A 2,4 -dichlorophenol degrading Pseudomonas strain GI241-1 was isolated from a soil sample. The dienelactone hydrolase gene, designated as dcpD which encodes dienelactone hydrolase involved in transforming cis-2-chloro-dienelactone into 2-chloromaleylacetic acid, was cloned from this bacterium strain. The gene cloning strategy was to construct genomic library after location of its neighbouring gene by Southem blot and to screen the aim transformant by dot blotting. Sequencing results showed that length of dcpD is 702bp. The sequence of dcpD and the deduced amino acid are different from the relative sequences registered in the GenBank.

The characteristic fingerprint of conventional dairy Nanhanshuishi was established by X-ray diffraction (XRD), based on similarity of caculation on public peaks by MATLAB software, and the feasibility of new dairy technology of microwave method was explored between XRD and the dissolution rate in artificial simulation gastric juices. The result showed that similarity of shared peak in XRD of conventional dairy Nanhanshuishi was > 95%, This XRD characteristic fingerprint of conventional dairy Nanhanshuishi had strong specificity, could be used to provide a reference for identification and quality evaluation. This study also showed that the similarity of microware dairy products and conventional dairy products was good, and the sample of microwave 15 min was the best, and new dairy method by the microwave could replace the traditional method.
Animals ; Chemistry, Pharmaceutical ; instrumentation ; methods ; Drugs, Chinese Herbal ; chemistry ; Medicine, Tibetan Traditional ; Microwaves ; Milk ; chemistry ; Plants, Medicinal ; chemistry ; Quality Control ; X-Ray Diffraction

Three butylphthalide derivatives were isolated from the Rhizome of Ligusticum chuanxiong using a series of isolation and purification approaches including macroporous resin, ODS-A column, Sephadex LH-20 and preparative HPLC. These structures were elucidated based on extensive spectroscopic data (UV, IR, HR-ESI-MS and NMR) and identified as (3Z,3aE)-(6R,7R,2'S)-6-hydroxy-7-(2-carboxyl-2-hydroxyethylthio)-3-(2-hydroxybutylidene)-4,5,6,7-tetrahydro-phthalide (1), (3Z,3aZ)-3-butylidene-6,7-dihydroxy-4,5,6,7-tetrahydro-phthalide 7-O-α-D-glucopyranosyl-(1→2)-β-D-fructo-furanoside (2) and 3-(3-β-D-glucopyranosyloxy-butylidene)-7-hydroxy-phthalide (3).

AIM:To observe the efficacy of intravitreal conbercept injection for chronic central serous chorioretinopathy (CSC).METHODS: Nine eyes of 9 patients diagnosed as chronic CSC between October 2015 to May 2016 were treated with an intravitreal injection of conbercept (0.5mg/0.05mL) (six patients were given the same does of intravitreal injection again at 1mo after the first injection).Follow-up observation was at 1, 2, and 6mo after injection.Observed indicators included best-corrected visual acuity (BCVA), intraocular pressure, optical coherence tomography (OCT), fundus fluorescein angiography (FFA), choroidal indocyanine green angiography (ICGA), macular fovea thickness (CMT), subfoveal choroidal thickness (SFCT).RESULTS:Seven of the 9 patients responded significantly to the drug, while 2 patients had no response.The CMT was 373.12±72.43μm at baseline, which decreased significantly to 332.05±67.13μm, 282.24±62.30μm and 225.56±71.08μm at 1, 2 and 6mo after the intravitreal injection.The mean thickness of SFCT was 422.11±64.82μm before treatment.The choroidal thickness of non-responsive patients before treatment was below average, respectively 353μm and 365μm.The SFCT of 1, 2, and 6mo after treatment was 391.45±75.24μm, 365.53±63.07μm, 355.40±66.65μm.Before treatment and 1mo after, there was no significant difference (P=0.074), but there was statistically significant (P<0.01) between those of before and 2mo and 6mo after.The mean BCVA of the prior treatment was 0.53±0.32, the after treatment was 0.65±0.20, there was no different between the two(P>0.05).CONCLUSION: Intravitreal conbercept injection in chronic CSC may have some effect in accelerating subertinal fluid resolution and decreasing the CMT.The SFCT within 6mo after treatment was significantly lower than pretreatment.The SFCT may be an indicator of whether patients respond.

