Identification of an NTF2-related factor that binds Ran-GTP and regulates nuclear protein export

Active transport of macromolecules between the nucleus and cytoplasm requires signals for import and export and their recognition by shuttling receptors. Each class of macromolecule is thought to have a distinct receptor that mediates the transport reaction. Assembly and disassembly reactions of receptor-substrate complexes are coordinated by Ran, a GTP-binding protein whose nucleotide state is regulated catalytically by effector proteins. Ran function is modulated in a noncatalytic fashion by NTF2, a protein that mediates nuclear import of Ran-GDP. Here we characterize a novel component of the Ran system that is 26% identical to NTF2, which based on its function we refer to as NTF2-related export protein 1 (NXT1). In contrast to NTF2, NXT1 preferentially binds Ran-GTP, and it colocalizes with the nuclear pore complex (NPC) in mammalian cells. These properties, together with the fact that NXT1 shuttles between the nucleus and the cytoplasm, suggest an active role in nuclear transport. Indeed, NXT1 stimulates nuclear protein export of the NES-containing protein PKI in vitro. The export function of NXT1 is blocked by the addition of leptomycin B, a compound that selectively inhibits the NES receptor Crm1. Thus, NXT1 regulates the Crm1-dependent export pathway through its direct interaction with Ran-GTP.     

A phylogenetically based secondary structure for the yeast telomerase RNA

BACKGROUND: Telomerase is a ribonucleoprotein complex whose RNA moiety dictates the addition of specific simple sequences onto chromosomes ends. While relevant for certain human genetic diseases, the contribution of the essential telomerase RNA to RNP assembly still remains unclear. Phylogenetic analyses of vertebrate and ciliate telomerase RNAs revealed conserved elements that potentially organize protein subunits for RNP function. In contrast, the yeast telomerase RNA could not be fitted to any known structural model, and the limited number of known sequences from Saccharomyces species did not permit the prediction of a yeast specific conserved structure. RESULTS: We cloned and analyzed the complete telomerase RNA loci (TLC1) from all known Saccharomyces species belonging to the "sensu stricto" group. Complementation analyses in S. cerevisiae and end mappings of mature RNAs ensured the relevance of the cloned sequences. By using phylogenetic comparative analysis coupled with in vitro enzymatic probing, we derived a secondary structure prediction of the Saccharomyces cerevisiae TLC1 RNA. This conserved secondary structure prediction includes a central domain that is likely to orchestrate DNA synthesis and at least two accessory domains important for RNA stability and telomerase recruitment. The structure also reveals a potential tertiary interaction between two loops in the central core. CONCLUSIONS: The predicted secondary structure of the TLC1 RNA of S. cerevisiae reveals a distinct folding pattern featuring well-separated but conserved functional elements. The predicted structure now allows for a detailed and rationally designed study to the structure-function relationships within the telomerase RNP-complex in a genetically tractable system.     

Fluorescent epsilon-ATP analogues for probing physicochemical properties of proteins. Synthesis, biochemical evaluation, and sensitivity to properties of the medium

Despite the significance of the elucidation of proteins' physicochemical parameters to understand various molecular phenomena, direct methods for measuring these parameters are not readily available. Here, we propose the use of 8-[p-amino-Ph]-epsilon-ATP, 3b, as a fluorescent probe for the elucidation of physicochemical parameters of binding sites in certain proteins. We synthesized novel fluorescent nucleotide analogues based on an extension of the epsilon-ATP scaffold. These analogues bear a primary or tertiary p-amino-phenyl moiety on the etheno-bridge. We explored the recognition of the fluorescent analogues by the target proteins: P2Y(1)-receptor (P2Y(1)-R) and NTPDase1. Based on the high affinity to the P2Y(1)-R (EC(50) 100nM), 3b proved a suitable probe for the investigation of this receptor. Next, we elucidated the dependencies of the absorption and emission spectra of 3b on environmental parameters, for establishing correlation equations. These equations will help determine the properties of the ATP-binding site from the spectral data of the protein-bound 3b. For this purpose, the sensitivity of the probe to acidity, dielectricity, H-bonding, viscosity, and to correlation between these parameters was determined. Thus, the pH-dependence of 3b emission intensity is bell shaped. At pH2.8 the quantum yield (phi) is enhanced 150-fold, as compared to neutral pH. The basic nitrogen atoms of 3b were assigned and pK(a) values were determined. A linear relationship was found between log phi and log viscosity, however, emission maxima (lambda(max)) remained constant. A linear relationship was found between both phi and lambda(max) and dielectricity, as measured in protic or aprotic solvents of comparable viscosity. pK(a)-like values were measured in acid-titrated alcohols with varying dielectricity but comparable viscosity, or with varying viscosity but comparable dielectricity. An inverse relationship and a linear relationship were found between the pK(a) values of 3b and the medium dielectricity and viscosity, respectively. These correlations help the calibration of properties of a protein ATP-binding site.     

