A temperature control system for quantitive polymerase chain reaction (PCR) is presented in the paper with both software and hardware configuration. The performance of the control system has been improved by optimizing the software and hardware design according to the system's properties. The control system has been proven to have a good repeatability and reliability as well as high control precision.
Equipment Design ; Microcomputers ; Polymerase Chain Reaction ; instrumentation ; Software ; Software Design ; Temperature

According to the designed specific primers of gene fragment based on the Salvia miltiorrhiza transcriptome data, with the method of reverse transcription polymerase chain reaction (RT-PCR), this study cloned full-length cDNA sequence of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase gene from Salvia miltiorrhiza bge.f.alba, this sequence is named as SmHDS and its GenBank registration number is KJ746807. SmHDS, 2 529 bp long, contains an ORF of 2 229 bp, encodes 742 amino acids, including 5' UTR 170 bp and 3' UTR 130 bp. Using bioinformatics software, having made a homology analysis of the obtained sequence, we can have a conclusion that SmHDS have a close genetic relationship with HDS of Salvia miltiorrhiza. Analysis result of prokaryotic expression revealed that in Escherichia coli, SmHDS expressed target proteins which in size are comparable with the protein predicted. Meanwhile, the 4 factors which can influence the protein expression were optimized, the 4 factors are inducing temperature, inducing time, IPTG concentrations and density of inducing host bacterium (A600). The optimal expression conditions of SmHDS were 30 degrees C until the A600 is 0.6, and add IPTG to a final concentration of 0.2 mmol x L(-1), and the induction time of 20 h. It provides theoretical basis for the further study of the function of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase in the biosynthesis of tanshinone compounds.
Cloning, Molecular ; DNA, Complementary ; genetics ; Diterpenes, Abietane ; biosynthesis ; Enzymes ; biosynthesis ; genetics ; Escherichia coli ; metabolism ; Plant Proteins ; biosynthesis ; genetics ; Salvia miltiorrhiza ; enzymology ; genetics

Soxhlet extraction is a common method of sample preparation. However, there has been no discussion about the efficiency of Soxhlet extraction from different batches and the factors that cause content fluctuation. In this study, Panax ginseng was selected as a model sample. Soxhlet extraction by means of a water bath, which has always been neglected, was identified as a novel key factor in the poor repeat-ability in different batches of Soxhlet extraction, as it can affect the siphon times and reflux time, which have been positively correlated with the ginsenoside contents. By substituting round bottom flasks in the same column, the relative standard deviation of the most fluctuated compound, ginsenoside Rb1, was decreased from 24.6% to 5.02%. Scanning electron microscopy analysis confirmed that the breakdown of the surface of the ginseng powder in the Soxhlet extraction led to a better dissolution of ginsenosides, indicating that chloroform may promote the extraction of ginsenosides by disrupting the cell structure. Moreover, 70% methanol was regarded as the better solvent for extracting the ginsenosides. Overall, this work offers a practical and effective protocol for improving the accuracy and repeatability of Soxhlet extraction methodology for ginsenosides and other analytes.

AIMTo study the transfection and anti-hepatitis B virus (HBV) effect of the co-modified hepatocytes-targeting cationic liposomes encapsulating anti-HBV antisense oligonucleotides (asON) , and to investigate the transfection mechanisms of the liposomes.

METHODSDipalmitoylphosphatidylcholine (DPPC) and 3beta-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) were used as the lipids, beta-sitosterol-beta-D-glucoside (sito-G) and Brij 35 were used to modify the liposomes. Flow cytometry (FCM), fluorescence microscopy and enzyme-linked immunosorbent assay (ELISA) were utilized to evaluate the transfection improvement of the asON encapsulated in the liposomes in primary rat hepatocytes and the antigens inhibition activity in HepG 2.2.15 cells. The transfection mechanisms were evaluated based on the influence of wortmannin, nigericin, and asialofetuin on the antigens inhibition in HepG 2.2.15 cells by ELISA.

RESULTSThe co-modification with sito-G and Brij 35 significantly improved the transfection of the liposomes in primary rat hepatocytes and antigens inhibition effect in HepG 2.2.15 cells. Both transfection efficiency and antigens inhibition effect showed to be concentration-dependent with the asON-encapsulating liposomes. In fluorescence microscopy, the transfected cells showed strong fluorescence in primary rat hepatocytes, especially in the nuclei. Wortmannin, nigericin and asialofetuin decreased the antigens inhibition of the asON-encapsulating liposomes to different levels. Cationic liposomes modification with sito-G and Brij 35 could improve the transfection and antigens inhibition effect of the asON. The transfection mechanisms of the co-modified liposomes included endocytosis and membrane fusion. The ligand sito-G was confirmed to be able to enhance asialoglycoprotein receptor (ASGPR)-mediated endocytosis.

