Evaluation of MMP-12 expression in chronic rhinosinusitis with nasal polyposis.

BACKGROUND: The purpose of this study was to evaluate the expression of MMP-12 in patients with chronic rhinosinusitis with polyps (CRSwNP). METHODOLOGY: Tissue samples from 37 patients with CRSwNP undergoing functional endoscopic sinus surgery and healthy mucosa specimens from 12 healthy controls were obtained intraoperatively. The mRNA and protein expression levels of MMP-12 were quantified by real-time polymerase chain reaction and Western blotting, respectively. RESULTS: mRNA levels of MMP-12 were significantly elevated in the CRSwNP tissue samples compared to those in control ones. The protein levels of MMP-12 showed a trend of increasing but with no statistical significance. CONCLUSIONS: Elevation of MMP-12 in patients with CRSwNP suggests its potential implication in the pathogenesis of the disease. The difference in the expression profile observed between mRNA and protein levels could be due to post-translational gene expression regulation. Our findings provide evidence that MMP-12 along with other MMPs may serve as a biomarker and therapeutic target in the management of the disease.

Histological and biochemical evidence related to the collagen quality in torn rotator cuff tendons.

This study investigates the histological background of torn rotator cuff tendons, evaluates the stability of newly synthesized collagen by measuring the hydro-xyproline content and attempts to correlate these findings with the clinical outcome after reconstruction of the rotator cuff. Sixty-one patients underwent reconstruction for a -rotator cuff tear. They were evaluated preoperatively with the Constant-Murley score, MRI and ultrasound. Biopsy samples were taken from chronic rotator cuff tears and histological analysis was performed. Hydroxyprolin presence was evaluated in various -tissues. Mean follow-up was 46 months. Histological analysis revealed collagen fragmentation and thinning (90.2% of patients), myxoid degeneration (88%), hyaline degeneration (50.8%), chondroid metaplasia (44.3%), calcification (24.7%), fatty infiltration (20.4%) and vascular proliferation (62.3%). Hydroxyproline was under-represented in newly synthesized collagen in 57% of patients. In the majority of the patients with a low hydroxyproline/collagen ratio the histological findings were abnormal. None of the findings was related to the clinical outcome with a statistical significance. Histological and biochemical findings reflected the poor quality of the tendon. The good clinical outcome did not depend on the histological or biochemical findings but rather on the meticulous surgical reconstruction and physical therapy.

Challenges in Identifying the Foot Motor Region in Patients with Brain Tumor on Routine MRI: Advantages of fMRI.

BACKGROUND AND PURPOSE: Accurate localization of the foot/leg motor homunculus is essential because iatrogenic damage can render a patient wheelchair- or bed-bound. We hypothesized the following: 1) Readers would identify the foot motor homunculus <100% of the time on routine MR imaging, 2) neuroradiologists would perform better than nonradiologists, and 3) those with fMRI experience would perform better than those without it. MATERIALS AND METHODS: Thirty-five attending-level raters (24 neuroradiologists, 11 nonradiologists) evaluated 14 brain tumors involving the frontoparietal convexity. Raters were asked to identify the location of the foot motor homunculus and determine whether the tumor involved the foot motor area and/or motor cortex by using anatomic MR imaging. Results were compared on the basis of prior fMRI experience and medical specialty by using Mann-Whitney U test statistics. RESULTS: No rater was 100% correct. Raters correctly identified whether the tumor was in the foot motor cortex 77% of the time. Raters with fMRI experience were significantly better than raters without experience at foot motor fMRI centroid predictions (13 ± 6 mm versus 20 ± 13 mm from the foot motor cortex center, P = 2 × 10(-6)) and arrow placement in the motor gyrus (67% versus 47%, P = 7 × 10(-5)). Neuroradiologists were significantly better than nonradiologists at foot motor fMRI centroid predictions (15 ± 8 mm versus 20 ± 14 mm, P = .005) and arrow placement in the motor gyrus (61% versus 46%, P = .008). CONCLUSIONS: The inability of experienced readers to consistently identify the location of the foot motor homunculus on routine MR imaging argues for using fMRI in the preoperative setting. Experience with fMRI leads to improved accuracy in identifying anatomic structures, even on routine MR imaging.

