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1.
目的: 构建α1亚基诱导表达、β2和γ2L亚基稳定表达的人源α1β2γ2L-GABAAR-CHO(Chinese hamster ovary)细胞株。方法: 从人cDNA文库中扩增α1、β2、γ2L亚基编码基因,分别构建亚基表达载体;将三个亚基表达载体共转染CHO-K1细胞,通过抗性筛选、膜电位检测法进行稳定表达克隆筛选;通过qPCR、Western blot对亚基表达进行鉴定;以激动剂GABA、阳性变构调节剂地西泮(diazepam,Dia)、拮抗剂荷包牡丹碱(bicuculine)为工具药,采用全细胞膜片钳方法及膜电位检测法对稳定表达细胞的药理学功能进行鉴定。结果: 经克隆筛选获得表达量较高的α1β2γ2L-GABAAR-CHO并对其亚基表达鉴定,结果显示该细胞稳定表达α1、β2、γ2L亚基,构建的α1β2γ2L-GABAAR-CHO细胞仅在加入四环素(tetracyclin)诱导的情况下表达α1亚基并与β2、γ2L组装成具有功能活性的α1β2γ2L-GABAAR;对其进行全细胞膜片钳检测研究发现,GABA可对其产生激动效应,引起α1β2γ2L-GABAAR-CHO细胞产生氯离子通道特征性电流变化,Dia可剂量依赖性地增强GABA对α1β2γ2L-GABAAR的激动效应;在膜电位检测研究中,获得GABA激动效应EC50为(177.72 ± 15.92)nmol/L,Dia变构效应EC50为(3.63±0.52)μmol/L,拮抗剂Bicuculine拮抗效应IC50为(538.83±29.55)nmol/L。结论: 通过采用诱导表达策略,成功构建了α1β2γ2L-GABAAR-CHO稳定表达细胞株,该细胞株具有对激动剂、阳性变构剂、拮抗剂特异性检测的药理学功能。  相似文献   

2.
The reaction of TiCl4 with Li2[(SiMe2)25-C5H3)2] in toluene at room temperature afforded a mixture of cis- and trans-[(TiCl3)2{(SiMe2)25-C5H3)2}] in a molar ratio of 1/2 after recrystallization. The complex trans-[(TiCl3)2{(SiMe2)25-C5H3)2}] was hydrolyzed immediately by the addition of water to THF solutions to give trans-[(TiCl2)2(μ-O){(SiMe2)25-C5H3)2}] as a solid insoluble in all organic solvents, whereas hydrolysis of cis-[(TiCl3)2{(SiMe2)25-C5H3)2}] under different conditions led to the dinuclear μ-oxo complex cis-[(TiCl2)2)(μ-O){(SiMe2)25-C5H3)2}] and two oxo complexes of the same stoichiometry [(TiCl)2(μ-O){(SiMe2)25-C5H3)2}]2(μ-O)2 as crystalline solids. Alkylation of cis- and trans-[(TiCl3)2{(SiMe2)25-C5H3)2}] with MgCIMe led respectively to the partially alkylated cis-[(TiMe2Cl)2{(SiMe2)25-C5H3)2}] and the totally alkylated trans-[(TiMe3)2{(SiMe2)25-C5H3)2}] compounds. The crystal and molecular structure of the tetranuclear oxo complex [(TiCl)2(μ-O){(SiMe2)25-C5H3)2}]2(μ-O)2 was determined by X-ray diffraction.  相似文献   

3.
The cyclopentadienyl osmium(II) complexes [(η5-C5H5)Os(PPh3)2X] [X = Br (1), CH3CN (2)] reacts with sodium azide (NaN3) to yield the corresponding azido complex [(η5-C5H5)Os(PPh3)2N3] (3). This undergoes [3+2] dipolar cycloaddition reaction with activated alkynes like dimethyl and diethyl acetylenedicarboxylate to yield triazolato complexes [(η5-C5H5)Os(PPh3)2{N3C2(CO2R)2}] [R = –CH2CH3 (4) and –CH3 (5)]. The complex 3 also reacts with nitriles such as tetracyanoethylene (TCE), fumaronitrile and p-nitrobenzonitrile to yield complexes of the type [(η5-C5H5)Os(PPh3)2{N4C2(CN)C(CN)2}] (6), [(η5-C5H5)Os(PPh3)2{N3C2HCN}] (7) and [(η5-C5H5)Os(PPh3)2{N4C(C6H4p-NO2)}] (8). These complexes were fully characterized on the basis of microanalyses, FT-IR and NMR spectroscopic data. The molecular structure of the representative complex [(η5-C5H5)Os(PPh3)2{N3C2(CO2CH2CH3)2}] (4) was determined by single crystal X-ray analysis.  相似文献   

