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1.
In vitro cycling rates of E. coli ribosomes and of elongation factors EF-Tu and EF-G have been obtained and these are compatible with translation rates in vivo. We show that the rate of translocation is faster than 50 s-1 and therefore that the EF-G function is not a rate limiting step in protein synthesis. The in vivo phenotype of some L7/L12 mutants could be accounted for by perturbed EF-Tu as well as EF-G functions. The S12 mutants that we studied were, in contrast, only perturbed in their EF-Tu function, while their EF-G interaction was not impaired in relation to wild type ribosomes. 相似文献
2.
Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium 总被引:13,自引:9,他引:4 下载免费PDF全文
Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel. 相似文献
3.
Host defense deficiency in newborn nonhuman primate lungs 总被引:2,自引:0,他引:2
A T Cheung G Kurland M E Miller E W Ford S A Ayin E M Walsh 《Journal of medical primatology》1986,15(1):37-47
We have investigated two major aspects of the pulmonary host defense mechanism--alveolar macrophage function as a "first line of bacterial defense" and induced neutrophil migration into the lung as a "back-up defense." Chemotactic and phagocytic/killing assays revealed a functional deficiency in the alveolar macrophages of newborn primates. Serial bronchoalveolar lavage investigations revealed diminished neutrophil migration into the newborn primate lung. The overall pulmonary host defense capability in newborn primates was deficient. The results of this investigation may have direct clinical relevance to the susceptibility of newborns to infections and pneumonia. 相似文献
4.
Ram ribosomes are defective proofreaders 总被引:11,自引:0,他引:11
We have studied the kinetics of poly(U) translation by three ribosomal ambiguity (Ram) mutants in an in vitro system with performance characteristics similar to those expressed in vivo. The leucine missense frequency supported by Ram ribosomes with tRNALeu2 increases between six and twelve-fold over that of wild-type ribosomes, while the corresponding increase with tRNALeu4 was between four and eight-fold, depending on the rpsD allele. We have used a steady-state assay for proofreading to identify the kinetic lesion responsible for the Ram phenotype. We were unable to detect any difference between Ram and wild-type ribosomes with respect to the initial kinetics of amino-acyl tRNA selection. All of the increased error rates could be associated with a decreased capacity of these Ram ribosomes to discard non-cognate aminoacyl-tRNA by proof reading. 相似文献
5.
Processivity errors of gene expression in Escherichia coli 总被引:11,自引:0,他引:11
Not all ribosomes that initiate translation of an mRNA sequence will successfully complete it and produce a full-length protein product. By comparing the amounts of lacZ monomer and lacZ dimer protein expressed from a plasmid in a strictly controlled assay, we calculate a dimer to monomer ratio of 0.76. We interpret this to mean that ribosomes have a 76% chance of completing the synthesis of a beta-galactosidase polypeptide. The remaining 24% of the initiated chains end in processivity accidents. For the wild-type, premature RNA polymerase termination is found to account for roughly one-third of the processivity accidents. For the hyperaccurate SmP mutant, we observe a processivity of 0.28, but the presence of streptomycin improves this to 0.50. Thus, the hyperaccuracy with respect to missense substitutions for this mutant is accompanied by a reduced processivity. Addition of streptomycin increase the first error class and reduces the second one. This finding is relevant to the optimization of ribosome function and the growth performance of ribosome mutants. 相似文献
6.
K. Lin I. Kurland L. Z. Xu A. J. Lange J. Pilkis M. R. El-Maghrabi S. J. Pilkis 《Protein expression and purification》1990,1(2):169-176
A number of mammalian enzymes have been expressed in Escherichia coli using the T7 RNA polymerase system, but the production of large amounts of these proteins has been limited by the low percentage of active enzyme that is found in the soluble fraction. In this report the effect of induction temperature was tested on the recovery of four rat liver enzymes, 6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase, fructose-2,6-bisphosphatase, glucokinase, and fructose-1,6-bisphosphatase. We also tested the effect using a host cell strain that contains a plasmid encoding T7 lysozyme, an inhibitor of T7 RNA polymerase. Large amounts of the first three enzymes accumulated in the cells after 4 h of induction at 37 degrees C, but only about 1-2% of the total expressed proteins were recovered in a soluble, active form. When the induction was carried out at 22 degrees C for 48 h with the pLysS strain, 20- to 30-fold higher amounts of the active expressed enzymes were recovered in the soluble fraction, even though the total accumulation and the rate of synthesis of these proteins were reduced. The optimal concentration of isopropyl-1-thio-beta-D-galactopyranoside required for induction was the same at both temperatures. On the other hand, the recovery of active fructose-1,6-bisphosphatase, a heat-stable enzyme, was 66% at 37 degrees C and was essentially unchanged at an induction temperature of 22 degrees C. Lowered induction temperature would appear to be of utility for enhanced recovery of active mammalian enzymes which are insoluble in E. coli cytosol at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
7.
Anthony T.W. Cheung Richard B. Moss Geoffrey Kurland Albin B. Leong William J. Novick 《Journal of medical primatology》1993,22(4):257-262
We have recently established a rhesus monkey model of chronic Pseudomonas aeruginosa (PA) endobronchitis by bronchoscopic instillation of PA-embedded agar beads. All experimental animals developed chronic neutrophilic endobronchitis similar to chronic PA endobronchitis in cystic fibrosis (CF). Histopathologic studies further confirmed similarities to chronic PA endobronchitis in CF, including marked peribronchial inflammation, epithelial damage, presence of degraded cilia and ciliary abnormalities, appearance of PA bacterial clusters, mucosal hyperplasia, goblet cell hypertrophy/hypersecretion, airway obstruction, alveolar abnormalities, bronchiectasis, and fibrosis. 相似文献
8.
Bohman K. Ruusala T. Jelenc P. C. Kurland C. G. 《Molecular genetics and genomics : MGG》1984,197(1):90-97
Molecular Genetics and Genomics - Comparisons in vivo and in vitro of wild-type and otherwise isogenic bacteria with five different mutant alleles of the gene (rpsL) specifying ribosomal protein... 相似文献
9.
23Na NMR relaxation rate measurements show that Na+ binds specifically to phosphatidylserine vesicles and is displaced partially from the binding site by K+ and Ca2+ but to a considerably less extent by tetraethylammonium ion. The data indicate that tetraethylammonium ion affects the binding of Na+ only slightly, by affecting the surface potential through its presence in the double layer, without competing for a phosphatidylserine binding site. Values for the intrinsic binding constant for the Na+-phosphatidylserine complex that would be consistent with the competition experiments (and the dependence of the relaxation rate on concentration of free Na+) fall in the range 0.4--1.2 M-1 with a better fit towards the higher values. We conclude that in the absence of competing cations in solution an appreciable fraction of the phosphatidylserine sites could be associated with bound Na+ at 0.1 M Na+ concentration. 相似文献
10.