To construct a system of genetic transformation suitable for Rhizopus oryzae, we constructed a single-exchange vector pBS-hygro carrying hygromycin B resistance gene (hph) as its selective marker using gene splicing by overlap extension PCR (SOE PCR) technique. We introduced this recombinant vector into Rhizopus oryzae AS 3.819 by PEG/CaCl2-mediated transformation of protoplast, electroporation of protoplast and germinated spores; and we studied the effects of hydrolysis time, field strength and spore germination time on transformation frequency. We conducted quantitative real-time PCR (qPCR) assay to determine the gene copy number of ldhA integrated in the genome of R. oryzae transformants and its effect on the stability of transformants. We successfully achieved R. oryzae transformants integrated with pBS-hygro-ldhA vector. The optimal hydrolysis time for protoplast production was 140 min, and the optimal field strength of electroporation pulse for protoplast was 13 kV/cm. The optimal germination time of spores for electroporation was 2.5 h, and the optimal field strength of electroporation pulse was 14 kV/cm. The transformation frequency of method based on germinated spores was generally higher than the methods based on protoplast. The qPCR test results suggested that transformants with high copy number of integration in a certain range were relatively stable. Our results provided basis and support for metabolic regulation and genetic engineering breeding of R. oryzae.
DNA, Recombinant ; Electroporation ; Genetic Engineering ; Genetic Vectors ; Hygromycin B ; Protoplasts ; Real-Time Polymerase Chain Reaction ; Rhizopus ; genetics ; Transformation, Genetic

1H NMR-based metabolomic approach combined with multivariate statistical analysis was used to evaluate the quality of 21 Farfarae Flos (FF) samples from different growth regions. Principal component analysis showed that wild and cultivated FF could be separated clearly, suggesting a big chemical difference existed between them. Supervised PLS-DA analysis indicated that the wild samples showed higher levels of secondary metabolites, such as bauer-7-ene-3β, 16α-diol, chlorogenic acid, rutin, 7-(3'-ethylcrotonoyloxy)-1α-(2'-methyl-butyryloxy)-3, 14-dehydro-Z-notonipetranone (EMDNT), tussilagone, β-sitosterol and sitosterone. This is consistent with traditional experience that the quality of wild samples are better than that of cultivated ones. The content of pyrrolizidine alkaloids senkirkine also differed greatly among samples from different habitats. The Pearson correlation analysis showed that senkirkine is positively correlated with 4, 5-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, 3,4-O-dicaffeoylquinic acid, rutin, kampferol analogues, to a statistically significant extent. The correlation between the toxic compounds and the bioactive components in FF should be further studied.
Chlorogenic Acid ; Drugs, Chinese Herbal ; chemistry ; Flowers ; chemistry ; Magnetic Resonance Imaging ; Magnetic Resonance Spectroscopy ; Metabolomics ; Quinic Acid ; analogs & derivatives ; Rutin ; Sitosterols ; Tussilago ; chemistry

It was difficult to prepare traditional Chinese medicine pellets due to the adverse characteristics of the herbal extract. In this study, Danshen extract (DS) powder mixed with different proportions of microcrystalline cellulose (MCC), lactose and starch were made into pellets by extrusion-spheronization. Particle size, span, bulk density, tapping density, compressibility, Hausner ratio and angle of repose were used to evaluate the micromeritic properties of mixing powders. Feret diameter, aspect ratio, yield, density and friability were used to evaluate the properties of the pellets. The correlations between micromeritic properties of raw material powders and the formability of their pellets were analyzed by cluster analysis, principal component analysis and partial least squares regression analysis. As a result, the particle size of the powders was negatively correlated with the size, density, yield, and was positively correlated with the friability of their pellets. The span, density, compressibility and angle of repose of the powders were positively correlated with the size, density, yield, and were negatively correlated with the friability of their pellets. So there were certain correlations between the micromeritic properties of raw material powders and the properties of their pellets prepared by extrusion-spheronization. This research provided a foundation for the technology and method of traditional Chinese medicine extract pellets.
Cellulose ; chemistry ; Drug Compounding ; methods ; Drug Implants ; chemistry ; Drugs, Chinese Herbal ; chemistry ; Excipients ; chemistry ; Lactose ; chemistry ; Medicine, Chinese Traditional ; methods ; Particle Size ; Powders ; chemistry ; Starch ; chemistry