4.
The quenching of the fluorescence of liver alcohol dehydrogenase (LADH) by molecular oxygen has been studied by both fluorescence lifetime and intensity measurements. This was done in the presence of 1 M acrylamide which selectively quenches the fluorescence of the surface tryptophan residue, Trp-15, thus allowing us to focus on the quenching of the deeply buried tryptophan, Trp-314, by molecular oxygen. Such studies yielded a Stern-Volmer plot of F0/F with a greater slope than the corresponding tau o/tau plot. This indicates that both dynamic and static quenching of Trp-314 occurs. The temperature dependence of the dynamic quenching of LADH by oxygen was also studied at three temperatures, from which we determined the activation enthalpy for the quenching of Trp-314 to be about 10 kcal/mol. The oxygen quenching of a ternary complex of LADH, NAD+ and trifluoroethanol was also studied. The rate constant for dynamic quenching of Trp-314 by oxygen was found to be approximately the same in the ternary complex as that in the unliganded enzyme.  相似文献   

5.
Recently, it has been shown that PKA-mediated phosphorylation of the β2-adrenergic receptor (β2-AR) by the cyclic AMP-dependent protein kinase (PKA) reduces its affinity for Gs and increases its affinity for Gi. Here we demonstrate that, like the β2-AR, the β1-AR is also capable of “switching” its coupling from Gs to Gi in a PKA-dependent manner. The β1-AR is capable of activating adenylate cyclase via Gs, and can also activate the extracellular-regulated kinases, p44 and p42 (ERK1/2). In transfected CHO cells, the observed β1-AR-mediated activation of ERK is both sensitive to pertussis toxin (PTX), indicating involvement of Gi/Go, and to the PKA inhibitor, H-89. β1-ARs with PKA phosphorylation sites mutated to alanines are unable to activate ERK. Mutating these same residues to aspartic acid, mimicking PKA phosphorylation, leads to a decrease in Gs-stimulated cAMP accumulation and an increase in PTX-sensitive ERK activation. These results strongly support the hypothesis that the β1-AR, like the β2-AR, can undergo PKA-dependent “Gs/Gi switching”.  相似文献   

6.
Catecholamines are viewed as major stimulants of diet- and cold-induced thermogenesis and of fasting-induced lipolysis, through the β-adrenoceptors (β123). To test this hypothesis, we generated β123-adrenoceptor triple knockout (TKO) mice and compared them to wild type animals. TKO mice exhibited normophagic obesity and cold-intolerance. Their brown fat had impaired morphology and lacked responses to cold of uncoupling protein-1 expression. In contrast, TKO mice had higher circulating levels of free fatty acids and glycerol at basal and fasted states, suggesting enhanced lipolysis. Hence, β-adrenergic signalling is essential for the resistance to obesity and cold, but not for the lipolytic response to fasting.  相似文献   

7.
Recently, evidence has accumulated in support of the heterologous expression of functional membrane proteins and their complexes on extracellular baculovirus particles (budded virus, BV). In this study, we attempted to apply this BV display system to detect G-protein-coupled receptor (GPCR) signaling. We infected Sf9 cells with a combination of four recombinant baculoviruses individually encoding the dopamine D1 receptor (DR-D1), G-protein -subunit (Gs), G-protein β1γ2 subunit dimer (Gβ1γ2), and adenylyl cyclase type VI (ACVI). The recovered BV fraction produced cAMP in response to the stimulation with dopamine. Co-expression of all three G-protein subunits in addition to receptor and ACVI led to a maximal response. BV co-expressing DR-D1, Gs, Gβ1γ2, and ACVI also responded to dopamine agonists and an antagonist. Furthermore, BV expressing two other Gs-coupled receptors together with Gs, Gβ1γ2, and ACVI also produced cAMP in response to their specific ligands. These results indicate the functional coupling of receptor, Gs and ACVI is reconstituted on BV. Since BV is essentially free of endogenous GPCRs, this BV co-display system should prove highly useful for the development of functional assay systems for GPCRs.  相似文献   

