Objective To investigate a potential role for ORF50 promoter of human herpesvirus 8(HHV 8)in reactivation of HHV 8 in primary effusion lymphoma(PEL)BC 3 cells by HIV 1 related inflammatory cytokine,hepatocyte growth factor/scatter factor(HGF/SF). Methods The persistent stimulation of BC 3 was performed by recombinant HGF/SF and treated BC 3 cells were collected at the 3 rd and 7 th day after persistent stimulation respectively. Northern blot, quantitative PCR(real time PCR), and electron microscopy(EM)were carried out to detect the expression of mRNA of minor capsid protein ORF26 and the presence of viral particles of HHV 8 from treated BC 3 cells. Co transfection of BC 3 and human umbilical veil endothelial cells (HUVECs) with HHV 8 ORF50 promoter driven luciferase construct built previously by us and recombinant expression plasmid named pEV containing HIV 1 Tat coding gene was performed and stimulation of co transfected cells with recombinant HGF/SF and/or HIV 1 recombinant gp120 was conducted at 24 hours after transfection to induce luciferase gene expression. Then relative luciferase activity unit(RLU) was calculated at 24 hours after stimulation. In the meantime, stimulation of co transfected cells with 12 O tetradecanoylphorbol 13 acetate(TPA) was performed to be used as positive control. Results It was found that HGF/SF induced an 4.1 folds increase in mRNA expression of ORF26 when it was added individually to BC 3 cells at the 7 th day after persistent stimulation. Viral particles of HHV 8 were readily identified in BC 3 cells stimulated with HGF/SF at the 7 th day with EM analysis. Recombinant HGF/SF could upregulate promoter activity of HHV 8 ORF50 promoter in BC 3 and HUVECs cells and however, HIV 1 Tat and gp120 failed to act synergistically with HGF/SF to enhance ORF50 promoter activity in HUVECs. Conclusions The results show that HIV 1 related inflammatory cytokine HGF/SF is responsible for the induction of HHV 8 lytic cycle replication and the induction may be, at least partly mediated by ORF50 promoter of HHV 8.

2600 Anthers from T0 modified cry1 Ac-transgenic rice lines of Minghui 81, an elite restoring line of commercial CMS indica hybrid rice, were cultured on SK3 media. 83 green plantlets were recovered, 43 double haploid (DH) and 40 haploid among them. Results of PCR analyzes indicated that 55 plants of 83 were harbored the cry1Ac gene, and the ratio of cry1Ac-positive against cry1Ac-negative was 2:1 (55/28). 36 putative transgenic DH plants were further confirmed by Southern blot. ELISA detection showed that Cry1Ac level in different transgenic rice plants of the same cry1Ac-DH clone was almost equal and the highest one amount to 0.25% of the total soluble protein. Pest insect-resistant bioassay at field trials demonstrated that some of the homozygous cry1Ac-transgenic rice plants not only showed high-level resistance against striped stem borer (Chilo suppressalis) but also retained elite agronomy characters. These results demonstrated that rice anther culture has a great value in rice molecular breeding.
Animals ; Bacillus thuringiensis ; genetics ; Bacterial Proteins ; genetics ; metabolism ; Bacterial Toxins ; Blotting, Southern ; Culture Techniques ; DNA, Plant ; genetics ; Endotoxins ; genetics ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Hemolysin Proteins ; Immunity, Innate ; genetics ; Moths ; growth & development ; Oryza ; genetics ; growth & development ; parasitology ; Plant Structures ; genetics ; growth & development ; parasitology ; Plants, Genetically Modified

OBJECTIVETo observe the effects of siwu tang on protein expression of bone marrow of blood deficiency mice and provide the theoretical and experimental basis for understanding the molecular mechanism of blood enriching function of siwu tang.

METHODBlood deficiency mice were established by using 3.5 Gy 60Co gamma-ray. With proteomic technologies including two-dimensional electrophoresis, image analysis, in-gel digestion, peptide mass fingerprinting and bioinformatics the proteins of bone marrow of blood deficiency mice were isolated, analyzed, and identified.

