Taxol is one of the most potent anti-cancer agents, which is extracted from the plants of Taxus species. Isopentenyl diphosphate isomerase (IPI) catalyzes the reversible transformation between IPP and DMAPP, both of which are the general 5-carbon precursors for taxol biosynthesis. In the present study, a new gene encoding IPI was cloned from Taxus media (namely TmIPI with the GenBank Accession Number KP970677) for the first time. The full-length cDNA of TmIPI was 1 232 bps encoding a polypeptide with 233 amino acids, in which the conserved domain Nudix was found. Bioinformatic analysis indicated that the sequence of TmIPI was highly similar to those of other plant IPI proteins, and the phylogenetic analysis showed that there were two clades of plant IPI proteins, including IPIs of angiosperm plants and IPIs of gymnosperm plants. TmIPI belonged to the clade of gymnosperm plant IPIs, and this was consistent with the fact that Taxus media is a plant species of gymnosperm. Southern blotting analysis demonstrated that there was a gene family of IPI in Taxus media. Finally, functional verification was applied to identify the function of TmIPI. The results showed that biosynthesis of β-carotenoid was enhanced by overexpressing TmIPI in the engineered E. coli strain, and this suggested that TmIPI might be a key gene involved in isoprenoid/terpenoid biosynthesis.

Objective To establish a post?treatment prognostic score model for newly diagnosed metastatic nasopharyngeal carcinoma, and to investigate the feasibility of stratified therapy. Methods A total of 263 eligible patients with newly diagnosed metastatic nasopharyngeal carcinoma from 2002 to 2010 were enrolled as subjects. The primary tumor was treated with conventional radiotherapy, three?dimensional conformal radiotherapy, or intensity?modulated radiotherapy, and radiation areas included nasopharyngeal tumor and cervical lymphatic drainage region. The metastatic bone tumor was mainly treated with conventional external radiotherapy, while the metastatic liver or lung tumor was mainly treated with surgical resection, radiotherapy, or radiofrequency ablation. The first?line therapy for most of patients was cisplatin?based combination chemotherapy. Factors including the general characteristics, tumor status, and therapy for patients were involved in multivariate analysis, and a prognostic model was established based on the n value (HR=en ) of the prognostic factors. Results The factors influencing the overall survival (OS) in patients were a Karnofsky performance score (KPS) not higher than 70(P= 0?? 00), multiple organ metastases (P=0?? 00), combination with liver metastasis (P= 0?? 00), a number of metastases not less than 2(P= 0?? 00), a level of lactate dehydrogenase (LDH) higher than 245 IU/ L (P= 0?? 00), a number of chemotherapy cycles ranging between 1 and 3( P= 0?? 00), a poor response for metastatic tumor ( stable disease or progressive disease)(P= 0?? 00), and primary tumor not treated with radiotherapy (P= 0?? 01). Based on the prognostic score, patients were divided into low?risk group (0?1?? 5 points), intermediate?risk group (2?? 0?6?? 5 points), and high?risk group (≥7?? 0 points), and the 5?year OS rates in the three groups were 59?? 0%, 25?? 1%, and 0%, respectively. Conclusions The prognostic score model based on the KPS, serum level of LDH, multiple organ metastases, combination with liver metastasis, and number of metastases can effectively predict the survival in patients. Active treatment including at least 4 chemotherapy cycles and radiotherapy for primary tumor can prolong the survival time of patients in the low?and intermediate?risk groups. However, patients in the high?risk group were mainly treated with palliative radiotherapy due to no improvement in the survival by radiotherapy for primary tumor.