Modulation of poly(A)-specific ribonuclease (PARN): current knowledge and perspectives.

Deadenylation is the exoribonucleolytic shortening of eukaryotic poly(A) tails. It is often the first and rate-limiting step for mRNA decay and translational silencing. The process is catalysed by a diversity of deadenylases, which provide robust and flexible means to control mRNA levels and gene expression. Poly(A)-specific ribonuclease (PARN) is a major mammalian deadenylase and the only known to concurrently bind the 5' cap-structure and the 3' poly(A), thus enhancing the degradation rate and amplifying its processivity. PARN is important during oocyte maturation, embryogenesis, early development, DNA damage, and in cell-cycle progression, but also in processes beyond mRNA metabolism, such as the maturation of snoRNAs. The enzyme also participates in nonsense-mediated mRNA decay and in the regulation of cytoplasmic polyadenylation. Importantly, PARN is involved in the degradation of several cancer-related genes, while its expression is altered in cancer. Apart from the direct interaction with the cap structure, several strategies regulate PARN activity, such as phosphorylation, interaction with RNA-binding proteins (RBPs), and natural nucleotides. Recent studies have focused on the regulation of its activity by synthetic nucleoside analogues with therapeutic potential. In this context, the wide repertoire of RBPs and molecules that regulate PARN activity, together with the established role of deadenylases in miRNA-mediated regulation of mRNA expression, suggest that mRNA turnover is more complex than it was previously thought and PARN holds a key role in this process. In this review, we highlight the importance of PARN during RNA's lifecycle and discuss clinical perspectives of modulating its activity.

Molecular phylogeny of VP1, 2A, and 2B genes of echovirus isolates: epidemiological linkage and observations on genetic variation.

Phylogenetic relationships between 37 echovirus clinical isolates, most of them originating from an aseptic meningitis outbreak during 2001 in Greece, were investigated by RT-PCR and sequencing. The generic primers 292 and 222 were used to amplify about 300 bp of the 5' end of VP1 while primers EUG3a, 3b, 3c, and EUC2 amplified the entire coding sequence of the 2A and 2B genes. Phylogenetic trees were constructed for each genomic region using the clinical isolates' sequences and those of the prototype echoviruses in order to investigate the correlation of part of VP1 with the serotype as well as the genetic variation of the echovirus genome in 2A and 2B. The phylogenetic grouping pattern of the clinical isolates revealed that there is a correlation of serotype and genotype in the part of VP1 that was investigated, while this pattern is disrupted in the adjacent genomic regions that were sequenced. Sequence analysis of the adjacent 2A and 2B genes provided a different pattern of phylogenetic relationships and strong evidence of epidemiological linkage of most of the clinical isolates.

Genomics and the evolution of aminoacyl-tRNA synthesis.

Translation is the process by which ribosomes direct protein synthesis using the genetic information contained in messenger RNA (mRNA). Transfer RNAs (tRNAs) are charged with an amino acid and brought to the ribosome, where they are paired with the corresponding trinucleotide codon in mRNA. The amino acid is attached to the nascent polypeptide and the ribosome moves on to the next codon. Thus, the sequential pairing of codons in mRNA with tRNA anticodons determines the order of amino acids in a protein. It is therefore imperative for accurate translation that tRNAs are only coupled to amino acids corresponding to the RNA anticodon. This is mostly, but not exclusively, achieved by the direct attachment of the appropriate amino acid to the 3'-end of the corresponding tRNA by the aminoacyl-tRNA synthetases. To ensure the accurate translation of genetic information, the aminoacyl-tRNA synthetases must display an extremely high level of substrate specificity. Despite this highly conserved function, recent studies arising from the analysis of whole genomes have shown a significant degree of evolutionary diversity in aminoacyl-tRNA synthesis. For example, non-canonical routes have been identified for the synthesis of Asn-tRNA, Cys-tRNA, Gln-tRNA and Lys-tRNA. Characterization of non-canonical aminoacyl-tRNA synthesis has revealed an unexpected level of evolutionary divergence and has also provided new insights into the possible precursors of contemporary aminoacyl-tRNA synthetases.

