多子小瓜虫感染后草鱼TLR信号通路基因 的表达动态

[目的]明确草鱼Toll样受体(Toll-like receptor,TLR)信号通路基因在防御多子小瓜虫(Ichthyophthirius multifiliis)感染过程中的免疫作用,为有效防控鱼类多子小瓜虫感染提供理论参考.[方法]以多子小瓜虫感染草鱼后,采用实时荧光定量PCR检测分析在不同时间点(感染后6 h、12 h、1 d、2 d、3 d、5 d和7 d)草鱼部分TLRs基因(TLR1、TLR2、TLR4、TLR9、TLR20和TLR21)、接头蛋白基因(MyD88和TRIF)、信号转导分子(IRAK4和IRAK1)及细胞因子(IL-1β、TNF-α和IFN)在其皮肤和脾脏中的表达动态变化.[结果]草鱼感染多子小瓜虫后,TLR1、TLR2、TLR9、TLR20和TLR21基因在皮肤和脾脏中的表达主要呈上调趋势,TLR4基因在皮肤中主要呈上调表达,在脾脏中则主要呈下调表达;TLR信号通路接头蛋白基因MyD88和TRIF的表达变化趋势明显不同,其中,MyD88基因的表达在感染后第1~3 d均呈显著上调趋势(P<0.05,下同),而TRIF基因的表达在整个试验过程中绝大多数时间点与对照组无显著差异(P>0.05);IRAK4和IRAK1在皮肤和脾脏中的表达也主要呈上调趋势,但在脾脏中IRAK4在感染后第1 d和IRAK1在感染后第6 h的表达呈显著下调趋势;IL-1β和TNF-α在绝大多数时间点均显著上调,但IFN的表达基本没有变化.[结论]草鱼TLR信号通路中的部分TLRs基因(TL1、TLR2、TLR4、TLR9、TLR20和TLR21)、接头蛋白基因(MyD88)、信号转导分子(IRAK4和IRAK1)及下游细胞因子(IL-1β和TNF-α)均参与防御多子小瓜虫感染的免疫反应,尤其是在感染早期和中期发挥关键作用.     

Structural basis of toll-like receptor 3 signaling with double-stranded RNA

Toll-like receptor 3 (TLR3) recognizes double-stranded RNA (dsRNA), a molecular signature of most viruses, and triggers inflammatory responses that prevent viral spread. TLR3 ectodomains (ECDs) dimerize on oligonucleotides of at least 40 to 50 base pairs in length, the minimal length required for signal transduction. To establish the molecular basis for ligand binding and signaling, we determined the crystal structure of a complex between two mouse TLR3-ECDs and dsRNA at 3.4 angstrom resolution. Each TLR3-ECD binds dsRNA at two sites located at opposite ends of the TLR3 horseshoe, and an intermolecular contact between the two TLR3-ECD C-terminal domains coordinates and stabilizes the dimer. This juxtaposition could mediate downstream signaling by dimerizing the cytoplasmic Toll interleukin-1 receptor (TIR) domains. The overall shape of the TLR3-ECD does not change upon binding to dsRNA.     

Crystal structure of human enterovirus 71

Enterovirus 71 is a picornavirus associated with fatal neurological illness in infants and young children. Here, we report the crystal structure of enterovirus 71 and show that, unlike in other enteroviruses, the "pocket factor," a small molecule that stabilizes the virus, is partly exposed on the floor of the "canyon." Thus, the structure of antiviral compounds may require a hydrophilic head group designed to interact with residues at the entrance of the pocket.     

Crystal structure of a lipid G protein-coupled receptor

The lyso-phospholipid sphingosine 1-phosphate modulates lymphocyte trafficking, endothelial development and integrity, heart rate, and vascular tone and maturation by activating G protein-coupled sphingosine 1-phosphate receptors. Here, we present the crystal structure of the sphingosine 1-phosphate receptor 1 fused to T4-lysozyme (S1P(1)-T4L) in complex with an antagonist sphingolipid mimic. Extracellular access to the binding pocket is occluded by the amino terminus and extracellular loops of the receptor. Access is gained by ligands entering laterally between helices I and VII within the transmembrane region of the receptor. This structure, along with mutagenesis, agonist structure-activity relationship data, and modeling, provides a detailed view of the molecular recognition and requirement for hydrophobic volume that activates S1P(1), resulting in the modulation of immune and stromal cell responses.     

