Objective To explore the expression pattern of Micall2a gene during the early development of zebrafish embryos and the effect of this gene on zebrafish vascular development.MethodsWhole embryo in situ hybridization was used to detect Micall2a expression levels at different stages of early embryo development of Tg (fli:GFP) transgenic (labeled with green fluorescent protein) and wild type zebrafish (AB). Micall2a gene expression was downregulated by microinjection of a morpholine antisense oligonucleotide, and real-time fluorescent quantitative PCR was used to detect mRNA expression of the gene at different developmental stages of zebrafish embryos. Laser confocal microscopy was used to observe and analyze vascular phenotypic changes in zebrafish after the downregulation of Micall2a. ResultsMicall2a was expressed in the brain, heart, and vascular system of zebrafish embryos at the 24th, 36th, and 48th hours post fertilization. The mRNA level of Micall2a increased after microinjection of morpholine antisense oligonucleotides, inhibiting vascular development in zebrafish embryos, resulting in internode angiogenesis defects in zebrafish. ConclusionDownregulation of Micall2a expression inhibits the development of blood vessels in zebrafish.

The aim of this study was to identify antigens for a vaccine or drug target to control rabbit coccidiosis. A combination of 2-dimensional electrophoresis, immunoblotting, and mass spectrometric analysis were used to identify novel antigens from the sporozoites of Eimeria stiedae. Protein spots were recognized by the sera of New Zealand rabbits infected artificially with E. stiedae. The proteins were characterized by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/TOF-MS) analysis in combination with bioinformatics. Approximately 868 protein spots were detected by silver-staining, and a total of 41 immunoreactive protein spots were recognized by anti-E. stiedae sera. Finally, 23 protein spots were successfully identified. The proteins such as heat shock protein 70 and aspartyl protease may have potential as immunodiagnostic or vaccine antigens. The immunoreactive proteins were found to possess a wide range of biological functions. This study is the first to report the proteins recognized by sera of infected rabbits with E. stiedae, which might be helpful in identifying potential targets for vaccine development to control rabbit coccidiosis.
Coccidiosis* ; Computational Biology ; Eimeria ; Electrophoresis ; HSP70 Heat-Shock Proteins ; Immunoblotting ; Mass Spectrometry ; Rabbits ; Sporozoites

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate in mavalonic acid pathway, which is the first committed step for isoprenoid biosynthesis in plants. However, it still remains unclear whether HGMR gene plays a role in the isoprenoid biosynthesis in Dendrobium officinale, an endangered epiphytic orchid species. In the present study, a HMGR encoding gene, designed as DoHMGR1 (GenBank accession JX272632), was identified from D. officinale using the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods, for the first time. The full length cDNA of DoHMGR1 was 2 071 bp in length and encoded a 562-aa protein with a molecular weight of 59.73 kD and an isoelectric point (pI) of 6.18. The deduced DoHMGR1 protein, like other HMGR proteins, constituted four conserved domains (63-561, 147-551, 268-383 and 124-541) and two transmembrane motifs (42-64 and 85-107). Multiple sequence alignment and phylogenetic analyses demonstrated that DoHMGR1 had high identity (67%-89%) to a number of HMGR genes from various plants and was closely related to Vanda hybrid cultivar, rice and maize monocots. Real time quantitative PCR (qPCR) analysis revealed that DoHMGR1 was expressed in the three included organs. The transcripts were the most abundant in the roots with 2.13 fold over that in the leaves, followed by that in the stems with 1.98 fold. Molecular characterization of DoHMGR1 will be useful for further functional elucidation of the gene involving in isoprenoid biosynthesis pathway in D. officinale.

OBJECTIVETo isolate and characterize endophytic fungi from seven Dendrobium species, and detect their antimicrobial activities.

METHODFungal endophytes were isolated by strictly sterile sample preparation and fungal identification methods were based on their ITS ribosomal DNA (ITS rDNA gene) sequences. The agar well diffusion method was then employed to evaluate the antimicrobial activity against six pathogenic organisms and the phylogenetic tree of active isolates was constructed by the MEGA.

