To establish single- and double-transfected transgenic cells stably expressing hMATE1, hMATE1 cDNA was cloned by RT-PCR from human cryopreserved kidney tissue, and subcloned into pcDNA3.1(+) plasmid by virtue of both HindIII and Kpn I restriction enzyme sites. Subsequently, the recombined pcDNA3.1(+)- hMATE1 plasmid was transfected into MDCK, MDCK-hOCT1 or MDCK-hOCT2 cells using Lipofectamine 2000 Reagent. After a 14-day-cultivation with hygromycin B at the concentration of 400 µg · mL(-1), all clones were screened with DAPI and MPP+ as substrates to identify the best candidate. The mRNA content of hMATE1, the cellular accumulation of metformin with or without cimetidine as inhibitor, or transportation of cimetidine was further valuated. The results showed that all of the three cell models over expressed hMATE1 mRNA. The cellular accumulation of metformin in MDCK-hMATE1 was 17.6 folds of the control cell, which was significantly inhibited by 100 µmol · L(-1) cimetidine. The transcellular transport parameter net efflux ratios of cimetidine across MDCK-hOCT1/hMATE1 and MDCK-hOCT2/hMATE1 monolayer were 17.5 and 3.65, respectively. In conclusion, cell models with good hMATE1 function have been established successfully, which can be applied to study the drug transport or drug-drug interaction involving hMATE1 alone or together with hOCT1/2 in vitro.
Animals ; Biological Transport ; Cimetidine ; pharmacology ; DNA, Complementary ; Dogs ; Drug Interactions ; Humans ; Madin Darby Canine Kidney Cells ; Metformin ; pharmacology ; Organic Cation Transport Proteins ; genetics ; metabolism ; Transfection

In this assay, the reaction kinetics, optimum temperature, pH and various influential factors of ATP microbial rapid detection reagent by bioluminescence were studied. The results showed that it's enough for detection system to have 40 ~ 50?g/mL D-Luciferin. The light production decreased fastest in the first minute of reaction, then began to decay slowly. The optimal reaction temperature was 24℃~25℃and the optimal pH was pH 7.2 -7.4 in the reaction system. In addition, when stored at 4℃for 45h, the dissolved reagent solution could keep its 86% activity. When preserved at 25℃, the enzyme activity decreased less for 1h, and degraded gradually as time went by and only left 53. 5% of its activity after 6. 5h. While stored at 33℃, the enzyme activity decreased quickly with the time and only left 59. 1% after 1. 5h. The result indicated that storage temperature was a very important influential factor to the activity of reagent Meanwhile, different chemical substance such as acid, alkali, salt and surfactants inhibited the ATP bioluminescent reaction. When the concentration of NaCl reached 1. 5g/L, it could inhibit 52. 5% light production. Triton X-100, acid, and alkali also had some effects on the reaction, while CTAB, SDS and TCA would inhibit the bioluminescent reaction seriously.

The interaction between Gliotoxin and bovine serum albumin (BSA) was studied by the fluo-rescence, Circular Dichroism (CD) and ultraviolet visible (UV-Vis) techniques. The fluorescent experiment showed that the intrinsic fluorescence of BSA was quenched by the binding of gliotoxin in a static quenching procedure, with an association constant of 7.2?103 L/mol and in hydropobic forces. And the CD spectrum revealed that gliotoxin effected the conformation of BSA by increased the mass of ?-helix.

BACKGROUND AND OBJECTIVEAs chemotherapy is generally used in the clinical treatment of cancer, the problem of multidrug resistance (MDR) of tumors against the chemotherapeutic agents becomes more and more serious. It has been the major cause for the failure of the chemotherapy. We detected the expressions of beta-catenin and tumor drug resistance related proteins, MRP2, P-gp, and Bcl-2, in esophageal squamous cell carcinoma (ESCC) to explore their function and correlation in the occurrence and development of MDR in ESCC.

METHODSWe used the tissue microarray technique, immunohistochemistry, and image analysis methods to detect the expressions of MRP2, P-gp, beta-catenin, and Bcl-2 proteins and analyze their relationships with clinical data in a ESCC tissue microarray consisting of 582 specimens of ESCC, 294 specimens of normal mucosa, 92 specimens of basal cell hyperplasia, and 87 specimens of dysplasia adjacent to cancer tissue.