Crystal structure of rhodopsin: A G protein-coupled receptor

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) respond to a variety of different external stimuli and activate G proteins. GPCRs share many structural features, including a bundle of seven transmembrane alpha helices connected by six loops of varying lengths. We determined the structure of rhodopsin from diffraction data extending to 2.8 angstroms resolution. The highly organized structure in the extracellular region, including a conserved disulfide bridge, forms a basis for the arrangement of the seven-helix transmembrane motif. The ground-state chromophore, 11-cis-retinal, holds the transmembrane region of the protein in the inactive conformation. Interactions of the chromophore with a cluster of key residues determine the wavelength of the maximum absorption. Changes in these interactions among rhodopsins facilitate color discrimination. Identification of a set of residues that mediate interactions between the transmembrane helices and the cytoplasmic surface, where G-protein activation occurs, also suggests a possible structural change upon photoactivation.     

Crystal structure of inhibitor-bound human 5-lipoxygenase-activating protein

Leukotrienes are proinflammatory products of arachidonic acid oxidation by 5-lipoxygenase that have been shown to be involved in respiratory and cardiovascular diseases. The integral membrane protein FLAP is essential for leukotriene biosynthesis. We describe the x-ray crystal structures of human FLAP in complex with two leukotriene biosynthesis inhibitors at 4.0 and 4.2 angstrom resolution, respectively. The structures show that inhibitors bind in membrane-embedded pockets of FLAP, which suggests how these inhibitors prevent arachidonic acid from binding to FLAP and subsequently being transferred to 5-lipoxygenase, thereby preventing leukotriene biosynthesis. This structural information provides a platform for the development of therapeutics for respiratory and cardiovascular diseases.     

Crystal structure analysis of deamino-oxytocin: conformational flexibility and receptor binding

Two crystal structures of deamino-oxytocin have been determined at better than 1.1A resolution from isomorphous replacement and anomalous scattering x-ray measurements. In each of two crystal forms there are two closely related conformers with disulfide bridges of different chirality, which may be important in receptor recognition and activation.     

Crystal structure of a naturally occurring dinucleoside monophosphate: uridylyl (3',5') adenosine hemihydrate

The crystal structure of uridylyl (3',5') adenosine hemihydrate has been analyzed by x-ray diffraction. The two independent molecules found in the asymmetric unit exhibit conformations that differ significantly from those found in double-helical RNA. The conformational information obtained from this analysis provides considerable insight into the possible conformations of nonhelical "loop" regions of transfer RNA, as well as single-stranded regions of nucleic acids in general.     

Adjuvant-enhanced antibody responses in the absence of toll-like receptor signaling

Innate immune signals mediated by Toll-like receptors (TLRs) have been thought to contribute considerably to the antibody-enhancing effects of vaccine adjuvants. However, we report here that mice deficient in the critical signaling components for TLR mount robust antibody responses to T cell-dependent antigen given in four typical adjuvants: alum, Freund's complete adjuvant, Freund's incomplete adjuvant, and monophosphoryl-lipid A/trehalose dicorynomycolate adjuvant. We conclude that TLR signaling does not account for the action of classical adjuvants and does not fully explain the action of a strong adjuvant containing a TLR ligand. This may have important implications in the use and development of vaccine adjuvants.     

Crystal Structure of the Polymer Electrolyte Poly(ethylene oxide)3:LiCF3SO3

Ionically conducting polymers (polymer electrolytes) are under intensive investigation because they form the basis of all solid-state lithium batteries, fuel cells, and electrochromic display devices, as well as being highly novel electrolytes. Little is known about the structures of the many crystalline complexes that form between poly(ethylene oxide) and a wide range of salts. The crystal structure is reported of the archetypal polymer electrolyte poly(ethylene oxide)(3):LiCF(3)SO(3), which has been determined from powder x-ray diffraction data. The poly(ethylene oxide) (PEO) chain adopts a helical conformation parallel to the crystallographic b axis. The Li(+) cation is coordinated by five oxygen atoms-three ether oxygens and one from each of two adjacent CF(3)SO(3)(-) groups. Each CF(3)SO(3)(-) in turn bridges two Li(+) ions to form chains running parallel to and intertwined with the PEO chain. There are no interchain links between PEO chains, and the electrolyte can be regarded as an infinite columnar coordination complex.     

