Continuously cropping obstacle restricts ginseng production and rational use of land resource severely, and autotoxicity is one of the most important factors. In our previous work, ginseng autotoxin degrading bacteria were isolated, in the present re- search, plate culturing method and traditional physiological and biochemical method were used to analyze biological indices and protective enzyme activities, in order to elucidate the mitigative effect of autotoxin degrading bacteria on autotoxicity of P. ginseng. Results indicated that, except for palmitic acid, autotoxicity of benzonic acid, diisobutyl phthalate, diisobutyl succinate, and 2,2-bis (4- hydroxyphenyl) propane on the growth of ginseng seeds was significantly alleviated after autotoxins degrading bacteria was inoculated, and which have no evident difference with control. Except for benzoic acid, enzyme activity of SOD, POD and CAT in other autotoxin degrading treatments decreased significantly. The present research showed that, microbial degradation could alleviate the autotoxicity of autotoxins on ginseng seeds effectively, and which will be helpful for the resolution of ginseng continuously cropping obstacle problem.
Bacteria ; metabolism ; Panax ; enzymology ; growth & development ; metabolism ; microbiology ; Toxins, Biological ; metabolism

Objective To construct Escherichia coli O157∶H7 T3SS effector NleF gene knockout mutant and its com-plementary strain, and probe its effects on bacterial growth and cell death .Methods T3SS Effector NleF gene knockout mutant ΔnleF was constructed with λ-Red homologous recombination .Complementary strain ΔnleF/NleF was constructed by transferring pET-24a(+)-NleF into ΔnleF competent cells.Wild type,ΔnleF and ΔnleF/NleF were cultured in LB and DMEM(10%FBS) respectively,D600 was measured every hour , and the growth curve was drawn .HeLa cells were infected with three kinds of strains , the supernatant of LDH release was detected with cytotoxicity detection kit ,and the cytotoxicity was calculated .Results ΔnleF and ΔnleF/NleF were constructed .The growth rates of wild type , ΔnleF and ΔnleF/NleF was not significantly different .Wild type O157 infection induced cell death .Cytotoxicity was increased as much in ΔnleF in-fected cells as in ΔnleF/NleF infected cells.Conclusion EHEC O157∶H7 T3SS Effector NleF has no significant effect on bacterial growth ,but might inhibit host cell death caused by bacterial infection .

Objective To construct a prokaryotic plasmid expressing the recombinant protein of enterohemorrhagic Escherichia coli(EHEC) effector NleB1 and to prepare the polyclonal antibody of mouse anti-NleB1.Methods The nleB1 (990 bp) gene was amplified from the genome EHEC O157∶H7 and cloned into the expression plasmid pET24a to construct the recombinant plasmid pET24a-nleB1 that was transformed into E.coli BL21(DE3).After induction with isopropylthio-gelactoside( IPTG) , the His-tag fusion proteins were purified by Ni+affinity chromatography and gel slices.The polyclonal antibody was prepared by immunizing BALB/c mice with purified recombinant proteins and analyzed by Western blotting and ELISA.Results The pET24a-nleB1 recombinant plasmid was successfully constructed, the fusion protein was ex-pressed and purified,and the polyclonal antibody was obtained by immunizing mice with purified fusion protein.Western blotting and ELISA staining demonstrated that the polyclonal antibody was successfully obtained.Conclusion The prepara-tion of the polyclonal antibody against EHEC O157∶H7 NleB1 will be of help for further studies on the function of NleB1 protein.

