Identification and characterization of a novel envelope protein in Rana grylio virus

Viral envelope proteins have been proposed to play significant roles in virus infection and assembly. In this study, an envelope protein gene, 53R, was cloned and characterized from Rana grylio virus (RGV), a member of the family Iridoviridae. Database searches found its homologues in all sequenced iricloviruses, and sequence alignment revealed several conserved structural features shared by virus capsid or envelope proteins: a myristoylation site, two predicted transmembrane domains and two invariant cysteine residues. Subsequently, RT-PCR and Western blot detection revealed that the transcripts encoding RGV 53R and the protein itself appeared late during infection of fathead minnow cells and that their appearance was blocked by viral DNA replication inhibitor, indicating that RGV 53R is a late expression gene. Moreover, immunofluorescence localization found an association of 53R with virus factories in RGV-infected cells, and this association was further confirmed by expressing a 53R-GFP fusion protein in pEGFP-N3/53R-transfected cells. Furthermore, detergent extraction and Western blot detection confirmed that RGV 53R was associated with virion membrane. Therefore, the current data suggest that RGV 53R is a novel viral envelope protein and that it may play an important role in virus assembly. This is thought to be the first report on a viral envelope protein that is conserved in all sequenced iridoviruses.

Inductive transcription and protective role of fish heme oxygenase-1 under hypoxic stress

Heme oxygenase-1 is the rate-limiting enzyme in the degradation of heme into biliverdin, carbon monoxide and free divalent iron. In this study, we cloned heme oxygenase isoform 1 (CaHO-1) from a hypoxia-tolerant teleost fish Carassius auratus. The full-length cDNA of CaHO-1 is 1247 bp and encodes a protein of 272 amino acids. RT-PCR and real-time PCR analysis indicated that CaHO-1 was predominantly transcribed in posterior kidney, head kidney, gill and intestine, and induction of gene transcription was observed predominantly in posterior kidney under hypoxic stress. Moreover, the hypoxia-induced transcription was confirmed in goldfish larvae and in in vitro cultured CAB cells. Fluorescence of the HO-1-GFP fusion protein revealed a cytoplasmic and plasma membrane localization, which was consistent with the putative transmembrane structure. Subsequently, we established a stably transfected CAB/pcDNA3.1-HO-1 cell line and a control CAB/pcDNA3.1 cell line, and found that the number of dead cells was obviously reduced in the pcDNA3.1-HO-1-transfected group following 4 days of hypoxic (1% O-2) treatment in comparison with numerous detached dead cells in the control pcDNA3.1-transfected cells. Furthermore, a significant cell viability difference between the two kinds of transfected cells during hypoxia-reoxygenation was revealed. Therefore, the data suggest that fish HO-1 might play a significant protective role in cells in response to hypoxic stress.

DE-71-induced apoptosis involving intracellular calcium and the Bax-mitochondria-caspase protease pathway in human neuroblastoma cells In vitro

Polybrominated diphenyl ethers (PBDEs) are used extensively as flame-retardants and are ubiquitous in the environment and in wildlife and human tissue. Recent studies have shown that PBDEs induce neurotoxic effects in vivo and apoptosis in vitro. However, the signaling mechanisms responsible for these events are still unclear. In this study, we investigated the action of a commercial mixture of PBDEs (pentabrominated diphenyl ether, DE-71) on a human neuroblastoma cell line, SK-N-SH. A cell viability test showed a dose-dependent increase in lactate dehydrogenase leakage and 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyl-tetrazolium bromide reduction. Cell apoptosis was observed through morphological examination, and DNA degradation in the cell cycle and cell apoptosis were demonstrated using flow cytometry and DNA laddering. The formation of reactive oxygen species was not observed, but DE-71 was found to significantly induce caspase-3, -8, and -9 activity, which suggests that apoptosis is not induced by oxidative stress but via a caspase-dependent pathway. We further investigated the intracellular calcium ([Ca2+](i)) levels using flow cytometry and observed an increase in the intracellular Ca2+ concentration with a time-dependent trend. We also found that the N-methyl d-aspartate (NMDA) receptor antagonist MK801 (3 mu M) significantly reduced DE-71-induced cell apoptosis. The results of a Western blotting test demonstrated that DE-71 treatment increases the level of Bax translocation to the mitochondria in a dose-dependent fashion and stimulates the release of cytochrome c (Cyt c) from the mitochondria into the cytoplasm. Overall, our results indicate that DE-71 induces the apoptosis of ([Ca2+](i)) in SK-N-SH cells via Bax insertion, Cyt c release in the mitochondria, and the caspase activation pathway.

