In the present study, terminal-restriction fragment length polymorphism (T-RFLP) technique was applied to assess the diversity and tissue distribution of the fungal endophyte communities of Alpinia officinarum collected from Longtang town in Xuwen county, Guangdong province, China, at which the pharmacological effect of the medicine plant is traditional considered to be the significantly higher than that in any other growth areas in China. A total of 28 distinct Terminal-Restriction Fragment (T-RFs) were detected with HhaI Mono-digestion targeted amplified fungal nuclear ribosomal internal transcribed spacer region sequences (rDNA ITS) from the root, rhizome, stem, and leaf internal tissues of A. officinarum plant, indicating that at least 28 distinct fungal species were able to colonize the internal tissue of the host plant. The rDNA ITS-T-RFLP profiles obtained from different tissues of the host plant were obvious distinct. And the numbers of total T-RFs, and the dominant T-RFs detected from various tissues were significantly different. Based on the obtained T-RFLP profiles, Shannon's diversity index and the Shannon's evenness index were calculated, which were significantly different among tissues (P < 0.05). Furthermore, two types of active chemicals, total volatile oils by water vapor distillation method and galangin by methanol extraction-HPLC method, were examined in the each tissue of the tested plant. Both of tested components were detected in all of the four tissues of the medicine plant with varying contents. And the highest was in rhizome tissue. Correlation analysis revealed there were significant negative correlations between both of the tested active components contents and calculated Shannon's diversity index, as well as the Shannon's evenness index of the fungal endophyte communities of the host plant (P = 0, Pearson correlation coefficient ≤ -0.962), and significant positive correlations between both of the tested active components contents and 325 bp dominant T-RF linkage to Pestalotiopsis (P = 0, Pearson correlation coefficient ≥ 0.975). In conclusion, A. officinarum is colonized by diverse fungal endophytes communities. The diversity of the fungal endophytes was found in the A. officinarum varied with differences of the tissue types of the host plants and was closely correlated with the accumulation of main active components, total volatile oils and galangin contents in the host plant tissue.
Alpinia ; chemistry ; microbiology ; Biodiversity ; China ; DNA, Fungal ; genetics ; DNA, Ribosomal ; genetics ; Drugs, Chinese Herbal ; analysis ; Endophytes ; classification ; genetics ; growth & development ; isolation & purification ; Fungi ; classification ; genetics ; growth & development ; isolation & purification ; Phylogeny ; Plants, Medicinal ; chemistry ; microbiology ; Polymorphism, Restriction Fragment Length

OBJECTIVE: To determine the optimum ultrasonic-assisted extracting process of total flavonoids from Zingiber mioga by response surface methodology, and then study the antioxidant activity of the extract. METHODS: On the basis of single factor experiments, a 4-factor, 3-level Box-Behnken center-united experiment was conducted. The Box-Behnken experiment analyzed and optimized the processing conditions by response surface methodology, then the antioxidant activity of the extract was determined. RESULTS: The optimum conditions at ultrasonic power of 300 W were as follows treatment temperature 60℃, ethanol concentration 85.99%, liquid-to-solid ratio 98.08 mL·g-1, treatment time 15 min. Under the optimum conditions, the extraction rate of the total flavonoids was 37.01%, with a bias of 4.28% compared with the predicted value of 35.49%; the reducing power of the total flavonoids from Zingiber mioga at the concentration of 1.038 7 mg·mL-1 was equal to that of vitamin C of 56.60 μg·mL-1; the ·OH radical scavenging ratio of the total flavonoids from Zingiber mioga was 73.07%, the DPPH radical scavenging ratio was 65.72%, and the inhibition rate of anti-lipid peroxidation was 49.94%. CONCLUSION: The total flavonoids from Zingiber mioga can be developed as a new type of antioxidant agents, and regression analysis and parameter optimization of the extracting process can be conducted by using response surface methodology.

