Objective To observe the effect of lentivirus vector-medicated enhanced green fluorescence protein (EGFP) transfection on in vitro corneal epithelial cells.Methods Primary corneal epithelial cells of rabbits were cultured and transfected with lentivirus vector into different multiplicity of infection (MOI) in order to find the best transfection dose.After 24,48,72,and 96 h of transfection,expression of EGFP was observed under microscope and detected by RT-PCR.The transfection rate of corneal epithelial cells was calculated in different MOI.Results EGFP was expressed in corneal epithelial cells 48 h after transfection,which increased with the increasing transfection time.The transfection rate of corneal epithelial cells was 4.5%?0.6%,30.4%?0.9%,51.9%?1.4%,and 75.4%?0.7%,respectively,when MOI was 1,10,50,and 100(P

Objective To investigate the short-term outcome of laparoscopic adjustable gastric banding (LAGB) for morbid obesity complicated with type 2 diabetes. Methods Eight morbidly obese patients with type 2 diabetes underwent LAGB from October 2006 to August 2007. The weight parameters, fasting (FBG) and 2-hour blood glucose (2hBG), medication for diabetes were assessed 1,3, 6 and 9 months after surgery. Results All of the patients lost weight, with a mean body mass index decreased from (38.7±7.5) kg/m2 before LAGB to (30.5±4.3) kg/m2 9 months after LAGB. The FBG and 2hBG were decreased significantly at month 6 and 9 after LAGB, with normal FBG and 2hBG in 4 patients. At month 9 after LAGB, 3 of 5 patients with insulin treatment before LAGB were changed to oral hypoglycemics, 1 was continuously administered with a reduced dose of insulin, and 4 patients stopped any medication. Conclusion LAGB is an effective procedure in the treatment of morbid obesity complicated with type 2 diabetes with a favorable short-term outcome.

Objective To investigate the effects of aldosterone on the expression of endothelin(ET)in cultured neonatal rat cardiac fibroblasts(CFs).Methods CFs were isolated by trypsin digestion.ET concentration in conditioned medium was measured by radioimmunoassay,intracellular ET-1 level was evaluated by immunofluorescence assay,and the expression of preproendothelin-1(ppET-1)was detected using reverse transcriptase-polymerase chain reaction(RT-PCR)method.Results Aldosterone(10-9,10-8,10-7 mol/L)induced a dose-dependent changes in ppET-1 mRNA expression,as well as ET-1 synthesis and secretion in CFs.Meanwhile,aldosterone(10-7 mol/L)time-related induced ppET-1 mRNA expression in CFs,which began to increase in 2 h and reached the highest level in 4 h,thereafter decreased.The effects of aldosterone(10-7 mol/L)were significantly inhibited by the pre-incubation with spironolactone(10-6 mol/L).Conclusion Aldosterone increases the expression of ppET-1 mRNA,ET-1 synthesis and secretion via mineralocorticoid receptor.

Objective To evaluate the effect of laparoscopic adjustable gastric banding(LAGB) in patients with obesity and obesity-related comorbidities.Methods From Oct.2006 to Dec.2007,50 morbidly obese patients including 11 cases with type 2 diabetes,3 with hypertension,15 with hyperlipidemia,28 with fatty liver,1 with obstructive sleep-apnea syndrome and 2 cases with gallstones underwent LAGB.The mean follow-up period for these patients was 11.2 months.ranging from 6 to 18 months.The weight loss,obesity-related comorbidities,outcomes and complications were evaluated.Results Mean BMI decreased significantly from preoperative(39±6)kg/m2 to postoperative(31±4)kg/m2,(28±7)kg/m2 and(27±7)kg/m2 respectively at 9,12 and 18 months(P＜0.05).The mean excess weight loss at 9,12 and 18 months postoperatively was 30%±11%、42%±13%and 45%±13% respectively.At 12 and 18 months,respectively,20%and 44%of patients had＞50%excess weight loss.The obesity-related comorbidities resolved or improved in 66%～100%of the patients at 12 and 18 months postoperatively.Complications occurred in 4 cases,among them 3 cases were cured conservatively and in 1 case reoperation was performed. Conclusions Based on short-term follow-up results,LAGB is a safe,effective and feasible technique in the treatment of patients with morbid obesity and obesity-related comorbidities.