Objectives To calculate the rate of pathological underestimation for core needle biopsy (CNB)- diagnosed intraductal papillary tumors, to analyze the clinical and imaging data of patients and to dis-cuss factors for underestimation. Methods A retrospective analysis of patients undergoing core needle biopsy and subsequent surgical excision was performed. 1359 female patients undergoing CNB from Jan. 2010 to Feb. 2013 in Comprehensive Breast Health Center of Ruijin hospital were analyzed. Clinical, radiological and histo-logical variables were assessed using the Chi-square test, Fisher’s exact test and a binary logistic regression model in order to predict pathological underestimation for tumors. Results There were 50 patients with CNB-di-agnosed intraductal papillary tumors. The overall underestimation rate was about 44%(22/50). CNB-diagnosed atypical papillary lesions (OR=15.164, 95% CI 1.49-170.443) and BI-RADS 5 by MRI (OR=26.766, 95% CI 2.409-297.440)were significantly related to underestimation in these patients. Conclusions Considering the high underestimation rate in CNB-diagnosed intraductal papillary tumors, routine surgical excision should be per-formed to avoid potential malignancy, especially for patients with high risk factors. MRI is helpful in these pa-tients to predict underestimation.

AIM: To investigate the effects of norepinephrine(NE)on vascular endothelial cell damage in-duced by lipopolysaccharides(LPS).METHODS: Human umbilical vein endothelial cells(HUVEC-12)were cultured with LPS at 100 mg/L to establish the cell damage model.Real-time PCR and Western blot were used to determine the ex-pressions of VE-cadherin at mRNA and protein levels.The levels of TNF-α,IL-1β,IL-2 and IL-10 in culture supernatant were measured by ELISA.The reactive oxygen species(ROS)production in the endothelial cells was detected by ROS as-say kit.RESULTS: LPS decreased both mRNA and protein levels of VE-cadherin accompanied by increased levels of TNF-α,IL-1β,IL-2 and intracellular ROS,and decreased level of IL-10 in the endothelial cells.NE reversed the expres-sion of VE-cadherin at mRNA and protein levels under the condition of LPS treatment in a dose -dependent manner,and al-so alleviated the intracellular oxidative stress.CONCLUSION: NE reverses the endothelial damage induced by LPS, which may be related to the up-regulation of VE-cadherin level and the decreases in oxidative stress and inflamatory media-tors.

OBJECTIVETo construct T vectors based on Xcm I recognition site and optimize the PCR fragments for its ligation.

METHODSWe firstly cloned the human histone H4 cDNA containing one Xcm I recognition site at both its 5' and 3' end into pCDNA 3.0 vector and then generated T vector with pCDNA 3.0 backbone by cutting the recombinant plasmid with Xcm I. To increase the ligation efficiency, the primers were firstly phosphorylated before DNA fragments amplification and then the PCR products were treated with Taq DNA polymerase and dATP after PCR amplification. Two DNA fragments with the length of 312 bp and 1 329 bp were ligated to it and the ligation mixture was transformed into E. coli DH5α competent cells and the positive rates of the transformants were evaluated by PCR and DNA agarose gel electrophoresis.

RESULTSOur results showed that the T vector produced by our method could ligate to the target DNA fragments with high efficiency. Besides, the phosphorylation state of the primers used for PCR amplification is also an important factor determining the cloning efficiency. What's more, as for longer DNA fragments, retreatment with Taq DNA polymerase and dATP after PCR amplification and purification could improve the ligation efficiency significantly.

CONCLUSIONOur protocol may overcome the dependence on blue/white screening to get positive clones and provide a potent way to generate T vectors and ligate them to the target PCR fragment.

Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; Genetic Vectors ; Histones ; genetics ; Humans ; Polymerase Chain Reaction ; methods

