Hyoscyamine 6 beta-hydroxylase (H6H) is the last rate-limiting enzyme directly catalyzing the formation of scopolamine in tropane alkaloids (TAs) biosynthesis pathway. It is the primary target gene in the genetic modification of TAs metabolic pathway. Full-length cDNA and gDNA sequences of a novel H6H gene were cloned from Datura arborea (DaH6H, GenBank accession numbers for cDNA and gDNA are KR006981 and KR006983, respectively). Nucleotide sequence analysis reveals an open reading frame of 1375 bp encoding 347 amino acids in the cDNA of DaH6H, while the gDNA of DaH6H contains four exons and three introns, with the highest similarity to the gDNA of H6H from D. stramonium. DaH6H also exhibited the most identity of 90.5% with DsH6H in amino acids and harbored conserved 2-oxoglutarate binding motif and two iron binding motifs. The expression level of DaH6H was highest in the mature leaf, followed by the secondary root, and with no expression in the primary root based on qPCR analysis. Its expression was inhibited by MeJA. DaH6H was expressed in E. coli and a 39 kD recombinant protein was detected in SDS-PAGE. Comparison of the contents of scopolamine and hyoscyamine in various TAs-producing plants revealed that D. arborea was one of the rare scopolamine predominant plants. Cloning of DaH6H gene will allow more research in the molecular regulatory mechanism of TAs biosynthesis in distinct plants and provide a new candidate gene for scopolamine metabolic engineering.
Cloning, Molecular ; DNA, Complementary ; Datura ; enzymology ; genetics ; Escherichia coli ; Hyoscyamine ; chemistry ; Mixed Function Oxygenases ; genetics ; Plant Leaves ; enzymology ; Plant Roots ; enzymology ; Recombinant Proteins ; genetics ; Scopolamine Hydrobromide ; chemistry

There are few articles or reports collecting evidence about Kudiezi injection from premarketing and postmarketing research or studies systematically. This article is an exact miniature of a systematical report about Kudiezi injection. We analyzed information from four aspects, such as quality control reports, non-clinical premarketing safety experiments, postmarketing research (efficacy studies, hospital information system data and national spontaneous reporting system data), and literature analysis. All the four aspects build an evidence body for Kudiezi injection in order to inform its safety use in clinical practice and further study.
Drugs, Chinese Herbal ; administration & dosage ; adverse effects ; Hospital Information Systems ; Humans ; Injections

Objective To analyze personality characteristics and psychological situation of children suffered from nephrotic syndrome of school age,and to provide clinical evidence for the children patients to be interfered fur- ther by psychological act.Methods EPQ,SAS and SDS were used to survey the personality and psychology of 30 cases of children suffered from nephrotic syndrome of school age and 28 cases of children who were in normal school age as statistic calculation comparison.Results The score of N,P,L inventory for the children suffered from nephrotie syndrome of school age was higher,and the score of E inventory was lower,which all had remarkably(P

Atropa belladonna is a medicinal plant and main commercial source of tropane alkaloids (TAs) including scopolamine and hyoscyamine, which are anticholine drugs widely used clinically. Based on the high throughput transcriptome sequencing results, the digital expression patterns of UniGenes representing 9 structural genes (ODC, ADC, AIH, CPA, SPDS, PMT, CYP80F1, H6H, TRII) involved in TAs biosynthesis were constructed, and simultaneously expression analysis of 4 released genes in NCBI (PMT, CYP80F1, H6H, TRII) for verification was performed using qPCR, as well as the TAs contents detection in 8 different tissues. Digital expression patterns results suggested that the 4 genes including ODC, ADC, AIH and CPA involved in the upstream pathway of TAs, and the 2 branch pathway genes including SPDS and TRII were found to be expressed in all the detected tissues with high expression level in secondary root. While the 3 TAs-pathway-specific genes including PMT, CYP80F1, H6H were only expressed in secondary roots and primary roots, mainly in secondary roots. The qPCR detection results of PMT, CYP80F1 and H6H were consistent with the digital expression patterns, but their expression levels in primary root were too low to be detected. The highest content of hyoscyamine was found in tender stems (3.364 mg x g(-1)), followed by tender leaves (1.526 mg x g(-1)), roots (1.598 mg x g(-1)), young fruits (1.271 mg x g(-1)) and fruit sepals (1.413 mg x g(-1)). The highest content of scopolamine was detected in fruit sepals (1.003 mg x g(-1)), then followed by tender stems (0.600 mg x g(-1)) and tender leaves (0.601 mg x g(-1)). Both old stems and old leaves had the lowest content of hyoscyamine and scopolamine. The gene expression profile and TAs accumulation indicated that TAs in Atropa belladonna were mainly biosynthesized in secondary root, and then transported and deposited in tender aerial parts. Screening Atropa belladonna secondary root transcriptome database will facilitate unveiling the unknown enzymatic reactions and the mechanisms of transcriptional control.
Alkaloids ; biosynthesis ; genetics ; metabolism ; Atropa belladonna ; genetics ; metabolism ; Gene Expression Regulation, Plant ; genetics ; Hyoscyamine ; genetics ; metabolism ; Plants, Medicinal ; genetics ; metabolism ; Scopolamine Hydrobromide ; metabolism ; Tropanes ; metabolism