The aim of this study was to investigate if transfusion of mesenchymal stem cells (MSC) could exhibit beneficial effects on rheumatoid arthritis. Human bone marrow MSC were intraperitoneally injected into Wistar rats with collagen-induced arthritis at a dose of 10(7) on the next day (preventive group) or 2 weeks (treatment group) after collagen II induction, once a week for 2 weeks (preventive group) or 4 weeks (treatment group). The control group was given normal saline (NS) at corresponding time. The symptom scorings were documented weekly from the second week of the induction. On week 6, the hind joints of the rats were pathologically examined and the activation status of splenocytes was analyzed by flow cytometry. The results showed that all the rats developed arthritis and subsequent joint abnormality. On the sixth week, symptom scores of the rats that received MSC preventive (9.5 ± 0.5) or therapeutic (9.4 ± 0.6) infusions had no significant difference between each other, but were significantly greater than those of the NS controls (7.6 ± 0.6, P < 0.05). Consistently, pathological examination on the involved knees showed that the synovitis and arthritis scorings of MSC treated rats were greatly elevated compared with NS controls. Furthermore, the ratios of CD86(+) cells in the spleens of MSC prevention, MSC treatment and NS control groups were (4.16 ± 1.48), (4.06 ± 1.97) and (4.15 ± 2.04) respectively, while those of CD11b/c(+)CD86(+) cells were (1.04 ± 0.68), (0.95 ± 0.56) and (0.98 ± 0.44), all of which were significantly higher than those of healthy controls [(0.97 ± 0.18) and (0.30 ± 0.17), P < 0.05 for both parameters]. It is concluded that MSC infusion has little beneficial effects on collagen-induced arthritis in rats, conversely, MSC therapy aggravated the damage of the involved joints, its underlying mechanisms need to be further investigated.
Animals ; Arthritis, Experimental ; pathology ; prevention & control ; therapy ; Bone Marrow Cells ; Cells, Cultured ; Humans ; Male ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; Rats ; Rats, Wistar ; Transplantation, Heterologous ; Treatment Outcome

To develop an analytic method for qualitative and quantitative analysis of dodecatetraenamides A, B in 42 samples of two official species of Asari Radix et Rhizoma( ARR) (37 samples of Asarum heterotropoides var. mandshuricum with different collection time and 5 samples of Asarum sieboldiivar. seoulense). The HPLC-IT-TOF-MS/MS methods for the qualitative and UPLC-PDA methods for the quantitative analysis were established. Dodecatetraenamides A, B were identified by comparing the retention time, UV absorption spectrum and quasi-molecular ion peak [ M + H]+ with the reference compound using HPLC-IT-TOF-MS/MS. The content of dodecatetraenamides A and B in ARR were determined by UPLC-PDA. The separation was successfully carried out on a ACQUITY UPLC BEH C18 (2.1 mm x 100 mm, 1.7 µm) column eluted with mobile phases of water (A) and acetonitrile (B) in gradient program (0-3 min, 35% B; 3-5 min, 35%-36% B; 5-6 min, 36%-43% B; 6 min-11 min 43% B; 11-12 min, 43%-100% B). The column temperature was 45 °C, and the detection wavelength was set at 254 nm. The flow rate was 0.6 mL · min(-1). On one level mass spectrometry scanning, the results showed that the quasi-molecular ion [M + H] + of both dodecatetraenamides A and B were m/z 248.20. The quantitative method with UPLC-PDA has made the baseline separation of the constituents, which were reported as mixtures in the most literatures. The average recovery of dodecatetraenamides A and B were 97.90% and 99.86%, the relative standard deviation were 0.4% and 1.1%, respectively. The contents of dodecatetraenamides A, B in all ARR samples was in the range of 0.11-3.89 and 0.24-6.65 mg · g(-1). Their contents reduced with the extension of storage time. Compared with the samples of 2013, the average content of the two constituents in the samples collected in year 2002-2003 reduced 34% and 36%, respectively (P < 0.05). Compared the A. sieboldii var. seoulense and A. heterotropoides var. mandshuricum with the same collective time and production area, the average contents of the two constituents in latter were up to (1.59 ± 0.75) mg · g(-1) and (2.90 ± 1.17) mg · g(-1), respectively, significantly higher than that in A. sieboldii var. seoulense (dodecatetraenamide A were (0.78 ± 0.52) mg · g(-1), dodecatetraenamide B were (1.69 ± 0.83) mg · g(-1)) (P < 0.05). The content of the dodecatetraenamide A in overground part was in the range of 0.11-0.33 mg · g(-1), dodecatetraenamide B was 0. 24-0.60 mg · g(-1), which were much lower than that of the underground part of ARR (dodecatetraenamide A was in the range of 0.73-3.89 mg · g(-1), dodecatetraenamide B was 2.11-6.24 mg · g(-1)). The method was certified to be simple, accurate and reliable and could be used for qualitative and quantitative analysis of dodecatetraenamide A and B in different species of ARR, also can be used for the comprehensive quality control of traditional Chinese medicine, Asari Radix et Rhizoma.
Amides ; chemistry ; Asarum ; chemistry ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Mass Spectrometry ; Molecular Structure ; Rhizome ; chemistry

