Diagnostic Imaging ; Echocardiography ; Humans ; Magnetic Resonance Imaging ; Myocarditis ; diagnosis ; Radionuclide Imaging

Objective To construct the bait expression plasmid pGBKT7-GR of glucocorticoid receptor(GR)binding domain.Methods The fragments of GR binding domain was amplified by RT-PCR,and then was cloned into pMD18-T.After being verified by sequencing,it was subcloned into the bait expression vector pGBKT7.Then the bait vector pGBKT7-GR was transformed into AH109 yeast cells and the expression of the bait protein was analyzed by Western blot.Toxicity and self-activation of the bait protein were detected.Results GR binding domain was amplified and cloned into pMD18-T and pGBKT7 successfully.The bait vector was transformed into AH109 yeast cells successfully,without toxicity or self-activation.The expression of the bait protein was confirmed by Western blot.Conclusion The successful construction of bait expression vector of glucocorticoid receptor binding domain lays the foundation for constructing small molecule ligand yeast three-hybrid system.

Objective To identify the interaction between nuclear localization signal-retinoic acid receptor α (NLS-RARα) and Ubiquilin 1(UBQLN1). Methods The recombination expression plasmids pGBKT7-NLS-RAR and pACT2-UBQLN1, which expressed bait-protein NLS-RARα and target protein UBQLN1 respectively, were cotransformated into yeast AH109. The interaction of the expression plasmids in the living cells was investigated by yeast two-hybrid assay. HA-tagged fusion protein (pCMV-HA-NLS-RARα) expression vector and Myc-tagged fusion protein (pCMV-Myc-JTV1) expression vector were constructed, identified, and cotransfected respectively into human embryo kidney 293 cells(HEK293). The interaction was detected by co-immunoprecipitation.Results Blue clones were found on yeast AH109 plate cotransformated with pGBKT7-NLS-RARα and pACT2-UBQLN1. Double restriction enzyme digestion showed that pCMV-HA-NLS-RARα and pCMV-Myc-JTV1 were successfully constructed. Then HA-NLS-RAR protein was immunoprecipitated by anti-HA polyclonal antibody and the expression of Myc-UBQLN1 was tested by Western blot with anti-Myc monoclonal antibody from immunoprecipitated complex. Conclusion The interaction between NLS-RARα and UBQLN1 can be verified by yeast two-hybrid assay and co-immunoprecipitation.

Anemia, Hemolytic ; etiology ; Child ; Female ; Humans ; Insecticides ; poisoning ; Trichlorfon ; poisoning

This study was aimed to verify the effects of staged sequential therapy on expressions of matrix metalloproteinase-9 (MMP-9) and its inhibitor TIMP-1 within lung tissues in asthmatic rats with the airway remodeling, by applying a series of tests such as the immunohistochemistry, western blot and real-time fluorescent quantitative PCR. SD rats were randomly divided into 7 groups, which were the asthmatic group (Group X), the normal group (Group Z), the No. 1 sequential therapy group (Group A1), the No. 2 sequential therapy group (Group A2), the No. 3 sequential therapy group (Group A3), the montelukast group (Group M), and the budesonide group (Group B). The asthmatic model was established in each group except Group Z, by sensitization with both ovalbumin (OVA) and aluminium hydroxide via injection at the 1st, 8th and 15th day in a 22-day duration, followed by OVA aerosol inhalation every other day for 8 weeks for asthma activation. At the 8th day after the asthmatic model was established, Group A1 was orally given Ma-Xing Er-Chen Tang (MXECT) during acute phase while given normal saline (NS) during catabasis and stable phase; Group A2 was given MXECT during acute phase, and given modified Jin-Shui Liu-Jun Jian (JSLJJ) during catabasis as well as given NS during stable phase; Group A3 was given MXECT during acute phase, and given modified JSLJJ during catabasis as well as given Liu-Wei Di-Huang (LWDH) Powders during stable phase;Group M was given salbutamol via aerosol inhalation after stimulation, while orally given montelukast during catabasis and stable phase; Group B was given salbutamol via aerosol inhalation after stimulation, while given inhaled budesonide during catabasis and stable phase; Group X was given NS. After the 7-week intervention, the immunohistochemistry, western blot and real-time quantitative PCR were applied to analyze the location and quantitative expression of MMP-9 and TIMP-1, so as to find out the biological mechanism on expressions of MMP-9 and TIMP-1 in lung tissues of asthmatic rats from molecular levels to gene levels. The results of immunohistochemistry, western blot and real-time fluorescent quantitative PCR showed as follows. Compared with Group Z, the contents of MMP-9 and TIMP-1 increased significantly within lung tissues in Group X. Compared with Group X, the contents of MMP-9 and TIMP-1 decreased within lung tissues of asthmatic rats in each treatment group. It was concluded that the expression of MMP-9 and TIMP-1 elevated during asthma. TCM staged sequential therapy can regulate the ratio between MMP-9 and its inhibitor so as to block the airway remodeling, by decreasing the expression of MMP-9 and its inhibitor within lung tissues in asthmatic rats. This is one of the important action mechanisms.

