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.     

Plasmid-coded degradation of ethylbenzene and 1-phenylethanol in Pseudomonas fluorescens

Abstract Pseudomonas fluorescens EB carries genes for the catabolism of ethylbenzene and 1-phenylethanol on a plasmid. The size of the plasmid as measured by analysis of agarose electrophoresis gels after restriction endonuclease hydrolysis, was 253–267 kb. By treatment with Mitomycin C, mutants of EB strain were obtained bearing a plasmid which had undergone an extensive deletion of about 80 kb. These mutants have lost the ability to grow on ethylbenzene and 1-phenylethanol as well as to synthesize meta-cleavage enzymes.     

Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition,bi-directionally from the primed protospacer

Clustered regularly interspaced short palindromic repeats (CRISPR), in combination with CRISPR associated (cas) genes, constitute CRISPR-Cas bacterial adaptive immune systems. To generate immunity, these systems acquire short sequences of nucleic acids from foreign invaders and incorporate these into their CRISPR arrays as spacers. This adaptation process is the least characterized step in CRISPR-Cas immunity. Here, we used Pectobacterium atrosepticum to investigate adaptation in Type I-F CRISPR-Cas systems. Pre-existing spacers that matched plasmids stimulated hyperactive primed acquisition and resulted in the incorporation of up to nine new spacers across all three native CRISPR arrays. Endogenous expression of the cas genes was sufficient, yet required, for priming. The new spacers inhibited conjugation and transformation, and interference was enhanced with increasing numbers of new spacers. We analyzed ∼350 new spacers acquired in priming events and identified a 5′-protospacer-GG-3′ protospacer adjacent motif. In contrast to priming in Type I-E systems, new spacers matched either plasmid strand and a biased distribution, including clustering near the primed protospacer, suggested a bi-directional translocation model for the Cas1:Cas2–3 adaptation machinery. Taken together these results indicate priming adaptation occurs in different CRISPR-Cas systems, that it can be highly active in wild-type strains and that the underlying mechanisms vary.     

Ethanol Administration in the Rat Decreases Prostacyclin Production by Isolated Brain Microvessels

The effects of short- and long-term ethanol administration to rats on basal levels and formation of prostacyclin (PGI2) measured as 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), and on lipid class content and fatty acid composition of isolated brain microvessels (BMV) were studied. After acute treatment (2 h, at the peak of plasma ethanol concentration) basal 6-keto-PGF1 alpha levels in BMV and release on incubation were reduced to 50% of control values. After chronic administration (15 days), PGI2 release was reduced to about 40% of control values, without changes in basal levels. Total lipid, phospholipid, and cholesterol levels in BMV, measured after prolonged administration of alcohol, were not modified. Also, only minor changes in the fatty acid composition of individual phospholipid classes were detected. The observed reduction of PGI2 synthesis in BMV thus could not be related to changes of the fatty acid precursor pool in the preparation. Precursor release and/or the biosynthetic pathways may be affected by ethanol administration.     

Ultrastructural cytochemical and autoradiographic demonstration of nonspecific esterase(s) in guinea pig basophils

We used ultrastructural autoradiographic and cytochemical methods to localize esterase activities in unstimulated guinea pig basophils and in basophils undergoing degranulation or recovery from degranulation. We used tritium-labeled diisopropylfluorophosphate (DFP) as a probe for serine enzymes and localized this probe by ultrastructural autoradiography to cytoplasmic granules of immature or mature unstimulated basophils, as well as to granules released by degranulating basophils. Ultrastructural cytochemistry using alpha naphthyl acetate (ANA) as substrate localized nonspecific esterase activity to extruded granules, either within the interiors of degranulation sacs or within granules completely separated from degranulating basophils. Extruded granules retained their esterase activity for as long as 24 hr after antigen-induced degranulation. The plasma membranes of unstimulated or degranulating basophils, as well as of basophils recovering from degranulation, displayed prominent cell surface ANA esterase ectoenzyme activity. Lipid bodies, organelles present in the cytoplasm of both control and recovering basophils, were also alpha naphthyl acetate esterase (ANAE)-positive. Thus, cytochemical and autoradiographic techniques localized esterase and/or [3H]-DFP-binding activities to cytoplasmic granules, lipid bodies, and cell surface of basophils, and these enzyme activities persisted during both degranulation and recovery from degranulation.     