CONCLUSIONCo-modified hepatocytes-targeting cationic liposomes would be a specific and effective carrier to transfer asON into hepatocytes.

Androstadienes ; pharmacology ; Animals ; Asialoglycoproteins ; pharmacology ; Cell Line, Tumor ; Cell Nucleus ; metabolism ; Cell Survival ; Cells, Cultured ; Endocytosis ; drug effects ; Female ; Fetuins ; Flow Cytometry ; Hepatitis B Antigens ; metabolism ; Hepatitis B virus ; genetics ; immunology ; Hepatocytes ; cytology ; drug effects ; metabolism ; Humans ; Liposomes ; Microscopy, Fluorescence ; Nigericin ; pharmacology ; Oligonucleotides, Antisense ; chemistry ; genetics ; Polyethylene Glycols ; chemistry ; Rats ; Rats, Wistar ; Sitosterols ; chemistry ; Transfection ; methods ; alpha-Fetoproteins ; pharmacology

Aged ; Arthroplasty, Replacement, Knee ; methods ; Female ; Femur ; surgery ; Humans ; Osteoarthritis ; surgery ; Osteotomy ; methods

Objective To explore the activation and function of Janus protein-tyrosine kinase (JAK)/ signal transducer and activator of transcription (STAT1) signal transduction pathway in kidney,lung and brain of MRL/lpr mice.Methods MRL/lpr mice with systemic lupus erythematosus (SLE) were studied at the age of 12 weeks up.Non-SLE MRL/lpr mice were used as controls.We used phosphospecific antibodies to detect STAT1 activation in kidney,lung and brain by immunohistochemistry and Western blots.Gene expression of the STAT induced feedback inhibitors of cytokine signaling 1 (SOCS-1) was investigated by SYBR green I real-time reverse transcriptase polymerase chain reaction (PCR).Results Phosphorylation of STAT1 protein was markedly activated in these three organs,although renal and pulmonary STAT1 activation were much more evidently activated.SOCS-1 gene expression increased in all three organs,while renal SOCS-1 gene expres- sion increased less than lung and brain.Conclusion The activation of JAK/STATI signal transduction path- way may be pathogenic in the organ involvement and progression of SLE.The pathogenesis of lupus nephritis may also be associated with the down-regulation of SOCS-1 feedback inhibition.

Objective To establish a LC-MS/MS method for pharmacokinetics study of methylergonovine in healthy Chinese volunteers after single and multiple-dose administration of methylergonovine maleate tablets. Methods 6-hydroxyflavone was used as internal standard.The separation was achieved on a Waters Xterra C18 (2. 1 mm × 100 mm, 3. 5 μm) with a mobile phase consisting of 0. 05％ ammonium acetate and methanol-acetonitrile (80:20) solution. At a flow rate of 0. 3 m L·min-1 within 12 min. Methylergonovine and 6-hydroxyflavone were measured by ESI in positive electron mode using multiple reaction monitoring (MRM) . The extracted ions monitored following MRM transitions were m/z 340. 3 →223. 2 for methylergonovine and m/z 239. 2 →129. 1 for 6-hydroxyflavone. Plasma samples were pretreated by liquid-liquid extraction. Results The calibration curve was linear within the range of 0. 1-20. 0 ng·m L-1. The Lower limit of quantitation was 0. 1 ng·m L-1 and CV％ of intra-and inter-day were less than 15％. The plasma samples were stable at room temperature (25 ℃) for 4 h, at-70℃ for 4 months and during three freeze-thaw cycles. There was no accumulationafter ultiple-dose of methylergonovine maleate tablets.Conclusion The method was proved to be accurate and sensitive suitable for the pharmacokinetics of methylergonovine in volunteers after oral administration of 0. 125, 0. 25, 0. 5 mg methylergonovine maleate tablets.

OBJECTIVETo detect the expression of the glucocorticoid receptor-alpha/beta (GR-alpha/beta) mRNA in nasal polyp and normal nasal mucosa and investigate the significance of receptor-alpha/beta in GC-insensitive and GC-sensitive patients.

METHODSThe expression of GR-alpha/beta mRNA in 40 samples of nasal polyp and 30 normal nasal mucosa was examined by using Fluorescent quantitative PCR.

