To obtain single chain variable fragment (scFv) and bivalent single chain variable fragment (bsFv) against transferrin receptor, up-stream and down-stream primers were designed according to the complementary sequences of FR1 region of variable heavy (VH) and FR4 of variable light (VL), respectively, which contained inter-linker G4S and the restriction endonuclease SfiI, AscI and NotI. Two pieces of scFv fragments were first amplified through PCR and then inserted into plasmid pAB1, which could express scFv protein once induced by IPTG in the host bacteria. To express scFv and bsFv, E. coli TG1 was cultured in LB broth and was induced by IPTG. The restriction enzyme digestion map and DNA sequencing demonstrated that scFv and bsFv genes were successfully inserted into the expression plasmid. SDS-PAGE and Western blotting revealed the protein band at 35kD and 60kD, which were consistent with the molecular weight of scFv and bsFv respectively. Flow cytometry showed that scFv and bsFv harbored the specific binding activity with TfR expressed in various tumor cells, and the avidity of bsFv was higher than that of the parent scFv.
Base Sequence ; Cloning, Molecular ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Genetic Vectors/genetics ; Hep G2 Cells ; K562 Cells ; Molecular Sequence Data ; Receptors, Transferrin/*immunology ; Recombinant Fusion Proteins/biosynthesis ; Recombinant Fusion Proteins/genetics ; Single-Chain Antibodies/*biosynthesis ; Single-Chain Antibodies/genetics

Objective To investigate the prevalence of glomerular microthrombosis in lupus nephritis (LN) and the significance of antibodies to anti-coagulation related factors and anti-phospholipid antibodies in glomerular microthrombosis (GMT).Methods Kidney biopsy specimens and plasma samples were obtained consecutively from 124 patients with LN. Kidney biopsy specimens were examined for the presence of glomerular microthrombi.Plasma samples from 25 LN patients with GMT (LN-GMT group) and 99 LN patients without GMT (LN-non-GMT group) were tested for lupus anticoagnlant (LA) and antibodies to cardiolipin (ACL),β2 glycoprotein I (β2GP I ),plasmin,thrombin,tissue plasminogen activator (t-PA) and Annexin A II.Results The prevalence of GMT in LN patients was about 20.2%.Compared to LN-non-GMT group,LN-GMT group had elevated SLE disease activity indices (SLEDAI),elevated activity and chronicity indices of kidney tissue injury,and elevated serum creatinine,blood urea nitrogen and proteinuria levels,and also had a higher frequency of hypertension (P＜0.01).The positive rates of LA,IgG class anti-β2GP I and anti-thrombin antibodies were higher in LN-GMT group than in LN-non-GMT group (P＜0.05).The positive rates of IgG class antibodies to ACL,plasmin,t-PA and Annexin A II in LN-GMT group were not statistically different from those in LN-non-GMT group (P＞0.05).No difference was found in the positive rate of any IgM class antibody between the two groups (P＞0.05).Conclusion This study has shown that GMT occurs approximately in 20.2% of the LN patients.Patients with GMT have more severe kidney tissue injury and more poor renal outcomes than patients without GMT.LA and antibodies to β2GP I and thrombin play a role in glomerular microthrombosis in lupus nephritis.

Objective To observe the post-systolic shortening (PSS) during isovolumic relaxation phase and its clinical significance in regional myocardium in chronic heart failure (CHF) patients.MethodsLeft ventricular regional myocardium movement in 60 CHF patients (CHF group) and 30 healthy volunteers (control group) were assessed with tissue velocity imaging (TVI). QLAB software was used to measure the systolic peak velocity (V_s), regional systolic time (T_s), post-systolic shortening velocity (V_(pss)) and post-systolic shortening time (T_(pss)) at the basal and middle levels of left ventricle. Results In CHF patients, the rate of isovolumic relaxation phase PSS was 34.44% both in basal and mid segments, the rate of pathological PSS was 29.44% and 29.72%, respectively. The rate of isovolumic relaxation phase PSS in control group was 26.11% and 20.56%, respectively; none pathological PPS occured. Compared with the physiological PSS of control group, the pathological PSS of CHF group had a higher peak velocity and a longer time (P<0.05). Conclusion The pathological PSS of CHF patients has high peak velocity and long duration, which may be one of the causes leading to the asynchronous movement of left ventricle in CHF.

