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Objective Establish detection method to measure Vibrio vulnificus rapidly and accurately.MethodsUsing flow cytometry(FCM)and a 5'-FITC fluorescent labeled aptamer with high binding affinity to detect Vibrio vulnificus rapidly.Measure a series of concentrations of Vibrio vulnificus to identify the Limit of Blank, Lower Limit of Detection, Linearity Range, etc.ResultsCombined application of FCM and the aptamer can detect Vibrio vulnificus rapidly with the duration less than 1 hour and lower limit of detection as low as 29 CFU/mL.Conclusion The aptamer targeting Vibrio vulnificus is an excellent detective element, while FCM can realize accurate quantitative detection.The detection method has great application potential.
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‘University and college students' innovative ability training plan’ were launched from 2009 in Second Military Medical University. The innovative thinking and practical ability of students were improved by participation in research projects, writing scientific papers, academic exchanges and other activities. Students' innovative thinking, practice ability and cooperative spirit were promoted and unifica-tion of teaching and learning was achieved.
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MicroRNA (miRNA) is a family of endogenous,non-coding small RNAs molecules that function as gene regulators.It has been revealed that miRNAs may play a critical role in many biological processes including cell proliferation,differentiation and apoptosis.Recent studies demonstrate that aberrant expression of miRNAs can lead to several human diseases even cancer.These tiny but potent molecules have the function as anti-oncogene or oncogene.Accordingly,further study of miRNAs has opened a novel avenue in the diagnosis and treatment of human cancer.
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This study is to investigate the influence and mechanism of action of asymmetrical dimethylarginine (ADMA) and the induced oxidative stress level on Alzheimer's disease (AD) incidence. ADMA concentration, nitric oxide, Abeta(40)/Abeta(42) ratio, inducible NO synthase (iNOS) activity and the concentrations of the induced free radicals including malondialdehyde (MDA), 3-nitrotyrosine (3-NT) and peroxynitrite (ONOO-) in the cerebrospinal fluid (CSF) from 34 neurologically normal controls and 37 AD patients were quantitatively determined and statistically compared. The results showed that the ADMA concentration significantly decreased in AD patients, and it showed negative correlation with the NO, iNOS activity, and showed positive correlation with MMSE score. ADMA concentration was negatively correlated with Abeta(40)/Abeta(42) ratio (P<0.01) with the observation that Abeta(40)/Abeta(42) ratio increased while ADMA level decreased in CSF in AD patients. The concentration levels of MDA, 3-NT and ROS significantly increased compared with the control with all the P values less than 0.05. These findings suggested that the ADMA disorder and the oxidative damage effect of the induced free radicals in CSF of AD patients are an important mechanism of AD incidence, and their joint regulation may provide new idea for the prevention and clinical treatment of AD.
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Objective To investigate the expression of CD40,CD40L and MMP9 in carotid atherosclerotic plaques and evaluate their roles in carotid atherosclerotic plaque stability.Methods Carotid atherosclerotic plaques were isolated in carotid eversion endarterectomy (GEE) in 37 patients with high-grade stenosis (>70%) including 20 stroke (A group) and 17 non-stroke patients (B group).The control group included samples of normal carotid artery from 11 normal individuals,The RNA expression levels of CD40,CD40 L and MMP9 in all A,B and control groups were quantitatively detected by real-time quantification polymerase chain reaction (PCR) and the protein expression levels were detected by Western blotting analysis.The expression and distribution of CD40,CD40L and MMP9 in carotid atherosclerotic plaques were detected by immunohistochemistry staining.Then correlations between CD40-CD40L and MMP9 were statistically analyzed.Results The relative CD40 mRNA level in high-grade stenosis of A group,B group and normal control were 2.41±0.43,1.03±0.38 and 0.31±0.12,respectively,and MMP9 mRNA 6.88±1.57,1.90±0.44 and 0.39±0.12,respectively.The levels of CD40 and MMP9 mRNA in A group were significantly higher than those in B group (P=0.000),the levels of CD40 and MMP9 in B group were significantly higher than those in controls (P=0.000).There was a linear correlation between CD40 and MMP9 mRNA (r=0.929,P=0.000).However,there were no significantly difference in mRNA levels of CD40L between carotid atherosclerosis and controls.The protein expression levels of CD40,CD40L and MMP9 in A group were significantly higher than those in B group (FCD40=104.100,P=0.000;FCD40L=129.932,P=0.000;FMMP9=13.565,P=0.021) and B group higher than normal controls (FCD40=115.848,P = 0.000;FCD40L= 30.482,P=0.005;FMMP9=35.557,P=0.004).The areas of positive staining of CD40,CD40L and MMP9 in immunochemistry study in A group were significantly higher than those in B group and B group was significantly higher than controls.There were linear correlations between positive staining areas Of CD40 and CD40L,CD40 and MMP9,CD40L and MMP9 (r=0.963,0.959,0.929,P=0.000).Expressions of CD40,CD40L and MMP9 were significantly higher in the shoulder areas of the atherosclerotic plaques than in other areas.Conclusions The CD40-CD40L has an important role in the formation of carotid atherosclerosis and plaque instability,probably by up-regulating MMP9.The expression of CD40L may be regulated by post-transcriptional modification to exert biological effects.
