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.
Amino Acid Sequence ; Cloning, Molecular ; Erythritol ; analogs & derivatives ; metabolism ; Gene Expression Regulation, Plant ; Molecular Sequence Data ; Nucleotidyltransferases ; chemistry ; genetics ; metabolism ; Phylogeny ; Plant Proteins ; chemistry ; genetics ; metabolism ; Protein Structure, Secondary ; Sequence Alignment ; Sugar Phosphates ; metabolism ; Tripterygium ; chemistry ; enzymology ; genetics

Vascular endothelial growth factor-D (VEGF-D) and vascular endothelial growth factor receptor-2, -3 (VEGFR-2, -3) with their corresponding signaling pathway play significant roles in the development of the embryonic vascular system and pathological lymphangiogenesis. The study was aimed to express and purify the GST-VEGF-D fusion protein, and to explore the angiogenesis effect of VEGF-D. The total RNA was extracted from human fetal lung tissue, and the mature form of VEGF-D was expanded by polymerase chain reaction (PCR), then the plasmid pGEX-5X-1/VEGF-D was reconstructed and the GST-VEGF-D fusion protein expressed in transformed E.coli BL21-DE3. The results showed that the molecular mass of this fusion protein was 38 kD and compassed more than 15% of the total bacteria proteins. The fusion protein was recognized by anti-GST and anti-VEGF-D antibodies. The soluble GST-VEGF-D fusion protein could interact with VEGFR-3/Fc and was able to stimulate the proliferation of human erythroleukemia cell line (HEL) cells. The data of chick embryo chorioallantoic membrane (CAM) experiments indicated that GST-VEGF-D could induce the CAM angiogenesis. It is concluded that the GST-VEGF-D fusion protein with biological activity was successfully expressed, and which may provide an experimental model for the investigation of the VEGF-D-induced angiogenesis and lymphangiogenesis.
Animals ; Chick Embryo ; Chorioallantoic Membrane ; blood supply ; Humans ; Neovascularization, Physiologic ; drug effects ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Vascular Endothelial Growth Factor D ; biosynthesis ; genetics ; pharmacology

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.
Amino Acid Sequence ; Cloning, Molecular ; Computational Biology ; Gene Expression Regulation, Plant ; Models, Molecular ; Molecular Sequence Data ; Phosphotransferases (Alcohol Group Acceptor) ; chemistry ; genetics ; metabolism ; Phylogeny ; Plant Proteins ; chemistry ; genetics ; metabolism ; Sequence Alignment ; Tripterygium ; chemistry ; enzymology ; genetics

OBJECTIVETo investigate the relationship between extracapsular spread (ECS) of cervical metastatic lymph node and the recurrence in patients with oral squamous cell carcinoma (OSCC).

METHODSThe medical records of 74 OSCC patients with histologically confirmed cervical lymph node metastasis were reviewed. They were divided into 2 groups, ECS positive (ECS+) and ECS negative (ECS-). The treatment results were followed up. Statistical analysis, with chi-square test, and multiple logistic regression was conducted.

RESULTSThe overall recurrence rates for pN+/ECS- and pN+/ECS+ patients were 47.6% and 75.0%, respectively, and the cervical recurrence rates for pN+/ECS- and pN+/ECS+ patients were 9.5% and 46.9%, respectively (P < 0.001). Multivariate analysis showed that ECS was one of the independent prognosis factors for cervical recurrence.

CONCLUSIONSExtracapsular spread significantly increased both overall and cervical recurrence rates, and ESC may be a prognosis factor for OSCC patients.

Adult ; Aged ; Aged, 80 and over ; Carcinoma, Squamous Cell ; pathology ; Female ; Follow-Up Studies ; Humans ; Logistic Models ; Lymph Nodes ; pathology ; Lymphatic Metastasis ; pathology ; Male ; Middle Aged ; Mouth Neoplasms ; pathology ; Neck ; pathology ; Neoplasm Recurrence, Local ; Prognosis ; Retrospective Studies

OBJECTIVETo investigate the effectiveness, safety and possible mechanism of recombinate human interleukin 11 (rhIL-11) in the treatment of chemotherapy-induced thrombocytopenia.

METHODSThirty-four patients (totally 76 cycles) with chemotherapy-induced thrombocytopenia received subcutaneous injection of rhIL-11 at the dose of 25 microg.kg(-1).d(-1) for 4 to 16 days. Serum IL-11 level was measured by ELISA, and IL-11 R alpha expression was detected by RT-PCR.

