Objective To compare the clinical efficacy and safety of domestic orlistat and imported orlistat in Chinese overweight and obese patients. Methods In a randomized, double-blinded and positive-controlled study, 228 adults (BMI 24-< 40 kg/m~2) evaluated at seven research centers were randomized to receive domestic orlistat or imported orlistat 120 mg 3 times a day with an energy-controlled diet for 24 weeks. Results After 24 weeks, domestic orlistat treated patients got significant weight-loss (5.0±3.7) kg, which was comparable with that of imported orlistat treated patients (4.5±3.5) kg (P=0.3922).Compared with the findings before treatment, there was significant decrease of systolic blood pressure (4.4±11.5)mm Hg (1 mm Hg=0.133 kPa) and serum levels of TC (0.54±0.79) mmol/L and LDL-C (0.32±0.64) mmol/L in the domestic orlistat treated group(compared with levels of baseline, P< 0. 0001). There was no significant difference between the two groups in the changes of blood pressure and lipid levels. Both groups had similar adverse event profiles, most of which were mild and transient gastrointestinal events. There were no serious adverse events in beth groups. Conclusions Domestic orlistat combined with a light low-energy diet promoted significant weight loss, which was comparable with that of imported orlistat after 24 weeks of treatment. There was also improvement in blood pressure and serum levels of TC and LDL-C. Domestic orlistat was as effective and safe as imported orlistat in the treatment of obesity.

AIM To establish the HPLC fingerprints of Yupingfeng Powder aqueous decoction and to determine the contents of nine constituents.METHODS The analysis of aqueous decoction was developed on a 30 ℃ thermostatic Hypersil ODS column (250 mm × 4.6 mm,5 μm),with the mobile phase comprising of acetonitrile-water flowing at 1.0 mL/min in a gradient elution manner,and the detection wavelength was set at 220 nm.RESULTS There were fifteen common peaks in the fingerprints of ten batches of samples,with the similarities of more than 0.95.Nine of them were identified as prim-O-glucosylcimifugin,calycosin-7-O-β-D-glucoside,cimifugin,4'-O-β-glucopyranosyl-5-O-methylvisamminol,psoralen,calycosin,sec-O-glucosylhamaudol,formononetin and atractylon,which showed good linear relationships within their own ranges (r ≥ 0.999 7),the average recoveries were 97.91％-99.81％ with the RSDs of 0.58％-1.27％.CONCLUSION This stable and reliable method can beused for the quality control of Yupingfeng Powder aqueous decoction.

BMP6 is a member of TGF-beta superfamily, represent more effective osteogenic activity. Two recombinant plasmids were constructed to expression rhBMP6 in mammalian cells, one contained the cDNA encoding the signal peptide, propeptide and mature peptide of human BMP6, wich was named pcDNA-BMP6, the other contained the recombinant DNA encoding the signal peptide, propeptide of human BMP2 and the mature peptide of BMP6, which was named pcDNA-BMP2/6. Transient expression in Cos7 cells demonstrated that the pcDNA-BMP2/6 produced more rhBMP6 than pcDNA-BMP6. For stable expression, the CHO-dhfr- cells were transfected with pcDNA-BMP2/6 and pSV2-dhfr, then screened by G418 and treated with MTX for targeting gene amplification. The partially purified rhBMP6 by heparin affinity chromatography was shown to possess bone induction activity tested by the induction of alkaline phosphatase activity in C2C12 cells.
Alkaline Phosphatase ; metabolism ; Animals ; Blotting, Western ; Bone Morphogenetic Protein 2 ; genetics ; Bone Morphogenetic Protein 6 ; genetics ; metabolism ; pharmacology ; CHO Cells ; COS Cells ; Cell Line ; Cercopithecus aethiops ; Cricetinae ; Cricetulus ; Gene Expression ; Humans ; Myoblasts ; cytology ; drug effects ; enzymology ; Protein Precursors ; genetics ; Protein Sorting Signals ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

