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 explore the expressions of endothelial nitric oxide synthase (eNOS) and cytochrome P450 (aromatase) in the testis of sexually mature male SD rats and their significance.

METHODSEighteen male SD rats, 6 five-week, 6 seven-week and 6 ten-week old, were selected for this study. Paraffin sections of the left testis were made and the expressions of eNOS and P450 observed by the immunohistochemical ABC method.

RESULTSPositive expressions of eNOS and P450 were found to be + + +, + and + + in the Leydig cells of the five-week, seven-week and ten-week old rats, respectively, and they were also observed in a few spermatocytes, though with no regularity.

CONCLUSIONIn the Leydig cells of sexually mature male SD rats, eNOS and P450 are differently expressed in different stages of sexual maturation, and they are correlated as well.

Animals ; Aromatase ; metabolism ; Male ; Nitric Oxide Synthase Type III ; metabolism ; Rats ; Rats, Sprague-Dawley ; Sexual Maturation ; Testis ; metabolism

Adenosine Triphosphate ; Atropine ; Child, Preschool ; Echocardiography, Stress ; methods ; Female ; Humans ; Infant ; Male ; Mucocutaneous Lymph Node Syndrome ; complications ; diagnostic imaging ; Myocardial Ischemia ; diagnostic imaging ; etiology

Objective To learn the current neglected situation and its impact factors on rural children in two provinces in Western China.Methods The investigation was conducted by using multistage stratified cluster sampling method in some parts of the Shaanxi province and the city of Chongqing.Results 1488 subjects,aged from 0 to 6,were recruited in the present study.Results showed that the total prevalence rates of neglected rural children in the two research sites were 31.59％ and 48.32 respectively.No significant difference was found on the prevalence of neglected for boys and girls ( x2 =0.86,U=1.51,P ＞ 0.05 ).The degrees of negligence in the older children showed a significant increase than in the younger children (x2 =13.36,F=33.45,P＜ 0.05 ).The prevalence and degree of negligence in families with three generations were lower than that of a single-parent families and remarried families (H=10.03,F=2.83,P＜0.05).The one-child-families were significantly lower than multiple children families,both in terms of prevalence and degree of negligence.Our data demonstrated the degrees of negligence in children whose parents were away from home,were higher than the children whose parents stayed with them ( x2=30.30,U=6.76,P＜0.05).The degree of negligence in Shaanxi was lower than that in Chongqing (x2=9.42,U=8.40,P＜0.05).Based on multivariate non-conditional logistic regression analysis,the determinants of stunting among the stranded children were as follows:parents were away from home (OR=1.54,95％ CI:1.20-1.97) ;structure of the family (OR=1.38,95％ CI:1.16-1.65) ; father' s occupation (OR=0.87,95％ CI:0.78-0.97) ; schooling of the children' s mother (OR=1.27,95％CI:1.07-1.52) ; relationship between children and their fathers (OR =1.43,95％ CI:1.07-1.91 ) etc.Conclusion The situation of negligence in children living in the rural areas,were serious in Shaanxi and Chongqing provinces,which called for the strengthening on the publicity and education of the issue.School and the society should also pay more attention to this problem.

OBJECTIVETo investigate the expressions of myogenic markers MyoD, myogenin,and desmin in skeletal muscle differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs).

METHODSMyogenic markers MyoD, myogenin,and desmin of hBM-MSCs cultured in vitro were detected by immunofluorescence and RT-PCR. A total of 21 8-to-10 week-old immunosuppressed mdx mice were transplanted with 1x107 passage 5 of hBM-MSCs. The mice were euthanized 2-24 weeks after transplantation,and gastrocnemius muscle were analyzed for human MyoD, myogenin,desmin,and dystrophin (Dys) expressions by immunohistochemistry and RT-PCR.

RESULTSThe numbers of MyoD-,myogenin-,and desmin-positive cells per 100 hBM-MSCs were 23.5∓5.3, 30.7∓6.2, and 28.4∓5.7, respectively. MyoD, myogenin, and desmin mRNA was observed in passage 5 of hBM-MSCs. After two weeks of hBM-MSCs transplantation,a small number of MyoD-and myogenin-positive cells were observed in skeletal muscle of mdx mice,and desmin-positive cells were observed 4 weeks after transplantation. Expressions of MyoD and myogenin were detected in the muscle of mdx mice 2-4 weeks after hBM-MSCs transplantation, which reached a peak 12-16 weeks later. Desmin was expressed in the muscle of mdx mice 4-8 weeks after transplantation,with much more expression after 16 weeks of transplantation. A small number of Dys-positive cell and Dys mRNA expression were presented in the muscle of mdx mice 4 and 8 weeks after hBM-MSCs transplantation,respectively. The expression of Dys in the muscle of mdx mice increased gradually after transplantation.

