasins A(1) and B(2), two biosynthetically related cytochalasins featuring novel structure and substituents, were isolated from the endozoic fungus Curvularia verruculosa which was associated with the deep-sea squat lobster Shinkaia crosnieri collected from the cold seep environment in South China sea. Their structures were elucidated by detailed interpretation of NMR spectroscopic and mass spectrometric data. The absolute configurations were confirmed by NOESY experiments as well as by DP4+ and ECD calculations. Differed from common cytochalasins, compound 1 is an uncommon secocytochalasin featuring the ester group cleaved between C-9 and C-23, and incorporating an additional oxygenated C4 unit which coupled with C-20 and C-22 to form a new substituted cyclohexenone moiety, while compound 2 contains an unusual 2–hydroxy-3-oxobutan-2-yl unit at C-22. Both compounds are distinctive from the commonly described cytochalasins. Compound 1 exhibited potent activity against angiotensinI-converting enzyme(ACE) whereas compound 2 showed antibacterial activity. Molecular docking simulations were performed to explore the intermolecular interaction of compounds 1 and 2 with ACE.

Revealing the F_actin Networks in Interphase Nuclei of Garlic Clove Cells by Confocal Fluorescence Microscopy

The interphase nuclei of parenchyma cells and epidermal cells of garlic ( Allium sativum L.) clove were labelled with rabbit anti_actin antibody and FITC_conjugated goat anti_rabbit IgG antibody. The authors observed results with fluorescence microscopy and confocal laser scanning microscopy. The nuclei showed prominent green_yellow fluorescence, indicating the presence of actin in the nuclei. Fluorescence examination with TRITC_phalloidin showed distinctive red fluorescence in the nuclei, indicating that F_actin is present in the nuclei. Confocal laser scanning microscopy indicated the presence of F_actin containing network structures in the nuclei, but the network structures were absent and the nuclei still showed red fluorescence when the cells were treated with cytochalasin D before fixation; the red fluorescence in the nuclei was hard to be observed when the cells were treated with unlabelled phalloidin before the cells were stained with TRITC_phalloidin. These results indicate that F_actin is in the nuclei and forms network structures in the nuclei of garlic cells.

细胞松弛素B和低温诱导华贵栉孔扇贝三倍体的产生(英文)

分别用细胞松驰素B（C．B）（浓度为0．5mg／L）和低温（10℃）处理诱导华贵栉孔扇贝Chlamysnobilis产生三倍体．抑制第一极体的三倍体诱导率达到44．1％和31．8％；而抑制第二极体的三倍体诱导率分别为90．2％和72．7％（胚胎初期检查）．随着C．B浓度的增加，孵化率下降；而随着温度的降低和休克时间的延长，非整倍体增多．

利用高速逆流色谱对一株内生真菌代谢物中抗肿瘤活性物质的分离

利用高速逆流色谱法对一株内生真菌活性代谢产物进行了分离。两相溶剂系统为正己烷-乙酸乙酯-甲醇-水(体积比为1∶3∶3∶3),固定相为系统上相,下相为流动相。从内生真菌的代谢物中分离得到一抗肿瘤细胞活性组分。经高效液相色谱分析,其纯度大于99%以上。其化学结构由ESI-MS、1H NMR和13C NMR确认为Cytochalasin N。

Chemical Components from the Fungus Engleromyces goetzei

A new ceramide, (2S,3S,4R, 10E)-2-[(2'R)-2'-hydroxytetracosanoyl amino]-10-octadecene-1, 3,4-triol (1), together with twelve known compounds, cerebroside A, cerebroside B, cerebroside D, cytochalasin D, epoxycytochalasin D, cytochalasin C, loganin, cerevisterol, ergosta-7,22-dien-3beta, 5alpha, 6alpha-triol, ergosta-4,6,8(14),22-tetraen-3-one, ergosterol peroxide and ergosta-5,7,22-trien-3-ol, was isolated from ethyl acetate fraction of Engleromyces goetzei P. Henn. The structure of new ceramide (1) was elucidated by spectral data and chemical method, especially by 2D-NMR techniques. All of the compounds except cytochalasin D were first obtained from this fungus.

