OBJECTIVETo establish a rapid, sensitive and efficient detection method for tobacco mosaic virus (TMV), and provide technical support of TMV detection of Rehmannia glutinosa f. hueichingensis. The virus-free plantlets could be produced on a large scale to ameliorate breed degeneration caused by viral disease.

METHODSpecific primers were designed based on the conserved region of coat protein(CP) gene of TMV. Immunocapture RT-PCR (IC-RT-PCR) was employed to detect TMV and the sequence of the products was detected.

RESULTThe expected nucleotide acid fragments were amplified by IC-RT-PCR. The homology of nucleotide acid sequence and amino acid sequence were 95.29% and 96.7% between the PCR products and the CP gene of TMV (accession number AY555269).

CONCLUSIONThe method was established for the detection of TMV in R. glutinosa f. hueichingensis by IC-RT-PCR. This detection combined molecular biology technology with immunology, was convenient for a quick, sensitive and simple detection of TMV.

Amino Acid Sequence ; Base Sequence ; Molecular Sequence Data ; Rehmannia ; virology ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Tobacco Mosaic Virus ; genetics ; immunology ; isolation & purification

Objective To construct a human tumor necrosis factor (hTNF-a) plasmid and identify it to optimize the fermentation conditions of hTNF-α protein so as to achieve high expression in Escherichia coli.Methods The gene of hTNF-a was cloned into pET24a vector to obtain the pET24a-hTNF-a expression plasmid that was transformed into Escherichia coli BL21(DE3),and the expression conditions of BL21 (DE3) were optimized.Results The plasmid of pET24a-hTNF-α was successfully constructed and identified by PCR and digestion,which was consistent with the target fragment hTNF-α.The plasmid was transformed into Escherichia coli BL21(DE3),the best induced expression conditions of Escherichia coli BL21 (DE3) were as follows:M9+LB medium,37℃,0.5 mmol/L IPTG,pH =7.5,and induction time was 5 h.The results showed that dry weight of the cells and the rate of TNF were increased by 2.56 times and 3.68 times,respectively,and the expression rate of hTNF-α was increased by 3.49 times from 9.38％ to 32.74％.Conclusion The optimal conditions for the expression of plasmid pET24a-hTNF-α in Escherichia coli were determined.

The aim of the present study was to investigate the effect of a short-term (3-day) simulated microgravity with and without daily dorsoventral gravitation (-G(x)) for 1 h on myogenic tone and vasoconstrictor responsiveness of the middle cerebral artery and mesenteric third-order small artery in rats. The tail-suspension (SUS) model was used to simulate cardiovascular deconditioning due to microgravity. Daily restoring to normal standing (STD) posture for 1 h was adopted to provide -G(x) as the countermeasure. Segments of middle cerebral artery and mesenteric third-order small artery were isolated and cannulated. Vascular diameters in response to increased intraluminal pressure (from 20 mmHg to 120 mmHg, by 20 mmHg steps) of isolated arteries under no-flow conditions were recorded by a Pressure Myograph System in both physiologic salt solution (PSS) (active diameter, Da) and calcium-free PSS (passive diameter, Dp). The myogenic tone was calculated by (Dp-Da)/Dpx100%. Vasoconstrictor responsiveness of the isolated middle cerebral artery to serotonin and that of small mesenteric artery to phenylephrine were assessed in the PSS under an intraluminal pressure of 40 mmHg. The results showed that SUS induced an enhancement of the myogenic tone and vasoconstrictor responsiveness in the isolated middle cerebral artery but a depression of those in the small mesenteric artery. Daily STD for 1 h prevented the depression of myogenic tone and vasoconstrictor responsiveness in the small mesenteric artery, but did not prevent the functional enhancement in the middle cerebral artery. These data suggest that a short-term simulated microgravity may result in different alterations in the function of the cerebral artery and the resistance vessel in the hind-body. Moreover, only the decrease of function in these resistance vessels, not in the cerebral arteries, can be prevented by such a countermeasure of daily STD for 1 h.
Animals ; Cerebral Arteries ; pathology ; Hindlimb Suspension ; Mesenteric Arteries ; pathology ; Pressure ; Rats ; Serotonin ; pharmacology ; Vascular Resistance ; Vasoconstriction ; Weightlessness Simulation

