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Aim To investigate the effects of cajanonic acid A (CAA) on lipid metabolism in murine 3T3-L1 adipocytes. Methods 3T3-L1 cells induced to differ-entiated into mature adipocytes were treated with CAA in different dosages for 48 h, then total lipids as well as triglyceride, free fatty acid and glycerol were meas-ured. The expression levels of genes related to lipid metabolism were quantitatively analyzed by real-time fluorescent quantitative polymearase chain reaction ( RTFQ-PCR) . Results Total lipids and triglyceride in 3T3-L1 adipocytes were markedly reduced by CAA. The release of free fatty acid and glycerol was lower than that of control. This coincided with decreased mRNA levels of the key enzymes involved in de novo lipogenesis ( acetyl CoA carboxylase and fatty acid syn-thase) , fatty acid uptake ( lipoprotein lipase) , and li-polysis ( hormone sensitive lipase and adipose triglycer-ide lipase ) . While the expression of fatty acid oxida-tive genes including acyl CoA oxidase and carnitine palmitoyl transferase1 was increased after CAA treat-ment. Conclusion CAA may inhibit lipogenesis and lipolysis,reduce circulating free fatty acid and improve the lipid metabolism in adipocytes by regulating gene expressions.
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<p><b>OBJECTIVE</b>To try to find the ways to enhance the expression of ADS gene encoding amorpha-4,11-diene synthase, a key enzyme in artemisinin biosynthesis pathway catalyzing the formation of amorpha-4,11-diene from farnesyl diphosphate, and accelerate the artemisinin synthesis, the promoter of ADS was isolated and characterized.</p><p><b>METHOD</b>5' untranslated regions of ADS were isolated from Artemisia annua with PCR. For functional characterization, the isolated fragment was fused with GUS reporter gene and introduced into Nicotiana tabacum by Agrobacterium-mediated transformation. The GUS expression regulated by 5' untranslated regions of ADS in transgenic N. tabacum under the normal or stressed conditions were detected by histochemical staining and quantitative spectrophotometry assay.</p><p><b>RESULT</b>The 2 448 bp DNA fragment upstream of ADS coding sequence was isolated from A. annua and introduced into N. tabacum. Histochemical staining showed that the isolated fragment conferred stable GUS expression in transgenic plants. The quantitative results showed that the GUS activity in transgenic tobacco plants treated by low-temperature (4 degrees C) and ultraviolet irradiation were 1. 6 and 2.2 folds higher than that in the controls.</p><p><b>CONCLUSION</b>It was suggested that the isolated fragment had promoter activity and maybe responsive to adverse environmental stresses.</p>
Subject(s)
5' Untranslated Regions , Genetics , Alkyl and Aryl Transferases , Genetics , Metabolism , Artemisia annua , Genetics , Gene Expression Regulation, Plant , Genetic Vectors , Genetics , Molecular Sequence Data , Promoter Regions, Genetic , GeneticsABSTRACT
OBJECTIVE To study the sterilization effect of on-wall oxygen humidifying inhalation sets.METHODS The different parts on on-wall oxygen humidifying inhalation sets were sterilized,then compared the result before and after sterilization.RESULTS The regular percents for bacterial examination of humidifying bottle were 17.14% before sterilization,and 100.00% after sterilization,the regular percents for bacterial examination of metallic guilloche were 34.29% before sterilization and 94.29% after sterilization,the regular percents for bacterial examination of vent tube in humidifying bottle were 8.57% before sterilization,and 97.14% after sterilization.The regular percents for bacterial examination of the different parts of oxygen humidifying inhalation sets after sterilization were higher than before it P
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OBJECTIVE To study the sterilization method for on-wall oxygen humidifying inhalation sets in hospital.METHODS The different parts of on-wall oxygen humidifying inhalation sets were sterilized with Jianzhishu disinfectant and Andofo povidone iodine disinfectant liquid.RESULTS The regular percents for bacterial examination of the different parts of oxygen humidifying inhalation sets after sterilization were higher than before,P
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Objective To investigate the expression patterns of artemisinin biosynthetic genes in Artemisia annua L.during the development stage and in different tissues,and to explore the mechanism of spatial and temporal modulators for artmisinin production.Methods The transcriptional profiles of artemisinin biosynthetic genes in the capital and cooperative pathways were quantitatively assayed by real time fluorescence quantitative-polymerase chain reaction(RTFQ-PCR) in different tissues of roots,stems,leaves and flowers,and in leaf-flourishing,pre-floral,and post-floral periods.Results The expression levels of the tested genes were extremely low in June,raised in July,and reached their peak values in August(before flowering),but dropped gradually in September(after blooming).In August,the transcription levels of the tested genes increased by 3 to 15 times compared to the lowestlevels.In particular,ADS and CYP71AV1 mRNA levels had the great elevation,which were 12 and 15 times as much as those in June.During the flowering period,the artemisinin biosynthetic genes mRNA expression was detectable in roots,stems,leaves and flowers,and the expression levels had no obvious difference except that ADS mRNA level in leaves was 2 times higher than that in other tissues(P
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Objective To construct a DNA vaccine capable of expressing S gene of hepatitis B virus(HBV) and evaluate the expression of recombinant S gene in vitro and in vivo. Methods A cloned S X gene fragment was inserted into a eukaryote expression vector to construct a recombinant plasmid. The S gene was transcribed in vitro and expressed in a transfected cell line, and the efficiency of HBsAg in eliciting anti HBs was evaluated in mice. Results The expression of S gene was confirmed by Northern blotting, Western blotting, and ELISA(for both antigen and antibody detection). Conclusions The recombination and expression of S gene is achieved successfully in vitro.
