Differential expression of the TwHMGS gene and its effect on triptolide biosynthesis in Tripterygium wilfordii / 中国天然药物

3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) is the first committed enzyme in the MVA pathway and involved in the biosynthesis of terpenes in Tripterygium wilfordii. The full-length cDNA and a 515 bp RNAi target fragment of TwHMGS were ligated into the pH7WG2D and pK7GWIWG2D vectors to respectively overexpress and silence, TwHMGS was overexpressed and silenced in T. wilfordii suspension cells using biolistic-gun mediated transformation, which resulted in 2-fold increase and a drop to 70% in the expression level compared to cells with empty vector controls. During TwHMGS overexpression, the expression of TwHMGR, TwDXR and TwTPS7v2 was significantly upregulated to the control. In the RNAi group, the expression of TwHMGR, TwDXS, TwDXR and TwMCT visibly displayed downregulation to the control. The cells with TwHMGS overexpressed produced twice higher than the control value. These results proved that differential expression of TwHMGS determined the production of triptolide in T. wilfordii and laterally caused different trends of relative gene expression in the terpene biosynthetic pathway. Finally, the substrate acetyl-CoA was docked into the active site of TwHMGS, suggesting the key residues including His247, Lys256 and Arg296 undergo electrostatic or H-bond interactions with acetyl-CoA.

The effects of TwHMGR overexpression on the biosynthesis of triptolide and celastrol in Tripterygium wilfordii / 药学学报

Tripterygium wilfordii 3-hydroxy-3-methylglutaryl coenzyme-A reductase (TwHMGR) is an important regulation site in terpenoids metabolic pathway in cytoplasm which is the first speed limit enzyme of MVA pathway. In order to investigate the effects of TwHMGR on the biosynthesis of triptolide and celastrol in Tripterygium wilfordii, the overexpression of TwHMGR (OE-HMGR) was studied in this paper. We cloned the full-length of TwHMGR to construct overexpression vector by Gateway technology then delivered the expression vector into Tripterygium wilfordii suspension cells by gene gun. qRT-PCR was used to detect the expression of TwHMGR:the expression of TwHMGR was increased to 1.75 folds over the control group (empty vector:pH7WG2D) in the overexpression group. The accumulation of triptolide and celastrol in the suspension cells of Tripterygium wilfordii was detected by UPLC, revealing that:the contents of triptolide and celastrol were increased to 163.93% and 190.04% of over the control group in the overexpression group. Based on these findings, the positive effect on the accumulation of active terpenoids, triptolide and celastrol in Tripterygium wilfordii was found and the results laid a foundation of the synthetic biology research on important active terpenoids in Tripterygium wilfordii.

Effect on biosynthesis of terpenoid active components through RNA interference with MCT gene in Tripterygium wilfordii / 药学学报

MCT is an important key enzyme in the terpenoid biosynthesis in MEP pathway. In this study, Gateway technology was used to construct RNAi vector of TwMCT, and a vector fragment with a size of 484 bp was obtained. The TwMCT RNAi vector was transferred into the suspension cells of Tripterygium wilfordii by gene gun. Accumulation of terpenoids was assayed by UPLC, and the result showed that the content of triptolide and celastrol in cells decreased by 23.4% and 42.8%, respectively, compared with the control group pK7GWIWG2D. Moreover, the gene expression of TwMCT and major genes in terpenoid biosynthesis pathway was detected by qRT-PCR, which demonstrated that the expression of TwMCT reduced by 29.2% relative to that of the control group pK7GWIWG2D, and the relative expression of TwDXR, TwGGPS, TwHMGR and TwHMGS diminished by 36.3%, 31.3%, 62.2%, and 29.1%, respectively, but the expression of TwDXS was up-regulated by 114.2%, and there was no significant change in TwFPS. Thus, it was verified in vivo that interference with TwMCT expression significantly inhibited the accumulation of triptolide and celastrol in Tripterygium wilfordii, laying a foundation for further exploring the regulation mechanism of MCT gene on the terpenoid biosynthesis in Tripterygium wilfordii.