This study was carried out to evaluate the anti-inflammatory and free radical scavenging activities of flavans from flex centrochinensis S. Y. Hu in vitro and their structure-activity relationship. LPS-stimulated RAW 264.7 macrophage was used as inflammatory model. MTT assay for cell availability, Griess reaction for nitric oxide (NO) production, the content of TNF-alpha, IL-1beta, IL-6 and PGE, were detected with ELISA kits; DPPH, superoxide anion and hydroxyl free radicals scavenging activities were also investigated. According to the result, all flavans tested exhibited anti-inflammatory effect in different levels. Among them, compounds 1, 3, 4 and 6 showed potent anti-inflammatory effect through the inhibition of NO, TNF-alpha, IL-lp and IL-6, of which 1 was the most effective inhibitor, however, 2 and 5 were relatively weak or inactive. The order of free radical scavenging activities was similar to that of anti-inflammatory activities. Therefore, these results suggest that 3, 4 and 6, especially of 1, were,in part responsible for the anti-inflammatory and free radical scavenging activity of Ilex centrochinensis. Hydroxyl group at 4'-position of B-ring plays an important role in the anti-inflammatory and free radical scavenging capacities.
Animals ; Anti-Inflammatory Agents, Non-Steroidal ; chemistry ; pharmacology ; Cell Line ; Cyclooxygenase 2 ; immunology ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Flavanones ; chemistry ; pharmacology ; Free Radical Scavengers ; chemistry ; pharmacology ; Ilex ; chemistry ; Interleukin-6 ; immunology ; Macrophages ; drug effects ; immunology ; Mice ; Nitric Oxide ; immunology ; Tumor Necrosis Factor-alpha ; immunology

A new thermophilic fungus J18 isolated from the soil samples was identified as Paecilomyces thermophila. This strain produced effectively xylanase utilizing several lignocellulosic materials in the solid-state fermentation (SSF) , and wheat straw was the best carbon source. The results of single-factor-experiment showed that the wheat straw of particle size 0. 3 mm ~ 0.45 mm, initial moisture content of 83% , initial pH of 7. 0 and cultivation temperature of 50℃were the optimal conditions for xylanase production. Under the optimized conditions, it produced 18 580 U/g dry substrate after 8 days of cultivation. Therefore, xylanase production by Paecilomyces thermophila in SSF possess great potential for commercial applications.

Objective To investigate the effect of acupuncture plus rehabilitation training on upper limb functional recovery in convalescent stroke patients.Methods One hundred and three convalescent stroke patients with upper limb motor dysfunction were randomized to a treatment group (54 cases) and a control groups (49 cases). Both group received conventional rehabilitation training. In addition, the treatment group received along-meridian syndrome differentiation-based acupuncture and the control group, sham acupuncture. Upper limb Fugl-Meyer Assessment (U-FMA) score, the Wolf Motor Function Test (WMFT) score, the modified Barthel Index (MBI) score and the 36-Item Short Form Health Survey (SF-36) score were recorded in the two groups of patients before and at the end of treatment and at the 3-month follow-up.Results In the two groups there were statistically significant differences in the U-FMA score, the WMFT score and the MBI score at the end of treatment and the 3-month follow-up compared with before (P>0.05 orP<0.01). At the end of treatment and the 3-month follow-up there were statistically significant differences in the U-FMA score, the WMFT score and the MBI score between the treatment and control groups (P<0.05 orP<0.01). The scores of SF-36 items except social functioning and bodily pain had statistically significant differences within the two groups at the end of treatment and the 3-month follow-up compared with before and between the two groups at the end of treatment and the 3-month follow-up (P<0.01 orP<0.05).Conclusion Acupuncture plus rehabilitation training can markedly improve upper limb function in stroke patients.

Human xanthine oxidase is considered to be a target for therapy of hyperuricemia. Cichorium intybus is a Chinese plant medicine which widely used in Xinjiang against various diseases. In order to screen the inhibitors of xanthine oxidase from C. intybus and to explore main pharmacological actions of cichory a compound collection of C. intybus was built via consulting related references about chemical research on cichory. The three-dimensional crystal structure of xanthine oxidase (PDB code: 1N5X) from Protein Data Bank was downloaded.. Autodock 4.2 was employed to screen the inhibitors of xanthine oxidase from cichory 70 compounds were found to possess quite low binding free energy comparing with TEI (febuxostat). C. intybus contains constituents possessing potential inhibitive activity against xanthine oxidase. It can explain the main pharmacological actions of cichory which can significantly lower the level of serum uric acid.
Chicory ; chemistry ; Databases, Protein ; Drugs, Chinese Herbal ; chemistry ; Enzyme Inhibitors ; chemistry ; Humans ; Molecular Docking Simulation ; Molecular Structure ; Xanthine Oxidase ; antagonists & inhibitors ; metabolism

OBJECTIVETo observe the influence of lead exposure on the activity of nitric oxide synthase (NOS) in different brain regions of rat.

