With the rapidly development of the biotechnology industry,large quantities of recombinant proteins are needed for specific therapeutic and diagnostic applications.Bacterial cells are most often used for the production of recombinant proteins.However,recombinant proteins expressed in the cytoplasm of bacteria are often misfolded as insoluble inclusion bodies and therefore inactive.To circumvent this problem,several eukaryotic expression systems have also been developed over the years,ranging from yeast to mammalian cell-based technologies.For many mammalian proteins,especially those secreted and modified posttranslationally,a more compatible expression system is highly desirable because proper folding or modification can only be provided with closely related cells,i.e.,mammalian cells.Large scale transient transfection of mammalian cells is a recent and powerful technology for the fast production of milligram amounts of recombinant proteins.Transient expression by means of extrachromosomal replication in COS cells is frequently used to check the functional integrity of genes/plasmids and to produce small quantities of cell supernatants containing the protein of interest.As it is allowed for easy and efficient purification,many recombinant proteins used for therapeutic and structural studies are naturally secreted or engineered to be secreted.The use of a proper signal peptide is one of the major determinants for the efficient secretion of heterologous proteins from mammalian cells.The noncatalytic C-terminal hemopexin-like domain of MMP-2,PEX,can block angiogenesis and tumor growth in vivo.Large quantities of biochemically active recombinant PEX are required for the study of their functions and biochemical properties,as well as for their industrial applications.For this purpose,the rat growth hormone,mouse IgG? chain and MMP-9 signal peptides were used for expression of PEX in COS7 cells,and their secretion efficiencies were compared by Western blotting and ELISA.Western-blotting of PEX protein from culture media,resulted in detection of proteins with the predicted molecular mass,which indicate that all of the signal sequences could direct PEX secretion successfully.The MMP-9 signal peptide seems to be superior to the signal peptides from IgG and rGH both in terms of extracellular yield and in terms of secretion efficiency.Thus,expression of pM9PEX construct resulted in higher yields of extracellular PEX and the majority of the produced PEX was secreted and not trapped intracellularly.To examine whether the observed difference in secretion yields is promoted at the transcriptional level,a RT-PCR analysis was performed at 6 h after transfection.The presence of mRNA transcripts of PEX was observed in all the DNA constructs.Moreover,semiquantitative reverse transcription(RT-PCR)results show that there were no significant differences in the expression levels of PEX among the constructs at 6 h after transfection.Though there was no difference in the expression levels of PEX at an early time point after transfection,the presence of an ER-targeting signal peptide sequence in the expression vector affected the trafficking of expressed proteins in the cells.Hence,the described difference in exported yields is probably promoted at the secretion level,rather than at the transcriptional level.Chick chorioallantoic membrane(CAM)bioassay show that the PEX protein purified from cell culture had biological activity to inhibit the angiogenesis.The MMP-9's signal peptide is used for the first time as leader sequence for secretion of foreign proteins.The results revealed that higher amounts of secreted PEX were obtained when vectors containing MMP-9 signal peptide were used and it is also indicated that MMP-9 signal sequence could be effective on promoting the secretion of other heterologous proteins in eukaryotic cells.

Human herpes simplex virus 1 (HSV-1) causes facial,ocular,and encephalitic disease and is associated with latent infection and cancer.Here,we developed a means of studying the pathogenesis of HSV-1 infection at the protein level by using the SELDI Protein Chip to detect changes of protein expression in Vero cells cultured in vitro.After infection with HSV-1 and culture for 12,24 or 48 h,cells were harvested and lysed.IMAC3 arrays were applied to SELDI-TOF-MS to detect proteomic differences before and after infection.The chip detected a series of differentially expressed protein peaks.Interestingly,both peaks at 16 912 Da and 17 581 Da corresponded precisely with the molecular mass of ISG 15,which may participate in antiviral activity during the process of infection.Thus,the results we obtained can serve as a basis to study the pathogenesis of HSV-1 and the interaction between the virus and its host.In addition,they can help in the discovery of new therapeutic targets for treatment of HSV-1 infection.