Bone morphogenetic protein 2(BMP-2) is a member of the of BMPs family, its osteoinductive capacity has already been demonstrated. We tried to express hBMP-2 in CHO cell. In this study, we inserted hBMP-2 cDNA into vector pCDNA3.1(+) to construct hBMP-2 eukaryotic expression vector pCDNA3.1(+)-hBMP-2. Recombinant Chinese hamster ovary (rCHO) cell line expressing high-level recombinant human bone morphogenetic protein 2(rhBMP-2) was constructed by co-transfecting the expression vectors pCDNA3.1(+)-hBMP-2 and plasmid pSV2-dhfr into dihydrofolate reductase (dhfr)-deficient CHO cells and the subsequent gene amplification in medium containing stepwise increments in methotrexate level such as 0.1 and 1 micromol/L. Western blot analyses showed a specific band of about 18 kD in reduced sample lane and a specific band of about 32 kD in non-reduced sample lane, this indicated that rCHO cells secret rhBMP-2 as a homodimeric glycoprotein form. Finally, we obtained a single clone cell strain expressing a high level (7.83 microg/24 h/10(6) cells) of rhBMP-2 tested by ELISA. Biological activity of rhBMP-2 was tested by the induction of alkaline phosphatase(ALP) activity in C2C12 cells. We treated C2C12 with different concentration of rhBMP-2 condition medium(CM) for 5d. The results showed that the rhBMP-2 could significantly increase the ALP activity of C2C12.
Alkaline Phosphatase ; biosynthesis ; Animals ; Blotting, Western ; Bone Morphogenetic Protein 2 ; biosynthesis ; chemistry ; isolation & purification ; pharmacology ; CHO Cells ; Cell Line ; Cricetinae ; Cricetulus ; Enzyme Induction ; drug effects ; Gene Expression ; Genetic Vectors ; genetics ; Humans ; Mice ; Recombinant Proteins ; biosynthesis ; chemistry ; isolation & purification ; pharmacology ; Solubility

BACKGROUNDBone morphogenetic protein-6 (BMP-6) is closely correlated with tumor differentiation and skeletal metastasis. Estrogen is considered as a stimulant for the initiation and promotion of breast cancer. Previous studies demonstrated that 17beta-estadiol (E2) can selectively increase the expression of BMP-6. This experiment is designed to detect the molecular mechanism of estrogen activating BMP-6 gene transcription in human estrogen receptor positive (ER+) breast cancer cell line MCF-7.

METHODSAfter the treatment of MCF-7 cells with E2 at different concentrations (10(-11) mol/L, 10(-9) mol/L, 10(-7) mol/L), the BMP-6 expression level was examined through real-time polymerase chain reaction. Through restriction enzyme digestion, human BMP-6 1.2 kb long promoter, BMP-6 0.7 kb long promoter was cloned into pGL-3 basic vector; after the treatment with 10(-7) mol/L E2, luciferase activities of the two promoters were detected. Site-directed mutagenesis was performed to obtain the mutant forms of estrogen response element half-site (1/2 ERE) element and Sp1 sites in the BMP-6 promoter, the activities of these mutant form promoters were detected following the methods mentioned above. Chromatin immunoprecipitation (ChIP) assay was also used to confirm the binding of estrogen receptor alpha (ERalpha) on BMP-6 promoter in the presence of E2.

RESULTSE2 dose dependently increased BMP-6 mRNA expression in human ER+ breast cancer cell line MCF-7. At a dose of 10(-7) mol/L E2, human BMP-6 1.2 kb promoter activity was increased by 90% compared with the control group treated with ethanol (P < 0.05). Both the 1/2 ERE response element mutant form and the Sp1 site mutant form of the BMP-6 promoter abolished the activation of the BMP-6 promoter's response to E2. Through ChIP assay, the binding of ERalpha on 1/2 ERE response element in BMP-6 promoter was further validated.

CONCLUSIONEstrogen induces BMP-6 expression in human ER+ breast cancer cell line MCF-7 through its receptor ERalpha binding on 1/2 ERE element in the BMP-6 promoter.

Bone Morphogenetic Protein 6 ; Bone Morphogenetic Proteins ; genetics ; Breast Neoplasms ; genetics ; Cell Line, Tumor ; Dose-Response Relationship, Drug ; Estradiol ; pharmacology ; Estrogen Receptor alpha ; physiology ; Female ; Humans ; Parathyroid Hormone-Related Protein ; secretion ; Promoter Regions, Genetic ; Transcriptional Activation ; drug effects

