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Cure of experimental botulism and antibotulismic effect of toosendanin

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Acta Pharmacologica Sinica 2004年第6期25卷 137-146页

作者： yu-liang shi2, Zhong-feng WANG key laboratory of neurobiology, shanghai institutes for biological sciences, chinese academy ofscience, shanghai200031, china 中科院上海生命科学院 Key laboratory of Neurobiology CAS 上海 200031

Botulinum neurotoxins (BoNTs), a group of bacterial proteins that comprise a light chain disulfide linked a heavy chain, are the most lethal biotoxins known to mankind. By inhibiting neurotransmitter release, BoNTs cause severe neuroparalytic disease, botulism. A series of important findings in the past 10 years which displayed the molecular targets of BoNTs and hence proposed a four-step action mechanism to explain BoNT intoxication greatly advanced the study of antibotulismic drug. In this article, we reviewed these progresses and anti-botulismic compounds found in recent years. These compounds function due to their facilitation on neurotransmitter release or to their interference on the binding, internalization, translocation, and endopeptidase activity of the toxins. Toosendanin is a triterpenoid derivative extracted from a digestive tract-parasiticide in chinese traditional medicine. chinese scientists have found that the compound is a selective prejunctional blocker. In spite of sharing some similar action with BoNT, toosendanin can protect botulism animals that have been administrated with lethal doses of BoNT/A or BoNT/B for several hours from death and make them restore normal activity. The neuromuscular junction preparations isolated from the rats that have been injected with toosendanin tolerate BoNT/A challenge. Toosendanin seems to have no effect on endopeptidase activity of BoNT, but blocks the toxin approach to its enzymatic substrate.

关键词： toosendanin botulinum toxins botulism synaptic transmission neurotransmitter triterpene chinese traditional medicine

Toosendanin interferes with pore formation of botulinum toxin type A in PC12 cell membrane

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Acta Pharmacologica Sinica 2006年第1期27卷 66-70页

作者： Mu-feng LI yu-liang shi Institute of physiology,shanghai institutes for biological sciences,chinese academy of sciences,shanghai 200031,china Instituteofphysiology ShanghaiInstitutesforBiologicalSciencesChineseAcademyofSciencesShanghai200031China

Aim:Botulinum neurotoxins (BoNT) abort the process of neurotransmitter re-lease at presynaptic motor nerve terminals,causing muscle *** abilityof botulinum toxin to produce its effect is dependent on the ability of the lightchain to cleave the SNARE proteins associated with transmitter ***-cation of the light chain protease through the heavy chain-formed channel is apivotal step in the intoxication *** (TSN),a triterpenoid de-rivative extracted from a chinese traditional medicine,has been demonstrated tobe an effective cure for experimental *** study was designed to explorethe antibotulismic mechanisms of ***:The inside-out single-channel recording patch-clamp technique was used to record the BoNT/A-in-duced currents in the presence and absence of ***:Channel formationwas delayed and the sizes of the channels were reduced in the TSN-treated PC12cell ***:The antibotulismic effect of TSN might occur viainterference with toxin translocation.

关键词： toosendanin botulinum toxin type A ion channel antibotulismic mechanism single channel recording

Toosendanin increases free-Ca2+ concentration in NG108-15 cells υia L-type Ca2+ channels

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Acta Pharmacologica Sinica 2004年第5期 55-59页

作者： Tong-hui XU, Jun DING, yu-liang shi2key laboratory of neurobiology, shanghai institutes for biological sciences,chinese academy of sciences, shanghai 200031, china

AIM: To examine if toosendanin (TSN) affects intracellular free-Ca2+ concentration ([Ca2+]i) in neuroblastomagliomahybrid cells (NG108-15 cells). METHODS: The [Ca2+]i was determined by laser-scanning confocal microscopicimaging technique in which Fluo-3 was used as Ca2+ indicator. RESULTS: TSN induced an increase in resting[Ca2+]i and in high K+-evoked Ca2+ transient in differentiated NG108-15 cells. The TSN-induced increase in [Ca2+]iwas dose-dependent and disappeared in CdCl2-, nifedipine-containing or Ca2+-free solution, and appeared afterwashing out the Ca2+ channel blockers or adding Ca2+. CONCLUSION: TSN increased [Ca2+]i in differentiatedNG108-15 cells. The [Ca2+]i enhancement was due to the influx of extracellular Ca2+ and related to L-type Ca2+channels.