Establishment of a genetic transformation system for hairy root culture of Erigeron breviscapus / 中国中药杂志

The electroporation method was performed to transfer plasmid DNA of PBI-1300 carrying GFP gene into Agrobacterium rhizogenes C₅₈C₁ strains. Mediated by A. rhizogenes C₅₈C₁, the GFP gene were transformed into Erigeron breviscapus aseptic leaves by leaf disc method, then the hairy roots were induced and the infected hairy roots were screened by hygromycin resistance. The chromosomal DNA of the hairy root was used as the templates for the PCR amplification with the GFP-specific primers and then the expected amplified DNA bands appeared, the green fluorescent of GFP in the cut hairy roots was observed by two-photon microscope. These results indicated that GFP gene was integrated into the genome of E. breviscapus and was expressed stably. This study laid the groundwork for foreign gene high-efficiency expression inthe genetic transformation system for hairy root culture of E. breviscapus.

Cloning and protein expression analysis of geranyl diphosphate synthase genes in Tripterygium wilfordii / 中国中药杂志

Based on the transcriptome data, the study cloned full-length cDNA of TwGPPS1 and TwGPPS2 genes from Tripterygium wilfordii suspension cells and then analyzed the bioinformation of the sequence and protein expression. The cloned TwGPPS1 has a 1 278 bp open reading frame (ORF) encoding a polypeptide of 425 amino acids. The deduced isoelectric point (pI) was 6.68, a calculated molecular weight was about 47.189 kDa. The full-length cDNA of the TwGPPS2 contains a 1 269 bp open reading frame (ORF) encoding a polypeptide of 422 amino acids. The deduced isoelectric point (pI) was 6.71, a calculated molecular weight was about 46.774 kDa.The entire reading frame of TwGPPS1,2 was cloned into the pET-32a(+) vector and expressed in E. coli BL21 (DE3) cells to obtain the TwGPPS protein, which laid a basis for further study on the regulation of terpenoid secondary metabolism and biological synthesis.

Cloning and protein expression of sterol-C-24-methyl transferase 2 in Tripterygium wilfordii / 药学学报

24-Alkyl sterols are the major players in the control of membrane component and plant growth. In this paper, we cloned an important rate-limiting enzyme:sterol-C-24-methyl transferase (SMT) in the sterol biosynthetic pathway according to the transcriptome data of Tripterygium wilfordii. suspension cells, whose full-length cDNA was 1 631 bp with an open reading frame of 1 080 bp, encoding a protein of 359 amino acids. It was estimated that theoretical isoelectric point (pI) was 6.43 and the molecular mass was 40.0 kDa. Bioinformatics analysis attributed the SMT gene to SMT2 family. The expression vector was constructed as the pMAL-c2x-TwSMT2 plasmid and the recombinant protein was expressed in E. coil BL21 (DE3) competent cells. After methyl jasmonate treatment, the relative expression level of TwSMT2 has improved significantly in 24 h. SDS-PAGE electrophoresis and Western Blot showed that protein of TwSMT2 in BL21 (DE3) strain was expressed after induction by IPTG. In this study, TwSMT2 was cloned for the first time and the recombinant protein was expressed, which lay the foundation for elucidation of the sterol biosynthetic pathway of Tripterygium wilfordii in the future.

Cloning and expression analysis of squalene synthase gene in Tripterygium wilfordii / 药学学报

In this paper, we cloned the full-length cDNA of TwSQS from Tripterygium wilfordii suspension cells (GenBank:KR401220) and performed the bioinformation and mRNA expression analysis. The expression after methyl jasmonate (MJ) treatment of the gene was detected by RT-PCR. The full-length cDNA of TwSQS was 1800 bp containing a 1242 bp open reading frame (ORF) encoding a polypeptide of 413 amino acids. The theoretical isoelectric point (pI) was 7.94 and the calculate molecular weight was about 47.20 kD. The relative expression level of TwSQS was deduced by MJ and reached the highest at 4 h after the treatment. The gene information we got in this study enriched the biosynthesis pathway of triterpenoids in Tripterygium wilfordii and laid foundation for further studies.

Cloning and bioinformatics analysis of geranylgeranyl diphosphate synthase gene of Tripterygium wilfordii / 中国中药杂志

A full-length cDNA of GGPPS gene from Tripterygium wilfordii suspension cells was obtained by use of RACE strategy (GeneBank: KM978333), and then analyzed by bioinformatics approaches. TwGGPPS cDNA has 1857 nucleotides and an open reading frame (ORF) encoding a protein of 514 amino acid residues. The deduced protein has isoelectric point (pI) of 7.85, a calculated molecular weight about 57.13 kD, 5 conserved domains and 2 functional domains. PSORT Prediction showed it was located at plasma membrane. Phylogenetic analysis demonstrated that TwGGPPS1 was similar to GGPPS from other species of plants. For the first time the cloning of geranylgeranyl diphosphate synthase gene from T. wilfordii was reported, it lays the foundation for further research of diterpenoids biosynthetic pathway.