RESULTSThe integral optical density (IOD) of MRP2 and Bcl-2, which was 195.7 +/- 175.9 x 10(3)) and 90.5 +/- 112.5 x 10(3)), respectively, was significantly higher in ESCC than in normal mucosa, which was 104.8 +/- 86.1 x103) and 25.2 +/- 46.6 x 10(3)), respectively (P < 0.01). The IOD of P-gp and beta-catenin, which was 57.7 +/- 75.5 x 10(3)) and 32.0 +/- 47.0 x 10(3)) respectively, was significantly lower in ESCC than in normal mucosa, which was 114.8 +/- 106.6 x 10(3)) and 46.1 +/- 35.7 x 10(3)), respectively (P < 0.01). According to the following order, well differentiated moderately differentiated poorly differentiated, the IOD of MRP2 increased in ESCC (P < 0.01). The IOD of beta-catenin was higher in poorly differentiated ESCC than in well or moderately differentiated ESCC (P < 0.01). The IOD of Bcl-2 was lower in well differentiated ESCC than in poorly and moderately differentiated ESCC (P < 0.01). The IOD of beta-catenin and Bcl-2 was higher in the ESCC of specimens with infiltration depths that were in membrane mucosa than those in the muscular layer or serous coat (P < 0.01). The IOD of Bcl-2 was significantly higher in cases with lymph node metastasis than in cases without (P < 0.01). Positive correlations which were respectively between the expressions of P-gp and MRP2, the expressions of P-gp and Bcl-2 were found (r = 0.288 and r = 0.253, respectively, P < 0.01).

CONCLUSIONSMRP2, P-gp, and Bcl-2 may be taken as prognostic factors for MDR of ESCC. beta-catenin may play an important role in carcinogenesis and progression of ESCC.

ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Adult ; Aged ; Aged, 80 and over ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Differentiation ; Esophageal Neoplasms ; metabolism ; pathology ; Female ; Humans ; Lymphatic Metastasis ; Male ; Middle Aged ; Multidrug Resistance-Associated Proteins ; metabolism ; Neoplasm Invasiveness ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Young Adult ; beta Catenin ; metabolism

Background and Objective:As chemotherapy is generally used in the clinical treatment of cancer,the problem of multidrug resistance(MDR)of tumors against the chemotherapeutic agents becomes more and more serious.It has been the major cause for the failure of the chemotherapy.We detected the expressions of β-catenin and tumor drug resistance related proteins,MRP2,P-gp,and Bcl-2,in esophageal squamous cell carcinoma (ESCC)to explore their function and correlation in the occurrence and development of MDR in ESCC.Methods:We used the tissue microarray technique,immunohistochemistry,and image analysis methods to detect the expressions of MRP2,P-gp,β-catenin,and Bcl-2 proteins and analyze their relationships with clinical data in a ESCC tissue microarray consisting of 582 specimens of ESCC,294 specimens of normal mucosa,92 specimens of basal cell hyperplasia,and 87 specimens of dysplasia adjacent to cancer tissue.Results:The integral optical density(IOD)of MRP2 and Bcl-2,which was 195.7±175.9(×10~3)and 90.5±112.5(×10~3),respectively,was significantly higher in ESCC than in normal mucosa.which was 1 04.8±86.1(×10~3)and 25.2±46.6(×10~3),respectively(P＜0.01).The IOD of P-gp and β-catenin,which was 57.7±75.5(×10~3)and 32.0±47.0(×10~3)respectively,was significantly lower in ESCC than in normal mucosa,which was 114.8±106.6(×10~3)and 46.1±35.7(×10~3),respectively(P＜0.01).According to the following order.well differentiated-moderately differentiated-poorly differentiated.the 1OD of M RP2 increased in ESCC(P＜0.01).The IOD of β-catenin was higher in poorly differentiated ESCC than in well or moderately differentiated ESCC(P＜0.01).The IOD of Bcl-2 was lower in well differentiated ESCC than in poorly and moderately differentiated ESCC(P＜0.01).The IOD of β-catenin and Bcl-2 was higher in the ESCC of specimens with infiltration depths that were in membrane mucosa than those in the muscular layer or serous coat(P＜0.01).The IOD of Bcl-2 was significantly higher in cases with Iymph node metastasis than in cases without (P＜0.01).Positive correlations which were respectively between the expressions of P-gp and MRP2,the expressions of P-gp and Bcl-2 were found(r=0.288 and r=0.253.respectively,P＜0.01).Conclusions:MRP2,P-gp,and Bcl-2 may be taken as prognostic factors for MDR of ESCC.β-catenin may play an important role in carcinogenesis and progression of ESCC.

