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Binding Properties of M-CAT Element in the Chicken Nicotinic Acetylcholine Receptor γ-Subunit Promoter

Chinese Journal of Biochemistry and Molecular Biology.2000;16(5):580-585.

Several M-CAT elements are present in the chicken nicotinic acetylcholine receptor γ-subunit promoter and required for the totipotency of this gene.To understand the binding properties of M-CAT element,the interaction of M-CAT motif,at the position -67/-61 relative to the initiation site of γ-promoter with nuclear proteins,were analyzed.Mutageneses and binding experiments demonstrated that the M-CAT core motif bound nuclear protein (s) from tested chicken tissues to generate a high-mobility complex;while the flanking sequence bound different factors to generate lower-mobility complexes with the exception of liver.Southwestern blotting showed that only a 30 kD protein was expressed in tested tissues of chicken embryo at day 13.These results suggest that a 30 kD factor can directly bind to M-CAT element in the manner of monomer,even homodimer and is ubiquitously expressed in tissues,while the binding of the factor(s) to flanking sequence should probably rely on the presence of a 30 kD ubiquitous factor.

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In vitro Selection of DNA Aptamer to Cyclosporin A by SELEX Method

Weibin LI ; Xiaopeng LAN ; Xiangyue YANG ; Changshao ZHOU

Chinese Journal of Biochemistry and Molecular Biology.2007;23(10):829-834.

To obtain the aptamers with high affinity and specificity for cyclosporin A(CsA),a synthesized 78 nt single stranded DNA(ssDNA)random library containing 35 random sequences flanked by invariant primer was subjected to 1 1 rounds of selection against CsA by SELEX protocol.Magnetic beads were used for target immobilization and the biotin-streptavidin-horseradish peroxidase system was employed for determining the binding affinity between the aptamers and CsA. After ten rounds of selection and amplification, with an increasing affinity for each round,the selected aptamers were cloned,sequenced and analyzed for their primaryand secondary structures.The 19 aptamers were divided into five groups based on primary sequence homology.Hairpin loop is the main motif in the predicted secondary structure and is supposed to be the binding part of the aptamers to CsA.The CsA-specific aptamers will be useful for enzyme-linked assays or immunofluorescence asses of CsA.

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An Improved Measurement of Fluorescence Resonance Energy Transfer to Analyse Protein-protein Interaction in Protein Homodimer

Fujun HAN ; Yongfeng LUO ; Jun XU

Chinese Journal of Biochemistry and Molecular Biology.2008;24(7):619-629.

Fluorescence resonance energy transfer(FRET)is increasingly used to study inter-and intramolecular interactions in living cells.Since being proportional to the concentration of the donor-acceptor complex.FRET value must be normalized to exclude the influence of the ratio and the concentration of donor and acceptor for comparison.Different from the intra.molecular FRET which is simplified by the fact that the COncentration of the donor is equal to that of the acceptor,the inter-molecular FRET is usually too complicated for most existing measurements to quantify exactly.We deduced the exact proportion of the donor-acceptor complex based on a unique characteristic of homodimer,a special kind of the intermolecular interaction,developed an exact quantification measurement of the FRET.We proved the novel method Can generate more reliable estimation of FRET value by comparison with other methods using a homodimer,estrogen receptor alpha(ERa),as a FRET pair.

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Effects of Divalent Mg2+ and Monovalent Na+ on Cleavage Reactions by Multiribozyme System in vitro

Shengli TIAN ; Shuo ZHENG ; Shide LIU ; Dongping XU ; Takao OHNUMA

Chinese Journal of Biochemistry and Molecular Biology.2008;24(7):612-618.

To better understand the cleavage efficiency of muhiribozyme system on its RNA substrate in the presence and absence of divalent magnesium and monovalent sodium ions.we constructed pGEM-Coat'A,pGEM-Coat'A196Rz plasmids and pGEM-MDRl target plasmid.They were applied to transcribe RNAs with SP6/T7 transcription kit.Cleavage reactions were carried out in cell-free system and reaction products were analyzed by electrophoresis on 6% denaturing polyacrylamide gels in TBS buffer.The gels were dried and exposed to X-ray films for autoradiography.The Image J software was employed to analyze the dried gels.The results indicated that the cleavage efficiency of the muhiribozyme was dependent on the concentration of divalent Mg2+.The cleavage products increased with the concentrations of divalent Mg2+ and were Mg2+ concentration and time dependent.No cleavage product was obtained in the presence of lower than 200 mmol/L Na+ alone.On the contrary,monovalent Na+ inhibited the Mg2+ -induced cleavage reaction in Na+ and Mg2+ coexistance.The cleavage rate was significantly lower than that observed with divalent Mg2+ alone.These results suggested that divalent Mg2+ was required for muhiribozyme on substrate cleavage reaction in the physical condition,whereas monovalent Na+ was not.

