Structural basis of Wnt recognition by Frizzled

Wnts are lipid-modified morphogens that play critical roles in development principally through engagement of Frizzled receptors. The 3.25 angstrom structure of Xenopus Wnt8 (XWnt8) in complex with mouse Frizzled-8 (Fz8) cysteine-rich domain (CRD) reveals an unusual two-domain Wnt structure, not obviously related to known protein folds, resembling a "hand" with "thumb" and "index" fingers extended to grasp the Fz8-CRD at two distinct binding sites. One site is dominated by a palmitoleic acid lipid group projecting from serine 187 at the tip of Wnt's thumb into a deep groove in the Fz8-CRD. In the second binding site, the conserved tip of Wnt's "index finger" forms hydrophobic amino acid contacts with a depression on the opposite side of the Fz8-CRD. The conservation of amino acids in both interfaces appears to facilitate ligand-receptor cross-reactivity, which has important implications for understanding Wnt's functional pleiotropy and for developing Wnt-based drugs for cancer and regenerative medicine.     

Wnt1和Frizzled2在性成熟前小鼠卵巢组织织定位及配受体关系鉴定

为揭示Wnt1和Frizzled(Fzd)2参与卵巢和卵泡发育的作用机制,选择12只3周龄小鼠,应用免疫组化研究Wnt1和Fzd2在卵巢组织内的定位,免疫共沉淀鉴定其蛋白间的配受体关系.结果表明:在健康初级、次级卵泡卵母细胞以及腔前和腔后卵泡近基膜颗粒细胞的胞质和胞膜中,Wnt1均表现较强的免疫染色,Fzd2在胞质内免疫染色;在卵泡腔周围颗粒细胞和透明带周围卵丘细胞中Wnt1免疫染色弱;从卵巢中心扩展或环绕生长卵泡的条形基质细胞中Wnt1和Fzd2免疫染色阳性,间质腺存在免疫染色细胞;在闭锁卵泡卵母细胞中Wnt1和Fzd2呈非均匀强染色;在卵母细胞裂解液的Wnt1或Fzd2免疫沉淀物的电泳图谱中,分别同时存在Fzd2和Wnt1蛋白条带.表明Wnt1定位于卵母细胞和近基膜颗粒细胞的胞质和胞膜中,Fzd2定位于胞质内:在卵巢基质细胞、卵泡膜细胞和间质腺细胞中存在Wnt1和Fzd2分布;Wnt1和Fzd2之间可能为配体-受体关系.     

Activity-dependent internalization of smoothened mediated by beta-arrestin 2 and GRK2

Binding of Sonic Hedgehog (Shh) to Patched (Ptc) relieves the latter's tonic inhibition of Smoothened (Smo), a receptor that spans the cell membrane seven times. This initiates signaling which, by unknown mechanisms, regulates vertebrate developmental processes. We find that two molecules interact with mammalian Smo in an activation-dependent manner: G protein-coupled receptor kinase 2 (GRK2) leads to phosphorylation of Smo, and beta-arrestin 2 fused to green fluorescent protein interacts with Smo. These two processes promote endocytosis of Smo in clathrin-coated pits. Ptc inhibits association of beta-arrestin 2 with Smo, and this inhibition is relieved in cells treated with Shh. A Smo agonist stimulated and a Smo antagonist (cyclopamine) inhibited both phosphorylation of Smo by GRK2 and interaction of beta-arrestin 2 with Smo. beta-Arrestin 2 and GRK2 are thus potential mediators of signaling by activated Smo.     

Regulation of receptor fate by ubiquitination of activated beta 2-adrenergic receptor and beta-arrestin

Although trafficking and degradation of several membrane proteins are regulated by ubiquitination catalyzed by E3 ubiquitin ligases, there has been little evidence connecting ubiquitination with regulation of mammalian G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) function. Agonist stimulation of endogenous or transfected beta2-adrenergic receptors (beta2ARs) led to rapid ubiquitination of both the receptors and the receptor regulatory protein, beta-arrestin. Moreover, proteasome inhibitors reduced receptor internalization and degradation, thus implicating a role for the ubiquitination machinery in the trafficking of the beta2AR. Receptor ubiquitination required beta-arrestin, which bound to the E3 ubiquitin ligase Mdm2. Abrogation of beta-arrestin ubiquitination, either by expression in Mdm2-null cells or by dominant-negative forms of Mdm2 lacking E3 ligase activity, inhibited receptor internalization with marginal effects on receptor degradation. However, a beta2AR mutant lacking lysine residues, which was not ubiquitinated, was internalized normally but was degraded ineffectively. These findings delineate an adapter role of beta-arrestin in mediating the ubiquitination of the beta2AR and indicate that ubiquitination of the receptor and of beta-arrestin have distinct and obligatory roles in the trafficking and degradation of this prototypic GPCR.     

