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71.
Leimkuhler S Freuer A Araujo JA Rajagopalan KV Mendel RR 《The Journal of biological chemistry》2003,278(28):26127-26134
Biosynthesis of the molybdenum cofactor involves the initial formation of precursor Z, its subsequent conversion to molybdopterin (MPT) by MPT synthase, and attachment of molybdenum to the dithiolene moiety of MPT. The sulfur used for the formation of the dithiolene group of MPT exists in the form of a thiocarboxylate group at the C terminus of the smaller subunit of MPT synthase. Human MPT synthase contains the MOCS2A and MOCS2B proteins that display homology to the Escherichia coli proteins MoaD and MoaE, respectively. MOCS2A and MOCS2B were purified after heterologous expression in E. coli, and the separately purified subunits readily assemble into a functional MPT synthase tetramer. The rate of conversion of precursor Z to MPT by the human enzyme is slower than that of the eubacterial homologue. To obtain insights into the molecular mechanism leading to human molybdenum cofactor deficiency, site-specific mutations identified in patients showing symptoms of molybdenum cofactor deficiency were generated. Characterization of a V7F substitution in MOCS2A, identified in a patient with an unusual mild form of the disease, showed that the mutation weakens the interaction between MOCS2A and MOCS2B, whereas a MOCS2B-E168K mutation identified in a severely affected patient attenuates binding of precursor Z. 相似文献
72.
Hawkins CJ Silke J Verhagen AM Foster R Ekert PG Ashley DM 《Apoptosis : an international journal on programmed cell death》2001,6(5):331-338
We have reconstituted the Apaf-1-activated apoptosis mechanism in Sacchromyces cerevisiae such that the presence of a constitutively active form of Apaf-1 together with both Caspase-9 and Caspase-3 results in yeast death. This system is a good model of the Apaf-1-activated pathway in mammalian cells: MIHA (XIAP/hILP), and to a lesser degree MIHB (c-IAP1/HIAP2) and MIHC (c-IAP-2/HIAP1) can inhibit caspases in this system, and protection by IAPs (inhibitor of apoptosis) can be abrogated by coexpression of the Drosophila pro-apoptotic proteins HID and GRIM or the mammalian protein DIABLO/Smac. Using this system we demonstrate that unlike DIABLO/Smac, other proteins which interact with mammalian IAPs (TAB-1, Zap-1, Traf-1 and Traf-2) do not act to antagonise IAP- mediated caspase inhibition. 相似文献
73.
74.
Laurent-Matha V Lucas A Huttler S Sandhoff K Garcia M Rochefort H 《Experimental cell research》2002,277(2):210-219
The cell surface binding, endocytosis, and lysosomal routing of procathepsin D (procath-D) in cancer cells are mostly independent of the mannose-6-phosphate (M6P) receptors. In an attempt to define the receptor involved, we intracellularly cross-linked procath-D with a 68-kDa protein that we identified with specific antibodies as prosaposin in human breast and ovarian cancer cell lines. In cancer cells, this protein-protein interaction was resistant to ammonium chloride or M6P treatment, indicating that it was independent of the M6P receptors. A similar interaction also occurred in the breast cancer cell culture medium between the secreted prosaposin and procath-D. Since these two precursors can be endocytosed, we then determined whether they were interacting with the same cell surface receptor. In fibroblasts, we confirmed that the endocytosis of these two proteins was different since it was generally mediated by the M6P receptors for procath-D and mostly by LRP (LDL receptor-related protein) for prosaposin. In breast cancer cells, prosaposin endocytosis was not detected, in contrast to procath-D endocytosis, suggesting that the majority of procath-D is not internalized as a complex with prosaposin. Moreover, RAP (receptor-associated protein), a ligand inhibiting LRP-mediated endocytosis, prevented internalization of prosaposin in 49-F rat fibroblasts, but did not affect procath-D M6P-independent internalization in MDA-MB231 cells. We conclude that in breast cancer cells, even though procath-D interacts intracellularly and extracellarly with prosaposin, it is endocytosed independent of prosaposin by a receptor different from the M6P receptors and the LRP. 相似文献
75.
