Objective To separate human γ-tubulin ring complexes (γTuRC) .Methods Cell lysates prepared from 293FT cells were separated using gel filtration chromatography .Then, the eluate fractions containing γTuRC or γ-tubulin small complexes (γTuSC ) were determined by immunoblotting .Results As the constitutive components of γTuRC,γ-tubulin,γ-tubulin complex protein 2 (GCP2), GCP3 and GCP4 were eluted and enriched in the fourth fraction .The molecular mass of eluates in the fourth fraction was about 2000 ×10 3 .Following γTuRC, the constitutive components ofγTuSC including γ-tubulin, GCP2 and GCP3 were eluted and enriched in the fourteenth fraction .The molecular mass of eluates in the fourteenth fraction was about 310 ×10 3 .Unassembled free components were washed out in the eighteenth and subsequent fractions .γTuRC could be detected in the corresponding fractions by negative-PAGE separation .ConclusionγTuRC and γTuSC were successfully separated from the unassembled free components in the fourth ( 4#) and fourteenth (14#) eluted fraction, respectively.The eluates containing ofγTuRC orγTuSC can be used for microtubule assembly research.

Objective To study the biological function of GP,VP30,NP and L proteins in Ebola virus(EBOV) infectious diseases,and to screen the host proteins which interact with GP,VP30,NP,and L proteins of EBOV from human liver cDNA library using yeast two-hybrid technique.Methods Recombinant PCR was used to construct bait plasmids pGBKT7-GP,pGBKT7-VP30,pGBKT7-NP and pGBKT7-L.Bait strains were combined with human liver cDNA library strains.The positive clones were analyzed by DNA sequencing and bioinformatics.A yeast recovery experiment was performed to further verify and exclude false positive results.Results We constructed bait plasmids pGBKT7-GP,pGBKT7-VP30,pGBKT7-NP and pGBKT7-L with the recombinant PCR method.Six host proteins which could interact with GP,VP30,NP,and L proteins were screened,including COMMD1,ALB,PSMD8,APOA2,CYP2E1,and HP.The yeast recovery experiment proved that COMMD1 and APOA2 might interact with NP protein.Conclusion A number of prey proteins which interact with GP,VP30,NP,and L proteins of EBOV are screened,which may provide reference for the research of EBOV.

Streptomycetes produce many antibiotics and are important model microorgansims for scientific research and antibiotic production. Metabolomics is an emerging technological platform to analyze low molecular weight metabolites in a given organism qualitatively and quantitatively. Compared to other Omics platform, metabolomics has greater advantage in monitoring metabolic flux distribution and thus identifying key metabolites related to target metabolic pathway. The present work aims at establishing a rapid, accurate sample preparation protocol for metabolomics analysis in streptomycetes. In the present work, several sample preparation steps, including cell quenching time, cell separation method, conditions for metabolite extraction and metabolite derivatization were optimized. Then, the metabolic profiles of Streptomyces coelicolor during different growth stages were analyzed by GC-MS. The optimal sample preparation conditions were as follows: time of low-temperature quenching 4 min, cell separation by fast filtration, time of freeze-thaw 45 s/3 min and the conditions of metabolite derivatization at 40 degrees C for 90 min. By using this optimized protocol, 103 metabolites were finally identified from a sample of S. coelicolor, which distribute in central metabolic pathways (glycolysis, pentose phosphate pathway and citrate cycle), amino acid, fatty acid, nucleotide metabolic pathways, etc. By comparing the temporal profiles of these metabolites, the amino acid and fatty acid metabolic pathways were found to stay at a high level during stationary phase, therefore, these pathways may play an important role during the transition between the primary and secondary metabolism. An optimized protocol of sample preparation was established and applied for metabolomics analysis of S. coelicolor, 103 metabolites were identified. The temporal profiles of metabolites reveal amino acid and fatty acid metabolic pathways may play an important role in the transition from primary to secondary metabolism in S. coelicolor.
Gas Chromatography-Mass Spectrometry ; Metabolic Networks and Pathways ; Metabolome ; Metabolomics ; methods ; Streptomyces coelicolor ; metabolism

