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71.
tRNA anticodon damage inflicted by the Kluyveromyces lactis γ-toxin underlies an RNA-based innate immune system that distinguishes self from nonself species. γ-toxin arrests the growth of Saccharomyces cerevisiae by incising a single phosphodiester 3' of the wobble base of tRNA(Glu(UUC)) to generate a break with 2',3'-cyclic phosphate and 5'-OH ends. Recombinant γ-toxin cleaves oligonucleotide substrates in vitro that mimic the anticodon stem-loop of tRNA(Glu). A single 2'-deoxy sugar substitution at the wobble nucleoside abolishes anticodon nuclease activity. To gain further insights to γ-toxin's substrate specificity, we tested deoxynucleoside effects at positions other than the site of transesterification. The results attest to a stringent requirement for a ribonucleoside at the uridine 5' of the wobble base. In contrast, every other nonwobble ribonucleoside in the anticodon loop can be replaced by a deoxy without significantly affecting γ-toxin's cleavage activity. Whereas either the 5' half or the 3' half of the anticodon stem can be replaced en bloc with DNA without a major effect, simultaneously replacing both strands with DNA interfered strongly, signifying that γ-toxin requires an A-form helical conformation of the anticodon stem. We purified γ-toxin mutants identified previously as nontoxic in vivo and gauged their anticodon nuclease activities in vitro. The results highlight Glu9 and Arg151 as candidate catalytic residues, along with His209 implicated previously. By analogy to other endoribonucleases, we speculate that γ-toxin drives transesterification by general acid-base catalysis (via His209 and Glu9) and transition-state stabilization (via Arg151). 相似文献
72.
Induction of long-term protective antiviral endogenous immune response by short neutralizing monoclonal antibody treatment
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Long-term immune control of viral replication still remains a major challenge in retroviral diseases. Several monoclonal antibodies (MAbs) have already shown antiviral activities in vivo, including in the clinic but their effects on the immune system of treated individuals are essentially unknown. Using the lethal neurodegeneration induced in mice upon infection of neonates by the FrCas(E) retrovirus as a model, we report here that transient treatment by a neutralizing MAb shortly after infection can, after an immediate antiviral effect, favor the development of a strong protective host immune response containing viral propagation long after the MAb has disappeared. In vitro virus neutralization- and complement-mediated cell lysis assays, as well as in vivo viral challenges and serum transfer experiments, indicate a clear and essential contribution of the humoral response to antiviral protection. Our observation may have important therapeutic consequences as it suggests that short antibody-based therapies early after infection should be considered, at least in the case of maternally infected infants, as adjunctive treatment strategies against human immunodeficiency virus, not only for a direct effect on the viral load but also for favoring the emergence of an endogenous antiviral immune response. 相似文献
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75.
Naip5/Birc1e and susceptibility to Legionella pneumophila 总被引:6,自引:0,他引:6
Genetic analysis in mice is a powerful approach for the identification of genes and proteins that have a key role at the interface of the host-pathogen interaction. The Lgn1 locus has been found to control the intracellular replication of Legionella pneumophila in murine macrophages. Using functional complementation in transgenic mice, the Naip5/Birc1e gene has been identified as responsible for the Lgn1 effect. The classification of Naip5/Birc1e as a member of the NLR protein family suggests that Naip5/Birc1e acts as an intracellular sensor of L. pneumophila. The nature of the signal transduced by Naip5/Birc1e in response to Legionella products is of great interest but is currently unknown. Here, several possible scenarios are presented. 相似文献
76.
Apuzzo S Abdelhakim A Fortin AS Gros P 《The Journal of biological chemistry》2004,279(32):33601-33612
The Pax3 protein has two DNA binding domains, a Paired domain (PD) and a paired-type Homeo domain (HD). Although the PD and HD can bind to cognate DNA sequences when expressed individually, genetic and biochemical data indicate that the two domains are functionally interdependent in intact Pax3. The mechanistic basis of this functional interdependence is unknown and was studied by protease sensitivity. Pax3 was modified by the creation of Factor Xa cleavage sites at discrete locations in the PD, the HD, and in the linker segment joining the PD and the HD (Xa172, Xa189, and Xa216) in individual Pax3 mutants. The effect of Factor Xa insertions on protein stability and on DNA binding by the PD and the HD was measured using specific target site sequences. Independent insertions at position 100 in the linker separating the first from the second helix-turn-helix motif of the PD and at position 216 immediately upstream of the HD were found to be readily accessible to Factor Xa cleavage. The effect of DNA binding by the PD or the HD on accessibility of Factor Xa sites inserted in the same or in the other domain was monitored and quantitated for multiple mutants bearing different numbers of Xa sites at each position. In general, DNA binding reduced accessibility of all sites, suggesting a more compact and less solvent-exposed structure of DNA-bound versus DNA-free Pax3. Results of dose response and time course experiments were consistent and showed that DNA binding by the PD not only caused a local structural change in the PD but also caused a conformational change in the HD (P3OPT binding to Xa216 mutants); similarly, DNA binding by the HD also caused a conformational change in the PD (P2 binding to Xa100 mutants). These results provide a structural basis for the functional interdependence of the two DNA binding domains of Pax3. 相似文献
77.
