Artemisnin is a novel sesquiterpene lactone with an internal peroxide bridge structure, which is extracted from traditional Chinese herb Artemisia annua L. (Qinghao). Recommended by World Health Organization, artemisinin is the first-line drug in the treatment of encephalic and chloroquine-resistant malaria. In the present study, transgenic A. annua plants were developed by overexpressing the key enzymes involved in the biosynthetic pathway of artemisinin. Based on Agrobacterium-mediated transformation methods, transgenic plants of A. annua with overexpression of both HDR and ADS were obtained through hygromycin screening. The genomic PCR analysis confirmed six transgenic lines in which both HDR and ADS were integrated into genome. The gene expression analysis given by real-time quantitative PCR showed that all the transgenic lines had higher expression levels of HDR and ADS than the non-transgenic control (except ah3 in which the expression level of ADS showed no significant difference compared with control); and the HPLC analysis of artemisinin demonstrated that transgenic A. annua plants produced artemisinin at significantly higher level than non-transgenic plants. Especially, the highest content of artemisinin was found in transgenic line ah70, in which the artemisinin content was 3.48 times compared with that in non-transgenic lines. In summary, overexpression of HDR and ADS facilitated artemisinin biosynthesis and this method could be applied to develop transgenic plants of A. annua with higher yield of artemisinin.
Artemisia annua ; genetics ; metabolism ; Artemisinins ; metabolism ; Biosynthetic Pathways ; Drugs, Chinese Herbal ; Mixed Function Oxygenases ; genetics ; Oxidoreductases ; genetics ; Plant Proteins ; genetics ; Plants, Genetically Modified ; genetics ; metabolism ; Plants, Medicinal ; genetics ; metabolism

OBJECTIVETo isolate exosomes from rabbit aqueous humor and to investigate their immunosuppression function.

METHODSAqueous humor was collected from 40 New Zealand rabbits and exosomes were isolated by fractional separation and ultracentrifugation methods; the morphology was studied with electron microscopy. The immunosuppressive-related proteins of exosomes were detected with Western blotting; their inhibitory effect on ConA-induced proliferation of T lymphocyte was estimation with CCK-8 cells proliferation assay.

RESULTSEight milliliters of aqueous humor were collected from 40 New Zealand rabbits and 200 μg exosomes was yielded. Under electron microscope, the exosomes had typical structure of lipid bi-layer with a diameter of 50-100 nm. The results of Western blotting showed that these exosomes expressed Hsp70, CD9 and Alix but not Grp94, presenting a typical exosomes protein profile. Moreover, exosomes expressed high level of TGF-β and significantly inhibited the proliferation of T lymphocytes.

CONCLUSIONImmunosuppressive exosomes can be isolated from rabbit aqueous humor, which may be involved in immunotolerance of the eye.

Animals ; Aqueous Humor ; immunology ; Cells, Cultured ; Exosomes ; immunology ; metabolism ; ultrastructure ; Female ; Immune Tolerance ; Male ; Rabbits ; T-Lymphocytes ; immunology

Atropa belladonna is a medicinal plant and main commercial source of tropane alkaloids (TAs) including scopolamine and hyoscyamine, which are anticholine drugs widely used clinically. Based on the high throughput transcriptome sequencing results, the digital expression patterns of UniGenes representing 9 structural genes (ODC, ADC, AIH, CPA, SPDS, PMT, CYP80F1, H6H, TRII) involved in TAs biosynthesis were constructed, and simultaneously expression analysis of 4 released genes in NCBI (PMT, CYP80F1, H6H, TRII) for verification was performed using qPCR, as well as the TAs contents detection in 8 different tissues. Digital expression patterns results suggested that the 4 genes including ODC, ADC, AIH and CPA involved in the upstream pathway of TAs, and the 2 branch pathway genes including SPDS and TRII were found to be expressed in all the detected tissues with high expression level in secondary root. While the 3 TAs-pathway-specific genes including PMT, CYP80F1, H6H were only expressed in secondary roots and primary roots, mainly in secondary roots. The qPCR detection results of PMT, CYP80F1 and H6H were consistent with the digital expression patterns, but their expression levels in primary root were too low to be detected. The highest content of hyoscyamine was found in tender stems (3.364 mg x g(-1)), followed by tender leaves (1.526 mg x g(-1)), roots (1.598 mg x g(-1)), young fruits (1.271 mg x g(-1)) and fruit sepals (1.413 mg x g(-1)). The highest content of scopolamine was detected in fruit sepals (1.003 mg x g(-1)), then followed by tender stems (0.600 mg x g(-1)) and tender leaves (0.601 mg x g(-1)). Both old stems and old leaves had the lowest content of hyoscyamine and scopolamine. The gene expression profile and TAs accumulation indicated that TAs in Atropa belladonna were mainly biosynthesized in secondary root, and then transported and deposited in tender aerial parts. Screening Atropa belladonna secondary root transcriptome database will facilitate unveiling the unknown enzymatic reactions and the mechanisms of transcriptional control.
Alkaloids ; biosynthesis ; genetics ; metabolism ; Atropa belladonna ; genetics ; metabolism ; Gene Expression Regulation, Plant ; genetics ; Hyoscyamine ; genetics ; metabolism ; Plants, Medicinal ; genetics ; metabolism ; Scopolamine Hydrobromide ; metabolism ; Tropanes ; metabolism