关键词： toosendanin intracellular calcium concentration confocal microscopy calcium indicator neuroblastomaglioma hybrid cells L-type Ca2+ channels

Transiently Expressed Short Hairpin RNA Targeting 126 kDa Protein of Tobacco Mosaic Virus Interferes with Virus Infection

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Acta Biochimica et Biophysica Sinica 2006年第1期38卷 22-28页

作者： Ming-Min ZHAO~(1,2),De-Rong AN~(1*),Jian ZHAO~2,Guang-Hua HUANG~3,Zu-Hua HE~4,and Jiang-Ye CHEN~3 ~1 College of Plant Protection,Northwest science and Technology University of Agriculture and Forestry,Yangling 712100,china ~2 College of Agriculture,Yangtze University,Jingzhou 434025,china ~3 State key laboratory of Molecular Biology,Institute of Biochemistry and Cell Biology,shanghai institutes for biological sciences, chinese academy of sciences,shanghai 200031,china ~4 State key laboratory of Plant Molecular Genetics,Institute of Plant physiology and Ecology,shanghai institutes for biological sciences, chinese academy of sciences,shanghai 200032,china College of Plant Protection Northwest Science and Technology University of Agriculture and Forestry Yang ling 712100 China College of Agriculture Yangtze University Jingzhou 434025 China State key laboratory of Molecular Biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China State key laboratory of Plant Molecular Genetics Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200032 China

RNA-interference (RNAi) silences gene expression by'guiding mRNA degradation in asequence-specific *** interfering RNA (siRNA),an intermediate of the RNAi pathway,has beenshown to be very effective in inhibiting virus infection in mammalian cells and cultured plant ***,wereport that Agrobacterium tumefaciens-mediated transient expression of short hairpin RNA (shRNA) couldinhibit tobacco mosaic virus (TMV) RNA accumulation by targeting the gene encoding the replication-asso-ciated 126 kDa protein in intact plant *** results indicate that transiently expressed shRNA efficientlyinterfered with TMV *** interference observed is sequence-specific,and time-and *** expressed shRNA corresponding to the TMV 126 kDa protein gene did not inhibit cucumbermosaic virus (CMV),an unrelated *** order to interfere with TMV accumulation in tobaccoleaves,it is essential for the shRNA constructs to be infiltrated into the same leaves as TMV *** support the view that RNAi opens the door for novel therapeutic procedures against virus *** propose that a combination of the RNAi technique and Agrobacterium-mediated transient expressioncould be employed as a potent antiviral treatment in plants.

关键词： tobacco mosaic virus 126 kDa protein RNA interference short hairpin RNA

Opioid receptor antagonists increase [Ca2+]i in rat arterial smooth muscle cells in hemorrhagic shock

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Acta Pharmacologica Sinica 2004年第3期25卷 141-146页

作者： Li KAI,Zhong-feng WANG2,yu-liang shi2,3,liang-ming LIU,De-yao HU Institute of Surgery,Department of Pharmacology,Third Military Medical University,Chongqing 400042 key laboratory of 2 neurobiology,shanghai institutes for biological sciences,chinese academy of sciences,shanghai 200031,china Institute of Surgery Department of Pharmacology Third Military Medical University 重庆 400042 中科院上海生命科学院 Key Laboratory of Neurobiology CAS 上海 200031