RESULTNinety-eight endophytic fungi obtained from seven Dendrobium spp., and among them twenty-four isolates, representing 11 genera and 14 species, displayed anti-microbial activities. The phylogenetic assay based on ITS-rDNA showed that 24 active isolates were sorted to 7 taxonomic orders: Hypocreales, Sordariales, Capnodiales, Eurotiales, Botryosphaeriales, Xylariales and Mucorales. The results of antimicrobial activity assay revealed that 1.02%, 10.2%, 18.4%, 1.02%, 1.02% and 10.2% of fermentation broths of 98 isolates displayed significant antimicrobial activities against E. coli, B. subtilis, S. aureus, C. albicans, C. neoformans and A. fumigatus, respectively. Four strains DL-R-3, DL-S-6, DG-R-10 and DN-S-1 displayed strong and broad antimicrobial spectrum.

CONCLUSIONEndophytic fungi associated with Dendrobium species have fungal diversity, and possess diverse antimicrobial activity.

Anti-Infective Agents ; metabolism ; pharmacology ; Aspergillus fumigatus ; drug effects ; Bacillus subtilis ; drug effects ; Base Sequence ; Biodiversity ; Candida albicans ; drug effects ; China ; Cryptococcus neoformans ; drug effects ; DNA, Fungal ; chemistry ; isolation & purification ; DNA, Ribosomal Spacer ; chemistry ; genetics ; Dendrobium ; microbiology ; physiology ; Endophytes ; classification ; genetics ; isolation & purification ; physiology ; Escherichia coli ; drug effects ; Fungi ; classification ; genetics ; isolation & purification ; physiology ; Microbial Sensitivity Tests ; Molecular Sequence Data ; Phylogeny ; Plant Roots ; microbiology ; physiology ; Plant Stems ; microbiology ; physiology ; Sequence Alignment ; Sequence Analysis, DNA ; Staphylococcus aureus ; drug effects

OBJECTIVETo set up a system for fluid suspension co-culture of Dendrobium officinale protocorm and living fungus MF24, so as to provide certain scientific evidence for industrial production of protocorm.

METHODWhether the protocorm culture system was suitable for the normal growth of MF24 fungus were studied, the growth of protocorm cultured alone and co-cultured with the fungus were researched under light and dark culture conditions, the biomass and proliferation times were determined, and HPLC method was used to analyze and compare the changes of 11 characteristic peak areas in D. officinale protocorm.

RESULTThe MF24 fungus could grow normally in the 6,7-V liquid medium used to culture the protocorm, and when it was cultured by 8-10 hours per day under 1 500 lx, the growth rate of the fungus was slowed. Protocorm could grow normally in light and dark culture conditions, and add the MF24 fungus in the early cultivation stages of protocorm, both inhibit the growth of each other. In the protocorm for the growth stability to add 5 diameter 9 mm fungi block, the protocorm growth and chemical composition type had no significant effect. However, under illumination, co-cultured for 5 days protocorm of which 10 compounds content decreased 13.64% to 138.47%, in dark conditions, co-cultured for 5 days protocorm of which 7 compounds increased by 0.71% to 12.82%, and 4 compounds slightly reduced by 3.03% to 14. 14% compared with the control.

CONCLUSIONUnder the appropriate condition, living fungus MF24 could co-culture with the D. officinale protocorm, and affected the latter's secondary metabolite levels.

Coculture Techniques ; methods ; Darkness ; Dendrobium ; cytology ; growth & development ; metabolism ; radiation effects ; Fungi ; growth & development ; metabolism ; physiology ; radiation effects ; Suspensions

The column chromatography on silica gel, sephadex LH-20 preparative HPLC were used to separate and purify the compounds from the stems of Dendrobium candidum. Twenty compounds were isolated and identified as 3,4'-dihydroxy-5-methoxybibenzyl(1), dihydroresveratrol(2), dendromoniliside E(3), denbinobin(4),2,4,7-trihydroxy-9, 10-dihydrophenanthrene(5), aduncin(6), (-)-loliolide(7), adenosine(8), uridine(9), guanosine(10), sucrose(11), 5-hydroxymethyl-furaldehyde(12), n-octacostyl ferulate(13), defuscin(14), n-triacontyl cis-p-coumarate(15), daucosterol(16), beta-sitosterol(17), hexadecanoic acid(18), hentriacontane(19), and heptadecanoic acid(20). Their structures were elucidated on the basis of spectroscopic data and physicochemical properties. All of the compounds were isolated from this plant for the first time.
Dendrobium ; chemistry ; Molecular Structure ; Plant Extracts ; chemistry ; isolation & purification

OBJECTIVETo compare the rutin and syringin content in tissue culturing seedlings and in botanical drug of Saussurea involucrata.