8.
The lithiation of indole, using a slight excess of n-butyl lithium in THF, followed by methylation and reaction with [Cr(CO)6] in refluxing dibutyl ether, resulted in the formation of [Cr(η6-N-methylindole)(CO)3] (1a) and [Cr(η6-N-methyl-2-methylindole)(CO)3] (1b). In contrast, lithiation of quinoline in THF, silylation and the subsequent reaction with [Cr(CO)6] under similar reaction conditions, afforded [Cr(η6-N-trimethylsilyl-2-butyl-1,2-dihydroquinoline)(CO)3] (2) and [Cr(η6-{2-butyl-1,2,3,4-tetrahydroquinoline})(CO)3] (3). The formation of [Cr(η6-2,2′-bis{N-methylindolyl})(CO)3] (4) implied lithiation at the 2-position of 1a. However, metallation at the 7-position was also indicated during the same reaction. In the presence of [Mn(CO)5Br], product 4 and the transmetallation product [Cr(η6-{7-(N-methylindolyl)Mn(CO)5})(CO)3] (5) were isolated. Reaction with titanocene dichloride gave [Cr(η6-{2-(N-methylindolyl)TiCp2Cl})(CO)3] (6), which slowly converted into [TiCp2{Cr(η6-2-(N-methylindolyl)(CO)3}2] (7).  相似文献   

9.
M R Eftink  K A Hagaman 《Biochemistry》1986,25(21):6631-6637
From measurements of the apparent phase and modulation fluorescence lifetime of liver alcohol dehydrogenase at multiple modulation frequencies (6, 18, and 30 MHz), the individual lifetimes and fractional intensities of Trp-314 and Trp-15 are calculated. Values of tau 314 = 3.6, tau 15 = 7.3, and f314 = 0.56, at 20 degrees C, are found. These values are in general agreement with values previously reported by Ross et al. [Ross, J.B.A., Schmidt, C.J., & Brand, L. (1981) Biochemistry 20, 4369] using pulse-decay methodology. In ternary complexes formed between the enzyme, NAD+ and either pyrazole or trifluoroethanol, the fluorescence lifetime of Trp-314 is found to be reduced, indicating that the binding of these ligands causes a dynamic quenching of this residue. The lifetime of Trp-314 is decreased more in the trifluoroethanol ternary complex than that with pyrazole. Also, the alkaline quenching transition of alcohol dehydrogenase is found to result in the selective, dynamic quenching of Trp-314. No change in the lifetimes of the two Trp residues is found upon selective removal of the active-site zinc atoms. From studies of the fluorescence anisotropy, r, of the enzyme as a function of added acrylamide (which selectively quenches the surface Trp-15 residue), the steady-state anisotropy of each residue is determined to be r314 = 0.26 and r15 = 0.21. In the ternary complexes the anisotropy of each residue increases slightly.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

10.
Resonance energy transfer from Trp-314 to ionized Tyr-286 was proposed (Laws, W. R., and Shore, J. D. (1978) J. Biol. Chem. 253, 8593-8597) as the mechanism for the observed decrease in protein fluorescence of liver alcohol dehydrogenase seen with alkaline pH, or with the formation of a ternary complex with NAD+ and trifluoroethanol. In the present study, ultraviolet difference spectra confirm the presence of ionized tyrosine not only in these two cases but also in the ternary complex with NADH and isobutyramide. Our results indicate that ternary complex formation, with either oxidized or reduced coenzyme, causes a conformational change leading to partial ionization of tyrosine residues in regions of the enzyme far from the active site.  相似文献   

11.
Reaction of sodium or potassium molybdate and excess malic acid in a wide range of pH values (pH 4.0–7.0) resulted in the isolation of two cis-dioxo-bis(malato)-Mo(VI) complexes, viz. Na3[MoO2H(S-mal)2] and K3[MoO2H(S-mal)2]·H2O (H3mal=malic acid). The sodium complex is also characterized by an X-ray structure analysis, showing that the mononuclear Mo units are linked together via very strong symmetric CO2···H··· O2C-hydrogen bond [2.432(5) Å], forming a polymeric chain. The molybdenum atoms are quasi-octahedrally coordinated by two cis-oxo groups and two bidentate malate ligands via its alkoxy and -carboxyl groups, while the β-carboxylic and carboxylate groups remain uncomplexed, as the coordination of vicinal carboxylate and alkoxide of homocitrate in FeMo cofactor of nitrogenase. The absolute configuration of the metal center in this S-malato complex is assigned as Λ and the homochirality within the chain is established as a homochiral form ···ΛS–ΛS–ΛS–ΛS···. It is proposed that the chiral configuration of the metal center in wild-type FeMo-co biosynthesis might be induced by the early coordination of the chiral R-homocitric acid, while a mixture of raceme might be obtained in the biosynthesis of NifV FeMo-cofactor. The absolute configuration of wild-type FeMo-cofactor is assigned as ΔR.  相似文献   