OBJECTIVE To investigate the hepatotoxicity mechanisms of ethanol extract of Radix Polygoni Multiflori (RPM) and Radix Polygoni Multiflori Praeparata (RPMP) by high-content screen assay.METHODS HepG2 cells were treated with RPM (10,25,50,100,200 and 300 mg·L-1) and RPMP (10,50,100,300,600 and 1200 mg· L-1) for 3-24 h,respectively.The cell viability was detected by a CellTiter-GloTM luminescent cell viability assay kit.Cell count,reactive oxygen species (ROS),mitochondrial membrane potential (MMP),glutathione (GSH),superoxide dismutase 2 (SOD2),activating transcription factor 4 (ATF4),apoptosis,and cell cycles were investigated by high-content screen assay.Besides,SOD2 and ATF4 levels were confirmed by Western blotting.RESULTS RPM 300 mg· L-1 showed nearly 48 ％ reduction in cell viability compared with cell control (P＜0.01),while RPMP had no significant effect at the same concentration.Both RPM and RPMP decreased the level of MMP (P＜0.05) but incresed levels of GSH,ROS,SOD2 and ATF4 significantly (P＜0.05).Besides,RPM 200 mg· L-1 significantly increased the expression of SOD2 (P＜0.05) at 3 h by high-content screen assay,and the enhanced expression of ATF4 was shown at 6 h (P＜0.05).RPMP 300 mg· L-1 markedly increased the expression of ATF4 at 6 h (P＜0.05),while the expression of SOD2 significantly increased at 24 h (P＜0.05).CONCLUSION Both RPM and RPMP have some cytotoxicity,and the cytotoxicity of RPM is stronger than that of RPMP.The hepatotoxicity mechanisms of RPM and RPMP may be related to cell apoptosis caused by long-term oxidative stress and endoplasmic reticulum stress.

Adult ; Gallbladder Diseases ; pathology ; Gastric Mucosa ; pathology ; Humans ; Male

OBJECTIVETo investigate the interaction between opioid receptor (OR) stimulation and adrenergic receptor (AR) stimulation in the isolated ischaemia/reperfusion (I-R) rat heart.

METHODSMale Sprague-Dawley rats were used for Langendoff isolated heart perfusion. Myocardial ischemia for 20 min was followed by 30 min of reperfusion, during which the kappa-OR agonist U50488h and beta(1)-AR agonist norepinephrine (NE) were administered.

RESULTS(1) 50488h antagonized the effect of NE in rising left ventricular systolic pressure (LVSP) in the early phase of myocardial ischemia at 10, 20, 30 min of reperfusion. (2) Arrhythmia scores in the I-R+NE+U50488h group were markedly lower than those in the I-R group during the 10 - 20 min reperfusion period. No significant differences in arrhythmia scores were found in either I-R+U50488h or I-R+NE group when compared with I-R group. (3) Compared with the I-R group, U50488h alone or plus NE decreased reperfusion heart rates after myocardial ischemia while NE alone showed no effect.

CONCLUSIONIt is suggested that the interaction in the signaling pathway between kappa-OR and beta(1)-AR occurred during myocardial I-R of rat heart.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer ; pharmacology ; Animals ; Male ; Myocardial Reperfusion ; Norepinephrine ; physiology ; Rats ; Rats, Sprague-Dawley ; Receptors, Adrenergic ; physiology ; Receptors, Opioid, kappa ; physiology ; Signal Transduction ; physiology

OBJECTIVETo establish a multiplex RT-PCR-based reverse dot blot hybridization technique to detect influenza viruses.

METHODSObtain the HA nucleotide sequences of seasonal influenza H1N1, seasonal influenza H3N2, influenza H1N1 and human avian influenza H5N1 from GenBank. Design primers in conservative district and probes t in high variable region respectively, after analyzing the HA nucleotide sequences of influenza virus through the Vector NTI 9.0. Establish and optimize multiple RT-PCR system by comparing amplification efficiency and specificity at different primer concentrations. Establish the reverse dot hybridization system after optimizing the concentration of probes. To compare the sensitivity and specificity of this technique and the general RT-PCR Method through extracting the viral RNA of the mentioned influenza virus which are to be the reference substance.

RESULTSSuccessfully establish a multiplex RT-PCR-based reverse dot blot hybridization technique for detecting influenza viruses. This technique is 100-1000 times more sensitive than gel electrophoresis method, and it has a good specificity.

CONCLUSIONSuccessfully established multiplex RT-PCR-based reverse dot blot hybridization technique for detecting influenza viruses.

Humans ; Influenza A Virus, H1N1 Subtype ; genetics ; isolation & purification ; Influenza A Virus, H3N2 Subtype ; genetics ; isolation & purification ; Influenza A Virus, H5N1 Subtype ; genetics ; isolation & purification ; Influenza, Human ; diagnosis ; virology ; Nucleic Acid Hybridization ; methods ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity