视觉细胞反应中方向选择性和取向选择性成分的分离

运动的光刺激常被用于研究视觉神经细胞的方向和取向选择性.但是,在细胞的反应中,两种因素同时存在且混杂在一起; 以往的各种分析方法均因未能有效地解决这一问题而不够准确,对此我们给出了理论和实例证明.本工作引入正余弦函数系对实验数据作最优回归分析.拟合程度好于以往的方法.并且从根本上分离了反应中的方向和取向选择性成分.使对这两种性质的准确定量成为可能.本文对此方法的内在含义作出了解释,并提出了对方向、取向选择性及最优方向、取向的理解.     

Influence of pore residues on permeation properties in the Kv2.1 potassium channel. Evidence for a selective functional interaction of K+ with the outer vestibule

The Kv2.1 potassium channel contains a lysine in the outer vestibule (position 356) that markedly reduces open channel sensitivity to changes in external [K(+)]. To investigate the mechanism underlying this effect, we examined the influence of this outer vestibule lysine on three measures of K(+) and Na(+) permeation. Permeability ratio measurements, measurements of the lowest [K(+)] required for interaction with the selectivity filter, and measurements of macroscopic K(+) and Na(+) conductance, were all consistent with the same conclusion: that the outer vestibule lysine in Kv2.1 interferes with the ability of K(+) to enter or exit the extracellular side of the selectivity filter. In contrast to its influence on K(+) permeation properties, Lys 356 appeared to be without effect on Na(+) permeation. This suggests that Lys 356 limited K(+) flux by interfering with a selective K(+) binding site. Combined with permeation studies, results from additional mutagenesis near the external entrance to the selectivity filter indicated that this site was located external to, and independent from, the selectivity filter. Protonation of a naturally occurring histidine in the same outer vestibule location in the Kv1.5 potassium channel produced similar effects on K(+) permeation properties. Together, these results indicate that a selective, functional K(+) binding site (e.g., local energy minimum) exists in the outer vestibule of voltage-gated K(+) channels. We suggest that this site is the location of K(+) hydration/dehydration postulated to exist based on the structural studies of KcsA. Finally, neutralization of position 356 enhanced outward K(+) current magnitude, but did not influence the ability of internal K(+) to enter the pore. These data indicate that in Kv2.1, exit of K(+) from the selectivity filter, rather than entry of internal K(+) into the channel, limits outward current magnitude. We discuss the implications of these findings in relation to the structural basis of channel conductance in different K(+) channels.     

Ionic permeation of lipid bilayer membranes mediated by a neutral,noncyclic Li+-selective carrier having imide and ether ligands. I. Selectivity among monovalent cations

Summary We have found that Simon's neutral, noncyclic, Li⁺-selective complexone, which has imide and ether ligands, renders lipid bilayer membranes selectively permeable to certain cations and anions. The present paper characterizes the ability of this molecule to carry monovalent cations; and we show it to be most selective for Li⁺ among the alkali cations, the first reconstitution of Li⁺-selective permeation in lipid bilayer membranes. This complexone acts as an equilibrium-domain carrier for Ag⁺> Li⁺>Tl⁺>Na⁺>NH ₄ ⁺ >Rb⁺>Cs⁺ over a wide range of experimental conditions. The major type of membrane-permeating species formed is a 21 carrier/cation complex dominant except at the lowest salt and carrier concentrations where a 11 carrier/cation, with a similar selectivity sequence, can be detected. Among the groupIa cations the selectivity sequence in bilayers, Li⁺>Na⁺>K⁺>Rb⁺>Cs⁺, is similar to that previously found for this molecule in thick solvent-polymer membrane electrodes. We find this carrier to be more selective to Ag⁺ than to any other monovalent cation yet studied. This high Ag⁺ selectivity is used, together with the dependence of the selectivity on the nature of the N-amide substitutents, to argue that the imide oxygens play a major role as ligands.     

胼胝体输入对猫皮层17/18交界区细胞方向和取向选择性的影响

用抑制性神经递质GABA阻断胼胝体输入、用微机控制的运动光棒作为视觉刺激,用金属电极胞外记录技术,研究猫皮层17/18区交界附近细胞方向选择性和取向选择性的变化.在被检测的48个细胞中,50%细胞的方向选择性强度,54.2%细胞的取向选择性强度发生了改变;约20%细胞的最优反应方向或.及最优取向发生了10-30°的偏移;共有56.2%细胞的方向选择性、58.3%细胞的取向选择性受到明确的影响.这些结果表明胼胝体对皮层细胞视觉反应的贡献是多方面的.     

Identification of two acetylcholinesterases in Pardosa pseudoannulata and the sensitivity to insecticides

Pardosa pseudoannulata is an important predatory enemy against insect pests, such as rice planthoppers and leafhoppers. In order to understand the insecticide selectivity between P. pseudoannulata and insect pests, two acetylcholinesterase genes, Pp-ace1 and Pp-ace2, were cloned from this natural enemy. The putative proteins encoded by Pp-ace1 and Pp-ace2 showed high similarities to insect AChE1 (63% to Liposcelis entomophila AChE1) and AChE2 (36% to Culex quinquefasciatus AChE2) with specific functional motifs, which indicated that two genes might encode AChE1 and AChE2 proteins respectively. The recombinant proteins by expressing Pp-ace1 and Pp-ace2 genes in insect sf9 cells showed high AChE activities. The kinetic parameters, V_max and K_m, of two recombinant AChE proteins were significantly different. The sensitivities to six insecticides were determined in two recombinant AChEs. Pp-AChE1 was more sensitive to all tested insecticides than Pp-AChE2, such as fenobucarb (54 times in K_i ratios), isoprocarb (31 times), carbaryl (13 times) and omethoate (6 times). These results indicated that Pp-AChE1 might be the major synaptic enzyme in the spider. By sequence comparison of P. pseudoannulata and insect AChEs, the key amino acid differences at or close to the functional sites were found. The locations of some key amino acid differences were consistent with the point mutation sites in insect AChEs that were associated with insecticide resistance, such as Phe331 in Pp-AChE2 corresponding to Ser331Phe mutation in Myzus persicae and Aphis gossypii AChE2, which might play important roles in insecticide selectivity between P. pseudoannulata and insect pests. Of course, the direct evidences are needed through further studies.     

Kinetic and structural insights into the binding of histone deacetylase 1 and 2 (HDAC1, 2) inhibitors

The structure–activity and structure–kinetic relationships of a series of novel and selective ortho-aminoanilide inhibitors of histone deacetylases (HDACs) 1 and 2 are described. Different kinetic and thermodynamic selectivity profiles were obtained by varying the moiety occupying an 11 Å channel leading to the Zn²⁺ catalytic pocket of HDACs 1 and 2, two paralogs with a high degree of structural similarity. The design of these novel inhibitors was informed by two ligand-bound crystal structures of truncated hHDAC2. BRD4884 and BRD7232 possess kinetic selectivity for HDAC1 versus HDAC2. We demonstrate that the binding kinetics of HDAC inhibitors can be tuned for individual isoforms in order to modulate target residence time while retaining functional activity and increased histone H4K12 and H3K9 acetylation in primary mouse neuronal cell culture assays. These chromatin modifiers, with tuned binding kinetic profiles, can be used to define the relation between target engagement requirements and the pharmacodynamic response of HDACs in different disease applications.     

Antibiotic activity and structural analysis of the scorpion-derived antimicrobial peptide IsCT and its analogs

IsCT is a non-cell-selective antimicrobial peptide isolated from the scorpion Opisthacanthus madagascariensis that has potent cytolytic activity against both mammalian and bacterial cells. To investigate the structure-activity relationships of IsCT and to design novel peptide antibiotics with bacterial cell selectivity, we synthesized several analogs of IsCT and determined their three-dimensional structures in solution by 2D-NMR spectroscopy. IsCT has a linear alpha-helical structure from Gly3 to Phe13, and [K7]-IsCT has a linear alpha-helical structure from Leu2 to Phe13. [K7, P8, K11]-IsCT, which has a bend in its middle region, exhibited the highest antibacterial activity without hemolytic activity, suggesting that its proline-induced bend is an important determinant of this selectivity. Tryptophan fluorescence showed that the high selectivity of [K7, P8, K11]-IsCT toward bacterial cells is closely correlated with its highly selective interaction with negatively charged phospholipids. Its potent activity against antibiotic-resistant bacteria suggests that [K7, P8, K11]-IsCT may serve as a promising lead candidate in the development of new peptide antibiotics.     