Isolation, characterization and genome sequence of a birnavirus strain from flounder Paralichthys olivaceus in China

A birnavirus strain, Paralichthys olivaceus birnavirus (POBV), was isolated and characterized from cultured flounder in China, and its complete genomic sequence was subsequently determined. The virus could induce cytopathic effects (CPE) in four of seven fish cell lines and was resistant to chloroform, 5-iodo-2'-deoxyuridine, acid and alkaline pH, and heat treatment. Purified virus particles had a typical icosahedral shape, with a diameter of approximately 55-60 nm. The genomic segments A and B of POBV were 3,091 and 2,780 bp in length and shared many of the features of the members of the family Birnaviridae. Segment A contained two partially overlapping ORFs encoding a polyprotein, pVP2-VP4-VP3, and a nonstructural protein, VP5, while segment B had only one ORF encoding for the VP1, a viral RNA-dependent RNA polymerase (RdRp). This is the first report about a birnavirus strain from a new non-salmonid host in China and its complete genome sequence.

Novel nanostructured biomaterials: implications for coronary stent thrombosis.

BACKGROUND: Nanomedicine has the potential to revolutionize medicine and help clinicians to treat cardiovascular disease through the improvement of stents. Advanced nanomaterials and tools for monitoring cell-material interactions will aid in inhibiting stent thrombosis. Although titanium boron nitride (TiBN), titanium diboride, and carbon nanotube (CNT) thin films are emerging materials in the biomaterial field, the effect of their surface properties on platelet adhesion is relatively unexplored. OBJECTIVE AND METHODS: In this study, novel nanomaterials made of amorphous carbon, CNTs, titanium diboride, and TiBN were grown by vacuum deposition techniques to assess their role as potential stent coatings. Platelet response towards the nanostructured surfaces of the samples was analyzed in line with their physicochemical properties. As the stent skeleton is formed mainly of stainless steel, this material was used as reference material. Platelet adhesion studies were carried out by atomic force microscopy and scanning electron microscopy observations. A cell viability study was performed to assess the cytocompatibility of all thin film groups for 24 hours with a standard immortalized cell line. RESULTS: The nanotopographic features of material surface, stoichiometry, and wetting properties were found to be significant factors in dictating platelet behavior and cell viability. The TiBN films with higher nitrogen contents were less thrombogenic compared with the biased carbon films and control. The carbon hybridization in carbon films and hydrophilicity, which were strongly dependent on the deposition process and its parameters, affected the thrombogenicity potential. The hydrophobic CNT materials with high nanoroughness exhibited less hemocompatibility in comparison with the other classes of materials. All the thin film groups exhibited good cytocompatibility, with the surface roughness and surface free energy influencing the viability of cells.

Molecular characterization and subcellular localization of Carassius auratus interferon regulatory factor-1