RESULTSForty cases were screened from the 80 cases after follow-up, including 26 cases of GC-sensitive and 14 cases of GC-insensitive. the levels of GR-beta mRNA in group of GC-insensitive [(5.72 +/- 0.58) x 10(2) copy/microg] were significantly higher than that of GC-sensitive [(4.82 +/- 0.28) x 10(2) copy/microg, t = -6.65, P < 0.01] and normal nasal mucosa [(4.44 +/- 0.35) x 10(2) copy/microg, t = -9.19, P < 0.01]. There was difference between group of GC-insensitive and other two groups of GC-sensitive and normal nasal mucosa (P < 0.01). The ratio of GR-alpha/GR-beta showed significant difference between the group of GC-sensitive (829.42 +/- 67.36) and that of GC-insensitive (535.70 +/- 89. 00, t = 11.74, P < 0.01).

CONCLUSIONSThe high expression of GR-beta mRNA and low expression of GR-alpha mRNA in nasal polyp show the status of "insensitive" in chronic sinusitis and nasal polyp. The GR-beta may play an important role in evaluating the effect of glucocorticoid in chronic sinusitis and nasal polyps.

Adult ; Case-Control Studies ; Chronic Disease ; Female ; Humans ; Male ; Nasal Mucosa ; metabolism ; Nasal Polyps ; metabolism ; RNA, Messenger ; genetics ; Receptors, Glucocorticoid ; metabolism ; Sinusitis ; metabolism

OBJECTIVETo clone, express and identify the mecA fragment which encoded penicillin binding protein 2a (PBP2a) from methicillin-resistant staphylococcus aureus (MRSA) isolated from patients by gene recombination method.

METHODSAccording to the sequence of mecA gene recorded in GenBank, the primer of mecA fragment which encoded amino acids 25 - 668 of PBP2a was designed. Then the mecA fragment was amplified by PCR and cloned into pQE30 plasmid. After being identified by enzyme digestion and sequencing, the recombinant plasmid was transferred into E. coli M15 [pREP4], and then its expression was induced by 1 mmol/L Isopropy-beta-D-Thiogalactoside (IPTG). The expression product was analyzed by SDS-PAGE, protein sequencing and mass spectroscopy.

RESULTSThe recombinant pQE30- mecA had been successfully constructed. The result of sequencing showed that the mecA fragment had 1932 bases, including 9 bases undergoing mutation. After being induced for 6 hours by IPTG, the soluble protein in M15 (pQE30- mecA), with a relative molecular weight of 74 x 10(3), was found by SDS-PAGE. The soluble protein had been confirmed to be PBP2a after identification.

CONCLUSIONThe soluble PBP2a of MRSA isolated from patients is expressed successfully by gene recombinant technology.

Base Sequence ; Cloning, Molecular ; Gene Expression ; Humans ; Methicillin Resistance ; genetics ; Methicillin-Resistant Staphylococcus aureus ; genetics ; isolation & purification ; Microbial Sensitivity Tests ; Penicillin-Binding Proteins ; genetics ; metabolism ; Peptide Synthases ; genetics ; metabolism ; Plasmids

OBJECTIVETo examine the effect of cinobufacini on rat thoracic aorta and its mechanism.

METHODSIsolated rat thoracic aorta was perfused and isometric tension was recorded by organ bath technique before and after cinobufacini treatment.

RESULTCinobufacini induced contraction of isolated thoracic aorta with or without endothelium in a concentration-dependent manner (at concentration of 2.5,5.0,7.5,10.0 g/L). The vasoconstriction effect of cinobufacini was more potent in endothelium-denuded aorta ring [(16.3+/-3.39)%, (52.5+/-7.70)%, (60.9+/-8.84)%, (69.2+/-11.34)%] than in endothelium-intact aorta ring [(6.2+/-2.07)%, (14.7+/-4.91), (17.6+/-5.86)%, (20.3+/-6.78)% (P<0.01)]. Its contractile effect was attenuated in Ca(2+)-free solution (about 1/10 of that in buffer with 1.25 mmol/L CaCl(2)) or by the treatment with verapamil (10(-7)mol/L), an L-type calcium channel antagonist. Cinobufacini induced contraction on the endothelium-intact rat aorta was augmented by pretreatment with L-NAME (10(-4)mol/L), a nitric oxide synthase inhibitor.

CONCLUSIONCinobufacini contracts rat thoracic aorta by opening the voltage-dependent Ca(2+) channel and increasing Ca(2+) influx into vascular smooth muscle. Cinobufacini can also stimulate the release of vascular relaxant factor, nitric oxide, from the endothelium and thus antagonize cinobufacini-induced contraction.

Animals ; Aorta, Thoracic ; drug effects ; Bufanolides ; pharmacology ; Endothelium, Vascular ; drug effects ; metabolism ; In Vitro Techniques ; Male ; Nitric Oxide ; biosynthesis ; Rats ; Rats, Sprague-Dawley ; Vasoconstriction ; drug effects ; Vasoconstrictor Agents ; pharmacology