纹外皮层PMLS区反馈投射对初级视皮层神经元反应特性的影响

神经系统中存在大量下行投射,与上行输入一起形成复杂的前馈与反馈回路,调控神经信号的传导和处理,但目前对皮层内反馈投射的功能作用认识还比较薄弱.通过微量注射抑制性神经递质γ-氨基丁酸(γ-aminobutyric acid,GABA),使猫纹外皮层后内侧外上雪氏区(area posteromedial lateral suprasylvian,PMLS)局部可逆性失活,使用胞外记录方法,研究初级视皮层17区神经元反应特性的变化.实验结果显示,PMLS区失活后,17区细胞对运动刺激的反应总体减弱,反应的相对稳定性基本不变,最高发放率/自发之比有所下降.与此同时,细胞的方向选择性指数减小,朝向选择性无显著变化.除少数"双向"反应细胞外,绝大部分细胞的最优方向基本不变.进一步分析发现,细胞对各个方向刺激的反应普遍下降,最优方向上的下降程度最大,是导致方向选择性减弱的主要原因.这些结果表明,PMLS区反馈投射可增强初级视皮层的方向选择性,而对朝向选择性影响有限.这一作用特点体现了PMLS区在皮层中偏重处理运动视觉信息的功能.     

KcsA: it's a potassium channel

Ion conduction and selectivity properties of KcsA, a bacterial ion channel of known structure, were studied in a planar lipid bilayer system at the single-channel level. Selectivity sequences for permeant ions were determined by symmetrical solution conductance (K(+) > Rb(+), NH(4)(+), Tl(+) > Cs(+), Na(+), Li(+)) and by reversal potentials under bi-ionic or mixed-ion conditions (Tl(+) > K(+) > Rb(+) > NH(4)(+) > Na(+), Li(+)). Determination of reversal potentials with submillivolt accuracy shows that K(+) is over 150-fold more permeant than Na(+). Variation of conductance with concentration under symmetrical salt conditions is complex, with at least two ion-binding processes revealing themselves: a high affinity process below 20 mM and a low affinity process over the range 100-1,000 mM. These properties are analogous to those seen in many eukaryotic K(+) channels, and they establish KcsA as a faithful structural model for ion permeation in eukaryotic K(+) channels.     

草地贪夜蛾对2种豆科蔬菜的取食和产卵选择性及其适应性研究

草地贪夜蛾Spodoptera frugiperda是一种多食性昆虫,为明确该害虫对不同寄主的选择性和适生性,本文比较了草地贪夜蛾对玉米Zea mays、豇豆Vigna unguiculata和四季豆Phaseolus vulgaris等3种寄主的取食及产卵偏好性,并分析了取食不同寄主对其生长发育及繁殖的影响。结果表明：草地贪夜蛾低龄幼虫（初孵幼虫和2龄）对玉米叶和豇豆叶表现出取食偏好性,而高龄幼虫（3龄和5龄）对3种寄主不同组织的取食选择性无明显差异;草地贪夜蛾取食3种寄主植物均可以完成世代发育,但取食豇豆叶和四季豆叶的幼虫历期、蛹历期显著变短,化蛹率、羽化率显著降低;取食豇豆叶对其蛹重、成虫寿命无显著影响,但取食四季豆叶的蛹重显著变轻、雄成虫寿命显著变短;种群生命表参数显示,草地贪夜蛾在3种寄主上的繁殖力表现为玉米叶（1 138.29）>豇豆叶（1 179.00）>四季豆叶（585.50）,处理间差异显著;取食2种非嗜好寄主的种群内禀增长率（rm）和周限增长率（λ）均显著降低,平均世代历期（T）显著延长,取食豇豆叶的雌雄性比显著降低;草地贪夜蛾对寄主玉米具有明显的产卵偏好性,选择豇豆和四季豆的产卵量仅占植物着卵量的4.19%和18.23%,显著低于玉米着卵量。结果表明草地贪夜蛾偏好选择玉米进行取食和产卵,但在豇豆和四季豆寄主植物上可以实现种群繁衍,当其种群密度大时存在转移为害豇豆和四季豆的潜在风险。     

