Detection of mutations in the FemXAB protein family in oxacillin-susceptible mecA-positive Staphylococcus aureus clinical isolates.

Objectives Methicillin-resistant Staphylococcus aureus (MRSA) strains that express the mecA gene but are oxacillin susceptible (OS-MRSA; oxacillin MIC

Effect of transosseous application of low-intensity ultrasound at the tendon graft-bone interface healing: gene expression and histological analysis in rabbits.

The present study investigates the effect of transosseous low-intensity pulsed ultrasound (LiUS) on the healing at tendon graft-bone interface, in molecular and histological level. The anterior cruciate ligament (ACL) in both knees of 52 New Zealand White rabbits was excised and replaced with the long digital extensor. A custom-made ultrasound transducer was implanted onto the medial tibial condyle, adjacent to the surface of the bone tunnel at both knees of the rabbits. The LiUS-treated right knees received 200-mus bursts of 1 MHz sine waves at a pulse repetition rate of 1 kHz and with 30 mW/cm(2) spatial-average temporal-average intensity for 20 min daily (study group), while the left knee received no LiUS (control group). Thirty-six rabbits were used to perform semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis from both study and control groups for transforming growth factor-beta1 (TGF-beta1), biglycan and collagen I. RT-PCR products showed statistically significant upregulation of biglycan and collagen I gene expression in the study group, while TGF-beta1 gene expression exhibited a bimodal profile. Histological examination performed in 16 rabbits from both groups supported the findings of the molecular analysis, indicating a faster healing rate and a more efficient ligamentization process after ultrasound treatment. These findings suggest that transosseous application of LiUS enhances the healing rate of the tendon graft-bone interface, possibly by affecting the expression levels of genes significant for the tendon to bone healing process.

On the role of an unusual tRNAGly isoacceptor in Staphylococcus aureus.

In the available Staphylococcus aureus genomes, four different genes have been annotated to encode tRNA(Gly) isoacceptors. Besides their prominent role in protein synthesis, some of them also participate in the formation of pentaglycine bridges during cell wall synthesis. However, until today, it is not known how many and which of them are actually involved in this essential procedure. In the present study we identified, apart from the four annotated tRNA(Gly) genes, a putative pseudogene which encodes and expresses an unusual fifth tRNA(Gly) isoacceptor in S. aureus (as detected via RT-PCR and subsequent direct sequencing analysis). All the in vitro transcribed tRNA(Gly) molecules (including the "pseudogene-encoded" tRNA(Gly)) can be efficiently aminoacylated by the recombinant S. aureus glycyl-tRNA synthetase. Furthermore, bioinformatic analysis suggests that the "pseudo"-tRNA(Gly(UCC)) identified in the present study and two of the annotated isoacceptors bearing the same anticodon carry specific sequence elements that do not favour the strong interaction with EF-Tu that proteinogenic tRNAs would promote. This observation was verified by the differential capacity of Gly-tRNA(Gly) molecules to form ternary complexes with activated S. aureus EF-Tu.GTP. These tRNA(Gly) molecules display high sequence similarities with their S. epidermidis orthologs which also actively participate in cell wall synthesis. Both bioinformatic and biochemical data suggest that in S. aureus these three glycylated tRNA(Gly) isoacceptors that are weak EF-Tu binders, possibly escape protein synthesis and serve as glycine donors for the formation of pentaglycine bridges that are essential for stabilization of the staphylococcal cell wall.

Recruitment of mitochondrial tRNA genes as auxiliary variability markers for both intra- and inter-species analysis: The paradigm of brown hare (Lepus europaeus).

We sequenced and analyzed the mitochondrial tRNA(Thr) and tRNA(Pro) genes from brown hare (Lepus europaeus) individuals of different geographic distribution and we investigated the role of various nucleotide substitutions that were detected. We compared these tRNAs with the respective available mitochondrial tRNA genes sequences within Lepus species and among mammals. The mutations that were detected represent specific and conserved polymorphisms that do not seem to affect the structural and functional features that are required for participation of tRNA molecules in mitochondrial protein synthesis. These changes however, possibly reflect on the evolutionary background of the species, which is based on the high intra-genomic variability and the evolutionary dynamic of the mitochondrial DNA. In an attempt to compare the phylogeny that is based on these specific tRNA genes with the phylogeny that is produced from sequencing data of the mitochondrial variable loop, we came up with results that indicate similar phylogeographic clusters. This observation implies that the tRNA mutations that were used for the present study have been well tolerated during evolution and they define an additional genetic and biochemical tag that can be used for such studies. Based on this notion and according to our results, we propose that mitochondrial tRNA genes can be used as valuable auxiliary molecular markers for contemporaneous and linked biochemical and genetic analyses.

