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 study the antalgic and antiphlogistic functions and mechanism of ronggudingtong (RGDT) plaster (traditional Chinese medicine).

METHODSThe painful models were established with hot plate test or acetic acid writhing and the inflammatory models were established with daubing dimethylbenzene on auricle or injecting formaldehyde in toe or synovial envelope to study the antalgic and antiphlogistic functions of RGDT Plaster. The total protein and leukotriene B4(LTB4) in inflammatory exudate were detected to investigate the antalgic and antiphlogistic mechanism of RGDT plaster. The mice were randomly divided into different groups (n = 11), on the basis of drug using, the indexes of pain threshold, swelling degree were observed. Sixty-six mice were used to establish gasbag synovitis model and randomly divided into normal control group,model control group, positive control group (Voltaren gel 0.8 mg/d)and low/medium/high dosage RGDT plaster treating groups(30 mg/d, 60 mg/d, 120 mg/d).

RESULTS30 mg/d, 60 mg/d,120 mg/d RGDT plaster could upgrade the pain thresholds, remit auricular and foot swelling (P < 0.05, P < 0.01), and degrade total protein and LTB4 in inflammatory exudates (P < 0.05, P < 0.01).

CONCLUSIONRGDT plaster has some antalgic and antiphlogistic functions, and one of the mechanisms is depressing synthesis of LTB4.

Analgesics ; pharmacology ; Animals ; Anti-Inflammatory Agents ; pharmacology ; Leukotriene B4 ; metabolism ; Medicine, Chinese Traditional ; Mice ; Pain ; drug therapy

After binding to its ligand, epidermal growth factor receptor (EGFR) dimerizes and is autophosphorylated. These events initiate the signal transduction process, which regulates a plethora of biologic activity. The duration and strength of these signals are controlled by many regulatory mechanisms, including downregulating activated EGFR primarily via endocytosis and ubiquitination-dependent lysomal degradation. The interaction between EGFR and the ubiquitin ligase Cbl/adaptor protein CIN85, as well as ESCRT complex recruitment play important roles in the process of downregulating EGFR. Tumorigenesis results when the de-sensitization process of EGFR is halted by its own mutation or a mutation that abrogates Cbl E3 ligase activity.
Adaptor Proteins, Signal Transducing ; metabolism ; Animals ; Down-Regulation ; Endocytosis ; Epidermal Growth Factor ; pharmacology ; Humans ; Mutation ; Proto-Oncogene Proteins c-cbl ; metabolism ; Receptor, Epidermal Growth Factor ; genetics ; metabolism ; Signal Transduction ; Ubiquitin ; metabolism

This study examined the anti-hepatitis B virus (HBV) effect of wild-type (WT) vacuolar protein sorting 4B (VPS4B) and its dominant negative (DN) mutant VPS4B-K180Q in vivo in order to further explore the relationship between HBV and the host cellular factor VPS4. VPS4B gene was amplified from Huh7 cells by RT-PCR and cloned into the eukaryotic expression vector pXF3H. Then, the VPS4B plasmid and the VPS4B-K180Q mutation plasmid were constructed by using the overlap extension PCR site-directed mutagenesis technique. VPS4B and HBV vectors were co-delivered into mice by the hydrodynamic tail-vein injection to establish HBV vector-based models. Quantities of HBsAg and HBeAg in the mouse sera were determined by ElectroChemiLuminescence (ECL). HBV DNA in sera was measured by real-time quantitative PCR. Southern blot analysis was used to assay the intracellular HBV nuclear capsid-related DNA, real-time quantitative PCR to detect the HBV-related mRNA and immunohistochemical staining to observe the HBcAg expression in the mouse liver tissues. Our results showed that VPS4B and its mutant VPS4B-K180Q could decrease the levels of serum HBsAg, HBeAg and HBV-DNA. In addition, the HBV DNA replication and the mRNA level of HBV in the liver tissues of treated mice could be suppressed by VPS4B and VPS4B-K180Q. It was also found that VPS4B and VPS4B-K180Q had an ability to inhibit core antigen expression in the infected mouse liver. Furthermore, the anti-HBV effect of mutant VPS4B-K180Q was more potent than that of wild-type VPS4B. Taken together, it was concluded that VPS4B and its DN mutant VPS4B-K180Q have anti-HBV effect in vivo, which helps develop molecular therapeutic strategies for HBV infection.