OBJECTIVE: To compare the results between auditory steady-state response (ASSR) and 40 Hz auditory event related potential (AERP), and explore the accuracy of hearing thresholds by using ASSR and AERP and the clinic forensic value. METHODS: Thirty seven ears were tested with pure-tone audiometer, 40Hz AERP and ASSR, respectively. All the volunteers in our study were awake during 40 Hz AERP test and ASSR test. RESULTS: Thresholds acquired with ASSR and 40Hz AERP test had a close correlativity and showed higher than those acquired with PTA test. There was no significant difference between the accuracy of ASSR and 40Hz AERP in estimating pure-tone thresholds. CONCLUSION After determining the correct value, ASSR can be used directly to evaluate hearing loss objectively.
Acoustic Stimulation ; Adult ; Audiometry, Evoked Response ; Audiometry, Pure-Tone/methods* ; Auditory Threshold ; Evaluation Studies as Topic ; Evoked Potentials, Auditory, Brain Stem/physiology* ; Female ; Hearing Loss, Sensorineural/physiopathology* ; Humans ; Male ; Middle Aged ; Predictive Value of Tests ; Sleep/physiology* ; Wakefulness ; Young Adult

OBJECTIVETo investigate the mechanisms of PTEN gene inactivation starting from DNA, mRNA and protein levels in ovarian cancers.

METHODSTumor tissue samples were obtained from 48 patients with epithelial ovarian cancers. Using four polymorphic markers (D10s541, D10s583, D10s1687 and D10s2491) within and flanking the PTEN gene located in chromosome 10q 23.3, polymerase chain reaction (PCR) and loss of heterozygosity (LOH) were introduced to examine LOH of PTEN gene; PCR-single strand conformation polymorphism (PCR-SSCP) was introduced to examine mutations of the fifth, sixth, seventh, and eighth exons of PTEN. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry (SP method) were applied to detect PTEN mRNA and PTEN protein expressions, respectively.

RESULTSLOH of PTEN gene was observed in 19 of 48 (39.6%) ovarian cancers. PTEN mutations were found only in 2 (4.2%) of the cases. Absence of PTEN mRNA expression was 18.8% (9 of 48). Immunostaining of 48 cancer samples revealed that 13 (27.1%) were PTEN immunostain negative. Of these 13 samples, only 2 (15.4%) had structural, biallelic inactivation by intragenic PTEN mutations and loss of the remaining wild-type allele; 7 (53.8%) showed evidence of LOH, 5 of these 7 samples showed deletion of PTEN mRNA expression, another 2 samples showed positive expression of PTEN mRNA; 4 (30.8%) tumors had neither PTEN gene mutation nor LOH but exhibited no PTEN protein expression, 2 of these 4 cases showed deletion of PTEN mRNA expression, another 2 showed positive expression of PTEN mRNA. For the cases of PTEN protein absent staining, the rate of LOH was 69.2% (9 of 13), higher than 28.6% (10 of 35) for the positive staining (P < 0.05).

CONCLUSIONSPTEN gene inactivation may contribute to epithelial ovarian carcinogenesis. There may be several mechanisms of PTEN gene inactivation in ovarian cancers. Protein expression deletions may be a significant mechanism.

Adult ; Aged ; Chromosomes, Human, Pair 10 ; Exons ; Female ; Gene Deletion ; Gene Expression Regulation, Neoplastic ; Genes, Tumor Suppressor ; Humans ; Loss of Heterozygosity ; Middle Aged ; Mutation ; Ovarian Neoplasms ; genetics ; metabolism ; PTEN Phosphohydrolase ; biosynthesis ; genetics ; Polymerase Chain Reaction ; Polymorphism, Single-Stranded Conformational ; RNA, Messenger ; biosynthesis ; genetics

OBJECTIVETo investigate the mRNA expression profiles of megakaryocytes (MKs) from human cord blood CD34+ cells ex vivo expanded using Solexa technique.

METHODSCD34+ Cells were isolated using density gradient centrifugation and magnetic activated cell sorting. Cultures were stimulated with recombinant human thrombopoietin (100 ng/ml). After 12 days, the MKs fraction was separated from the non-MKs fraction using an anti-CD41 monoclonal antibody by immunomagnetic sorting. The mRNA expression of MKs and non-MKs was detected by Solexa sequencing.

RESULTSWe obtained 3 773 147 and 3 533 805 Tags from MKs and non-MKs, respectively. The amounts of unambiguous tags were 3 291 132 and 2 967 947 and those of distinct tags were 197 769 and 245 318. The expression of 1161 genes was up-regulated and that of 902 genes down-regulated. The expression of 2717 tags was up-regulated and that of 1519 tags down-regulated.

