Objective To observe the effects of menopause on prothrombotic profiles in ovariectomized rats .Methods Thirty two healthy female SD rats of 9 to 10 months were divided into 4 groups, the control shamed and the observation groups ovariectomized .Rats in the baseline group and the early menopause group were sacrificed one week later , and the control and late menopause group 10 weeks later.The prothrombotic profiles were detected including plasma FIB , ATⅢ activity, PAI-1 levels, D-D level, vWF levels and NO concentration, TXA2 and PGI2 levels.Results In early menopause, plasma FIB increased dramatically while ATⅢactivity remained lit-tle changed.PAI-1 demonstrated an increasing trend .vWF significantly increased but NO significantly decreased .In later menopausal stage, PAI-1 increased dramatically but FIB somewhat decreased .Plasma ATⅢactivity significantly increased and vWF still remained a high level.NO increased a little.In both early and later stage, TXA2 and PGI2 simultaneously increased while D -D showed little change between groups .Conclusion Menopause plays different roles in different aspects of thromoembolism , resulting in increased risk in early menopause due to prothrombotic state and decreased risk in later menopause when new balances between profiles were established .

Objective To study effects of raloxifene (RLX) with different doses of conjugated equine estrogen(CEE) on prothrombotic profiles in the ovariectomized rats model.Methods Total of 32healthy female SD rats at age of 9 to 10 months were equally divided into every 8 rats at 4 groups randomly.One week after ovariectomized,they were treated by drugs,including control group with placebo(0.9％ Nacl intragastric administration),RLX group with RLX 6 mg/(kg · d),RLX and low CEE group with RLX 6 mg/(kg · d) + CEE 0.07 mg/(kg · d)and RLX and high CEE group with RLX 6 mg/(kg · d) + CEE 0.5mg/(kg· d)for 10 weeks before death.Thrombin turbidimetry method was used to evaluate the plasma fibrinogen(FIB),transmitting substrate method for antithrombin Ⅲ (AT Ⅲ) activity,double-antibody sandwich ELISA for plasminogen activator inhibitor 1 (PAI-1),D-dimer (D-D) and yon Willebrand factor (vWF) and nitrate reductase method for nitric oxide(NO).Results (1) Coagulation and anticoagulation indicators:it was observed (1.62 ± 0.22) g/L FIB at control group,(2.02 0.54) g/L at RLX group,(1.97 ±0.16) g/L at RLX and low CEE group,(2.00 0.18) g/L at RLX and high CEE group.There was a statistically significant difference between control group and any one of treatment groups(P ＜ 0.05) and no statistical significance among those three treatment groups (P ＞ 0.05).No significant change was observed in plasma AT Ⅲ activity among groups (P ＞ 0.05).(2) Fibrinolytic and anti-fibrinolytic indicators:it was observed (14.1 2.8) μg/L PAI-1 at control group,(20.0 ±3.3) μg/L at RLX group,(41.5 ±5.5) μg/L at RLX and low CEE group,(38.9 ± 6.0) μg/L at RLX and high CEE group.A remarkable increase was observed between control group and any one of treatment groups(P ＜0.05).But there was no significant difference of D-D among groups (P ＞ 0.05).(3) Endothelial function:it was (43 ± 7) ％ vWF at control group,(49±5)％ at RLX group,(46±6)％ at RLX and low CEE group,(36±5)％ at RLX and high CEE group.The vWF of RLX and high CEE group was the lowest among all groups (P ＜0.05).There was no difference of NO among groups (P ＞ 0.05).Conclusions In the different links of thrombosis,RLX gives different fuction and may increase the risk.CEE plays a synergism role in the matter of fibrinolysis and anti-fibrinolysis with RLX,further giving rise to thrombosis effect of RLX.And it also has a protective role in the function of vascular endothelium to some extent.But this only works with high dose.

Objective:To retrospectively investigate the regulation of cervical and posterior to level V (PLV) lymph node metastasis on clinical target delineation in radiotherapy for nasopharyngeal carcinoma (NPC). Methods:A total of 428 NPC cases from February 2013 to April 2016 were subjected to enhanced CT scan from the base of the skull to the clavicle for pathological diagnosis. A deputy chief physician and an attending physician assessed the nodal distribution in each level in accordance with the RTOG guidelines proposed in 2013. The central point of the metastatic lymph nodes of PLV in the patients were recreated proportionally on the CT images of a stan-dard patient with N0 NPC in reference to the normal anatomy of the PLV area. SPSS 19.0 was used to analyze the correlation between PLV and the other levels. Moreover, the nodal location and characteristics of PLV were analyzed. Results:Among the 428 patients, 381 (89.0%) showed nodal involvement. The top four metastatic probabilities were presented as follows:Ⅱb (75.2%),Ⅶa (60.3%),Ⅱa (59.6%), andⅢ(42.0%). Up to 21 (4.9%) patients exhibited nodal involvement of PLV with 32 nodes. The mean vertical distance of all central points of PLV from the anterior border of the trapezius was 16 mm. Correlation analysis indicated the nodal involvement of PLV with the ipsilateral level Va (P=0.001). Conclusion:NPC showed a high probability of nodal metastasis. Nodes were mostly metasta-sized from the upper to the lower level, as well as from the proximal to the distal area. The leap metastasis rate was very low. The nod-al involvement of PLV correlated with the ipsilateral metastasis of level Va. Thus, the ipsilateral delineation of the posterior border of level V should be contoured to 25 mm far from the anterior surface of the trapezius during the nodal involvement of level Va.

AIM: To explore the role of calcineurin i n th e expression of NF-?B and the neurotoxicity in cultured cortical neurons treate d with interleukin-1? (IL-1?) and NMDA. METHODS: The cultured rat cortical neurons were used in the expe riment, damage of neurons was induced by interleukin-1?(IL-1?) or excitator y amino acid (NMDA). The degree of neuron damage was examined with the methods o f MTT assay and LDH releasing rate assay, as well as the Annexin V and PI immuno fluorescence. The expression of NF-?B p65 on the neurons was tested by the West ern blot analysis. RESULTS: Viability of neurons was obviously lower in the IL-1? group and NMDA group respectively than that in control group (P0.05). Annexin V and PI immunofluoresc ence showed that IL-1? mainly induced the neuron apoptosis, and NMDA induced th e neuron necrosis. CONCLUSIONS: The calcineurin mediates the higher expression of N F -?B p65 and neuron damage induced by IL-1?, but not play a critical role in th e necrosis induced by NMDA in the cultured cortical neurons. These results indic ate that calcineurin is the key molecule in the apoptotic signaling pathway.

Objective To observe the treatment effect of Shah Mette Lo/fluticasone inhalation(Seretide)for stable stage of chronic obstructive pulmonary disease (COPD).Methods 50 patients with COPD were randomly divided into treatment group and control group.The treatment group was given Seretide (specifications of 250μg)1 -2 ceiling/time,2 times/day,after 24 weeks of treatment,the control group was given 2mg of regular salbutamol com-bined with budesonide 1mg atomization inhalation,2 times daily.The clinical symptoms were compared before and aftertreatment.Results In the treatment group,acute seizure frequency was significantly reduced,cough,wheezing singing decreased or disappeared,self -care ability improved,the control group had no obvious change.The control group:before treatment FEV1 (1.1 ±0.4)L;FEV1 % predicted value(22.5 ±5.1 )%;FEV1 /FVC%:(25.3 ±5.8)%;after treatment FEV1 (1.5 ±0.7)L;FEV1 % predicted value(29.4 ±6.2)%;FEV1 /FVC%:(32.8 ±6.6)%.The treatment group:before treatment FEV1 (1.1 ±0.6)L;FEV1 % predicted value (24.5 ±5.6)%;FEV1 /FVC%:(26.7 ±6.6)%;after treatment FEV1 (1.7 ±0.6)L;FEV1 % predicted value(33.4 ±7.8)%;FEV1 /FVC%:(37.8 ± 8.6)%.Conclusion In patients with stable COPD inhaled Seretide treatment can significantly improve lung func-tion,and it is better than sardine an alcohol combined with budesonide inhalation therapy.

Pichia pastoris is one of the most important systems used in the field of molecular biology for the expression of recombinant proteins. The system has advantages of high expression, high stability, high secretion, easy high-density fermentation and low cost. Many factors affect the expression of recombinant protein, such as gene copy number, codon usage preference, type of promoter, molecular chaperones, glycosylation, signal peptide and fermentation process. In this review, research advances of the above aspects are summarized, which lay a foundation for improving the expression of recombinant proteins in P. pastoris.
Fermentation ; Gene Dosage ; Glycosylation ; Molecular Chaperones ; Pichia ; metabolism ; Promoter Regions, Genetic ; Protein Sorting Signals ; Recombinant Proteins ; biosynthesis

Lanosterol synthase is encoded by the erg7 gene and catalyzes the cyclization of 2, 3-oxidosqualene, which is a rate-limiting step of the inherent mevalonate (MVA)/ergosterol metabolic pathway in Saccharomyces cerevisiae. The intermediate 2, 3-oxidosqualene is also the precursor of triterpenoids. Therefore, the cyclization of 2, 3-oxidosqualene is the key branch point of ergosterol and triterpenoids biosynthesis. Down-regulation of 2, 3-oxidosqualene metabolic flux to ergosterol in S. cerevisiae may redirect the metabolic flux toward the triterpenoid synthetic pathway reconstructed by the synthetic biology approach. To construct erg7 knockout cassette harboring the loxP-Marker-loxP element, long primers were designed, which were homologous to the sequences of both erg7 ORF and plasmid pUG66. The cassette was transformed into diploid wild strain INVSc1 by LiAc/SS Carrier DNA/PEG method and then erg7 gene haploid deficient mutant was obtained by homologous recombination. The results of semiquantitative PCR and real-time quantitative PCR revealed that erg7 expression level in erg7 gene haploid deficient mutant is one time lower than that in wild strain. The results of TLC and HPLC showed that the ergosterol content in deficient mutant decreased to 42% of that in wild strain.
Chromatography, High Pressure Liquid ; DNA Primers ; Down-Regulation ; Ergosterol ; metabolism ; Haploidy ; Intramolecular Transferases ; genetics ; Polymerase Chain Reaction ; Saccharomyces cerevisiae ; genetics ; Squalene ; analogs & derivatives ; metabolism

Objective　To evaluate the effect of phacoemulsification and intraocular lens implantation with topical anesthesia in out-patients department.Methods　Phacoemulsification and intraocular lens implantation were performed successively in 132 patients(157 eyes) with topical anesthesia.Results　Phacoemulsification and intraocular lens implantation were successfully performed in 152 eyes(96.8%)with topical anesthesia and 5 eyes(3.2%)need retrobulbar anesthesia additionally.The visual acuity and complications after operation in the topical anesthesia were the same as that performed previously under retrobular anesthesia.Conclusions　Phacoemulsification and intraocular lens implantation may be performed with topical anesthesia through scleral tunnel incision.It is convenient for us to perform the operations on the out-patient patients with cataract.

Objective To observe the expressions of inducible nitric oxide synthase(iNOS) in the progress of rat subchronic fluorosis,and analyse the mechanism of nitric oxide(NO) free radical injnry in bone.Methods Male wistar rats were divided randomly by body weight into two groups.i.e.sodium fluoride group and control group.Sodium fluoride group was given drinking water with 150 mg/L sodium fluoride,and control group was given tap water only.The animals were bred for 24 weeks.Every four weeks some rats were killed.The contents of serum and bone fluoride were examined and analyzed.The levels of serum NO were determined by Griess Reagent.The expressions of iNOS mRNA and protein were analyzed by RT-PCR and immunohistochemistry.Results The serum NO contents significantly increased(t=9.36,P＜0.01) in NaF-treated rats after 8 weeks[(19.94±3.04)nmol/L],but significantly decreased(t=10.47,4.46,P＜0.01) after 20 weeks[(11.55±3.54)nmol/L]and 24 weeks[(20.83±2.49)nmol/L],compared with control group[(9.11±1.21,31.13±3.93,33.10±7.37)nmol/L].The expression of iNOS mRNA significantly increased(t=13.09,4.82,14.23,4.64,7.82,5.29,P＜0.01)in rats treated with sodium fluoride[(1.87±0.11),(1.87±0.78),(1.90±0.29),(1.93±0.67),(1.88±0.38),(1.84±0.03)],compared with control group[(0.41±0.25),(0.30±0.17),(0.18±0.06),(0.63±0.15),(0.66±0.04),(0.65±0.55)],and these proteins mainly appeared in hyperplasie zone and hypertrophic zone cells of epiphyseal plate,cartilages,articular cartilage cells,osteoblasts and ligament cells.Conclusions High dose fluoride might persistentlv induce the expressions of iNOS and catalyze synthesis of NO,then regulates osteoblast and osteoclast activitv and finally influences bone turnover.

Objective To observe the expressions of matrix metal proteinases-13(MMP-13) mRNA and tissue inhibitor of metal protease- 1 (TIMP- 1) mRNA and analyse the molecular mechanism of bone matrix degradation in the progress of rat subchronic fluorosis. Methods Male Wistar rats were randomly divided into two groups according to body weight, i.e. sodium floride group and control group. Rats in the sodium fluoride group were given drinking water containing 150 mg/L F-, and the animals in the control group were given tap water. The animals were bred for 24 weeks. Every 4 weeks some rats were killed. The change of obsteoclst was observed by transmission electron microscope. The expression levels of MMP-13 mRNA and TIMP-I mRNA were analyzed by RT-PCR. Results The number of lysesome and the synthesis of lysosoma enzyme in osteeclast were decreased. The expression of MMP-13 mRNA was significantly increased(t=2.29,2.41,3.07,2.52, 3.15,2.22, P<0.05) in rats treated with sodium fluoride (1.87±0.67,1.87±0.75,1.90±0.73,1.93±0.86,1.88±0.61,1.84±0.53), compared with control group(1.24±0.39, 1.19±0.27,1.07±0.22, I. 15 ~ 0.17, 1.17±0.18, 1.20±0.62). The expression of TIMP-1 mRNA was significantly increased (t=2.69,2.19,2.68,2.46,2.43,2.96, P<0.05) in rats treated with sodium fluoride(1.89±0.77,1.70±0.85,1.61±0.82,1.81±0.84,1.70±0.74, 2.06±0.96), compared with control group (1.07±0.39,0.87±0.49,0.71±0.48,0.99±0.43,0.95±0.46,0.89±0.57). Conclusion High dose fluoride might persistently induce the expressions of MMP-13 mRNA and TIMP-1 mRNA and may be involved in bone turnover.