Interferon (IFN)-regulatory transcription factor-1 (IRF-1) has been studied in mammals and fish but little is known about the relationship between its gene structure and nuclear 'ion of IRF-1 protein. In this study, a cDNA encoding Carassius auratus IRF-1 (CaIRF-1) was isolated from an interferon-producing cell line, C. ouratus blastulae embryonic (CAB) cells, exposed to UV-inactivated grass carp hemorrhagic virus (GCHV). The CaIRF-1 genomic locus exhibits exon-intron arrangements similar to those of other vertebrate IRF-1 loci, with nine exons and eight introns, although together with pufferfish IRF-1, CaIRF-1 distinguishes itself from other vertebrate IRF-1 genes by a relatively compact genomic size. Similar to the known IRF-1 genes, CaIRF-1 is ubiquitously expressed, and is upregulated in vitro and in vivo in response to virus, Poty I:C, or CAB INF-containing supernatant (ICS). Subcellular localization analysis confirms the nuclear distribution of CaIRF-1 protein, and reveals two nuclear localization signals (NILS), any one of which is sufficient for nuclear translocation of CaIRF-1. One NLS Locates to amino acids 117-146, and appears to be the structural and functional equivalent of the NLS in mammalian IRF-1. The second NLS (amino acids 73-115) is found within the DNA-binding domain (DBD) of CaIRF-1, and contains two regions rich in basic amino acids (''(KDKSINK101)-K-95" and ''(75)KTWKANFR(82)"). In comparison with mammalian IRF-1, in which the corresponding amino acid stretch does not seem to drive nuclear translocation, five conserved basic amino acids (K-75, K-78, R-82, K-95, and K-101) and one non-conserved basic amino acid (K-97) are present in this NLS from CaIRF-1. This observation suggests that K97 Of CaIRF-1 might be essential for the function of its second NLS, wherein the six basic aminoacids might cooperate to drive CaIRF-1 to the nucleus. Therefore, the current study has revealed a new nuclear localization motif in the DBD of a vertebrate IRF-1. (C) 2007 Elsevier Ltd. All rights reserved.

Mitochondrion-mediated apoptosis induced by Rana grylio virus infection in fish cells

A fish cell line, fathead minnow (FHM) cell, was used to investigate the alteration of mitochondrial dynamics and the mechanism of apoptosis under Rana grylio virus (RGV) infection. Microscopy observations, flow-cytometry analysis and molecular marker detection revealed the apoptotic fate of the RGV-infected cells. Some typical apoptotic characteristics, such as chromatin condensation, DNA fragmentation and mitochondrial fragmentation, were observed, and significantly morphological changes of mitochondria, including size, shape, internal structure and distribution, were revealed. The mitochondria in RGV-infected cells were aggregated around the viromatrix, and the aggregation could be blocked by colchicine. Moreover, the Delta psi m collapse was induced, and caspase-9 and caspase-3 were activated in the RGV-infected cells. In addition, NF-kappa B activation and intracellular Ca2+ increase were also detected at different times after infection. The data revealed the detailed dynamics of mitochondrion-mediated apoptosis induced by an iridovirus, and provided the first report on mitochondrial fragmentation during virus-induced apoptosis in fish cells.

The innate immune response to grass carp hemorrhagic virus (GCHV) in cultured Carassius auratus blastulae (CAB) cells

Virus infection of mammalian cells activates an innate antiviral immune response characterized by production of interferon (IFN) and the subsequent transcriptional upregulation of IFN-stimulated genes (ISGs) by the JAK-STAT signaling pathway. Here, we report that a fish cell line, crucian carp (Carassius auratus L.) blastulae embryonic (CAB) cells, can produce IFN activity and then form an antiviral state after infection with UV-inactivated grass carp hemorrhagic virus (GCHV), a double-stranded (ds) RNA virus. From UV-inactivated GCHV-infected CAB cells, 15 pivotal genes were cloned and sequenced, and all of them were shown to be involved in IFN antiviral innate immune response. These IFN system genes include the dsRNA signal sensing factor TLR3, IFN, IFN signal transduction factor STAT1, IFN regulatory factor IRF7, putative IFN antiviral effectors Mx1, Mx2, PKR-like, Viperin, IFI56, and other IFN stimulated genes (ISGs) IFI58, ISG15-1, ISG15-2, USP18, Gig1 and Gig2. The identified fish IFN system genes were highly induced by active GCHV, UV-inactivated GCHV, CAB IFN or poly(I).poly(C), and showed similar expression patterns to mammals. The data indicate that an IFN antiviral innate immune response similar to that in mammals exists in the UV-inactivated GCHV-infected CAB cells, and the IFN response contributes to the formation of an antiviral state probably through JAK-STAT signaling pathway. This study provides strong evidence for existence of IFN antiviral innate immune response in fish, and will assist in elucidating the origin and evolution of vertebrate IFN system. (c) 2006 Elsevier Ltd. All rights reserved.