Crystal structure of the extracellular segment of integrin alpha Vbeta3

Integrins are alphabeta heterodimeric receptors that mediate divalent cation-dependent cell-cell and cell-matrix adhesion through tightly regulated interactions with ligands. We have solved the crystal structure of the extracellular portion of integrin alphaVbeta3 at 3.1 A resolution. Its 12 domains assemble into an ovoid "head" and two "tails." In the crystal, alphaVbeta3 is severely bent at a defined region in its tails, reflecting an unusual flexibility that may be linked to integrin regulation. The main inter-subunit interface lies within the head, between a seven-bladed beta-propeller from alphaV and an A domain from beta3, and bears a striking resemblance to the Galpha/Gbeta interface in G proteins. A metal ion-dependent adhesion site (MIDAS) in the betaA domain is positioned to participate in a ligand-binding interface formed of loops from the propeller and betaA domains. MIDAS lies adjacent to a calcium-binding site with a potential regulatory function.     

Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8

Double-stranded ribonucleic acid (dsRNA) serves as a danger signal associated with viral infection and leads to stimulation of innate immune cells. In contrast, the immunostimulatory potential of single-stranded RNA (ssRNA) is poorly understood and innate immune receptors for ssRNA are unknown. We report that guanosine (G)- and uridine (U)-rich ssRNA oligonucleotides derived from human immunodeficiency virus-1 (HIV-1) stimulate dendritic cells (DC) and macrophages to secrete interferon-alpha and proinflammatory, as well as regulatory, cytokines. By using Toll-like receptor (TLR)-deficient mice and genetic complementation, we show that murine TLR7 and human TLR8 mediate species-specific recognition of GU-rich ssRNA. These data suggest that ssRNA represents a physiological ligand for TLR7 and TLR8.     

Primary structure of the human chorionic somatomammotropin (HCS) molecule

The complete amino acid sequence of the human chorionic somatomammotropin molecule been proposed; and then compared with that of human growth hormone and ovine lactogenic hormone.     

Crystal structure of a complex between the catalytic and regulatory (RIalpha) subunits of PKA

The 2.0-angstrom structure of the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) catalytic subunit bound to a deletion mutant of a regulatory subunit (RIalpha) defines a previously unidentified extended interface. The complex provides a molecular mechanism for inhibition of PKA and suggests how cAMP binding leads to activation. The interface defines the large lobe of the catalytic subunit as a stable scaffold where Tyr247 in the G helix and Trp196 in the phosphorylated activation loop serve as anchor points for binding RIalpha. These residues compete with cAMP for the phosphate binding cassette in RIalpha. In contrast to the catalytic subunit, RIalpha undergoes major conformational changes when the complex is compared with cAMP-bound RIalpha. The inhibitor sequence docks to the active site, whereas the linker, also disordered in free RIalpha, folds across the extended interface. The beta barrel of cAMP binding domain A, which is the docking site for cAMP, remains largely intact in the complex, whereas the helical subdomain undergoes major reorganization.     

A crystal structure of the complex between human complement receptor 2 and its ligand C3d

The interaction of complement receptor 2 (CR2)--which is present on B cells and follicular dendritic cells--with its antigen-bound ligand C3d results in an enhanced antibody response, thus providing an important link between the innate and adaptive immune systems. Although a cocrystal structure of a complex between C3d and the ligand-binding domains of CR2 has been published, several aspects of this structure, including the position in C3d of the binding interface, remained controversial because of disagreement with biochemical data. We now report a cocrystal structure of a CR2(SCR1-2):C3d complex at 3.2 angstrom resolution in which the interaction interfaces differ markedly from the previously published structure and are consistent with the biochemical data. It is likely that, in the previous structure, the interaction was influenced by the presence of zinc acetate additive in the crystallization buffer, leading to a nonphysiological complex. Detailed knowledge of the binding interface now at hand gives the potential to exploit the interaction in vaccine design or in therapeutics directed against autoreactive B cells.     

