• 【新型抗CD4单克隆抗体将人免疫缺陷病毒感染和CD4细胞融合与病毒结合分离开来。】 复制标题 收藏 收藏
    DOI:10.1084/jem.172.4.1233 复制DOI
    作者列表:Healey D,Dianda L,Moore JP,McDougal JS,Moore MJ,Estess P,Buck D,Kwong PD,Beverley PC,Sattentau QJ
    BACKGROUND & AIMS: :Human immunodeficiency virus (HIV) binds to cells via an interaction between CD4 and the virus envelope glycoprotein, gp120. Previous studies have localized the high affinity binding site for gp120 to the first domain of CD4, and monoclonal antibodies (mAbs) reactive with this region compete with gp120 binding and thereby block virus infectivity and syncytium formation. Despite a detailed understanding of the binding of gp120 to CD4, little is known of subsequent events leading to membrane fusion and virus entry. We describe two new mAbs reactive with the third domain of CD4 that inhibit steps subsequent to virus binding critical for HIV infectivity and cell fusion. Binding of recombinant gp120 or virus to CD4 is not inhibited by these antibodies, whereas infection and syncytium formation by a number of HIV isolates are blocked. These findings demonstrate that in addition to virus binding, CD4 may have an active role in membrane fusion.
    背景与目标: 人类免疫缺陷病毒(HIV)通过CD4和病毒包膜糖蛋白gp120之间的相互作用与细胞结合。先前的研究已经将gp120的高亲和力结合位点定位在CD4的第一个域,并且与该区域反应的单克隆抗体(mAb)与gp120结合竞争,从而阻断了病毒的感染性和合胞体的形成。尽管对gp120与CD4的结合有详细的了解,但对导致膜融合和病毒进入的后续事件知之甚少。我们描述了与CD4的第三个域具有反应性的两个新的单克隆抗体,可抑制继病毒结合后对HIV感染性和细胞融合至关重要的步骤。重组gp120或病毒与CD4的结合不受这些抗体的抑制,而许多HIV分离株的感染和合胞体形成却被阻止。这些发现表明,除病毒结合外,CD4可能在膜融合中发挥积极作用。
  • 【差异磷酸肌醇与G蛋白门控K通道的成分结合。】 复制标题 收藏 收藏
    DOI:10.1007/s00232-006-0014-5 复制DOI
    作者列表:Thomas AM,Brown SG,Leaney JL,Tinker A
    BACKGROUND & AIMS: :The regulation of ion channels and transporters by anionic phospholipids is currently very topical. G protein-gated K(+) channels from the Kir3.0 family are involved in slowing the heart rate, generating late inhibitory postsynaptic potentials and controlling hormone release from neuroendocrine cells. There is considerable functional precedent for the control of these channels by phosphatidylinositol 4,5-bisphosphate. In this study, we used a biochemical assay to investigate the lipid binding properties of Kir3.0 channel domains. We reveal a differential binding affinity to a range of phosphoinositides between the C termini of the Kir3.0 isoforms. Furthermore, the N terminus in addition to the C terminus of Kir3.4 is necessary to observe binding and is decreased by the mutations R72A, K195A and R196A but not K194A. Protein kinase C phosphorylation of the Kir3.1 C-terminal fusion protein decreases anionic phospholipid binding. The differential binding affinity has functional consequences as the inhibition of homomeric Kir3.1, occurring after M3 receptor activation, recovers over minutes while homomeric Kir3.2 does not.
    背景与目标: :阴离子磷脂对离子通道和转运蛋白的调节目前非常热门。 Kir3.0家族的G蛋白门控的K()通道参与减慢心率,产生晚期抑制的突触后电位并控制神经内分泌细胞的激素释放。磷脂酰肌醇4,5-双磷酸酯对这些通道的控制有相当大的功能先例。在这项研究中,我们使用生化分析来研究Kir3.0通道域的脂质结合特性。我们揭示了对Kir3.0亚型的C末端之间的一系列磷酸肌醇的不同结合亲和力。此外,除了观察Kir3.4的C末端外,N末端对于观察结合也是必需的,并且由于突变R72A,K195A和R196A而不是K194A而降低。 Kir3.1 C端融合蛋白的蛋白激酶C磷酸化减少了阴离子磷脂的结合。差异结合亲和力具有功能性后果,因为抑制M3受体激活后发生的同源Kir3.1抑制会在数分钟内恢复,而同源Kir3.2不会恢复。
  • 【II类主要组织相容性复合物超抗原结合域的溶液结构。】 复制标题 收藏 收藏
    DOI:10.1006/bbrc.1997.6692 复制DOI
    作者列表:Jablonsky MJ,Subramaniam PS,Johnson HM,Russell JK,Krishna NR
    BACKGROUND & AIMS: We have used 600 MHz 1H NMR spectroscopy data to determine the solution structure of a 31-residue domain of a murine class II major histocompatibility (MHC) protein. This domain, I-Ab(beta)-(60-90), binds to the superantigen staphylococcal enterotoxin A. Distance geometry and dynamical simulated annealing calculations were performed using NOESY- and COSY-deduced constraints. I-Ab(beta)-(60-90), which is mostly alpha-helical, is more similar to the corresponding region of the class II MHC protein HLA-DR1 than to the class I MHC protein HLA-A2. Arg-72 and Arg-80 lie on the same side of the helix and face away from the antigenic peptide binding groove. His-81, implicated in both superantigen and peptide binding, is located midway between the surface defined by Arg-72/Arg-80 and residues that define the inside of the peptide binding groove, allowing for its participation in both types of binding.