Child ; Cross Infection ; microbiology ; Disease Outbreaks ; Humans ; Methicillin-Resistant Staphylococcus aureus ; classification ; drug effects ; genetics

Objective To investigate the pathogens and their antibiotic resistance in patients with peritoneal dialysis‐related peritonitis .Methods The clinical data including pathogens ,antibiotic resistance profile of 213 patients with peritoneal dialysis‐related peritonitis who were treated in our peritoneal dialysis center from January 2011 to December 2013 were analyzed retrospectively .Results Dialysate culture was positive for 132 (62 .0% ) of the 213 cases ,resulting in a total of 140 strains of microorganisms ,including 84 strains of gram positive cocci ,37 strains of gram‐negative bacilli ,10 strains of fungus and 9 strains of gram positive bacilli . Coagulase‐negative Staphylococcus was the most common gram positive bacteria while Escherichia coli was the most common gram negative bacteria isolated from the effluent .The prevalence of methicillin‐resistant S .aureus and methicillin‐resistant coagulase negative Staphylococcus was 14 .3% (1/7) and 43 .2% (19/44) ,respectively . About 44 .4% (8/18) of the E .coli and K . pneumoniae isolates produced extended spectrum beta‐lactamases .All the gram‐positive cocci were sensitive to vancomycin and linezolid and slightly resistant to chloramphenicol (6 .3% ) , moxifloxacin (8 .5% ) , and rifampicin (9 .5% ) , but highly resistant to cefazolin (90 .0% ) ,followed by ampicillin (76 .7% ) ,oxacillin (71 .2% ) and penicillin (69 .7% ) . Coagulase negative Staphylococcus isolates were sensitive to vancomycin , linezolid , tigecycline , quinupristin‐dalfopristin and daptomycin ,but all resistant to cefazolin and ampicillin ,and highly resistant to penicillin (91 .9% ) and oxacillin (82 .5% ) .All the gram‐negative bacilli were sensitive to meropenem ,ertapenem ,cefoperazone‐sulbactam and tigecycline .About 80 .6% and 65 .5% of the gram‐negative bacilli were resistant to ampicillin and peperacillin ,respectively .E .coli isolates were sensitive to meropenem ,ertapenem and piperacillin‐tazobactam but highly resistant to ampicillin (81 .3% ) and piperacillin (71 .4% ) . Conclusions Gram‐positive cocci especially Staphylococcus and gram negative bacteria E .coli are major pathogens in peritoneal dialysis‐related peritonitis .Adequate microbiological culture and suitable antimicrobial therapy are key to successful treatment of the peritonitis associated with peritoneal dialysis .

To obtain single chain variable fragment (scFv) and bivalent single chain variable fragment (bsFv) against transferrin receptor, up-stream and down-stream primers were designed according to the complemenary sequences of FR1 region of variable heavy (VH) and FR4 of variable light (VL), respectively, which contained inter-linker G4S and the restriction endonuclease Sill, AscI and NotI. Two pieces of scFv fragments were first amplified through PCR and then inserted into plasmid pAB 1, which could express scFv protein once induced by IPTG in the host bacteria. To express scFv and bsFv, E. coli TG1 was cultured in LB broth and was induced by IPTG The restriction enzyme digestion map and DNA sequencing demonstrated that scFv and bsFv genes were successfully inserted into the expression plasmid. SDS-PAGE and Western blotting revealed the protein band at 35kD and 60kD, which were consistent with the molecular weight of scFv and bsFv respectively.Flow cytometry showed that scFv and bsFv harbored the specific binding activity with TfR expressed in various tumor cells, and the avidity of bsFv was higher than that of the parent scFv.

OBJECTIVETo investigate the effect of demethylating agent 5-Aza-CdR (5-aza-2'- deoxycytidine) on demethylation and transcription-regulating of RASSF1A gene in gastric cancer cell SGC7901 in vitro, as well as on the growth inhibition of cells.

METHODSAfter SGC7901 cells were treated with 5-Aza-CdR, MTT assay, flow cytometry, and Annexin V-FITC staining were performed to analyze the cell proliferation, cell cycle and apoptotic rate respectively. Methylation- specific PCP (MSP), RT-PCR and Western blotting were used to detect methylation state, expression of mRNA and protein of RASSF1A gene.

RESULTSAfter SGC7901 cells were treated with different concentrations of 5-Aza-CdR, the cell growth was inhibited(P<0.05), the cell cycle was blocked at G(1) phase, and the apoptotic rate increased significantly(P<0.05). Hypermethylation was detected in the promoter region of RASSF1A gene in SGC7901 cells, and no expression of RASSF1A mRNA and protein was found. After treated with 5-Aza-CdR, demethylation occurred in RASSF1A gene,which subsequently induced re-expression of this gene at both mRNA and protein level.

