OBJECTIVETo investigate the effect of glycogen synthase kinase-3beta (GSK-3beta) on the proliferation of human ovarian cancer cells.

METHODSTwo human ovarian cancer cell lines SKOV3 and ES-2 were analysed for the expression of GSK-3beta and phosphorylated GSK-3beta (pGSK-3beta) by Western blot analysis. Cell growth curve analysis done by cell count was used to investigate the effect of GSK-3beta inhibitors on the growth of SKOV3 and ES-2 cells. Four plasmids, namely, GSK-3betaS9A, GID5-6, GID5-6LP and the control vector, were cotransfected respectively with the green fluorescent protein (GFP) into SKOV3 cells by electroporation, and then BrdU incorporation assay was adopted to analyse the role of GSK-3beta activity in the proliferation of ovarian cancer cells. After transfection, G418 was added to the medium to select those stably transfected cells, which were used to investigate the long term effect of GSK-3beta activity change on the proliferation of ovarian cancer cells by colony formation assay.

RESULTSBoth SKOV3 and ES-2 cells expressed GSK-3beta, though the expression level of pGSK-3beta was lower in SKOV3 than in ES-2 cells. GSK-3beta inhibitors attenuated the growth of SKOV3 and ES-2 cells. Transfection with GSK-3betaS9A to upregulate the GSK-3beta activity resulted in the increase of BrdU incorporation in SKOV3 cells compared with that in the control vector. On the contrary, transfection with GID5-6 to downregulate GSK-3beta activity decreased the BrdU incorporation in SKOV3 cells, compared with that in GID5-6LP, which is a control vector of GID5-6. Stable transfection with GSK-3betaS9A increased the colony number while stable transfection with GID5-6 decreased the colony number, compared with each control vector.

CONCLUSIONGSK-3beta can promote the proliferation of ovarian cancer cells. Inhibition of GSK-3 p may become a potential theraputic

Blotting, Western ; Cell Line, Tumor ; Cell Proliferation ; Female ; Glycogen Synthase Kinase 3 ; antagonists & inhibitors ; genetics ; metabolism ; Glycogen Synthase Kinase 3 beta ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Indoles ; pharmacology ; Lithium Chloride ; pharmacology ; Maleimides ; pharmacology ; Microscopy, Fluorescence ; Ovarian Neoplasms ; enzymology ; genetics ; pathology ; Phosphorylation ; drug effects ; Plasmids ; genetics ; Serine ; genetics ; metabolism ; Time Factors ; Transfection ; beta Catenin ; metabolism

OBJECTIVETo construct the delta-pIRES2-EGFP plasmid and investigate its expression in HEK293 cells.

METHODSFull length cDNA of rat delta opioid receptor gene amplified from rat brain tissues using reverse transcription and nested PCR was cloned into pMD20 T vector. The delta cDNA was inserted into pIRES2-EGFP plasmid to construct the recombinant eukaryotic plasmid delta-pIRES2-EGFP, which was transfected into HEK293 cells via Lipofectamine2000. The expression of delta was examined under fluorescence microscope.

RESULTSThe recombinant delta-pIRES2-EGFP plasmid was successfully constructed, and high expression of delta was detected in HEK293 cells transfected by the plasmid.

CONCLUSIONdelta-pIRES2-EGFP has been successfully cloned, which shows high expression of delta in HEK293 cells.

Animals ; DNA, Complementary ; genetics ; Gene Expression ; Genetic Vectors ; Green Fluorescent Proteins ; biosynthesis ; genetics ; HEK293 Cells ; Humans ; Plasmids ; Polymerase Chain Reaction ; Rats ; Rats, Sprague-Dawley ; Receptors, Opioid, delta ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection

Objective To investigate the occurrence and risk factors of vascular access infection(VAI) in hemodialysis outpatients.Methods Prospective surveillance method,monitoring methods was formulated and adopted by referring to the relevant guidelines and regulations at home and abroad,targeted surveillance was performed among all outpatients receiving hemodialysis in a hospital from June 1,2014 to May 31,2016.Results A total of 584 outpatients received hemodialysis from June 1,2014 to May 31,2016,with 64 203 times of vascular access,79 patients developed 85 cases of infection,case incidence of VAI was 1.32％.36 cases(42.35％) were infection at vascular puncture sites,49 (57.65％) were vascular access-related bloodstream infection.Among patients with different types of vascular access,incidence of VAI was the highest among patients with artificial vascular graft(19.67％),followed by those with non tunneled central venous catheter(4.91％),with tunneled central venous catheter (0.73％),and with arteriovenous fistula(0.09％).Age＞ 60 years,hemodialysis time＞ 1 year,diabetes,and hypertension were risk factors for VAI in outpatients with hemodialysis(all P＜0.05).39 strains of pathogens were isolated from 49 patients with vascular access-related bloodstream infection,including 36 (92.31％) gram positive bacteria,mainly Staphylococcus aureus (n =30,6 of which were methicillin-resistant Staphylococcus aureus);3 (7.69 ％) gram-negative bacteria.Conclusion Strengthening prospective targeted surveillance can better understand the status,characteristics,and risk factors of VAI in hemodialysis outpatients,it is conducive to taking targeted prevention and control measures,thus reduce the incidence of VAI in hemodialysis outpatients.

The recombined adenovirus DNA was transfected into 293 cells for packing and amplification of replication-deficient Ad-CMV-E6/E7, Ad-K14 -E6/E7 virus was purified by CsCl density gradient centrifugation , recombined adenovirus Ad-CMV-E6/E7, Ad-K14 -E6/E7 were used as experimental group, while pAd-CMV and pAdtrack-K14 were used as control group. Four of them were injected through one main vein of nude mice tail respectively. These mice were then treated with 0.05 mg 17beta-estradiol over 12 weeks. Mice were anaesthesiaed with 2.5% Avertint and the vagina, mammary gland, ovaries and uterus were dissected and fixed in 3.75% paraformaldehyde overnight at 4 degrees C. Paraffin-embedded sections, HE staining and identification of P53 and Bcl-2 protein via immunohistochemistry were performed. The expression of E6/E7 was verified by RT-PCR in different tissue of nude mice. HE staining showed evident hyperplasy in cervix-uterus transformation zone of experimental group 2. The expression of mutant P53 and Bcl-2 were higher than control group via immunohistochemical S-P method in uterus stroma-cell. Western blotting also showed that E6 protein was expressed. The expression of E6/E7 was higher than control group by human cytokeratin promoter 14 and hyperlasy changes were detected in epithelial tissue of cervix-uterus transformation zone.
Adenoviridae ; genetics ; Animals ; Blotting, Western ; Cell Line ; Female ; Genital Diseases, Female ; pathology ; virology ; Genitalia, Female ; pathology ; virology ; Humans ; Immunohistochemistry ; Mammary Glands, Animal ; metabolism ; pathology ; Mice ; Mice, Nude ; Oncogene Proteins, Viral ; genetics ; metabolism ; Ovary ; metabolism ; pathology ; Papillomaviridae ; metabolism ; physiology ; Papillomavirus E7 Proteins ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Repressor Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Suppressor Protein p53 ; metabolism ; Uterus ; metabolism ; pathology ; Vagina ; metabolism ; pathology

OBJECTIVETo improve Madigan prostatectomy (MPC) for a much satisfactory effect in open surgery.

METHODSA total of 52 patients with benign prostatic hyperplasia (BPH) were treated using MPC. The MPC procedure was modified by exposing anterior prostatic urethra near the bladder neck and conjunction with cystotomy. This modified procedure preserved prostatic urethra intact and could also deal with intracystic lesions at the same time.

RESULTSThe intact of prostatic urethra was kept completely or almost for 48 cases. The hemorrhage amount during modified procedure was a less. The mean operative time was 120 minutes. The 35 patients had been followed up for 1 - 12 months. The average Qmax was 18.9 ml/s. The cystourethrography revealed that the urethra and bladder neck were intact in 8 patients postoperatively. Furthermore, the prostatic urethra was obviously wider after modified MPC.

CONCLUSIONSThe modified MPC can reduce the urethra injury and enlarge the MPC indications. The modified technique is easy to perform with little complications and much more satisfactory clinical result. The modified MPC is highly recommended.

Aged ; Humans ; Male ; Middle Aged ; Prostatectomy ; methods ; Prostatic Hyperplasia ; surgery

Objective To observe the effect of changing grain and selenium supplementation for 1-year on control of children's Kaschin-Beck disease in Qinghai province. Methods Epidemiology, clinical and right-hand X-ray examination were carried out on children aged 7 - 12 years in 2008. Patients were diagnosed and divided into 3 groups by village, control group from Xinjianping village in Guide county, changing grain group from Xiemalang village in Guide county and supplying salt with selenium and iodine group from Shanglujuan and Xialujuan villages in Xinghai county. One year before and after the treatment, right-hand X-ray photograph (including carpal bones)was taken and child hair samples were collected, selenium was detected by 2,3-diaminonaphthalene fluorescence spectrophotometry. Results After 1 year prevention and control, the detectable rate of X-ray in control group was raised from 4.88%(2/41) to 12.20%(5/41) , the detection rate in changing grain group was declined from 17.54%(10/57) to 5.26%(3/57), and from 13.51%(10/74) to 5.41%(4/74) in supplying salt with selenium and iodine group. In changing grain group, there were 10 patients, 7 cases were cured, 2 patients stable, 1 case progressed,no new case;in supplying salt with selenium and iodine group of 10 patients, 7 were cured, 3 patients stable, 1 new diagnosed case;in control group, 2 patients stable, 2 new diagnosed metaphysis cases, 1 new diagnosed metaphyseal case. Compared with control group, the difference was statistically significant between changing grain group and supplying salt with selenium and iodine group(x2 = 5.49,4.14, all P ＜ 0.05). After 1 year control and prevention,hair selenium contents in control group and changing grain group were increased from (107.15 ± 42.30), (125.30 ±40.30)μg/kg to (108.32 ± 35.67), (135.38 ± 65.24)μg/kg, the difference was statistically insignificant(t = 0.01,0.68, all P ＞ 0.05), and selenium contents in supplying salt with selenium and iodine group were obviously increased from (95.62 ± 43.42)μg/kg to (197.64 ± 97.08)μg/kg (t = 5.41, P ＜ 0.05). Conclusion Changing grain and supplying selenium can prevent and control children's Kaschin-Beck disease.