Objective To explore the value of SOD activity in diagnosis of central nervous system leukemia (CNSL )by detec-ting SOD activity of cerebrospinal fluid of patients with CNSL .Methods The cerebrospinal fluid of 55 patients from department of hematology of Nanfang hospital of southern medical university were collected from January 2008 to January 2009 ,in which 30 pa-tients suffered with central nervous system leukemia (CNSL group) ,the other 25 patients suffered with acute leucemia without im-paired central nervous system(control group) .The SOD activity of cerebrospinal fluid was detected by the xanthine oxidase meth-od ,while the routine test ,biochemistry test and cell smear of cerebrospinal fluid was detected .Results There were statistics differ-ence in the level of white cell and protein in cerebrospinal fluid between CNSL and control group (P<0 .05) ,but with no difference in the level of cerebrospinal fluid pressure ,glucose ,chlorine(P>0 .05) .There was statistics difference in the level of SOD activity between CNSL and control group(P<0 .05) .The white cell quantity and the protein level in cerebrospinal fluid had negative corre-lation with the activity of SOD ,(r=0 .871 ,P=0 .000 ;r=0 .518 ,P=0 .003) .The activity of SOD in the cerebrospinal fluid had sta-tistics difference before and after intrathecal chemotherapy (P<0 .05) .The activity of SOD in the cerebrospinal fluid whose under 45 year-old (755 .64 ± 345 .77) ,which was significant lower than that of the paitents whose equal with or above 45 year-old (1 420 .49 ± 307 .69)(P<0 .05) .Conclusion The changes of the SOD activity in the cerebrospinal fluid had relation with central nervous system leukemia ,and the SOD activity might be a auxiliary diagnosis index used in central nervous system leukemia by revi-sing age factor .

Objective To investigate the therapeutic effect of intracerebral transplantation of mesenchymal stem cells(MSCs) derived from human umbilical cord blood(UCB) on hypoxic-ischemic brain damage(HIBD) in neonatal rat.Methods Twenty samples of human UCB were collected from healthy full-term newborns.MSCs were isolated from human UCB by density gradient centrifugation and purified by adhere cell selection method.For transplantation,P3 human UCB-derived MSCs were labeled by the 5-bromo-2-deoxyuridine (BrdU).Thirty SD rats of 7 d were built for neonatal HIBD model.One rat died and others were divided into transplant group(n=18) and control group(n=11).At the third day after building models,human UCB-derived MSCs were injected into left cortex in transplant group,while PBS of the same volume was injected into the same site in control group at the same time.The seventh day after transplantation,6 rats of transplant group were sacrificed to prepare brain tissue sections.The survival,migration and differentiation of the transplanted cells were investigated by brain tissue immunohistochemical analysis,and nervous function of 2 groups were evaluated by modified neurological severity score(mNSS) on the first,7th,14th,21th and 28th day after transplantation.Results MSCs were isolated from 5 of 20 human UCB samples.Immunocytochemical analysis of brain tissue showed that the transplanted human UCB-derived MSCs could survive and migrate around by the center of transplant site.There were (12.67?2.73)% of MSCs differentiated into astrocyte-like cells.mNSS showed that the score of transplant group was lower than that of control group on the first,7th,14th,21th and 28th day,and the differences of score points between 2 groups on the 14th,21th and 28thday were statistically significant(Pa

HK-2 cells were cultured with various concentrations of glucose and insulin for 12,24,48,72 h.Transforming growth factor-?_1(TGF-?_1) protein in supematant was measured by ELISA,while TGF-?_1 mRNA expression was assessed by RT-PCR.Data showed that high concentration of glucose and insulin up-regulated the expression of TGF-?_1 in HK-2 cells through different pathways.