RESULTSiwu tang could reverse 10 up-regulated and 4 down-regulated proteins of blood deficiency mice bone marrow. Seven of the proteins were likely to be lymphocyte specific protein 1, proteasome 26S ATPase subunit 4, hematopoietic cell protein-tyrosine phosphatase, glyceraldehyde-3-phosphate dehydrogenase, growth factor receptor binding protein 14 and lgals12 respectively.

CONCLUSIONSiwu tang can regulate the protein expression of bone marrow of blood deficiency mice and thus promote the growth and differentiation of hematopoietic cells and then exert its effects on blood enriching function.

Adaptor Proteins, Signal Transducing ; Animals ; Drugs, Chinese Herbal ; pharmacology ; Female ; Medicine, Chinese Traditional ; Mice ; Mice, Inbred C57BL ; Phosphoproteins ; blood ; Proteins ; metabolism ; Proteome ; metabolism ; Proteomics ; methods ; Radiation Injuries, Experimental ; blood ; Whole-Body Irradiation

Objective To analyze three different classification criteria, the clinical characteristics of antiphospholipid syndrome(APS)in a cohort of Chinese patients. Methods From January 1996 to October 2006, APS patients diagnosed with different classification criteria were retrospectively studied. Results There were totally 120 APS patients fulfilled at least one criterion, One hundred and one patients fulfilled the 1988 Asherson criteria, 96 patients fulfilled the 1999 Sapporo criteria, and 115 patients fulfilled the 2006 Sydney criteria. The ratio of male to female in a cohort of 115 definite APS patients was 1 to 10.5. The mean period of the disease until entry into the study was 82.6 months, the mean age at study entry was(41?12)years. Ninety patients had thrombosis episodes, among which the most common presenting manifestations were deep venous thrombosis, stroke and skin vasculitis. Forty-six of 92 married women in our cohort had fetal morbidity. Catas- trophic APS occurred in 7 patients. The presence of anticardiolipin antibodies(aCL)was detected in 86 pa- tients, anti-beta-2 glycoproteinⅠantibodies in 58 patients and lupus anticoagulant(LA)in 27 patients. Conclusion The most common presenting manifestations are deep venous thrombosis, stroke and cutaneous manifestations. The sensitivity of Sydney classification criteria is improved by adding anti-beta-2 glycopreteinⅠantibody as one of the laboratory criteria. However, primary APS patients who only presented with thrombo- cytupenia and positive laboratory tests could not satisfy this criterion. In addition, the significance of autoanti- bodies to some coagulant factors in APS needs further study.

To study the effect of Siwu decoction on the function and expression of P-glycoprotein (P-gp) in Caco-2 cells. The Real-time quantitative poly-merase chain reaction (Q-PCR) was used to analyze the mRNA expression of MDR1 gene in Caco-2 cells. Flow cytometer was used to study the effect of Siwu decoction on the uptake of Rhodamine 123 in Caco-2 cells, in order to evaluate the efflux function of P-gp. Western blotting method was used to detect the effect of Siwu decoction on the P-gp protein expression of Caco-2 cells. Compared with the blank control group, after Caco-2 incubation with Siwu decoction at concentrations of 3.3, 5.0, 10.0 g x L(-1) for 24, 48, 72 h, the mRNA expression of MDR1 was up-regulated, suggesting the effect of Siwu decoction in inducing the expression of MDR1. After the administration with Siwu decoction in Caco-2 cells for 48 h, the uptake of Rhodamine 123 in Caco-2 cells decreased by respectively 16.6%, 22.1% (P < 0.05) and 45.4% (P < 0.01), indicating that the long-term administration of Siwu decoction can enhance the P-gp efflux function of Caco-2 cells. After the incubation of Caco-2 cells with Siwu decoction for 48 h, the P-gp protein expression on Caco-2 cell emebranes, demonstrating the effect of Siwu decoction in inducing the protein expression of P-gp.
ATP Binding Cassette Transporter, Sub-Family B ; genetics ; metabolism ; ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; metabolism ; Caco-2 Cells ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Up-Regulation ; drug effects