OBJECTIVETo prepare monoclonal antibodies (McAbs) against human mesenchymal stem cells (hMSCs) and to study their biological characteristics.

METHODSBALB/C mice were immunized with pooled hMSCs. McAbs were prepared by hybridoma technique and their biological characteristics were analyzed by indirect immunofluorescence, immunohistochemistry and flow cytometry.

RESULTFive hybridoma cell lines were successfully established, which secret McAbs specifically against hMSCs. Investigations showed that all these McAb reacted only to hMSCs and had no cross-reaction to other human cells, the relative affinities of 5 McAbs were 1x10(6) (ZUB1), 1x10(5) (ZUB4), 1x10(6) (ZUC3), ZUE12 (1x10(5)) and 1x10(5) (ZUF10), respectively. Isotype analysis showed that ZUB1, ZUE12, ZUF10 against the same isotype, while ZUC3, ZUB4 against other two different isotypes alone. Flow cytometric analysis showed that the positive expression rate of cultured hMSCs was 87.39% (ZUB1), 88.07% (ZUB4), 88.12% (ZUC3), 69.89% (ZUE12) and 83.67% (ZUF10).

CONCLUSIONThe prepared five McAbs can specifically react against hMSCs, which can be used for selection and study of hMCSs.

Animals ; Antibodies, Monoclonal ; biosynthesis ; Antibody Specificity ; Bone Marrow Cells ; cytology ; immunology ; Fluorescent Antibody Technique ; methods ; HL-60 Cells ; Humans ; Hybridomas ; secretion ; Immunoglobulin G ; immunology ; Immunoglobulin M ; immunology ; Mesenchymal Stromal Cells ; cytology ; immunology ; Mice ; Mice, Inbred BALB C ; Rats

Colectomy ; Colorectal Neoplasms/surgery* ; Humans ; Laparoscopy ; Treatment Outcome

OBJECTIVETo provide new germplasm materials for breeding new varieties of Pseudostellaria heterophylla.

METHODThe method of single plant selection was adopted, with the comparative experiments being carried out under the same conditions in Shibing county. The 8 plants of Shibing SB-4 were compared respectively with factor analysis for 27 phenotypic traits and 8 yield traits, and single factor variance analysis for the contents of polysaccharides.

RESULTUsing factor analysis, 27 phenotypic traits were classified into 7 principal divisors and 8 yield traits were simplified into 3 principal divisors. The 4 strains of P. heterophylla, ZT-01, ZT-02, ZT-06 and ZT-07, performed better than others in the phenotypic traits, and ZT-01, ZT-02, ZT-03 and ZT-07 in the yield traits. The contents of polysaccharides of ZT-01, ZT-02, ZT-05 and ZT-08 showed significantly higher value.

CONCLUSIONThere is significant difference among the 8 strains of P. heterophylla in phenotypic traits, yield traits and quality traits, making it possible to select certain strains for different purposes. ZT-01 and ZT-02 can be breaded further. ZT-06 and ZT-07 were used as ornamental cultivars for its great phenotypic traits. ZT-03 with good resistance and high yield was taken as resistant variety, and ZT-05 would face next selection on the basis of its high content of polysaccharide.

Breeding ; Caryophyllaceae ; chemistry ; growth & development ; China ; Phenotype ; Polysaccharides ; analysis

OBJECTIVETo construct the eukaryotic expression plasmid containing mouse vasoactive intestinal peptide(VIP) gene with biological activities.

METHODSVIP cDNA including the sequences of signal peptide was cloned from mouse thymus by RT-PCR, and then inserted into the mammalian expression vector pcDNA3.1 between Hind III and EcoR I restriction sites. COS-7 cells were transfected with pcDNA3. 1-VIP using liposome, the expression of VIP was identified by Western blot and ELISA. Supernatant of transfected cell culture was added to LPS-stimulated macrophages and the TNF-alpha production in cell medium was observed by ELISA.

