[Objective] To explore the culture conditions of inducing callus from tissue of Aristolochia contorta Bge (ACB). [Methods] The leaves of ACB were used as the explants. Basic medium (including 1/2 MS medium, MS medium and modified MS medium) containing corresponding phyto-hormones was applied for the induction of ACB callus. The influerices of different culture conditions such as three kinds of basic medium, different pH values and addition of different kinds of phyto-hormones at different concentrations into the basic medium respectively or simultaneously, on the callus induction of ACB were observed. [Results] (1) After the three kinds of basic medium were added with the phytohormones of 0.1mg/L kinetin (KT) and 0.2mg/L ?-naphthalene acetic acid (NAA) , a large amount of ACB calluses were induced in the MS medium and modified MS medium, while ACB calluses did not occur in the 1/2 MS medium. (2) When the pH value of the culture medium composed of modified MS medium and 0.1mg/L KT and 0.2mg/L NAA was at 5.8, 7.0 and 8.0, a large amount of ACB calluses were induced in the medium with pH value being 7.0 and 8.0 while a few calluses occurred in the medium with pH value being 5.8. (3) ACB calluses were induced in modified MS medium with NAA added , but calluses did not occur in modified MS medium with KT added. When the modified MS medium was added with different kinds of phyto-hormones at different concentrations, ACB calluses were loose in the medium with high-concentration NAA and this did not benefit to the differentiation of buds for too fast cell division, and the callus induction rate was low in the medium with low-concentration NAA. The optimized culture medium was modified MS medium with 1mg/L KT and 0.5mg/L NAA added. [Conclusion] The optimum culture conditions of inducing callus from ACB tissues are: MS medium or modified MS medium with 1mg/L KT and 0.5 mg/L NAA added being the culture medium, and pH value being 7.0-8.0.

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.

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.

Objective To investigate the variation of the thioredoxin system (Trx),and the role of it in transient out-ward potassium current (Ito) channels in left ventricular myocytes of diabetes mellitus (DM) in rats. Methods Forty-five SD rats were divided into DM group and control group. DM group were treated with streptozotocin (STZ) to induce DM model. The values of left ventricular end diastolic diameter (LVEDD), end-systolic diameter (LVESD), fractional shortening (LVFS), ejection fraction (LVEF) and heart rate (HR), QRS duration and corrected QT (QTc) interval were detected by echocardiogra-phy (UCG) and electrocardiogram (ECG) in two groups. The left ventricular myocardial tissue samples were taken to detect the Trx,glutaredoxin (GRX),thioredoxin reductase (TrxR) and glutathione reductase (GR) by using UV spectrophotometer. The level of free thiol (P-SH) of total cardiac protein was detected by 5, 5′-dithio-bis-2-nitrobenzoic acid method. Ito of the cardiomyocytes was recorded by whole-cell patch-clamp method. After being incubated in vitro with insulin(Ins), treated with TrxR inhibitor-auranofin(AF) and 13-cis-retinoic acid(RA), the changes of Ito of the cardiomyocytes were observed. Results Compared with control group, the values of heart rate (HR), left ventricular minor axis decurtaion rate (LVFS), left ventricular ejection fraction (LVEF) and TrxR were lower in DM group. The values of LVEDD, LVESD, QRS and QTc inter-vals, Trx, Grx and P-SH were higher in DM group than those of control group. Ito density was significantly higher in DM+Ins group than that of DM group, Ins+RA group and Ins+AF group when the stimulation voltage ≥ 0 mV (P ＜ 0.05). Conclusion The impaired Trx system in diabetic rat myocardium was the electrophysiological basis of the reduced ventric-ular function and arrhythmia. And Ins was able to reverse the decreased Ito of cardiomyocytes in DM rats.

AIM:To investigate the effect of compound of cordyceps sinensis(CS) and tripterygium hypoglaucum on survival time of the heterologous human-pig graft skin.METHODS: CS solution were used to hatch the skin graft and CS was used locally.Ciclosporin A(CsA) and normal saline solution(NS) were used in control.Besides,skin HE staining was detected and CD4,CD8,IL-2 and IL-10 were also examined before and after operation.RESULTS: The survival time of the graft skin treated with CS was(21.20?3.19) days,while the control group treated with NS was(10.17?1.94) days(P

Objective To further investigate direct effects of dipeptidyl peptidase-4(DPP4) on calcification and to identify responsible signaling pathways in human vascular smooth muscle cells (HVSMC). Methods The effect of DPP-4 on calcification of HVSMC was observed by alizarin red, and Western blot was used to detect whether DPP4 induced calcification-related protein expressions through extracellular signal-regulated kinases 1/2 (ERK1/2) pathway. Results The Alizarin red staining results showed that calcified nodules in DPP4 group were significantly increased as compared with control group, similar to calcification group.The protein expressions of osteoprotegerin (OPG), osteopontin (OPN), Runt-related transcription factor 2 (RUNX2), and bone morphogenetic protein 2 (BMP2) were stimulated by DPP4 in a concentration- and time-dependent manner. The phosphorylation level of ERK1/2 was significantly increased after DPP4 incubation for 15 min (P<0.05). PD98059, an ERK1/2 inhibitor, significantly lowered DPP4-stimulated expressions of calcification-related proteins (P<0.05). Conclusion DPP4 may promote the calcification of HVSMC through ERK1/2 signaling pathways.

OBJECTIVETo 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.

METHOD5' 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.

RESULTThe 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.

