Objective Insulin-like growth factor-1 receptor (IGF-1R),similar to insulin receptor,is one of the families of re- ceptor tyrosine kinases.,which has been found to be overexpressed in a variety of cancer.It is the main proliferation and survival sig- nal molecule in cancer cell and plays an important role in cancer growth and progress.Blocking signal transduction of IGF-1R by vari- ous strategies can suppress tumor growth and induce regression of established tumor.This study is to construct the siRNA expression vector targeting IGF-1R and to evaluate its ability to induce cell apoptosis in human lung cancer cells.Methods Two siRNA expres- sion vector,pENTR/U6-shRNA-1 and pENTR/U6-shRNA-2 targeting IGF-1R,were constructed using pENTR/U6 vector,and a vector targeting hieiferase gene,pENTR/U6-shRNA-Iuc,was constructed as control.After vectors were transfected into A549 for 48h, knockdown of IGF-1R mRNA and protein and Akt phosphorylation were accessed,and DNA ladder and flow cytometry were used for cell apoptosis.Results siRNA expression vectors targeting IGF-1R were successfully constructed,which was confirmed by PCR and DNA sequencing,pENTR/U6-shRNA-1 and pENTR/U6-shRNA-2 demonstrated the expression were (22.1?2.5) % and (80.1? 3.9) % in IGF-1R mRNA level,(15.2?3.1)% and (47.1?4.1)% in protein level,respectively,compared with pENTR/U6- shRNA-luc.Suppression of IGF-1R by pENTR/U6-shRNA-1 blunted Akt phosphorylation,increased cell apoptosis induced by 3% ethanol,and retained 77.5 % A549 cells in the G0/G1 phase.Conclusion siRNA expression vector targeting IGF-1R can effectively suppress the expression of IGF-1R expression in A549.This study suggests that DNA vector-based RNAi has the potential to be effec- tive and practical cancer gene therapy strategy.

Peptide cyclization, a pivotal approach to modifying linear precursors of proteins and pepticles, has been used to enhance their biological activities and serum stabilities. Recently, sortase A (SrtA) from Staphyloccus aureus becomes a promising new technology for efficiently incorporating site specific modifications into proteins, conjugating the cell surface and cyclizing the linear peptides. In this study, we constructed two recombinant expression systems, one with chitin binding domain and the other with six-histidine tag and chitin binding domain on the N-terminal of SrtA, separately. The results of enzymatic kinetics indicate that the two recombinant tags do not impair the transpeptidase activity of SrtA compared with the standard reaction reported under the same reaction condition. The two synthesized peptides with N-ternimal three glycines and C-terminal penta-amino acid motif, LPETG, were cyclized using immobilized and recycled SrtA. The SrtA-based cyclization promises to represent a simple method for easy and efficient enzymatic synthesis of large cyclic peptides.
Aminoacyltransferases ; metabolism ; Bacterial Proteins ; metabolism ; Cyclization ; Cysteine Endopeptidases ; metabolism ; Enzymes, Immobilized ; metabolism ; Kinetics ; Peptides ; metabolism ; Peptides, Cyclic ; biosynthesis ; Staphylococcus aureus ; enzymology

The meridians belong to zang-fu organs inside and connect with the extremities outside. They keep information exchange among different tissues and make various organs of the human body as an organic whole. If the meridians are blocked and the information exchange makes mistake, the body will produce pathologic changes. Because the location of the blocked part is difficult to identify, in this paper it is explained that selection of distal acupoints is the best way to dredge the nodes of disease, and the theoretical basis is closely related with the theories of "the root cause and symptoms of a disease", "Genjie", and "Jing, Xing, Shu, Jing, He", and also implies the thought of "to cure a disease must treat the root of the disease".
Acupuncture Points ; Acupuncture Therapy ; Humans ; Medicine, Chinese Traditional ; Meridians

Objective:To explore the perivenous blocking management strategy for portal vein-superior mesenteric vein (PSMV) resection and reconstruction and its effect on postoperative complications in patients undergoing pancreaticoduodenectomy (PD).Methods:The data of 137 patients with pancreatic cancer treated with PD in Beijing Chaoyang Hospital Affiliated to Capital Medical University, Chaoyang Central Hospital, the Second Hospital of Chaoyang, Rizhao Central Hospital, the Second People's Hospital of Binzhou from January 2010 to December 2020 were collected. There were 83 males and 54 females with an average age of 61.8 years. There were 42 patients in the reconstruction group and 95 patients in the control group. The main intraoperative indexes and postoperative complications were compared between the two groups with the aim to review our experience in PSMV resection and reconstruction by using the perivenous blocking management strategy.Results:PD was successfully completed in 137 patients in the reconstruction group, the PSMV blocking time was 15-120 min, with a median of 30 min. The operation time 380 (330, 465) min, intraoperative blood loss 725 (500, 1000) ml, and postoperative hospital stay 21.0 (16.0, 28.0) d in the reconstruction group were significantly higher than those of control group [305 (280, 340) min, 400 (300, 500) ml and 18.0 (14.0, 24.5) d] (all P<0.05). The reoperation rate and perioperative mortality were 4.8% (2/42) and 2.4% (1/42) in the reconstruction group, while 2.1% (2/95) and 1.0% (1/95) in the control group, respectively. There was no significant difference between the two groups (both P>0.05). The incidence of pancreatic fistula, peritoneal effusion and infection, pulmonary infection of the reconstruction group was significantly higher than those of the control group ( P<0.05). There was no significant difference in the incidence of postoperative bleeding, delayed gastric emptying, biliary fistula, incision infection, reoperation between the two groups ( P>0.05). Conclusions:PSMV resection and reconstruction significantly increased the incidences of complication after PD, including pancreatic fistula, peritoneal effusion/infection and pulmonary infection. The perivenous blocking management strategy significantly promoted smooth postoperative recovery and effectively reduced morbidity rates of postoperative bleeding and mortality after PSMV resection and reconstruction in PD.

Objective To investigate the effect of 4-hydroxytamoxifen on the expression of pituitary tumor transforming gene (PTTG) in GH3 prolactinoma cells. Methods RT-PCR and Western blotting were employed to detect the expressions of PTTG mRNA and protein in human GH3 prolaetinoma cells. Different concentrations of estradiol (E2) or 4-hydroxytamoxifen (OHTam) were addedl into the hormone-depleted medium, and the viable cell number and expression levels of PTTG mRNA and protein were measured. Results The growth of OH3 prolaetinoma cells was significantly inhibited in hormone-depleted medium. E2 at a concentration of 1×10<'-8> mol/L obviously promoted the cell growth, the effect of which was inhibited by the application of OHTam (1×10<'-6> mol/L) to cause slowed cell growth. The expressions of PTTG at both the mRNA and protein levels were detected in detected in untreated GH3 prolactinoma cells, and OHTam at the concentration of 1×10<'-6> mol/L significantly inhibited their expressions. Conclusion The growth of GH3 cells is estrogen-dependant and can be inhibited by estrogen antagonist OHTam, which also results in reduced expression of PTTG gene in the cells.

OBJECTIVETo developed a method for the determination of thiabendazole residues in barbary wolfberry fruit by ion exchange chromatography (IEC) with fluorescence detection.

METHODThe samples were extracted with 0.1% H3PO4. Chromatographic analysis was performed on a Supelcosil LC-SCX (4.6 mm x 250 mm, 5 microm) column eluted with 0.1 mol x L(-1) KH2PO4 (pH 3.0)-acetonitrile (70:30) and detection at lamdaex = 307 nm and lamdaem = 359 nm.

RESULTThiabendazole in measured samples was separated completely. The calibration curve was linear at the range of 0.0005-0.02 mg x L(-1) with good precision and accuracy.

CONCLUSIONThe proposed method was satisfactorily applied to the analysis of thiabendazole residues in barbary wolfberry fruit.

Chromatography, Ion Exchange ; methods ; Fruit ; chemistry ; Lycium ; chemistry ; Reproducibility of Results ; Spectrometry, Fluorescence ; Thiabendazole ; analysis ; chemistry

OBJECTIVETo evaluate the safety and reliability of percutaneous internal fixation for pelvic ring injuries with cannulated screws.

METHODSForty-eight patients (21 male and 27 female, aged from 17 to 61 years with an average age of 38 years) with unstable pelvic ring injuries were treated with closed reduction and percutaneous cannulated screws fixation under C-arm fluoroscopic guidance. According to Tile's classification, the patients were classified into type B1 in 4 cases, B2.1 in 8, B2.2 in 10, B3 in 4, C1 in 11, C2 in 7 and C3 in 4. Among them, 39 patients were treated with anterior and posterior fixation, 4 were treated with anterior fixation, and 5 were treated with posterior fixation alone. Anteroposterior, inlet and outlet X-ray radiographs and CT scans of the pelvis were taken preoperatively to evaluate the stability and deformities, and after surgery the plain radiographs and CT scans were taken to evaluate the reduction and the location of screws.

RESULTSThe average operative time was 55 minutes (range, 15 to 95 minutes), and the average intraoperative blood loss was 60 ml (range, 15 to 150 ml), no patient accepted blood transfusion during or after operation. All 48 patients were inserted 157 cannulated screws (mean 3.3, range 2 to 8 per patient). Forty-two patients (135 screws) underwent postoperative pelvic CT scan and 91.11% (123 screws) of them was considered in optimal location; 7 screws penetrated the wall of pelvis and acetabulam because of overlength (<0.5 cm) or deviation, 5 screws interfered with the sacral canal or foramen. Fortunately, these 12 screws did not cause any symptom to the patients. The average follow-up period was 13 months (range 8 to 49 months), the displacement of injured pelvis was satisfactorily corrected in 45 patients (93.75%) and the fractures were healed at one stage. Among all patients, 40 cases (83.33%) had returned to their original works, 4 were still in the process of recovery at the last follow-up and the other 4 were unemployed as sciatic nerve injury or amputation. According to Lindahl improved standard of functional assessment of pelvic injury, the result was excellent in 35 cases, good 10 and fair 3, the average score was 78.7.

CONCLUSIONWith better understanding of the pelvic anatomy, and under C-arm fluoroscopic guidance, treatment of closed reduction and percutaneous cannulated screw internal fixation for unstable pelvic ring injuries is a safe, reliable and feasible method. The clinical outcome is satisfactory.

Adolescent ; Adult ; Bone Screws ; Female ; Fracture Fixation, Internal ; methods ; Humans ; Male ; Middle Aged ; Minimally Invasive Surgical Procedures ; methods ; Pelvic Bones ; injuries

OBJECTIVETo investigate the biological behaviors and chemosensitivity of non-small cell lung cancer (NSCLC) cell line A549 after IGF-IR gene silencing by RNA interference (RNAi) in vitro.

METHODSTwo plasmids siRNA 1 and 2 expressing IGF-IR siRNA with human U6 promoter were constructed,and an unrelated siRNA was used as negative control. NSCLC A549 cells were transfected with sequence-specific siRNA or unrelated siRNA as control. Quantitative RT-PCR and Western blot were used to detect the expression of IGF-IR. NSCLC A549 cells were transfected with siRNA and treated with DDP. MTT assay and flow cytometry were used to assess the effects of IGF-IR silencing on tumor cell proliferation and chemosensitivity.

RESULTTransfection of NSCLC cells with siRNA resulted in reduction of IGF-IR mRNA expression by 78.9 % and protein production by 89.8%. The decrease in IGF-IR levels caused significant growth inhibition of A549 cells both at 48 h and at 72 h, and decrease of the IC50 of DDP at 24 h, 48 h and at 72 h. Flow cytometry showed that 77.5% of A549 cells retained in G0/G1 phase.

CONCLUSIONThe sequence specific suppression of IGF-IR gene expression by RNAi enhances sensitivity to DDP in NSCLC cell.

Carcinoma, Non-Small-Cell Lung ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Gene Silencing ; Humans ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Plasmids ; genetics ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Receptor, IGF Type 1 ; metabolism ; Transfection

According to previous studies of SARS-CoV (Severe acute respiratory syndrome coronavirus), a variety of novel accessory genes have been identified in SARS-CoV genome, which were interspersed the structural genes of SARS-CoV and considered to be unique to the SARS-CoV genome. The predicted unknown proteins (PUPs) encoded by the accessory genes might play important roles in the SARS-CoV infection. Three of those genes, called X4, X5 and ORF10, were synthesized and introduced into E. coli to induce expression. SDS-PAGE and Western blotting revealed that the three genes have been expressed in E. coli. The induction of SARS PUPs genes expression in different temperatures, induction times, IPTG concentrations and A values of E. coli cells were performed. The optimal induction condition of SARS-CoV PUPs genes was characterized according to the orthorgonal analysis. The ratio of recombinant proteins of PUPs to total proteins is as follows: X4, 20%; X5, 27.8%; ORF10, 68.5% under the optimum conditions.
Escherichia coli ; genetics ; metabolism ; Gene Expression Regulation, Viral ; Genes, Viral ; Genetic Vectors ; Genome, Viral ; Open Reading Frames ; Recombinant Proteins ; genetics ; metabolism ; SARS Virus ; genetics ; Temperature ; Time ; Viral Matrix Proteins ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism

Taxol is one of the most potent chemotherapeutic agents known, showing excellent activity against a range of cancers. In addition to anticancer, taxol has the effect of preventing graft arteriosclerosis, antiscaring formation and inhibiting angiogenesis. There are five possible routes to industrialize taxol production: isolation from the bark of the yew species, total synthesis, semisynthesis, tissue or cell culture, endophytic fungal fermentation and metabolism engineering. There are at least 14 genes related to the taxol biosynthesis had been cloned from yews and functionally expressed in different hosts. The combinational expression system of taxol makes progress as the clarification of biosynthetic pathway of taxol.
Antineoplastic Agents, Phytogenic ; biosynthesis ; chemical synthesis ; chemistry ; isolation & purification ; Arteriosclerosis ; prevention & control ; Genes, Plant ; Genetic Engineering ; Neoplasms ; drug therapy ; Paclitaxel ; biosynthesis ; chemical synthesis ; chemistry ; isolation & purification ; Taxoids ; chemical synthesis ; chemistry ; Taxus ; chemistry ; genetics