OBJECTIVETo inhibit the expression of connexin43 (Cx43) in the human corpus cavernosum penis smooth muscle cells by small interfering RNA (siRNA) and detect the gap junction intercellular communication (GJIC), and to investigate the application of siRNA technology in the gap junction of corpus cavernosum penis smooth muscle cells and its role in the penile erection process.

METHODSWith the help of the software of Ambion Corporation, the specific recombinant plasmids with siRNA targeting human Cx43 gene were constructed. The recombinant plasmids having been stably transferred into human corpus cavernosum penis smooth muscle cells for 48 hours, semi-quantitive reverse transcription polymerase chain reaction (RT-PCR) and Western blotting techniques were used to examine the inhibitory effects of siRNA on the expressions of the Cx43 gene and protein, in comparison with the siRNA negative control and the blank control group, respectively. The GJIC was detected by scrape-loading and fluorescence dye transfer experiments through the fluorescence microscope.

RESULTSThe results of enzyme digestion analysis and DNA sequencing showed that the recombinant plasmid pSilencer 1.0-U6-siRNA-Cx43 was successfully constructed. The relative levels of Cx43 mRNA and protein expression in the smooth muscle cells were (0.45 +/- 0.08)% and (0.56 +/- 0.06)% after successful transfer of the recombinant plasmid. However, the expression levels of mRNA and protein were (0.72 +/- 0.04)% and (0.80 +/- 0.08)% in the negative siRNA transfer group, and (0.74 +/- 0.09)% and (0.77 +/- 0.11)% in the blank control, respectively, with a significant difference (P < 0.05). The GJIC also decreased significantly.

CONCLUSIONsiRNA can significantly inhibit the expression of Cx43 and block the GJIC in the human corpus cavernosum penis smooth muscle cells. siRNA technology plays an important role in penile erection and flaccidity.

Blotting, Northern ; Cells, Cultured ; Connexin 43 ; biosynthesis ; genetics ; Humans ; Intercellular Junctions ; Male ; Myocytes, Smooth Muscle ; physiology ; Penis ; cytology ; metabolism ; RNA, Small Interfering ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

Because c-Src and iNOS are key regulatory enzymes in tumorigenesis, a new series of 4-heterocycle amine-3-quinolinecarbonitriles as potent dual inhibitors of both enzymes were designed, synthesized and evaluated as multiple targets agents in cancer therapy. All compounds were evaluated by two related enzyme inhibition assays and an anti-proliferation assay in vitro. The results showed that most compounds inhibited c-Src and iNOS well. The best compound 33 inhibited both enzymes with the IC50 values of 0.0484 micromol x L(-1) and 34.5 micromol x (-1), respectively. Some of the compounds also showed moderate anti-proliferation activities at 10 micromol x L(-1) against colon cancer HT-29 and liver cancer HepG2 cell lines.
Aniline Compounds ; chemical synthesis ; chemistry ; pharmacology ; Antineoplastic Agents ; chemical synthesis ; chemistry ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Drug Delivery Systems ; Drug Design ; Humans ; Nitric Oxide Synthase Type II ; antagonists & inhibitors ; metabolism ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; metabolism ; Quinolines ; chemical synthesis ; chemistry ; pharmacology ; src-Family Kinases

UNLABELLEDA Special "Fever and Cough" Clinic was set up at the Children's Hospital Affiliated to Capital Institute of Pediatrics for children with symptoms of fever and cough in late April when the severe acute respiratory syndrome (SARS) epidemic was at its peak in Beijing to separate the children with fever from others during their visit to the Outpatient Department.

OBJECTIVEFor patients with fever, normal or low count of white blood cell and with suspected pneumonia suggested by X-ray, it was urgent to determine the etiological agents of the diseases before they were admitted to the hospital.