OBJECTIVE：To prov ide reference for the improvement of quality standards of Shiwei yipi granules. METHODS ： According to the general rules of 2015 edition of Chinese Pharmacopeia （part Ⅳ），microscopic identification was used to identify Massa Medicata Fermentata and Galli Gigerii Endothelium Corneum ；TLC method was used to qualitatively identify Crataegi Fructus and Semen Raphani ；the content of sinapine thiocyanate in Semen Raphani was determined by HPLC. RESULTS ：The microscopic characteristics were obvious for Massa Medicata Fermentata （palisade cells of testa and stone cells of testa ）and Galli Gigerii Endothelium Corneum （irregular fragments ）. The same fluorescent spots of Crataegi Fructus and Semen Raphani were displayed at the same position of ursolic acid ，sinapine thiocyanate control and Semen Raphani reference substance. The linear range of sinapine thiocyanate was 23.27-9 574.42 ng （r＝1.000 0）. The LOD and LOQ were of 0.50 μ g/mL and 1.68 μ g/mL， respectively. RSDs of precision ，repeatability，intermediate precision and stability tests （24 h）were all less than 1.5％. The average recoveries were 99.40％-100.89％（RSDs were 0.18％-0.49％，n＝3）. The contents of sinapine thiocyanate in 3 batches of Shiwei yipi granules were 0.086 4-0.220 6 mg/g，the average was 0.168 4 mg/g. CONCLUSIONS ：The identification method of Massa Medicata Fermentata ，Galli Gigerii Endothelium Corneum ，Crataegi Fructus and Semen Raphani in Shiwei yipi granules as well as the method for content determination of sinapine thiocyanate in Semen Raphani are established successfully. The content of Semen Raphani in the Shiwei yipi granules is no less than 0.16 mg/g calculated by sinapine thiocyanate （C16H24NO·5 SCN）.

Objective To construct erythromycin-overproducing mutants by tandemly expressing S-adenosylmethionine synthetase gene metK, Vitreoscilla hemoglobin gene vhbS and pleiotropic regulatory gene adpA in Saccharopolyspora eryth-raea.Methods Through PEG-mediated protoplast transformation , the integrative plasmid carrying metK, vhbS and adpA was respectively introduced into erythromycin-producing wild-type strain S.erythraea A226 and industrial strain WB .The engineered strains were generated by apramycin resistance screening and PCR identification .The erythromycin production was compared in original strains and their mutants by the inhibition test of Bacillus subtilis and HPLC analysis .Results and Conclusion Four A226-derived mutants A226-P1-P4 and three WB-derived mutants WB-P1-P3 were independently obtained.Compared with wild-type strain A226, the relative erythromycin titer of the four engineered strains A 226-P1-P4 was increased from 8%to 25%by scoring the growth-inhibition zones .Further HPLC analysis showed that the four mutants had increased erythromycin A yield by 64%-94%.Likewise, the relative erythromycin titer and erythromycin A yield of the three engineered strains WB-P1-P3 were enhanced by 6%-10%and 31%-62%, respectively, in comparison with the original strain WB.The results show the universality of enhancing erythromycin productionvia tandem expression of metK, vhbS and adpA in S.erythraea.

Epidermal growth factor receptor (EGFR) and its ligands (EGF and TGFalpha) are over-expressed in a variety of tumors. Immunization EGF-carrier protein inhibits tumor growth through abrogating binding of EGF to EGFR. Here, a chimeric protein of EGF and TGFalpha (E5T) was genetically fused to Staphylococcal enterotoxin A (SEA), a bacterial superantigenic protein which promotes humoral B cell response through enhancement of Ag-specific CD4 T cells activity. The resulted fusion proteins were expressed in Escherichia coli and purified though metal chelating affinity chromatography. Immunization of E5T-mSEA fusion protein in mice induced production of high titers antibodies, which recognize both EGF and TGFalpha. Anti- E5T-mSEA serum at dilution of 1:10 significantly inhibited growth of A431 cell lines but had little effect on 293T cell lines.
Amino Acid Sequence ; Animals ; Cancer Vaccines ; biosynthesis ; immunology ; Cell Line, Tumor ; Enterotoxins ; biosynthesis ; genetics ; Epidermal Growth Factor ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Humans ; Immunization ; Mice ; Mice, Inbred C57BL ; Molecular Sequence Data ; Random Allocation ; Receptor, Epidermal Growth Factor ; antagonists & inhibitors ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Transforming Growth Factor alpha ; biosynthesis ; genetics