Human glutamate decarboxylase 65(hGAD65) is an enzyme that catalyzes the transformation of L-glutamic acid into ?-aminobutyric acid.It has been found that Type 1 diabetes mellitus(T1DM)is an autoimmune disease,in which pancreatic islet ?-cells are destroyed due to immune response mediated by autoantigen.hGAD65 is considered as a key autoantigen of the autoimmune response,so anti-hGAD65 antibody(hGAD65-Ab) is the most effective and specific immune marker for T1DM diagnosis,and hGAD65 can be used to detect hGAD65-Ab in serum of T1DM patients.The hGAD65 gene was cloned into pET32a(+),then the recombinant plasmid with hGAD65 was transformed into E.coli BL21(DE3) and expressed by IPTG induction.The fusion protein containing thioredox,hexahistidine and hGAD65(Trx-hGAD65) was mostly insoluble,but the band of soluble Trx-hGAD65 could also be detected by SDS-PAGE,and it was a great improvement compared with the results reported.Trx-hGAD65 was isolated from lysate and purified by immobilized metal ion affinity chromatography(IMAC).After enterokinase digestion and IMAC purification,hGAD65 with high purity was obtained.Detection of thin-layer chromatography(TLC) showed that both Trx-hGAD65 and hGAD65 had enzymatic activity,whereas Trx-hGAD65 had better stability.Furthermore,it was confirmed that Trx-hGAD65 was able to conjugate with hGAD65-Ab in the serum of T1DM patients by ELISA assay.In conclusion,Trx-hGAD65 instead of hGAD65 can be used for T1DM diagnosis,and its application in prophylaxis and therapy of T1DM is expectable.

Objective To discuss the clinical value of transabdominal sonography after bowl preparation in diagnosis of ileocecal valve syndrome ( IVS) .Methods The ultrasonic features of IVS in 37 cases were summerized and correlated with the follow-up findings after conservative treatment or the pathologic results after operation .Twenty-eight cases were confirmed by follow-up and 9 cases by operative pathology.Results Among the 37 cases of IVS,28 were idiopathic IVS (75.7%,28/37) and 9 were secondary IVS (24.3%, 9/37%).For the secondary cases, the primary diseases included 5 acute appendicitis,2 Meckel diverticulum,1 terminal ileitis and 1 carcinoma of ascending colon .The diagnostic accuracy rate of ultrasound was 89.2%(33/37).Misdiagnosis rate was 10.8%(4/37),including 1 case of idiopathic and 3 cases of secondary IVS .The IVS ultrasonic images coulde be displayed clearly using 7.0-10.0 MHz probes.In fasting examination,three ultrasonic characteristic signss were found in interminal ileum region at the right lower abdomen .And these features were bagel-shaped sign [91.9%(34/37),average size (1.9 ±1.6) cm ×(0.8 ±0.3) cm],short sleevelet-shaped sign [91.9% (34/37,average size (2.1 ± 0.4)cm ×(1.3 ±0.2) cm],and rose-shaped sign [83.8% (31/37),average size (1.4 ±0.2) cm × (1.0 ±0.2) cm].The shapes of some signs were changeable when the probe compressed .In the case of idiopathic IVS ,several pathologic changes could be seen on sonography after intestinal tract filling of oral 20%mannitol,including slight thickened mucosa and submucosa of erminalileum ,enlarged ieocecal valve and the crocodile-mouth sign.Conclusions Transabdominal ultrasonic examination with high frequency probe after bowl preparation plays an important role in diagnosis of IVS .The method is simple and accurate and should be recommended and applied clinically .