12.
The complexes RuC(CCPh)=CPhC(CCPh)=CPh(CO)3(NMe3) (3), Ru2μ-C(CCPh)=CPhC(CCPh)=CPh(CO)6 (1), Ru2μ-[C(CCPh)=CPh]2CO(CO)6 (2), Ru33-PhC2CCPh)(μ-CO)(CO)9 (4) and Ru44-PhC2CCPh)(CO)12 (5) have been isolated from reactions between PhC2C2Ph and Ru3(CO)12 or RU3(CO)10(NCMe)2. The molecular structures of complexes 1, 2, 3 and 5 have been determined from single-crystal X-ray studies. All complexes have precedents in similar products obtained from reactions involving mono-ynes; in the present cases, each alkyne fragment retains a phenylethynyl (PhCC---) group as a non-coordinated substituent.  相似文献   

13.
In a search for novel analogues of β3-adrenoceptor (AR) agonists relaxing the bladder for treatment of urinary dysfunction, 2-[4-(2-{[(1S,2R)-2-hydroxy-2-(4-hydroxyphenyl)-1-methylethyl]amino}ethyl)phenoxy]-2-methylpropionic acids (1a–e), into which a fibrate-like structure had been incorporated, were synthesised. Compound 1a was found to be a selective β3-AR agonist in functional assays using the ferret detrusor (β3-AR), rat uterus (β2-AR), and rat atrium (β1-AR); β3: EC50=7.8 nM, β2: IC50=7,300 nM, β1: EC20=23,000 nM. The introduction of a chlorine atom or methyl substituent at the ortho-position on the phenyl ring of 1a further improved β3-AR selectivity. In an in vivo study, 1a lowered intrabladder pressure (ED50=31 μg/kg) in rats, without increasing heart rate, in keeping with the in vitro results. Consequently, it is proposed that 1a and its analogues (1b–e), possess β3-AR agonistic activity in the absence of undesirable β1- or β2-AR mediated actions, and may be useful for clinical treatment and pharmacological studies.  相似文献   

14.
Reactions of [Rh(COD)Cl]2 with the ligand RN(PX2)2 (1: R = C6H5; X = OC6H5) give mono- or disubstituted complexes of the type [Rh2(COD)Cl22−C6H5N(P(OC6H5)2)2}] or [RhCl{ν2−C6H5 N(P(OC6H5)2)2 }]2 depending on the reaction conditions. Reaction of 1 with [Rh(CO)2Cl]2 gives the symmetric binuclear complex, [Rh(CO)Cl{μ−C6H5N(P(OC6H5)2)2} 2, whereas the same reaction with 2 (R = CH3; X = OC6H5) leads to the formation of an asymmetric complex of the type [Rh(CO)(μ−CO)Cl{μ−CH3N(P(OC6H5)2)2}2 containing both terminal and bridging CO groups. Interestingly the reaction of 3 (R = C6H5, X = OC6H4Br−p with either [Rh(COD)Cl]2 or [Rh(CO)2Cl]2 leads only to the formation of the chlorine bridged binuclear complex, [RhCl{ν2−C6H5N(P(OC6H4Br−p)2)2}]2. The structural elucidation of the complexes was carried out by elemental analyses, IR and 31P NMR spectroscopic data.  相似文献   

15.
The cell-binding abilities of a recombinant, RGD-containing peptide from foot-and-mouth disease virus (FMDV) have been characterized in HeLa and BHK cells. This peptide represents the aa sequence of the solvent-exposed G-H loop of protein VP1 which is involved in cell recognition and infection. The efficiency of the viral motif in promoting cell attachment and spreading is comparable to that shown by fibronectin or vitronectin. Cell binding is inhibited by a monoclonal antibody directed against a viral, RGD-involving B-cell epitope and also by sera against vitronectin (Vβ35) and fibronectin (5β1) receptors. In addition, a synthetic RGD peptide, which is a ligand for both integrins, prevents the cell binding mediated by the FMDV domain. These data demonstrate that the FMDV RGD motif is a potent ligand for cell-receptor integrins and sufficient to promote cell attachment to susceptible cells mainly through the vitronectin receptor.  相似文献   

16.
The preparation and structure–activity relationships (SARs) of potent agonists of the human β3-adrenergic receptor (AR) derived from a 4-aminopiperidine scaffold are described. Examples combine human β3-AR potency with selectivity over human β1-AR and/or human β2-AR agonism. Compound 29s was identified as a potent (EC50=1 nM) and selective (greater than 400-fold over β1- with no β2-AR agonism) full β3-AR agonist with in vivo activity in a transgenic mouse model of thermogenesis.  相似文献   

