891 resultados para Delay lock loops
Resumo:
Colicin D has long been thought to stop protein synthesis in infected Escherichia coli cells by inactivating ribosomes, just like colicin E3. Here, we show that colicin D specifically cleaves tRNAsArg including four isoaccepting molecules both in vivo and in vitro. The cleavage occurs in vitro between positions 38 and 39 in an anticodon loop with a 2′,3′-cyclic phosphate end, and is inhibited by a specific immunity protein. Consistent with the cleavage of tRNAsArg, the RNA fraction of colicin-treated cells significantly reduced the amino acid-accepting activity only for arginine. Furthermore, we generated a single mutation of histidine in the C-terminal possible catalytic domain, which caused the loss of the killing activity in vivo together with the tRNAArg-cleaving activity both in vivo and in vitro. These findings show that colicin D directly cleaves cytoplasmic tRNAsArg, which leads to impairment of protein synthesis and cell death. Recently, we found that colicin E5 stops protein synthesis by cleaving the anticodons of specific tRNAs for Tyr, His, Asn, and Asp. Despite these apparently similar actions on tRNAs and cells, colicins D and E5 not only exhibit no sequence homology but also have different molecular mechanisms as to both substrate recognition and catalytic reaction.
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Autocrine ligands are important regulators of many normal tissues and have been implicated in a number of disease states, including cancer. However, because by definition autocrine ligands are synthesized, secreted, and bound to cell receptors within an intrinsically self-contained “loop,” standard pharmacological approaches cannot be used to investigate relationships between ligand/receptor binding and consequent cellular responses. We demonstrate here a new approach for measurement of autocrine ligand binding to cells, using a microphysiometer assay originally developed for investigating cell responses to exogenous ligands. This technique permits quantitative measurements of autocrine responses on the time scale of receptor binding and internalization, thus allowing investigation of the role of receptor trafficking and dynamics in cellular responses. We used this technique to investigate autocrine signaling through the epidermal growth factor receptor by transforming growth factor alpha (TGFα) and found that anti-receptor antibodies are far more effective than anti-ligand antibodies in inhibiting autocrine signaling. This result indicates that autocrine-based signals can operate in a spatially restricted, local manner and thus provide cells with information on their local microenvironment.
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Interlocked feedback loops may represent a common feature among the regulatory systems controlling circadian rhythms. The Neurospora circadian feedback loops involve white collar-1 (wc-1), wc-2, and frequency (frq) genes. We show that WC-1 and WC-2 proteins activate the transcription of frq gene, whereas FRQ protein plays dual roles: repressing its own transcription, probably by interacting with the WC-1/WC-2 complex, and activating the expression of both WC proteins. Thus, they form two interlocked feedback loops: one negative and one positive. We establish the physiological significance of the interlocked positive feedback loops by showing that the levels of WC-1 and WC-2 determine the robustness and stability of the clock. Our data demonstrate that with WC-1 being the limiting factor in the WC-1/WC-2 complex, the greater the levels of WC-1 and WC-2, the higher the level of the FRQ oscillation and the more robust the overt rhythms. Our data also show that, despite considerable changes in the levels of WC-1, WC-2, and FRQ, the period of the clock has been limited to a small range, suggesting that the interlocked circadian feedback loops are also important for determining the circadian period length of the clock.
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The chicken pineal gland contains the autonomous circadian oscillator together with the photic-input pathway. We searched for chicken pineal genes that are induced by light in a time-of-day-dependent manner, and isolated chicken homolog of bZIP transcription factor E4bp4 (cE4bp4) showing high similarity to vrille, one of the Drosophila clock genes. cE4bp4 was expressed rhythmically in the pineal gland with a peak at very early (subjective) night under both 12-h light/12-h dark cycle and constant dark conditions, and the phase was nearly opposite to the expression rhythm of cPer2, a chicken pineal clock gene. Luciferase reporter gene assays showed that cE4BP4 represses cPer2 promoter through a E4BP4-recognition sequence present in the 5′-flanking region, indicating that cE4BP4 can down-regulate the chick pineal cPer2 expression. In vivo light-perturbation studies showed that the prolongation of the light period to early subjective night maintained the high level expression of the pineal cE4bp4, and presumably as a consequence delayed the onset of the induction of the pineal cPer2 expression in the next morning. These light-dependent changes in the mRNA levels of the pineal cE4bp4 and cPer2 were followed by a phase-delay of the subsequent cycles of cE4bp4/cPer2 expression, suggesting that cE4BP4 plays an important role in the phase-delaying process as a light-dependent suppressor of cPer2 gene.
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Protein-protein interactions typically are characterized by highly specific interfaces that mediate binding with precisely tuned affinities. Binding of the Escherichia coli cochaperonin GroES to chaperonin GroEL is mediated, at least in part, by a mobile polypeptide loop in GroES that becomes immobilized in the GroEL/GroES/nucleotide complex. The bacteriophage T4 cochaperonin Gp31 possesses a similar highly flexible polypeptide loop in a region of the protein that shows low, but significant, amino acid similarity with GroES and other cochaperonins. When bound to GroEL, a synthetic peptide representing the mobile loop of either GroES or Gp31 adopts a characteristic bulged hairpin conformation as determined by transferred nuclear Overhauser effects in NMR spectra. Thermodynamic considerations suggest that flexible disorder in the cochaperonin mobile loops moderates their affinity for GroEL to facilitate cycles of chaperonin-mediated protein folding.