5-芳甲基-4-叔丁基-2-氨基噻唑的合成与表征

溴化铜在乙醇中溴化4,4-二甲基-1-芳基-3-戊酮,得到α-溴化产物不经分离直接与硫脲环合制得9种2-氨基噻唑类新化合物,收率21.9%~92.9%.目标物2-氨基噻唑类化合物经核磁谱、红外光谱、质谱测定其结构.用单晶X-射线衍射测定了化合物1g.HBr的晶体结构.化合物1g.HBr晶体属单斜晶系,空间群为P2(1)/c,晶胞参数为:a=0.96 713(5)nm,b=1.558 25(8)nm,c=1.306 87(7)nm,Z=4,V=1.665 34(15)nm3,Dc=1.577 g/cm3,F(000)=800,R1=0.0313,ωR2=0.0757,S=1.024.     

水-乙醇-硫酸铵体系及在分离提取天然产物中的应用

水-乙醇-硫酸铵构成的双水相体系,具有不使用有毒有机溶剂、萃取条件温和的特点.使用测定上下相电导和折光的方法,测定了分相前乙醇含量为30%和40%的溶液分相后上下相的组成;上下相的组成和硫酸铵的加入量有关.分相前乙醇含量为40%的溶液,分相后上相的乙醇含量可达55%,同时上相中仍含约0.03 g/ml的硫酸铵;下相乙醇含量在10%左右,硫酸铵的含量约为0.45 g/ml.     

Synthesis and biological activities of N-dichloroacetyl-2-n-propyl-1-oxa-4-aza-spiro-4, 5-decane

The title compound was synthesized and characterized by IR, 1HNMR and elemental analysis. The preliminary biological test showed that the compound protected maize against injury by imazethapyr to some extent.     

DNA甲基化抑制剂DAC处理对鸡Wnt10a基因表达及其启动子甲基化的影响

Wnt10a基因在细胞增殖、分化及迁移中发挥着重要作用,参与调控机体生长发育的诸多生物学过程,但对鸡Wnt10a基因表达的表观遗传调控机制尚不清楚.为探究DNA甲基化对Wnt10a基因表达的影响,以DMSO处理为对照组,5-Aza-2'-deoxycytidine (DAC)处理为试验组,分别用0.25、0.5、1.0...     

Binding of DCC by netrin-1 to mediate axon guidance independent of adenosine A2B receptor activation

Netrins stimulate and orient axon growth through a mechanism requiring receptors of the DCC family. It has been unclear, however, whether DCC proteins are involved directly in signaling or are mere accessory proteins in a receptor complex. Further, although netrins bind cells expressing DCC, direct binding to DCC has not been demonstrated. Here we show that netrin-1 binds DCC and that the DCC cytoplasmic domain fused to a heterologous receptor ectodomain can mediate guidance through a mechanism involving derepression of cytoplasmic domain multimerization. Activation of the adenosine A2B receptor, proposed to contribute to netrin effects on axons, is not required for rat commissural axon outgrowth or Xenopus spinal axon attraction to netrin-1. Thus, DCC plays a central role in netrin signaling of axon growth and guidance independent of A2B receptor activation.     

Beta-arrestin 2 mediates endocytosis of type III TGF-beta receptor and down-regulation of its signaling

beta-Arrestins bind to activated seven transmembrane-spanning (7TMS) receptors (G protein-coupled receptors) after the receptors are phosphorylated by G protein-coupled receptor kinases (GRKs), thereby regulating their signaling and internalization. Here, we demonstrate an unexpected and analogous role of beta-arrestin 2 (betaarr2) for the single transmembrane-spanning type III transforming growth factor-beta (TGF-beta) receptor (TbetaRIII, also referred to as betaglycan). Binding of betaarr2 to TbetaRIII was also triggered by phosphorylation of the receptor on its cytoplasmic domain (likely at threonine 841). However, such phosphorylation was mediated by the type II TGF-beta receptor (TbetaRII), which is itself a kinase, rather than by a GRK. Association with betaarr2 led to internalization of both receptors and down-regulation of TGF-beta signaling. Thus, the regulatory actions of beta-arrestins are broader than previously appreciated, extending to the TGF-beta receptor family as well.     

Wnt5a control of cell polarity and directional movement by polarized redistribution of adhesion receptors

Mechanisms by which Wnt pathways integrate the organization of receptors, organelles, and cytoskeletal proteins to confer cell polarity and directional cell movement are incompletely understood. We show that acute responses to Wnt5a involve recruitment of actin, myosin IIB, Frizzled 3, and melanoma cell adhesion molecule into an intracellular structure in a melanoma cell line. In the presence of a chemokine gradient, this Wnt-mediated receptor-actin-myosin polarity (W-RAMP) structure accumulates asymmetrically at the cell periphery, where it triggers membrane contractility and nuclear movement in the direction of membrane retraction. The process requires endosome trafficking, is associated with multivesicular bodies, and is regulated by Wnt5a through the small guanosine triphosphatases Rab4 and RhoB. Thus, cell-autonomous mechanisms allow Wnt5a to control cell orientation, polarity, and directional movement in response to positional cues from chemokine gradients.     