Crystal and molecular structure of adenosine 3',5'-cyclic phosphate

The structure of adenosine 3',5'-cyclic phosphate has been determined by single-crystal x-ray diffraction. The two molecules in the asymmetric unit show different conformation about the glycosidic bond, while other structural details are essentially the same. The furanose rings are puckered with the C(4') atom out of the best four-atom plane. The bond lengths and angles appear to be normal.     

凡纳滨对虾Toll样受体基因cDNA片段的克隆及序列分析

对凡纳滨对虾(Litopenaeus vannamei)Toll样受体基因的cDNA片段进行了克隆.从凡纳滨对虾提取肌肉、鳃和肝胰腺中的总RNA,根据黑腹果蝇(Drosophila melanogaster)Toll样受体的TIR区设计引物,从鳃中逆转录扩增得到cDNA序列,进行测序.所得序列结果与其他物种的已知TIR及TLR氨基酸序列进行聚类分析建立系统进化树,并进行氨基酸序列同源性比较.结果表明所得序列与斑节对虾(Penaeus monodon)、赤拟谷盗(Tribolium castaneum)、果蝇(Drosophila melanogaster)、意大利蜜蜂(Apis mellifera)、线虫(Caenorhabditiselegans)、紫海胆(Strongylocentrotus purpuratus)的Toll样基因TIR区域的氨基酸序列有较高的相似性.     

Crystal structure of the human two-pore domain potassium channel K2P1

Two-pore domain potassium (K(+)) channels (K2P channels) control the negative resting potential of eukaryotic cells and regulate cell excitability by conducting K(+) ions across the plasma membrane. Here, we present the 3.4 angstrom resolution crystal structure of a human K2P channel, K2P1 (TWIK-1). Unlike other K(+) channel structures, K2P1 is dimeric. An extracellular cap domain located above the selectivity filter forms an ion pathway in which K(+) ions flow through side portals. Openings within the transmembrane region expose the pore to the lipid bilayer and are filled with electron density attributable to alkyl chains. An interfacial helix appears structurally poised to affect gating. The structure lays a foundation to further investigate how K2P channels are regulated by diverse stimuli.     

Crystal structure of human ZAG, a fat-depleting factor related to MHC molecules

Zn-alpha2-glycoprotein (ZAG) is a soluble protein that is present in serum and other body fluids. ZAG stimulates lipid degradation in adipocytes and causes the extensive fat losses associated with some advanced cancers. The 2.8 angstrom crystal structure of ZAG resembles a class I major histocompatibility complex (MHC) heavy chain, but ZAG does not bind the class I light chain beta2-microglobulin. The ZAG structure includes a large groove analogous to class I MHC peptide binding grooves. Instead of a peptide, the ZAG groove contains a nonpeptidic compound that may be implicated in lipid catabolism under normal or pathological conditions.     

凡纳滨对虾Toll样受体基因cDNA片段的克隆及序列分析

对凡纳滨对虾(Litopenaeus vannamei)Toll样受体基因的cDNA片段进行了克隆.从凡纳滨对虾提取肌肉、鳃和肝胰腺中的总RNA,根据黑腹果蝇(Drosophila melanogaster)Toll样受体的TIR区设计引物,从鳃中逆转录扩增得到cDNA序列,进行测序.所得序列结果与其他物种的已知TIR及TLR氨基酸序列进行聚类分析建立系统进化树,并进行氨基酸序列同源性比较.结果表明所得序列与斑节对虾(Penaeus monodon)、赤拟谷盗(Tribolium castaneum)、果蝇(Drosophila melanogaster)、意大利蜜蜂(Apis mellifera)、线虫(Caenorhabditiselegans)、紫海胆(Strongylocentrotus purpuratus)的Toll样基因TIR区域的氨基酸序列有较高的相似性.