Microcystin-RR-induced accumulation of reactive oxygen species and alteration of antioxidant systems in tobacco BY-2 cells

Microcystins are cyclic heptapeptide hepatoxins produced by cyanobacteria. It has been shown that microcystins have adverse effects on animals and on plants as well. Previous researches also indicated that microcystins were capable of inducing oxidative damage in animals both in vivo and in vitro. In this study, tobacco BY-2 suspension cell line was applied to examine the effects of microcystin-RR on plant cells. Cell viability and five biochemical parameters including reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT), glutathione peroxide (GPX) and peroxide dismutase (POD) were investigated when cells were exposed to 50 mg/L microcystin-RR. Results showed that microcystin-RR evoked decline of the cell viability to approximately 80% after treating for 144 h. ROS levels, POD and GPX activities of the treated cells were gradually increased with a time dependent manner. Changes of SOD and CAT activities were also detected in BY-2 cells. After 168 h recovery, ROS contents, POD, GPX and CAT activities returned to normal levels. These results suggest that the microcystin-RR can cause the increase of ROS contents in plant cells and these changes led to oxidant stress, at the same time, the plant cells would improve their antioxidant abilities to combat mirocystin-RR induced oxidative injury. (c) 2005 Elsevier Ltd. All rights reserved.

Dioxin-like components in human breast milk collected from Hong Kong and Guangzhou

The H4IIE rat hepatoma cell line was employed as a cell model to screen 7-ethoxyresorufin O-deethylase (EROD)-TCDD equivalents (EROD-TEQ) of human breast milk samples collected from Hong Kong and Guangzhou, China. The screening methods employed a 96-well plate spectrofluorometer-EROD assay. For cell-line validation, our results demonstrated a dose-dependent increase in the Ah receptor-mediated response (i.e., CYP1A1 mRNA and EROD) of the cells upon exposure to a number of known Ah receptor agonists, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzothiophene, benzo[a]pyrene, and beta-naphthaflavone. TCDD induced CYP1A1 mRNA and EROD was in a close positive correlation (r = 0.98). For the screening of dioxin-like compounds, breast milk samples collected during lactation weeks 3-5 were used. One hundred (from Hong Kong) and 48 (from Guangzhou) breast milk samples were assayed, of which 65% and 68% of the samples, respectively, showed detectable dioxin-like activities using the H4IIE cell EROD screening method. For sixty-five samples from Hong Kong the mean EROD-TEQ values ranged from 58.1 to 96.5 pg/g of milk fat for those aged 21-36 years while 32 samples from Guangzhou had mean values of 98.8-202.1 pg/g of milk fat. In comparisons of the EROD-TEQ values for different age groups from both cities, there were no significant differences (P < 0.05). However, the mean and median EROD-TEQ values of the Guangzhou population were in general higher than those of the Hong Kong population. The results of the present study indicate that it is feasible to use the H4IIE cell-line as a model for screening dioxin-like compounds in human breast milk. In addition, the method is rapid and cost-effective, particularly for a routine and high-throughput sample screening analysis, compared to the costly and time-intensive chemical analytical techniques. (C) 2003 Elsevier Inc. All rights reserved.

Retention of the developmental pluripotency in medaka embryonic stem cells after gene transfer and long-term drug selection for gene targeting in fish