    背景与目标: 我们已经使用600 MHz 1H NMR光谱数据确定了鼠类II类主要组织相容性(MHC)蛋白的31个残基域的溶液结构。该域I-Abβ-(60-90)与超抗原葡萄球菌肠毒素A结合。使用NOESY和COSY推导的约束条件进行距离几何结构和动态模拟退火计算。 I-Abβ-(60-90)主要是α-螺旋,与II类MHC蛋白HLA-DR1的相应区域相比,与I类MHC蛋白HLA-A2的对应区域更为相似。 Arg-72和Arg-80位于螺旋的同一侧,背对抗原肽结合槽。 His-81涉及超抗原和肽的结合,位于Arg-72 / Arg-80定义的表面与定义肽结合槽内部的残基之间的中间位置,允许其参与两种结合类型。 br>
  • 【牙龈卟啉单胞菌主动调节β2整联蛋白的粘附活性,并促进与巨噬细胞的结合和内在化。】 复制标题 收藏 收藏
    DOI:10.1128/IAI.00784-06 复制DOI
    作者列表:Hajishengallis G,Wang M,Harokopakis E,Triantafilou M,Triantafilou K
    BACKGROUND & AIMS: :In monocytes, the fimbriae of the oral pathogen Porphyromonas gingivalis activate cross talk signaling from Toll-like receptor 2 (TLR2) to the beta2 integrin CD11b/CD18, leading to the induction of the high-affinity state of the latter receptor. CD14 plays an important role in this "inside-out" proadhesive pathway by binding fimbriae and facilitating the activation of TLR2 and phosphatidylinositol 3-kinase signaling. In its high-affinity state, CD11b/CD18 mediates monocyte adhesion to endothelial cells and transmigration to sites of infection. We have now shown that P. gingivalis fimbriae function as both an activator and a ligand of CD11b/CD18; thus, fimbriae proactively promote their own binding to monocytes. Indeed, treatments that interfered with fimbria-induced activation of CD11b/CD18 (i.e., blockade of CD14, TLR2, or phosphatidylinositol 3-kinase signaling) also suppressed the cell binding activity of fimbriae, which was largely inducible and CD11b/CD18 dependent. Development of a recombinant inside-out signaling system in Chinese hamster ovary cells confirmed the ability of fimbriae to activate CD14/TLR2 signaling and induce their own CD11b/CD18-dependent binding. Induction of this proadhesive pathway by P. gingivalis fimbriae appeared to take place in lipid rafts. Indeed, methyl-beta-cyclodextrin, a cholesterol-sequestering agent that disrupts lipid raft organization, was found to inhibit the fimbria-induced assembly of CD14/TLR2 signaling complexes and the activation of the high-affinity state of CD11b/CD18. Experiments using macrophages from mice deficient in various pattern recognition receptors indicated that the receptors involved in the inside-out proadhesive pathway (CD14, TLR2, and CD11b/CD18) are important for mediating P. gingivalis internalization within macrophages. It therefore appears that P. gingivalis proactively modulates beta2 integrin adhesive activity for intracellular uptake.
    背景与目标: :在单核细胞中,口腔病原体牙龈卟啉单胞菌的菌毛激活了从Toll样受体2(TLR2)到β2整联蛋白CD11b / CD18的串扰信号,从而导致了后者受体的高亲和力状态的诱导。 CD14通过结合菌毛并促进TLR2和磷脂酰肌醇3激酶信号的激活,在此“由内而外”的前粘附途径中起重要作用。在其高亲和力状态下,CD11b / CD18介导单核细胞与内皮细胞的粘附并转移到感染部位。现在我们已经显示,牙龈卟啉单胞菌菌毛既是CD11b / CD18的激活剂又是其配体;因此,菌毛主动地促进其自身与单核细胞的结合。实际上,干扰菌毛诱导的CD11b / CD18活化(即阻断CD14,TLR2或磷脂酰肌醇3-激酶信号传导)的治疗也抑制了菌毛的细胞结合活性,其在很大程度上是诱导性的并且是CD11b / CD18依赖性的。中国仓鼠卵巢细胞中一种由内而外的重组信号系统的开发证实了菌毛具有激活CD14 / TLR2信号并诱导其自身CD11b / CD18依赖性结合的能力。齿龈假单胞菌对这种前粘性途径的诱导似乎发生在脂质筏中。实际上,已发现甲基-β-环糊精(一种可破坏脂质筏组织的胆固醇替代剂)可抑制菌毛诱导的CD14 / TLR2信号复合物的装配以及CD11b / CD18的高亲和力状态的激活。使用来自缺乏各种模式识别受体的小鼠的巨噬细胞进行的实验表明,参与由内而外的前粘附途径的受体(CD14,TLR2和CD11b / CD18)对于介导巨噬细胞内牙龈卟啉单胞菌的内化作用很重要。因此,似乎牙龈卟啉单胞菌主动地调节β2整联蛋白的粘附活性以用于细胞内摄取。
  • 【E-选择素的代谢变化是由携带唾液酸化的刘易斯A抗原的粘蛋白结合引起的。】 复制标题 收藏 收藏
    DOI:10.1006/bbrc.1997.6683 复制DOI
    作者列表:Nakada H,Inoue M,Yamashina I
    BACKGROUND & AIMS: Mucin-type glycoproteins carrying sialylLeA antigens (SL-GP) were isolated from the ascites fluid of a patient with colorectal cancer. SL-GP bound to E-selectin on endothelial cells in Ca2+- and sialylLeA antigen-dependent manners. To examine the metabolic change in E-selectin caused by ligation, endothelial cells were labeled with 32P-phosphate or 35S-Met and 35S-Cys. Phosphorylation at one or more serine residues of E-selectin was elevated by ligation with SL-GP but not with sialylLeA hexasaccharide. Pulse-labeling of E-selectin with 35S-Met and 35S-Cys in the presence of SL-GP indicated that the degradation of E-selectin was accelerated by SL-GP ligation, but labeling after pre-ligation with SL-GP revealed an increase in the synthesis of E-selectin. The synthesis may reflect compensation for the E-selectin degraded on pre-ligation. These results indicate that the overall metabolism of E-selectin was enhanced by the ligation of SL-GP, with degradation and synthesis being apparently balanced.