CONCLUSIONIt was suggested that the isolated fragment had promoter activity and maybe responsive to adverse environmental stresses.

5' Untranslated Regions ; genetics ; Alkyl and Aryl Transferases ; genetics ; metabolism ; Artemisia annua ; enzymology ; genetics ; Gene Expression Regulation, Plant ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Promoter Regions, Genetic ; genetics

Objective To prospectively summary the piercing-out position,direction,length and piercing-in position of perforator,and investigate the feasibility of preoperative design and optimization of the anterolateral thigh flap and its clinical application.Methods All 58 cases of anterolateral thigh flaps were designed and taken from the lateral thigh area from January,2014 to January,2016.Portable Doppler ultrasound was used before an operation to detect the piercing-out position (point P) of perforators.The direction and length (lower subcutaneous segment of perforators) of perforators after leaving piercing-out position were observed during the operation.And the piercing-in positions (point P') on superficial fascia and the dermis were observed.Based on this,we added line B (anterior superior spine-lateral femoral epicondyle) and line C (anterior superior spine-the middle point of superior border of patella) in the lateral and anterior side of original ilium-patella line in the thigh (line A),respectively.Results All perforators found in 58 cases before and during the operations were located on line A or between line A and line B.No perforators were found between line A and line C.Perforators walked toward the anterior medial side after leaving the muscle membrane.The perforator vascular subcutaneous segment (distance between point P and point P') was (2.02±0.23) cm.There was rectus muscle branch in the descending branch of lateral femoral circumflex artery,while no rectus muscle cutaneous branch was seen.20 cases were designed by one-line method,12 cases were designed by two-line method,while 26 cases were designed by three-line method.Conclusion Advanced three-line method is beneficial to detect of the perforators on the anterior thigh lateral region and to reduce the intraoperative injury perforator vessels at the puncture point.Clinical application of the anterior lateral thigh flap is simple and reliable.

Objective : To investigate the behavior problems and influencing factors of school-age students from the third to the sixth grade in Zhongshan,and to provide evidence for early intervention of behavior problems in children. Methods : According to the proportion of population in urban area and township in Zhongshan,students of Grade Three to Six from eight primary schools（three in urban area and five in township）were recruited by stratified sampling method. The behavior problems in children were assessed by the Conners Parent Symptom Questionnaire（PSQ）. Sociodemographic information,family discipline and so on was investigated by a general questionnaire. The influencing factors for behavior problems were analyzed by a logistic regression model. Results : A total of 2 292 questionnaires were issued,and 2 236 valid questionnaires were recycled,with an effective rate of 97.56%. The positive rate of behavioral problems was 11.72%. The results of multivariate logistic regression analysis showed that the risk factors for behavior problems were females（OR=1.594,95%CI：1.170-2.171）,birth asphyxia（OR=2.372,95%CI：1.320-4.261）,main family discipline（laissez-faire：OR=3.326,95%CI：1.450-7.630;doting：OR=3.244,95%CI：1.867-5.638;autocratic：OR=2.609,95%CI：1.584-4.296,mixed：OR=2.313,95%CI：1.669- 3.207）,less than four hours per week for father-child communication（OR=1.551,95%CI：1.052-2.286）,negative life events（OR=2.188,95%CI：1.448-3.308）,living in township（OR=2.031,95%CI：1.330-3.102）,academic performance （average：OR=2.786,95%CI：1.868-4.156;poor：OR=6.665,95%CI：3.236-13.727;very poor：OR=25.068,95%CI：5.786-108.617）;the protective factors were occupation of mother as civil servants or professional personnel（OR=0.449,95%CI：0.238-0.844）and higher grades（Grade Five：OR=0.496,95%CI：0.339-0.727;Grade Six：OR=0.468,95%CI：0.309-0.710）. Conclusion Females,birth asphyxia,main family discipline,less communication between father and child,occupation of mother,negative life events,place of residence,academic performance and grade were the influencing factors for behavior problems.

A 1 270 bp full-length cDNA fragment was obtained from the Schistosoma japonicum (Chinese strain) adult cDNA library after the '3' and 5' ends of the incomplete expression sequence tag (EST) of hypoxanthine-guanine phosphoribosyltransferase of Schistosoma japonicum (SjHGPRT) were amplified by the anchored PCR with 2 pairs of primer that were designed according to the published incomplete SjHGPRT EST and the sequence of multiclone sites of library λgt1 1 vector. Sequence analysis indicated that this fragment, with an identity of 82% to hypoxanthine-guanine phosphoribosyltransferase ofSchistosoma mansoni (SmHGPRT), contained a complete open reading frame(ORF). The deduced amino acid sequence showed 83% identity to that of SmHGPRT. This fragment was cloned into the prokaryotic expression vector pQE30, and subsequently sequenced and expressed in Escherichia coli. SDS-PAGE revealed that M of the recombinant protein was about 28 ku. Western-blot analysis showed that the recombinant protein was recognized by the polyclonal antisera from rabbits immunized with Schistosoma japonicum adult worm antigen. Mice vaccinated with recombinant protein revealed significant worm burden, liver eggs per gram (LEPG), fecal eggs per gram (FEPG) and intrauterine eggs of the female worms reduction percentage, compared with the controls. Taken together, the SjHGPRT full-length cDNA can be cloned and expressed in E. coli as a recombinant protein that elicited immunity against the challenge infection with Schistosoma japonicum, indicating its potential as a partia1 protection vaccine candidate.