Ionic effects on bumetanide binding to the activated Na/K/2Cl cotransporter: Selectivity and kinetic properties of ion binding sites

Summary The loop diuretic bumetanide binds specifically to the Na/K/2Cl cotransporter of many cell types including duck erythrocytes. Membranes isolated from these erythrocytes retain the ability to bind bumetanide when cells are exposed to cotransport activity stimuli prior to membrane isolation. An extensive study of the effects of ions on specific [³H]bumetanide binding to such membranes is presented here and compared to the activity of these ions in supporting transport function in intact cells. Both Na⁺ and K⁺ enhanced bumetanide binding in a saturable manner consistent with a single-site interaction. The K _m for each ion was dependent on the concentration of the other cation suggesting heterotropic cooperative interactions between the Na⁺ and K⁺ binding sites. Na⁺ and K⁺ were partially replaceable, with the selectivity of the Na⁺ site being Na⁺ > Li⁺ > NH ₄ ⁺ ; N-methyl-d-glucamine⁺, choline⁺ and tetramethylammonium⁺ also supported a small amount of specific binding when substituted for Na⁺. The selectivity of the K⁺ site was K⁺ Rb⁺ > NH ₄ ⁺ > Cs⁺; N-methyl-d-glucamine⁺, choline⁺ and tetramethylammonium⁺ were inactive at this site. The results of transport experiments revealed a slightly different pattern. Li⁺ could partially substitute for Na⁺ in supporting coteansport, but other monovalent cations were completely inactive. The order of potency at the K⁺ site was NH ₄ ⁺ > K⁺ Rb⁺ > Cs⁺ other monovalent cations. The effect of Cl^- on bumetanide binding was biphasic, being stimulatory at low [Cl^-] but inhibitory at high [Cl^-]. As this implies the existence of two Cl^- binding sites (termed Cl _H and Cl _L for the high- and low- affinity sites, respectively) each phase was examined individually. Cl^- binding to Cl _H could be described by a rectangular hyperbola with a K _m of 2.5 mm, while kinetic analysis of the inhibition of bumetanide binding at high [Cl^-] revealed that it was of a noncompetitive type (K _i = 112.9 mm). The selectivity of anion binding to the two sites was distinct. Cl _H was highly selective with Cl^- > SCN^- > Br^-; F^-, NO ₃ ^- , ClO ₄ ^- , MeSO ₄ ^- , gluconate^- and SO ₄ ^2- were inactive. The efficacy of anion inhibition of binding to Cl _L was ClO ₄ ^- > I^- > SCN^- > NO₃ > Cl^-; F^-, MeSO ₄ ^- , gluconate^-, and SO ₄ ^2- were inactive. Thus, Cl _H is much more selective than Cl _L and largely accounts for the specificity of the system with respect to anion transport. SO ₄ ^- , NO ₃ ^- , I^-, SCN^- and ClO ₄ ^- did not support cotransport when bound to Cl _L and the latter three anions were inhibitory. Mg²⁺ was found to stimulate binding at a narrowly defined peak around 1.5 mm, but was inhibitory at higher concentrations. Other divalent cations caused a similar inhibition of bumetanide binding but did not exert a stimulatory effect at 1.5 mm. Divalent cations have little effect on cotransport in intact cells at concentrations up to 20 mm, suggesting that their effects on diuretic binding reflect interactions at internally disposed sites. Bumetanide binding was optimal at a pH of 7.8–8.1 and declined sharply as the pH was lowered towards 6. The titration curve correlated well with the effect of pH on cotransport in intact cells; the inhibitory effect of low pH suggests that protonation of the cotransporter may inhibit its function.We thank Drs. Brad Pewitt, John Westley and Mrinalini Rao for discussion, Sara Leung and Artelia Watson for their excellent technical assistance, and Dr. R.J. Turner for his gift of [³H] bumetanide. This work was supported in part by Cystic Fibrosis Center grant #CF RO11 7-04.     