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Complete Sequencing and Analysis of mtDNA in Phlaeoba albonema Zheng

Hongwen SHI ; Fangmei DING ; Yuan HUANG

Chinese Journal of Biochemistry and Molecular Biology.2008;24(7):604-611.

The complete mitochondrial genome sequence of Phlaeoba albonema Zheng Was determined by using L-PCR and conserved primers walking sequencing.The obtained genome sequence is 15657 bp in size.containing 13 protein-coding genes,2 ribosomal RNA and 22 transfer RNA genes.All the 37 genes are conserved in the same orientations as observed in Locusta migratoria.11202 bp of the mtDNA are coding for proteins,1486 bp for tRNAs,1312 bp for rRNA large subunit(1rRNA),and 844 bp for rRNA small subunit(srRNA).The A+T-rich region is 728 bp in size.The genes overlapping sequences are 41 bp in total and are spreading over 9 locations(1-8 bp at each site).A total of 126 bp intergenic spacer sequences are scattered in 21 regions at the size of 1 to 20 bp,where the largest 20 bp region iS located between the tRNALys and ATP8 genes.The predicted secondary structures of both srRNA and lrRNA were compared with that of Ruspolia dubia,and the patterns of base pairs in tRNA anticodon stem and A/T,C/G bias of protein-coding genes in different strands were discussed.

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Characterization and Amino Acid Sequence of y3,an Antiviral Protein from Mushroom Coprinus comatus

Liping WU ; Zujian WU ; Dong LIN ; Fang FANG ; Qiying LIN ; Lianhui XIE

Chinese Journal of Biochemistry and Molecular Biology.2008;24(7):597-603.

Many proteins from mushrooms can be used as potential antiviral agents and in plant protection.An antiviral protein,designated as y3,Was isolated from fruiting bodies of the fungus Coprinus comatus by fastflow ion-exchange column chromatography coupled with high-resolution molecular sieve chromatography.This glycoprotein was detected in both fruiting body and mycelium by Western blot analysis.According to its Nterminal sequence.an amino acid sequence and a partial cDNA sequence of the y3 gene were obtained.Protein y3 at 2.0μg/ml achieved 50.0% inhibition of tobacco mosaic virus(TMV,20μg/ml)lesions in Nicotiana glutinosa leaves.It also inhibited multiplication of TMV in Nicotiana tabacum Var.K326.

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Comparative Proteome Analysis of Plasma Membrane from Different Differential Nasopharyngeal Carcinoma Cell Lines

Tingting SHENG ; Lijun ZHANG ; Xiaohui LIU ; Xiaofang JIA ; Jixian XIONG ; Quanyuan HE ; Rui CAO ; Xia PENG ; Ni SHI ; Songping LIANG

Chinese Journal of Biochemistry and Molecular Biology.2008;24(8):712-718.

A subcellular proteomic method was applied to investigate the protein expression profiles of nasopharyngeal carcinoma (NPC) cell lines,CNE1 and CNE2,at various differentiation levels.Plasma membrane (PM) proteins were obtained by Percoll density grade centrifugation and subjected to twodimensional electrophoresis (2DE) followed by PDQuest software analysis.Nine proteins expressed with more than two folds difference were identified by MALDI-TOF-TOF,of which functions involved in cell differentiation,signal transduction,and metabolism.Half of these proteins,such as galectin-1 and annexin Ⅱ,were analyzed with real-time quantitative PCR or Westem blotting.We have tested a proteomic method to study differentiated NPCs at different levels and found several proteins that might be related to their biological characteristics.

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Coordinative Effects of Salt and Alkali Stresses on Sunflower Antioxidative Enzymes

Yanmin SHENG ; Jinzhi YIN ; Decheng SHI ; Deli WANG

Chinese Journal of Biochemistry and Molecular Biology.2008;24(8):704-711.