Mechanical properties of hepatocellular carcinoma cells

AIM: To study the viscoelastic properties of human hepatocytes and hepatocellular carcinoma (HCC) cells under cytoskeletal perturbation, and to further to study the viscoelastic properties and the adhesive properties of mouse hepatoma cells (HTC) in different cell cycle. METHODS: Micropipette aspiration technique was adopted to measure viscoelastic coefficients and adhesion force to collagen coated surface of the cells. Three kinds of cytoskeleton perturbing agents, colchicines (Col), cytochalasin D (CD) and vinblastine (VBL), were used to treat HCC cells and hepatocytes and the effects of these treatment on cell viscoelastic coefficients were investigated. The experimental results were analyzed with a three-element standard linear solid. Further, the viscoelastic properties of HTC cells and the adhesion force of different cycle HTC cells were also investigated. The synchronous G(1) and S phase cells were achieved through thymine-2-desoryriboside and colchicines sequential blockage method and thymine-2-desoryriboside blockage method respectively. RESULTS: The elastic coefficients, but not viscous coefficient of HCC cells (K(1)=103.6+/-12.6N.m(-2), K(2)=42.5 +/ 10.4N.m(-2), mu=4.5 +/- 1.9Pa.s), were significantly higher than the corresponding value for hepatocytes (K(1)=87.5 +/- 12.1N.m(-2), K(2)=33.3+/-10.3N.m(-2), mu=5.9+/-3.0Pa.s, P【0.01). Upon treatment with CD, the viscoelastic coefficients of both hepatocytes and HCC cells decreased consistently, with magnitudes for the decrease in elastic coefficients of HCC cells (K(1): 68.7 N.m(-2) to 81.7N.m(-2), 66.3% to 78.9%; K(2): 34.5N.m(-2) to 37.1N.m(-2), 81.2% to 87.3%, P【0.001) larger than those for normal hepatocytes (K(1): 42.6N.m(-2) to 49.8N.m(-2), 48.7% to 56.9%; K(2): 17.2N.m(-2) to 20.4N.m(-2), 51.7% to 61.3%, P【0.001). There was a little decrease in the viscous coefficient of HCC cells (2.0 to 3.4Pa.s, 44.4 to 75.6%, P【0.001) than that for hepatocytes (3.0 to 3.9Pa.s, 50.8 to 66.1% P【0.001). Upon treatment with Col and VBL, the elastic coefficients of hepatocytes generally increased or tended to increase while those of HCC cells decreased. HTC cells with 72.1% of G(1) phase and 98.9% of S phase were achieved and high K(1), K(2) value and low mu value were the general characteristics of HTC cells. G(1) phase cells had higher K(1) value and lower mu value than S phase cells had, and G(1) phase HTC cells had stronger adhesive forces ((275.9 +/- 232.8) x 10(-10)N) than S phase cells ((161.2 +/- 120.4) x 10(-10)N, P【0.001). CONCLUSION: The difference in both the pattern and the magnitude of the effect of cytoskeletal perturbing agent on the viscoelastic properties between HCC cells and hepatocytes may reflect differences in the state of the cytoskeleton structure and function and in the sensitivity to perturbing agent treatment between these two types of cells. Change in the viscoelastic properties of cancer cells may affect significantly tumor cell invasion and metastasis as well as interactions between tumor cells and their micro-mechanical environments.

TETRAPLOID INDUCTION BY INHIBITING MITOSIS I IN SCALLOP CHLAMYS FARRERI

Tetraploid induction was carried out by inhibiting mitosis I in fertilized eggs ofChlamys farreri. Mitosis I was blocked with cold shock (5–7°C), Cytochalasin B (0.75 mg/L) and 6-dimethylaminopurine (6-DMAP) (60–75 mg/L) when 60% fertilized eggs released polar body II at 20°C. At 4-cells embryo stage, the ploidy was determined by counting chromosome number. In control groups, most embryos were diploids (72.22%) and aneuploids (24.78%). In Cytochalasin B, cold shock and 6-DMAP treated groups, tetraploids were respectively 10.51%, 4.08%, and 13.34%; aneuploids were 43.10%, 35.93% and 29.16%, and triploids were 7.84%, 8.52% and 18.33%. At D-larva stage, ploidy was determined by flow cytometry (FCM). The ploidy analysis of day 2 larvae showed diploids in control group and also in three treated groups. Juvenile scallops (0.2–0.3cm) which were harvested in two control groups and two CB treated groups were all diploids through checking ploidy individually by FCM.

Cytochalasin B loaded nano/microparticles:preparation and in vitro drug release evaluation

Cytochalasin B (cytoB) loaded nanoparticles (NPs) and microspheres (MSs) were formed using an emulsification / solvent evaporation technique.Biodegradable poly(lactic co glycolic acid)(PLGA) and poly(L lactic acid)(PLLA)were used as carrier and were compared in terms of drug release rate and initial burst.The PLGA nanoparticles and microparticles of graded diameter,150 nm,500 nm,1 μm,5 μm,10 μm and 20 μm,is formulated.Nanoparticles of 150 500 nm diameter were obtained by high energy sonication,whereas larger size particles were obtained by normal homogenization.The degree of initial burst release varied depending on particle size and polymer matrix.For the PLGA matrix,about 50% of the entrapped drug released from the 150 nm nanoparticles,whereas only 10% released from the 20 μm particles within the first 24 hours.On the other hand,cytoB release from 150 nm PLLA nanoparticles was much slower and showed less burst effect than the PLGA ones with same size.The results suggest that desired release profile can be achieved by properly selecting the matrix material and particle size.

