Covalent inhibition of hAChE by organophosphates causes homodimer dissociation through long-range allosteric effects

Acetylcholinesterase (EC 3.1.1.7), a key acetylcholine-hydrolyzing enzyme in cholinergic neurotransmission, is present in a variety of states in situ, including monomers, C-terminally disulfide-linked homodimers, homotetramers, and up to three tetramers covalently attached to structural subunits. Could oligomerization that ensures high local concentrations of catalytic sites necessary for efficient neurotransmission be affected by environmental factors? Using small-angle X-ray scattering (SAXS) and cryo-EM, we demonstrate that homodimerization of recombinant monomeric human acetylcholinesterase (hAChE) in solution occurs through a C-terminal four-helix bundle at micromolar concentrations. We show that diethylphosphorylation of the active serine in the catalytic gorge or isopropylmethylphosphonylation by the R_P enantiomer of sarin promotes a 10-fold increase in homodimer dissociation. We also demonstrate the dissociation of organophosphate (OP)-conjugated dimers is reversed by structurally diverse oximes 2PAM, HI6, or RS194B, as demonstrated by SAXS of diethylphosphoryl-hAChE. However, binding of oximes to the native ligand-free hAChE, binding of high-affinity reversible ligands, or formation of an S_P-sarin-hAChE conjugate had no effect on homodimerization. Dissociation monitored by time-resolved SAXS occurs in milliseconds, consistent with rates of hAChE covalent inhibition. OP-induced dissociation was not observed in the SAXS profiles of the double-mutant Y337A/F338A, where the active center gorge volume is larger than in wildtype hAChE. These observations suggest a key role of the tightly packed acyl pocket in allosterically triggered OP-induced dimer dissociation, with the potential for local reduction of acetylcholine-hydrolytic power in situ. Computational models predict allosteric correlated motions extending from the acyl pocket toward the four-helix bundle dimerization interface 25 Å away.     

Spatial and temporal expressions of prune reveal a role in Müller gliogenesis during Xenopus retinal development

The development of stratified retinal cell architecture is highly conserved in all vertebrates, implying that a common fundamental molecular mechanism is involved in the generation of the organized retina. However, the detailed molecular mechanisms of retinal development are not fully understood. Here we have identified the Xenopus ortholog of prune and show that it is expressed in both differentiating and differentiated retinal domains during development. Interestingly, these spatial and temporal expression patterns coincide with the expression of prune binding partners, the NM23 family members. Overexpression of prune in retinal precursor cells significantly increases the ratio of Müller glial cells as observed by modulation of NM23 activity (Mochizuki et al., 2009). However, a mutated form of prune that has replacement of four aspartate (D) residues (D'Angelo et al., 2004), essential for phosphodiesterase activity, does not exhibit gliogenic activity. Our observations suggest that Xenopus prune may regulate Müller gliogenesis through phosphodiesterase-mediated regulation of NM23 family members.     

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson''s disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients^1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise^3,5.In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)⁸, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice^9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer¹¹. More recent studies have generated intra-nigral lesions with both a low mortality rate >80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia^11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse¹⁵. Whilst this model has proven useful in the assessment of potential neuroprotective agents¹⁶, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion^{17, 18}.Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes >95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.     

Crystallographic conformers of actin in a biologically active bundle of filaments

Actin carries out many of its cellular functions through its filamentous form; thus, understanding the detailed structure of actin filaments is an essential step in achieving a mechanistic understanding of actin function. The acrosomal bundle in the Limulus sperm has been shown to be a quasi-crystalline array with an asymmetric unit composed of a filament with 14 actin-scruin pairs. The bundle in its true discharge state penetrates the jelly coat of the egg. Our previous electron crystallographic reconstruction demonstrated that the actin filament cross-linked by scruin in this acrosomal bundle state deviates significantly from a perfect F-actin helix. In that study, the tertiary structure of each of the 14 actin protomers in the asymmetric unit of the bundle filament was assumed to be constant. In the current study, an actin filament atomic model in the acrosomal bundle has been refined by combining rigid-body docking with multiple actin crystal structures from the Protein Data Bank and constrained energy minimization. Our observation demonstrates that actin protomers adopt different tertiary conformations when they form an actin filament in the bundle. The scruin and bundle packing forces appear to influence the tertiary and quaternary conformations of actin in the filament of this biologically active bundle.     