OBJECTIVE To explore the protective effect and mechanism of Longshengzhi capsules on cerebral ischemia- reperfusion injury in rats. METHODS The model of middle cerebral artery occlusion （MCAO） was established by using the improved thread occlusion method. The experiment was divided into six groups： sham surgery group （only separating blood vessels without inserting thread plugs， given the same volume of normal saline）， model group （modeling， given the same volume of normal saline）， nimodipine group （positive control， modeling， dose of 20 mg/kg）， and low-dose， medium-dose， and high-dose groups of Longshengzhi capsules （modeling， doses of 0.72， 1.44 and 2.88 g/kg， respectively）， with 10 mice in each group. Each group was given corresponding medication solution/normal saline by gavage， once a day， for 7 consecutive days. One hour after the last administration， the Zea Longa scoring method was used to score the neurological deficits in each group of rats， and the ABC enzyme-linked immunosorbent assay was used to detect the serum levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in rats； TTC staining was used to observe the volume of cerebral infarction in rats and calculate the cerebral infarction volume ratio. Hematoxylin eosin staining was used to observe the pathological changes in the brain tissue of rats. Immunohistochemical staining was used to detect the positive expression of NLRP3 protein in the brain tissue of rats. Real-time fluorescence quantitative PCR was used to detect mRNA relative expressions of Toll-like receptor 4 （TLR4） and nuclear factor-κB （NF-κB） in the brain tissue of rats. Western blot assay was adopted to detect the relative expressions of TLR4， NLRP3 and phosphorylated NF-κB （p-NF-κB） protein in the brain tissue of rats and its intracellular NF-κB protein. RESULTS Compared with the sham surgery group， the neural dysfunction score， serum levels of TNF-α and IL-6， cerebral infarction volume ratio， relative expression levels of NF-κB and TLR4 mRNA， as well as protein relative expressions of TLR4， NLRP3 and p-NF-κB in the brain tissue， and relative protein expression of intracellular NF-κB were increased significantly in the model group （P＜0.01）； the enlarged gap and significant edema were observed in cortical nerve cells of brain tissue in rats， with a large amount of inflammatory cell infiltration； the positive expression of NLRP3 protein in brain tissue of rats obviously increased. Compared with the model group， the levels of the above indicators in the medium-dose and high-dose groups of Longshengzhi capsules， as well as the Nimodipine group， were reversed to varying degrees， and most differences were statistically significant （P＜0.05 or P＜0.01）； the pathological morphology observation showed a significant improvement， and the positive expression of NLRP3 protein in the brain tissue of rats was obviously reduced. CONCLUSIONS Longshengzhi capsules may inhibit TLR4/NF-κB/NLRP3 signaling pathway and neuroinflammatory response， thereby achieving a protective effect against cerebral ischemia-reperfusion injury in rats.

This article systematically sorted out and researched the name， origin， harvesting and other aspects of Rhapontici Radix by referring to ancient materia medica， medical books and prescription books， combined with modern literature， in order to provide a reference basis for the development of the famous classical formulas containing this herb. According to the results of the herbal textual research， it can be seen that all the generations of the materia medica have taken Loulu as the proper name， and there are also aliases such as Luligen， Laowenghua and Jiahao. The mainstream base of Rhapontici Radix recorded in the past dynasties was the present Compositae plant Rhaponticum uniflorum， which is mostly used as medicine with roots. Since the Tang dynasty， the stems and leaves of Siphonostegia chinensis have been used as Rhapontici Radix in the northern region. Until modern times， Qizhou Pharmacognosy began to differentiate it into two categories， Qizhou Loulu and Yuzhou Loulu， according to the commodity circulation at that time， producing area and origin， of which Yuzhou Loulu is the roots of Echinops latifolius， a plant of Compositae family. In ancient times， the quality of Loulu was based on "the one that comes out of Shanzhou is the best". However， in modern times， the quality of Qizhou Loulu is better if the surface is black， neat， sturdy， firm， not broken， and without a withered heart， while the quality of Yuzhou Loulu is better if the branches are thick and long with an earthy-brown surface， solid texture and neat in length. In ancient times， most of the harvesting and processing of Loulu was "harvesting the roots in lunar August and drying them in the shade"， while in modern times， the roots are mostly excavated in the spring and autumn， and dried in the sun. Its ancient method of processing is to mix and steam with licorice， nowadays， it is prepared by removing impurities， washing， moistening thoroughly， cutting into thick slices and drying in the sun， and then taking the raw products as medicine. Based on the research conclusion， it is suggested that when developing and utilizing the famous classical formulas containing Loulu， the background of the formula should be verified， and if the original formula indicated the requirement of processing， it should be processed according to the requirement， but if not， it is recommended to use raw products as medicine.

The research studies the effect of different fertilization treatments on yield and accumulation of secondary metabolites of Codonopsis pilosula by using single factor randomized block design, in order to ensure reasonable harvesting time and fertilization ratio, and provide the basis for standardized cultivation of C. pilosula. According to the clustering results, the nitrogen fertilizer benefitted for the improvement of root diameter and biomass of C. pilosula. The phosphate fertilizer could promote the content of C. pilosula polysaccharide. The organic fertilizers could increase the content of lobetyolin. With the time going on, C. pilosula's yield, polysaccharide and ehanol-soluble extracts increased while the content of lobetyolin decreased. According to various factors, October is a more reasonable harvest period. Organic fertilizers are more helpful to the yield and accumulation of secondary metabolites of C. pilosula.

OBJECTIVE To study the content changes of 5 chemical compositions in water extract and ethanol precipitate of different processed products of Psoralea corylifolia， and to preliminarily evaluate its hepatotoxicity. METHODS The water extracts from crude product of P. corylifolia and processed products by Leigong method， running water rinsing method， and salt stir-frying method were prepared， as well as the ethanol precipitates of processed products by Leigong method and salt stir-frying method were prepared. The contents of psoralenoside， isopsoralenoside， psoralen， isopsoralen and bakuchiol were determined by high- performance liquid chromatography and compared. The median lethal concentration （LC50） and maximum non-lethal concentration （MNLC） of each sample to wild-type zebrafish juveniles were calculated after 72 h of treatment with different concentrations of water extracts from raw product and processed products by running water rinsing method， Leigong method and salt stir-frying method， different concentrations of ethanol precipitates from processed products by Leigong method and salt stir-frying method， and the acetaminophen was used as the positive control. The basic morphology of wild-type zebrafish juveniles and the liver phenotype of transgenic zebrafish juveniles were observed after 72 h of treatment with the above samples （MNLC）. Pearson correlation analysis was used to evaluate the correlation between component content and hepatotoxicity. RESULTS Compared with the water extract of raw products， the contents of psoralenoside and isopsoralenoside in the water extract of different processed products were generally decreased （P＜0.05）， while the contents of psoralen， isopsoralen and bakuchiol in the ethanol precipitate of Leigong method and salt stir-frying products were significantly increased （P＜0.05）. The LC50 of water extracts of crude product and processed products by running water rinsing method， Leigong method， salt stir-frying method， and ethanol precipitates of processed products by Leigong method and salt stir- frying method were 2.45， 5.00， 5.38， 1.55， 2.36， 0.64 g/L （calculated by crude drug）， and MNLC were 2.21， 4.53， 5.02， 1.37， 2.13， 0.53 g/L （calculated by crude drug）. Compared with the blank control group， the zebrafish juveniles in each sample treatment group showed different degrees of deformity， the liver relative fluorescence intensity was significantly weakened （P＜0.05 or P＜ 0.01）. Fat-soluble components such as bakuchiol， isopsoralen and psoralen were highly correlated with liver fluorescence intensity （R 2＞0.7）. CONCLUSIONS The processed products of P. corylifolia mainly compose of psoralenoside and isopsoralenoside after water extraction， the contents of psoralen， isopsoralen and bakuchiol increase after alcohol precipitation， and the hepatotoxicity is positively correlated with the contents of liposoluble compositions in P. corylifolia.