Mitochondrial tRNA mutations: clinical and functional perturbations.

During the last decade, there has been a progressive accumulation of reports that connect the identification of specific mitochondrial tRNA gene mutations to severe disorders in human. As a result, mitochondrial tRNA genes and their products have emerged as novel and essential molecular markers for wide biochemical and genetic screenings among different human populations. So far, 139 pathogenic and 243 polymorphic mt tRNA mutations have been described and they have become the foreground of numerous case reports. Given the complexity of mitochondrial genetics and biochemistry, the clinical manifestations of mitochondrial disorders are extremely heterogeneous. They range from lesions of single tissues or structures to more severe impairments including myopathies, encephalomyopathies, cardiomyopathies, or complex multisystem syndromes. Moreover, the exact mechanisms by which biochemical cascades can be dramatically affected by mitochondrial tRNA mutations still remain uncharacterized. However and regardless of the vast amount of information that daily emerges, only few efforts have been carried out to systematically record all the mitochondrial tRNA-associated pathogenic mutations or polymorphisms. In this report, we summarize all the clinical phenotypes associated with mitochondrial tRNA pathogenic mutations that have been reported so far. In a next step we describe in detail all the pathogenic and polymorphic mutations that have been recorded so far and we categorize them per tRNA species and per associated disease. Finally, we discuss the impact of the frequency of mitochondrial tRNA mutations in general population surveys and we preview any relevant implications on the essential functional integrity of mitochondrial biochemical pathways.

A single tRNA base pair mediates bacterial tRNA-dependent biosynthesis of asparagine.

In many prokaryotes and in organelles asparagine and glutamine are formed by a tRNA-dependent amidotransferase (AdT) that catalyzes amidation of aspartate and glutamate, respectively, mischarged on tRNAAsn and tRNAGln. These pathways supply the deficiency of the organism in asparaginyl- and glutaminyl-tRNA synthtetases and provide the translational machinery with Asn-tRNAAsn and Gln-tRNAGln. So far, nothing is known about the structural elements that confer to tRNA the role of a specific cofactor in the formation of the cognate amino acid. We show herein, using aspartylated tRNAAsn and tRNAAsp variants, that amidation of Asp acylating tRNAAsn is promoted by the base pair U1-A72 whereas the G1-C72 pair and presence of the supernumerary nucleotide U20A in the D-loop of tRNAAsp prevent amidation. We predict, based on comparison of tRNAGln and tRNAGlu sequence alignments from bacteria using the AdT-dependent pathway to form Gln-tRNAGln, that the same combination of nucleotides also rules specific tRNA-dependent formation of Gln. In contrast, we show that the tRNA-dependent conversion of Asp into Asn by archaeal AdT is mainly mediated by nucleotides G46 and U47 of the variable region. In the light of these results we propose that bacterial and archaeal AdTs use kingdom-specific signals to catalyze the tRNA-dependent formations of Asn and Gln.

RNA-mediated therapeutics: from gene inactivation to clinical application.

The specific targeting and inactivation of gene expression represents nowadays the goal of the mainstream basic and applied biomedical research. Both researchers and pharmaceutical companies, taking advantage of the vast amount of genomic data, have been focusing on effective endogenous mechanisms of the cell that can be used against abnormal gene expression. In this context, RNA represents a key molecule that serves both as tool and target for deploying molecular strategies based on the suppression of genes of interest. The main RNA-mediated therapeutic methodologies, deriving from studies on catalytic activity of ribozymes, blockage of mRNA translation and the recently identified RNA interference, will be discussed in an effort to understand the utilities of RNA as a central molecule during gene expression.