OBJECTIVETo investigate the feasibility of using cartilage link protein of hyaluronic acid (HA-CLP) for defining the tumor boundary in a mouse model of lung carcinoma.

METHODSLung carcinoma was induced in KM mice by chemical carcinogenesis. HA-CLP separated from bovine cartilage and purified by affinity chromatography was labeled with (125)I for autoradiography. Immunohistochemical analysis and Western blotting were used to examine the efficiency of HA-CLP in defining the boundaries of the lung tumors.

RESULTSWith autoradiography, the clearest image of lung cancer was obtained at 2 h. With immunohistochemical method, the tumor boundary was the most clearly displayed at 2 h when the strongest signals of HA-CLP was detected; Western blotting also showed the clearest bands of HA-CLP at 2 h.

CONCLUSIONHA-CLP has the immunogenicity of HABP, and can efficiently indicate lung tumor boundary in autoradiography and immunohistochemistry.

Animals ; Autoradiography ; methods ; Extracellular Matrix Proteins ; metabolism ; pharmacology ; Female ; Hyaluronic Acid ; metabolism ; Immunohistochemistry ; Iodine Radioisotopes ; Lung Neoplasms ; radiotherapy ; Male ; Mice ; Proteoglycans ; metabolism ; pharmacology ; Radiotherapy, Image-Guided ; methods

OBJECTIVETo determine the optimal cytokine combinations with hepatic growth factor (HGF) that results in the most significant simultaneous in vitro expansion of cc-kit(+)Lin(-) cells derived from the bone marrow.

METHODSC-kit(+)Lin(-) cells were isolated from mouse bone marrow using a high-gradient magnetic cell sorting system (MACS) and expanded in the presence of stem cell factor (SCF), FLt-3 ligand (FL), leukemia inhibitor factor (LIF) thrombopoietin (TPO) and different concentrations of HGF for 7days in a liquid culture system. The total cell number and Annexin-V-positive cell number were counted, and the antigen expressions were studied with fluorescence-activated cell sorting (FACS).

RESULTSIn each group, c-kit(+)Lin(-) cells were expanded effectively and rapidly by 2 to 8 folds. Addition of 10 ng/ml HGF into SCF+FL+LIF+TPO resulted in the most significant expansion of c-kit(+)Lin(-) and total cells by 8.00 and 45.43 folds, respectively, with cell apoptosis rate of 17.42 %. But as the concentration of HGF increased, the c-kit(+)Lin(-) cells and the apoptosis rate decreased.

CONCLUSIONHGF at10 ng/ml shows optimal synergistic effect with SCF, FL, LIF and TPO in expansion of c-kit(+)Lin(-) cells, and excessive HGF may induce cell differentiation.