Cloning and expression analysis of 4- (cytidine-5-diphospho) -2-C-methyl-D-erythritol kinase gene in Tripterygium wilfordii / 中国中药杂志

4-(Cytidine-5-diphospho) -2-C-methyl-D-erythritol kinase is a key enzyme in the biosynthesis pathway of terpenoids. According to the transcriptome database, the specific primers were designed and used in PCR. The bioinformatic analysis of the sequenced TwCMK gene was performed in several bioinformatics software. The Real-time fluorescence quantification polymerase chain reaction (RT-qPCR) were used to detect the expression levels of TwCMK from T. wilfordii after elicitor MeJA supplied. The results showed that the full length of TwCMK cDNA was 1 732 bp encoding 387 amino acids. The theoretical isoelectric point of the putative TwCMK protein was 5.79 and the molecular weight was about 42.85 kDa. MeJA stimulated the rising of TwCMK expression in suspension cell and signally impacted at 24 h. The research provides a basis for further study on the regulation of terpenoid secondary metabolism and biological synthesis.

Cloning and expression analysis of 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase gene in Tripterygium wilfordii / 中国中药杂志

To clone the 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (TwMCT) full length cDNA from Tripterygium wilfordii, the specific primers were designed according to the transcriptome data and the LCPCR were carried out. After a series of bioinformatics analysis on the TwMCT, the MeJA induced expression content were investigated by real-time fluorescence quantification polymerase chain reaction (RT-qPCR). The result showed that the full of TwMCTcDNA was 1 318 bp nucleotides encoding 311 amino acids. The molecular weight of the deduced TwMCT protein was about 34.14 kDa and the theoretical isoelectric point was 8.65. Result of the RT-qPCR analysis indicated that the content of TwMCT mRNA expression in T. wilfordii suspension cell was rising after treating with MeJA and reached the maximum in 24 h. Cloning and analyzing TwMCT gene from T. wilfordii provided gene element for studying the function and expression regulation of secondary metabolites.

Cloning and expression analysis of a acetyl-CoA U-acetyltransferase gene (TwAACT) from Tripterygium wilfordii / 中国中药杂志

In this study, based on the transcriptome data, we cloned the full-length cDNAs of TwAACT gene from Tripterygium wilfordii suspension cells, and then analyzed the bioinformation of the sequence, detected the genetic differential expression after being induced by methyl jasmonate (MeJA) by RT-PCR. The full-length cDNA of the TwAACT was 1 704 bp containing a 1 218 bp open reading frame (ORF) encoding a polypeptide of 405 amino acids (GeneBank accession No. KP297934). The deduced isoelectric point (pI) was 6.10, a calculated molecular weight was about 41.20 kDa, and online prediction showed that TwAACT had two catalytic active sites. After the induction of MeJA, the relative expression level of TwAACT increased rapidly. The expression level of TwAACT was highest at 24 h. TwAACT was cloned firstly, that laid the foundation for identifying thegene and illustrating thebiosynthesis mechanism and its synthetic biology.

Safety and efficacy of anisodamine on prevention of contrast induced nephropathy in patients with acute coronary syndrome / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Previous studies have proved the renal protective effects of anisodamine in patients with septic shock. The aim of this study was to investigate anisodamine for the prevention of contrast induced nephropathy (CIN) in patients with acute coronary syndrome (ACS).</p><p><b>METHODS</b>Consecutive ACS patients undergoing elective percutaneous coronary intervention (PCI) were randomly assigned to one of two groups: patients in the anisodamine group (ANI group) were assigned to receive intravenous infusions of anisodamine by an adjusted-dose (0.1 - 0.2 µg × kg(-1)× min(-1)) from the PCI procedure to 24 hours after PCI, and the control group (CON group) received 0.9% isotonic saline of the same volume. All patients were hydrated for 6 to 12 hours before and 12 hours after PCI. Blood samples were taken on the day of PCI and at 24, 48 and 72 hours after PCI to measure the serum creatinine (SCr).</p><p><b>RESULTS</b>A total of 177 patients were involved in the study, 88 in the ANI group and 89 in the CON group. In both groups, the SCr concentrations significantly increased after PCI, with the peak value occurring at 48 hours. At 72 hours, the SCr concentration in the ANI group retuned to the baseline level (P > 0.05), but the SCr concentration in CON group was still higher than baseline level (P < 0.01). The SCr concentrations at 48 and 72 hours after PCI were much lower in the ANI group than those in the CON group (both P < 0.01). The estimated glomerular filtration rate (eGFR) significantly decreased after PCI, the lowest value occurred at 48 hours. In the ANI group, the eGFR at 72 hours was similar to the baseline level. In the CON group, the eGFR failed to return to baseline at 72 hours (P < 0.01). The eGFR at 24, 48 and 72 hours after PCI were higher in the ANI group (all P < 0.05). The incidence of CIN in the ANI group was lower than that in the CON group within 72 hours after PCI (P < 0.05). The results of multiple Logistic regression proved that both diabetes and left ventricular ejection fraction (LVEF) were independent predictors of CIN, and treatment with anisodamine was an independent preventive factor of CIN (OR 0.369 and 95%CI 0.171 to 0.794, P = 0.011). No serious side effects were found in the ANI group.</p><p><b>CONCLUSION</b>Intravenous infusion of anisodamine during and after elective PCI may safely prevent the occurrence of CIN in ACS patients.</p>