Stimulation of arachidonic acid metabolism by CCl4 in isolated rat hepatocytes

Arachidonic acid metabolism was evaluated in isolated rat hepatocytes after CCl4 exposure. CCl4 induced dose-dependently the synthesis and release of prostacyclin (PGI2) and thromboxane (TXB2). Treatment with prostaglandin E2 (PGE2) 30 min after exposure to CCl4, significantly reduced the cell damage as well as the release of TXB2 from the cells.     

Dietary n-9, n-6, and n-3 fatty acids modify linoleic acid more than arachidonic acid levels in plasma and platelet lipids and minimally affect platelet thromboxane formation in the rabbit

We have studied the effects of semisynthetic diets containing 5% by weight (12% of the energy) of either olive oil (70% oleic acid, OA) or corn oil (58% linoleic acid), or fish oil (Max EPA, containing about 30% eicosapentaenoic, EPA C 20:5 n-3, plus docosahexaenoic, DHA C 22:6 n-3, acids, and less than 2% linoleic acid), fed to male rabbits for a period of five weeks, on plasma and platelet fatty acids and platelet thromboxane formation. Aim of the study was to quantitate the absolute changes of n-6 and n-3 fatty acid levels in plasma and platelet lipid pools after dietary manipulations and to correlate the effects on eicosanoid-precursor fatty acids with those on platelet thromboxane formation. The major differences were found when comparing the group fed fish oil and depleted linoleic acid vs the other groups. The accumulation of n-3 fatty acids in various lipid classes was associated with modifications in the distribution of linoleic acid and arachidonic acid in different lipid pools. In platelets maximal incorporation of n-3 fatty acids occurred in phosphatidyl ethanolamine, which also participated in most of the total arachidonic acid reduction occurring in platelets, and linoleic acid, more than archidonic acid, was replaced by n-3 fatty acids in various phospholipids. The archidonic acid content of phosphatidyl choline was unaffected and that of phosphatidyl inositol only marginally reduced. Thromboxane formation by thrombin stimulated platelets did not differ among the three groups, and this may be related to the minimal changes of arachidonic acid in phosphatidyl choline and phosphatidyl inositol.     

Pericentric inversion of chromosome 19 in three families

Summary Pericentric inversion of chromosome 19 has been found in several members of three unrelated families from a restricted geographical region. In one of the families, an additional pericentric inversion of chromosome 9 was observed. Reproductive problems, multiple abortions in two families and a neonatal death in the third, were present. A review of previously described cases is included, and the genetic risk connected with this type of rearrangement is also discussed.     

Unilateral reduction mammaplasty: sculpturing the breast from the undersurface

Various traditional mammaplasty techniques have been suggested for unilateral breast reduction, and an inverted-T incision is still the most popular approach. However, unilaterally performed traditional techniques can rarely provide long-lasting symmetry because the operated and the unoperated breasts react differently to aging, weight changes, and pregnancy. Considerable residual scarring, interference with clinical and mammographic evaluation, and limited versatility are all major drawbacks of traditional procedures. We have performed unilateral mammaplasties on 47 patients with various types of congenital and acquired asymmetries, reducing and sculpturing the breast from the undersurface by means of minimal incisions, always avoiding horizontal scarring in the inframammary crease. Through a vertical infra-areolar incision, the breast is completely detached from the underlying pectoralis fascia and hooked up, thus completely exposing the undersurface of the mammary cone. The breast can thereafter be reshaped according to the size and shape of the contralateral breast by means of a discoid resection and/or selective sectoral removal of excessive subcutaneous tissues; modifications of the basic discoid resection can increase anterior projection of the new breast mound and can change the inclination of the anteroposterior breast axis on the anterior chest wall both on the horizontal and vertical planes. The results show that if criteria for patient selection are carefully respected, the procedure can provide long-lasting symmetry with minimal residual scarring and fully preserve the breast anatomy, function, and vascularization.