Cytochalasin D,a tropical fungal metabolite,inhibits CT26 tumor growth and angiogenesis

Objective:To investigate whether eytochalasin D can induce antitumor activities in a tumor model.Methods:Murine CT26 colorectal carcinoma cells were cultured hi vitro and cytochalasin D was used as a cytotoxic agent to detect its capabilities of inhibiting CT26 cell proliferation and inducing cell apoptosis by MTT and a TUNEL-based apoptosis assay.Murine CT26 tumor model was established to observe the tumor growth and survival time.Tumor tissues were used to detect the mierovessel density by immunohistochemistry.In addition,alginate encapsulated tumor cell assay was used to quantify the tumor angiogenesis in vivo.Results:Cytochalasin D inhibited CT26 tumor cell proliferation in lime and dose dependent manner and induced signiflcanl CT26 cell apoptosis,which almost reached the level induced by the positive control nuclease.The optimum effective dose of cytochalasin D for in vivo therapy was about 50 mg/kg.Cytochalasin D in vivo treatment significandy inhibited tumor growth and prolonged the survival times in CT26 tumor-bearing mice.The results of immunohistochemistry analysis and alginate encapsulation assay indicated that the cytochalasin D could effectively inhibited tumor angiogenesis. Conclusions:Cytochalasin D inhibits CT26 tumor growth potentially through inhibition of cell proliferation,induction of cell apoptosis and suppression of tumor angiogenesis.

Cytochalasin B induced triploidy in Penaeus chinensis

Feartilized eggs of Penaeus chinensis were treated with cytochalasin B (C. B ) to induce triploid.A number of them were cultured to the stage of on larvae. The C. B concentrations employed were 0. 1～2. 5 mg/dm3. The treatments started at 5～20 min after fertilization and lasted for various periods of time from 10 to 40 min. The highest inducing rate was 62. 5%.It was obvious that haaer C. B concentration gave stronger inhibition on the releas of polar bodies,and the higher the C. B concentration was, the more abnormal and aneuploid embryos were obtained. The success in inducing tripled in P.chinensis provided a posibility of polyploid breding of this species.

Relevances of microfilament and microtubule to the adhesion properties of the HCC onto the Collagen IV/ Laminin coated surface

The adhesion properties of both hepatocytes and hepatocellular carcinoma cells (HCCs) onto the Coll IV/laminin coated surface were measured by means of micropipette aspiration technique. Further,relevances of microfilament and microtubule systems to the adhesion properties of the HCCs on Coll IV/laminin coated dishes were investigated by using cytoskeletal agents colchicine and cytochalasin D. The results showed: cytochalasin D, which inhibits microfilament polymerization, had great inhibitory effect on adherence of both kinds of cells to Coll IV/ Laminin substratum (about 70%～90%). At the same time, HCCs extension obviously decreased and focal contacts almost vanished after being treated with cytochalasin D, in contrast, there was no significant effect on the formation of extention of hepatocytes. Colchicine, which inhibits microtubular polymerization, had different effects on both cells: Compared with untreated groups, the adhesion forces of HCC cells decreased and those of hepatocytes increased. These data suggested that, in these tumor cells, microfilament system are crucial for adherence, and abnormal cytoskeletons of tumor cells may be basis of their abnormal adhesion properties.

In situ Localization and Isolation of Actin Filaments from Pollen Tubes of Amaryllis vittata Ait

Actin filaments (AFs) in un-fixed pollen tubes of Amaryllis vittata Ait were visualized after TRITC-phalloidin staining with DMSO as a permeabilising agent. Typically, strands or hundles of microfilaments (Mfs) were distributed in the extreme tip as well as pollen tubes in a form of network.Fluorescent granules or circles of various sizes were frequently found that continued with the filamentous structures. In addition, a more brightly stained structure, possibly Mf organizing center, was observed. Treatment of pollen tubes with cytochalasin D(CD)for increasing time intervals (5-40 minutes) caused gradual reduction of strands until flurescent granules filled up the pollen tubes. Mcanwhile, cytoplasmie streaming was inhibited completely. Though closely associated with vegetative nuclei (VN) and generative cells (GC), AFs were not found in the cytoplasm of GC.Mg++concentration greatly affected the isolated Mfs.