RESULTSThe cloned VIP cDNA was confirmed by enzyme digestion and DNA sequencing. The expression of VIP was detected in the pcDNA3. 1-VIP transfected COS-7 cells by Western blot and ELISA. The VIP in culture supernatant potently inhibited TNF-alpha production by LPS-induced Macrophages in vitro.

CONCLUSIONThe eukaryotic expression plasmid that expresses biological active murine VIP has been constructed successfully.

Animals ; Base Sequence ; COS Cells ; Cercopithecus aethiops ; Cloning, Molecular ; DNA, Complementary ; genetics ; Eukaryotic Cells ; metabolism ; Mice ; Molecular Sequence Data ; Plasmids ; Recombinant Proteins ; biosynthesis ; genetics ; Recombination, Genetic ; Reverse Transcriptase Polymerase Chain Reaction ; Vasoactive Intestinal Peptide ; biosynthesis ; genetics

OBJECTIVETo study the protective mechanisms of the astragaloside against ischemia-reperfusion lung injury in rats.

METHODSIschemia-reperfusion lung injury was induced in SD rats. Astragalus armour glucoside was dissolved in 1% of sodium carboxymethyl cellulose at different concentrations (8, 6, and 3 mg/ml) was intragastrically administered in the rats at the dose of 1 ml/100 g. Cellular and subcellular structural changes in the lung tissue were observed at the end of the experiment using optical and transmission electron microscope, with the wet/dry ratio of the lung tissue and myeloperoxidase (MPO) activity measured.

RESULTSThe wet/dry ratio and myeloperoxidase activity in the lung tissue were significantly higher in the model group than in the sham-operated group (P<0.05), and were significantly lowered by the treatment with astragalus armour glucoside at different doses (P<0.01 or 0.05), and the effect was especially obvious in rats receiving a moderate dose. Pulmonary capillary expansion, erythrocyte leakage and exudate in the alveolar space with obvious pathological changes in the type I and II epithelial cells were observed in model group. Pulmonary capillary expansion was reduced in rats treated with high, medium and low dose of Astragalus armour glucoside, and the medium dose group showed the most obvious effect, in which no edema fluid in the alveolar space or erythrocyte leakage was found with also reduced type II lung epithelial cell degranulation.

CONCLUSIONAstragaloside has obvious antioxidant effect in rats with ischemia-reperfusion lung injury, and a medium dose produces the best effect.

Animals ; Female ; Ischemic Preconditioning ; Lung ; drug effects ; pathology ; Male ; Phytotherapy ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; drug therapy ; pathology ; Saponins ; pharmacology ; therapeutic use ; Triterpenes ; pharmacology ; therapeutic use

Electroencephalography ; Female ; Humans ; Hyperbilirubinemia, Neonatal ; physiopathology ; Hypoxia-Ischemia, Brain ; physiopathology ; Infant, Newborn ; Infant, Newborn, Diseases ; physiopathology ; Male ; Retrospective Studies

BACKGROUNDThe benefit of neoadjuvant chemotherapy in the management of head and neck squamous cell carcinomas (HNSCC) still remains controversial. The aim of this meta-analysis is to evaluate the role of the neoadjuvant chemotherapy with the cisplatin and fluororacil (PF) regimen in enhancing the overall survival of and decreasing locoregional relapse and distant metastasis in HNSCC patients.

METHODSMedline and manual searches were performed to identify all published randomized controlled trials (RCTs) investigating the efficacy of the neoadjuvant chemotherapy with the PF regimen. Outcomes assessed by meta-analysis included locoregional relapse, distant metastasis, and overall survival. The odds ratio was the principle measurement of effect, which was calculated as the treatment group (chemotherapy plus locoregional treatment) versus the control group (locoregional treatment alone) and was presented as a point estimate with 95% confidence intervals (CI).

RESULTSEight RCTs were adopted for analysis. The meta-analysis showed that the odds ratio for the locoregional relapse was 0.92 (0.70 - 1.22, 95% CI), which was not statistically significant. The odds ratios for distant metastasis and overall survival were 0.47 (0.33 - 0.68, 95% CI) and 1.28 (1.01 - 1.62, 95% CI) respectively, which were both statistically significant.

CONCLUSIONSNeoadjuvant chemotherapy with the PF regimen in HNSCC patients has no effect on locoregional relapse. However, it shows a small but significant benefit in reducing distant metastasis and improving the overall survival.

Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Carcinoma, Squamous Cell ; drug therapy ; mortality ; Chemotherapy, Adjuvant ; Cisplatin ; administration & dosage ; Fluorouracil ; administration & dosage ; Head and Neck Neoplasms ; drug therapy ; mortality ; Humans ; Neoadjuvant Therapy ; Randomized Controlled Trials as Topic