A computational study of elongation factor G (EFG) duplicated genes: diverged nature underlying the innovation on the same structural template

Background

Elongation factor G (EFG) is a core translational protein that catalyzes the elongation and recycling phases of translation. A more complex picture of EFG''s evolution and function than previously accepted is emerging from analyzes of heterogeneous EFG family members. Whereas the gene duplication is postulated to be a prominent factor creating functional novelty, the striking divergence between EFG paralogs can be interpreted in terms of innovation in gene function.

Methodology/Principal Findings

We present a computational study of the EFG protein family to cover the role of gene duplication in the evolution of protein function. Using phylogenetic methods, genome context conservation and insertion/deletion (indel) analysis we demonstrate that the EFG gene copies form four subfamilies: EFG I, spdEFG1, spdEFG2, and EFG II. These ancient gene families differ by their indispensability, degree of divergence and number of indels. We show the distribution of EFG subfamilies and describe evidences for lateral gene transfer and recent duplications. Extended studies of the EFG II subfamily concern its diverged nature. Remarkably, EFG II appears to be a widely distributed and a much-diversified subfamily whose subdivisions correlate with phylum or class borders. The EFG II subfamily specific characteristics are low conservation of the GTPase domain, domains II and III; absence of the trGTPase specific G2 consensus motif “RGITI”; and twelve conserved positions common to the whole subfamily. The EFG II specific functional changes could be related to changes in the properties of nucleotide binding and hydrolysis and strengthened ionic interactions between EFG II and the ribosome, particularly between parts of the decoding site and loop I of domain IV.

Conclusions/Significance

Our work, for the first time, comprehensively identifies and describes EFG subfamilies and improves our understanding of the function and evolution of EFG duplicated genes.     

Synthesis and biological activity of bimorpholine and its carbanucleoside

A new enantiomerically pure carbacyclic nucleoside analogue with bimorpholine as a nonaromatic nucleobase was synthesized. The nucleoside analogue and bimorpholine were tested for cytotoxicity using an MTT assay and the xCELLigence System. Both assays revealed that compound 3 was highly cytotoxic at a 50 μM concentration while the cytotoxic effect of compound 1 was much less prominent. No antiretroviral activity was detected for this compound. In contrast, it acted as a potent inhibitor of hepatitis C virus (HCV) replication. Most likely this effect originates largely from the cytotoxicity of the compound; however, it is possible that a specific mechanism of HCV inhibition also exists.     

Male Infertility Workup Needs Additional Testing of Expressed Prostatic Secretion and/or Post-Massage Urine

The male factor accounts for almost 50% of infertility cases. Inflammation may reduce semen quality via several pathways, including oxidative stress (OxS). As male infertility routinely is assessed using semen analysis only, the possible presence of non-leukocytospermic asymptomatic inflammatory prostatitis may be overlooked. We compared local and systemic OxS levels in male partners of infertile couples with different inflammation patterns in their genital tract and/or oligospermia.Subjects (n=143) were grouped according to inflammation in their semen, expressed prostatic secretion (EPS), and/or post-massage urine (post-M). Systemic (8-isoprostanes in urine) and local (diene conjugates and total antioxidant capacity in seminal plasma) OxS was measuredThe levels of OxS markers were significantly elevated in both severe inflammation groups – leukocytospermic men and subjects whose inflammation was limited only to EPS and/or post-M. Comparison between oligospermic and non-oligospermic men with genital tract inflammation, and oligozoospermic men with or without inflammation in the genital tract indicated that inflammation but not oligospermia status had significant impact on the measured OxS markers.Hence, a high leukocyte count in prostate-specific materials (EPS, post-M), even in absence of clear leukocytopsermia, is an important source of local and systemic OxS that may be associated with male infertility and affect general health. We suggest including the tests for detection of inflammation of the prostate into the workup of infertile men as was suggested in the WHO 1993 recommendation.     