RESULTSThe mean baseline platelet count before chemotherapy was (135.0 +/- 54.3) x 10(9)/L for the 1st cycle and (259.4 +/- 64.5) x 10(9)/L for the 2nd cycle. The time to administer rhIL-11 was 7 to 16 days (median 12 days) in the 1st cycle and 4 to 10 days (median 6 days) in the 2nd, respectively (P < 0.05). The duration of post-chemotherapy platelet count below 50 x 10(9)/L was 7 to 13 days (median 10 days) for the 1st cycle and 3 to 8 days (median 5 days) for the 2nd, respectively (P < 0.05). Platelet count reached 300 x 10(9)/L or above in 30 chemotherapy cycles. The maximum platelet count was found to appear at D10 to D 17 (median D14), and negatively correlated with the pre-chemotherapy serum IL-11 level after administration of rhIL-11. Major adverse reactions included edema, headache, muscle and joint pain.

CONCLUSIONrhIL-11 is effective and safe for the treatment of chemotherapy-induced thrombocytopenia, with a relatively slow but sustained effect on the recovery of platelet count. Pre-chemotherpy serum IL-11 level might predict the efficacy of rhIL-11.

Adult ; Aged ; Aged, 80 and over ; Antineoplastic Combined Chemotherapy Protocols ; adverse effects ; therapeutic use ; Breast Neoplasms ; drug therapy ; Carcinoma, Non-Small-Cell Lung ; drug therapy ; Female ; Humans ; Injections, Subcutaneous ; Interleukin-11 ; administration & dosage ; adverse effects ; blood ; Lung Neoplasms ; drug therapy ; Male ; Middle Aged ; Platelet Count ; Recombinant Proteins ; administration & dosage ; adverse effects ; Thrombocytopenia ; chemically induced ; drug therapy ; Treatment Outcome

Cellular senescence is one of the important steps against tumor. This study was to observe the characteristics of boningmycin induced senescence of human tumor cells. MIT method and clone formation assay were used to detect the growth-inhibitory effect. Cellular senescence was detected with senescence-associated beta-galactosidase staining. Cell cycle distribution and accumulation of intracellular reactive oxygen species (ROS) were analyzed with flow cytometry. Protein expression was detected by Western blotting. The results showed that the growth-inhibitory effect of boningmycin was obviously stronger on human oral epithelial carcinoma KB cells than that on non-small cell lung cancer A549 cells. Comparison to the similar action of doxorubicin, that boningmycin induced the features of cellular senescence in both cell lines, its due to the arrest at G2/M phase and an increase of ROS level. The molecular senescence marker P21 increased significantly after boningmycin treatment at a dosage of 0.1 micromol x L(-1), whereas a higher concentration of it induced apoptosis. The results indicated that cellular senescence induced by boningmycin was one of its mechanisms in tumor suppression.
Antibiotics, Antineoplastic ; administration & dosage ; pharmacology ; Apoptosis ; drug effects ; Bleomycin ; administration & dosage ; analogs & derivatives ; pharmacology ; Carcinoma, Non-Small-Cell Lung ; metabolism ; pathology ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cellular Senescence ; drug effects ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Dose-Response Relationship, Drug ; Doxorubicin ; pharmacology ; Humans ; KB Cells ; Lung Neoplasms ; metabolism ; pathology ; Poly(ADP-ribose) Polymerases ; metabolism ; Reactive Oxygen Species ; metabolism ; Tumor Suppressor Protein p53 ; metabolism

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.
Amino Acid Sequence ; Cloning, Molecular ; Farnesyltranstransferase ; chemistry ; genetics ; metabolism ; Molecular Sequence Data ; Phylogeny ; Plant Proteins ; chemistry ; genetics ; metabolism ; Sequence Alignment ; Sequence Homology, Amino Acid ; Tripterygium ; chemistry ; enzymology ; genetics

OBJECTIVETo explore the effect and mechanism of tagalsin on hepatoma cells.

METHODSThe animal models were established by transplanting H(22) mouse hepatoma cells to mouse liver, and ten days later the mice were randomly divided into five groups: blank group, carmofur positive group and tagalsin groups, including low-dose, middle-dose and high-dose groups. Then medicine or oil was given to the mice by gastric gavage in consecutive 5 days with a 2-days interval as a course of treatment, two courses in all. All mice were killed at 24 hours after medication, and the survival period, ascites conditions, aggressive conditions intra- or extra-liver, weight changes, tumor volume and spleen index of the tumor-bearing mice were observed. Pathological changes of the tumors were examined. Apoptotic factors p53 and Bcl-2 protien and mRNA were detected by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR).

RESULTStagalsin inhibited the hepatoma growth effectively without influencing spleen index to some extent. The tumor inhibition rate of tagalsin low, middle and high dose groups were 17.9%, 63.1% and 71.8%, respectively. Immunohistochemical results showed that the p53 and Bcl-2 protein positive cell counts of the positive control and experimental groups were significantly lower than those of the blank group (P < 0.01). RT-PCR results showed that the p53 mRNA expression was significantly enhanced and Bcl-2 mRNA expression was decreased in the positive control groups and tagalsin treatment groups, especially in the high dose group, compared with those of the blank group (P < 0.05).