Cysteinyl-tRNA synthetase is not essential for viability of the archaeon Methanococcus maripaludis.

The methanogenic archaea Methanocaldococcus jannaschii and Methanothermobacter thermautotrophicus contain a dual-specificity prolyl-tRNA synthetase (ProCysRS) that accurately forms both prolyl-tRNA (Pro-tRNA) and cysteinyl-tRNA (Cys-tRNA) suitable for in vivo translation. This intriguing enzyme may even perform its dual role in organisms that possess a canonical single-specificity cysteinyl-tRNA synthetase (CysRS), raising the question as to whether this latter aminoacyl-tRNA synthetase is indeed required for cell viability. To test the postulate that all synthetase genes are essential, we disrupted the cysS gene (encoding CysRS) of Methanococcus maripaludis. The knockout strain was viable under normal growth conditions. Biochemical analysis showed that the pure M. maripaludis ProCysRS was capable of forming Cys-tRNA, implying that the dual-specificity enzyme compensates in vivo for the loss of CysRS. The canonical CysRS has a higher affinity for cysteine than ProCysRS, a reason why M. maripaludis may have acquired cysS by a late lateral gene transfer. These data challenge the notion that all twenty aminoacyl-tRNA synthetases are essential for the viability of a cell.

Coronary arteriographic findings in symptomatic and asymptomatic subjects with coronary artery calcification.

The relation of coronary artery calcification with the presence of symptoms of coronary artery disease and its angiographic severity is not clear. We studied 37 apparently healthy, asymptomatic subjects that were found by digital cinefluoroscopy to have coronary calcium and compared to age- and sex-matched group of patients with coronary calcium and symptomatic coronary artery disease. Normal coronary arteries and non-obstructive lesions only were found in 12/37 (32.4%) and 11/37 (29.7%) asymptomatic subjects vs. 1/37 (2.7%) and 2/37 (5.4%) patients; P<0.001 and P<0.012, respectively. Obstructive lesions were more rare in asymptomatic subjects than in patients, 14/37 (37.8%) vs. 34/37 (91.9%) (P<0.0001), as well as total occlusions, 2/37 (5.4%) vs. 10/37 (27%) (P<0.024). Median worst lesion stenosis was 30% in asymptomatic subjects and 95% in patients (P<0.0001). In asymptomatic usual cardiovascular risk subjects, coronary calcium detection by digital cinefluoroscopy is accompanied by a relatively high probability of obstructive disease, although less severe angiographically than in age- and sex-matched catheterized patients with symptomatic coronary artery disease.

Protein synthesis: twenty three amino acids and counting.

The genetic code can be interpreted during translation as 21 amino acids and three termination signals. Recent advances at the interface of chemistry and molecular biology are extending the genetic code to allow assignment of new amino acids to existing codons, providing new functional groups for protein synthesis.

Intravenous magnesium sulfate versus diltiazem in paroxysmal atrial fibrillation.