Ultrafine powder and cell wall-broken powder of herbal medicine lack of the morphological characters and microscopic identification features. This makes it hard to identify herb's authenticity with traditional methods. We tested ITS2 sequence as DNA barcode in identification of herbal medicine in ultrafine powder and cell wall-broken powder in this study. We extracted genomic DNAs of 93 samples of 31 representative herbal medicines (28 species), which include whole plant, roots and bulbs, stems, leaves, flowers, fruits and seeds. The ITS2 sequences were amplified and sequenced bidirectionally. The ITS2 sequences were identified using Basic Local Alignment Search Tool (BLAST) method in the GenBank database and DNA barcoding system to identify the herbal medicine. The genetic distance was analyzed using the Kimura 2-parameter (K2P) model and the Neighbor-joining (NJ) phylogenetic tree was constructed using MEGA 6.0. The results showed that DNA can be extracted successfully from 93 samples and high quality ITS2 sequences can be amplified. All 31 herbal medicines can get correct identification via BLAST method. The ITS2 sequences of raw material medicines, ultrafine powder and cell wall-broken powder have same sequence in 26 herbal medicines, while the ITS2 sequences in other 5 herbal medicines exhibited variation. The maximum intraspecific genetic-distances of each species were all less than the minimum interspecific genetic distances. ITS2 sequences of each species are all converged to their standard DNA barcodes using NJ method. Therefore, using ITS2 barcode can accurately and effectively distinguish ultrafine powder and cell wall-broken powder of herbal medicine. It provides a new molecular method to identify ultrafine powder and cell wall-broken powder of herbal medicine in the quality control and market supervision.
Cell Wall ; DNA Barcoding, Taxonomic ; DNA, Plant ; genetics ; DNA, Ribosomal Spacer ; genetics ; Drugs, Chinese Herbal ; analysis ; Phylogeny ; Plants, Medicinal ; classification ; genetics ; Powders ; Quality Control

OBJECTIVETo study erythrocyte oxidative stress status and its association with left to right shunt congenital heart disease (CHD) in children.

METHODSA total of 31 children with left to right shunt CHD were enrolled, including 7 cases of atrial septal defect (ASD), 12 ventricular septal defect (VSD), 4 patent ductus arteriosus (PDA), 6 patent foramen ovale (PFO), and 2 complete endocardial cushion defect. Twenty healthy age-matched (1 month to 3 years old) children severed as the control group. The contents of superoxide dismutase (SOD) and malonaldehyde (MDA) in erythrocytes were determined using ELISA. ESR was measured by Westergen. PaO(2) and PaCO(2) were measured by Blood Gas Analyzer (GEM Premier 3000).

RESULTSThe MDA content in erythrocytes in the CHD group was significantly higher, in contrast, SOD content was significantly lower than that in the control group (P<0.05). The CHD children with heart failure had more decreased SOD and more increased MDA contents compared with the control group (P<0.01). The SOD level was the highest in the PFO group and was the lowest in the complete endocardial cushion defect group. The SOD level in the PFO group was significantly higher than that in the ASD, VSD and complete endocardial cushion defect groups (P<0.05). The MDA level was the highest in the VSD group and was the lowest in the complete endocardial cushion defect group. There were significant differences in the MDA level among CHD subgroups (P<0.05). The ESR was negatively correlated to the SOD level (r=-0.191, P<0.05), while positively correlated to PaO(2) level in CHD children (r=0.216, P<0.05). There was a negative correlation between SOD and MDA levels (r=-0.312, P<0.05).

CONCLUSIONSOxidative stress exists in children with left to right shunt CHD. The SOD and MDA contents in erythrocytes can be used as markers for the assessment of severity of the disease.

Blood Gas Analysis ; Blood Sedimentation ; Child, Preschool ; Erythrocytes ; metabolism ; Female ; Heart Defects, Congenital ; metabolism ; Humans ; Infant ; Male ; Malondialdehyde ; blood ; Oxidative Stress ; Superoxide Dismutase ; blood

Objective To explore the effect of aspirin(ASP) combined with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) inducer TIC10 on apoptosis and autophagy of cervical cancer Siha and HeLa cells. Methods HPV16 positive squamous carcinoma Siha cells and HPV18 positive cervical adenocarcinoma HeLa cells were selected as research objects. Two cell lines were treated with ASP,TIC10 or two drugs combination. The effects of ASP and TIC10 on cell proliferation were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay,cell migration ability was detected by cell scratch assay,cell clone formation ability was detected by plate clone formation assay,and the levels of cleaved Caspase-3/8,LC3A/B,Beclin1 were detected by Western Blot. Results The MTT showed that the proliferations of Siha and HeLa cells were inhibited by APS in a concentration dependent manner (all P < 0. 01). After treated with TIC10 combined with ASP for 48 h,the cell proliferation inhibition rates of Siha and HeLa cells were higher than thosetreated with TIC10 alone (all P < 0. 01). After treated with TIC10 or ASP for 24 h, the migration rates of Siha and He- La cells in the experimental groups [ASP: (19. 61 ± 1. 17) ％,(23. 75 ± 0. 78) ％; TIC10: (16. 89 ± 1. 47) ％,(20. 59 ± 2. 01) ％]were lower than those of control group [(41. 18 ± 2. 01) ％,(40. 83 ± 3. 77) ％],all P < 0. 01. After two weeks of treatmentof ASP and TIC10,the CFE(colony-forming efficiency) rates of Siha and HeLa cells in the experimental groups [ASP: (24. 93 ± 2. 12) ％, (26. 47 ± 3. 30) ％, TIC10: (17. 33 ± 1. 50) ％,(19. 13 ± 4. 99) ％]were lower than those of control group [(69. 60 ± 3. 54) ％,(68. 40 ± 4. 20) ％],all P < 0. 01. Western blot assays showed that the expression of cleaved Caspase-3/8 and autophagy related protein LC3A/B,Beclin1 were higher than those of control group (all P < 0. 01). Conclusion ASP increases cervical cancer cells apoptosis with TRAIL inducer TIC10 through autophagy enhancement.