Engineered CD4- and CXCR4-using simian immunodeficiency virus from African green monkeys is neutralization sensitive and replicates in nonstimulated lymphocytes 下载免费PDF全文
König RR Flory E Steidl S Neumann J Coulibaly C Holznagel E Holzammer S Norley S Cichutek K 《Journal of virology》2002,76(21):10627-10636
During human immunodeficiency virus type 1 (HIV-1) infection, disease progression correlates with the occurrence of variants using the coreceptor CXCR4 for cell entry. In contrast, apathogenic simian immunodeficiency virus (SIV) from African green monkeys (SIVagm), specifically the molecular virus clone SIVagm3mc, uses CCR5, Bob, and Bonzo as coreceptors throughout the course of infection. The influence of an altered coreceptor usage on SIVagm3mc replication was studied in vitro and in vivo. The putative coreceptor binding domain, the V3 region of the surface envelope (SU) glycoprotein, was replaced by the V3 loop of a CD4- and CXCR4-tropic HIV-1 strain. The resulting virus, termed SIVagm3-X4mc, exclusively used CD4 and CXCR4 for cell entry. Consequently, its in vitro replication was inhibited by SDF-1, the natural ligand of CXCR4. Surprisingly, SIVagm3-X4mc was able to replicate in vitro not only in interleukin-2- and phytohemagglutinin-stimulated but also in nonstimulated peripheral blood mononuclear cells (PBMCs) from nonhuman primates. After experimental infection of two pig-tailed macaques with either SIVagm3-X4mc or SIVagm3mc, the coreceptor usage was maintained during in vivo replication. Cell-associated and plasma viral loads, as well as viral DNA copy numbers, were found to be comparable between SIVagm3mc and SIVagm 3-X4mc infections, and no pathological changes were observed up to 14 months postinfection. Interestingly, the V3 loop exchange rendered SIVagm3-X4mc susceptible to neutralizing antibodies present in the sera of SIVagm3-X4mc- and SIVagm3mc-infected pig-tailed macaques. Our study describes for the first time a successful exchange of a V3 loop in nonpathogenic SIVagm resulting in CD4 and CXCR4 usage and modulation of virus replication in nonstimulated PBMCs as well as sensitivity toward neutralization. 相似文献
76.
Structural investigation of the binding of a herpesviral protein to the SH3 domain of tyrosine kinase Lck 总被引:4,自引:0,他引:4
Schweimer K Hoffmann S Bauer F Friedrich U Kardinal C Feller SM Biesinger B Sticht H 《Biochemistry》2002,41(16):5120-5130
Herpesvirus saimiri codes for a tyrosine kinase interacting protein (Tip) that interacts with both the SH3 domain and the kinase domain of the T-cell-specific tyrosine kinase Lck via two separate motifs. The activation of Lck by Tip is considered as a key event in the transformation of human T-lymphocytes during herpesviral infection. We investigated the interaction of proline-rich Tip peptides with the LckSH3 domain starting with the structural characterization of the unbound interaction partners. The solution structure of the LckSH3 was determined by heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy using 44 residual dipolar couplings in addition to the conventional experimental restraints. Circular dichroism spectroscopy proved that the polyproline helix of Tip is already formed prior to SH3 binding and is conformationally stable. NMR titration experiments point out three major regions of the Tip-Lck interaction comprising the RT loop, the n-src loop, and a helical turn preceding the last strand of the beta-sheet. Further changes of the chemical shifts were observed for the N- and C-terminal beta-strands of the SH3 domain, indicating additional contacts outside the proline-rich segment or subtle structural rearrangements transmitted from the binding site of the proline helix. Fluorescence spectroscopy shows that Tip binds to the SH3 domains of several Src kinases (Lck, Hck, Lyn, Src, Fyn, Yes), exhibiting the highest affinities for Lyn, Hck, and Lck. 相似文献
77.
Plastids are the site of the reductive and the oxidative pentose phosphate pathways, which both generate pentose phosphates as intermediates. A plastidic transporter from Arabidopsis has been identified that is able to transport, in exchange with inorganic phosphate or triose phosphates, xylulose 5-phosphate (Xul-5-P) and, to a lesser extent, also ribulose 5-phosphate, but does not accept ribose 5-phosphate or hexose phosphates as substrates. Under physiological conditions, Xul-5-P would be the preferred substrate. Therefore, the translocator was named Xul-5-P/phosphate translocator (XPT). The XPT shares only approximately 35% to 40% sequence identity with members of both the triose phosphate translocator and the phosphoenolpyruvate/phosphate translocator classes, but a higher identity of approximately 50% to glucose 6-phosphate/phosphate translocators. Therefore, it represents a fourth group of plastidic phosphate translocators. Database analysis revealed that plant cells contain, in addition to enzymes of the oxidative branch of the oxidative pentose phosphate pathway, ribose 5-phosphate isomerase and ribulose 5-phosphate epimerase in both the cytosol and the plastids, whereas the transketolase and transaldolase converting the produced pentose phosphates to triose phosphates and hexose phosphates are probably solely confined to plastids. It is assumed that the XPT function is to provide the plastidic pentose phosphate pathways with cytosolic carbon skeletons in the form of Xul-5-P, especially under conditions of a high demand for intermediates of the cycles. 相似文献
78.