OBJECTIVE: To optimize the ultrasonic-assisted extraction process of isoflavones from Pueraria lobata (Wild.) Ohwi using response surface methodology and measure the reducing power. METHODS: On the basis of single factor experiments, a four factors and five levels Box-Behnken experiment was designed. Then the reducing power of isoflavones was measured. RESULTS: The optimum conditions for the ultrasonic-assisted extraction process of isoflavone from Pueraria lobata (Wild.) Ohwi at ultrasonic power of 300 W were as follows: ethanol concentration 75%, liquid-solid ratio 35 mL · g-1, extraction time 35 min, and treatment temperature 50℃. Under the optimum conditions, the yield of polysaccharide from Pueraria lobata (Wild.) Ohwi was 24.18%, and the relative error between experimental value and predicted value was only 3.02%; the EC50 of the reducing power of isoflavones reached 0.135 mg · mL-1. CONCLUSION: The response surface methodology is suitable for regression analysis and optimization of the ultrasonicassisted extraction of polysaccharide from Pueraria lobata (Wild.) Ohwi.

0.05)between the two groups.Conclu- sion The long-term outcomes of e-CHB is not markedly different compared with that of e+CHB.

Objective To compare the diagnostic value of myocardial perfusion imaging (MPI) and 64 multi-slice spiral CT (64-MSCT) for coronary artery disease (CAD). Methods Fifty-two patients with suspected or known CAD were included in the study. Each patient underwent both stress and rest MPI,MSCT as well as conventional coronary angiography (CAG) within 1 month. The stress and rest MPI were scored by a 5-grade criteria (0 ～ 4) based on 17 coronary artery segments. The difference between summed stress and rest scores ＞ 1 was defined as myocardial ischemia. Stenosis in one main vessel or one main branch of the main vessel ≥50% was defined as myocardial ischemia by MSCT. CAG was used as the reference for comparison. Statistical analysis was performed using SPSS 13. 0 software. Kappa value was used to test the accordance of MPI and MSCT results. X2 test was used to evaluate the difference between MPI and MSCT results. Results The patient-based sensitivity, specificity, positive and negative predictive values and accuracy of MPI and MSCT for the diagnosis of CAD were 86.7% (26/30), 77.3% ( 17/22),83.9% (26/31), 81.0% ( 17/21), 82.7% (43/52) and 83.3% ( 25/30), 86.4% ( 19/22), 89.3%( 25/28), 79.2% ( 19/24), 84.6% (44/52), respectively. The vessel-based sensitivity, specificity, positive and negative predictive values and accuracy of MPI and MSCT were 74.5% (38/51), 81.0% (85/105 ), 65.5% (38/58), 86.7% ( 85/98), 78.8% ( 123/156 ) and 90.2% (46/51 ), 88.6% ( 93/105 ),79.3 % (46/58), 94.9% (93/98), 89.1% ( 139/156), respectively. There was no statistically significant difference between MPI and MSCT for either patient or lesion-based diagnosis (X2 =0.44, 0.21, both P ＞0.05 ). 96.0% (24/25) patients with both abnormal MPI and MSCT positive were valified by CAG while 83.3% (15/18) patients with both MPI and MSCT negative were excluded by CAG. Conclusions Both MPI and MSCT are reliable diagnostic modalities for CAD. They also provide complementary diagnostic value to each other.

OBJECTIVETo evaluate the role of RNA interference (RNAi) in silencing the enhanced green fluorescent protein (eGFP) expression in 293T and Mel cells.

METHODSNested-PCR was used to amplify H1 promoter from human 293T cells for driving RNAi synthesis. RNAi vectors (TR1) for silencing the eGFP expression was constructed. The eGFP vector and RNAi vector (TR1) were then co-transfected into the 293T and Mel cells, in which the silencing effect on eGFP expression was investigated by fluorescence microscopy, reverse transcription-PCR(RT-PCR), fluorescence-assited cell sorting(FACS) analysis and real-time RT-PCR.

RESULTSRNAi could effectively reduce more than 50 percent of eGFP expression in 293T cells as well as in Mel cells.

CONCLUSIONThe RNAi vector constructed in this way paper can effectively inhibit eGFP expression in cells.