AIM: To examine the effects of opioid receptor antagonists and norepinephrine on intracellular free Ca2+ concentra- tion ([Ca2+]i) in mesenteric arterial (MA) smooth muscle cells (SMC) isolated from normal and hemorrhagic shocked rats in the vascular hyporesponse stage. METHODS: The rat model of hemorrhagic shock was made by withdrawing blood to decrease the artery mean blood pressure to 3.73-4.26 kPa and keeping at the level for 3 h. [Ca2+]i of vascular smooth muscle cells (VSMC) were detected by the laser scan confocal microscopy. RESULTS: In the hyporesponse VMSC of rats in hemorrhagic shock, selective δ-, κ-, and μ-opioid receptor antagonists (naltrindole, nor-binaltorphimine, and β-funaltrexamine, 100 nmol/L) as well as norepinephrine 5 μmol/L significantly increased [Ca2+]i by 47 %±13 %, 37 %±14 %, 33 %±10 %, and 54 %±17 %, respectively, although their effects were lower than those in the normal rat cells (the increased values were 148 %±54 %, 130 %±44 %, 63 %±17 % and 110 %±38 %, respectively); and the norepinephrine-induced increase in [Ca2+]i was further augmented by three δ-, κ-, and μ-opioid receptor antagonists (50 nmol/L, respectively) application (from 52 %±16 % to 99 %±29 %, 146 %±54 % and 137 %±47 %, respectively). CONCLUSION: The disorder of [Ca2+]i regulation induced by hemorrhagic shock was mediated by opioid receptor and α-adrenoceptor, which may be partly responsible for the vascular hyporesponse, and the opioid receptor antagonists improved the response of resistance arteries to vascu- lar stimulants in decompensatory stage of hemorrhagic shock.

关键词： hemorrhagic shock vascular smooth muscle opioid receptors calcium

MicroRNA in cell differentiation and development

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science china(Life sciences) 2009年第3期52卷 205-211页

作者： shi Yi & JIN YouXin State key laboratory of Molecular Biology,shanghai Institute of Biochemistry and Cell Biology,shanghai institutes for biological sciences,chinese academy of sciences,shanghai 200031,china State key laboratory of Molecular Biology Shanghai Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China

The regulation of gene expression by microRNAs(miRNAs) is a recently discovered pattern of gene regulation in animals and *** have been implicated in various aspects of animal development and cell differentiation,such as early embryonic development,neuronal development,muscle development,and lymphocyte development,by the analysis of genetic deletions of individual miRNAs in *** studies show that miRNAs are key regulators in animal development and are potential causes of human *** we review some recent discoveries about the functions of miRNAs in cell differentiation and development.

关键词： miRNA embryonic development neuronal development muscle development lymphocyte development

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Substitutions of the Conserved Gly47 Affect the CF1 Inhibitor and Proton Gate Functions of the Chloroplast ATP Synthase ε Subunit

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Acta Biochimica et Biophysica Sinica 2005年第7期37卷 453-462页

作者： Hui DONG Zhang-Lin NI Jia-Mian WEI Institute of Plant physiology & Ecology,shanghai institutes for biological sciences,chinese academy of sciences,shanghai 200032,china Institute of Plant physiology & Ecology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200032 China

The conserved residue Gly47 of the chloroplast ATP synthase ε subunit was substituted withLeu,Arg,Ala and Glu by site-directed *** process generated the mutants εG47L,εG47R,εG47A and εG47E,*** the ε variants showed lower inhibitory effects on the soluble CF1（-ε）Ca2+-ATPase compared with wild-type ε.In reduced conditions,εG47E and εG47R had a lower inhibitoryeffect on the oxidized CF1（-ε）Ca2+-ATPase compared with wild-type ε.In contrast,εG47L and εG47Aincreased the Ca2+-ATPase activity of soluble oxidized CF1（-ε）.The replacement of Gly47 significantlyimpaired the interaction between the subunit ε and γ in an in vitro binding *** study showed thatall ε variants were more effective in blocking proton leakage from the thylakoid *** enhancedATP synthesis of the chloroplast and restored ATP synthesis activity of the reconstituted membranes to alevel that was more efficient than that achieved by wild-type ε.These results indicate that the conservedGly47 residue of the ε subunit is very important for maintaining the structure and function of the ε subunitand may affect the interaction between the ε subunit,β subunit of CF1and subunit Ⅲ of CF_o,therebyregulating the ATP hydrolysis and synthesis,as well as the proton translocation role of the subunit Ⅲ of CF_o.