CONCLUSIONStagalsin can inhibit the growth of mouse hepatoma cells significantly. The mechanism of its anti-tumor effect may work via up-regulating the wild type p53 gene expression and down-regulating Bcl-2 gene expression and thus regulating tumor cell apoptosis.

Animals ; Body Weight ; drug effects ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Diterpenes ; pharmacology ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; pharmacology ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Mice ; Neoplasm Transplantation ; Plants, Medicinal ; chemistry ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Random Allocation ; Rhizophoraceae ; chemistry ; Tumor Suppressor Protein p53 ; genetics ; metabolism

ObjectiveTo investigate the iodine nutritional status of pregnant women,newborn heel blood thyroid stimulating hormone(TSH) level and their relationship with urinary iodine(UI) level during pregnancy in Zhoupu and Kangqiao districts of Pudong New Area of Shanghai.Methods A total of 993 urinary samples(the first,second and third trimesters of pregnancy were 200 people,respectively),breast feeding(193 people) and non-pregnant women (200 people) in Zhoupu and Kangqiao districts of Pudong New area were collected from Apr 2009 to Dec 2010.Two hundred copies of neonatal heel blood samples were collected.Median of UI was measured by arsenic-cerium catalysis.TSH in neonatal heel blood was analyzed 72 h after birth by time resolved fluoroisnmunoassay(TRFIA).ResultsMedian UI of all pregnant women was 161.35 μg/L,and that in third trimesters of pregnancy( 126.35 μg/L) was lower than that of the first,the second,the breast feeding and non-pregnant women (178.80,180.50,167.90,163.40 μg/L,all P＜ 0.05).The percentage of UI level less than 150 μg/L in the third trimester[57.5％(115/200) ] was higher than that of the first[39.0％(78/200) ],the second[39.5％(79/200) ],the breast feeding [ 16.6％ (32/193) ] and non-pregnant women [ 23.0％ (46/200) ],respectively (all P ＜ 0.05).The percentage of UI level higher than 300 μg/L in the first [9.0％(18/200)],the second[8.0％(16/200) ] and the third trimester [ 5.0％ ( 10/200 ) ] of pregnancy was lower than that of the breast feeding [ 20.2％ (39/193) ] and nonpregnant [20.5％(41/200) ] women,respectively(all P ＜ 0.05).The level of neonatal heel blood TSH was(2.92 ± 1.83)mU/L,the range was 0.01 - 9.76 mU/L,11.0％(22/200) of the neonates heel blood TSH level(5 mU/L)exceeded the ratio of World Health Organization (WHO) standard ( ＜ 3％ ) suitable for iodine nutrition.Conclusions The overall level of iodine nutrition among pregnant women in Zhoupu and Kangqiao districts of Pudong New Area of Shanghai is in the appropriate range,but the pregnant women in the third trimester is in mild iodine deficiencies,and the neonates in these districts may be prone to iodine deficiency.Monitoring of iodine nutrition of pregnant women should be strengthened and iodine supplementation should be done scientifically.

AIM:To explore the regulatory effect of chemokine CCL 3 on exosome secretion from human bone marrow mesenchymal stem cells(hBMSCs).METHODS: hBMSCs were stimulated with chemokine CCL 3 at different concentrations in vitro.The proliferation of hBMSCs was measured by CCK-8 assay and viable cell counting.Exosome se-cretion from hBMSCs was qualitatively analyzed by transmission electron microscope(TEM)and flow cytometry, and the quantitative analysis was carried out by flow cytometry and nanoparticle tracking analysis(NTA).RESULTS:Compared with control group,the viability of the hBMSCs detected by CCK-8 assay was increased when hBMSCs were treated with CCL3(P<0.05).The results of viable cell counting demonstrated that the number of hBMSCs was raised in CCL 3 group in a dose-dependent manner(P<0.05).The results of flow cytometry showed that hBMSCs expressed 3 CCL3-related spe-cific receptors,CCR1,CCR5 and CCR9.Compared with control group,the fluorescence intensity of CCR9 in CCL3 group was obviously enhanced.However,no significant difference of fluorescence intensity for CCR 5 and CCR1 was observed be-tween the 2 groups.The results of NTA demonstrated that the secretion capacity of CCL 3-induced hBMSCs was far less than that in control group(P<0.05).However, the microvesicles larger than 100 nm in CCL3 groups were increased(P<0.05).The above results indicated that the higher concentration of CCL 3 induced the lower secretion of exosomes.In addi-tion,the results of flow cytometry demonstrated that CCL 3-induced hBMSCs showed lower quantity of CD 9 +exosomes than those in control group(P<0.01).CONCLUSION:CCL3 promotes the proliferation of hBMSCs but depresses the secre-tion of exosomes in a dose-dependent manner.CCL3 affects the size distribution of exosomes and increases the number of nonfunctional microvesicles of larger than 100 nm in size.CCL3 induces the expression of CCR9 in hBMSCs.