An indirect estimate of bimodal gillnet selectivity of smelt

This study showed that it is possible to model a bimodal gillnet selectivity curve without knowing how individual fish have been caught. A bimodal model based on a modified normal density distribution was fitted over the length ranger of smelt Osmerus eperlanus from 8·9 to 17·0 cm. The model had eight parameters, and the height of the second peak was allowed to change with fish length. The coefficient of determination ( r ²) was 0·915 and the residuals were distributed symmetrically against variable axis. Corrected relative length distribution did not differ statistically from the relative length distribution of the trawl catch. In earlier studies of other species, pooled relative efficiency of multimesh gillnets composed by a geometric series mesh-size combination has been constant on the average. Because the model increased along fish length, the pooled relative efficiency was increasing also with fish length. Therefore, it is suggested that the gillnet catches of smelt always have to be corrected for gillnet selectivity.     

Preparation of a Fludarabine Intermediate via Selective Alkylation of 2-Fluoroadenine

The reaction between 2-fluoroadenine (3) and 1,3,5-tri-O-benzyl-1-α-d-chloroarabinofuranose (4) with potassium t-amylate was evaluated in various solvents to afford 9-β-d-(2,3,5-tri-O-benzyl-arabinofuranosyl)-2-fluoroadenine (5) and the corresponding α-anomer (6). In addition, 7-β-d-(2,3,5-tri-O-benzyl-arabinofuranosyl)-2-fluoroadenine (7) and an unusual “bis-fluoroadenine” nucleoside (8) were isolated as by-products. The highest anomeric ratio (β/α > 10) and conversion (>80%) were observed with the highly polar solvent sulfolane. This reaction was demonstrated on gram scale as a practical laboratory synthesis of 5, a known intermediate in the synthesis of fludarabine.     

Asymmetric imine-ene reactions: Diastereofacial selective reactions with chiral glyoxylate-derivedα-imino esters and asymmetric catalysis of enantiofacial selective reactions with prochiralα-imino esters

Summary The diastereofacial selective imine-ene reactions with-imino esters, prepared from (–)-8-phenylmenthyl glyoxylate, are shown to provide an efficient entry to the asymmetric synthesis of-amino acids. The feasibility study of the asymmetric catalysis is also reported on the enantiofacial selective ene reactions with prochiral-imino esters.     

Na+ block and permeation in a K+ channel of known structure

The effects of intracellular Na(+) were studied on K(+) and Rb(+) currents through single KcsA channels. At low voltage, Na(+) produces voltage-dependent block, which becomes relieved at high voltage by a "punchthrough" mechanism representing Na(+) escaping from its blocking site through the selectivity filter. The Na(+) blocking site is located in the wide, hydrated vestibule, and it displays unexpected selectivity for K(+) and Rb(+) against Na(+). The voltage dependence of Na(+) block reflects coordinated movements of the blocker with permeant ions in the selectivity filter.     

猫后内侧上雪氏区视神经元对运动棒反应的取向和方向特征

猫后内侧上雪区（ｐｏｓｔｅｒｏｍｅｄｉａｌｌａｔｅｒａｌｓｕｐｒａｓｙｌｖｉａｎａｒｅａ,ＰＭＬＳ）的绝大多数神经元（１７１／２００）对运动棒的取向调谐,６２％（１２４／２００）细胞的取向调谐宽度（半高波宽）小于９０°：按方向选择性和取向选择性可分辨出几类特征明显的细胞类型：１、强取向和强方向选择性细胞;２、强取向调谐的双向选择细胞;３、弱取向调谐的强方向选择细胞;４、无取向无方向选择性细胞;以及５、特征不明显的或中间类型细胞。它们与最近光学记录揭示的鹰猴中颞叶视区（ｍｉｄｄｌｅｔｅｍｐｏｒａｌｖｉｓｕａｌａｒｅａ,ＭＴ）的组织有很好的吻合。     

The responses to illusory contours of neurons in cortex areas 17 and 18 of the cats

Responses to illusory contours (ICs) were sampled from neurons in cortical areas 17 and 18 of the anesthetized cats. For ICs sensitive cells, the differences of receptive field properties were compared when ICs and real contour stimuli were applied. Two hundred orientation or direction selective cells were studied. We find that about 42 percent of these cells were the ICs sensitive cells. Although their orientation or direction tuning curves to ICs bar and real bars were similar, the response modes (especially latency and time course) were different. The cells' responses to ICs were independent of the spatial phases of sinusoidal gratings, which composed the ICs. The cells' optimal spatial frequency to composing gratings the ICs was much higher than the one to moving gratings. Therefore, these cells really responded to the ICs rather than the line ends of composing gratings. For some kinds of velocity-tuning cells, the optimal velocity to moving ICs bar was much lower than the optimal velocity to moving     

Potassium-dependent changes in the conformation of the Kv2.1 potassium channel pore.