Carbapenem Non-Susceptible Enterobacteriaceae in Quebec,Canada: Results of a Laboratory Surveillance Program (2010–2012)

The emergence and spread of carbapenemase-producing Enterobacteriaceae (CPE) represent a major public health concern because these bacteria are usually extensively resistant to most antibiotics. In order to evaluate their dissemination in Quebec, a surveillance program was introduced in 2010. We report the molecular and epidemiological profiles of CPE isolates collected. Between August 2010 and December 2012, a total of 742 non-duplicate isolates non-susceptible to carbapenems were analysed. AmpC β-lactamase and metallo-β-lactamase production were detected by Etest and carbapenemase production by the modified Hodge test (MHT). Antibiotic susceptibility profiles were determined using broth microdilution or Etest. Clonality of Klebsiella pneumoniae carbapenemase (KPC) strains was analyzed by pulsed-field gel electrophoresis (PFGE). The presence of genes encoding carbapenemases as well as other β-lactamases was detected using PCR. Of the 742 isolates tested, 169 (22.8%) were CPE. Of these 169 isolates, 151 (89.3%) harboured a bla _KPC gene while the remaining isolates carried bla _SME (n = 9), bla _OXA-48 (n = 5), bla _NDM (n = 3), and bla _NMC (n = 1) genes. Among the 93 KPC strains presenting with a unique pattern (unique PFGE pattern and/or unique antibiotics susceptibility profile), 99% were resistant to ertapenem, 95% to imipenem, 87% to meropenem, 97% to aztreonam, 31% to colistin and 2% to tigecycline. In 19 patients, 2 to 5 KPC strains from different species or with a different PFGE pattern were isolated. CPE strains were present in the province of Quebec with the majority of strains harbouring KPC. Alternately, SME, OXA-48 and NMC containing strains were rarely found.     

A Side by Side Comparison of Bruker Biotyper and VITEK MS: Utility of MALDI-TOF MS Technology for Microorganism Identification in a Public Health Reference Laboratory

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has emerged as a rapid, highly accurate, and cost-effective method for routine identification of a wide range of microorganisms. We carried out a side by side comparative evaluation of the performance of Bruker Biotyper versus VITEK MS for identification of a large and diverse collection of microorganisms. Most difficult and/or unusual microorganisms, as well as commonly encountered microorganisms were selected, including Gram-positive and negative bacteria, mycobacteria, actinomycetes, yeasts and filamentous fungi. Six hundred forty two strains representing 159 genera and 441 species from clinical specimens previously identified at the Laboratoire de santé publique du Québec (LSPQ) by reference methods were retrospectively chosen for the study. They included 254 Gram-positive bacteria, 167 Gram-negative bacteria, 109 mycobacteria and aerobic actinomycetes and 112 yeasts and moulds. MALDI-TOF MS analyses were performed on both systems according to the manufacturer’s instructions. Of the 642 strains tested, the name of the genus and / or species of 572 strains were referenced in the Bruker database while 406 were present in the VITEK MS IVD database. The Biotyper correctly identified 494 (86.4%) of the strains, while the VITEK MS correctly identified 362 (92.3%) of the strains (excluding 14 mycobacteria that were not tested). Of the 70 strains not present in the Bruker database at the species level, the Biotyper correctly identified 10 (14.3%) to the genus level and 2 (2.9%) to the complex/group level. For 52 (74.2%) strains, we obtained no identification, and an incorrect identification was given for 6 (8.6%) strains. Of the 178 strains not present in the VITEK MS IVD database at the species level (excluding 71 untested mycobacteria and actinomycetes), the VITEK MS correctly identified 12 (6.8%) of the strains each to the genus and to the complex/group level. For 97 (54.5%) strains, no identification was given and for 69 (38.7%) strains, an incorrect identification was obtained. Our study demonstrates that both systems gave a high level (above 85%) of correct identification for a wide range of microorganisms. However, VITEK MS gave more misidentification when the microorganism analysed was not present in the database, compared to Bruker Biotyper. This should be taken into account when this technology is used alone for microorganism identification in a public health laboratory, where isolates received are often difficult to identify and/or unusual microorganisms.     