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Objective To investigate the effects of human interlukin-13 (hIL-13) on the expression of E- selectin and intercellular adhesion molecule-1(ICAM-1) on bovine aortic endothelial cells(BAECs) stimulated by tumor necrosis factor alpha(TNF-?), and to provide experimental basis for hIL-13 inducing immunity endure and relieving the repulsion reaction of xenograft. Methods BAECs were co-cultured with different concentrations of hIL-13 for 2 h and followed by co-cultured with 4 ng/ml TNF-? for 6 h or 18 h. The expressions of E-selectin and ICAM-1 on BAECs were detected by Cell-ELISA. The effect of hIL-13 on activity of BAECs was detected by MTT colorimetry.Results BAECs pretreateded with hIL-13 could inhibit the expression of E-selectin and ICAM-1 induced by TNF-?, and showed a does-dependent manner from 5 ng/ml to 20 ng/ml of hIL-13 (P
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Aim To analyze THANK gene expression in peripheral blood mononuclear cells(PBMC) stimulated with different stimulators and to clone whole length human THANK gene. Methods PBMC were conventionally isolated and cultured in RPMI1640 containing 10％ FCS. After stimulated with LPS,TNF α ,IL 2,IFN γ ,PHA or PMA,the THANK gene expression in PBMCs was analyzed by RT PCR and THANK cDNA was cloned. Result RT PCR detection showed that THNAK gene expressed in PBMCs after stimulated with interferon γ for 3 days, whereas THANK'expression could not be detected after stimulated with LPS,TNF α ,IL 2,IFN γ ,PHA or PMA respectively. Then THANK gene was cloned by cloning PCR product and sequenced. Conclusion Human THANK gene is cloned successfully, thus providing the possibility for further research of THANK'function.
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Objective: To prepare highly purified THANK protein. Methods: THANK was efficiently expressed in E.coli as inclusion bodies. After bacteria were lysed under ultrasonication, TE buffer,1% TritonX and 2 mol /L urea was used to efficiently extract inclusion bodies. After denaturation with 8 mol/L urea,THANK was partially purified by Sephacryl S-200 chromatography, and then subsequent refolding step was optimized for a maximal recovery of biological active protein. The renatured THANK was purified to homogeneity with Q Sephadex Fast Flow gel filtration and then desalted by Sephadex G-25 chromatography. Results: The purity of biologically active THANK was above 97% in SDS-PAGE densitometric studies. Conclusion: An effective method of denaturation, renaturation and purification of recombinant THANK is established.
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Objective: To clone THANK gene and express its extracelluar fragment. Methods: Using RNA isolated from HL 60 cell lines, THANK cDNA was amplified by RT PCR. The fragment was linked to pMD18 T vector and sequenced, and then the extracellular fragment of THANK was subcloned into pET vector and THANK protein expression was induced.Results: A 858 bp DNA fragment was amplified and the cDNA sequence was identical with the published sequence encoding THANK gene. Western blot showed that THANK protein with a relative molecular weight of 2.6?10 4 was expressed. Conclusion: Human THANK gene was cloned and expressed successfully, which provides a base of further research of THANK gene. [
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Various chemical and gas-liquid chromatographic analyses indicate that the lipid A backbone of the lipopolysaccharide (LPS) isolated from Bacteriodes fragilis NCTC 9343 is chemically constituted by a ?1, 6-interlinked D-gluco-samine disaccharide. It is phosphorylated at its 1-position by a glycosidic-linkage while the ester-bound phosphate present generally in other lipid A is depleted. The lipid A is lower fatty acylated in the amount of 5.2 fatty acids per lipid A molecule (of which 0.73 forms 3-acyloxyacyl groups). The LPS containing such a lipid A component has been shown endowing with a weaker endotoxicity.