关键词： chloroplast ATP synthase ε subunit GST pull-down assay site-directed mutagenesis

In Vitro Reassembly of Tobacco Ribulose-1,5-bisphosphate Carboxylase/Oxygenase from Fully Denatured Subunits

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Acta Biochimica et Biophysica Sinica 2006年第10期38卷 737-745页

作者： Zhen-Hua YONG Gen-yun CHEN* Jiao-Nai shi Da-Quan XU Institute of Plant physiology and Ecology,shanghai institutes for biological sciences,chinese academy of sciences,and Graduate School of the chinese academy of sciences,shanghai 200032,china Institute of Plant physiology and Ecology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences and Graduate School of the Chinese Academy of SciencesShanghai 200032 China

It has been generally proved impossible to reassemble ribulose-1,5-bisphosphate carboxylase/oxygenase （Rubisco） from fully denatured subunits in vitro in higher plant,because large subunit of fullydenatured Rubisco is liable to precipitate when the denaturant is removed by common methods of directdilution and one-step *** our experiment,the problem of precipitation was resolved by an improvedgradual dialysis method,which gradually decreased the concentration of ***,fully denaturedRubisco subunits still could not be reassembled into holoenzyme using gradual dialysis unless chaperonin 60was *** restored activity of reassembled Rubisco was approximately 8% of natural *** of reassembled Rubisco increased greatly when heat shock protein 70 was present in the *** and Mg2+ were unnecessary for in vitro reassembly of Rubisco,and Mg2+ inhibited the *** reassembly was weakened when ATP,Mg2+ and K+ existed together in the reassembly process.

关键词： Rubisco reassembly gradual dialysis chaperonin 60 Hsp70

A Viral Protein Suppresses siRNA-directed Interference in Tobacco Mosaic Virus Infection

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Acta Biochimica et Biophysica Sinica 2005年第4期37卷 248-253页

作者： Ming-Min ZHAO De-Rong AN Guang-Hua HUANG~1 Zu-Hua HE~2 Jiang-Ye CHEN~1 College of Plant Protection,North West science and Technology University of Agriculture and Forestry,Shaanxi 712100,china ~1State key laboratory of Molecular Biology,Institute of Biochemistry and Cell Biology,shanghai institutes for biological sciences. chinese academy of sciences,shanghai 200031,china ~2State key laboratory of Plant Molecular Genetics.Institute of Plant physiology and Ecology,shanghai institutes for biological sciences.chinese academy of sciences,shanghai 200032.china College of Plant Protection North West Science and Technology University of Agriculture and ForestryShaanxi 712100 China State key laboratory of Molecular Biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200031 China State key laboratory of Plant Molecular Genetics Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200032 China

Plant viruses encode suppressors of post-transcriptional gene silencing (PTGS), an adaptivedefense response that limits virus replication and its spread in *** helper component proteinase (HC-Pro) of the potato virus A (PVA,genus Potyvirus) suppresses PTGS of silenced ***,the effectof HC-Pro on siRNA-directed interference in the tobacco mosaic virus (TMV) was examined by using atransient Agrobacterium tumefaciens-based delivery system in intact *** was shown that the interferenceeffect was completely blocked by co-infiltration with HC-Pro plus siRNA constructs in both systemic andhypersensitive *** the system host,all plants agro-infiltrated with HC-Pro plus siRNA constructsdisplayed the same symptoms as the negative ***,TMV RNA accumulation was found to beabundant in the upper leaves using reverse transcriptase-PCR (RT-PCR) and Northern blot *** thecontrary,plants agro-infiltrated with the siRNA construct alone were free of ***,ourstudy suggests that the transient expression of HC-Pro inhibited the siRNA-directed host defenses againstTMV infection.

关键词： siRNA HC-Pro tobacco mosaic virus suppressor