ABSTRACT
OBJECTIVE：To estab lish the fingerprint ，analyze the monosaccharide composition and content ，investigate the inhibitory effects of the polysaccharide from Desmodium styracifolium on α-glucosidase in vitro . METHODS ：Polysaccharide from D. styracifolium was prepared by water extraction and ethanol precipitation. After hydrolyzed by TFA and derived by PMP ，HPLC method was adopted to establish the fingerprint （using glucose peak as reference ），and analyze the constituent and content of monosaccharide. The content determination was performed on Phenomenex Luna C 18 column with mobile phase consisted of acetonitrile-0.05 mol/L potassium phosphate （pH adjusted to 6.8 with sodium hydroxide ）in gradient elution at the flow rate of 0.8 mL/min. The detection wavelength was set at 250 nm，and column temperature was set at 30 ℃. The sample size was 10 μL. Using acarbose as control ，PNPG assay was used to investigate the α-glucosidase inhibitory activity of polysaccharide from D. styracifolium. RESULTS ：There were 9 common peaks in HPLC fingerprints of 18 batches of samples ，and the similarity of 15 batches of samples was higher than 0.90. Totally 7 peaks were identified as mannose ，rhamnose，galacturonic acid ，glucose， galactose，xylose and arabinose. The contents of rhamnose ，galacturonic acid ，glucose，galactose and arabinose were 0.471-2.092， 1.379-8.919，2.560-35.679，1.194-6.905，0.566-4.158 mg/g，respectively. Based on rhamnose ，the molar ratios of the other four monosaccharides were 1.58-4.07，2.26-19.95，2.20-4.21 and 1.31-2.86，respectively. The inhibitory activity of polysaccharide from D. styracifolium on α-glucosidase increased with the increase of dose ，and the half inhibitory concentrations of it was 0.70 mg/mL， lower than 7.76 mg/mL of acarbose （positive control ）. CONCLUSIONS ：Glucose is the main component of D. styracifolium polysaccharide in different batches ，and the contents of monosaccharides are different. The polysaccharide from D. styracifolium have significant inhibitory activity on α-glucosidase，which is better than that of acarbose.
ABSTRACT
Objective@#To investigate the migration and invasion behaviors of Hep-2 after the targeted knockdown of yes-associated protein (YAP).@*Methods@#Hep-2 cells were knock-downed for YAP by shRNA as YAP-shRNA group, Hep-2 treated with non-specific shRNA as YAP-NC group, and Hep-2 with no treatment as control. Glucose uptake and lactate production in the cells were examined to assess Warburg effect. The migration and invasion behaviors of cells in three groups were observed. The expressions of vimentin and E-cadherin were detected by RT-PCR and Western Blot. The statistical software GraphPad Prism 7.0 was used to analyze significance of data. Two tailed Student′ s t-tests was used to determine significance when only two groups were compared. P values of less than 0.05 was considered statistically significant.@*Results@#Downregulation of YAP led to a obvious decrease in glucose uptake [(18.51±1.72)%] and lactate production [103.40±8.32] in Hep-2 cells compared with control [(41.20±1.11)% and 743.69±19.49, t=19.20 and 52.33, respectively, both P<0.01] and YAP-NC group [(39.60±0.78)% and 705.22±17.20, t=19.34 and 54.56, respectively, both P<0.01]. Compared with the control group (78.32±4.04) and YAP-NC group (77.28±3.11), the scratch healing ability of Hep-2 cells was significantly decreased in YAP-shRNA group (44.71±4.68). The P value was less than 0.01 (t=9.42 and 10.04). The number of cells with YAP-shRNA (33.30±4.19) passing through compartments was remarkable fewer than the control group (133.71±6.72) and YAP-NC group (126.32±4.21). The P value was less than 0.01 (t=21.96 and 27.13). The expression of E-cadherin protein in cells of YAP-shRNA group (6.16±0.11) was up-regulated compared with control (0.97±0.10, t=35.70, P<0.01) and YAP-NC group (1.13±0.09, t=36.28, P<0.01), while the expression of vimentin protein in cells of YAP-shRNA group (1.08±0.09) was down-regulated compared with control (5.67±0.12, t=29.91, P<0.01) and YAP-NC group (5.51±0.12, t=29.04, P<0.01).@*Conclusions@#The down-regulation of YAP in Hep-2 inhibits the migration and invasion of cells via suppressing Warburg and EMT program.