Dioscin has a wide range of biological effects and broad application prospects. However the studies concerning the toxicology and mechanism of dioscin is small. This article is to study the hepatotoxicity of dioscin and the effect of dioscin treatment on expression of aryl hydrocarbon receptor (AhR) mRNA and CYP1A mRNA and protein in HepG2 cells in vitro. Dioscin 0.5-32 µmol · L(-1) exposed to HepG2 cells for 12 h, cell viability was examined by CCK-8 assay and the release rate of lactate dehydrogenase (LDH) was to evaluate cell membrane damage. HepG2 cells morphologic changes were quantified by inverted Microscope, and the effect on production of reactive oxygen species (ROS) was detected by flow cytometry. The mRNA expression of CYP1A and AhR was evaluated by RT-RCR. The protein expression of CYP1A1 was detected by western blot. The cell viability was significantly inhibited after HepG2 cells were exposed to dioscin 0.5-32 µmol · L(-1). Compared with the control, the LDH release rate and ROS were significantly increased. The expression of CYPlA and AhR mRNA was increased. The expression of CYP1Al protein was increased after dioscin treatment, and resveratrol, an AhR antagonist, could downregulate the expression of CYP1A1. It follows that large doses dioscin has potential hepatotoxicity. The possible mechanism may be dioscin can active aryl hydrocarbon receptor (AhR) and induce the expression of CYP1A.
Cell Survival ; drug effects ; Chemical and Drug Induced Liver Injury ; etiology ; Cytochrome P-450 CYP1A1 ; genetics ; Diosgenin ; analogs & derivatives ; toxicity ; Hep G2 Cells ; Humans ; L-Lactate Dehydrogenase ; secretion ; RNA, Messenger ; analysis ; Reactive Oxygen Species ; metabolism ; Receptors, Aryl Hydrocarbon ; genetics

To research the effect of Ginseng Radix et Rhizoma and Aconiti Lateralis Radix Praeparata compatibility on cardiac toxicity in rats by UPLC-Q-TOF/MS, and explore the endogenous markers and molecule mechanism. Different compatibility of Shenfu decoction were given to male Wistar rats at dosage of 20 g · kg(-1) for 7 days, collected the serum, and analyze the endogenous metabolites effected by Shenfu formulation by principal component analysis and partial least-squares analysis. Results showed that content of glutathione, phosphatidylcholine and citric acid decreased in mixed-decoction group, while ascorbic acid, uric acid, D-galactose, tryptophan, L-phenylalanine increased. The results showed cardiac toxicity of Aconiti Lateralis Radix Praeparata in Shenfu mixed-decoction. Shenfu co-decoction group showed a similar or weaker trend compared with control group, but most of them do not have a statistically significant. The results indicated the scientific basis of Shenfu compatibility by comparison of co-decoction group with mixed-decoction group. Shenfu compatibility can reduce cardiac toxicity induced by Aconiti Lateralis Radix Praeparata, and citric acid, glutathione, phosphatidyl choline, uric acid might be regarded as potential markers of cardiotoxicity.
Animals ; Biomarkers ; Cardiotoxicity ; Drugs, Chinese Herbal ; toxicity ; Glutathione ; blood ; Least-Squares Analysis ; Male ; Metabolomics ; methods ; Principal Component Analysis ; Rats ; Rats, Wistar

OBJECTIVETo explore the effect of Astragalus membranaceus (AM) on T-helper cell type 1 (Thl) specific transcription factor T-box expressed in T cells (T-bet) expression and Thl/Th2 equilibrium.

METHODSThe levels of T-bet mRNA in peripheral blood mononuclear cells (PBMCs) from 15 patients with asthma and 15 healthy subjects were determined by reverse transcription-polymerase chain reaction (RT-PCR). PBMCs in asthma patients were incubated with AM and then the concentration of interferon gamma (IFN-gamma) and interleukin-4 (IL-4) in the supernate before and after AM intervention were determined by ELISA. The numbers of CD4 + CCR3 + and CD4 + CCR5 + cells were counted by flow cytometry.

RESULTSThe expression of T-bet mRNA and the level of IFN-gamma were lower, but level of serum IL-4 was higher in asthma patients when compared with those in healthy subjects respectively. After AM (60 microg/ml) intervention, the former two parameters raised and showed a positive correlation between them, while the level of IL-4 was decreased. The mean percentage of CD4 + CCR3 + cells in asthma patients was significantly higher but that of CD4 + CCR5 + cells was lower when compared with those in healthy subjects respectively. After AM intervention, the abnormal change in the two indexes was improved to certain extent, showing a reversing status of Th2 polarization.