BACKGROUND: Drugs currently available for the acute treatment of paroxysmal atrial fibrillation have significant limitations. We assessed the safety and effectiveness of intravenous magnesium sulfate versus diltiazem therapy in patients with prolonged episodes of paroxysmal atrial fibrillation. METHODS: In a prospective randomized trial, 46 symptomatic patients presenting with paroxysmal atrial fibrillation were given intravenous magnesium sulfate (n=23) or diltiazem (n=23) therapy. Primary outcome measures were effects on ventricular rate control and proportion of patients restored to sinus rhythm at 6 h after initiation of treatment. RESULTS: There were no differences in baseline characteristics between the two groups. Both forms of treatment were well tolerated, with no adverse clinical events. Both drugs had similar efficacy in reducing the ventricular rate at the first hour of treatment (P<0.05) with a tendency toward a further decrease during infusion times of 2 (P<0.01), 3, 4, 5 and 6 h, respectively (P<0.001). However, at the end of the 6-h treatment period, restoration of sinus rhythm was observed in a significantly higher proportion of patients in the magnesium group compared with the diltiazem group [13 of 23 patients, (57%), versus five of 23 patients, (22%), P=0.03]. CONCLUSIONS: Magnesium sulfate favorably affects rate control and seems to promote the conversion of long lasting episodes of paroxysmal atrial fibrillation to sinus rhythm, representing a safe, reliable and cost-effective alternative treatment strategy to diltiazem.

Extensive deproteinization of Dictyostelium discoideum RNase P reveals a new catalytic activity.

Nuclear Dictyostelium discoideum RNase P was subjected to vigorous deproteinization procedures. After treatment with proteinase K followed by phenol extraction of samples containing D. discoideum RNase P activity, a new enzymatic activity was recovered. The proteinase K/phenol/SDS treated enzyme cleaves Schizossacharomyces pombe tRNAser (supS1), D. discoideum tRNASer and tRNALeu precursors several nucleotides upstream of the cleavage site of RNase P, liberating products with 5'-hydroxyl ends. This activity seems to be associated with one or two RNA molecules copurifying with D. discoideum RNase P activity as judged by its inhibition in the presence of micrococcal nuclease, which is in contrast to its resistance to proteinase K/phenol/SDS treatment.

Methanococcus jannaschii prolyl-cysteinyl-tRNA synthetase possesses overlapping amino acid binding sites.

The protein translation apparatus of Methanococcus jannaschii possesses the unusual enzyme prolyl-cysteinyl-tRNA synthetase (ProCysRS), a single enzyme that attaches two different amino acids, proline and cysteine, to their cognate tRNA species. Measurement of the ATP-PP(i) exchange reaction revealed that amino acid activation, the first reaction step, differs for the two amino acids. While Pro-AMP can be formed in the absence of tRNA, Cys-AMP synthesis is tRNA-dependent. Studies with purified tRNAs indicate that tRNA(Cys) promotes cysteine activation. The k(cat) values of wild-type ProCysRS for tRNA prolylation (0.09 s(-1)) and cysteinylation (0.02 s(-1)) demonstrate that both aminoacyl-tRNAs are synthesized with comparable rates, the cysteinyl-tRNA synthetase activity being only 4.5-fold lower than prolyl-tRNA synthetase activity. Kinetic analysis of ProCysRS mutant enzymes, generated by site-directed mutagenesis, shows glutamate at position 103 to be critical for proline binding, and proline at position 100 to be involved in cysteine binding. The proximity in ProCysRS of amino acid residues affecting binding of either cysteine or proline strongly suggests that structural elements of the two amino acid binding sites overlap.

Effect of peptidyltransferase inhibitors on ribonuclease P activity from Dictyostelium discoideum. Effect of antibiotics on RNase P.

The effect of several peptidyltransferase inhibitors on ribonuclease P activity from Dictyostelium discoideum was investigated. Among the inhibitors tested puromycin, amicetin and blasticidin S revealed a dose-dependent inhibition of tRNA maturation. Blasticidin S and amicetin do not compete with puromycin for the same site on the enzyme, suggesting the existence of distinct antibiotic binding sites on D. discoideum RNase P. Inhibition experiments further indicate that binding sites for blasticidin S and amicetin overlap.

A dual-specificity aminoacyl-tRNA synthetase in the deep-rooted eukaryote Giardia lamblia.