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Anti-viral drug treatment of human immunodeficiency virus type I (HIV-1) and hepatitis B virus (HBV) infections causes rapid reduction in plasma virus load. Viral decline occurs in several phases and provides information on important kinetic constants of virus replication in vivo and pharmacodynamical properties. We develop a mathematical model that takes into account the intracellular phase of the viral life-cycle, defined as the time between infection of a cell and production of new virus particles. We derive analytic solutions for the dynamics following treatment with reverse transcriptase inhibitors, protease inhibitors, or a combination of both. For HIV-1, our results show that the phase of rapid decay in plasma virus (days 2-7) allows precise estimates for the turnover rate of productively infected cells. The initial quasi-stationary phase (days 0-1) and the transition phase (days 1-2) are explained by the combined effects of pharmacological and intracellular delays, the clearance of free virus particles, and the decay of infected cells. Reliable estimates of the first three quantities are not possible from data on virus load only; such estimates require additional measurements. In contrast with HIV-1, for HBV our model predicts that frequent early sampling of plasma virus will lead to reliable estimates of the free virus half-life and the pharmacological properties of the administered drug. On the other hand, for HBV the half-life of infected cells cannot be estimated from plasma virus decay.
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Recognition of peptides bound to class I major histocompatibility complex (MHC) molecules by specific receptors on T cells regulates the development and activity of the cellular immune system. We have designed and synthesized de novo cyclic peptides that incorporate PEG in the ring structure for binding to class I MHC molecules. The large PEG loops are positioned to extend out of the peptide binding site, thus creating steric effects aimed at preventing the recognition of class I MHC complexes by T-cell receptors. Peptides were synthesized and cyclized on polymer support using high molecular weight symmetrical PEG dicarboxylic acids to link the side chains of lysine residues substituted at positions 4 and 8 in the sequence of the HLA-A2-restricted human T-lymphotrophic virus type I Tax peptide. Cyclic peptides promoted the in vitro folding and assembly of HLA-A2 complexes. Thermal denaturation studies using circular dichroism spectroscopy showed that these complexes are as stable as complexes formed with antigenic peptides.
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A characteristic feature of all myosins is the presence of two sequences which despite considerable variations in length and composition can be aligned with loops 1 (residues 204-216) and 2 (residues 627-646) in the chicken myosin-head heavy chain sequence. Recently, an intriguing hypothesis has been put forth suggesting that diverse performances of myosin motors are achieved through variations in the sequences of loops 1 and 2 [Spudich, J. (1994) Nature (London) 372, 515-518]. Here, we report on the study of the effects of tryptic digestion of these loops on the motor and enzymatic functions of myosin. Tryptic digestions of myosin, which produced heavy meromyosin (HMM) with different percentages of molecules cleaved at both loop 1 and loop 2, resulted in the consistent decrease in the sliding velocity of actin filaments over HMM in the in vitro motility assays, did not affect the Vmax, and increased the Km values for actin-activated ATPase of HMM. Selective cleavage of loop 2 on HMM decreased its affinity for actin but did not change the sliding velocity of actin in the in vitro motility assays. The cleavage of loop 1 and HMM decreased the mean sliding velocity of actin in such assays by almost 50% but did not alter its affinity for HMM. To test for a possible kinetic determinant of the change in motility, 1-N6-ethenoadenosine diphosphate (epsilon-ADP) release from cleaved and uncleaved myosin subfragment 1 (S1) was examined. Tryptic digestion of loop 1 slightly accelerated the release of epsilon-ADP from S1 but did not affect the rate of epsilon-ADP release from acto-S1 complex. Overall, the results of this work support the hypothesis that loop 1 can modulate the motor function of myosin and suggest that such modulation involves a mechanism other than regulation of ADP release from myosin.
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DNA conformational changes are essential for the assembly of multiprotein complexes that contact several DNA sequence elements. An approach based on atomic force microscopy was chosen to visualize specific protein-DNA interactions occurring on eukaryotic class II nuclear gene promoters. Here we report that binding of the transcription regulatory protein Jun to linearized plasmid DNA containing the consensus AP-1 binding site upstream of a class II gene promoter leads to bending of the DNA template. This binding of Jun was found to be essential for the formation of preinitiation complexes (PICs). The cooperative binding of Jun and PIC led to looping of DNA at the protein binding sites. These loops were not seen in the absence of either PICs, Jun, or the AP-1 binding site, suggesting a direct interaction between DNA-bound Jun homodimers and proteins bound to the core promoter. This direct visualization of functional transcriptional complexes confirms the theoretical predictions for the mode of gene regulation by trans-activating proteins.
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Stability of the first-order neutral delay equation x’ (t) + ax’ (t – τ) = bx(t) + cx(t – τ) with complex coefficients is studied, by analyzing the existence of stability switches.
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This letter presents a method to model propagation channels for estimation, in which the sampling scheme can be arbitrary. Additionally, the method yields accurate models, with a size that converges to the channel duration, measured in Nyquist periods. It can be viewed as an improvement on the usual discretization based on regular sampling at the Nyquist rate. The method is introduced in the context of multiple delay estimation using the MUSIC estimator, and is assessed through a numerical example.
Resumo:
Different non-Fourier models of heat conduction, that incorporate time lags in the heat flux and/or the temperature gradient, have been increasingly considered in the last years to model microscale heat transfer problems in engineering. Numerical schemes to obtain approximate solutions of constant coefficients lagging models of heat conduction have already been proposed. In this work, an explicit finite difference scheme for a model with coefficients variable in time is developed, and their properties of convergence and stability are studied. Numerical computations showing examples of applications of the scheme are presented.