    背景与目标: 从患有结肠直肠癌的患者的腹水中分离出带有唾液酸LeA抗原的粘蛋白型糖蛋白(SL-GP)。 SL-GP以Ca2-和sialylLeA抗原依赖性方式与内皮细胞上的E-选择素结合。为了检查由连接引起的E-选择蛋白的代谢变化,用32P-磷酸或35S-Met和35S-Cys标记内皮细胞。通过与SL-GP而非唾液酸LeA六糖连接,可增强E-选择蛋白一个或多个丝氨酸残基的磷酸化作用。在SL-GP存在下用35S-Met和35S-Cys脉冲标记E-选择素表明SL-GP连接可加速E-选择素的降解,但与SL-GP预连接后的标记显示E-选择素的降解E-选择素的合成增加。该合成可以反映对在预连接时降解的E-选择蛋白的补偿。这些结果表明SL-GP的连接增强了E-选择素的整体代谢,降解和合成明显平衡。

  • 【一种用于改进对冷冻电子显微照片及其局部区域进行分类的功率谱可视化的新方法。】 复制标题 收藏 收藏
    DOI:10.1016/j.jsb.2006.06.014 复制DOI
    作者列表:Jonić S,Sorzano CO,Cottevieille M,Larquet E,Boisset N
    BACKGROUND & AIMS: :In a context of automation of cryo-electron microscopy, we developed a novel method for improving visibility of diffraction rings in the power spectra of cryo-electron micrographs of vitreous ice (without carbon film or high concentration of diffracting material). We used these enhanced spectra to semi-automatically detect and remove micrographs and/or local areas introducing errors in the global 3D map (drifted and charged areas) or those unable to increase global signal-to-noise ratio (non-diffracting areas). Our strategy also allows a detection of micrographs/areas with a strong astigmatism. These images should be removed when using algorithms that do not correct astigmatism. Our sorting method is simple and fast since it uses the normalized cross-correlation between enhanced spectra and their copies rotated by 90 degrees. It owes its success mainly to the novel pre-processing of power spectra. The improved visibility also allows an easier visual check of accuracy of sorting. We show that our algorithm can even improve the visibility of diffraction rings of cryo-electron micrographs of pure water. Moreover, we show that this visibility depends strongly on ice thickness. This algorithm is implemented in the Xmipp (open-source image processing package) and is freely available for implementation in any other software package.
    背景与目标: :在低温电子显微镜自动化的背景下,我们开发了一种新颖的方法,用于改善玻璃冰(无碳膜或高浓度衍射材料)的低温电子显微镜照片的功率谱中衍射环的可见性。我们使用这些增强的光谱来半自动检测和移除显微照片和/或局部区域,从而在全局3D地图(漂移和带电区域)或无法提高全局信噪比的区域(非衍射区域)引入误差。我们的策略还允许检测具有强烈散光的显微照片/区域。当使用无法校正像散的算法时,应删除这些图像。我们的排序方法简单快捷,因为它使用增强光谱与其旋转90度的副本之间的归一化互相关。它的成功主要归功于新颖的功率谱预处理。改进的可见性还可以使目视检查的准确性更加容易。我们证明了我们的算法甚至可以提高纯水冷冻电子显微照片的衍射环的可见性。此外,我们证明了这种能见度在很大程度上取决于冰的厚度。该算法在Xmipp(开源图像处理软件包)中实现,可免费用于任何其他软件包中。
  • 【纤连蛋白与成纤维细胞和纤连蛋白的III1组件结合所需的N端I型模块。】 复制标题 收藏 收藏
    DOI:10.1042/bj3230051 复制DOI
    作者列表:Sottile J,Mosher DF