METHODSThroat swabs or nasopharyngeal aspirate specimens were collected from those patients and common respiratory virus antigens including influenza virus A and B, respiratory syncytial virus, adenovirus, parainfluenza virus types I, II, and III were tested by indirect immunofluorescent assay. The patients with atypical pneumonia diagnosed by X-ray and evidences of common respiratory virus infection were admitted to the regular ward for children with respiratory diseases. Children with pneumonia demonstrated by X-ray and negative for common respiratory viruses were admitted to the isolated ward for suspected SARS patients for the first step and further viral etiological studies were requested. RT-PCR was performed for those patients to detect gene fragments of human metapneumovirus (HMPV), rhinovirus (RhV) and enterovirus (EV) in their specimens. Nested RT-PCR was also developed to detect SARS coronavirus gene fragment from the specimens. Primer sequences for SARS virus detection with the PCR were selected according to the primer sequences published online by WHO on April 18, 2003. All the primers derived from the sequence at the 1b frame of coronavirus replicase gene and products with a size of 368 or 348 bp were expected with 2 different primer pairs.

RESULTSAmplicons with the sizes of 368 bp and 348 bp were obtained from a throat swab specimen collected from a 17 years old girl, who was admitted to the isolated ward because of high fever (39.5 degrees C) for 7 days, cough for 2 days, low WBC count, and pneumonia shown by X-ray when she visited the "Fever and Cough" Clinic, and without known history of contact with probable SARS patient. Antigens for the common respiratory viruses were all negative, RT-PCR for HMPV, RhV and EV were also negative while RT-PCR with different primer pairs for SARS virus were all positive which indicated that SARS coronavirus gene fragments were amplified from the specimen from this girl. The amplified fragment with a size of 368 bp was sequenced and the sequence was compared with those in the GenBank. The sequence shared 100% homology with the sequences from 1b frame of replicase genes from all 17 of SARS coronaviruses published in the GenBank so far, and shared very low homology with 2 reference strains of human coronavirus as well as other animal coronaviruses. The serum collected before her discharge from the hospital (19 days after the onset of the disease) showed SARS specific IgM and IgG antibodies.

CONCLUSIONThese data indicate that the patient was a confirmed case of SARS. It is of great importance to differentiate SARS patients from those infected with common respiratory viruses during SARS epidemic, especially for pediatric patients, because most of the patients visiting the outpatient department present with the symptoms of fever, cough and normal WBC count. The data mentioned above indicate that antigen and gene detections for those common respiratory viruses are useful methods for the differentiation to avoid the spread of SARS.

Adolescent ; Amino Acid Sequence ; Antibodies, Viral ; analysis ; China ; Female ; Fluorescent Antibody Technique, Indirect ; Humans ; Molecular Sequence Data ; Reverse Transcriptase Polymerase Chain Reaction ; SARS Virus ; genetics ; immunology ; Sequence Analysis, DNA ; Sequence Homology, Amino Acid ; Severe Acute Respiratory Syndrome ; diagnosis ; virology

OBJECTIVETo evaluate the effects of two gastric cancer screening schemes for early detection of gastric cancer in a high-risk population.

METHODSA cluster random sampling method was used to select local residents aged 40-69 years from Linqu County, Shandong Province. "Serum pepsinogen initial screening combined with further endoscopic examination (PG scheme)" and "direct endoscopic examination (endoscopy scheme)" were conducted. The associations between screening schemes and detection rates of gastric cancer, and early gastric cancer/high-grade intraepithelial neoplasia were evaluated by unconditional logistic regression analysis.

RESULTSOverall, 3654 and 2290 participants completed PG and endoscopy schemes, respectively. A total of 11 (0.30%) cases of gastric cancer and 10 (0.27%) cases of high-grade intraepithelial neoplasia were detected by PG scheme, of which 7 (0.19%) cases were early gastric cancer. While, 19 (0.83%) cases of gastric cancer and 10 (0.44%) cases of high-grade intraepithelial neoplasia were detected by endoscopy scheme, with 12 (0.52%) cases of early gastric cancer. Compared with the PG scheme, the endoscopy scheme had a significantly higher detection rates of gastric cancer (OR = 2.83, 95%CI 1.34-5.98), and early gastric cancer/high-grade intraepithelial neoplasia (OR = 2.12, 95%CI 1.12-4.02).

CONCLUSIONSThe endoscopy scheme is more effective in the detection of gastric cancer in a high-risk population, particularly for early gastric cancer/high-grade intraepithelial neoplasia than the PG scheme.

Adult ; Aged ; Carcinoma ; blood ; diagnosis ; Carcinoma in Situ ; blood ; diagnosis ; Early Detection of Cancer ; methods ; Female ; Gastroscopy ; Humans ; Male ; Mass Screening ; methods ; Middle Aged ; Pepsinogen A ; blood ; Stomach Neoplasms ; blood ; diagnosis