The voltage-gated K+ channel, Kv2.1, conducts Na+ in the absence of K+. External tetraethylammonium (TEAo) blocks K+ currents through Kv2.1 with an IC50 of 5 mM, but is completely without effect in the absence of K+. TEAo block can be titrated back upon addition of low [K+]. This suggested that the Kv2.1 pore undergoes a cation-dependent conformational rearrangement in the external vestibule. Individual mutation of lysine (Lys) 356 and 382 in the outer vestibule, to a glycine and a valine, respectively, increased TEAo potency for block of K+ currents by a half log unit. Mutation of Lys 356, which is located at the outer edge of the external vestibule, significantly restored TEAo block in the absence of K+ (IC50 = 21 mM). In contrast, mutation of Lys 382, which is located in the outer vestibule near the TEA binding site, resulted in very weak (extrapolated IC50 = approximately 265 mM) TEAo block in the absence of K+. These data suggest that the cation-dependent alteration in pore conformation that resulted in loss of TEA potency extended to the outer edge of the external vestibule, and primarily involved a repositioning of Lys 356 or a nearby amino acid in the conduction pathway. Block by internal TEA also completely disappeared in the absence of K+, and could be titrated back with low [K+]. Both internal and external TEA potencies were increased by the same low [K+] (30-100 microM) that blocked Na+ currents through the channel. In addition, experiments that combined block by internal and external TEA indicated that the site of K+ action was between the internal and external TEA binding sites. These data indicate that a K+-dependent conformational change also occurs internal to the selectivity filter, and that both internal and external conformational rearrangements resulted from differences in K+ occupancy of the selectivity filter. Kv2.1 inactivation rate was K+ dependent and correlated with TEAo potency; as [K+] was raised, TEAo became more potent and inactivation became faster. Both TEAo potency and inactivation rate saturated at the same [K+]. These results suggest that the rate of slow inactivation in Kv2.1 was influenced by the conformational rearrangements, either internal to the selectivity filter or near the outer edge of the external vestibule, that were associated with differences in TEA potency.     

Novel potent and selective pyrazolylpyrimidine-based SYK inhibitors

Hybridisation of amino-pyrimidine based SYK inhibitors (e.g. 1a) with previously reported diamine-based SYK inhibitors (e.g. TAK-659) led to the identification and optimisation of a novel pyrimidine-based series of potent and selective SYK inhibitors, where the original aminomethylene group was replaced by a 3,4-diaminotetrahydropyran group. The initial compound 5 achieved excellent SYK potency. However, it suffered from poor permeability and modest kinase selectivity. Further modifications of the 3,4-diaminotetrahydropyran group were identified and the interactions of those groups with Asp512 were characterised by protein X-ray crystallography. Further optimisation of this series saw mixed results where permeability and kinase selectivity were increased and oral bioavailability was achieved in the series, but at the expense of potent hERG inhibition.     

Fraction of the dark current carried by Ca(2+) through cGMP-gated ion channels of intact rod and cone photoreceptors

The selectivity for Ca(2+) over Na(+), PCa/PNa, is higher in cGMP-gated (CNG) ion channels of retinal cone photoreceptors than in those of rods. To ascertain the physiological significance of this fact, we determined the fraction of the cyclic nucleotide-gated current specifically carried by Ca(2+) in intact rods and cones. We activated CNG channels by suddenly (<5 ms) increasing free 8Br-cGMP in the cytoplasm of rods or cones loaded with a caged ester of the cyclic nucleotide. Simultaneous with the uncaging flash, we measured the cyclic nucleotide-dependent changes in membrane current and fluorescence of the Ca(2+)-binding dye, Fura-2, also loaded into the cells. The ratio of changes in fura-2 fluorescence and the integral of the membrane current, under a restricted set of experimental conditions, is a direct measure of the fractional Ca(2+) flux. Under normal physiological salt concentrations, the fractional Ca(2+) flux is higher in CNG channels of cones than in those of rods, but it differs little among cones (or rods) of different species. Under normal physiological conditions and for membrane currents 相似文献