Cell Line ; Flow Cytometry ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; Humans ; RNA Interference ; Reverse Transcriptase Polymerase Chain Reaction

Adult ; Antiviral Agents ; administration & dosage ; therapeutic use ; Drug Therapy, Combination ; Female ; Hepatitis B e Antigens ; Hepatitis B, Chronic ; drug therapy ; immunology ; Humans ; Interferon-alpha ; administration & dosage ; therapeutic use ; Male ; Nucleotides ; administration & dosage ; therapeutic use ; Polyethylene Glycols ; administration & dosage ; therapeutic use ; Recombinant Proteins ; administration & dosage ; therapeutic use ; Treatment Outcome ; Young Adult

Adolescent ; Adult ; Female ; Humans ; Leg Bones ; abnormalities ; surgery ; Male ; Middle Aged ; Soft Tissue Injuries ; surgery ; Surgical Flaps

OBJECTIVEThe aim of this study was to investigate the changes in mTOR activity and survivin expression in liver cancer cell line HepG2 cells treated with tamoxifen.

METHODSSurvivin transcription level and p70S6K was demonstrated by PCR, dual-luciferase reporter assay and Western blot analysis, respectively, and the apoptosis in the HepG2 cells was detected by flow cytometry.

RESULTSTamoxifen leads to apoptosis of the cells and reduction in survivin expression, as well as a dramatic reduction in the activated form of p70S6K. Treating HepG2 cells with rapamycin, a specific mTOR inhibitor, significantly reduced the survivin protein level but not affected the survivin transcription, indicating that tamoxifen and rapamycin were synergistic in regards to down-regulation of survivin expression in hepatocellular carcinoma cells.

CONCLUSIONSOur results suggest that tamoxifen down-regulates survivin expression in HepG2 cells and it is mediated by transcriptional and post-transcriptional level via PI3K/Akt/mTOR pathway to induce apoptosis.

Antibiotics, Antineoplastic ; pharmacology ; Antineoplastic Agents, Hormonal ; pharmacology ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Down-Regulation ; Drug Synergism ; Hep G2 Cells ; Humans ; Inhibitor of Apoptosis Proteins ; genetics ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; RNA, Messenger ; metabolism ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Signal Transduction ; Sirolimus ; pharmacology ; TOR Serine-Threonine Kinases ; antagonists & inhibitors ; metabolism ; Tamoxifen ; pharmacology

OBJECTIVETo assess the diagnostic value of 8 equations using different variables for determining the estimated glomerular filtration rate (eGFR) in patients with cardiovascular diseases.

METHODSGFR was estimated in 208 patients with cardiovascular diseases by (99m)Tc-DTPA dynamic renal imaging, and the eGFR was derived from 8 equations using different variables.

RESULTSIn patients with chronic kidney disease (CKD) stages 1-3, the eGFR calculated suing serum creatinine (SCr)-based equation was better correlated to GFR estimated by (99m)Tc-DTPA renal imaging than that derived from cystatin C (Cys C)-based equations, whereas in patients with CKD stages 4 and 5, the estimates by the latter equation showed a better correlation to GFR. Compared with (99m)Tc-DTPA renal imaging, MDRD-based equation and simple MDRD equation resulted in a higher eGFR in patients with CKD stages 4 and 5, the Rule equation had a lower eGFR in CKD stages 1 and 2, the Macisaac equation yielded a higher eGFR in CKD stages 2-5, and the Tan equation showed a higher eGFR in CKD stages 2 and 3. In patients with mild renal dysfunction, the Scr-based equation had a higher AUC(ROC) than Cys C-based equation, which was reversed in patients with severe renal dysfunction; the AUC(ROC) of the two equations were comparable in patients with moderate renal dysfunction. Compared with (99m)Tc-DTPA renal imaging, the modified MDRD equation and Arnal-Dade equation showed no significant difference in the eGFR in patients with CKD stages 1-5.

CONCLUSIONModified MDRD equation (or simple MDRD equation) and Arnal-Dade equation are superior to other calculation methods for estimating the GFR in Chinese patients with cardiovascular disease.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Cardiovascular Diseases ; physiopathology ; Child ; Chronic Disease ; Creatinine ; blood ; Cystatin C ; blood ; Female ; Glomerular Filtration Rate ; physiology ; Humans ; Kidney Diseases ; physiopathology ; Male ; Middle Aged ; Young Adult