    BACKGROUND & AIMS: Assembly of fibronectin fibrils occurs at the surface of substrate-attached cells and is mediated by the first to the fifth type I modules in the N-terminal 70 kDa portion of the molecule. The first type III module (III1) of fibronectin, not present in the 70 kDa portion, contains a conformation-dependent binding site for the 70 kDa N-terminal region of fibronectin, suggesting that the III1 module on cell-surface fibronectin may serve as a binding site for fibronectin's N-terminus on substrate-attached cells. To explore this possiblility, we compared the ability of mutant recombinant 70 kDa proteins containing deletions of one or several of the first five type I modules to bind to fibroblasts and to III1. Proteins containing the fourth and fiftBiomolecular Chemistry and Medicine, University of Wisconsin, Madison, WI 53706U.S.A. Assembly of fibronectin fibrils occurs at the surface of substrate-attached cells and is mediated by the first to the fifth type I modules in the N-terminal 70 kDa portion of the molecule. The first type III module (III1) of fibronectin, not present in the 70 kDa portion, contains a conh as 70 kDa deletion mutants lacking I4 and I5 also bound to the cell surface, and deletion mutants lacking I1-3 and I4-5 both competed only partially for binding of 125I-labelled fibronectin or 70 kDa protein. These data indicate that the N-terminal part of fibronectin binds to III1 via I4 and I5 and that interactions in addition to that of I4 and I5 with III1 are important for cell-surface-mediated fibronectin polymerization.

    背景与目标: 纤连蛋白原纤维的组装发生在附着有底物的细胞表面,并由分子的N端70 kDa部分中的第一至第五类I模块介导。纤连蛋白的第一个III型模块(III1)(不存在于70 kDa的部分中)包含纤连蛋白70 kDa N端区域的构象依赖性结合位点,表明细胞表面纤连蛋白上的III1模块可以作为底物附着细胞上纤连蛋白N末端的结合位点。为了探讨这种可能性,我们比较了包含前五个I型模块中一个或多个缺失的突变重组70 kDa蛋白与成纤维细胞和III1结合的能力。含有第四和第五种蛋白质的蛋白质生物分子化学和医学,威斯康星大学麦迪逊分校,威斯康星州53706纤连蛋白原纤维的组装发生在附着有底物的细胞表面,并由分子的N端70 kDa部分中的第一至第五类I模块介导。纤连蛋白的第一个III型模块(III1)(不存在于70 kDa的部分)包含一个conh,因为缺少I4和I5的70 kDa缺失突变体也与细胞表面结合,而缺少I1-3和I4-5的缺失突变体都存在仅部分竞争结合125 I标记的纤连蛋白或70 kDa蛋白。这些数据表明纤连蛋白的N末端部分通过I4和I5与III1结合,并且除了I4和I5之外,与III1的相互作用对于细胞表面介导的纤连蛋白聚合也很重要。
  • 【通过凝血酶刺激的血小板结合诱导白细胞中细胞因子的表达。】 复制标题 收藏 收藏
    DOI:10.1161/01.cir.95.10.2387 复制DOI
    作者列表:Neumann FJ,Marx N,Gawaz M,Brand K,Ott I,Rokitta C,Sticherling C,Meinl C,May A,Schömig A
    BACKGROUND & AIMS: BACKGROUND:Activated platelets tether and activate myeloid leukocytes. To investigate the potential relevance of this mechanism in acute myocardial infarction (AMI), we examined cytokine induction by leukocyte-platelet adhesion and the occurrence of leukocyte-platelet conjugates in patients with AMI.