CONCLUSIONSMKs and non-MKs have remarkably different mRNA expression profiles. The differential gene-encoded products may be involved in cellular development, adhesion, apoptosis metabolism, intra- and intercellular signal transduction, and immune response. Further studies on this topic may clarify the expression mechanism, signal transduction, and regulation mechanisms.

Antigens, CD34 ; Cells, Cultured ; Fetal Blood ; cytology ; Humans ; Megakaryocytes ; cytology ; metabolism ; RNA, Messenger ; genetics ; Transcriptome

OBJECTIVETo construct replication selective adenovirus AdhepE1 targeting human melanoma and observe its specific killing of human melanoma cells in vitro.

METHODSAdenovirus E1 region, the murine tyrosinase promoter and enhancer DNA sequences were acquired respectively by PCR cloning. The shuttle plasmid of replication-selective adenovirus targeting human melanoma was constructed by DNA recombination. Replication-selective adenovirus AdhepE1 was generated by homologous recombination. The human melanoma cell line SK-Mel-1 and hepatocellular carcinoma cell line HepG2 were attacked separately by lower dose of AdhepE1. Change of cell morphology was observed and the surviving cells were calculated. The expression of E1A was assayed by RT-PCR to verify the specific-replication of AdhepE1.

RESULTSReplication selective adenovirus AdhepE1 targeting human melanoma was acquired by PCR. Human melanoma cell line SK-Mel-1 was sensitive to oncolytic killing of AdhepE1 whereas HepG2 was little responsive. The results of RT-PCR suggested that AdhepE1 replicated specifically in human melanoma cells.

CONCLUSIONAdhepE1 can selectively kill human melanoma cells.

Adenoviridae ; genetics ; Animals ; Cell Line, Tumor ; Genetic Therapy ; Humans ; Liver Neoplasms ; therapy ; Melanoma ; therapy ; virology ; Mice ; Reverse Transcriptase Polymerase Chain Reaction ; Virus Replication

BACKGROUNDDiabetes has become one of the most common chronic diseases and the third leading cause of death in China. Many programs have been initiated at national and local levels to address the illness. However, the effect of these programs in daily outpatient clinics is still unclear. The objective of this study was to investigate the management status of type 2 diabetes mellitus (T2DM) and factors associated with it in diabetes clinics of tertiary hospitals in Beijing.

METHODSA cross-sectional survey was conducted in six tertiary hospitals in Beijing. Control criteria were defined based on 2007 China guideline for type 2 diabetes (CGT2D).

RESULTSA sample of 1151 patients, age (60.8 ± 9.2) years, and with a median disease duration of 7.3 years was included. The hemoglobin A1c (HbA1c) mean level was (7.15 ± 1.50)%, the percentage of patients achieving the targets for HbA1c was 37.8%, blood pressure 65.6%, triglyceride (TG) 48.8%, high-density lipoprotein (HDL) 59.2%, low-density lipoprotein (LDL) 34.0%, and total cholesterol (TC) 42.0%. The factors independently associated with glycemic control were diabetes duration (odds ratio (OR) = 0.95; 95% confidence interval (CI): 0.919 - 0.982, P < 0.01), body mass index (BMI) (OR = 0.914, 95%CI: 0.854 - 0.979, P = 0.01) and smoking (OR = 0.391, 95%CI: 0.197 - 0.778, P < 0.01). The factors independently associated with blood pressure control were BMI (OR = 0.915, 95%CI: 0.872 - 0.960, P < 0.01) and male gender (OR = 0.624, 95%CI: 0.457 - 0.852, P < 0.01). The factor independently associated with LDL control was education level (OR = 1.429, 95%CI: 1.078 - 1.896, P = 0.013).

CONCLUSIONSThe management status of T2DM patients in tertiary hospitals in Beijing has improved remarkably. However, there is still room for further improvement to reach the guideline target. Long diabetes duration, high BMI, smoking, male gender and low education level were independently associated with poor metabolic control.

Adolescent ; Adult ; Aged ; Blood Glucose ; metabolism ; Blood Pressure ; China ; Cholesterol ; blood ; Diabetes Mellitus, Type 2 ; blood ; metabolism ; Female ; Glycated Hemoglobin A ; metabolism ; Hospitals ; statistics & numerical data ; Humans ; Lipoproteins, HDL ; blood ; Lipoproteins, LDL ; blood ; Male ; Middle Aged ; Triglycerides ; blood ; Young Adult