METHODSBy establishing a series of rat models exposed to different low levels of lead (drinking water containing 0.025%, 0.050%, 0.075% of lead acetate) during developing period, NOS activities in hippocampus, cerebellum, cerebral cortex and brain stem were studied.

RESULTSOn the 21st day after birth, NOS activities in hippocampus of three levels of lead exposed groups [(1.53 +/- 0.20), (1.66 +/- 0.23), (1.88 +/- 0.32) U/mg pro respectively], and in cerebellum [(0.87 +/- 0.24), (0.85 +/- 0.09), (0.91 +/- 0.18) U/mg pro respectively] were significantly lower than those of control group [(2.36 +/- 0.18), (1.41 +/- 0.18) U/mg pro, respectively, P < 0.01]. NOS activities in cerebral cortex of 0.075% group [at 7, 14, 21 d of age [(1.29 +/- 0.14), (1.03 +/- 0.15), (0.69 +/- 0.10) U/mg pro] were significantly lower than those in control group [(2.54 +/- 0.31), (1.64 +/- 0.22), (1.24 +/- 0.14) U/mg pro respectively], and 0.025% group [(2.42 +/- 0.19), (1.59 +/- 0.17), (1.27 +/- 0.12) U/mg pro respectively], and 0.050% group [(2.56 +/- 0.53), (1.77 +/- 0.19), (1.24 +/- 0.10) U/mg pro respectively, P < 0.05]. There were no significant differences among control, 0.025%, and 0.050% groups (P > 0.05). Lead exposure had no influence on NOS activity in brain stem at the same age (P > 0.05).

CONCLUSIONNOS activities in hippocampus, cerebellum and cerebral cortex were inhibited by low level lead exposure and the degree of the effect was related to Pb exposure time and/or level of Pb exposed.

Animals ; Brain ; drug effects ; enzymology ; Brain Stem ; drug effects ; enzymology ; Cerebellum ; drug effects ; enzymology ; Cerebral Cortex ; drug effects ; enzymology ; Dose-Response Relationship, Drug ; Female ; Hippocampus ; drug effects ; enzymology ; Lead ; toxicity ; Nitric Oxide Synthase ; metabolism ; Rats ; Rats, Sprague-Dawley ; Time Factors

Objective To survey the arsenic content of drinking water in Jingzhou City, to provide basis for prevention and control of endemic arsenic disease. Methods According to historical data, the .arsenic content of water was detected in 357 villages from 6 counties of Jianglin, Songzi, Gongan, Shishou, Jianli, Honghushi in Jinzhou City in 2007 and 2008, The past have been found to have high arsenic water villages, villages known to have high concentration of arsenic were put into census. Villages not found to have high-arsenic wells were sampled 10 percent of the whole water resources at five directions of east, west, south, north and the center. Using sampling investigation, water arsenic was determined by half -quantitative fast reagent kit. All samples of water with arsenic exceeding the standard were re-determined using silver diethyl dithiocarbamate using silver diethyl dithiocarbamate colorimetric mothod. Survey on the disease was carried out in the villages with arsenic exeeeding the standard. Results All 6074 water samples was inspected. Arsenic in 210 samples outnumbered 0.05 mg/L, 51 natural villages were high arsenic areas;The maximum level of arsenic content in drinking water was 0.7 mg/L 3.2% (152/4784) of the wells no deeper that 30 meters and 4.9%(58/1184) between 30 to 100 m had arsenic exceeding the standard The water arsenic content was normal when the wells was deeper that 100 m. The abnormal percentages of water arsenic was related with the depth of wells with a significant difference(χ2 = 12.29,P < 0.01). Medical examination 84 064 residents in 51 villages having high arsenic water 31 neighboring villages was made, No Patients was found suffering from endemic arsenic poisoning. Conclusions High arsenic source has been found in Jingzhou City ,but no endemic arsenic poisoning patient in Jingzhou City. It is suggested that necessary preventive measures should be taken in high arsenic area, low-arsenic water should be spotted or high arsenic water improved. Moreover, wells should be drilled for more than 100 meters or more in depth.

The combination effect of high hydrostatic pressure and moderate heat on the inactivation kinetics of Bacillus coagulans spore in phosphate buffer and UHT(Ultra High Temperature)whole milk was investigated.The pressure come-up time and corresponding log-reduction of spore inactivation were considered during pressure-thermal treatment.Bacillus coagulans spore had a much higher resistance to pressure in UHT whole milk than in phosphate buffer.Survival data were modeled using the linear,Weibull and log-logistic models to obtain relevant kinetic parameters.The tailing phenomenon occurred in all survival curves,indicating the linear model was not adequate for describing these curves.The log-logistic model produced best fits to survival curves,following by Weibull model.