Animals ; Bone Marrow Cells ; cytology ; drug effects ; metabolism ; Cell Count ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Flow Cytometry ; Hepatocyte Growth Factor ; pharmacology ; Mice ; Mice, Inbred BALB C ; Proto-Oncogene Proteins c-kit ; metabolism

BACKGROUND: The thermo-sensitive composite hydrogels have gained increasing interest in bone regeneration domain due to their biomimetic extracellular matrix (ECM) structure, good biocompatibility, minimal invasive performance and in situ molding. OBJECTIVE:To prepare a thermo-sensitive injectable alginate/α-tricalcium phosphate (α-TCP)/collagen I (Alg/TCP/Col) composite hydrogel and explore its characterization. METHODS:Ca-carrying interdigitation-fusion vesicles (Ca-IFVs) were prepared. The liposomes carrying the optimal concentration of calcium ions were selected for the following experiments by investigating their encapsulation efficiency and drug loading rate. Alg/TCP/Col precursor solution (Alg or Alg/TCP precursor solution) was mixed with Ca-IFVs at 37℃ in different proportions (5, 10, 15, 20) to prepare thermosensitive hydrogels. The structure, rheology behavior, volume swelling ratio, and mechanical properties of the composite hydrogel were observed. MC3T3-E1 cells were co-cultured with Alg/TCP/Col, Alg, and Alg/TCP hydrogels, respectively. Then, morphology of the cells was observed by confocal microscopy at 1, 3, 7 days after co-culture. RESULTS AND CONCLUSION:(1) The pore size of the freeze-dried hydrogel was 50-100 μm, and TCP particles uniformly adhered to the surface of the Alg/TCP hydrogel surface. The Alg/TCP/Col hydrogel was a dense aggregate with collagen fibers in contrast to the Alg/TCP hydrogel. (2) The Alg/TCP/Col hydrogel exhibited a suitable phase transition temperature (Tm) between 35-39℃. (3) The volume swelling ratio of the hydrogel was increased with the increase of Ca-IFVs size. When the α-TCP complex was added into the Alg/TCP hydrogel, the swelling ratio decreased slightly. Alg/TCP/Col hydrogel exhibited a higher swelling ratio than the Alg/TCP hydrogel. (4) When the mixture ratio of precursor solution to liposome was 10, the compressive modulus of Alg/TCP/Col hydrogel and Alg/TCP hydrogel was significantly higher than that of the Alg hydrogel (P<0.05). (5) When the mixture ratio of precursor solution to liposome was 10, round MC3T3-E1 cells were observed on the Alg hydrogel; the cells on the surface of the Alg/TCP hydrogel were scattered and tended to extend; the cells on the surface of the Alg/TCP/Col hydrogel had a stress-extended morphology, and grew into the hydrogel, and meanwhile, the cell number increased significantly. To conclude, the liposome-mediated Alg/TCP/Col has good mechanical properties and cytocompatibility.

Objective To observe the effects of acupuncture at "Sanyinjiao" (SP6) and "Baihui" (GV20) on behaviors and contents of monoamine neurotransmitter dopamine (DA), noradrenalin (NE) and 5-hydroxytrypamine (5-HT) in prefrontal cortex (PFC) of attention deficit hyperactivity disorder (ADHD) rats; To discuss mechanism of action of acupuncture for prevention and treatment of ADHD. Methods 4-week-old SHR rats were randomly divided into the model group, Western medicine group and acupuncture group, and WKY rats with the same age were selected as the normal control group, with 10 rats in each group. Acupuncture was applied to "Sanyinjiao" (SP6) and "Baihui" (GV20) of rats in acupuncture group for 15 min. The rats in Western medicine group was given MPH (ritalin) for gavage, and rats were treated for 4 weeks in the dark-phase. Open field test (OFT), elevated plus maze (EPM) and novel object recognition test (NORT) were conducted to evaluate the spontaneous activity, impulsivity and learning-memorial ability of rats individually at the end of treatment. Contents of DA, NE and 5-HT in PFC were detected by HPLC. Results Compared with the normal control group, all of the behavior parameters of rats in model group were significantly higher (P＜0.05), and the contents of DA, NE and 5-HT in PFC of model group were lower than those of normal control group (P＜0.05). Compared with the model group, acupuncture group showed shorter movement distance, less rearing and fewer grooming activities in the OFT; percentages of times of entering the open arms and staying duration were reduced in the total period in the EPM; preference index in the NORT was elevated (P＜0.05); the contents of NE and 5-HT in PFC of acupuncture group increased significantly (P＜0.05). Conclusion Acupuncture at "Sanyinjiao" (SP6) and "Baihui" (GV20) canreduce the spontaneous activity and impulsivity, and improve learning and memory ability of ADHD model rats, which may be related to elevated contents of NE and 5-HT in the PFC.