Evaluation of the renal replacement therapy on the liver transplant patients with acute renal failure / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To analyze the preoperative risk factors on liver transplant recipients with acute renal failure(ARF), and to evaluate renal replacement therapy (RRT) as a transitonary therapy before liver transplantation.</p><p><b>METHODS</b>Liver transplant recipients with acute renal failure treated with renal replacement therapy between January 1st, 2001 and January 1st, 2008 in our center were retrospected. Clinical characteristics, the kinds of RRT and prognosis were analyzed; Logistic regression was applied to analyze the parameters that can forecast the motality of the liver transplant recipients with acute renal failure.</p><p><b>RESULTS</b>Of the patients who received RRT, 30% survived to liver transplantation, 67.5% died while waiting for liver transplantation. The dead had a higher multiple organ dysfunction score (MODS), and lower mean arterial pressure than those survived to liver transplantation. There was no significant difference in the duration of RRT between continuous renal replacement therapy (CRRT) patients and hemodialysis patients. CRRT patients had a higher MODS, lower mean arterial pressure, lower serum creatinine than hemodialysis patients. Lower mean arterial pressure was statistically associated with higher risk of mortality.</p><p><b>CONCLUSION</b>Though mortality was high, RRT helps part (30%) of patients survive to liver transplantation. Therefore, considering the high mortality without transplantation, RRT is acceptable for liver transplant recipients with ARF.</p>

Isolation and identification of arboviruses from mosquito pools in some regions of Liaoning province,China / 中华流行病学杂志

Objective To isolate and identify arboviruses from mosquito pools in some regions of Liaoning province.Methods Mosquitoes were collected from Shenyang,Yingkou,Panjin,Jinzhou and Dandong cities of Liaoning province in 2006.Viruses were isolated by inoculating the specimens onto C6/ 36 and BHK-21cells.The new isolates were identified using serological and molecular biological methods.Results 5410 mosquitoes were collected from the five cities in total.Three isolates produced CPE in C6/ 36 cell and five isolates produced CPE in both C6/36 and BI-IK-21 cell.Three isolates (LN0684,LN0688 and LN0689) were identified as Banna virus and one isolate (LN0636) was identified as Getah virus.Phylogenetic analysis showed that the three Banna virus strains were clustered into the same evolution branch as the other Chinese isolates.The identity of nucleotide sequence was between 91.2% and 94.7%,compared with other Banna virus strains.The new isolated Getah virus was clustered into the same branch with the strain of South Korea (swine).The identity of nucleotide sequence was 99.2%,when comparing with the strain of South Korea and was 95% to 99% with the strains fi'om Russia,mainland of China and Taiwan region.Conehmion Eight virus isolates,including three Banna virus,one Getah virus and four unknown virus strains were isolated from mosquitoes in Liaoning province.Banna virus and Getah virus were reported for the first time in Liaoning province,while Getah virus showed the highest nucleotide homology with the South Korea strains.