17.
The new inorganic 1D coordination polymer [Cu2(H3tea)24-pma)]n has been prepared, via self-assembly in aqueous medium, from copper(II) nitrate, triethanolamine (H3tea), pyromellitic acid (H4pma) and lithium hydroxide, and characterized by IR spectroscopy, elemental and single-crystal X-ray diffraction analyses. This compound and the related 2D polymer [Cu2(μ-H2tea)23-Na2(H2O)4}(μ6-pma)]n · 10nH2O are shown to mimic the alkane partial oxidation activity of the multicopper particulate methane monooxygenase, acting as catalysts precursors for the peroxidative oxidation of cyclohexane into cyclohexanol and cyclohexanone, by hydrogen peroxide (as green oxidant) and at room temperature in acidic MeCN/H2O medium. An overall yield (based on cyclohexane) of 29% has been achieved.  相似文献   

18.
Two new multi-cobalt-containing polyoxotungstates K4Na6Co2(H2O)12{Co(H2O)4[Co2(H2O)10Co4(H2O)2(B--SiW9O34)2]2} · 40H2O (1) and K10Na2[Co4(H2O)2(GeW9O34)2] · 20H2O (2) have been obtained by the routine synthetic reactions in aqueous solution. The polyoxoanion framework of 1 consists of two sandwich-type polyoxoanions [Co4(H2O)2(B--SiW9O34)2]12− connected together by a [CoO2(H2O)4] cluster to constitute the sandwich dimer, and then, four isolated Co(H2O)5 cations coordinate to the dimer through four μ2-O atoms. The polyoxoanion 2 is isomorphic to the sandwich-type polyoxoanion [Co4(H2O)2(B--SiW9O34)2]12− in 1. The magnetic property of compound 1 has been studied by measuring its magnetic susceptibility in the temperature range 2.0–300.0 K, indicating the existence of intramolecular ferromagnetic Co–Co interactions, and, the electrochemical properties of 1 and 2 are detected in the pH 4 buffer solution.  相似文献   

19.
The 1:1 complex of copper (II) and human serum albumin (HSA) slowly reacts with radiolytically generated O2- radical-anion at a rate constant of 6.1×106 M-1 s-1. Absorbance and fluorescence spectroscopies demonstrate that addition of an equimolar portion of quercetin (QH2) to the solution of the copper (II)-HSA complex induces a relocalization of the copper resulting in a ternary copper (II)-QH2-HSA complex. This form of quercetin slowly oxidizes in air-saturated solutions. A 10-fold excess urate, a plasma antioxidant, cannot displace copper (II) bound to HSA. In N2O-saturated solutions the ternary complex form of QH2 can repair the urate radical with a rate constant of 2.7×106 M-1 s-1 by an electron transfer reaction similar to that observed in the absence of copper (II). In O2-saturated solutions and in the absence of copper, HSA-bound QH2 fails to repair the urate radical because of the fast competitive reaction of O2- with urate radicals. However, addition of equimolar copper (II) restores the electron transfer from QH2 to the urate radical. These contrasting results are tentatively explained either by an enhanced reactivity of copper (II) with O2- in the ternary complex or by direct production of quercetin radicals via a copper-catalyzed reduction of the O2- radicals by QH2.  相似文献   

20.
A new compound containing a cubane tungsten chalcogenide cluster [W43-Te)4(CN)12]6− and Ca2+ complex units has been prepared by the reaction of aqueous solution of K6[W43-Te)4(CN)12] · 5H2O with the solution of a Ca(NO3)2 and phen(1,10-phenanthroline) (1:2 molar ratio) in a solvent mixture of H2O/EtOH. The structure of [{Ca(phen)2(H2O)}{Ca(phen)(H2O)4}{Ca(phen)2(H2O)3}][W4Te4(CN)12] · 5H2O 1 has been determined by X-ray crystallography. Compound 1 contains [{Ca(phen)(H2O)4}{Ca(phen)2(H2O)3}][W43- Te)4(CN)12] units bridged by {Ca(phen)2(H2O)}2+ units to form an one-dimensional zigzag chain structure. Interestingly, compound 1 showed a heterogeneous catalytic activity in the transesterification of a range of esters with methanol under the mild conditions. Moreover, it can be reused without any loss of activity through 10 runs with ester.  相似文献   

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