山林权属纠纷调处方法比较研究

调处山林权属纠纷的常用方法有协商解决、行政裁决和司法裁决.协商解决的适用以山林权属纠纷的性质不属于单方违法行为和当事人自愿及以尊重事实为基础;行政裁决方法的运用以经过当事人协商没有解决和未经人民法院审理为条件;司法裁决方法的运用则以必经行政裁决程序和在规定期限内向人民法院起诉为前提.认为协商解决方法有利于协议的遵守和执行,有利于增强团结和促进社会的稳定,但调处的时间较长,耗费的财力和物力较多;行政裁决方法效率较高,能及时有效地处理山林纠纷,抑制"无理争山"现象,但易造成当事人之间的对立情绪,行政裁决后的执行效果不佳;司法裁决方法最具有公平性和公正性,所费人力财力物力最少,但裁决后的执行没有保障.根据浙江省的实际情况,从管理体制和解决机制以及政策和法律等视角,提出了在实践中正确运用3种调处方法的具体措施.表2参4     

2-氨基-5-取代基-4-噻唑基膦酸酯及衍生物的合成

报道了2-氨基-5-取代基-4-噻唑基膦酸酯及衍生物的合成,讨论了其 IR 和1~HNMR特征,并证明其对棉花枯萎病和小麦赤霉病菌有抑制活性。     

5-苄基-4-叔丁基-2-苄亚氨基噻唑的合成及其对环氧合酶-2的抑制活性

5-苄基-4-叔丁基-2-氨基噻唑与芳香醛缩合制备了11 种5-苄基-4-叔丁基-2-苄亚氨基噻唑.结构经1H NMR和元素分析确证, 并用单晶 X-射线衍射测定了化合物Ⅰc的晶体结构.化合物Ⅰc晶体属单斜晶系, 空间群为 P21/n,晶胞参数为:a= 1.437 53(11) nm,b= 0.986 65(8) nm,c=1.465 65(12) nm,β=114.256 0(10)°;Z＝4,V＝1.895 3(3) nm3, Dc＝1.349 g/cm3, F(000)＝808, R1＝0.050 7, wR2＝0.116 3,S＝1.03.体外筛选结果表明,化合物Ⅰb, Ⅰk 在10 μmol/L 浓度下对COX-2的抑制率超过50%.     

N2O5 oxidizes chloride to Cl2 in acidic atmospheric aerosol

Molecular chlorine (Cl2) is an important yet poorly understood trace constituent of the lower atmosphere. Although a number of mechanisms have been proposed for the conversion of particle-bound chloride (Cl-) to gas-phase Cl2, the detailed processes involved remain uncertain. Here, we show that reaction of dinitrogen pentoxide (N2O5) with aerosol-phase chloride yields Cl2 at low pH (<2) and should constitute an important halogen activation pathway in the atmosphere.     

Imaginal discs secrete insulin-like peptide 8 to mediate plasticity of growth and maturation

Developing animals frequently adjust their growth programs and/or their maturation or metamorphosis to compensate for growth disturbances (such as injury or tumor) and ensure normal adult size. Such plasticity entails tissue and organ communication to preserve their proportions and symmetry. Here, we show that imaginal discs autonomously activate DILP8, a Drosophila insulin-like peptide, to communicate abnormal growth and postpone maturation. DILP8 delays metamorphosis by inhibiting ecdysone biosynthesis, slowing growth in the imaginal discs, and generating normal-sized animals. Loss of dilp8 yields asymmetric individuals with an unusually large variation in size and a more varied time of maturation. Thus, DILP8 is a fundamental element of the hitherto ill-defined machinery governing the plasticity that ensures developmental stability and robustness.     

桃树砧木品种筑波4号筑波5号对爪哇根结线虫的抗性

为探明桃树砧木品种筑波4号(Prunus persica'tsukuba-4')和筑波5号(P.persica'tsukuba-5')的抗根结线虫性能和抗原价值,对实生钵苗采用人工接种、根系次氯酸钠-酸性品红染色等方法研究了其对爪哇根结线虫(Meloidogyne javanica)的抗性.结果表明:接种后30～40 d,90株筑波4号中9株有线虫侵入,1株产生根结,侵入根中的13条线虫有11条未发育,2条发育至3龄阶段,无雌成虫形成;90株筑波5号中11株有线虫侵入,5株产生根结,侵入根中的24条线虫20条未发育,4条发育至3龄阶段,无雌成虫形成.表明2品种高抗爪哇根结线虫;实生苗个体间存在显著的抗性分离现象,但分离范围较小,只存在免疫和高抗2种基因型;免疫株率分别为98.9%和94.4%;对爪哇根结线虫均具有极强的抗侵入与抗发育能力,接种侵入率分别为0.014%和0.027%,根中线虫分别有15.4%和16.7%发育至3龄幼虫阶段,未形成成虫.筑波4号、筑波5号是极优异的抗爪哇根结线虫桃砧木品种和抗原植物种质资源.