Pulldown assay is an in vitro method for studies of protein-protein interactions, in which tagged proteins are usually expressed as the bait to enrich other proteins that could bind to them. In this technology, the GST tag is broadest used for its modest size and hydrophilic property. In most cases, the GST tag could increase the hydrophility of the fusion protein and help to avoid the formation of inclusion bodies. However, in the other few cases, the target protein may be strongly hydrophobic or have complicated structures that were hard to fold and assemble in correct conformations without champerons, and even the existence of GST tag could not make them soluble. These proteins were always expressed as inclusion bodies and had no functions. LMO2 was a small molecular weight and insoluble protein, in this study, GST system and MBP system were used to express GST-LMO2 and MBP-LMO2 fusion proteins, respectively. We found that GST-LMO2 fusion protein was expressed as inclusion bodies whereas MBP-LMO2 fusion protein was expressed in soluble form. Moreover, the production rate of MBP-LMO2 was also much higher than GST-LMO2. Then MBP-LMO2 fusion proteins and renatured GST-LMO2 fusion proteins were used as bait in pulldown assay to study the interaction between LMO2 and endogenous GATA1 in K562 cells. Western blot analyses showed that both of these proteins could bind to endogenous GATA1 in K562 cells, but recovered GATA1 protein by MBP-LMO2 fusion protein was much more than GST-LMO2 fusion protein. These results suggest that using of MBP system is a helpful attempt in the case of studying small molecular weight, strong hydrophobic proteins.
Adaptor Proteins, Signal Transducing ; Carrier Proteins ; chemistry ; Chemical Precipitation ; DNA-Binding Proteins ; chemistry ; GATA1 Transcription Factor ; chemistry ; Genetic Vectors ; Glutathione Transferase ; chemistry ; Humans ; K562 Cells ; LIM Domain Proteins ; Maltose-Binding Proteins ; Metalloproteins ; chemistry ; Protein Binding ; Protein Interaction Domains and Motifs ; Protein Renaturation ; Proto-Oncogene Proteins ; chemistry ; Recombinant Fusion Proteins ; genetics ; metabolism

Bone morphogenetic protein 2 (BMP2), which belongs to the transforming growth factor-beta (TGF-beta) superfamily, is a multifunctional molecule with distinct abilities to induce bone formation. BMP2 has been identified to have eminent pharmaceutical importance for clinical application. We previously constructed stable cell line in Chinese hamster ovary cells (CHO) that highly expressed recombinant human BMP2 (rhBMP2). For large-scale production of the recombinant protein used in clinical application, it is critical to have both high expression and stability of the protein. In the present study, the stability of the cell line (rCHO(hBMP2)-C8) with the highest expression, as well as the stability of rhBMP2 protein were investigated systematically. We cultured the rCHO (hBMP2)-C8 cell line in the presence or absence of MTX for two months, the cell growth and rhBMP2 production characteristics were examined during the culture; we found the duration that the rCHO(hBMP2)-C8 cell line could secret rhBMP2 continually into the serum-free medium. Moreover, we detected the temperature sensitivity of rhBMP2 in culture medium. This study will contribute to our understanding for further producing rhBMP2 by large-scale culture technology.
Animals ; Bone Morphogenetic Protein 2 ; biosynthesis ; genetics ; CHO Cells ; Cell Culture Techniques ; methods ; Cricetinae ; Cricetulus ; Culture Media ; Genetic Vectors ; genetics ; Humans ; Recombinant Proteins ; biosynthesis ; genetics

Linalool is an important monoterpene, and widely used in food, pharmaceutical and cosmetic industry. The low concentration in plants and the difficulties in extraction restrict its large scale production. Saccharomyces cerevisiae can provide the monoterpene precursor, geranyl diphosphate (GPP) through its endogenous isoprenoid pathway. Therefore, it could be used as the host for monoterpene production. However, the weak metabolic flux through the isoprenoid pathway leads to the insufficient supply of GPP, and results in low monoterpene productivity. In order to increase the metabolic flux, we constructed the integrated expression plasmid pRS305-tHMG1 and free expression plasmid pYLIS-IDI1 to enhance the expression levels of isopentenyl diphosphate isomerase (IDI1) and a truncated 3-hydroxyl-3-methylglutaryl-CoA reductase gene (tHMG1). The two plasmids were separately transformed into S. cerevisiae CEN.PK2-1C, resulting in strains LS01 and LS02. The plasmid pYLIS-IDI1 was further transformed into strain LS01, resulting in strain LS03. GC-MS analysis showed that the linalool concentration was increased by 1.3 times and reached (127.71 +/- 7.68) microg/L. In conclusion, enhancement of the supply of GPP precursors through the regulation of isoprenoid pathway could increase the linalool production in S. cerevisiae.
Biosynthetic Pathways ; genetics ; Butadienes ; metabolism ; Hemiterpenes ; metabolism ; Monoterpenes ; metabolism ; Pentanes ; metabolism ; Saccharomyces cerevisiae ; genetics ; metabolism