Embryonic stem (ES) cells provide a unique tool for introducing random or targeted genetic alterations, because it is possible that the desired, but extremely rare recombinant genotypes can be screened by drug selection. ES cell-mediated transgenesis has so far been limited to the mouse. In the fish medaka (Oryzias latipes) several ES cell lines have been made available. Here we report the optimized conditions for gene transfer and drug selection in the medaka ES cell line MES1 as a prelude for gene targeting in fish. MES1 cells gave rise to a moderate to high transfection efficiency by the calcium phosphate co-precipitation (5%), commercial reagents Fugene (11%), GeneJuice (21%) and electroporation (>30%). Transient gene transfer and CAT reporter assay revealed that several enhancers/promoters and their combinations including CMV, RSV and ST (the SV40 virus early gene enhancer linked to the thymidine kinase promoter) were suitable regulatory sequences to drive transgene expression in the MES1 cells. We show that neo, hyg or pac conferred resistance to G418, hygromycin or puromycin for positive selection, while the HSV-tk generated sensitivity to ganciclovir for negative selection. The positive-negative selection procedure that is widely used for gene targeting in mouse ES cells was found to be effective also in MES1 cells. Importantly, we demonstrate that MES1 cells after gene transfer and long-term drug selection retained the developmental pluripotency, as they were able to undergo induced differentiation in vitro and to contribute to various tissues and organs during chimeric embryogenesis.

Characterisation of a pathogenic virus isolated from marine threadfin fish (Eleutheronema tetradactylus) during a disease outbreak

An unknown virus was isolated from massive mortality of cultured threadfin (Eleutheronema tetradactylus) fingerlings. The virus replicated in BF-2 fish cell line and produced a plaque-like cytopathic effect. Electron micrographs revealed non-enveloped, icosahedral particles approximately 70-80 nm in diameter composed of a double capsid layer. Viroplasms and subviral particles approximately 30 run in diameter and complete particles of 70 nm in diameter were also observed in the infected BF-2 tissue culture cells. The virus was resistant upon pH 3 to 11 and ether treatment. It is also stable to heat treatment (3 h at 56 T). Replication was not inhibited by 5-iododeoxyuridine (5-IUdR). Acridine orange stain revealed typical reovirus-like cytoplasmic inclusion bodies. Electrophoresis of purified virus revealed 11 segments of double-stranded RNA and five major structural polypeptides of approximately 136, 132, 71, 41 and 33 kDa. Based on these findings, the virus isolated was identified to belong to the genus Aquareovirus and was designated as threadfin reovirus. This virus differed from a majority of other aquareovirus by its increase in virus infectivity upon exposure to various treatments such as high and low pH, heat (56 degreesC), ether and 5-IUdR. The RNA and virion protein banding pattern of the threadfin reovirus was shown to differ from another Asian isolate, the grass carp hemorrhage reovirus (GCV). Artificial injection of the threadfin reovirus into threadfin fingerlings resulted in complete mortality, whereas sea bass (Lates calcarifer) fingerlings infected via bath route showed severe mortality within a week after exposure. These results indicate that the threadfin virus is another pathogenic Asian aquareovirus isolate that could cross-infect into another marine fish, the sea bass. (C) 2002 Elsevier Science B.V. All rights reserved.

Development of a positive-negative selection procedure for gene targeting in fish cells

The gene targeting technique is a powerful tool for analyzing functions of cloned genes and for generating transgenic animals with site-directed integration of foreign genes. In order to develop this technique in fish, positive-negative selection (PNS) and homologous recombination vectors were constructed, and their expression was examined in fish cells. A vector (pNK) for PNS consists of the neomycin resistance gene (neo) as a positive selectable marker gene and the herpes simplex virus (HSV) thymidine kinase (tk) gene as a negative selectable marker gene. Positive selection with geneticin (G418) of epithelioma papulosum of carp (EPC) cells transfected with linearized pNK vector yielded 350 colonies, while double selection of transfected EPC cells with G418 and gancyclovir (Gc) resulted in nearly complete cell death, demonstrating that the PNS procedure is effective in fish cells. Homologous recombination vectors consist of the Xiphophorus melanoma receptor kinase (X mrk(Y)) gene as homologous sequence in addition to the neo and tk genes. Conditions for homologous recombination vector transfection and drug selection were established. After verification of the feasibility of expression of homologous recombination vectors in EPC cells, the first gene targeting experiments were attempted in the Xiphophorus melanoma cell line, PSM. Positive-negative selection of the targeting vector-transfectants led to a low enrichment in this particular cell line. The reasons for the low enrichment in PSM cells were discussed. (C) 2002 Elsevier Science B.V. All rights reserved.