CONCLUSIONhBM-MSCs have the potential of myogenic differentiation in vitro and contribute to myogenic conversion in xenogeneic animal,during which the up-regulation of MyoD and myogenin expressions may play an important role.

Animals ; Biomarkers ; Bone Marrow Cells ; cytology ; metabolism ; Cell Differentiation ; Cells, Cultured ; Desmin ; metabolism ; Humans ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Mice ; Mice, Inbred mdx ; Muscle, Skeletal ; cytology ; metabolism ; MyoD Protein ; metabolism ; Myogenin ; metabolism ; Up-Regulation

OBJECTIVETo introduce the WHO 2000 diagnostic criteria of biopsy of colorectal intraepithelial neoplasia and carcinoma and to enhance diagnostic accuracy and avoid overdiagnosis and underdiagnosis.

METHODThe postoperative pathological examination and preoperative biopsy in 56 patients diagnosed as colorectal intraepithelial neoplasia and carcinoma before operation from January 2001 to October 2005 were compared retrospectively.

RESULTSAmong the 56 cases, 16 patients were diagnosed by preoperative biopsy as carcinoma in situ, intramucosal carcinoma and adenocarcinoma, but according to the new standard, of them 14 cases should be revised to be higher grade colorectal intraepithelial neoplasia.

CONCLUSIONSStrictly adhere to the new WHO criteria, colorectal intraepithelial neoplasia and carcinoma can be diagnosed properly, but for the cases that submucosal muscular layer would not presented in biopsy, the diagnosis should be made by combining clinical findings and various examination results so as to avoid underdiagnosis and delay of treatment.

Adult ; Aged ; Biopsy ; Carcinoma ; diagnosis ; pathology ; Carcinoma in Situ ; diagnosis ; pathology ; Colon ; pathology ; Colorectal Neoplasms ; diagnosis ; pathology ; Diagnosis, Differential ; Female ; Humans ; Male ; Middle Aged ; Preoperative Care ; Rectum ; pathology ; Retrospective Studies

OBJECTIVETo assess the distribution frequency of Gly82Ser polymorphism of receptor for advanced glycation end products (RAGR) gene and investigate its association with type 2 diabetic Chinese patients.

METHODSThe allele frequencies and genotype distribution of Gly82Ser polymorphism of RAGE gene were compared in a case-control study of 194 type 2 diabetic and 546 non-diabetic subjects. PCR-restriction fragment length polymorphism (PCR-RFLP) was used for detection of the genotype variants.

RESULTSIn general Chinese population and type 2 diabetic Chinese patients, the most frequent genotype and allele of RAGR gene Gly82Ser polymorphism were genotype GG and allele G, whose frequency distribution were significantly higher than those in other countries (P<0.01). No significantly difference in the genotype frequencies or allele frequencies of Gly82Ser polymorphism were found between the diabetic patients and non-diabetic subjects (P>0.05).

CONCLUSIONGly82Ser polymorphism of RAGE gene does not demonstrate any association with type 2 diabetes in Chinese patients, but high genotype and allele frequencies of Gly82Ser polymorphism occur in Chinese population and type 2 diabetic Chinese patients.

Aged ; Amino Acid Substitution ; Asian Continental Ancestry Group ; genetics ; China ; Diabetes Mellitus, Type 2 ; ethnology ; genetics ; Female ; Gene Frequency ; Genotype ; Glycine ; genetics ; Humans ; Male ; Middle Aged ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Polymorphism, Restriction Fragment Length ; Receptor for Advanced Glycation End Products ; Receptors, Immunologic ; genetics ; Serine ; genetics

OBJECTIVETo understand the characteristics of embB gene mutation of Mycobacterium tuberculosis (MTB) isolates from tuberculosis patients in Chongqing, and the value of embB306 as a molecular marker used to diagnose ethambutol (EMB)-resistant MTB strains.

METHODSDirect sequencing was used to analyze the polymorphism of embB mutation in 51 EMB-resistant MTB strains and 50 EMB-sensitive MTB strains. And diagnostic testing was used to evaluate the value of embB306 as a molecular marker of EMB -resistant MTB strains as compared with the traditional sensitivity test.