CONCLUSIONAM could increase the expression of T-bet mRNA and Thl cytokines such as IFN-Y, and might reverse the Th2 predominant status in asthma patients.

Adult ; Asthma ; drug therapy ; immunology ; Astragalus membranaceus ; Cell Polarity ; Cross-Sectional Studies ; Female ; Humans ; Interferon-gamma ; biosynthesis ; blood ; Interleukin-4 ; blood ; Male ; Middle Aged ; Phytotherapy ; RNA, Messenger ; analysis ; Receptors, CCR3 ; Receptors, CCR5 ; blood ; Receptors, Chemokine ; blood ; T-Box Domain Proteins ; genetics ; Th1 Cells ; drug effects ; immunology ; Up-Regulation

OBJECTIVETo screen active components in Compound Danshen (CD) based on pregnane X receptor-cytochrome P450 3A4 (PXR-CYP3A4).

METHODSBy using PXR-CYP3A stable transfection human hepatoblastoma G2 (HepG2) cell lines engineering cell strain combined reporter genes technology, active components that induce or inhibit PXR-CYP3A4 paths in CD were screened, and confirmed at the level of enzymic activities. The experiment was divided into the positive control group (RIF 10 micro mol/L), the DMSO group (DMSO 0.1%), each dose of treatment groups (ginsenoside Rc, Rf, Rb2, Rg2, F2, F1, tanshinone I , isoborneol 5, 10, 25, 50, 100, and 200 micro mol/L; each with six duplicates). Cells medium was removed 36, 48, and 60 h after treatment. The activity of CYP3A4 was then determined in the supernant and the fold induction was calculated.

RESULTSCompared with the DMSO group, the fold induction increased when ginsenoside Rc, Rf, Rb2, Rg2, F2, F1, tanshinone I , and isoborneol 50 and 100 micro mol/L was respectively intervened for 36, 48, and 60 h (P <0.05). When cells were treated with isoborneol 200 micro mol/L for 48 and 60 h,the fold induction of ginsenoside Rb2, Rg2, and F1 was significantly higher than that of the RIF group (P <0.05). Enzymic activity results showed that ginsenoside Rc, Rf, Rb2, F2, and F1 could increase the enzyme activity of CYP3A4 at 48 h (P <0.05).

CONCLUSIONGinsenoside Rc, Rf, Rb2, F2, F1, tanshinone I, and isoborneol in DC could induce CYP3A4 enzymes.

Cytochrome P-450 CYP3A ; metabolism ; Diterpenes, Abietane ; Drugs, Chinese Herbal ; chemistry ; Genes, Reporter ; Ginsenosides ; metabolism ; Hep G2 Cells ; Humans ; Receptors, Steroid ; metabolism ; Salvia miltiorrhiza ; Transfection

To study the effect of Panax notoginseng saponins (PNS) on liver drug metabolic enzyme activity, mRNA and protein expressions in rats. Male Wistar rats were randomly divided into nine groups. After administration of the test drugs, their liver microsomes, liver total RNA and total protein were extracted to detect the regulating effect of PNS on liver drug metabolic enzyme activity-related subtype enzymatic activity, mRNA and protein expression by substrate probe, quantitative PCR and Western Blot technology. The result of this experiment was that PNS could significantly induce CYP1A2 and CYP2E1 enzyme activity, mRNA expression, CYP2E1 protein expression level. PNS significantly induced CYP3A mRNA expression, but with no significant effect in CYP3A enzyme activity level. PNS had no significant effect CYP1A1 and CYP2B mRNA expressions and enzyme activity levels. PNS had selective regulations on different P450 subtypes, and the major subtypes were CYP1A2 and CYP2E1. In clinical practice, particularly in the combination with CYP1A2 and CYP2E1 metabolism-related drugs, full consideration shall be given to the possible drug interactions in order to avoid potential toxic and side effects. Meanwhile, whether the induction effect of CYP2E1 gets involved in ginsenoside's effect incavenging free radicals deserves further studies.
Animals ; Cytochrome P-450 Enzyme System ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Liver ; drug effects ; enzymology ; Male ; Microsomes, Liver ; drug effects ; enzymology ; Panax notoginseng ; chemistry ; Rats, Wistar ; Saponins ; pharmacology