唾液酸转移酶Ⅰ（ST6GalⅠ）及甲胎蛋白（AFP）上肝癌特异糖链在肝细胞肝癌细胞系和肝病患者血液中的表达

肝癌（HCC）是一种重要的恶性肿瘤，具有高发病率和不良预后的特点，在中国有很多的肝癌患者。早期发现和精确区分肝癌与其他肝病，对于肝癌的临床诊断治疗有十分重要的作用。由于肝脏所处位置较深，以及检测仪器和手段的限制，肝癌的早期诊断相对困难，所以比较好的血清学标记物应用于早期区分肝癌和其他肝病显得尤为重要。目前应用较多的标记物是甲胎蛋白AFP。由于单独的AFP其敏感性和特异性并不高，更为理想的检测手段正在研究中。 Beta-galactoside alpha-2,6-sialyltransferase Ⅰ(ST6GalⅠ)是唾液酸转移酶家族中的一员，主要调控蛋白的唾液酸化。有研究发现ST6Gal在肝癌中表达升高，而且随着肝实体瘤的恶化而可能被释放到外周血中。所以血液中的ST6GalⅠ水平可能是一种有潜力的肝癌指标。另外，有研究显示，AFP的糖链结构在肝癌和其他肝病中是不同的，如果能用AFP上肝癌特异糖链作为检测目标，联合已有的检测体系，可能有助于早期精确诊断肝癌。 目的：我们选择ST6Gal Ⅰ和AFP上肝癌特异糖链TF（Thomsen-Friedenreich）抗原，H2血型抗原，Lewis Y血型抗原作为主要的研究内容，通过对其在肝癌细胞系和肝病血液中的表达研究，探讨其应用于肝癌早期诊断的可能。 方法：1）用合成的ST6Gal Ⅰ多肽免疫兔子，制备抗ST6Gal Ⅰ多克隆抗体；2）通过免疫细胞化学，ELISA和western blot等方法对制作的兔多克隆抗体进行效价分析；3）通过western blot方法对肝癌、肝硬化和正常人的血浆和血清中ST6Gal表达进行研究；4）通过免疫沉淀的方法对肝癌细胞系和肝癌血浆中AFP上肝癌特异糖链进行研究。 结果：1）制备的兔抗人ST多克隆抗血清，效价可达1:400000，能够通过免疫细胞化学和western blot检测到肝癌细胞系中的ST6GalⅠ；2）所选的3例肝癌血浆和2例肝癌血清中都有ST6GalⅠ阳性，在所选的2例肝硬化血浆和2例肝硬化血清中，分别只有1例有ST6GalⅠ的阳性，在3例正常人血浆中有2例有ST6GalⅠ阳性；3）AFP上肝癌特异的糖链TF抗原、H2抗原、Lewis Y抗原可以通过免疫沉淀的方法在肝癌病人血浆和肝癌细胞系HepG2中检出。 结论：制备的兔抗人ST6GalⅠ多克隆抗体具有较高效价，并可以检测到自然状态和变性状态的目标蛋白，可以应用于ST6GalⅠ的研究；通过western blotting对肝病血液的检测，发现在肝癌、肝硬化和正常人血液中的ST6GalⅠ的表达有差异，为ST6GalⅠ作为一种肿瘤标记物进行血清学检测提供了可能的前提；免疫沉淀的结果，证实了在肝癌病人的AFP上有TF、H2、LewisY等糖链的存在，为AFP上肝癌特异糖链作一种区分肝癌和其他良性肝病的标记物提供了证据。