The purpose of this study was to isolate and purify polysaccharide from Gynura divaricata and analyze its monosaccharide composition. A water-soluble crude polysaccharide was obtained by hot water extraction, ethanol precipitation and deproteinization after degreasing. The crude polysaccharide then purified with DEAE-Sepharose Fast Flow column chromatography and dialysis. The monosaccharide composition and structure were analyzed by HPLC, UV spectrophotometer and 1H-NMR. The results showed that the purity and molecular weight of GDPS-2 and GDPS-3 were 87.3%, 2.03 x 10(4) Da and 90.9%, 4.29 x 10(4) Da, respectively. The UV spectrophotometer and 1H-NMR data suggested that glycosidic bond of GDPS-2 and GDPS-3 were a type. Both GDPs-2 and GDPs-3 were homogeneous polysaccharides, and GDPs-2 was mainly composed of glucuronic acid and xylose at a molar ratio of 1.1:0.63. GDPs-3 was mainly composed of rhamnose, glucuronic acid, galactose, xylose and galacturonic acid at a molar ratio of 0.32:6.0:0.21:1.75:4.3.
Asteraceae ; chemistry ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Magnetic Resonance Spectroscopy ; Molecular Weight ; Polysaccharides ; chemistry ; isolation & purification

AIMTo study the mechanisms of protection of bcl-2 gene transfection against heat-stressed cardiomyocytes.

METHODSCardiomyocytes were isolated and cultured. bcl-2 was transfected into cardiomyocytes with Lipofectamine transfection methods. The cardiomyocytes were stressed by heat. The change of H+ -ATPase synthesis activity of cardiomyocytes mitochondria caused by bcl-2 transfection was measured by chemical radiation method. The changes of Caspase 3 activity of cardiomyocytes caused by bcl-2 transfection was measured by fluorometric analysis.

RESULTSbcl-2 transfection could increase the H+ -ATPase synthesis activity of cardiomyocytes mitochondria under heat stress at 41 degrees C and 43 degrees C and could decrease the Caspase 3 activity of cardiomyocytes under heat stress at 41 degrees C and 43 degrees C.

CONCLUSIONThe protection effect of bcl-2 transfection on heat-stressed cardiomyocytes may be associated with preserved H+ ATPase synthesis activity of cardiomyocytes mitochondria and the activity of Caspase 3 of cardiomyocytes.

Animals ; Caspase 3 ; metabolism ; Cells, Cultured ; Genes, bcl-2 ; Heat-Shock Response ; genetics ; Myocytes, Cardiac ; cytology ; metabolism ; Proton-Translocating ATPases ; metabolism ; Rats ; Transfection

To enhance the quality and efficiency of ozagrel by investigating the differences between the ozagrel polymorphs in bioavailability. Solid ozagrel in different polymorph forms were orally administered to SD rats. An HPLC method was established to determinate plasma level of ozagrel. The bioavailabilities of two polymorph forms were calculated and compared. The pharmacokinetic parameters of ozagrel, were as follows: Cmax was 32.72 ± 17.04 and 34.01 ± 19.13 mg · L(-1), respectively; AUC0-t was 61.14 ± 14.76 and 85.56 ± 18.08 mg · L(-1) · h, respectively; t½ was 1.53 ± 0.51 and 4.73 ± 3.00 h, respectively. There was no significant difference in pharmacokinetic parameters between form I and II polymorphs of ozagrel while the t½ of form II is longer, which indicates that the use of form II polymorph as pharmaceutical product may prolong the effective action time in clinics. This would help the polymorph quality control in drug production.
Animals ; Biological Availability ; Chromatography, High Pressure Liquid ; Methacrylates ; chemistry ; pharmacokinetics ; Rats ; Rats, Sprague-Dawley

Objective:To compare the therapeutic efficacy and safety of Hyper-CVAD/MA regimen and CHOP/CHOP-like regimen in the treatment of peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS). Methods:The 78 primary PTCL-NOS patients who were initially diagnosed and treated in Tianjin Medical University Cancer Institute and Hospital and Tianjin Union Medical Center from June 2004 to June 2012 were retrospectively analyzed. The patients were then divided into two groups:Hyper-CVAD/MA group (n=21) and CHOP/CHOP-like group (n=57). Curative efficacies and toxicities were analyzed by Chi-square test, and survival was estimated by Ka-plan-Meier method. Results: In the Hyper-CVAD/MA group, complete response (CR) was 42.9%, overall response rate (ORR) was 85.7%, median progression-free survival (PFS) was 20 months, and the three-year overall survival (OS) was 56.9%. In the CHOP/CHOP-like group, the CR, ORR, and three-year OS were 28.1%, 59.6%, and 49.6%, respectively, and the median PFS was 13 months. Compara-tive analysis showed that the ORR and three-year OS were statistically significant (P<0.05), but the relapse rates (57.1%versus 77.2%) and three-year OS were similar (P>0.05). The incidence rates ofⅢ/Ⅳneutrocytopenia and thrombocytopenia in Hyper-CVAD/MA group (66.7%and 61.9%, respectively) were significantly higher than those of the CHOP/CHOP-like group (22.8%and 14.0%, respec-tively) (P<0.05). Conclusion:Hyper-CVAD/MA regimen can achieve satisfactory efficacy in parents with PTCL-NOS, and toxicity can be controlled with granulocyte colony stimulating factor (G-CSF).

