Antibacterial Biphenanthrenes from the Fibrous Roots of Bletilla striata.

Four new 9',10'-dihydro-biphenanthrenes, including an unprecedented 1,2'-linked biphenanthrene, 4,7,3',5'-tetramethoxy-9',10'-dihydro(1,2'-biphenanthrene)-2,7'-diol (1), a new 1,3'-linked biphenanthrene, 4,7,7'-trimethoxy-9',10'-dihydro(1,3'-biphenanthrene)-2,2',5'-triol (2), and two new 1,1'-linked biphenanthrenes, 4,7,4'-trimethoxy-9',10'-dihydro(1,1'-biphenanthrene)-2,2',7'-triol (3) and 4,7,3',5'-tetramethoxy-9',10'-dihydro(1,1'-biphenanthrene)-2,2',7'-triol (4), as well as two known biphenanthrenes (5, 6), were isolated from a 95% ethanol extract of the fibrous roots of Bletilla striata. Their structures were determined by spectroscopic and spectrometric methods. Atropisomerism of these compounds was considered based on their chiral optical properties and potential energy surface scans at the ab initio HF/3-21G level, which revealed their racemic mixture form. Compounds 2-6 showed potent antibacterial activities against six Gram-positive bacterial strains.

Lasiodiplodia sp. ME4-2, an endophytic fungus from the floral parts of Viscum coloratum, produces indole-3-carboxylic acid and other aromatic metabolites.

BACKGROUND: Studies on endophytes, a relatively under-explored group of microorganisms, are currently popular amongst biologists and natural product researchers. A fungal strain (ME4-2) was isolated from flower samples of mistletoe (Viscum coloratum) during a screening program for endophytes. As limited information on floral endophytes is available, the aim of the present study is to characterise fungal endophytes using their secondary metabolites. RESULTS: ME4-2 grew well in both natural and basic synthetic media but produced no conidia. Sequence analysis of its internal transcribed spacer rDNA demonstrated that ME4-2 forms a distinct branch within the genus Lasiodiplodia and is closely related to L. pseudotheobromae. This floral endophyte was thus identified as Lasiodiplodia sp. based on its molecular biological characteristics. Five aromatic compounds, including cyclo-(Trp-Ala), indole-3-carboxylic acid (ICA), indole-3-carbaldehyde, mellein and 2-phenylethanol, were found in the culture. The structures of these compounds were determined using spectroscopic methods combined with gas chromatography. To the best of our knowledge, our work is the first to report isolation of these aromatic metabolites from a floral endophyte. Interestingly, ICA, a major secondary metabolite produced by ME4-2, seemed to be biosynthesized via an unusual pathway. Furthermore, our results indicate that the fungus ME4-2 is a potent producer of 2-phenylethanol, which is a common component of floral essential oils. CONCLUSIONS: This study introduces a fungal strain producing several important aromatic metabolites with pharmaceutical or food applications and suggests that endophytic fungi isolated from plant flowers are promising natural sources of aromatic compounds.

Evidence for nitrite-dependent anaerobic methane oxidation as a previously overlooked microbial methane sink in wetlands.

The process of nitrite-dependent anaerobic methane oxidation (n-damo) was recently discovered and shown to be mediated by "Candidatus Methylomirabilis oxyfera" (M. oxyfera). Here, evidence for n-damo in three different freshwater wetlands located in southeastern China was obtained using stable isotope measurements, quantitative PCR assays, and 16S rRNA and particulate methane monooxygenase gene clone library analyses. Stable isotope experiments confirmed the occurrence of n-damo in the examined wetlands, and the potential n-damo rates ranged from 0.31 to 5.43 nmol CO2 per gram of dry soil per day at different depths of soil cores. A combined analysis of 16S rRNA and particulate methane monooxygenase genes demonstrated that M. oxyfera-like bacteria were mainly present in the deep soil with a maximum abundance of 3.2 × 10(7) gene copies per gram of dry soil. It is estimated that ∼0.51 g of CH4 m(-2) per year could be linked to the n-damo process in the examined wetlands based on the measured potential n-damo rates. This study presents previously unidentified confirmation that the n-damo process is a previously overlooked microbial methane sink in wetlands, and n-damo has the potential to be a globally important methane sink due to increasing nitrogen pollution.

Distribution and diversity of nitrite-dependent anaerobic methane-oxidising bacteria in the sediments of the Qiantang River.