BACKGROUNDHeme oxygenase-1 (HO-1) can be induced by inflammatory cytokines, oxidation, ischemia, hypoxia, and endotoxins. As a "graft survival protective gene," HO-1 is a hot spot in organ transplantation research. However, the role of HO-1 gene expression in the function of human colon adenocarcinoma cell line (Caco-2) cells has not been reported previously.

METHODSThe role of HO-1 in the proliferation and migration of Caco-2 cells was analyzed using a stable HO-1 expression plasmid. We constructed a recombinant adeno-associated virus plasmid containing the HO-1 gene, heme oxygenase 1 (HMOX1), which was transfected into Caco-2 intestinal cells. We identified a number of target genes by global microarray analysis combined with real-time polymerase chain reaction (PCR) and chromatin immunoprecipitation assay.

RESULTSOur results showed that significant HO-1 upregulation was demonstrated in the Caco-2 cells after HO-1 transfection. Restoration of HO-1 expression promoted proliferation and invasion in vitro. The CTNND1 gene, a member of the armadillo protein family, was identified as a direct HO-1 target gene.

CONCLUSIONOverexpression of HO-1 promotes Caco-2 cell proliferation and migration by targeting the CTNND1 gene.

Caco-2 Cells ; Catenins ; genetics ; Cell Movement ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Heme Oxygenase-1 ; genetics ; physiology ; Humans ; Real-Time Polymerase Chain Reaction

Cardiac fibrosis occurs after pathological stimuli to the cardiovascular system. One of the most important factors that contribute to cardiac fibrosis is angiotensin II (Ang II). Accumulating studies have suggested that reactive oxygen species (ROS) plays an important role in cardiac fibrosis and sodium tanshinone IIA sulfonate (STS) possesses antioxidant action. We therefore examined whether STS depresses Ang II-induced collagen type I expression in cardiac fibroblasts. In this study, Ang II significantly enhanced collagen type I expression and collagen synthesis. Meanwhile, Ang II depressed matrix metalloproteinase-1 (MMP-1) expression and activity. These responses were attenuated by STS. Furthermore, STS depressed the intracellular generation of ROS, NADPH oxidase activity and subunit p47(phox) expression. In addition, N-acetylcysteine the ROS scavenger, depressed effects of Ang II in a manner similar to STS. In conclusion, the current studies demonstrate that anti-fibrotic effects of STS are mediated by interfering with the modulation of ROS.
Acetylcysteine/pharmacology ; Angiotensin II/*antagonists & inhibitors/pharmacology ; Animals ; Blotting, Western ; Cells, Cultured ; Collagen Type I/*metabolism ; Drugs, Chinese Herbal/*pharmacology ; Fibroblasts/*drug effects/metabolism ; Free Radical Scavengers/pharmacology ; Matrix Metalloproteinase 1/metabolism ; Myocardium/*cytology ; NADPH Oxidase/metabolism ; Oxidative Stress/drug effects ; Phenanthrenes/*pharmacology ; Rats ; Rats, Wistar ; Reactive Oxygen Species/metabolism

By electrophysiological methods, cultured Drosophila embryonic and larval central neurons have been widely used to study ion channels, neurotransmitter release and intracellular message regulation. Voltage-activated K(+) channels play a crucial role in repolarizing the membrane following action potentials, stabilizing membrane potentials and shaping firing patterns of cells. In this study, a mechanical vibration-isolation system was used to produce a sufficient number of acutely dissociated larval central neurons, of which the majority were type II neurons (2~5 microm in diameter). Using patch clamp technique, the whole-cell K(+) currents in type II neurons were characterized by containing a transient 4-AP-sensitive current (I(A)) and a more slowly inactivating, TEA-sensitive component (I(K)). According to their kinetic properties, five types of whole-cell K(+) currents were identified. Type A current exhibited primarily fast transient K(+) currents that activated and inactivated rapidly. The majority of the neurons, however, slowly inactivated K(+) currents with variable inactivation time course (type B current). Type C current, being present in a small number of the cells, was mainly composed of noninactivating components. Some of the neurons expressed both transient and slow inactivating components, but the slowly inactivating components could reach more than 50% of the peak current (type D current). Type E current showed distinct voltage-dependent activation properties, characterized by its bell-shaped activation curve. Type E current was inhibited by application of Ca(2+)-free solution or 0.1 mmol/L Cd(2+). Moreover, this novel current ran down much more rapidly than other types. These results indicate that different K(+) channels, which have different kinetic and pharmacological properties, underlie the whole-cell K(+) currents in type II neurons of Drosophila larval central nervous system.
Action Potentials ; Animals ; Cell Separation ; methods ; Drosophila ; metabolism ; physiology ; Larva ; cytology ; Membrane Potentials ; Neurons ; metabolism ; physiology ; Patch-Clamp Techniques ; Potassium ; physiology