从子囊菌炭球菌中分离的活性成分L-696,474和cytochalasin D对植物病原真菌的活性(英文)

在我国云南省高等真菌中寻找新的天然农用抗真菌活性物质时,发现炭球菌子实体甲醇提取物具有较强的离体抗真菌活性。采用生物活性追踪和化学分离相结合的方法,从中分离得到2个具有较强农用抗真菌活性化合物,经红外、质谱、氢谱和碳谱确定其为L-696,474和cytochalasin D。在离体和活体条件下分别测定了L-696,474和cytochalasin D对10种植物病原菌的菌丝抑制率和2种植物病原菌的孢子萌发率,并评价了其对小麦白粉病的治疗和保护作用。结果表明:小麦纹枯病菌、油菜菌核病菌对L-696,474最为敏感,浓度为10μg/mL时菌丝抑制率分别为83.5%和82.9%;小麦赤霉病菌、小麦全蚀病菌、油菜菌核病菌、小麦叶枯病菌和苹果炭疽病菌对cytochalasin D最为敏感,浓度为10μg/mL时菌丝抑制率分别为83.6%、82.4%、81.9%、80.5%和79.7%。L-696,474和cytochalasin D在200μg/mL时完全抑制苹果炭疽病的孢子萌发;在500μg/mL时对小麦白粉病10天后的治疗作用分别为74.3%和85.7%,保护作用为67.1%和79.5%。

1株花柳珊瑚来源真菌Curvularia lunata产cytochalasin B发酵优化及其化学表观遗传修饰研究

目的通过对中国南海花柳珊瑚来源真菌Curvularia lunata(RA16-1)进行发酵优化以提高该菌的cytochalasin B产量,并加入组蛋白去乙酰化酶抑制剂丁酸钠以考察对该菌次级代谢产物的影响。方法运用单因素试验设计,对菌株产cytochalasin B的碳源、氮源、初始pH值、盐浓度等发酵条件进行优化;运用柱色谱层析技术对加入丁酸钠的真菌Curvularia lunata(RA16-1)发酵产物进行分离,以及核磁与质谱技术对分离的化合物进行结构鉴定。结果单因素试验表明最优发酵条件为:碳源为蔗糖(30g·L^(-1))、氮源为蛋白胨(30g·L-1)、发酵时间为96h、初始pH为5.0、盐浓度为2%。此外,从加入丁酸钠的发酵产物中分离得到了4个化合物,其中2个化合物(化合物2和3)为首次从该物种中获得。结论在优化条件下,cytochalasin B的产量可达到2.3g·L^(-1)。组蛋白去乙酰化酶抑制剂丁酸钠的加入后使得该菌发酵产物的结构类型发生较大变化。

A new cytochalasin from endophytic Phomopsis sp. IFB-E060

AIM: To study the chemical constituents of the solid culture of the endophyte Phomopsis sp. IFB-E060 in Vatica mangachapoi. METHOD: Isolation and purification were performed through silica gel column chromatography, gel filtration over Sephadex LH-20, ODS column chromatography, and HPLC. Structures of the isolated compounds were elucidated by a combination of spectroscopic analyses(UV, CD, IR, MS, 1D, and 2D NMR). The cytotoxicity of the isolates was evaluated in vitro by the MTT method against the human hepatocarcinoma cell line SMMC-7721. RESULTS: Five compounds were isolated from the solid culture of the endophyte Phomopsis sp. IFB-E060 and their structures were identified as 18-methoxy cytochalasin J(1), cytochalasin H(2),(22E, 24S)-cerevisterol(3), ergosterol(4), and nicotinic acid(5). Compound 1 had an inhibition rate of 24.4% at 10 μg·mL-1 and 2 had an IC50 value of 15.0 μg·mL-1, while a positive control 5-fluorouracil had an inhibition rate of 28.7% at 10 μg·mL-1. CONCLUSION: 18-Methoxy cytochalasin J(1), produced by endophytic Phomopsis sp. IFB-E060, is a new cytochalasin with weak cytotoxicity to the human hepatocarcinoma cell line SMMC-7721.

Effects of microfllamentous depolymerization by cytochalasin B on cAMP-PKA pathway

Diacylglycerol (DG) and cAMP, two intracellular second messengers, play importantroles in responding to the stimulation of extracellular signal. DG activates protein kinaseC (PKC), and cAMP activates protein kinase A (PKA). Activation of these

FFECTS OF OOPLASM CYTOSKELETON ON MIGRATION AND DEVELOPMENT OF SPERM-NUCLEI IN MOUSE EGGS

Longo and CHEN elucidated in 1985 that the origin of polarity of mouse oocytes, the germinal vesicle breakdown and resumption of meiosis are temporospatially related to one another, and further demonstrated the responsibility of microfilaments for the peripheral movement of the meiotic apparatus including chromosomes. Recently, it has been shown by CHEN et al. that the chromosomal movement of the mitotic apparatus at the ear-

THE EFFECTS OF CYTOCHALASIN B ON THE SWELLING, CONTRACTION AND OXIDATIVE PHOSPHORYLATION IN ISOLATED MUNG BEAN MITOCHONDRIA

Cytochalasin B(CB)was a depolymeriziner agent of F-aetin.Therefore a lot of plant cell motilities related to action,such as cytoplasmic streaming,pollen