Gros L Maksimenko AV Privezentzev CV Laval J Saparbaev MK 《The Journal of biological chemistry》2004,279(17):17723-17730
Lipid peroxidation generates aldehydes, which react with DNA bases, forming genotoxic exocyclic etheno(epsilon)-adducts. E-bases have been implicated in vinyl chloride-induced carcinogenesis, and increased levels of these DNA lesions formed by endogenous processes are found in human degenerative disorders. E-adducts are repaired by the base excision repair pathway. Here, we report the efficient biological hijacking of the human alkyl-N-purine-DNA glycosylase (ANPG) by 3,N(4)-ethenocytosine (epsilonC) when present in DNA. Unlike the ethenopurines, ANPG does not excise, but binds to epsilonC when present in either double-stranded or single-stranded DNA. We developed a direct assay, based on the fluorescence quenching mechanism of molecular beacons, to measure a DNA glycosylase activity. Molecular beacons containing modified residues have been used to demonstrate that the epsilonC.ANPG interaction inhibits excision repair both in reconstituted systems and in cultured human cells. Furthermore, we show that the epsilonC.ANPG complex blocks primer extension by the Klenow fragment of DNA polymerase I. These results suggest that epsilonC could be more genotoxic than 1,N(6)-ethenoadenine (epsilonA) residues in vivo. The proposed model of ANPG-mediated genotoxicity of epsilonC provides a new insight in the molecular basis of lipid peroxidation-induced cell death and genome instability in cancer. 相似文献
78.
Mammalian Vangl1 and Vangl2 are highly conserved membrane proteins that have evolved from a single ancestral protein Strabismus/Van Gogh found in Drosophila. Mutations in the Vangl2 gene cause a neural tube defect (craniorachischisis) characteristic of the looptail (Lp) mouse. Studies in model organisms indicate that Vangl proteins play a key developmental role in establishing planar cell polarity (PCP) and in regulating convergent extension (CE) movements during embryogenesis. The role of Vangl1 in these processes is virtually unknown, and the molecular function of Vangl1 and Vangl2 in PCP and CE is poorly understood. Using a yeast two-hybrid system, glutathione S-transferase pull-down and co-immunoprecipitation assays, we show that both mouse Vangl1 and Vangl2 physically interact with the three members of the cytoplasmic Dishevelled (Dvl) protein family. This interaction is shown to require both the predicted cytoplasmic C-terminal half of Vangl1/2 and a portion of the Dvl protein containing PDZ and DIX domains. In addition, we show that the two known Vangl2 loss-of-function mutations identified in two independent Lp alleles associated with neural tube defects impair binding to Dvl1, Dvl2, and Dvl3. These findings suggest a molecular mechanism for the neural tube defect seen in Lp mice. Our observations indicate that Vangl1 biochemical properties parallel those of Vangl2 and that Vangl1 might, therefore, participate in PCP and CE either in concert with Vangl2 or independently of Vangl2 in discrete cell types. 相似文献
79.
New metallopolymers were generated efficiently on Pt and ITO (indium-tin oxide) glass slides by repeated scan oxidation of Ru and Fe complexes containing pyrrolostyryl bipyridine ligands. The UV-Vis spectrum and voltammogram of Ru-based polymer films showed blue-shifts of the MLCT absorption bands and increased oxidation potentials indicating conjugation cut in the π-extended system. In contrast, formation of polypyrrole occurred in Fe-based polymers due to oxidation of pyrrole groups by the initially oxidised metal centres. The coated ITO electrodes displayed promising wide absorption domains for use in light conversion devices. 相似文献
80.
Renate J Scheibe Gerolf Gros Seppo Parkkila Abdul Waheed Jeffrey H Grubb Gul N Shah William S Sly Petra Wetzel 《The journal of histochemistry and cytochemistry》2006,54(12):1379-1391
Expression of membrane-bound carbonic anhydrases (CAs) of CA IV, CA IX, CA XII, and CA XIV has been investigated in the mouse heart. Western blots using microsomal membranes of wild-type hearts demonstrate a 39-, 43-, and 54-kDa band representing CA IV, CA IX, and CA XIV, respectively, but CA XII could not be detected. Expression of CA IX in the CA IV/CA XIV knockout animals was further confirmed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Cardiac cells were immunostained using anti-CA/FITC and anti-alpha-actinin/TRITC, as well as anti-CA/FITC and anti-SERCA2/TRITC. Subcellular CA localization was investigated by confocal laser scanning microscopy. CA localization in the sarcolemmal (SL) membrane was examined by double immunostaining using anti-CA/FITC and anti-MCT-1/TRITC. CAs showed a distinct distribution pattern in the sarcoplasmic reticulum (SR) membrane. CA XIV is predominantly localized in the longitudinal SR, whereas CA IX is mainly expressed in the terminal SR/t-tubular region. CA IV is present in both SR regions, whereas CA XII is not found in the SR. In the SL membrane, only CA IV and CA XIV are present. We conclude that CA IV and CA XIV are associated with the SR as well as with the SL membrane, CA IX is located in the terminal SR/t-tubular region, and CA XII is not present in the mouse heart. Therefore, the unique subcellular localization of CA IX and CA XIV in cardiac myocytes suggests different functions of both enzymes in excitation-contraction coupling. 相似文献