Barth TF Rinaldi N Brüderlein S Mechtersheimer G Sträter J Altevogt P Möller P 《Cell communication & adhesion》2002,9(1):1-14
Pleural cavities are lined by a polarized monolayer of mesothelial cells (MC). During pleuritis, MC are shed into effusions, and pleural obstruction may occur. Integrins are cell surface receptors mediating interactions with extracellular matrix (ECM) proteins. The distribution of beta 1-, beta 3-, beta 4-integrins and fibronectin and laminin in normal and chronically inflamed pleura and in/on MC from pleural effusions was examined by immunomorphology and flow cytometry. Adhesion assays of MC to fibronectin and laminin were performed. In situ, resting MC expressed beta 1-, beta 3-, and beta 4-, and alpha v-subunits. Activated MC were beta 1- and alpha v-positive and also expressed alpha 3 and alpha 6; beta 4 was confined to the basal surface of MC; beta 3 was absent. Floating MC from effusions neoexpressed alpha 5 and reexpressed beta 3. In vitro, MC surface expressed beta 1, beta 3, alpha 3, alpha 5, alpha 6, alpha v, and also alpha 1 and alpha 2. In normal pleura, fibronectin and laminin were components of the basement membrane. In pleuritis, the basement membrane was desintegrated. Instead, newly formed fibronectin/laminin containing fibrils extended into the submesothelial connective tissue. Floating MC freshly isolated from effusions carried fibronectin and laminin on their surface and showed specific binding to these ECM proteins. Binding was blocked by anti-beta 1 or anti-alpha 5 and anti-alpha 6 antibodies, respectively. MC incubated with fibronectin showed a clear shift to the S phase, while laminin had no effect. In conclusion, activated and detached MC progressively enrich their integrin repertoire. By capturing soluble fibronectin and laminin and by matrix-mediated bridging, readhering MC may contribute to pleural obstruction. Further, soluble fibronectin bound to alpha 5 beta 1 might be life-sustaining for floating MC by driving cells into cell cycle. 相似文献
79.
Intracellular bacteria of the genus Caedibacter limit the reproduction of their host, the freshwater ciliate Paramecium. Reproduction rates of infected strains of paramecia were significantly lower than those of genetically identical strains that had lost their parasites after treatment with an antibiotic. Interference competition occurs when infected paramecia release a toxic form of the parasitic bacterium that kills uninfected paramecia. In mixed cultures of infected and uninfected strains of either P tetraurelia or of P novaurelia, the infected strains outcompeted the uninfected strains. Infection of new host paramecia seems to be rare. Infection of new hosts was not observed in either mixtures of infected with uninfected strains, or after incubation of paramecia with isolated parasites. The competitive advantages of the host paramecia, in combination with their vegetative reproduction, makes infection of new hosts by the bacterial parasites unnecessary, and could be responsible for the continued existence of "killer paramecia" in nature. Caedibacter parasites are not a defensive adaptation. Feeding rates and reproduction of the predators Didinium nasutum (Ciliophora) and Amoeba proteus (Amoebozoa, Gymnamoebia) were not influenced by whether or not their paramecia prey were infected. Infection of the predators frequently occurred when they preyed on infected paramecia. Caedibacter-infected predators may influence competition between Paramecium strains by release of toxic parasites into the environment that are harmful to uninfected strains. 相似文献
80.
Vogel S Piantedosi R O'Byrne SM Kako Y Quadro L Gottesman ME Goldberg IJ Blaner WS 《Biochemistry》2002,41(51):15360-15368
We reported previously that mice lacking plasma retinol-binding protein (RBP) are phenotypically normal except that they display impaired vision at the time of weaning. This visual defect is associated with greatly diminished eyecup levels of retinaldehyde and is reversible if the mutants are maintained for several months on a vitamin A-sufficient diet. Here we provide a biochemical basis for the visual phenotype of RBP-deficient mice. This phenotype does not result from inadequate milk total retinol levels since these are not different for RBP-deficient and wild-type mice. The eye, unlike all other tissues that have been examined, takes up dietary retinol very poorly. Moreover, compared to other tissues, the eye displays a strong preference for retinol uptake when retinol is delivered bound to RBP. The poor uptake of dietary retinol by the eye coupled with its marked ability to take up retinol from RBP, we propose, provides a basis for the impaired vision observed in weanling RBP-deficient mice. Further study of the mutants suggests that the impaired vision is reversible because the eyes of mutant mice slowly acquire sufficient retinol from the low levels of retinol present in their circulation either bound to albumin or present in lipoprotein fractions. Thus, the eye is unlike other tissues in the body in that it shows a very marked preference for acquiring retinol needed to support vision from the retinol-RBP complex and is unable to meet adequately its retinol need through uptake of recently absorbed dietary retinol. This provides an explanation for the impaired vision phenotype of RBP-deficient mice. 相似文献