Retro-inversion of certain cell-penetrating peptides causes severe cellular toxicity

Cell-penetrating peptides (CPPs) are a promising group of delivery vectors for various therapeutic agents but their application is often hampered by poor stability in the presence of serum. Different strategies to improve peptide stability have been exploited, one of them being "retro-inversion" (RI) of natural peptides. With this approach the stability of CPPs has been increased, thereby making them more efficient transporters. Several RI-CPPs were here assessed and compared to the corresponding parent peptides in different cell-lines. Surprisingly, treatment of cells with these peptides induced trypsin insensitivity and rapid severe toxicity in contrast to L-peptides. This was measured as reduced metabolic activity and condensed cell nuclei, in parity with the apoptosis inducing agent staurosporine. Furthermore, effects on mitochondrial network, focal adhesions, actin cytoskeleton and caspase-3 activation were analyzed and adverse effects were evident at 20 μM peptide concentration within 4 h while parent L-peptides had negligible effects. To our knowledge this is the first time RI peptides are reported to cause cellular toxicity, displayed by decreased metabolic activity, morphological changes and induction of apoptosis. Considering the wide range of research areas that involves the use of RI-peptides, this finding is of major importance and needs to be taken under consideration in applications of RI-peptides.     

Trapping the Pasture Odorscape Using Open-Air Solid-Phase Micro Extraction,a Tool to Assess Grassland Value

Besides supporting cattle feeding, grasslands are home to a diversity of plants and insects that interact with each other by emitting volatile compounds. The aim of this work was to develop a method to determine permanent grassland odorscape and relate it to flower-visiting insects. Two grasslands were chosen for their contrasting levels of botanical diversity, resulting from differing grazing managements. Measurements were made over two periods of three consecutive days at the beginning of grazing, and just after the cows had left the plots. Volatile compounds were trapped using solid-phase microextraction (SPME) fibers exposed eight hours a day in three exclosures per plot, and then analyzed by gas-chromatography-mass spectrometry (GC-MS). Insects were trapped using pan traps and a net, sorted and counted. The open air SPME method yielded volatile compound profiles that were richer than maize field profiles, comprising the common green leaf volatiles (GLV) and more specific ones. Differences between the odorscapes of the two grasslands were found, but they were not as marked as expected from their botanical composition. By contrast, there were sharp differences between the two periods, resulting from the combined effects of changes in weather conditions, plant phenological stage and grazing progress. Several correlations between insect counts and volatile compounds were found. Although their correlation coefficients were low, some of them were confirmed when tested by Spearman rank correlation, and could be logically explained. This method of grassland odorscape deserves to be developed because it can provide information on many aspects of grassland function and on the stresses that grassland plants undergo.     

Different sensitivity of H69 modification enzymes RluD and RlmH to mutations in Escherichia coli 23S rRNA

Nucleoside modifications are introduced into the ribosomal RNA during the assembly of the ribosome. The number and the localization of the modified nucleosides in rRNAs are known for several organisms. In bacteria, rRNA modified nucleosides are synthesized by a set of specific enzymes, the majority of which have been identified in Escherichia coli. Each rRNA modification enzyme recognizes its substrate nucleoside(s) at a specific stage of ribosome assembly. Not much is known about the specificity determinants involved in the substrate recognition of the modification enzymes. In order to shed light on the substrate specificity of RluD and RlmH, the enzymes responsible for the introduction of modifications into the stem-loop 69 (H69), we monitored the formation of H69 pseudouridines (Ψ) and methylated pseudouridine (m3Ψ) in vitro on ribosomes with alterations in 23S rRNA. While the synthesis of Ψs in H69 by RluD is relatively insensitive to the point mutations at neighboring positions, methylation of one of the Ψs by RlmH exhibited a much stronger sensitivity. Apparently, in spite of synthesizing modifications in the same region or even at the same position of rRNA, the two enzymes employ different substrate recognition mechanisms.