RESULTSAll 34 of 51 EMB-resistant strains (66.7%) and 3 of 51 EMB-sensitive strains (6%) had had embB306 mutation. The embB306 mutation rate in EMB-resistant strains coming from previously treated case was 87.5%, showing significantly higher than that from new cases (48.1%, P < 0.01); embB306 mutation rate was increased with the number of the resistant drugs; embB306 mutation serving as a marker to diagnose EMB-resistant MTB strains comparing with the traditional sensitivity test, had the rate of sensitivity = 66.7%, specificity = 94.0%, accuracy = 80.2% and Youden index = 60.7%.

CONCLUSIONembB306 mutation should be the main mechanism of MTB resistance to EMB in Chongqing, showing an association with the history of the treated and numbers of the resistant drugs. embB306 mutation should be a good marker to diagnose EMB-resistant MTB strains.

China ; DNA Mutational Analysis ; DNA, Bacterial ; genetics ; Genes, Bacterial ; Humans ; Mutation ; Mycobacterium tuberculosis ; drug effects ; genetics ; isolation & purification ; Pentosyltransferases ; genetics ; Tuberculosis, Multidrug-Resistant ; microbiology

OBJECTIVETo investigate the relationship of angiogenesis and the Ang family members/ receptor (Ang/Tie2) in hemangioma.

METHODSExpression of Ang1, Ang2 and the receptor Tie2 was detected with immunohistochemical SP method and RT-PCR method in 17 cases of proliferating hemangioma, 13 involuting cases and 10 cases of normal children skin.

RESULTSThe expression of Ang2 and Tie2 was higher markedly in proliferating hemangiomas than in involuting hemangiomas (P < 0.01), and was rare or negative in normal skin. Expression of Ang1 was rare or negative both in hemangioma and normal skin without significant difference between them (P > 0.05).

CONCLUSIONAng/Tie2 system may play an important role in the proliferating and involuting process of hemangioma.

Angiopoietin-1 ; metabolism ; Angiopoietin-2 ; metabolism ; Child, Preschool ; Hemangioma ; metabolism ; pathology ; Humans ; Infant ; Receptor, TIE-2 ; metabolism

OBJECTIVETo investigate the effect of bone marrow stem cell transplantation (BMT) on the diaphragm muscles of mdx mice, a mouse model of Duchenne muscular dystrophy (DMD).

METHODSThe bone marrow-derived stem cells form male SD rats was transplanted through the tail vein into 18 female 8-week-old mdx mice, which were sacrificed at 4, 8 and 12 weeks after BMT (6 at each time point), respectively. The diaphragm muscles of the mice were subjected to HE staining, immunofluorescence detection of dystrophin, reverse transcription (RT)-PCR analysis of dystrophin mRNA transcripts and PCR analysis of Sry (sex-determining region on the Y chromosome) gene, with age-matched female C57 mice and untreated mdx mice as the controls.

RESULTSThe proportion of centrally nucleated fibers (CNF) in the diaphragm muscle of the recipient mdx mice was (15.58+/-0.91) %, (12.50+/-1.87) % and (10.17+/-1.17) % at 4, 8 and 12 weeks after BMT, respectively, significantly smaller than that of untreated mdx mice [(19.5+/-1.87) %], and the fibers after BMT showed less inflammatory infiltration. Compared with the untreated mice, the recipient mdx mice showed green fluorescence on significantly more diaphragm muscle cell membranes [with the proportion of dystrophin-positive fibers of (1.00+/-0.32) %, (6.00+/-1.05) % and (11.92+/-1.11) % at 4, 8, and 12 weeks after BMT]. RT-PCR of dystrophin mRNA also demonstrated significantly higher relative levels of dystrophin in the recipient mdx mice (0.19+/-0.05, 0.26+/-0.06 and 0.36+/-0.04 at 4, 8 and 12 weeks after BMT) than in untreated mdx mice, and Sry gene was present in the recipient mice.

CONCLUSIONBMT can partially restore dystrophin expression and ameliorate the pathology in the diaphragm muscles of mdx mice, and has great potential to produce general therapeutic effect in patients with DMD.

Animals ; Bone Marrow Transplantation ; methods ; Diaphragm ; metabolism ; pathology ; Dystrophin ; biosynthesis ; genetics ; Female ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Inbred mdx ; Muscular Dystrophy, Duchenne ; metabolism ; pathology ; surgery ; Rats ; Rats, Sprague-Dawley ; Transplantation, Heterologous