ABSTRACT
Objective@#To analyze the genetic characteristics of rubella virus isolated from 2012 to 2015 in Guizhou province.@*Methods@#A total of 390 cases of suspected measles were collected from Guizhou measles network laboratory from 2012 to 2015 and 25 cases of rubella cases were diagnosed. Rubella virus isolation was performed using Vero/SLAM cells. The presence of rubella viral RNA was detected using Real-time RT-PCR after RNA extraction from infected tissue culture cells. Fragments of 480 bp and 633 bp nucleotides of E1 genes of the isolates were amplified by RT-PCR and the PCR products were sequenced and spliced. The phylogenetic tree was conducted based on the 739 bp nucleotide sequences of E1 genes and gene characteristic analysis was performed.@*Results@#There were 19 cases of rubella outbreaks and 6 cases of rubella sporadic cases in 25 cases of suspected rubella cases. There were 11 males (44.0%) and 14 females (56.0%). The mean age and standard deviation were (12.3±3.9) years. A total of 10 rubella strains were isolated. The results of phylogenetic analysis showed that 7 strains of rubella virus isolates belonged to genotype 1E and the other belonged to genotype 2B. The nucleotide acid and amino acid homology among 7 strains 1E genotype were 99.0%-100% and 100% respectively. 2B genotype of 3 strains of nucleotide and amino acid homology were 99.4%-100% and 99.5%-100% respectively. Ten strains of rubella virus were not mutated in the E1 glycoprotein gene, Asn 177 and Asn 209 N-type glycosylation sites and E1 antigen epitopes between 213 and 285aa.Among them, 7 strains of 1E genotype had a mutation from leucine to phenylalanine in 338 amino acid, 2 strains of 2B genotype at 377 amino acids from valine to alanine.@*Conclusion@#Rubella virus epidemic was caused by 1E and 2B genotypes in Guizhou from 2012 to 2015.Ten strains of rubella virus were highly conserved in nucleotide and amino acid sequences and there was no variation of important functional sites.
ABSTRACT
Objective To analyze the genetic characteristics of measles virus strains causing two outbreaks in Guizhou province from November 2014 to March 2015. Methods Throat swab samples collect-ed from measles cases in two outbreaks were inoculated into Vero/SLAM cells. Viral RNAs were extracted from positive cultures. Nucleoprotein genes were amplified by using reverse transcription-polymerase chain reaction ( RT-PCR) and the PCR products were sequenced and analyzed. Results Eleven strains of wild-type measles virus were isolated from the two measles outbreaks and all of them belonged to H1a sub-geno-type. Phylogenetic analysis showed that those strains were clustered into two distinct branches. Differences in nucleotide and amino acid genetic distances between the 11 strains of measles virus and the WHO reference strain of H1a sub-genotype (Chin9322) were 1. 1%-1. 6% and 0. 7%-3. 4%, respectively. Compared with the reference strain Chin9322 and Guizhou epidemic strains in recent years, six strains showed amino acid sequence mutations in 47 ( G to S) , 82 ( S to G) and 122 ( R to K) sites and two strains had a mutation in 98 ( P-L) site. Conclusion H1a sub-genotype measles virus was the predominant pathogen causing two measles outbreaks in Guizhou province during 2014 to 2015. Moreover, it was also a predominant sub-geno-type circulating in China and Guizhou province. Different measles virus strains of H1a sub-genotype contin-ued to be prevalent in Guizhou province. This study provided some scientific data for the control and elimina-tion of measles in Guizhou province.