Based on the characteristics of salt.alkalinized soil in northeastem China,twenty-five kinds of salt alkaline conditions with different salinities and pH were simulated by mixing NaCl,NaHCO3,Na2SO4,and Na2CO3,in various proportions and applied to sunflower (Helianthus annuus L.) seedlings to investigate the coordinative effects of salt and alkali stresses on its antioxidant enzyme system.The soil was conditioned with a salt concentration range between 50 to 250 mmol/L and pH values from 7.12 to 10.46.Several physiological indexes of stressed seedlings were measured,including the activities of superoxide dismutase (SOD),catalase (CAT),and pemxidase (POD),as well as the content of malondialdehyde (MDA).The results showed that the responses of the antioxidant enzyme system in sunflowers were influenced by salinity and alkalinity,which all three antioxidant enzymes exhibited a rise-drop pattern as salinity increased,whereas their responses to alkalinity appeared to be diverse:decreased for SOD and CAT,and increase for POD along when increasing alkalinity.A two-way analysis of variance (ANOVA) showed that the effects of salinity and alkalinity on the activities of the three enzymes were significant (P＜0.001).The effect of salinity on POD and SOD was greater than that of alkalinity,whereas the effect of alkalinity on CAT was greater than that of salinity.The interrelation of salinity and alkalinity on each antioxidant enzyme was significant (P＜0.001) except for SOD.The correlation and stepwise regression analyses between the activity of the antioxidant enzyme system and the MDA content were significant to difierent extend,SOD was a dominant factor,and POD was neglectable.

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Hemolytic Effects of Melittin Involve Activity Change of Two Types of Enzymes on RBC Membrane

Yahua ZHAO ; Riqing LI ; Wei ZHANG ; Yangsheng ZHONG ; Zucheng LIANG ; Jianrong LIN

Chinese Journal of Biochemistry and Molecular Biology.2008;24(6):522-530.

The effects of melittin on the activities of Na+-K+-ATPase and glucose-6-phosphate dehydrogenase (G-6-PD) which are on the membrane of red blood cell (RBC) are chosen as the index of this study. The possible target sites of these effects through enzyme activity determination by spectrophotometry are investigated, and the hemolytic process and the activity change of these two types of enzymes on the RBC membrane are discussed. The results show that the main mode of melittin inhibition to the activity of enzymes on the RBC membrane is the coexistence of adhesion/insertion form and free-state form, and the effect of the former is more stronger than the latter. The K+ binding site of Na+-K+-ATPase is one of the target sites of melittin. The membrane-insertion process of melittin synchronizes with the action of melittin on this enzyme. Melittin slowly inhibits the catalysis of G-6-PD through the action on G-6-P and NADP, and the extent in which melittin forms tetramers isclosely related to the enzyme activity. EDTA inhibits the aggregation of melittin, and interferes with its action on G-6-P. The biochemical mechanisms of melittin effects on the substrate G-6-P and the coenzyme NADP are similar, and the inhibition of melittin is not related to the structure of G-6-PD.

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Enzymatic Synthesis of Endomorphin by Immobilized Papain on Sodium Alginate-Chitosan

Ping HE ; Zhuolie HUANG ; Chunyi LI ; Weijia CHEN ; Guanghong WU ; Zhizhan CHU

Chinese Journal of Biochemistry and Molecular Biology.2008;24(5):426-431.

The enzymatic synthesis of endomorphins were carried out by immobilized papain on sodium alginate-chitosan (IPSAC). The reaction has been carried out in two steps. First, Trp-Phe-NH2 was obtained by Boc-Trp-OH coupling with Phe-NH2 using IPSAC catalyst in microaqueous acetonitrile system with a 27.8 % yielding as determined by high performance liquid chromatography (HPLC). The catalytic properties of IPSAC were examined by studying the dependence of pH, ionic strength, solution content, reaction temperature,enzyme loading and reaction time over the yields. The results of orthogonal experiments indicated that pH was the most significant factor that influenced this synthesis. Second, Boc-Tyr-Pro-OMe and Trp-Phe-NH2 were suspended into the microaqueous acetonitrile to produce Tyr-Pro-Trp-Phe-NH2 (endomorphin-1) with a 35.2% yield.

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