Rhizosphere persistence of three Pythium oligandrum strains in tomato soilless culture assessed by DNA macroarray and real-time PCR

In tomato soilless culture, plant-disease optimal control and growth promotion are achieved when the rhizosphere is heavily colonized by the biocontrol agent Pythium oligandrum. Discrepancies in performance are generally attributed to the poor persistence of P. oligandrum on roots. In this study, three selected strains of P. oligandrum were introduced into the rhizosphere of greenhouse-grown tomato plants, and their persistence was assessed by DNA macroarray hybridization and real-time PCR. The experimental data from DNA detection and plate counting were compared. PCR-based methods detected P. oligandrum throughout the 6-month growing season, whereas plate counting indicated its presence only over the first 3 months. Moreover, the DNA array method provided information about the various Pythium species present in the rhizosphere: P. dissotocum was frequently detected on roots of plants, without distinction between plants inoculated or not inoculated with the antagonist. The detection of other Pythium species was noticed sporadically (P. ultimum, P. sylvaticum and P. intermedium), independent of the treatment. Even though the yield enhancement is not significant throughout the entire growing season, data obtained from epidemiological studies demonstrate an enhancement of P. oligandrum persistence on the rhizosphere of plants and less use of mycoparasitism.     

Rapid measurement of deuterium-labeled long-chain fatty acids in plasma by HPLC-ESI-MS

Imbalanced fatty acid metabolism contributes significantly to the increased incidence of metabolic disorders. Isotope-labeled fatty acids (2H, 13C) provide efficient means to trace fatty acid metabolism in vivo. This study reports a new and rapid method for the quantification of deuterium-labeled fatty acids in plasma by HPLC-MS. The sample preparation protocol developed required only hydrolysis, neutralization, and quenching steps followed by high-performance liquid chromatography-electrospray ionization-mass spectrometry analysis in negative ion mode using single ion monitoring. Deuterium-labeled stearic acid (d7-C18:0) was synthesized to reduce matrix interference observed with d5 analog, which improved the limit of detection (LOD) significantly, depending on the products analyzed. Linearity > 0.999 between the LOD (100 nM) and 30 microM, accuracy > 90%, precision > 88%, and adequate recovery in the dynamic range were obtained for d7-C18:0 and d7-oleic acid (C18:1). Upon oral dosing of d7-C18:0 in rats, the parent compound and its desaturation and beta-oxidation products, d7-C18:1 and d7-C16:0, were circulating with a maximal concentration ranging from 0.6 to 2.2 microM, with significant levels of d7-fatty acids detected for up to 72 h.     

In vitro biotransformations of the prostaglandin D2 (DP) antagonist MK-0524 and synthesis of metabolites

Metabolites of the potent DP antagonist, MK-0524, were generated using in vitro systems including hepatic microsomes and hepatocytes. Four metabolites (two hydroxylated diastereomers, a ketone and an acyl glucuronide) were characterized by LC-MS/MS and 1H NMR. Larger quantities of these metabolites were prepared by either organic synthesis or biosynthetically to be used as standards in other studies. The propensity for covalent binding was assessed and was found to be acceptable (<50 pmol-equiv/mg protein).