6 resultados para William I, King of the Netherlands, 1772-1843.
em National Center for Biotechnology Information - NCBI
Resumo:
We introduced disulfide bonds to lock the integrin αLβ2 I domain in predicted open, ligand binding or closed, nonbinding conformations. Transfectants expressing αLβ2 heterodimers containing locked-open but not locked-closed or wild-type I domains constitutively adhered to intercellular adhesion molecule-1 (ICAM-1) substrates. Locking the I domain closed abolished constitutive and activatable adhesion. The isolated locked-open I domain bound as well as the activated αLβ2 heterodimer, and binding was abolished by reduction of the disulfide. Lovastatin, which binds under the conformationally mobile C-terminal α-helix of the I domain, inhibited binding to ICAM-1 by αLβ2 with wild-type, but not locked-open I domains. These data establish the importance of conformational change in the αL I domain for adhesive function and show that this domain is sufficient for full adhesive activity.
Resumo:
Hemochromatosis (HC) is an inherited disorder of iron absorption, mapping within the human major histocompatibility complex (MHC). We have identified a multigene system in the murine MHC that contains excellent candidates for the murine equivalent of the human HC locus and implicate nonclassical class I genes in the control of iron absorption. This gene system is characterized by multiple copies of two head-to-head genes encoded on opposite strands and driven by one common regulatory motif. This regulatory motif has a striking homology to the promoter region of the beta-globin gene, a gene obviously involved in iron metabolism and hence termed beta-globin analogous promoter (betaGAP). Upstream of the betaGAP sequence are nonclassical class I genes. At least one of these nonclassical class I genes, Q2, is expressed in the gastrointestinal tract, the primary site of iron absorption. Also expressed in the gastrointestinal tract and downstream of the betaGAP motif is a second set of putative genes, termed Hephaestus (HEPH). Based on these observations, we hypothesized that the genes that seem to be controlled by the betaGAP regulatory motifs would be responsible for the control of Fe absorption. As a test of this hypothesis, we predicted that mice which have altered expression of class I gene products, the beta2-microglobulin knockout mice, [beta2m(-/-)], would develop Fe overload. This prediction was confirmed, and these results indicate beta2m-associated proteins are involved in the control of intestinal Fe absorption.
Resumo:
Transcription by RNA polymerase I in Saccharomyces cerevisiae requires a series of transcription factors that have been genetically and biochemically identified. In particular, the core factor (CF) and the upstream activation factor (UAF) have been shown in vitro to bind the core element and the upstream promoter element, respectively. We have analyzed in vivo the DNAse I footprinting of the 35S promoter in wild-type and mutant strains lacking one specific transcription factor at the time. In this way we were able to unambiguously attribute the protections by the CF and the UAF to their respective putative binding sites. In addition, we have found that in vivo a binding hierarchy exists, the UAF being necessary for CF binding. Because the CF footprinting is lost in mutants lacking a functional RNA polymerase I, we also conclude that the final step of preinitiation-complex assembly affects binding of the CF, stabilizing its contact with DNA. Thus, in vivo, the CF is recruited to the core element by the UAF and stabilized on DNA by the presence of a functional RNA polymerase I.
Resumo:
We have investigated the dynamic properties of the switch I region of the GTP-binding protein Ras by using mutants of Thr-35, an invariant residue necessary for the switch function. Here we show that these mutants, previously used as partial loss-of-function mutations in cell-based assays, have a reduced affinity to Ras effector proteins without Thr-35 being involved in any interaction. The structure of Ras(T35S)⋅GppNHp was determined by x-ray crystallography. Whereas the overall structure is very similar to wildtype, residues from switch I are completely invisible, indicating that the effector loop region is highly mobile. 31P-NMR data had indicated an equilibrium between two rapidly interconverting conformations, one of which (state 2) corresponds to the structure found in the complex with the effectors. 31P-NMR spectra of Ras mutants (T35S) and (T35A) in the GppNHp form show that the equilibrium is shifted such that they occur predominantly in the nonbinding conformation (state 1). On addition of Ras effectors, Ras(T35S) but not Ras(T35A) shift to positions corresponding to the binding conformation. The structural data were correlated with kinetic experiments that show two-step binding reaction of wild-type and (T35S)Ras with effectors requires the existence of a rate-limiting isomerization step, which is not observed with T35A. The results indicate that minor changes in the switch region, such as removing the side chain methyl group of Thr-35, drastically affect dynamic behavior and, in turn, interaction with effectors. The dynamics of the switch I region appear to be responsible for the conservation of this threonine residue in GTP-binding proteins.
Resumo:
Predictions of earthquakes that are based on observations of precursory seismicity cannot depend on the average properties of the seismicity, such as the Gutenberg-Richter (G-R) distribution. Instead it must depend on the fluctuations in seismicity. We summarize the observational data of the fluctuations of seismicity in space, in time, and in a coupled space-time regime over the past 60 yr in Southern California, to provide a basis for determining whether these fluctuations are correlated with the times and locations of future strong earthquakes in an appropriate time- and space-scale. The simple extrapolation of the G-R distribution must lead to an overestimate of the risk due to large earthquakes. There may be two classes of earthquakes: the small earthquakes that satisfy the G-R law and the larger and large ones. Most observations of fluctuations of seismicity are of the rate of occurrence of smaller earthquakes. Large earthquakes are observed to be preceded by significant quiescence on the faults on which they occur and by an intensification of activity at distance. It is likely that the fluctuations are due to the nature of fractures on individual faults of the network of faults. There are significant inhomogeneities on these faults, which we assume will have an important influence on the nature of self-organization of seismicity. The principal source of the inhomogeneity on the large scale is the influence of geometry--i.e., of the nonplanarity of faults and the system of faults.
Resumo:
N-type Ca2+ channels mediate Ca2+ influx, which initiates fast exocytosis of neurotransmitters at synapses, and they interact directly with the SNARE proteins syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) through a synaptic protein interaction (synprint) site in the intracellular loop connecting domains II and III of their α1B subunits. Introduction of peptides containing the synprint site into presynaptic neurons reversibly inhibits synaptic transmission, confirming the importance of interactions with this site in synaptic transmission. Here we report a direct interaction of the synprint peptide from N-type Ca2+ channels with synaptotagmin I, an important Ca2+ sensor for exocytosis, as measured by an affinity-chromatography binding assay and a solid-phase immunoassay. This interaction is mediated by the second C2 domain (C2B) of synaptotagmin I, but is not regulated by Ca2+. Using both immobilized recombinant proteins and native presynaptic membrane proteins, we found that the synprint peptide and synaptotagmin competitively interact with syntaxin. This interaction is Ca2+-dependent because of the Ca2+ dependence of the interactions between syntaxin and these two proteins. These results provide a molecular basis for a physical link between Ca2+ channels and synaptotagmin, and suggest that N-type Ca2+ channels may undergo a complex series of Ca2+-dependent interactions with multiple presynaptic proteins during neurotransmission.