    METHODS AND RESULTS:We obtained peripheral venous blood samples in 20 patients with AMI before and daily for 5 days after direct percutaneous transluminal coronary angioplasty (PTCA) and in 20 patients undergoing elective PTCA. Throughout the study period, CD41 immunofluorescence of leukocytes (flow cytometry) revealed increased leukocyte-platelet adhesion in patients with AMI compared with control patients (mean +/- SE of fluorescence [channels] before PTCA: 77 +/- 16 versus 35 +/- 9; P = .003). In vitro, thrombin-stimulated fixed platelets bound to neutrophils and monocytes. Within 2 hours, this resulted in increased mRNA for interleukin (IL),1 beta, IL-8, and monocyte chemoattractant protein (MCP)-1 in unfractionated leukocytes. After 4 hours, IL-1 beta and IL-8 concentration of the cell-free supernatant had increased by 268 +/- 36% and 210 +/- 7%, respectively, and cellular MCP-1 content had increased by 170 +/- 8%. Addition of activated platelets to adherent monocytes had a similar effect and was associated with nuclear factor-kappa B activation. Inhibition of binding by anti-P selectin antibodies reduced the effect of activated platelets on cytokine production.

    CONCLUSIONS:In patients with AMI, leukocyte-platelet adhesion is increased. Binding of activated platelets induces IL-1 beta, IL-8, and MCP-1 in leukocytes. Our findings suggest that leukocyte-platelet adhesion contributes to the regulation of inflammatory responses in AMI.

    背景与目标: 背景:激活血小板束缚并激活髓样白细胞。为了研究该机制在急性心肌梗塞(AMI)中的潜在相关性,我们研究了白细胞-血小板粘附引起的细胞因子诱导以及AMI患者中白细胞-血小板结合物的发生。

    方法和结果:我们在20例AMI患者中,分别在直接经皮腔内冠状动脉成形术(PTCA)之前和之后5天以及每天20例接受择期PTCA的患者中获取了外周静脉血。在整个研究过程中,与对照组相比,AMI患者的白细胞CD41免疫荧光(流式细胞仪)显示白细胞-血小板粘附增加(PTCA之前的平均[/ SE]荧光[通道]:77 /-16对35 /-9; P = 0.003)。在体外,凝血酶刺激的固定血小板与嗜中性粒细胞和单核细胞结合。在2小时内,这导致未分离的白细胞中白介素(IL),1β,IL-8和单核细胞趋化蛋白(MCP)-1的mRNA增加。 4小时后,无细胞上清液的IL-1 beta和IL-8浓度分别增加268 /-36%和210 /-7%,细胞MCP-1含量增加170 /-8% 。将活化的血小板添加至粘附的单核细胞具有相似的作用,并且与核因子-κB活化有关。抗P选择素抗体抑制结合可降低活化血小板对细胞因子产生的影响。

    结论:在AMI患者中,白细胞-血小板粘附增加。活化血小板的结合在白细胞中诱导IL-1β,IL-8和MCP-1。我们的发现表明,白细胞-血小板粘附有助于调节AMI中的炎症反应。

  • 【膜联蛋白V与脂质体结合后的构象适应性:时间分辨荧光研究。】 复制标题 收藏 收藏
    DOI:10.1006/bbrc.1997.6596 复制DOI
    作者列表:Follenius-Wund A,Piémont E,Freyssinet JM,Gérard D,Pigault C
    BACKGROUND & AIMS: The fluorescence intensity decay of the single tryptophan residue, Trp-187, of free annexin V is described by the sum of three lifetime components (5.4, 1.3, and 0.4 ns), which may be correlated to three ground-state classes of Trp conformers. The two major classes (44 and 48%) are embedded in the protein matrix. When annexin V binds to calcium and liposomes made of dioleoylphosphatidylcholine and dioleoylphosphatidylserine, similar results are obtained whatever the (10-200) lipid ratio. The Trp fluorescence decay is fitted with only two components (6.9-7.2 and 2.0-2.2 ns). Decay-associated spectra reveal that the longest lifetime of bound annexin V can be related to Trp residues (60%) located in a partially polar environment, which could correspond to the protein-membrane interface. The shortest lifetime is attributed to Trp residues (40%) which reside in a hydrophobic surroundingthese Trp residues would penetrate into the phospholipid membrane and contribute to the stabilization of the 2D-array of annexin V molecules.