Nitrite-dependent anaerobic methane oxidation (n-damo) process was reported to be mediated by "Candidatus Methylomirabilis oxyfera", which belongs to the candidate phylum NC10. M. oxyfera-like bacteria have been detected in lake ecosystems, while their distribution, diversity and abundance in river ecosystems have not been well studied. In this study, both the 16S rRNA and the pmoA molecular biomarkers confirmed the presence of diverse NC10 phylum bacteria related to M. oxyfera in a river ecosystem-the Qiantang River, Zhejiang Province (China). Phylogenetic analysis of 16S rRNA genes demonstrated that the recovered M. oxyfera-like sequences could be grouped into several distinct clusters that exhibited 89.8% to 98.9% identity to the M. oxyfera 16S rRNA gene. Similarly, several different clusters of pmoA gene sequences were observed, and these clusters displayed 85.1-95.4% sequence identity to the pmoA gene of M. oxyfera. Quantitative PCR showed that the abundance of M. oxyfera-like bacteria varied from 1.32 ± 0.16 × 10(6) to 1.03 ± 0.12 × 10(7) copies g (dry weight)(-1). Correlation analysis demonstrated that the total inorganic nitrogen content, the ammonium content and the organic content of the sediment were important factors affecting the distribution of M. oxyfera-like bacterial groups in the examined sediments. This study demonstrated the distribution of diverse M. oxyfera-like bacteria and their correlation with environmental factors in Qiantang River sediments.

Antitumor activity of bacterial exopolysaccharides from the endophyte Bacillus amyloliquefaciens sp. isolated from Ophiopogon japonicus.

The endophytic bacterium, MD-b1, was isolated from the medicinal plant Ophiopogon japonicas and identified as the Bacillus amyloliquefaciens sp. with 99% similarity based on the partial sequence analysis of 16S rDNA. Exopolysaccharides were extracted from the endophyte for the evaluation of its antitumor activity against gastric carcinoma cell lines (MC-4 and SGC-7901). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and microscopy were performed to estimate the cell viability and morphological changes of the MC-4 and SGC-7901 cells following treatment with the exopolysaccharides at 14, 22 and 30 µg/µl. The results revealed that the exopolysaccharides displayed concentration-dependent inhibitory effects against the MC-4 and SGC-7901 cells, with an IC50 of 19.7 and 26.8 µg/µl, respectively. The exopolysaccharides also induced morphological abnormalities in the cells. These effects indicated the the exopolysaccharides had an antitumoral mechanism of action associated with the mitochondrial dysfunction of the treated cells. This is the first study to investigate the endophytic microorganism isolated from O. japonicas and also the first discovery of such antitumoral exopolysaccharides derived from the genus Bacillus. This provides a promising and reproducible natural product source with high therapeutic value for anticancer treatment, thereby facilitating the development of new anticancer agents.

A PVP-extract fungal protein of Omphalia lapideacens and its antitumor activity on human gastric tumors and normal cells.

Omphalia lapidescens is an important medicinal fungus as well as traditional Chinese medicine used for disease treatment. It is mainly used as a vermifuge for anthelmintic therapy, but it has not been hitherto reported to possess antitumor activity. In this study, a purified bioactive protein in O. lapidescens (pPeOp) was obtained using polyvinylpyrrolidone (PVP) followed by gel filtration chromatography. To evaluate the in vitro antitumor activity of pPeOp in human gastric tumor cells (MC-4 and SGC-7901) and normal cells (MC-1), MTT assay and FCM assay were used and the morphological changes, cell viability, cell death rate and cell apoptosis rate of MC-4, SGC-7901 and MC-1 cells were estimated. The results showed that pPeOp could significantly reduce the cell viability of MC-4 and SGC-7901 cells in a concentration-dependent manner, with IC50 values of 236.05 and 156.28 µg/ml, respectively. The morphological observation also indicated a similar result. In FCM assays, a significant increase of cell death rate and cell apoptosis rate of the tumor cells were observed, indicating probable necrosis-inducing effects and/or apoptosis-inducing effects of pPeOp. Importantly, there was no significant effect of pPeOp on MC-1 cells in each assay, showing that pPeOp has no adverse effects on the normal cells. In conclusion, pPeOp is a newly discovered bioactive protein in O. lapidescens and this is the first report on antitumor activity of such a fungal protein. This may provide a meaningful basis for developing a new protein drug for treatment against cancer, especially gastric cancer.

[Study of the property of lipids reducing of curcumin on hyperlipidemia mice after fermented by Monascus purureus].

OBJECTIVE: To study the lipids reducing property of curcumin on Hyperlipidemia mice after fermented by Monascus purureus. METHODS: The stain Monascus purureus was used for microbial transformation, and both substrate control and strain control were set. The mice were reared with high lipid and cholesterol feed for 15d to establish the Hyperlipidemia models. The models were treated with fermented curcumin in 500 mg/kg, 200 mg/kg, 100 mg/kg, substrate control and strain control in 500 mg/kg. Positive and Normal group were treated with natural saline. The general situation was observed and the changes of TG, TC, HDL-C levels in serum and liver were tested after 10 d. RESULTS: Fermented curcumin could significantly reduce the serum TC, TG of Hyperlipidemia mice all in high, middle and low doses. Serum TC was reduced by 38.7%, 34.5%, 32.7% and TG was reduced by 38.3%, 28.6%, 30.1%, respectively while substrate control and strain control had no effect. Fermented curcumin also could reduce the TC, TG in liver but no effect of curcumin substrate at the same dose. CONCLUSION: The property of lipids reducing of curcumin is significantly enhanced after fermentation.

[Effect of proteins extracted from mycelia of Omphalia lapidescens on inhibiting H, liver cancer in mice and regulating immune function].