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[Objective] To obtain the tyrosine decarboxylase gene (tyrDC) from Aristolochia contorta Bge.and to assay its cDNA sequence and homologous analysis, thus to remove its nephrotoxieity. [Methods] The primers designed by referring to the conservative amino acid sequences of known plant tyrDC were used to amplify a fragment of cDNA from Aristolochia contorta Bge.by reverse transcription-polymerase chain reaction (RT-PCR). The amplified cDNA sequence was cloned and sequenced to design a pair of specific primers and to amplify a full-length tyrDC cDNA from Aristolochia contorta Bge.by rapid amplification of cDNA ends (RACE). [Results] The length of cloned tyrDC cDNA is 1678 base pairs (bp), which comprises an open reading frame ( ORF) of 1551 bp encoding 516 amino acids and a downstream untranslated region (3'UTR) of 127 bp. The results of sequence comparison indicated that the amino acid sequence deduced from the nucleotide sequence of tyrDC from Aristolochia contorta Bge.shares 76% homology with issued tyrosine decarboxylase of Papaver somniferum L. and 79% homology with tyrosine /DOPA decarboxylase from Thalictrum flavum subsp. glaucum. [Conclusion] The full-length tyrDC cDNA has been amplified from Aristolochia contorta Bge. and the homologous retrieve of tyrosine decarboxylase reveals an extensive sequence similarity among tyrosine decarboxylases of different plants. This will provide evidence for the romoval of nephrotoxicity of Aristolochia contorta Bge. .
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Objective To increase artemisinin yield in transgenic Artemisia annua plants by regulating metabolic affluxion through metabolic pathway engineering. Methods The gene targeting vector was constructed by squalene synthase (SS) gene of A. annua, green fluorescent protein (GFP) gene, and cytosine deaminase (CodA) gene, and the vector was introduced into Agrobacterium tumefaciens by freeze-thawing procedure. A. annua was transformed through Leaf Disk method and regenerated transgenic plants were screened by the “Step-by-Step Selection”. Results Among the transgenic A. annua plants emitting green fluorescence after expression of GFP gene, the exogenous GFP gene rather than endogenous SS gene was detected in one transgenic plant by PCR as well as hybridization of PCR products. The preliminary data showed that the wild-type SS gene was replaced by mutated SS gene in the transgenic A. annua plant. Conclusion Gene targeting of squalene synthases of A. annua is successful.
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Objective To explore the feasibility of utilizing the cytosine deaminase A (CodA) gene as an effective negative selectable marker in Artemisia annua for gene targeting.Methods The PCR procedure was employed to amplify CodA gene from Escherichia coli.After being cloned and sequenced, the gene was inserted into a plant expression vector, pROKⅡ, and then introduced into Agrobacterium tumefaciens LBA4404 (pAL4404).The leaf disks of A.annua were transformed by the co-cultivation protocol, after which the transformed calli were selected and green shoots of A. annua were regenerated on N6 medium supplemented with 25 ?g/mL Kanamycin (Kan).Then the Kan-resistant transgenic shoots were transplanted onto the MS medium containing 500 ?g/mL 5-fluocytosine (5-FC) plus 25 ?g/mL Kan and continuously cultured for up to two weeks.Results The transgenic shoots have totally died while untransformed shoots still kept normal growth, indicating that A.annua cells introduced into the CodA gene had conferred an expected negative selection phenotype.When detected by RT-PCR, the transgenic shoots displayed a CodA-positive amplified band, but untransformed shoots gave no such CodA-specific amplified pattern.This result suggested that CodA gene had transcribed into corresponding mRNA in A.annua cells with furtherly verifying the result of phenotypic assay.Conclusion The CodA gene can be utilized as an effective negative selectable marker in A.annua for gene targeting.
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Object To explore the feasibility of breeding genetic modified (GM) medicine by expressing human cytokine in transgenic Chinese materia medica Methods Human interferon ? gene and RANTES gene available from the amplification in vitro were enzymatically excised, recoveried, and inserted into intermediate vectors The recombinants were identified by double enzyme digestion of EcoRⅠand HindⅢ The plasmids were extracted from Escherichia coli and introduced into A tumefaciens, and the transformants harboring binary vectors were screened by addition of antibiotics of kanamycin (Km) and rifampicin (Rif), and the explants of M charantia and P vulgaris were transformed by co cultivation of leaf disks with A tumefaciens strain Results RT PCR was applied to detect the transient expression of human interferon ? gene and RANTES gene in transformed medicinal herbal calli Conclusion The expression of recombinant human interferon ? gene and RANTES gene in transgenic M charantia and P vulgaris cells was firstly reported, which opens an alternative road to antivirus, especially anti AIDS virus, by using transgenic Chinese materia medica