CONCLUSIONDemethylating agent 5-Aza-CdR can regulate demethylation and re-expression of RASSF1A gene in gastric cancer cell SGC7901,and inhibit its growth.

Azacitidine ; analogs & derivatives ; pharmacology ; Cell Line, Tumor ; DNA Methylation ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Stomach Neoplasms ; genetics ; metabolism ; Tumor Suppressor Proteins ; metabolism

Objective To evaluate the capability and accuracy of multi-shce spiral computed tomography(MSCT)in detecting atherosclerotic plaques in nonstenotic coronary arteries with reference to the findings of intravascular ultrasound(IVUS)in a segment analysis.Methods Both IVUS exams and 16-row MSCT scans were performed on 35 consecutive patients among whom 30 patients had successful MSCT scans.A total of 94 coronary segments without significant coronary stenoses were paired-analyzed both on IVUS and MSCT segment by segment.The plaques were classified as calcified,fibrotic and soft types according to the echogeneity on IVUS.Plaque attenuation on MSCT was measured and expressed by Hounsfield units(HU).Results When referred to IVUS,MSCT had a sensitivity of 82.1%(46/56)and specificity of 89.5% (34/38),respectively in detectiong any plaques.For the detection of calcified plaques,the sensitivity and specificity were 92.1%(35/38)and 96.4%(54/56),respectively.For the detection of mixed and noncalcified plaques,MSCT had sensitivity of 73.2%(30/41)and specificity of 88.7%(47/53).But for the detection of the noncalcified plaque,the sensitivity was 66.7%(12/18). According to the findings On IVUS,the plaques were classified as calcified(n=19),fibrotic(n=19)and soft(n=16).The CT attenuation of calcified plaques was(489?169)HU(196 to 817 HU),fibrotic plaques(69?21)HU(25 to 117 HU)and soft plaques(23?18)HU(-12 to 47 HU).Nonparametric Kruskal-Wallis test revealed a significant difference of plaque attenuation among the three groups(P

OBJECTIVETo investigate the effects of dexamethasone on expression of matrix metalloproteinase-9 (MMP-9) in rats with acute lung injury induced by phosgene.

METHODSThe rats were randomly divided into 3 groups: normal control group that consists of the rats with air exposure, phosgene group that consists of the rats with phosgene exposure and dexamethasone group that consists of the rats with phosgene exposure after 2.5 mg/kg dexamethasone being injected. Wet and dry ratio of the lung (W/D) was calculated, and leukocyte count and total protein content of bronchoalveolar lavage fluid (BALF) were recorded at 2 h after exposure. The concentrations of MMP-9 in the serum and BALF were measured by enzyme-linked immunosorbent assay. The pathologic changes of lung tissues were observed under light microscopy. The immunohistochemistry and the RT-PCR were used to detect the contents of MMP-9 in the lung tissue.

RESULTSCompared with phosgene group, the lung W/D, protein content and WBC count in of BALF dexamethasone group was significantly decreased (P < 0.01). MMP-9 levels of the serum and BALF in dexamethasone group were (4.799 +/- 0.043) microg/L and (15.052 +/- 0.029) microg/L, respectively, which were significantly lower than those [(9.439 +/- 0.100) and (20.640 +/- 0.446) microg/L] in phosgene group (P < 0.01). Compared with phosgene group (2.789 +/- 0.282),the expression level (1.183 +/- 0.260) of lung MMP-9 mRNA in dexamethasone group was significantly lower than that in phosgene group (P < 0.01). Histological experimental results showed the marked hyperemia and thickening of alveolar walls and stroma cells infiltrating and more visible alveolar structure damage of alveolar walls in phosgene group while the alveolar structure and the alveolar walls were clear and slightly thickened with inflammatory cells in dexamethasone group. Immunohistochemical results showed that MMP-9 protein expression levels of lung and bronchus tissues in normal control group and dexamethasone group were weakly positive, which in phosgene group were strongly positive.

CONCLUSIONDexamethasone has a beneficial effects on acute lung injury induced by phosgene in rats due to the inhibiting MMP-9.

Acute Lung Injury ; chemically induced ; drug therapy ; metabolism ; Animals ; Bronchoalveolar Lavage Fluid ; Dexamethasone ; therapeutic use ; Disease Models, Animal ; Male ; Matrix Metalloproteinase 9 ; metabolism ; Phosgene ; toxicity ; Rats ; Rats, Sprague-Dawley