    背景与目标: 游离膜联蛋白V的单个色氨酸残基Trp-187的荧光强度衰减由三个寿命成分(5.4、1.3和0.4 ns)的总和来描述,这三个成分可能与Trp构象异构体的三个基态类别相关。蛋白质基质中嵌入了两个主要类别(44%和48%)。当膜联蛋白V结合钙和由二油酰基磷脂酰胆碱和二油酰基磷脂酰丝氨酸制成的脂质体时,无论脂质比率为(10-200),都可获得相似的结果。 Trp荧光衰减仅适合两个分量(6.9-7.2和2.0-2.2 ns)。衰变相关光谱表明,结合的膜联蛋白V的最长寿命可能与位于部分极性环境中的Trp残基(60%)有关,这可能与蛋白质-膜界面相对应。最短的寿命归因于Trp残基(40%),该残基位于疏水的周围,这些Trp残基会渗入磷脂膜并有助于膜联蛋白V分子的2D阵列的稳定化。

  • 【二硝基苯胺与apicomplexan和动素体α-微管蛋白的结合和相互作用。】 复制标题 收藏 收藏
    DOI:10.1021/jm060472+ 复制DOI
    作者列表:Mitra A,Sept D
    BACKGROUND & AIMS: :Despite years of use as commercial herbicides, it is still unclear how dinitroanilines interact with tubulin, how they cause microtubule disassembly, and why they are selectively active against plant and protozoan tubulin. In this work, through a series of computational studies, a common binding site of oryzalin, trifluralin, and GB-II-5 on apicomplexan and kinetoplastid alpha-tubulin is proposed. Furthermore, to investigate how dinitroanilines affect tubulin dynamics, molecular dynamics simulations of Leishmania alpha-tubulin with and without a bound dinitroaniline are performed. The results obtained provide insight into the molecular mechanism by which these compounds interact with tubulin and function to prevent microtubule assembly. Finally, to aid in the design of effective parasitic microtubule inhibitors, several novel dinitroaniline analogues are evaluated. The location of the binding site and the relative binding affinities of the dinitroanilines all agree well with experimental data.
    背景与目标: 尽管多年用作商业除草剂,但仍不清楚二硝基苯胺如何与微管蛋白相互作用,它们如何引起微管分解以及为什么它们对植物和原生动物微管蛋白具有选择性活性。在这项工作中,通过一系列的计算研究,提出了米黄素,三氟拉林和GB-II-5在apicomplexan和运动质体α-微管蛋白上的共同结合位点。此外,为了研究二硝基苯胺如何影响微管蛋白动力学,进行了结合和不结合二硝基苯胺的利什曼原虫α-微管蛋白的分子动力学模拟。获得的结果提供了对这些化合物与微管蛋白相互作用并防止微管装配的分子机制的深入了解。最后,为帮助设计有效的寄生微管抑制剂,对几种新型的二硝基苯胺类似物进行了评估。二硝基苯胺的结合位点的位置和相对结合亲和力都与实验数据很好地吻合。
  • 【环氧二十碳三烯酸通过鸟嘌呤核苷酸结合蛋白激活冠状动脉平滑肌中的K通道。】 复制标题 收藏 收藏
    DOI:10.1161/01.res.80.6.877 复制DOI
    作者列表:Li PL,Campbell WB
    BACKGROUND & AIMS: Epoxyeicosatrienoic acids (EETs) are endothelium-derived arachidonic acid metabolites of cytochrome P450. They dilate coronary arteries, open K+ channels, and hyperpolarize vascular smooth muscles. However, the mechanisms of these smooth muscle actions remain unknown. This study examined the effects of EETs on the large-conductance Ca(2+)-activated K+ channel (KCa) in smooth muscle cells of small bovine coronary arteries. In cell-attached patch-clamp experiments, 11,12-EET produced a 0.5- to 10-fold increase in the activity of the KCa channels when added in concentrations of 1, 10, and 100 nmol/L. In the inside-out excised membrane patch mode, 11,12-EET was without effect on the activity of the KCa channel unless GTP (0.5 mmol/L) or GTP and ATP (1 mmol/L) were added to the bath solution. In the presence of GTP and ATP, the increase in the KCa channel activity with 11,12-EET in inside-out patches was comparable to that in cell-attached patches. This effect of 11,12-EET in inside-out patches was blocked by the addition of GDP-beta-S (100 mumol/L). In outside-out patches, 11,12-EET also increased the KCa channel activity when GTP and ATP were added to the pipette solution. The addition of a specific anti-Gs alpha antibody (100 nmol/L) in the pipette solution completely blocked the activation of the KCa channels induced by 11,12-EET. An anti-G beta gamma or anti-Gi alpha antibody was without effect. We conclude that 11,12-EET activates the KCa channels by a Gs alpha-mediated mechanism. This mechanism contributes to the effects of EETs as endothelium-derived hyperpolarizing factors to hyperpolarize and relax arterial smooth muscle.

