Mutational analyses of human eIF5A-1-identification of amino acid residues critical for eIF5A activity and hypusine modification

The eukaryotic translation initiation factor 5A (eIF5A) is the only protein that contains hypusine [N-epsilon-(4-amino-2-hydroxybutyl)lysine], which is required for its activity. Hypusine is formed by post-translational modification of one specific lysine (Lys50 for human eIF5A) by deoxyhypusine synthase and deoxyhypusine hydroxylase. To investigate the features of eIF5A required for its activity, we generated 49 mutations in human eIF5A-1, with a single amino acid substitution at the highly conserved residues or with N-terminal or C-terminal truncations, and tested mutant proteins in complementing the growth of a Saccharomyces cerevisiae eIF5A null strain. Growth-supporting activity was abolished in only a few mutant eIF5As (K47D, G49A, K50A, K50D, K50I, K50R, G52A and K55A), with substitutions at or near the hypusine modification site or with truncation of 21 amino acids from either the N-terminus or C-terminus. The inactivity of the Lys50 substitution proteins is obviously due to lack of deoxyhypusine modification. In contrast, K47D and G49A were effective substrates for deoxyhypusine synthase, yet failed to support growth, suggesting critical roles of Lys47 and Gly49 in eIF5A activity, possibly in its interaction with effector(s). By use of a UBHY-R strain harboring genetically engineered unstable eIF5A, we present evidence for the primary function of eIF5A in protein synthesis. When selected eIF5A mutant proteins were tested for their activity in protein synthesis, a close correlation was observed between their ability to enhance protein synthesis and growth, lending further support for a central role of eIF5A in translation.

Aditivos na ensilagem do capim-elefante. II. Qualidade das silagens amostradas por dois métodos

Silages of Pennisetum purpureum Schum. cv. Guaçu prepared with 0, 8, 16 and 24% of ground ear com with husks, wheat bran and saccharin, dry weight of additive/wet weight of green chop basis. The experimental design was a randomized blocks one in split-plot; the plots were the additives and levels, and the sub-plots the sampling methods. The material was ensiled using plastic vessels in middle of which holed pvc pipes (3 inches diameter) were put. These pipes (one per vessel) had the same length as the height of the vessels, and were filled at the same time and compacted the same way as the vessels. The first method of sampling used the material ensiled inside the pvc pipe, which was lifted out from the vessel at the moment of the silo opening. The other sampling method, normaly used in digestibility trials, consisted of samples composed by daily sub-samples collected in the vessels. The pvc sampling method was more efficient because it sampled a profile of the whole silage. All of the silages showed high percentages of lactic acid and low percentages or even absence of butyric acid, though in all silages high ammoniacal-N percentages were detected.

Cellulolytic activity of wild type and mutant Trichoderma pseudokoningii

Strains of Trichoderma pseudokoningii are promising objects for genetic studies and cellulase production. Auxotrophic mutants with deficiencies in the biosynthesis of aminoacids, nucleotides and vitamins (up to five markers) in addition to morphological aspects like conidial colour were obtained from two strains of double auxotrophic mutants using UV radiation. In order to compare the cellulolytic capabilities of the T. pseudokoningii (wild type strain), some of its mutants and T. reesei QM9414 we performed semiquantitative cellulase assays and quantitative determination of the enzymes exoglucanase and endoglucanase. The semiquantitative test showed that the strains with minimal mycelial growth rate were better producers. Both tests revealed that two of the studied mutants, TG3 and TG4 presented a yield higher than the wild type, reaching 30% more exoglucanase and 70% more endoglucanase. These results indicate that the wild type was improved for cellulase production. Highly significant values of correlation were found for exoglucanase and endoglucanase activities, suggesting that these enzymes may be co-regulated in T. pseudokoningii.

Mapping eIF5A binding sites for Dys1 and Lia1: In vivo evidence for regulation of eIF5A hypusination

The evolutionarily conserved factor eIF5A is the only protein known to undergo hypusination, a unique posttranslational modification triggered by deoxyhypusine synthase (Dys1). Although eIF5A is essential for cell viability, the function of this putative translation initiation factor is still obscure. To identify eIF5A-binding proteins that could clarify its function, we screened a two-hybrid library and identified two eIF-5A partners in S. cerevisiae: Dys1 and the protein encoded by the gene YJR070C, named Lia1 (Ligand of eIF5A). The interactions were confirmed by GST pulldown. Mapping binding sites for these proteins revealed that both eIF5A domains can bind to Dys1, whereas the C-terminal domain is sufficient to bind Lia1. We demonstrate for the first time in vivo that the N-terminal α-helix of Dys1 can modulate enzyme activity by inhibiting eIF5A interaction. We suggest that this inhibition be abrogated in the cell when hypusinated and functional eIF5A is required. © 2003 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Influence of the collection methodology on the Apis mellifera venom composition: Peptide analysis