Cysteinyl-tRNA (Cys-tRNA) is essential for protein synthesis. In most organisms the enzyme responsible for the formation of Cys-tRNA is cysteinyl-tRNA synthetase (CysRS). The only known exceptions are the euryarchaea Methanococcus jannaschii and Methanobacterium thermoautotrophicum, which do not encode a CysRS. Deviating from the accepted concept of one aminoacyl-tRNA synthetase per amino acid, these organisms employ prolyl-tRNA synthetase as the enzyme that carries out Cys-tRNA formation. To date this dual-specificity prolyl-cysteinyl-tRNA synthetase (ProCysRS) is only known to exist in archaea. Analysis of the preliminary genomic sequence of the primitive eukaryote Giardia lamblia indicated the presence of an archaeal prolyl-tRNA synthetase (ProRS). Its proS gene was cloned and the gene product overexpressed in Escherichia coli. By using G. lamblia, M. jannaschii, or E. coli tRNA as substrate, this ProRS was able to form Cys-tRNA and Pro-tRNA in vitro. Cys-AMP formation, but not Pro-AMP synthesis, was tRNA-dependent. The in vitro data were confirmed in vivo, as the cloned G. lamblia proS gene was able to complement a temperature-sensitive E. coli cysS strain. Inhibition studies of CysRS activity with proline analogs (thiaproline and 5'-O-[N-(l-prolyl)-sulfamoyl]adenosine) in a Giardia S-100 extract predicted that the organism also contains a canonical CysRS. This prediction was confirmed by cloning and analysis of the corresponding cysS gene. Like a number of archaea, Giardia contains two enzymes, ProCysRS and CysRS, for Cys-tRNA formation. In contrast, the purified Saccharomyces cerevisiae and E. coli ProRS enzymes were unable to form Cys-tRNA under these conditions. Thus, the dual specificity is restricted to the archaeal genre of ProRS. G. lamblia's archaeal-type prolyl- and alanyl-tRNA synthetases refine our understanding of the evolution and interaction of archaeal and eukaryal translation systems.

Inhibition of eukaryotic ribonuclease P activity by aminoglycosides: kinetic studies.

The effect of several aminoglycoside antibiotics on ribonuclease P (RNase P) was investigated using an in vitro experimental system from Dictyostelium discoideum. Detailed kinetic analysis showed that all aminoglycosides tested (tobramycin, gentamicin, kanamycin, paromomycin, neomycin) behave as classical non-competitive inhibitors, with neomycin being the strongest inhibitor. The inhibition effect is attributed to the electrostatic competition of the cationic aminoglycosides with magnesium ions required for catalysis. Increasing Mg(2+) ion concentrations reduced the effect of aminoglycosides on RNase P activity. Detailed kinetic analysis showed that aminoglycosides compete with Mg(2+) for common binding sites on RNase P holoenzyme.

Secretion of virulence determinants by the general secretory pathway in gram-negative pathogens: an evolving story.

Secretion of proteins by the general secretory pathway (GSP) is a two-step process requiring the Sec translocase in the inner membrane and a separate substrate-specific secretion apparatus for translocation across the outer membrane. Gram-negative bacteria with pathogenic potential use the GSP to deliver virulence factors into the extracellular environment for interaction with the host. Well-studied examples of virulence determinants using the GSP for secretion include extracellular toxins, pili, curli, autotransporters, and crystaline S-layers. This article reviews our current understanding of the GSP and discusses examples of terminal branches of the GSP which are utilized by factors implicated in bacterial virulence.

The adaptor hypothesis revisited.

As originally postulated in Crick's Adaptor hypothesis, the faithful synthesis of proteins from messenger RNA is dependent on the presence of perfectly acylated tRNAs. The hypothesis also suggested that each aminoacyl-tRNA would be made by a unique enzyme. Recent data have now forced a revision of this latter point, with an increasingly diverse array of enzymes and pathways being implicated in aminoacyl-tRNA synthesis. These unexpected findings have far-reaching implications for our understanding of protein synthesis and its origins.