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

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

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.

With the use of computer image processing technology , a new method was proposed for studying the contact characteristics of the subtalar joint. The results showed the total subtalar articulation area was 9.52±0.40 cm2. On neutral position and under 600N load, the contact area of the subtalar joint was 2.00±0.11cm2. The contact area of the posterior articulation was significantly larger than that of the anterior and medial articulation (p<0.01). The average contact pressure was 19.3±1.38N, the force transmitted by the subtalar was 389.16±28.75N, which accounted for about 64.86% of the applied shank load(600N), and 69.39% of the force was transmitted by the posterior articulation. The posterior articulation plays an important role in the load. The fracture line of the calcaneus often appears in this area.

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

To study the effects of Qingdai compound on proliferation and apoptosis of K562 cells, as well as the expression of bcr/abl and JWA mRNA, K562 cells were treated in culture with different concentrations of Qingdai compound (2.5, 5, 7.5, 10 and 20 mg/ml) and harvested at 24 hours. Then morphological changes were observed by light microscopy (LM); expressions of bcr/abl and JWA were detected with semi-quantitative RT-PCR. The results showed that morphological changes were observed as the increment of the Qingdai compound concentration. Inhibition effects on proliferation and apoptosis in K562 cells were seen. A concentration-dependent decreases were found in bcr-abl and JWA mRNA expression of K562 cells. Qingdai compound partially inhibited proliferation and induced apoptosis of K562 cells. Expressions of both bcr/abl and JWA, which took part in cell proliferation and apoptosis, were down-regulated in a dose dependent manner. In conclusion, Qingdai compound can partially inhibit the expressions of bcr/abl and JWA genes in K562 cells, and the clinical effect of Qingdai compound on CML may be associated with apoptosis of leukemic cells.
Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; pharmacology ; Fusion Proteins, bcr-abl ; genetics ; Gene Expression Regulation, Neoplastic ; drug effects ; Heat-Shock Proteins ; genetics ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; K562 Cells ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction