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Identification and characterization of glnA Promoter and its corresponding Trans-regulatory Protein GlnR in the Rifamycin SV Producing Actinomycete,Amycolatopsis mediterranei U32

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Acta Biochimica et Biophysica Sinica 2006年第12期38卷 831-843页

作者： Hao YU~(1&) Wen-Tao PENG~1 Yang LIU~1 Ting WU~2 Yu-Feng YAO~1 Ming-Xue cUI~1 Wei-Hong JIANG~1 Guo-Ping ZHAO~(1,3,4*) 1 Laboratory of Molecular Microbiology,Institute of Plant Physiology and Ecology,shanghai Institutes for Biological sciences,chinese Academy of sciences,shanghai 200032,c.ina 2 department of medicinal chemistry,{6.,fudan University,shanghai 200032,c.ina 3 State Key Laboratory of Genetic Engineering & department of Microbiology,sc.ool of Life science,fudan University,shanghai 200433,c.ina 4 Laboratory of Disease and Health Genomics,chinese National Human Genome center at shanghai,shanghai 201203,c.ina Laboratory of Molecular Microbiology Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200032 China department of medicinal chemistry School of Pharmacy Fudan University Shanghai 200032 China State Key Laboratory of Genetic Engineering & department of Microbiology School of Life Science Fudan University Shanghai 200433 China Laboratory of Disease and Health Genomics Chinese National Human Genome Center at Shanghai Shanghai 201203 China

The genetic requirements for the transcription of glnA,encoding the major glutaminesynthetase in a rifamycin SV-producing Amycolatopsis mediterranei strain,U32,were *** experiments showed that the promoter of U32 glnA （pglnA） was likely to have two transcriptioninitiation sites:P1and P2,located 157 and 45 nucleotides （nt） upstream of the translational start codon,*** mobility shift and DNase I footprinting analyses revealed a 30 bp cis-element locatedat 45 to 75 nt downstream of P1,or 38 to 6. nt upstream of *** sequence of the cis-element displayedhigh similarity to the corresponding regions of pglnA from Streptomyces coelicolor and *** as a reporter gene,the expression levels of U32 pglnA and its deletion derivatives under differentnitrogen-source conditions were analyzed by detecting the catechol dioxygenase activities in ***54,*** J508 and *** FS10 （glnR mutant）.These in vivo studies showed that theactivation of U32 pglnA in *** required GlnR,and its binding to the U32 pglnA was furtherconfirmed by the gel mobility shift *** and heterologous expression of the U32 glnR allowed usto detect the in vitro interaction between the U32 GlnR and the corresponding pglnA *** shown by in vivo glnR inactivation and complementation indicated that GlnR is essential for theactive transcription of glnA in U32.

关键词： glnA promoter cis-element glnR Amycolatopsis mediterranei

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Role of Apolipoprotein A-I in Protecting against Endotoxin Toxicity

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Acta Biochimica et Biophysica Sinica 2004年第6期36卷 419-424页

作者： Juan MA, Xue-Ling LIAO, Bin LOU, and Man-Ping WU* department of Biochemistry, sc.ool of pharmacy, fudan University, shanghai 200032, c.ina department of Biochemistry School of Pharmacy Fudan UniversityShanghai 200032 China

High density lipoprotein (HDL) binds lipopolysaccharide (LPS or endotoxin) and neutralizes its toxicity. We investigated the function of Apolipoprotein A-I (ApoA-I), a major apolipoprotein in HDL, in this process. Mouse macrophages were incubated with LPS, LPS+ApoA-I, LPS+ApoA-I+LFF (lipoprotein-free plasma fraction d>1.210 g/ml), LPS+HDL, LPS+HDL+LFF, respectively. MTT method was used to detect the mortality of L-929 cells which were attacked by the release-out cytokines in LPS-activated macrophages. It was found that ApoA-I significantly decreased L-929 cells mortality caused by LPS treatment (LPS vs. LPS+ApoA-I, Pct became even more significant when LFF was utilized (LPS vs. LPS+ApoA-I+LFF, Pcant difference between LPS+ApoA-I+LFF and LPS+HDL+LFF treatment, indicating that ApoA-I was the main factor. We also investigated in vivo effects ofApoA-I on mouse mortality rate and survival time after LPS administration. We found that the mortality in LPS+ApoA-I group (20%) and in LPS+ApoA-I+LFF group (10%) was signifi- cantly lower than that in LPS group (80%) (Pctively); the survival time was (43.20 ± 10.13) h in LPS+ApoA-I group and (46.80 ± 3.79) h in LPS+ApoA-I+LFF group, which were significantly longer than that in LPS group (16.25 ± 17.28) h (Pcarried out in vitro binding study to investigate the binding capacity of ApoA-I and ApoA-I+LFF to fluorescence labeled LPS (FITc-LPS). It was shown that both ApoA-I and ApoA-I+LFF could bind with FITc-LPS, however, the binding capacity of ApoA-I+LFF to FITc-LPS (6..47 ± 8.06. was significantly higher than that of ApoA-I alone (24.35 ± 3.70) (Pct against LPS; (2) LFF enhances the effect of ApoA-I; (3) ApoA-I is the major contributor for HDL anti-endotoxin function.

关键词： Apolipoprotein-I lipopolysaccharide (LPS) macrophages FITc-lipopolysaccharide (FITc-LPS) L-929 cells

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Distribution of nimodipine in brain following intranasal administration in rats

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Acta Pharmacologica Sinica 2004年第4期25卷 124-129页

作者： Qi-zhi ZHANG, Xin-guo JIANG2, chun-hua WU department of Pharmaceutics, sc.ool ofpharmacy, fudan University, shanghai 200032, c.ina department of Pharmaceutics School of Pharmacy Fudan University

AIM: To determine whether nasally applied nimodipine (NM) could improve its systemic bioavailability and be transported directly from the nasal cavity to the brain. METHODS: NM was administered nasally, intravenously (iv), and orally to male Sprague-Dawley rats. At different times post dose, blood, cerebrospinal fluid (cSF), and brain tissue samples were collected, and the concentrations of NM in the samples were analyzed byHPLc. RESULTS: Oral systemic bioavailability of NM in rats was 1.17 %, nasal dosing improved bioavailibility to 6..4 %. Following intranasal administration, NM concentrations in olfactory bulb (OB) within 30 min post dose were found significant higher than in the other brain tissues. However, similar NM levels in different brain regions were observed after iv injection. AUc in cSF and OB from the nasal route was 1.26.and 1.39 fold compared with the iv route, respectively. The brain-to-plasma AUc ratios were significantly higher after nasal administration than after iv administration (PcONcLUSION: Nasally administered NM could markedly improve the bioavailability and a fraction of the NM dose could be transported into brain via the olfactory pathway in rats.

关键词： intranasal administration nimodipine olfactory bulb olfactory pathways

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Induction of systemic lupus erythematosus syndrome in BALB/c mice by immunization with active chromatin

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Acta Pharmacologica Sinica 2004年第6期25卷 105-109页

作者： Hong LI, Yun-yi ZHANG, Ya-nan SUN, Xi-yi HUANG, Yong-feng JIA, Duan LI2 department of Pharmacology, sc.oolof pharmacy, fudan University, shanghai 200032, c.ina department of Pharmacology School of Pharmacy Fudan University Shanghai 200032 China

AIM: To establish an animal model for systemic lupus erythematosus (SLE)-like syndrome in mice. METHODS: BALB/c mice were immunized with active chromatin isolated from conA-actived syngeneic spleno-lymphocytes. Plasma samples of mice were tested by enzyme-linked immunosorbent assays (ELISA) for the presence of IgG anti-dsDNA, -ssDNA, and anti-histone antibodies. Tumor necrosis factor-α (TNF-α) in serum was measured by ELISA. Spleno-lymphocyte proliferation assays and the levels of interferon-γ (IFN-γ) in supernatants were tested respectively. Proteinuria was measured. Kidneys were examined by direct immunohistochemical method and light microscopy. RESULTS: Anti-ds DNA, ssDNA, and histone antibodies were induced in active chromatin-immu- nized mice, the proliferation response of splenocytes to conA and LPS were reduced, levels of interferon-γ in supernatants and TNF-α in serum were lowered. Lupus nephritis was assessed by the presence of Ig deposits, glomerular pathology and proteinuria. cONcLUSION: The active chromatin-induced SLE-like mouse model was similar to idiopathic SLE in human.

关键词： systemic lupus erythematosus chromatin autoantibody tumor necrosis factor interferon type II B-lymphocytes T-lymphocytes female enzyme-linked immunosorbent assay

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Structural comparisons of meptazinol with opioid analgesics

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Acta Pharmacologica Sinica 2005年第3期26卷 334-338页

作者： Wei LI Jing-lai HAO Yun TANG Yan cHEN Zhui-bai QIU~2 department of medicinal chemistry,sc.ool of pharmacy,fudan University shanghai 200032,c.ina departmentofmedicinalchemistry SchoolofPharmacyFudanUniversityShanghai200032China

Aim:To investigate the mechanism of action of a potent analgesic,(±)-***:The structures of meptazinol enantiomers were compared with opioidpharmacophore and ***:Neither enantiomer of meptazinol fittedany patterns among the opioid pharmacophore and tramadol,although they didshare some structural and pharmacological ***,the structuresuperpositions implied that both enantiomers of meptazinol might share somesimilar analgesic mechanisms with typical opiate ***:Meptazinol should have a different mechanism of action to known analgesics,which would be helpful in further investigations of meptazinol in the search fornon-addictive analgesics.

关键词： meptazinol tramadol opioid analgesics pharmacophore

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Bacterial Type Ⅰ Glutamine Synthetase of the Rifamycin SV Producing Actinomycete,Amycolatopsis mediterranei U32,is the Only Enzyme Responsible for Glutamine Synthesis under Physiological conditions

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Acta Biochimica et Biophysica Sinica 2006年第12期 821-830页

作者： Wen-Tao PENG~(1&) Jin WANG~1 Ting WU~2 Jian-Qiang HUANG~1 Jui-Shen cHIAO~1 Guo-Ping ZHAO~(1,3,4) 1 Laboratory of Molecular Microbiology,Institute of Plant Physiology and Ecology,shanghai Institutes for Biological sciences,chinese Academy of sciences,shanghai 200032,c.ina 2 department of medicinal chemistry,{6.,fudan University,shanghai 200032,c.ina 3 State Key Laboratory of Genetic Engineering/department of Microbiology,sc.ool of Life science,fudan University,shanghai 200433,c.ina 4 Laboratory of Health and Disease Genomics,chinese National Haman Genome center at shanghai,shanghai 201203,c.ina

The structural gene for glutamine synthetase,glnA,from Amycolatopsis mediterranei U32 wascloned via screening a genomic library using the analog gene from Streptomyces *** clone wasfunctionally verified by complementing for glutamine requirement of an Escherichia coli glnA null mutantunder the control of a lac *** analysis showed an open reading frame encoding a protein of46. amino acid *** deduced amino acid sequence bears significant homologies to other bacterialtype Ⅰ glutamine synthetases,specifically,71% and 72% identical to the enzymes of *** and Myco-bacterium tuberculosis,*** of this glnA gene in *** U32 led to glutamineauxotrophy with no detectable glutamine synthetase activity in *** contrast,the cloned glnA+gene cancomplement for both phenotypes in *** thus suggested that in *** U32,the glnA geneencoding glutamine synthetase is uniquely responsible for in vivo glutamine synthesis under our laboratorydefined physiological conditions.

关键词： glnA glutamine synthetase Amycolatopsis mediterranei rifamycin SV

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crystal Structure of 3-methyl-1, 2, 3, 4, 4a, 4b, 9a, 15, 15a,15b-decahydro-(4,5);(4a,15)-dimethano-tricyclo(4, 3, 1, 0^(2,7)) deca 〔8, 9, 1, 10-a, b, c〕pyrido〔4,3-a〕-acridin-6.en-9-ol-8-one

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chinese Journal of Structural chemistry 1997年第3期16卷 186-190页

作者： HU chun-Hua cHEN Jian ZHENG Pei-Ju(Research center of Analysis and Measurement, fudan University, shanghai 200433)ZENG Yi-Bin QIU Zhui-Bai YANG Jie(department of medicinal chemistry, shanghai Medical University, {6.) 复旦大学分析测试研究中心上海上海医科大学药物化学系上海

The title compound, c25H24N2O2, a new rearrangement byproduct,has been found in the O-demethylation of 7a-o-amino pheny1-6. 14-endo-ethenotetrahydro thebaine. Its structure was determined by X-ray crystallography. Its crystal isorthorhombic, space group P212121, Mr= 384. 48 with a= 9. 128(2), b= 11. 953 (3),c=17. 422(4) A, V= 1901 (1) A3, Dc= 1. 34 g/cm3, Z= 4, A(MoKa)=0. 71073A,μ= 0. 8 cm-1, F(000) = 816. R= 0. 048, Rw= 0. 051. The molecule is mainly a closedstructure except three extended legs. It consists of a lot of multi-membered rings suchas five- and six- membered rings.

关键词： crystal structure 6 14-endo-ethenotetrahydro thebaine

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2-甲氧基雌二醇合成方法的改进

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复旦学报（医学版） 2008年第4期35卷 621-623页

作者：蒋皓夏鹏复旦大学药学院药物化学教研室上海200032

目的合成新生血管抑制剂2-甲氧基雌二醇。方法以雌二醇为原料经过双乙酰化,四氯化锆催化下发生2-位乙酰基重排,溴苄保护3-位羟基,Baeyer-Villiger氧重排反应后产物再经过碱水解,2-位甲醚化,最后用盐酸脱去3-位苄基共6.合成2-甲氧基雌... 详细信息

目的合成新生血管抑制剂2-甲氧基雌二醇。方法以雌二醇为原料经过双乙酰化,四氯化锆催化下发生2-位乙酰基重排,溴苄保护3-位羟基,Baeyer-Villiger氧重排反应后产物再经过碱水解,2-位甲醚化,最后用盐酸脱去3-位苄基共6.合成2-甲氧基雌二醇。结果将文献报道路线缩短了一步,与原文献相比将改进的6.反应总收率提高了10%。合成获得的2-甲氧基雌二醇成品进行了结构确证。结论通过改进2-甲氧基雌二醇的合成工艺,简化操作,缩短反应时间,与原文献相比改进后的合成条件更温和更环保,更有利于规模化生产需要。

关键词： 2-甲氧基雌二醇新生血管抑制剂抗肿瘤工艺改进

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Effect of MePEG molecular weight and particle size on in vitro release of tumor necrosis factor-α-loaded nanoparticles

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Acta Pharmacologica Sinica 2005年第2期26卷 242-249页

作者： chao FANG,Bin SHI,Yuan-ying PEI~2 department of Pharmaceutics,sc.ool of pharmacy,fudan University,shanghai 200032,c.ina department of Pharmaceutics School of Pharmacy Fudan University Shanghai 200032 China

Aim:To study the in vitro release of recombinant human tumor necrosis factor-alpha(rHuTNF-α)encapsulated in poly(methoxypolyethyleneglycol cyanoacry-late-co-n-hexadecyl cyanoacrylate)(PEG-PHDcA)nanoparticles,and investigatethe influence of methoxypolyethyleneglycol(MePEG)molecular weight and par-ticle ***:Three sizes(approximately 80,170,and 240 nm)of PEG-PHDcA nanoparticles loading rHuTNF-α were prepared at different MePEG mo-lecular weights(Mr=2000,5000,and 10000)using the double emulsion *** in vitro rHuTNF-α release was studied in PBS and rat ***:Ahigher burst-release and cumulative-release rate were observed for nanoparticleswith higher MePEG molecular weight or smaller particle size.A decreased cu-mulative release of rHuTNF-α following the initial burst effect was found in PBS,while the particle sizes remained constant and MePEG *** contrast,inrat plasma,slowly increased cumulative-release profiles were obtained after theburst *** a 5-h incubation in rat plasma,more than 50% of the PEG-PHDcA nanoparticles ***:The MePEG molecular weightand particle size had an obvious influence on rHuTNF-α release,rHuTNF-αreleased from PEG-PHDcA nanoparticles in a diffusion-based pattern in PBS,but in a diffusion and erosion-controlled manner in rat plasma.

关键词： tumor necrosis factor-alpha stealth nanoparticles polyethylene glycols poly(methoxypolyethyleneglycol cyanoacrylate-con-hexadecyl cyanoacrylate)

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Artificial pulmonary surfactant as a carrier for intratracheally instilled insulin

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Acta Pharmacologica Sinica 2007年第5期28卷 744-750页

作者： Ying JI,chen LIU,Yuan-ying PEI~2department of Pharmaceuties,sc.ool of pharmacy,fudan University,shanghai 200032,c.ina departmentofPharmaceutics SchoolofPharmacyFudanUniversityShanghai200032China

Aim:The relative bioavailabilities and effects on lung injury alleviation of 4 insu-lin-artificial pulmonary surfactant (INS-APS) preparations were studied in *** relationship between the minimal surface tension (γmin) of INS-APS andthe absorption of insulin was also ***:Four formulations ofAPS[1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPc)/lecithin/palmitic acid(PA),DPPc/1-hexadecanol (Hex)/tyloxapol (Tyl),DPPc/L-α-phosphatidyl-DL-glyc-erol sodium salt (PG),DPPc/Tyl]were prepared by thin-film sonication methodand direct *** γminof 4 APS dispersions was examined with andwithout INS by pulsating with a bubble surface *** vivo experimentswere performed in which serum glucose change and the insulin level were mea-sured by an enzymatic glucose reagent kit and a radioimmunology assay kit afterIT to *** reduction in lung injury by INS-APS following 7 d of consecutiveadministration was evaluated by the pulmonary edema index (the weight ratio ofwet lung to dry lung) and histopathology ***:The γminof allAPS dispersions were below 10 mN/*** was no significant difference (P>0.05) between the γ(min)of APS and the corresponding *** vivo experi-ments showed a significant glucose level decrease and insulin absorptionincrease (Pce of APS,compared to the insulin solution *** the results,we found that the pulmonary edema index values of all the INS-APS groups were significant lower (Pcant differences (P>0.05) between INS/DPPc/Tyl,INS/DPPc/PG,and the control *** pulmonary edema indices and histopatho-logical observation indicated that INS-APS could alleviate lung ***:The most potent hypoglycemic effect and insulin absorption increase in this studywere obtained with INS/DPPc/*** to the results,there was a linearcorrelation between the γminand relative bioavailability of INS-

关键词： pulmonary drug delivery system pharmacokinetics pharmacodynamics hypoglycemic effect minimal surface tension absorption pulmonary edema index histopathology examination

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