OBJECTIVETo study the effect of inhibiting the expression of endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1) on proliferation of human umbilical vein endothelial cell line ECV304.

METHODSAfter constructing and transfecting EGFP-EOLA1 fusion protein expressive vector into ECV304 cells, the transfected cells was cultured in M199 containing G418 for 5 weeks to screen the cell line stable expression EGFP-EOLA1 fusion protein. Oligonucleotides targeting EOLA1 at different sites were synthesized and inserted into pSinencer3.1/H1 vector. Then, the recombinant vector was transfected into the cultured ECV304 cells and the inhibiting effect to target gene EOLA1 was investigated by observing the green fluorescence in transfected cells under inverted fluorescent microscope and by Western blot assay. The proliferation of ECV304 cells was numbered when the expression of EOLA1 in ECV304 cells was inhibited by RNA interference.

RESULTSThe ECV304 cell line stably expressing EGFP-EOLA1 fusion protein was constructed and the siEOLA1 interfere vectors can knock down EOLA1 gene expression specially. When blocking the expression of EOLA1 in ECV304 cells,the proliferation of cells slowed down.

CONCLUSIONEOLA1 maybe has a role on the proliferation of cells.

Blotting, Western ; Cell Line ; Cell Proliferation ; Down-Regulation ; genetics ; Humans ; Membrane Proteins ; genetics ; Phenotype ; RNA Interference ; RNA, Small Interfering ; genetics ; metabolism ; Recombinant Proteins ; genetics

2,4-Dichlorophenol is toxic and biorefratory organic pollutant. A 2,4-dichlorophenol degrading bacterial strain GT241-1, identified as Pseudomonas sp., was isolated from soil samples which was collected from drainage area of several 2,4-dichlorophenol producing factories. Strain GT241-1 had strong 2,4-dichlorophenol degrading ability, it could decompose 91% 2, 4-dichlorophenol of 90 mg/L within 48 hours at 25 - 30 degrees C, and could utilize 2,4-dichlorophenol, 2,4-dichlorophenoxyacetic acid (2,4-D), benzoate and catechol as sole carbon and energy source. Southern blot showed that 2,4-dichlorophenol hydroxylase gene (dcpA) of strain GT241-1 locates on the about 10kb EcoR I/Xba I fragment. This fragment was recovered, linked to the vecter pUC19 and transformed into the E. coli DH5alpha. A aim transformant, Z539, was obtained by dot blotting from about 1200 transformants. PCR and the sequencing results shew that the whole dcpA gene is contained within the 10kb EcoR I /Xba I fragment of pZ539. This fragment was shortened to about 2.4kb by HindmIII. The shorted fragment was subcloned to vecter pRSET-B to get a transformant BS1-12. The subcloned fragment was sequenced. Sequencing results showed that the whole length of the subcloned fragment containing dcpA is 2389bp and the nucleotide span of coding region is from number 276 to number 2072 (1797 bp), with ATG and TAA as start and stop codon respectively. The sequence analysis of dcpA and the deduced amino acid encoded by dcpA showed that they are different from the relative sequences registered in the GenBank. The subcloned fragment carry the promoter of dcpA, this can deduce from the fact that the upflow length of dcpA coding region is 275bp, and further confirmed by the 2,4-dichlorophenol hydroxylase activity measurement results. The 2,4-dichlorophenol hydroxylase activity of transformant Z539 and BS1-12 were detected, the results showed these transformants have 2,4-dichlorophenol hydroxylase activity. By comparison, the activity of these transformants were lower than that of the strain GT241-1.
Amino Acid Sequence ; Bacterial Proteins ; genetics ; metabolism ; Biodegradation, Environmental ; Chlorophenols ; metabolism ; Cloning, Molecular ; Environmental Pollutants ; metabolism ; Mixed Function Oxygenases ; genetics ; metabolism ; Molecular Sequence Data ; Pseudomonas ; enzymology ; genetics ; isolation & purification ; Soil Microbiology