Taxol is one of the most potent anti-cancer agents, which is extracted from the plants of Taxus species. Isopentenyl diphosphate isomerase (IPI) catalyzes the reversible transformation between IPP and DMAPP, both of which are the general 5-carbon precursors for taxol biosynthesis. In the present study, a new gene encoding IPI was cloned from Taxus media (namely TmIPI with the GenBank Accession Number KP970677) for the first time. The full-length cDNA of TmIPI was 1 232 bps encoding a polypeptide with 233 amino acids, in which the conserved domain Nudix was found. Bioinformatic analysis indicated that the sequence of TmIPI was highly similar to those of other plant IPI proteins, and the phylogenetic analysis showed that there were two clades of plant IPI proteins, including IPIs of angiosperm plants and IPIs of gymnosperm plants. TmIPI belonged to the clade of gymnosperm plant IPIs, and this was consistent with the fact that Taxus media is a plant species of gymnosperm. Southern blotting analysis demonstrated that there was a gene family of IPI in Taxus media. Finally, functional verification was applied to identify the function of TmIPI. The results showed that biosynthesis of β-carotenoid was enhanced by overexpressing TmIPI in the engineered E. coli strain, and this suggested that TmIPI might be a key gene involved in isoprenoid/terpenoid biosynthesis.
Amino Acid Sequence ; Carbon-Carbon Double Bond Isomerases ; genetics ; Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; Paclitaxel ; biosynthesis ; Phylogeny ; Plant Proteins ; genetics ; Taxus ; enzymology ; genetics

Objective To evaluate the efficacy and safety of olopatadine hydrochloride for the treatment of chronic idiopathic urticaria (CIU). Methods A multicentre, double-blind, randomized, parallel-group, controlled clinical trial was conducted. A total of 144 patients with CIU from 3 research centers were enrolled into this study, and randomly and equally divided into a test group and a control group. The test group administrated olopatadine hydrochloride 5 mg twice a day for 28 consecutive days, while the control group administrated levocetirizine hydrochloride 5 mg in the forenoon and a placebo tablet of olopatadine hydrochloride 5 mg in the afternoon for 28 consecutive days. The symptom score reducing index(SSRI)served as the primary outcome, and global assessment score for efficacy and total response rates as the secondary outcome. Results Totally, 137 patients completed the trial, including 70 in the test group and 67 in the control group. As intention-to-treat analysis showed, there were no significant differences in the total response rate between the test group and control group on day 7 (64.29% (45/70)vs. 56.72%(38/67), P > 0.05), 14(82.86%(58/70)vs. 74.63%(50/67), P > 0.05), or 28(87.14%(61/70)vs. 77.61%(52/67), P >0.05)after start of treatment. The SSRI was significantly higher in the test group than in the control group after 4 weeks of treatment(82.67% ± 22.70% vs. 70.51% ± 32.07%, P < 0.05). In addition, no significant difference was observed in the incidence of adverse reactions between the test group and control group(33.80%(24/71)vs. 27.94%(19/68), P > 0.05), and adverse reactions mainly included lethargy, dry mouth, fatigue, etc. Conclusion Olopatadine hydrochloride is effective and safe for the treatment of CIU.

AIM:To observe the effects of sevoflurane preconditioning on brain injury in hypoxic mice and its possible mechanism. METHODS:Male C57BL/6J mice were randomly divided into control (C) group, hypoxia (H) group,2% sevoflurane preconditioning for 30 min + hypoxia(S1+H) group,2% sevoflurane preconditioning for 60 min+hypoxia (S2+H) group and 4% sevoflurane preconditioning for 30 min + hypoxia(S3+H) group. The hypoxia model was established by continuous inhalation of(6.5±0.1)% O2for 24 h. The sevoflurane preconditioning treatments,S1,S2 and S3,were conducted by inhalation of 2% sevoflurane for 30 min,2% sevoflurane for 60 min and 4% sevoflurane for 30 min,respectively,with the carrier of(21.0±0.5)% O2,followed by washout for 15 min and then hypoxia treatment. The histological changes of the hippocampal CA1 area were observed under light microscope and transmission electron micro-scope(TEM),and serum lactate dehydrogenase (LDH) activity was measured by colorimetric method. Furthermore, the protein levels of erythropoietin (EPO) and vascular endothelial growth factor(VEGF) in brain tissue homogenate were ex-amined by ELISA,and the content of malondialdehyde(MDA) and the activity of superoxide dismutase(SOD) and gluta-thione peroxidase(GPx) were measured by microplate reader. RESULTS:After hypoxia for 24 h,cell edema or pyknosis in the hippocampal CA1 area was observed in H group. Sevoflurane preconditioning reduced hypoxic injury, and the cell ultrastructure under TEM was significantly improved in S2+H group. Compared with C group,the serum LDH activity and the levels of EPO,VEGF and MDA in brain tissues were significantly increased in H group,while the activity of SOD and GPx decreased. After sevoflurane pretreatment,the serum LDH activity and the levels of EPO and VEGF in brain tissues were lower than those in H group,and the most significant difference was observed in S2+H group. Moreover, the MDA content and SOD activity decreased,and the GPx activity increased in the sevoflurane preconditioning groups. CONCLU-SION:Sevoflurane preconditioning attenuates brain injury in hypoxic mice by regulating antihypoxic protein synthesis and reducing oxidative stress.

OBJECTIVETo investigate the effect of diet-induced obesity on the apoptosis of testicular spermatogenic cells in pubertal male rats.

METHODSForty healthy male rats were equally and randomly divided into a control and a high-fat group, the former fed on normal diet, while the latter high-fat and high-calorie diet. The testes of the rats were harvested at the end of 10 weeks for detection of total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) in the peripheral blood with the automatic biochemical analyzer. Pathological changes of the testis were observed under the light microscope, the apoptosis of the testicular cells detected by TUNEL, the expressions of Bcl-2 and Bax proteins determined by immunohistochemistry, and those of Bcl-2 mRNA and Bax mRNA measured by RT-PCR.

RESULTSThe levels of TC, TG, LDL-C and HDL-C were significantly higher in the high-fat group (5.17 +/- 0.17, 1.18 +/- 0.09, 1.76 +/- 0.11 and 5.08 +/- 0.18) than in the control (1.38 +/- 0.12, 0.39 +/- 0.05, 0.97 +/- 0.07 and 0.75 +/- 0.06) (P < 0.05), so was the apoptotic index of spermatogenic cells (37.17 +/- 2.74 versus 5.16 +/- 0.81, P < 0.01), and the apoptotic spermatogenic cells were mainly spermatogonia and spermatocytes. The expressions of Bax protein and Bax mRNA were markedly higher in the high-fat group (153.26 +/- 8.74 and 1.08 +/- 0.12) than in the control (101.81 +/- 6.14 and 0.37 +/- 0.04) (P < 0.01), while those of Bcl-2 protein and Bcl-2 mRNA remarkably lower in the former (139.26 +/- 7.21 and 0.46 +/- 0.05) than in the latter (159.37 +/- 8.96 and 1.05 +/- 0.11) (P < 0.01).

CONCLUSIONDiet-induced obesity can increase the apoptosis of spermatogenic cells in the rat testis, which may be associated with the reduced expression of Bcl-2 and elevated expression of Bax.

Animals ; Apoptosis ; Diet ; Lipids ; blood ; Male ; Obesity ; etiology ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Wistar ; Testis ; cytology ; pathology ; bcl-2-Associated X Protein ; metabolism

Objective To observe the clinical efficacy of herb-partitioned fire therapy in treating stomachache due to cold-deficiency. Method Sixty patients with stomachache due to cold-deficiency were randomized into a treatment group and a control group, 30 cases each. The control group was intervened by Western and Chinese medications, based on which, the treatment group was given herb-partitioned fire therapy. The clinical efficacies of the two groups were observed, as well as the symptoms scores before and after the treatment. Result There was a significant difference in the clinical efficacy between the two groups (P<0.01). The symptoms scores dropped after the intervention in both groups, and the score in the treatment group was significantly lower than that in the control group (P<0.01). Conclusion Herb-partitioned fire therapy plus medication is effective in treating stomachache due to cold-deficiency, and it can produce a more significant efficacy than medication alone.

Objective To investigate the effects of advanced glycation end-products(AGEPs)on the function of normal keratinocytes in vitro so as to explore the role of AGEPs in impaired wound healing. Methods Normal rat keratinocytes were incubated with different concentrations of AGEPs.After 48 hours of culturing,the cell proliferation rates were measured by MTT colorimetric determination.The cell cycle distributions and apoptosis were analyzed with flow cytometry,and the migration was investigated by 24-well fluorimetric cell migration assay kit by exposing to 100?g/ml AGEPs.Nuclear extracts from these cells were examined for binding of nucleotides containing NF-?B consensus by immunocytochemistry and EMSA in vitro.Results The proliferations of normal keratinocytes were significantly arrested and many cells were induced to early apoptosis compared with control ones(P＜0.05)by exposing to AGEPs for 48 hours. Meanwhile AGEPs also irritated keratinocytes migration compared with control ones(P＜0.05).Inhibiting the activation of NF-?B could partly recover the proliferation of keratinocytes,reverse apoptosis and attenu- ate migration.Conclusion AGEPs are correlated with the migration,proliferation and apoptosis of kera- tinocytes by NF-?B.