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The lipid A component of Re-lipopolysaccharide from S.typhimurium SL 1102 was prepared by hydrolysis with a 0.1 mol/L acetate buffer.Chemical as well as advanced l3C-,and 31P-NMR investigations revealed a ?1' ,6-interlinked D-glucosamine diasaccharide, which is substituted by two phosphate(P)groups, one being esterlinked to the non-reducing residue,and another bound glycosidically to the C1 of the reducing residue, is as the central backbone.Its structure is shown as follows:-P-GlcN(?1', 6)GlcN-P-The amino(2 and 2')and hydroxy(3 and 3')groups of the backone are substituted by 3-OH-14: 0, and unhydroxy fatty acids appear to be the subtituents of the hydroxy group of 3-OH-14 :0, in the means of formation of 3-acyloxyacyl ester and amides.
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Superoxide anion releasing from activated murine macrophages stimulated with recombinant human interferon-? (IFN-?), tumour necrosis factor-? (TNF-?) and bacterial lipopolysaccharidc were studied. The results showed that both of the cytokines enhanced superoxide anion release in a dose and time - dependent manner, but lipopolysaccharide had no such an effect as compared with IFN-? and TNF-?. It was also shown that O2- generation from macrophages was highly enhanced after primed with IFN-? or TNF-? for 24 hours. It was concluded from these data that cytokines released from macrophages and lymphocytes during inflammatory reactions could promote O2- generation which may play a crucial role in destruction or kill of intracellular pathogenic bacteria, and tumour cells.
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Objective: To establish p53 trancated muteins group and analyse their expression and effects on p53 related downstream genes in HeLa cells. Methods: Using PCR-based site-directed mutagenesis technique, a group of P53 muteins with the N- and/or C-terminals differently trancated were set up. The plasmids with p53 mutein genes were used to transfect HeLa cells, the p53 muteins and some p53 related downstream genes' expression were analyzed by RT-PCR. Results: The p53 muteins were expressed in HeLa cells and they had effects on some of p53 related downstream genes. Conclusion: The P53 muteins may be a good tool to analyze P53 function and has effects on p53 related downstream genes' expression model.
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The ocean is a huge resource of organisms and about 80% of the world species live in it.Marine organisms synthesize various structurally-unique and pharmacologically-active metabolites which differ from those derived from the land organisms and serve as lead compounds for drug development.More than 15 000 new compounds have been isolated and identified from marine animals,plants and microorganisms since decades ago,and drugs like cephalosporins,ziconotide(Prialt),cytarabine(AraC) and vidarabine(AraA) were developed using these compounds as precursors.
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A typical smooth-form lipopolysaccharide (LPS) isolated from Salmonella abortus equi was fractionated into a S (smooth)-and a R (rough)-fraction and their serological and biological properties were investigated. It was shown that S- fraction expressed an O-antigenicity while R-fraction predominately a Rb-antigenicity. The R-fraction was endowed with higher bioactivities than the S-fraction in lethal toxicity, local Shwartzman reaction and pyrogenicity. Both S-and R-fractions were active in inducing mitogenicity to murine spleen cells. A reconstituted LPS preparation with fractionated S-fraction and a trace amount of R-fraction (1% of the original preparation) exhibited a same extent of lethal toxicity as that of the original one. Antr-Ra and -Rb antiserum with a liter of 1?4096 showed a .highly effective protection against the lethal challenge of LPS, indicating that the R-fraction in the natural LPS preparations plays a critical role to the lethality of LPS.
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Effects of Lycium barbarian polysaccharide (LBP), Astragalus polysaccharide (APS), polysaccharide of Acanthopanax senticosus (PAS) and polysaccharide of lipopolysaccharide (PS) on cytotoxicities of LAK cells from splenocytes of C57BL/6 mices were observed with 18 h 125IUdR release assay. Our study demonstrated that the polysaccharides alone were shown to induce no cytotoxicities. When combined with rIL-2, all of 4 kinds of the polysaccharides augrnented LAK activities in dose-dependent manner. The augmentations reached the maximum at 0.01-0.1 mg ? ml-1 LBP, 0.01 mg ? ml-1 APS and PAS, 0.01 mg ? ml-1 PS. They were found to increase LAK activities in short range of rIL-2 concentrations (250-1000U? ml-1). If the polysaccharides were used beyond the suitable concentrations, they were shown to inhibit LAK cell activities.