Objective To explore the relationship between heart rate variability (HRV) and deceleration capacity (DC) in children with idiopathic ventricular premature contraction of different origins. Methods The clinical data from 155 children with idiopathic ventricular premature contraction were retrospectively analyzed. According to the age, the children were divided into young children group (

Alveolar epithelial type II (AT II) cells are essential for lung development and remodeling, as they are precursors for type I cells and also produce other non-repair cells (fibroblasts). Progenitor cells are believed to possess capability of multi-potent transdifferentiation, which is closely related to the niche, suggesting the importance of establishment of a lung progenitor cell niche model. We hypothesized that pulmonary surfactant-associated protein A (SPA) suicide gene system would cause AT II cell to kill itself through apoptosis and leave its niche. In vitro, the recombinant adeno-associated virus vectors-SPA-thymidine kinase (rAAV-SPA-TK) system was established to get targeted apoptotic AT II cells. The apoptosis of AT II cells was detected by using MTT. The results showed that cloned SPA gene promoter had specific transcriptional activity in SPA high expression cells, and SPA high expression cells (H441) transfected with TK gene had higher sensitivity to ganciclovir (GCV) than SPA low expression cells (A549). In vivo, increased apoptosis of AT II cells induced by GCV in rAAV-SPA-TK system was observed by TUNEL. Finally, the successful packaging and application of rAAV-SPA-TK system provide experimental basis to get specific lung progenitor cell (AT II) niche in vitro and in vivo.

The cDNA encoding human Vascular Endothelial Growth Factor 165 (VEGF165) was amplified using RT-PCR from human tonsil tissue and cloned into eukaryotic expression vector pcDNA3.1 (+). The recombinant plasmid pcDNA/V was transferred into 293 cells mediated by liposome and the cells stably expressing VEGF were selected under the pressure of G418. ELISA and Western blotting demonstrated that the eukaryotic expression vector pcDNA/V was successfully constructed and its corresponding protein could be expressed efficiently in vitro. Chick Charioallantoic Membrane (CAM) bioassay showed that recombinant protein has biological activity of hVEGF. Model rats with acute myocardial ischemia were used to further study the expression of VEGFin vivo. The model rats were divided randomly into three groups: control group, pcDNA3.1 (+) group and pcDNA/V group. 50microL naked plasmid DNA or saline was intramyocardially injected at three sites into the border zone of infarction. The hearts of rats were excised and fixed histologically, then the infarction sizes were studied by immunohistochemical staining and electron microscope after four weeks. Immunohistochemical staining for VEGF appeared to be negative in control and pcDNA3.1 (+) groups. In pcDNA/V group, myocardial cells in infarction border zone showed positive staining for VEGF in cytoplasm. Ultrastructural anaylsis showed that there were visible hyperplasia of vascular endothilium in pcDNA/V group. The control and pcDNA3.1 (+) groups showed less capillary hyperplasia. In this study, VEGF165 gene was successfully cloned and its protein expressed in vitro and in vivo was of bioactivity, which provides a basis for the further study of biological functions of human VEGF.
Animals ; Cell Line ; Chickens ; Chorioallantoic Membrane ; blood supply ; Disease Models, Animal ; Genetic Therapy ; Humans ; Male ; Myocardial Infarction ; metabolism ; pathology ; therapy ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; biosynthesis ; genetics ; therapeutic use ; Transfection ; Vascular Endothelial Growth Factor A ; biosynthesis ; genetics

OBJECTIVETo evaluate the outcomes and factors related to weight loss after laparoscopic gastric bypass(LGBP) in obese patients.

METHODSForty-one obese patients who underwent LGBP from May 2010 to December 2011 in the First Affiliated Hospital of Nanjing Medical University were followed up. The operative time, intraoperative complications, postoperative complications, preoperative body mass index(BMI), postoperative BMI, and excess weight loss rate(EWL) were determined and their correlation with efficacy were analyzed.

RESULTSAll the surgeries were successful without conversions or perioperative deaths. The average operative time was (229±96)min, intraoperative blood loss was(15±3) ml, postoperative hospital stay was(5.7±1.7) d. Patients were followed up for 3-12 months. The average EWL at 1, 3, 6, 9, and 12 months after operation was 24.2%, 45.6%, 60.1%, 66.5% and 69.0%. The EWL was negatively correlated with preoperative BMI(P<0.01), but not correlated with age, gender, and waist-hip ratio(all P>0.05). Postoperative short-term EWL did not differ between central obesity patients and peripheral obesity patients, and before and after standardized treatment(both P>0.05). After standardization, however, operative time and postoperative hospital stay were significantly reduced(P<0.01).

CONCLUSIONSLGBP is an effective and feasible treatment for obesity patients. Short-term efficacy after surgery is negatively correlated with preoperative BMI. Standardization may reduce operative time and postoperative hospital stay, but not associated with improved short-term outcomes.

Adolescent ; Adult ; Female ; Gastric Bypass ; methods ; Humans ; Laparoscopy ; Male ; Middle Aged ; Obesity, Morbid ; surgery ; Retrospective Studies ; Treatment Outcome ; Young Adult

OBJECTIVETo discuss the mechanism of traditional Chinese medicines reducing phlegm and resolving masses in treatment of iodine deficiency-induced goiter by observing the expression of growth factors and the balance-regulating mechanism of proliferation and apoptosis.

METHOD180 four-week-old Wistar rats were selected to establish the iodine deficiency model. After the modeling, the rats were randomly divided into six groups: the normal control group, the model control group, the iodine group, the phlegm compound group, the L-T4 group and the phlegm compound and L-T4 group. At the 21st day and 77th day after administration, 15 rats in each group were killed to collect specimens. Doses were calculated and adjusted according to body surface area and body weight. TT3, TT4 radioimmunoassay, TSH, immunoradiometric method were adopted. Fas, FasL and PCNA protein expressions are detected using immunohistochemical methods.

RESULTCompared with the normal group and the model group, the expressions of fas and FasL in the phlegm Group significantly increased, the expressions of fas and FasL in the phlegm and L-T4 group were also increased significantly. The expression of fas in the L-T4 Group was significantly lower than that of the L-T4 group and the phlegm compound and L-T4 group. Compared with the normal group, the expression of PCNA of the phlegm group and the phlegm and L-T4 group was significantly lower. Compared with the model group, the expression of PCNA of the iodine group, the phlegm groups and the phlegm and L-T4 group were significantly lower. Compared with the normal group, the expression of VEGF in the iodine group significantly decreased after treatment. Compared with the iodine group, the expression of VEGF in the phlegm group and the L-T4 group significantly reduced. Compared with the normal group, the expression of TGF-beta1 in the model group and the phlegm group significantly increased. Compared with model group, the expression of TGF-beta1 in the iodine group significantly reduced. Compared with the phlegm group, the expression of TGF-beta1 in the phlegm compound and L-T4 group was significantly reduced.

CONCLUSIONTraditional Chinese medicines reducing phlegm and resolving masses can completely recover goiter by promoting apoptosis of thyroid cells, inhibiting their proliferation and the expression of growth factors and enhancing the expression of TGF-beta, without causing injury on thyroid cells.

Animals ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Gene Expression ; drug effects ; Goiter ; drug therapy ; genetics ; metabolism ; Humans ; Male ; Proliferating Cell Nuclear Antigen ; genetics ; metabolism ; Rats ; Rats, Wistar ; Thyroid Hormones ; secretion ; Vascular Endothelial Growth Factor A ; genetics ; metabolism