OBJECTIVETo detect changes of Foxp3 expression in the decidua in patients with preeclampsia and investigate the correlation of Foxp3-924 (rs2232365) polymorphisms with preeclampsia.

METHODSFrom October 2011 to December 2012, 252 normal pregnant women and 156 preeclampsia patients of Han nationality from the same geographic region were tested for Foxp3-924 genotypes by polymerase chain reaction with sequence-specific primer (PCR-SSP). Sixty-eight of the patients with preeclampsia (33 with mild and 35 with severe preeclampsia) and 30 of the normal pregnant women were also examined for Foxp3 expression in the decidua using immunohistochemical method.

RESULTSFoxp3 positive expression rates in the decidua was 51.52% in mild preeclampsia and 28.57% in severe preeclampsia cases, significantly lower than that in the control group (86.67%, P<0.05). In preeclampsia patients, the frequencies of Foxp3-924G/G, G/A, and A/A genotypes were 0.1346, 0.4615 and 0.4038, respectively, and the frequencies of Foxp3-924A and Foxp3-924 G were 0.6346 and 0.3654, respectively. The genotype frequencies of Foxp3-924G/G, G/A and A/A in the control group were 0.1508, 0.4087 and 0.4405, respectively, and the frequencies of Foxp3-924 A and Foxp3-924 G were 0.6448 and 0.3552, respectively. No significant differences were found in the gene frequencies of Foxp3-924G/A between preeclampsia patients and the control group (P>0.05).

CONCLUSIONThe expression level of Foxp3 in the placental tissue of preeclampsia patients is significantly lower than that in normal pregnant women, suggesting that lowered Foxp3 expression decreases the immunosuppressive function and causes imbalance of immune tolerance between maternal-fetal to induce preeclampsia. Foxp3-924 polymorphisms is not significantly correlated with the occurrence of preeclampsia.

Case-Control Studies ; Female ; Forkhead Transcription Factors ; genetics ; metabolism ; Gene Frequency ; Genotype ; Humans ; Placenta ; metabolism ; Polymorphism, Genetic ; Pre-Eclampsia ; genetics ; Pregnancy

OBJECTIVETo study the incidence and the risk factors of dysphagia after ischemic stroke in Chengdu City, China.

METHODSReview was made with unified questionnaire on dysphagia after ischemic stroke in 563 patients from four hospitals, from January to December 2005. Over 20 risk factors related to dysphagia after ischemic stroke were analyzed by logistic regression.

RESULTSThe total incidence of dysphagia after ischemic stroke was 13.3% in Chengdu. Data from logistic regression analysis indicated that gender, morbidity frequency, basal nuclei, periventricular lesion, hypertension, pneumonia, hyperlipemia, cervical spondylosisi, internal carotid artherosclerosis were closely related to dysphagia after ischemic stroke,with OR values as 0.416, 0.489, 0.327, 0.454, 2.187, 2.146, 0.352, 0.242 and 0.273 respectively.

CONCLUSIONThe total incidence was lower than literature published from home and abroad. Gender, morbidity frequency, basal nuclei, periventricular lesion, hypertension, pneumonia, hyperlipemia, cervical spondylosisi and internal carotid artherosclerosis seemed to be risk factors to dysphagia after ischemic stroke.

Aged ; Aged, 80 and over ; Brain Ischemia ; complications ; Carotid Artery Diseases ; complications ; China ; epidemiology ; Deglutition Disorders ; epidemiology ; etiology ; Female ; Humans ; Hyperlipidemias ; complications ; Hypertension ; complications ; Logistic Models ; Male ; Middle Aged ; Pneumonia ; complications ; Risk Factors ; Sex Factors ; Spondylosis ; complications