OBJECTIVETo systematically evaluate the efficacy and safety of kidney-tonifying traditional Chinese medicine in the treatment of male infertility.

METHODSBased on the principles and methods of Cochrane systematic reviews, we searched CNKI, VIP, and Wanfang databases from inception to December 2012 for randomized controlled clinical trials addressing the treatment of male infertility with kidney-tonifying traditional Chinese medicine. According to the inclusion and exclusion criteria and retrieval strategies, we extracted the data, evaluated the quality of the included literature, and conducted meta-analysis using the RevMan 5. 2 software.

RESULTSTwenty trials involving 2,272 patients were included, and the sample size of each study was from 60 to 270 cases. All the studies were graded as of poor quality, with Jadad scores of no more than 3 points. The results of meta-analysis showed that the total effectiveness rate of traditional Chinese medicine versus Western medicine on male infertility was RR = 1.71, 95% CI 1.19-2.47, and that of Chinese-Western combined therapy versus Western medicine was RR = 1.15, 95% CI 1.01-1.30. Both traditional Chinese medicine and Chinese-Western combined therapy showed a significantly better total effectiveness than Western medicine alone in improving the pregnancy rate without serious adverse reactions.

CONCLUSIONDue to the poor methodological quality and high heterogeneity of the included studies, the evidence for the efficacy and safety of kidney-tonifying traditional Chinese drugs in the treatment of male infertility is of but limited value, and further validation is needed by more high-quality studies.

Drugs, Chinese Herbal ; therapeutic use ; Female ; Humans ; Infertility, Male ; drug therapy ; Kidney ; Male ; Medicine, Chinese Traditional ; Pregnancy ; Randomized Controlled Trials as Topic

Adolescent ; Bacteria ; isolation & purification ; Child ; Child, Preschool ; Humans ; Infant ; Infant, Newborn ; Respiratory Tract Infections ; microbiology

OBJECTIVETo analysis the changes of two chemical constituents, namely 2, 3-dihydro-3, 5- dihydroxy-6-methyl-4H-pyran-4-one (DDMP) and 5-hydryoxymethyl-furfural (5-HMF) produced in Radix Polygoni Multiflori after processing, with processing time, and to determine the contents of 5-HMF in samples of Radix Polygoni Multiflori and Radix Polygoni Multiflori preparata.

METHODAn HPLC method was applied with a Zobax SB-C18 (3.9 mm x 150 mm, 5 microm) column by a elution using methanol-water (10: 90) as the mobile phase. The detection was set at UV 280 nm.

RESULTThe contents of DDMP were increasing with the processing time until 24 hour, followed by a decrease until 60 hour process. The contents of 5-HMF were increasing gradually throughout the 60 hour steaming process. The contents of 5-HMF in 11 samples of Radix Polygoni Multiflori preparata were from 0.013% to 0.101%, and only one in 4 samples of Radix Polygoni Multiflori containing trace amount of 5-HMF.

CONCLUSIONThe chemical components in Radix Polygoni Multiflori were changed during the processing procedures. Therefore, the processing of Radix Polygoni Multiflori should be controlled and standardized.

Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; chemistry ; Polygonaceae ; chemistry ; Reproducibility of Results

OBJECTIVETo investigate the role of EPHA2 in regulating apoptosis, proliferation and vasculogenic mimicry of osteosarcoma cells, by gene silencing through RNA interference.

METHODSEPHA2-siRNA plasmids were achieved by gene cloning. The plasmids were transfected into human osteosarcoma cells (MG63). The expression level of EPHA2 protein was measured by Western blotting. The proliferation, apoptosis and vasculogenic mimicry features of osteosarcoma MG63cells were assessed by light microscopy, MTIP assay, flow cytometry, annexin V-FITC/PI and HE staining, respectively.

RESULTSThe EPHA2-siRNA plasmid was confirmed by DNA sequencing. After treatment with Sequence-specific siRNA targeted EPHA2, the protein level of the transfected group decreased significantly. As compared to non-siRNA transfected cells, the transfected group showed lower proliferation, higher and earlier apoptosis and less osteosarcoma-generated vasculogenic mimicry.

CONCLUSIONEPHA2 gene may be involoved in apoptosis and proliferation of osteosarcoma cells, and may be necessary for vasculogenic mimicry. Down-regulation of EPHA2 expression by sequence-specific siRNA may be considered as a new option in the treatment of EPHA2 over-expressing cancer including osteosarcoma in future.

Apoptosis ; Bone Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Neovascularization, Pathologic ; pathology ; Osteosarcoma ; metabolism ; pathology ; Plasmids ; RNA Interference ; RNA, Small Interfering ; genetics ; Receptor, EphA2 ; genetics ; metabolism ; Transfection