    背景与目标: 环氧二十碳三烯酸(EET)是内皮细胞色素P450的花生四烯酸代谢产物。它们扩张冠状动脉,打开K通道,并使血管平滑肌超极化。但是,这些平滑肌动作的机制仍然未知。这项研究检查了EETs对小牛冠状动脉平滑肌细胞中大电导Ca(2)激活的K通道(KCa)的影响。在贴有细胞的膜片钳实验中,当以1,10和100 nmol / L的浓度添加时,11,12-EET使KCa通道的活性增加0.5至10倍。在由内而外的切膜模式下,除非将GTP(0.5 mmol / L)或GTP和ATP(1 mmol / L)添加到浴液中,否则11,12-EET对KCa通道的活性没有影响。在存在GTP和ATP的情况下,由内而外的贴片中11,12-EET的KCa通道活性的增加与细胞附着的贴片中的KCa通道活性的增加相当。通过添加GDP-β-S(100 mumol / L),阻止了由内而外的11,12-EET的这种作用。在外向斑块中,将GTP和ATP添加到移液器中时,11,12-EET也增加了KCa通道活性。在移液器中添加特异性抗Gsα抗体(100 nmol / L)完全阻断了11,12-EET诱导的KCa通道的激活。抗Gβγ或抗Giα抗体无效。我们得出的结论是11,12-EET通过Gs alpha介导的机制激活了KCa通道。这种机制有助于将EETs作为内皮源的超极化因子来使动脉平滑肌超极化和松弛。

  • 【谷氨酸脱羧酶65(GADA)抗体的测量:与[35S] GAD 65-配体结合测定相比,两种新的125I测定。】 复制标题 收藏 收藏
    DOI: 复制DOI
    作者列表:Borg H,Fernlund P,Sundkvist G
    BACKGROUND & AIMS: Recently, 65-kDa glutamic acid decarboxylase (GAD 65) antibodies (GADA) have been introduced as autoimmune markers in blood to confirm the diagnosis of insulin-dependent diabetes mellitus (IDDM). In this study, to evaluate two new assays that use 125I-labeled GAD 65, we assayed samples from 100 children with recent onset of diabetes and 100 control children, the results were compared with those of a [35S]GADA assay and with results for islet cell antibodies (ICA), the conventional autoimmune marker. Receiver operating characteristic (ROC) curve analysis showed one of the new assays (from RSR) to be more sensitive (P = 0.01) than the comparison ([35S]GADA) assay, whereas the second new assay (from Elias) was less sensitive (P < 0.001). The GADA frequency at 97.5% specificity was greatest in the comparison assay63 of 100 vs 41 of 100 (P < 0.01) and 53 of 100 (P = 0.16) in the RSR and Elias assays, respectively. Almost all GADA-positive patients had ICA, but one-third of the ICA-positive patients was GADA-negative. Accordingly, adding GADA analysis results to ICA testing increased the frequency of detection of autoimmune markers only slightly (from 81% to 85%). In conclusion, at 97.5% specificity the [35S]GADA assay seemed to be more efficient than the 125I assays, although the difference was significant only for the Elias 125I assay. Antigen-specific antibodies other than GADA may explain the difference in GADA and ICA frequencies.

    背景与目标: 最近,已经引入65 kDa的谷氨酸脱羧酶(GAD 65)抗体(GADA)作为血液中的自身免疫标记,以确认对胰岛素依赖型糖尿病(IDDM)的诊断。在这项研究中,为了评估使用125I标记的GAD 65的两种新检测方法,我们检测了100例最近患糖尿病的儿童和100例对照儿童的样品,将结果与[35S] GADA检测的结果进行了比较,并得出了胰岛细胞抗体(ICA),传统的自身免疫标记。接收者工作特征(ROC)曲线分析显示,一种新的测定法(来自RSR)的灵敏度(P = 0.01)比比较([35S] GADA)测定法灵敏,而第二种新的测定法(来自Elias)灵敏度低(P <0.001)。在RSR和Elias分析中,分别在100和41中的比较中63相对于100中的41(P <0.01)和100中的53(P = 0.16),在97.5%特异性下的GADA频率最大。几乎所有GADA阳性患者都患有ICA,但是ICA阳性患者中有三分之一是GADA阴性。因此,将GADA分析结果添加到ICA测试中,仅略微增加了自身免疫标记物的检测频率(从81%增至85%)。结论是,[35S] GADA分析的特异性为97.5%,似乎比125I分析更有效,尽管差异仅对Elias 125I分析有意义。 GADA以外的抗原特异性抗体可以解释GADA和ICA频率的差异。

