Optimization of speed and accuracy of decoding in translation

The speed and accuracy of protein synthesis are fundamental parameters for understanding the fitness of living cells, the quality control of translation, and the evolution of ribosomes. In this study, we analyse the speed and accuracy of the decoding step under conditions reproducing the high speed of translation in vivo. We show that error frequency is close to 10⁻³, consistent with the values measured in vivo. Selectivity is predominantly due to the differences in k_cat values for cognate and near-cognate reactions, whereas the intrinsic affinity differences are not used for tRNA discrimination. Thus, the ribosome seems to be optimized towards high speed of translation at the cost of fidelity. Competition with near- and non-cognate ternary complexes reduces the rate of GTP hydrolysis in the cognate ternary complex, but does not appreciably affect the rate-limiting tRNA accommodation step. The GTP hydrolysis step is crucial for the optimization of both the speed and accuracy, which explains the necessity for the trade-off between the two fundamental parameters of translation.     

Parasite accessory cell interactions in murine leishmaniasis. I. Evasion and stimulus-dependent suppression of the macrophage interleukin 1 response by Leishmania donovani

Interleukin 1 (IL 1) is a principal mediator of the host immune response to microbial challenge. Accessory cells of the monocyte-macrophage series are a major source of this cytokine and are also chronically parasitized by protozoa of the genus Leishmania. This suggests that characterization of the macrophage IL 1 response to Leishmania would increase our understanding of the regulation of host immunity to these organisms. In the present study, the macrophage IL 1 response to Leishmania donovani was examined because infections with this organism have findings consistent with parasite-specific T cell unresponsiveness. Cytokine activity was measured either by direct stimulation or by co-stimulation of thymocytes. Conditioned media from BALB/c resident peritoneal macrophages infected with amastigotes of L. donovani contained no more IL 1 than did supernatant fluids of control cells. In contrast, supernatants from cells stimulated with lipopolysaccharide or heat-killed Listeria monocytogenes had significantly increased cytokine content. Resident cells infected with L. donovani for 4 hr before being stimulated with Listeria demonstrated a suppressed IL 1 response (approximately 40% of Listeria alone) to this secondary particulate stimulus. In contrast, the secondary response of leishmania-preinfected cells to lipopolysaccharide was not affected. To examine whether accessory cell nonresponsiveness to L. donovani (with respect to IL 1) was related to the state of macrophage activation, elicited peritoneal macrophages obtained by injection of proteose peptone were also studied. These cells responded to stimulation with lipopolysaccharide and fixed Staphylococcus aureus with increases in intracellular, membrane, and secreted cytokine activities. In contrast, L. donovani failed to induce any of these activities. This was found to be the case irrespective of whether amastigotes were alive or killed or opsonized with specific antibodies. Elicited cells preinfected with Leishmania responded normally to secondary stimulation with lipopolysaccharide, but not S. aureus (64% of Staphylococcus alone). In addition, attachment and penetration of L. donovani promastigotes and their subsequent conversion to amastigotes within macrophages failed to induce IL 1 synthesis. The findings of this study indicate that L. donovani has the ability to both evade and suppress the macrophage IL 1 response. Because this monokine provides an obligatory signal during macrophage-dependent T cell activation, evasion of signal transduction for IL 1 synthesis may be related to defects in cell-mediated immunity which occur during infections with this organism.     

Gabaergic Pharmacological Activity of Propofol Related Compounds as Possible Enhancers of General Anesthetics and Interaction with Membranes

Phenol compounds, such as propofol and thymol, have been shown to act on the GABA_A receptor through interaction with specific sites of this receptor. In addition, considering the high lipophilicity of phenols, it is possible that their pharmacological activity may also be the result of the interaction of phenol molecules with the surrounding lipid molecules, modulating the supramolecular organization of the receptor environment. Thus, in the present study, we study the pharmacological activity of some propofol- and thymol-related phenols on the native GABA_A receptor using primary cultures of cortical neurons and investigate the effects of these compounds on the micro viscosity of artificial membranes by means of fluorescence anisotropy. The phenol compounds analyzed in this article are carvacrol, chlorothymol, and eugenol. All compounds were able to enhance the binding of [³H]flunitrazepam with EC₅₀ values in the micromolar range and to increase the GABA-evoked Cl^? influx in a concentration-dependent manner, both effects being inhibited by the competitive GABA_A antagonist bicuculline. These results strongly suggest that the phenols studied are positive allosteric modulators of this receptor. Chlorothymol showed a bell-type effect, reducing its positive effect at concentrations >100 μM. The concentrations necessary to induce positive allosteric modulation of GABA_A receptor were not cytotoxic. Although all compounds were able to decrease the micro viscosity of artificial membranes, chlorothymol displayed a larger effect which could explain its effects on [³H]flunitrazepam binding and on cell viability at high concentrations. Finally, it is suggested that these compounds may exert depressant activity on the central nervous system and potentiate the effects of general anesthetics.