滇金足草、凋缨菊和长喙吴萸的化学成分研究

本论文对滇金足草（Goldfussia yunnanensis）、凋缨菊（Camchaya loloana）和长喙吴萸（Evodia vestia）的化学成分进行了研究，通过色谱分离得到40个化合物。主要基于波谱数据鉴定了它们的结构，其中10个为新化合物。 1．从滇金足草地上枝叶的95%乙醇提取物中共分离鉴定了16个化合物：泽漆内酯A（1）、18-羟基泽漆内酯A（2）、18-氧代泽漆内酯A（3）、18-羟基-3-O-β-D-吡喃葡萄糖-泽漆内酯A（4）、3-O-β-D-吡喃葡萄糖-泽漆内酯A（5）、3-O-β-D-吡喃半乳糖-泽漆内酯A（6）、6-E-肉桂酰哈巴俄苷（7）、E-哈巴俄苷（8）、5，6-异亚丙二氧基哈巴俄苷（9）、β-谷甾醇（10）、β-胡萝卜苷（11）、齐墩果酸（12）、肉桂酸（13）、麦角固醇（14）、硬脂酸（15）和丁二酸（16）。其中2-7为新化合物。5，6-异亚丙二氧基哈巴俄苷（9）以人工产物形式得到。 2．从凋缨菊地上枝叶的95%乙醇提取物中分离并鉴定了13个化合物：凋缨菊内酯A~C (17-19)、1β-乙酰基凋缨菊内酯C(20)、b-谷甾醇（10）、β-胡萝卜苷（11）、羽扇豆醇（21）、桦木醇（22）、桦木酸（23）、芥子醇（24）、紫丁香苷（25）、咖啡酸（26）和熊果酸（27）。其中化合物17-20为桉叶烷内酯类新化合物。化合物17、18、20对细胞株HepG2的GI50依次为7.80、7.08、4.99 µg/mL。 3．从长喙吴萸（E. vestia）地上枝叶的95%乙醇提取物中分离并鉴定了13个化合物：佛手内酯（28）、花椒毒素（29）、异茴芹内酯（30）、七叶内酯（31）、东莨宕素（32）、瑞香素（33）、异紫花前胡内酯(34)、茵芋碱（35）、山刈碱（36）、白鲜碱（37）、黄柏酮（38）、柠檬苦素（39）和对羟基苯甲醛（40）。 4．综述了1990—2007年期间从菊科植物中发现的桉叶烷-12，6内酯的化学结构、生物活性、生物转化及化学合成方面的研究进展。 Phytochemical investigation on Goldfussia yunnanensis, Camchaya loloana, and Evodia vestia, led to the isolation of 40 compounds, 10 of which were new ones. 1. Six new compounds were isolation from 95% ethanolic extract of the aerial parts of G. yunnanensis, and identified as 18-hydroxyhelioscopinolide A (2), 18-oxohelioscopinolide A (3), 18-hydroxy-3-O-β-D-glucopyranosylhelioscopinolide A (4), 3-O-β-D-glucopyranosylhelioscopinolide A (5),3-O-β-D-Galactopyranosyl helioscopinolide A (6), 6-O-trans-cinnamoyl E-harpagoside (7). The known compounds isolated were helioscopinolide A (1), E-harpagoside A (8), 5,6-isopropylidene E-harpagoside A (9), β-sitosterol (10), β-daucosterol (11), oleanolic acid (12), cinnamic acid (13), ergosterol (14), stearic acid (15) and succinic acid (16). Compound 9 was an artifact. 2. Four new compounds, loloanolides A – C (17 - 19) and 1β-acetoxy-loloanolide C (20), were isolation from 95% ethanolic extract of the aerial parts of C. loloana. The known ones were β-sitosterol (10), β-daucosterol (11), lupeol (21), betulin (22), betulinic acid (23), sinapyl (24), syringin (25), caffeic acid (26) and ursolic acid (27). The GI50 values of compounds 17, 18 and 20 to HepG2 cell line were 7.80, 7.08 and 4.99 µg/mL, respectively. 3. Thirteen were isolated from 95% ethanolic extract of the aerial parts of E. vestia for the first time. They were determined to be bergapten (28), xanthotoxin (29), isopimpinellin (30), esculetin (31), scopoletin (32), daphnetin (33), marmesin (34), skimmianine (35), confusameline (36), dictamine (37), obacunone (38), limonin (39) and p-hydroxy phenyl aldehyde (40). 4. The structures, biological activities, biotransformation and chemical syntheses of eudesmane-12, 6-olides from the Asteraceae during 1990-2007 were reviewed.