METHODThe HPLC with Hydro-RP C18 (4.6 mm x 250 mm, 5 microm) column was used, a mixture of acetonitrile-water (5:95) was used as a mobile phase, with flow rate of 1 mL x min(-1), column temperature at 25 degrees C and detection wavelength at 220 nm.

RESULTThe effective constituents of tissue culturing seedlings were almost similar to the botanical drug. And syringin in tissue culturing seedlings was increased 4.35 times.

CONCLUSIONIt has a good prospect to acquire high-quality S. involucrata by tissue culturing seedlings.

Chromatography, High Pressure Liquid ; Glucosides ; analysis ; Phenylpropionates ; analysis ; Rutin ; analysis ; Saussurea ; chemistry ; growth & development ; Seedlings ; chemistry

BACKGROUND: Bone mineral content(BMC) can be determined by many methods, which are different in detecting position, clinical significance and differentiation between normal group and people with osteoporosis.OBJECTIVE: To establish a normal BMC standard by observing lumbar BMC with quantitative computerized tomography(QCT) measurement in Shenzhen women, so as to provide basis for clinical prevention and treatment of osteoporosis in the region.DESIGN: Randomized controlled, observational and comparative study taking normal women as subjects.SETTING: Medical imaging department of a hospital at district level.PARTICIPANTS: A total of 120 women aged 30 to 69 years, who received physical examination in the Central Hospital of Longgang district in Shenzhen from September 2000 to March 2002, were enrolled in this study. They were divided into four groups: 30 - 39 age group, 40 - 49 age group, 50 - 59 age group and 60 - 69 age group with 30 in each.METHODS: Trabecular and cortical BMC of lumber bodies(L1-3) were measured with QCT software so as to establish a standard of normal BMC in Shenzhen women and compare it with that of other regions.MAIN OUTCOME MEASURES: The mean BMC in Shenzhen women, and comparison with that of other regions at home and abroad.RESULTS: The results of QCT showed linear correlation between BMC and bone ash weight, which could be expressed by the following linear regression equation: ash weight =0. 92432 × BMC + 39. 0633. Lumbar BMC loss increased with age in Shenzhen perimenopausal women. The annual loss of spongy bone and compact bones was 1.38% and 0. 84%, respectively. BMC of women aged 50 to 59 years was[ (135.31 ± 18.36) mg/cm3], obviously higher than that of women in Changchun city, Beijing city and the United States [(120.21 ±37.40), (116.7 ±26.6), and(119.5 ±27.1) mg/cm3]( t = 2. 002, 3. 383, 3. 636, P ＜ 0.05 - 0. 01 ) . Moreover, BMC of women aged 30 to 39 years was also obviously higher than that of corresponding American women( t = 3.119, P ＜ 0.01 ). No significant difference was found in BMC among women of the other age groups in these regions( P ＞ 0. 05).CONCLUSION: This is the first time in our country to establish a standard of normal BMC in perimenopausal women with QCT measurement, which provides basis for early prevention and treatment of osteoporosis as well as evaluation of prognosis and fracture risk.

The pharmacological activity of Mycena dendrobii Fan et Guo, a new species of endophytic fungus was studied. It was revealed that the mycelia methanol extracts and the fermentation liquid ethanol extracts of Mycena dendrobii showed anglgesic effect to mice, which have the correlations to that of the traditional Chinese medicine ‘shihu’. The fermentation liquid ethanol extracts of Mycena dendrobii showed excitation effect to central nervous system of mice. Then the effective parts of anglgesic effect was determined.

Purpose　The aim is to study the conditions of preparation and regeneration of Gliocladium sp. (F) protoplast. Methods　 Different enzyme systems, enzymolysis time, osmotic pressure stabilizers were studied to investigate their influence on the productivity and regenerating rate of Gliocladiumsp. F protoplasts. The HPLC method was used to determine EP (6,22-diene-5,8-epidioxy ergosta-3-hydroxy) content.Results　The higher productivity of protoplasts was obtained when mycelia of strain F growing for 60 hours was digested at 28℃ for 4 hours by solution containing 2% cellulase and 2% helicase dissolved in 0.5 mol/L mannitol and the medium containing 0.5mol/L mannitol as osmotic pressure stabilizer would be suitable for protoplast regeneration. According to the EP productivity detected by HPLC, high positive rate of the regenerated strains growing in the medium containing 0.5mol/L mannitol could be got. Conclusion　The results will promote the research of strain F mutantgenesis and will be helpful for obtaining the strain more effectively biosythesizing compound EP.