Objective To explore the clinical characteristics,diagnosis,treatment and misdiagnosis matter of children with idiopathic pulmonary hemosiderosis(IPH).Methods The data of clinical characteristics,laboratory examination,treatment and follow-up of 21 children admitted from Jun.1993 to May 2007 were retrospectively analyzed,included 9 males,12 females,aged 1-14 years old,course of di-sease were 1-6 months.Twenty-one patients were diagnosed as IPH by chest X-ray radiography,CT scan,bone marrow biopsy,hemosiderin-laden macrophages in either sputum or gastric juice or bronchoalveolar lavage fluid.Then,therapy and prognosis of IPH were analyzed.Results All patients had varied degrees of anemia,11(52.38%) children had cough,9 (42.86%) children had fever,6(28.50%) cases had shortage of orexia,4( 19.05%) children had hemoptysis.Chest X-ray radiography and CT scan demonstrated diffuse patchy,nodular,reticulate pattern.Eighteen children received bone marrow biopsy and presented hyperplastic erythropoiesis,4(22.2%) cases were accompanied iron deficiency anemia.Nineteen (90.80%) cases shown the presence of hemosiderin-laden macrophages in either sputum or gastric juice or bronchoalveolar lavage fluid.Twenty-one misdiagnosed patients consisted of bronchopneumonia combined anemia(8 cases),lung tuberculosis combined anemia(5 cases),nulli-iron anemia(4 cases),hemolytic anemia(3 cases),myelodysplastic syndrome(1 case)and received corticosteroid therapy.Four cases of all patients were associatated with large-dose human-?-globulin and 3 cases were associatated with vincristine therapy.The therapeutic effect was significant.Eighteen patients were followed-up,3 patients were of which cured and had stopped treatment for over 2 years,11 patients presented clinically persistent remission,4 patients were recurred and aggravated.Conclusions Early diagnosis and long-term therapy of corticosteroid are very important for controlling acute onset,lessening the frequency of IPH recurrence and improving prognosis of the disease.

Objective To analyze if there are membrane estrogen receptors(ER)in endometrial carcinoma and if there is some relationship between membrane ER and nuclear ER.Methods The cell membrane and total cell ER?and ER?expressions of high and moderate differentiation endometrial carcinoma cells(Ishikawa and HEC-1A cells)were analyzed.Intermittent immunofluorescence dyeing and fluorescent microscopy were carried out with the ceils treated with polyformaldehyde and Triton X-100. Intermittent immunofluorescence dyeing and flow cytometry were carried out with the live cells and the cells treated with Triton X-100 respectively.Results There were fluorescences on the membrane of the Ishikawa and HEC-1A cells which were treated with polyformaldehyde.When the cells were treated with Triton X- 100,the fluorescences were also seen inside the cells.The fluorescence intensity of ER?and ER?in Ishikawa cell membrane(1.09?0.21,1.27?0.33)was stronger than the control,but there were no significant differences(P＞0.05).When treated with Triton X-100,the total cell fluorescence intensity of ER?and ER?in Ishikawa cell(4.21?0.34,4.69?1.96)was stronger than the membrane(P＜0.05). The ER?and ER?fluorescence intensity of HEC-1A cell membrane(1.58?0.13,1.49?0.04)were stronger than the control(P＜0.05).The fluorescence intensity of ER?and ER?of the HEC-1A cell (2.34?0.33,2.52?0.15)was stronger than the membrane also(P＜0.05).The membrane ER?fluorescence intensity of Ishikawa was lower than HEC-1A(P=0.028).But the total cell ER?fluorescence intensity of Ishikawa was higher than HEC-1A(P=0.002).Conclusions There are membrane ER on endometrial carcinoma cells Ishikawa and HEC-1A.The membrane ER must have some similarity to the nuclear receptor.There is no direct correlation between the quantity of the membrane ER and nuclear ER.

Objective To explore the clinical significance of the blood lactic dehydrogenase(LDH), ?_2-microglobulin(?_2-MG),D-dimer measuring in the diagnosis and treatment of Non-Hodgkin lymphoma. Methods In 40 cases with NHL,LDH was measured by L-P continuous monitoring method,?_2-MG was measured by luminescent immunoassay,D-dimer was measured by immunoturbidimettic assay.Results The levels of the blood LDH,?_2-MG and D-dimer in patients with NHL were higher than those of in the controls(P 0.05).Con- clusion The levels of blood LDH,?_2-MG,D-dimer can be taken as an auxiliary clinical index to diagnose, classify the phase,evaluate the effectiveness of treatment and prognosis in the NHL patients,and have impor- tant clinical significance.