植物向光性反应的研究进展

本文对近年来有关植物向光性反应的研究结果作一综述:1)向光素和隐花色素是植物向光反应中的主要光受体,光敏色素在植物向光性反应中也起一定的作用;2)对植物的光辐照度-弯曲度曲线的分析,可知植物的正向光性运动有两种反应,即第一次正向光性弯曲和第二次正向光性弯曲;3)拟南芥(Arabidopsisthaliana)和水稻(Oryza sativa)等植物的根系具有负向光性的特性,根的负向光性倾斜生长角度为负向光性生长和向重性生长相互作用的矢量和;4)生长素的胞间运输依赖于生长素载体,生长素载体的不对称分布和动态运动是生长素极性运输和向性运动的分子基础.     

Formation of Polar Bundles of Pili and the Behavior of Azospirillum brasilenseCells in a Semiliquid Agar

This paper describes the formation of single polar bundles of pili on Azospirillum brasilensecells, the twitching motility of cell aggregates, and a new type of social behavior—the dispersal of bacterial cells in semiliquid agar associated with the formation of granular inclusions (the so-called Gri⁺phenotype)—which is an alternative to swarming (the Swa phenotype). The wild-type A. brasilensecells occurring in a semiliquid agar may show either the Swa⁺Gri^–, or Swa^–Gri^–, or Swa^–Gri⁺phenotype. The formation of single polar flagella (Fla) or polar bundles of pili may reflect two alternative states of A. brasilensecells. The components of the Fla system may be involved in the regulation of the phenotypic variation of azospirilla.     

Spectral organization of the eye of a butterfly,Papilio

Journal of Comparative Physiology A - This review outlines our recent studies on the spectral organization of butterfly compound eyes, with emphasis on the Japanese yellow swallowtail butterfly,...     

水稻穗颈维管束及穗部性状的QTL分析

以籼稻 (OryzasativaL .ssp .indicaZYQ8)和粳稻 (O .sativassp .japonicaJX17)的杂交F1代花培加倍的DH群体为材料考察了该群体的穗颈节大小维管束数、一次枝梗数、每穗颖花数、穗颈节顶部直径和穗长 ,并用该群体构建的分子图谱进行数量性状座位 (QTL)分析。检测到控制大维管束的 3个QTL (qLVB_1、qLVB_6和qLVB_7)分别位于第 1、第 6和第 7染色体 ;控制小维管束的 2个QTL (qSVB_4和qSVB_6 )分别位于第 4和第 6染色体 ;控制一次枝梗的 4个QTL (qPRB_4a、qPRB_4b、qPRB_6和qPRB_7)分别位于第 4(2个 )、第 6和第 7染色体 ;每穗颖花数的 3个QTL (qSPN_4a、qSPN_4b和qSPN_6 )分别位于第 4(2个 )和第 6染色体上 ;穗颈节顶部直径的 5个QTL (qPTD_2、qPTD_5、qPTD_6、qPTD_8和qPTD_12 )分别位于第 2、第 5、第 6、第 8和第 12染色体 ;穗长的 3个QTL (qPL_4、qPL_6和qPL_8)分别位于第 4、第 6、第 8染色体上。其中qLVB_6、qSVB_6、qSPN_6、qPTD_6和qPL_6均位于第 6染色体的G12 2_G1314b之间 ;qPL_8和qPTD_8位于第 8染色体的GA40 8_BP12 7a之间 ;qPRB_4a和qSPN_4a位于第 4染色体的G177_CT2 0 6之间 ;qPL_4和qSPN_4b位于第 4染色体CT40 4_CT5 0 0之间 ;qSVB_4所在的区间与qPL_4、qSPN_4b和qPRB_4b所在的区间相邻。     

Conformation and ion channel properties of a five-helix Bundle protein.

The primary amphipathic peptide Ac-Met-Gly-Leu-Gly-Leu-Trp-Leu-Leu-Val-Leu10-Ala-Ala-Ala-Leu-Gln-Gly-Ala-Lys-Lys-Lys20-Arg-Lys-Val-NH-CH2-CH2-SH called SPM was able to induce formation of ion channels into planar lipid bilayers with main conductance values of 75 and 950 pS in 1 M KCl. The 75 pS value can be attributed to an aggregate composed of five monomers since the corresponding five-unit bundle (5-SPM) also presented a 70 pS channels under the same conditions. The upper 950 pS level would be generated by a hexameric aggregate. Ion channels induced by both SPM and its pentameric bundle are slightly cation selective but not voltage-dependent. The structural studies showed that the SPM and 5-SPM possess mainly an alpha-helical structure (approximately 40%) and are strongly embedded in the bilayer. This behaviour and the strong hydrophobic interactions occurring between helices in the bundle induce a strong stabilization of 5-SPM in the bilayer and would be responsible for the stepwise current fluctuations observed during the incorporation of 5-SPM into the membrane.