OBJECTIVE: To explore the effect of proteins extraceed from mycelia of Omphalia lapidescens on inhibiting H22 liver cancer in vivo. METHODS: 50 hepatoma 22 tumor bearing mice models were divided into five groups randomly:control group( CG), cyclophosphamide group, and 3 groups of incremental Hepatoma 22 dosages (5, 3, 1 mg/kg). All groups were i.v. with drugs once a day. After 8 consecutive days, the concentrations of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in serum were detected by enzyme-linked immunoabsorbent assay (ELISA). The weight changes of tumor, thymus, liver, heart, spleen, lung and kidney were observed. RESULTS: It showed the tumors' weight were significant heavier in CG than in EGs. The tumor-inhibition rate (IR) was 36.4% in high dosage group,which was lower than 43.2% in cyclophosphamide group. The spleen mass of proteins groups increased significantly. The concentration of IFN-gamma in serum of proteins groups increased as CG, but IL-4 in inverse direction. The observations of thymus, liver, heart, lung and kidney in EGs were the same as CG. CONCLUSION: The proteins extracted from mycelium of Omphalia lapidescens can inhibit the growth of tumour and enhance the immune function of H22 tumor-bearing mice.

[Study on endophyte and the growth stages of Ophiopogon japonicus].

OBJECTIVE: To study on the relationship between the endophyte and the life cycle of Ophiopogon japonicus in Zhejiang. METHODS: Sample roots of Ophiopogon japonicus at different growth stages were thoroughly washed and cut into small fragments, then cleared (removing cytoplasmic contents from cells) using hot 10% KOH and stained with acid fuchsin (alternative stain). The hyphae, the arbuscular and the vesicular of endophyte were examined. RESULTS: The hyphae appeared and grew in the seedling stage, the hyphae grew into arbuscular in the root tuber generating stage and vesicular in the stage of root tuber expanding period. CONCLUSION: The endophytes in Ophiopogon japonicus appeared in forms of hyphae, arbuscular and vesicular at different growth stages to meet the needs of Ophiopogon japonicus developing.

[Isolation and fermentation culture of fungi from Cordyceps soofifera].

OBJECTIVE: To study the fungi isolated from Cordyceps sobolifera and its fermentation culture. METHODS: The fungi was isolated and identified by its hypha and spores. Three liquid media were used in the culture. RESULTS: Pure culture was gained and the fungi was identified to be Paecilomyces cicadae. The fungi can grow best in liquid media: egg 110 g + silkworm powder 30 g + VB1 2 tahlets + MgSO4 x 7H2O 0.5 g + K2HPO4 1 g + H2O 1000 ml, in which every litre can produce 135 g wet hypha after cultured 4 d. CONCLUSION: Paecilomyces cicadae from Cordyceps sobolifera can be cultured in liquid media.

[Complete HBV DNA clone and sequence from serum samples of severe hepatitis B patients].

OBJECTIVE: To study the association between hepatitis B virus (HBV) mutants and the pathogenesis of severe hepatitis B by full-length HBV genome. METHODS: Serum samples from 10 severe hepatitis B patients were collected in our hospital. Serum HBV DNAs were extracted using DNA mini Kit, and amplified by LA Taq DNA polymerase to yield full-length HBV DNA. PCR products were isolated and cloned into vector pUCm-T, then transfected into DH-5 alpha cells. Positive clones were selected and checked by digestion, and full-length HBV DNAs were sequenced. RESULTS: 4 cases were cloned into vector pUCm-T successfully and completed the full-length sequencing. Among them, 3 cases had a G to A mutation at nucleotide 1896 in pre-C region and 1 had a double mutation of T1762-A1764 in the core promoter region. Some amino acid changes occurred within the known CTL, B or T cell epitopes of the PrS2 and C regions. CONCLUSIONS: This method could serve to study the relationship between HBV genome and the pathogenesis of severe hepatitis B.

Determination of serum carbohydrate-deficient transferrin in the diagnosis of alcoholic liver disease.

BACKGROUND: Alcoholic liver disease (ALD) is a serious and potentially fetal consequence of alcohol use. The diagnosis of ALD is based on alcohol consumption, physical signs and symptoms, and laboratory tests. The aim of the present study was to assess the reliability of carbohydrate-deficient transferrin (CDT) in the diagnosis of ALD. METHODS: According to the diagnostic criteria for ALD by the Chinese Medical Association in 1995, 76 patients with ALD, 55 patients with alcoholism, 32 patients with nonalcoholic liver disease (NALD), and 27 healthy subjects (controls) were studied. Serum CDT was assayed by isoelectric focusing immunofixation and Comassie blue staining. The levels of alamine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma glutamyltransferase (GGT) were also examined. RESULTS: The positive rate of CDT in the patients with ALD was 93.4%(71/76), which was higher than that in those with alcoholism (52.7%, 29/55, P<0.001), in those with NALD(9.4%, 3/32, P<0.001), and in healthy controls, respectively. The sensitivity and specificity of CDT for ALD was 93.4% and 71.9%, respectively. CONCLUSION: CDT may help diagnose alcoholic liver disease.