Total RNA was extracted from leaf of Suaeda hetroptera kitag, then the CMO ( choline monooxygenase) cDNA was amplified using the reverse transcriptase polymerase chain reaction ( RT-PCR) method and cloned into pMD-T-simple vector. The positive clones from the Blue/White Screen were sequenced. After confirming its validity, the CMO gene fragment was cloned into pBI121 vector. Double enzyme restriction and PCR analysis indicated that the pBI121/CMO recombinant plasmid was successfully constructed.

Objective To explore the diagnostic significance of differential cell count in induced sputum to chronic cough and assessment of airway inflammation.Methods The sputum of 335 chronic cough patients were induced.Differential cell counts were measured in these samples.The side effects were observed during the induced procedure.The final diagnosis was made based on clinical manifestation and examination findings including pulmonary function tests,provocation test,induced sputum cell differentials, etc.Results The cause of chronic cough was defined in 322 patients.The six most important causes of cough were typical asthma(TA,n=84),eosinophilic bronchitis (EB,n=62),atopic cough (AC,n= 42),cough variant asthma (CVA,n=40),gastroesophageal reflux cough(GERC,n=37),rhinitis and/ or paranasal sinusitis (PNDs,n=32),and others and indefinite cause (n=25,13).Percentage of eosinophils were significantly increased in the induced sputum of AC,EB,CVA,and GERC patients (0.005,0.052,0.059,0.234) compared with those in other causes and the healthy controls (0) (P

Acute promyelocytic leukemia (APL) is characterized by the generation of the prototypic promyelocytic leukemia-retinoicacid receptor alpha (PML-RARα), an oncogenic fusion protein due to chromosomal translocation. In a human myeloid cell line,PML-RARα is cleaved by neutrophil elastase (NE) to produce the mutational PML [nuclear localization signal (NLS) deletion andRARα (NLS-RARα, containing NLS of PML), both of which may play an important role in APL pathogenesis. The yeast two-hybridtechnique was used to screen the intracellular proteins interacting with NLS-RARα, which may be involved in NLS-RARα signaling. The NLS-RARα coding sequence was amplified by polymerase chain reaction method and was cloned into the bait plasmid pGBKT7vector, which, after the confirmation by sequencing, was transformed into yeast AH109 and the subsequent expression of bait plasmidwas proved by Western-blot. The transformed yeast AH109 was mated with yeast Y187 (containing leukemia cDNA library plasmidspACT2) in medium. Diploid yeast was plated on synthetic dropout nutrient medium containing X-α-gal for screening. After beingreintroduced into yeast AH109 and sequenced to verify the expression of ORF, eight positive colonies were obtained, among whichonecontaining JTV-1 was cloned. The interaction between NLS-RARα and JTV-1 was further supported by indirect immunofluorescence,GST pull-down and co-immunoprecipitation, respectively. These findings brought some new clues for the further exploration ofNLS-RARα signaling to APL.

OBJECTIVETo screen the protein interacting with retinoic acid receptor variant protein (RAR alpha-V) via the yeast two-hybrid technique (YTHT) and to find out the targets protein and study its biological function.

METHODSThe bait vector of pGBKT7-RAR alpha-V was constructed for screening proteins interacting with RAR alpha-V in K562 cell cDNA expression library via YTHT. The protein-protein interaction was confirmed with re-transformation in yeast and GST pull-down in vitro.

RESULTSThe bait vector was successfully constructed without toxicity, leakage and self-activation. Sixteen proteins were screened by YTHT and eight positive clones were identified by re-transformation in yeast. The interaction between RAR alpha-V and JTV-1 was confirmed by GST pull-down in vitro.

CONCLUSIONSThere are some kinds of proteins interacting with RAR alpha-V in cell. The biological dysfunction caused by certain protein-protein interaction may be involved in the pathogenesis of leukemia.

Adult ; Aged ; Endotoxemia ; Endotoxins ; blood ; Female ; Humans ; Inflammation Mediators ; blood ; Liver Diseases ; blood ; therapy ; Liver, Artificial ; Male ; Middle Aged ; Plasma Exchange ; Serum ; chemistry ; Young Adult