Differences of venom peptide composition as function of two collection methodologies, electrical stimulation (ES) and reservoir disruption (RD), were analyzed by reverse-phase HPLC in Apis mellifera races - A. m. adansonii, A. m. ligustica and Africanized honeybee. The analyses were performed through determination of the relative number and percentage of each molecular form associated to the peaks eluted by chromatography. Comparison of these profiles revealed qualitative and quantitative differences related to the venom collection methodology as well to the three races analyzed. In contrast to data usually found for venom proteins, the three races presented a major number of peaks or molecular forms when venom was collected by ES. Besides, in general, the relative concentration of each peak was higher for ES in relation to RD. That indicates the presence of molecular precursors in the venom obtained by RD. The presence/absence pattern of the peaks, such as their relative concentrations showed a closer similarity between A. m. adansonii and the Africanized honeybees than that observed between these and A. m. ligustica. The obtained data allowed a discussion about the differences in the relative concentration of each venom component according to the collection methodology, and finally the biological action of the venom in different races. So, these results, apart from being useful to establish a peptide profile for each bee race as a function of the venom collection methodology, pointed out once more that the chromatographic techniques are a great tool for the identification of A. mellifera subspecies.

eIF5A: Uma proteína essencial para a viabilidade celular cuja função permanece obscura

The putative translation initiation factor 5A (eIF5A) is a highly abundant and conserved protein in all eukaryotes and archaebacteria. This factor is essential for cell viability and is the only cellular protein known to contain the unusual amino acid residue hypusine. In Saccharomyces cerevisiae eIF5A is expressed in aerobic conditions by the gene TIF51A. Although eIF5A has been known for almost 30 years, the biological role of this protein is still obscure. This article reviews the research on the function of eIF5A, discussing the evidence for its involvement in various steps of mRNA metabolism, including translation initiation, nucleocytoplasmic transport and mRNA decay. Moreover, it indicates other studies that have associated eIF5A with cell proliferation and cell cycle progression. Finally, this review presents recent results obtained in our laboratory that reemphasize the role of eIF5A in the translation scenario. Further experiments will be necessary to define the role played by eIF5A in the translational machinery.

Pectin and pectinases: Production, characterization and industrial application of microbial pectinolytic enzymes

Pectinases are a big group of enzymes that break down pectic polysaccharides of plant tissues into simpler molecules like galacturonic acids. It has long been used to increase yields and clarity of fruit juices. Since pectic substances are a very complex macromolecule group, various pectinolytic enzymes are required to degrade it completely. These enzymes present differences in their cleavage mode and specificity being basically classified into two main groups that act on pectin smooth regions or on pectin hairy regions. Pectinases are one of the most widely distributed enzymes in bacteria, fungi and plants. This review describes the pectinolytic enzymes and their substrates, the microbial pectinase production and characterization, and the industrial application of these enzymes. © Pedrolli et al.; Licensee Bentham Open.

Chaos induced by turbulent and erratic functions

Let (X, d) be a compact metric space and f: X → X a continuous function and consider the hyperspace (K(X), H) of all nonempty compact subsets of X endowed with the Hausdorff metric induced by d. Let f̄: K(X) → K (X) be defined by f̄(A) = {f(a)/a ∈ A} the natural extension of f to K(X), then the aim of this work is to study the dynamics of f when f is turbulent (erratic, respectively) and its relationships.

Measurement of the charge ratio of atmospheric muons with the CMS detector

We present a measurement of the ratio of positive to negative muon fluxes from cosmic ray interactions in the atmosphere, using data collected by the CMS detector both at ground level and in the underground experimental cavern at the CERN LHC. Muons were detected in the momentum range from 5 GeV/. c to 1 TeV/. c. The surface flux ratio is measured to be 1.2766±0.0032(stat.)±0.0032(syst.), independent of the muon momentum, below 100 GeV/. c. This is the most precise measurement to date. At higher momenta the data are consistent with an increase of the charge ratio, in agreement with cosmic ray shower models and compatible with previous measurements by deep-underground experiments. © 2010.

Measurement of the muon stopping power in lead tungstate

A large sample of cosmic ray events collected by the CMS detector is exploited to measure the specific energy loss of muons in the lead tungstate (PbWO4) of the electromagnetic calorimeter. The measurement spans a momentum range from 5 GeV/c to 1 TeV/c. The results are consistent with the expectations over the entire range. The calorimeter energy scale, set with 120 GeV/c electrons, is validated down to the sub-GeV region using energy deposits, of order 100 MeV, associated with low-momentum muons. The muon critical energy in PbWO4 is measured to be 160+5 -68 GeV, in agreement with expectations. This is the first experimental determination of muon critical energy. © 2010 IOP Publishing Ltd and SISSA.

Commissioning of the CMS experiment and the cosmic run at four tesla

The CMS Collaboration conducted a month-long data-taking exercise known as the Cosmic Run At Four Tesla in late 2008 in order to complete the commissioning of the experiment for extended operation. The operational lessons resulting from this exercise were addressed in the subsequent shutdown to better prepare CMS for LHC beams in 2009. The cosmic data collected have been invaluable to study the performance of the detectors, to commission the alignment and calibration techniques, and to make several cosmic ray measurements. The experimental setup, conditions, and principal achievements from this data-taking exercise are described along with a review of the preceding integration activities. © 2010 IOP Publishing Ltd and SISSA.

Performance of the CMS Level-1 trigger during commissioning with cosmic ray muons and LHC beams

The CMS Level-1 trigger was used to select cosmic ray muons and LHC beam events during data-taking runs in 2008, and to estimate the level of detector noise. This paper describes the trigger components used, the algorithms that were executed, and the trigger synchronisation. Using data from extended cosmic ray runs, the muon, electron/photon, and jet triggers have been validated, and their performance evaluated. Efficiencies were found to be high, resolutions were found to be good, and rates as expected. © 2010 IOP Publishing Ltd and SISSA.