Preparations of the specific antibodies against exon 2 and exon 3 of human tau protein / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To prepare the specific antibodies against exon 2 and exon 3 of human tau protein.</p><p><b>METHODS</b>Sequences encoding exon 2 and exon 3 of human tau protein were amplified from human peripheral blood DNA and cloned into a prokaryotic expression vector pGEX-2T. Fusion proteins GST-E2 and GST-E3 were expressed and purified from E. coli system. The antisera were elicited by immunization of the purified fusion proteins to rabbits and mice. The specific antibodies were purified by Protein G/A and CNBr-activated sepharose 4B coupled with GST protein. The specificity and sensitivity of the purified antibodies were evaluated by Western blotting and ELISA.</p><p><b>RESULTS</b>Recombinant fusion proteins GST-E2 and GST-E3 were expressed and purified from E. coli, which showed Mr. 29 x 10(3). Various antisera were collected from the immunized experimental animals. Reliable immunoreactive specificity and titers of the purified antibodies against exon 2 and exon 3 of human tau protein were confirmed by Western blotting and ELISA after serial purification processes.</p><p><b>CONCLUSION</b>Four specific antibodies against exon 2 and exon 3 of human tau protein have been successfully prepared, which provides basis for analyzing the role of tau in neurodegenerative diseases.</p>

Effect of anti-ICOS monoclonal antibody combined with low-dose CsA on chronic rejection of heart grafts in rats / 中南大学学报(医学版)

OBJECTIVE@#To evaluate the effect of anti-inducible costimulator monoclonal antibody (anti-ICOS-Ab) combined with low-dose cyclosporine (CsA) on the survival quality and chronic rejection of heart allografts in rats.@*METHODS@#The rats' heterotopic cardiac transplantation model was established by Ono's method. The recipient rats were randomly divided into an isotransplantation control group and an allotransplantation experiment group. The experiment group was re-classified into a placebo group, a normal-dose CsA group, an anti-ICOS-Ab group, a low-dose CsA group, and an anti-ICOS-Ab combined with low-dose CsA group. The survival time of grafts was monitored. The cardiac grafts were harvested for histological analysis. Flow cytometric analysis was employed to detect the population of CD25+CD4+ in peripheral lymphocytes from recipients with a long-term surviving graft.@*RESULTS@#The survival time of the cardiac allografts in CsA-treated groups was significantly longer than that in placebo group (P0.05). Compared with the normal-dose CsA group, the chronic rejection lesions of the anti-ICOS-Ab combined with low-dose CsA treatment group significantly were alleviated in the long-term survival grafts, and the proportion of CD4+CD25+ regulatory T cell increased in peripheral blood.@*CONCLUSION@#The anti-ICOS-Ab combined with low-dose CsA can prolong the survival of cardiac allografts and alleviate the chronic rejection significantly. The high expression level of CD4+CD25+ regulatory T cell is beneficial to the long-term survival of grafts.

Genotype 1 JEV was isolated again from Liaoning Province, China, 2007 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To isolate Japanese encephalitis virus (JEV) from mosquitoes collected in Liaoning province and analysis the genotype of new isolated JEV strains and the characters of nucleotide and amino acid in the E gene.</p><p><b>METHODS</b>Collected mosquitoes in Dandong Liaoning Province in August, 2006. Virus isolation was using issue culture cells. Isolated viruses were identified by using serological and molecular methods.</p><p><b>RESULTS</b>Two new JEV strains, LNDG07-02 and LNDG07-16, were isolated from 1500 mosquitoes were belonging to genotype 1. The identity of nucleotide and amino acid of E gene between new JEV strains and live attenuated vaccine strain SA14-14-2 were 87.8-88% and 97.2%, respectively. Total 11 amino acid sites were differences in E gene between new isolates and SA14-14-2. However, there were no differentiation between the new JEV strains and the isolates in Donggang 2002.</p><p><b>CONCLUSION</b>Genotype 1 JEV was isolated again from Donggang, since the first isolation of this genotype in 2002. Genotype 1 JEV continues in existence in Donggang Liaoning Province.</p>

Effect of Splenectomy in Prognosis of Human Liver Transplantation / 中国普外基础与临床杂志

Objective To explore the effect and indication of splenectomy in liver transplantation.Methods From January 2001 to April 2006,260 patients underwent piggyback orthotopic liver transplantation(PBOLT),and 28 patients had undergone combined PBOLT and splenectomy(splenectomy group).These patients were compared to 56 randomly selected non-splenectomy patients from the same transplant period,meaningly two controls were se- lected for every non-spleneetomy case.Two groups were analyzed with respect to rate of infection and survival rate, as well as biopsy-proven acute allograft rejection within 30 days after transplantation.Results Rate of infection in the splenectomy group was higher than that in the non-splenectomy patients(85.7% vs 55.4%,P