  • 【鉴定和分离粘附性糖蛋白纤连蛋白的胶原结合片段。】 复制标题 收藏 收藏
    DOI:10.1073/pnas.76.3.1160 复制DOI
    作者列表:Hahn LH,Yamada KM
    BACKGROUND & AIMS: :We have identified and purified a polypeptide region containing the collagen-binding site of the adhesive glycoprotein fibronectin. Chicken cellular fibronectin isolated from cultured embryonic fibroblasts was permitted to bind to gelatin coupled to agarose beads and was then digested extensively with chymotrypsin. A prominent 40,000-dalton fragment of fibronectin consisting of a single polypeptide chain was detected by sodium dodecyl sulfate/polyacrylamide gel electrophoresis of material remaining bound to the gelatin-agarose. This fragment appeared within 10 min after the digestion was initiated and persisted for more than 20 hr. This proteolytic fragment was isolated in electrophoretically pure form and retained its affinity for collagen. Plasma fibronectins from chicken and human blood also contained collagen-binding proteolytic fragments of similar size. This finding suggest that the collagen-binding sites of cellular and plasma fibronectins are homologous.
    背景与目标: :我们已经鉴定并纯化了包含粘附性糖蛋白纤连蛋白的胶原蛋白结合位点的多肽区域。从培养的胚胎成纤维细胞中分离出的鸡细胞纤连蛋白被允许结合到与琼脂糖珠上偶联的明胶上,然后用胰凝乳蛋白酶进行广泛消化。通过十二烷基硫酸钠/聚丙烯酰胺凝胶电泳,检测仍与明胶-琼脂糖结合的物质,检测到由单个多肽链组成的纤连蛋白突出的40,000道尔顿片段。该片段在开始消化后的10分钟内出现,并持续超过20小时。该蛋白水解片段以电泳纯的形式分离,并保留了其对胶原蛋白的亲和力。来自鸡和人血的血浆纤连蛋白也含有相似大小的胶原结合蛋白水解片段。该发现表明细胞和血浆纤连蛋白的胶原结合位点是同源的。
  • 【勘误表“生物碱与DNA的结合:光物理和量热法” [J.光化学。感光油。 B:生物学。 130(2014)272-280]。】 复制标题 收藏 收藏
    DOI:10.1016/j.jphotobiol.2015.03.003 复制DOI
    作者列表:Sarkar S,Bhadra K
    BACKGROUND & AIMS: -2
    背景与目标: -2
  • 【有效的,选择性的和口服活性的基于蒽酰胺的Xa因子抑制剂的合成及其构效关系:弱碱性磺胺嘧啶基团作为新型S4结合元素的应用。】 复制标题 收藏 收藏
    DOI:10.1016/j.ejmech.2012.10.005 复制DOI
    作者列表:Pandya V,Jain M,Chakrabarti G,Soni H,Parmar B,Chaugule B,Patel J,Jarag T,Joshi J,Joshi N,Rath A,Unadkat V,Sharma B,Ajani H,Kumar J,Sairam KV,Patel H,Patel P
    BACKGROUND & AIMS: :A novel series of potent and efficacious factor Xa inhibitors which possesses sulfoximine moiety as novel S4 binding element in anthranilamide chemotype has been identified. Lead optimization at this novel P4 group led to many potent factor Xa inhibitors with excellent anticoagulant activity in human plasma. Selected compounds were dosed orally in rats and checked for their ex vivo prothrombin time prolonging activity, which resulted in identification of compound 5-chloro-N-(5-chloropyridin-2-yl)-2-(4-(N-(2-(diethylamino)acetyl)-S-methylsulfonimidoyl)benzamido)benzamide (18f). The detailed pharmacokinetic evaluation and subsequent metabolism study of 18f suggested the presence of an active metabolite. The compound 18f and its active metabolite 18b demonstrated excellent in vivo efficacy in both arterial and venous thrombosis model in rats and were found to be highly selective against related serine proteases. Based on this promising profile, compound 18f was selected for further evaluation.
    背景与目标: :已经鉴定了一系列有效且有效的因子Xa抑制剂,其具有硫肟亚胺部分作为邻氨基苯甲酰胺化学型中的新的S4结合元件。在这个新颖的P4组中进行的前导优化导致了许多有效的Xa抑制剂在人血浆中具有出色的抗凝活性。在大鼠中口服选择的化合物并检查其离体凝血酶原时间延长活性,从而鉴定出化合物5-氯-N-(5-氯吡啶-2-基)-2-(4-(N-(2 -(二乙氨基)乙酰基)-S-甲基磺酰亚胺基)苯甲酰胺基)苯甲酰胺(18f)。 18f的详细药代动力学评估和随后的代谢研究表明存在活性代谢物。化合物18f及其活性代谢物18b在大鼠的动脉和静脉血栓形成模型中均显示出优异的体内功效,并且被发现对相关的丝氨酸蛋白酶具有高度选择性。基于这一有前途的概况,选择了化合物18f进行进一步评估。

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