ABSTRACT
Objective To identify the rubella virus circulated in Guizhou province in 2012 ,and confirm the genotype and analyze the genetic characterization of rubella virus isolated .Methods The throat swab samples were collected from suspected rubella cases during the rubella outbreak and sporadic in 2012 .Rubella virus isolation was performed using Vero/SLAM cells .The presence of vi-ral RNA was detected using real-time RT-PCR after RNA extraction from infected tissue culture cells .Fragments of 944 nucleotides of E1 genes of the isolates were amplified by RT-PCR ,the PCR products were directly sequenced and analyzed .The phylogenetic trees based on the 739 nucleotide sequences 1E was conducted with the rubella viruses isolated in Guizhou province in 2012 and 32 WHO reference sequences representing 13 genotypes .Results 5 rubella virus strains were isolated in Guizhou province for the first time .The results of phylogenetic analysis showed that all 5 rubella virus isolates belong to genotype 1E .The nucleotide acid and a-mino acid homology among 5 rubella virus isolates were 99 .8% -100 .0% and 100 .0% respectively .Compared with the WHO ref-erence strain (Chinese strain T14-CH-02) ,the nucleotide acid and amino acid homology were 98 .7% -98 .9% and 100 .0% respec-tively ;while compared with another WHO reference strain (Malaysia strains M1-MAL-01) ,the nucleotide acid and amino acid ho-mology were 97 .5% -97 .6% and 99 .5% .Conclusion Genotype 1E rubella virus was the predominant genotype in Guizhou prov-ince in 2012 ,which was similar to the other provinces .This study accumulated the molecular epidemiological baseline date in Guizhou province .
ABSTRACT
<p><b>OBJECTIVE</b>To explore the genetic diversity and relationship of different Alpinia officinarum germplasm.</p><p><b>METHOD</b>Amplified fragment length polymorphism (AFLP) markers were developed to analyze genetic polymorphism in A. officinarun from eight resources. The amplified fragments were used as primary matrix with NTSYSpc-2.11F software to analyze the similarity between the A. officinarum germplasm and to construct the genetic phylogenetic tree.</p><p><b>RESULT</b>A total of 1,120 fragments were genotyped using AFLP with eight prime combinations. Analysis identified 1,044 polymorphic fragments, accounting for 92.57% of the total detected variation. Genetic phylogenetic tree analysis indicates that three categories can be divided among the eight resources of A. officinanrum.</p><p><b>CONCLUSION</b>Significant polymorphism and genetic diversity can be observed among A. officinarum germplasm resources.</p>
Subject(s)
Alpinia , Classification , Genetics , Amplified Fragment Length Polymorphism Analysis , Genetic Markers , Genetic Variation , Genotype , PhylogenyABSTRACT
[Objective] To improve the way of establishing myocardial ischemia reperfusion rat model.[Methods]30 SD rats,quality(220?20)g,male,randomly split into normal model group and improved model group,improved established model ligation with a 0.6mm diameter line,and drawn after 30min.Compare such a method with normal model.[Results] In the context of keeping experimental stability,the reduced heart rate,wake time and survival rate of the way of improved established model respectively are(50?20)/min,(15?5)min,86.67%,but it is just(120?20)/min,(80?20)min,66.67% of the normal model group.[Conclusion]The improved myocardial ischemia reperfusion model offers significant superiority to the former one.
ABSTRACT
Objective To explore the effect of light chain gene library slauffling on the affinity of phage engineered antibody against HBsAg. Methods Human antibody light chain gene repertoire generated by RT-PCR from human peripheral blood lymphocyte, was inserted into the phagmid containing Fd gene to construct the phage antibody sublibrary, from which the light chain gene matching the heavy chain Fd gene was screened. Results After three rounds of selection by biopanning, eight clones with higher absorbency than that of original clone at 490nm in ELISA were obtained, indicating that the affinity of chain shuffled phage antibodies (phab) was improved (A_ 490 from 0.43?0.09 to 1.24?0.10). DNA sequencing showed three of the five V_L genes were ? type, and the other two were ? type. Conclusions The phab obtained possesses the specificity of anti-HBsAg property.