Characteristics of the functional state of rat tracheal and bronchial smooth muscle at different stages of alveolitis

In isolated tracheal smooth muscle preparations in normal rats and in rats with experimental fibrotic alveolitis, responses to electrical field stimulation of nervous and muscle fibers were studied. At stimulation of muscles or nerves of tracheal preparations without intramural ganglia in rats with acute alveolitis, parameters of smooth muscle contractions did not practically differ from those in normal rats. In rats with fibrotic alveolitis the amplitude and rate of muscle contraction decreased, while the response latent period (LP) increased. At stimulation of preganglionic nerve fibers of the tracheal preparations with intramural ganglia in rats with acute alveolitis, the value and rate of smooth muscle contraction decreased, while the response LP increased. After transition into chronic phase of the disease (fibrotic alveolitis), a partial restoration of the response parameters took place. In rats with acute alveolitis, the repeated stimulation of the nerve fibers led to an increase of amplitude and a decrease of rate of tracheal smooth muscle contractions. In rats with fibrotic alveolitis, the repeated stimulation caused a decrease of amplitude and rate of contractions and an increase of the response LP.     

Influence of individual domains of the translation termination factor eRF1 on induction of the GTPase activity of the translation termination factor eRF3

Translation termination in eukaryotes is governed by two proteins, belonging to the class-1 (eRF1) and class-2 (eRF3) polypeptide release factors. eRF3 catalyzes hydrolysis of GTP to GDP and inorganic phosphate in the ribosome in the absence of mRNA, tRNA, aminoacyl-tRNA and peptidyl-tRNA but needs the presence of eRF1. It's known that eRF1 and eRF3 interact with each other in vitro and in vivo via their C-terminal regions. eRF1 consists of three domains - N, M, and C. In this study we examined the influence of individual domains of the human eRF1 on induction of the human eRF3 GTPase activity in the ribosome in vitro. It was shown that none of the N-, M-, C- and NM-domains induces eRF3 GTPase activity in presence of the ribosomes. MC-domain does induce GTPase activity of eRF3 but four times less efficient than full-length eRF1, therefore, MC-domain (and very likely M-domain) binds to the ribosome in the presence of eRF3. Based on these data and taking into account the data available in literature, a conclusion was drawn that the N domain of eRF1 is not essential for eRF1-dependent induction of the eRF3 GTPase activity. A working hypothesis is formulated, postulating that GTPase activity eRF3 during the translation termination is associated with the intermolecular interactions of GTP/GDP, GTPase center of the large ribosomal subunit (60S), MC-domain of eRF1, C-terminal region and GTP-binding domains of eRF3, but without participation of the N-terminal region of eRF3.     

Role of the leader sequence in tobacco pectin methylesterase secretion

We report that unprocessed tobacco pectin methylesterase (PME) contains N-terminal pro-sequence including the transmembrane (TM) domain and spacer segment preceding the mature PME. The mature portion of PME was replaced by green fluorescent protein (GFP) gene and various deletion mutants of pro-sequence fused to GFP were cloned into binary vectors and agroinjected in Nicotiana benthamiana leaves. The PME pro-sequence delivered GFP to the cell wall (CW). We showed that a transient binding of PME TM domain to endoplasmic reticulum membranes occurs upon its transport to CW. The CW targeting was abolished by various deletions in the TM domain, i.e., anchor domain was essential for secretion of GFP to CW. By contrast, even entire deletion of the spacer segment had no influence on GFP targeting.     

Structure and dynamics in solution of the stop codon decoding N-terminal domain of the human polypeptide chain release factor eRF1

The high-resolution NMR structure of the N-domain of human eRF1, responsible for stop codon recognition, has been determined in solution. The overall fold of the protein is the same as that found in the crystal structure. However, the structures of several loops, including those participating in stop codon decoding, are different. Analysis of the NMR relaxation data reveals that most of the regions with the highest structural discrepancy between the solution and solid states undergo internal motions on the ps-ns and ms time scales. The NMR data show that the N-domain of human eRF1 exists in two conformational states. The distribution of the residues having the largest chemical shift differences between the two forms indicates that helices α2 and α3, with the NIKS loop between them, can switch their orientation relative to the β-core of the protein. Such structural plasticity may be essential for stop codon recognition by human eRF1.