Objective To establish a quick,economical and reproducible method for high-quality RNA extraction from pancreas.Methods We utilized TRIzol Reagent and liquid nitrogen to isolate total RNA from the rat pancreas.The RNA quality was determined by detection of its content and optic density(A) at 260/280nm,and electrophoresis in 1% non-denatured agarose gel.Then reverse transcription-polymerase chain reaction(RT-PCR) was performed to detect expression of the pancreas-specific genes.Results The content of the total RNA extracted from the rat pancreas reached 3-6?g/mg pancreatic tissues,and A260/280 ratio was 1.75-1.89.Electrophoresis of the total RNA showed 28S and 18S rRNA bands with clear smear between them.The RT-PCR products of pancreas-specific genes including insulin 1,glucagon,?-amylase and housekeeping gene ?-actin all exhibited clear bands on 1% agarose gel,which were located in the expected positions,respectively.Conclusion These results suggest that we have successfully isolated the high-quality and intact RNA from the rat pancreas with TRIzol Reagent and liquid nitrogen.The extracted total RNA can be used in RT-PCR for pancreatic gene expression.

OBJECTIVETo observe the effect of Compound Zhajin Granule (CZG) on Toll-like re-ceptor 4 (TLR4) signaling pathway in high-fructose corn syrup induced NASH mice.

METHODSThirty 6-week-old male C3H mice were divided into the high fat and high fructose (HFHFr) group (n = 20) and the control group (n = 10) according to body weight. Mice in the HFHFr group ate high fat diet and drank 20% fructose water, while those in the control group ate common diet and drank common water. After 8 weeks mice in the HFHFr group were divided into two group according to body weight, the HFHFr group and the CZG group, 10 in each group. Mice in the CZG group were fed with high fat forage and 20% fructose water, and administered with 50 mL/kg 12. 8% CZG (prepared by hawthorn, Radix Curcumae, Alisma Orientale, Fritillaria Thunbergii, Silybum Marianum, peach seed in the ratio of 3:1.5:1.5:2:1.5:2:1) by gastrogavage. Mice in the HFHFr group were fed in the same way and daily administered with equal volume of distilled water by gastrogavage. Sixteen weeks later all mice were sacrificed. Body weight, liver wet weight, liver function, and lipid metabolism were detected. Pathological changes of liver tissues were assessed by HE staining, oil red O staining, and Masson staining. Expressions of TLR4, myeloid differentiation factor 88 (MyD88), tumor necrosis factor-alpha (TNF-α) were detected using immunohistochemical staining and real-time fluorescent quantitative PCR.

RESULTSBody weight, alanine aminotransferase (ALT), aspartate aminotransferase (AST) were obviously lower in the CZG group than in the HFHFr group (P < 0.05); oil red O stained area and density were decreased more in the CZG group than in the control group. HE staining showed ballooning inflammation was reduced more in the CZG group than in the HFHFr group. Masson staining was negative. Positive rates of TLR4 and MyD88 and mRNA expressions were significantly lower in the CZG group than in the HFHFr group (all P < 0.05).

CONCLUSIONCZG could significantly inhibit TLR4 signaling pathway of liver in NASH mice.

Alanine Transaminase ; metabolism ; Animals ; Aspartate Aminotransferases ; metabolism ; Diet, High-Fat ; Drugs, Chinese Herbal ; pharmacology ; Fructose ; administration & dosage ; adverse effects ; Inflammation ; Lipid Metabolism ; Male ; Mice ; Mice, Inbred C3H ; Myeloid Differentiation Factor 88 ; metabolism ; Non-alcoholic Fatty Liver Disease ; drug therapy ; Signal Transduction ; drug effects ; Toll-Like Receptor 4 ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism