N-glycosylation in sugarcane

The N-linked glycosylation of secretory and membrane proteins is the most complex posttranslational modification known to occur in eukaryotic cells. It has been shown to play critical roles in modulating protein function. Although this important biological process has been extensively studied in mammals, much less is known about this biosynthetic pathway in plants. The enzymes involved in plant N-glycan biosynthesis and processing are still not well defined and the mechanism of their genetic regulation is almost completely unknown. In this paper we describe our first attempt to understand the N-linked glycosylation mechanism in a plant species by using the data generated by the Sugarcane Expressed Sequence Tag (SUCEST) project. The SUCEST database was mined for sugarcane gene products potentially involved in the N-glycosylation pathway. This approach has led to the identification and functional assignment of 90 expressed sequence tag (EST) clusters sharing significant sequence similarity with the enzymes involved in N-glycan biosynthesis and processing. The ESTs identified were also analyzed to establish their relative abundance.

Photosynthetic performance of freshwater Rhodophyta in response to temperature, irradiance, pH and diurnal rhythm

Responses of net photosynthetic rates to temperature, irradiance, pH/inorganic carbon and diurnal rhythm were analyzed in 15 populations of eight freshwater red algal species in culture and natural conditions. Photosynthetic rates were determined by oxygen concentration using the light and dark bottles technique. Parameters derived from the photosynthesis-irradiance curves indicated adaptation to low irradiance for all freshwater red algae tested, confirming that they tend to occur under low light regimes. Some degree of photoinhibition (β = -0.33-0.01 mg O2 g-1 DW h-1 (μmol photons m-2 s-1)-1) was found for all species/populations analyzed, whereas light compensation points (lc) were very low (≤ 2 μmol photons m- photons s-1) for most algae tested. Saturation points were low for all algae tested (lk = 6-54 μmol photons m-2 S-1; lS = 20-170 μmol photons m-2 s-1). Rates of net photosynthesis and dark respiration responded to the variation in temperature. Optimum temperature values for net photosynthesis were variable among species and populations so that best performances were observed under distinct temperature conditions (10, 15, 20 or 25°C). Rates of dark respiration exhibited an increasing trend with temperature, with highest values under 20-25°C. Results from pH experiments showed best photosynthetic performances under pH 8.5 or 6.5 for all but one species, indicating higher affinity for inorganic carbon as bicarbonate or indistinct use of bicarbonate and free carbon dioxide. Diurnal changes in photosynthetic rates revealed a general pattern for all algae tested, which was characterized by two relatively clear peaks, with some variations around it: a first (higher) during the morning (07.00-11.00 hours.) and a second (lower) in the afternoon (14.00-18.00 hours). Comparative data between the 'Chantransia' stage and the respective gametophyte for one Batrachospermum population revealed higher values (ca 2-times) in the latter, much lower than previously reported. The physiological role of the 'Chantransia' stage needs to be better analyzed.

Taxonomy and distribution of Paralemanea (Lemaneaceae, Rhodophyta) in Central Mexico

Three collections of Paralemanea from Central Mexico included two species. Paralemanea mexicana is large (length ≥ 4.0 cm; diameter > 400 μm) and generally branched (≥ 40 % of plants branched), with whorled branches, of first to second order. Paralemanea annulata is small (length < 5.0 cm ; diameter < 500 μm), generally unbranched (≤ 5 % of plants branched), with branches of first order. Spermatangial sori contained obovoid spermatangia, formed from cells of the outer cortical layers, extending above the thallus surface. Carpogonial branches are described for the first time in P. mexicana. They develop on lateral filaments at nodes or internodes and have ovoid to globular cells, abundantly branched at the basal portion, penetrating the cortex towards the thallus surface. Carposporophytes are sessile on the inner portion of the cortex and produce carpospores in chains of up to twelve. The 'Chantransia' stage was observed in P. mexicana. Paralemanea annulata is described for the first time from Mexico and P. mexicana is endemic from this country. Both species were collected in cold (temperature 12-16°C), acidic (pH 5.5-6.0), shallow (depth 1-60 cm) and moderate to fast flowing waters (> 35 cm s-1), in shaded or partly shaded river segments, on rocky substrata (mostly bedrock).

Genetic interactions of yeast eukaryotic translation initiation factor 5a (eIF5A) reveal connections to poly(A)-binding protein and protein kinase C signaling

The highly conserved eukaryotic translation initiation factor eIF5A has been proposed to have various roles in the cell, from translation to mRNA decay to nuclear protein export. To further our understanding of this essential protein, three temperature-sensitive alleles of the yeast TIF51A gene have been characterized. Two mutant eIF5A proteins contain mutations in a proline residue at the junction between the two eIFSA domains and the third, strongest allele encodes a protein with a single mutation in each domain, both of which are required for the growth defect. The stronger tif51A alleles cause defects in degradation of short-lived mRNAs, supporting a role for this protein in mRNA decay. A multicopy suppressor screen revealed six genes, the overexpression of which allows growth of a tif51A-1 strain at high temperature; these genes include PAB1, PKC1, and PKC1 regulators WSC1, WSC2, and WSC3. Further results suggest that eIFSA may also be involved in ribosomal synthesis and the WSC/PKC1 signaling pathway for cell wall integrity or related processes.

Photosynthetic characteristics of charophytes from tropical iotic ecosystems

Responses of photosynthetic rates, determined by oxygen evolution using the light and dark bottles technique, to different temperatures, irradiances, pH, and diurnal rhythm were analyzed under laboratory conditions in four charophyte species (Chara braunii Gmelin, C. guairensis R. Bicudo, Nitella subglomerata A. Braun and Nitella sp.) from Iotic habitats in southeastern Brazil. Parameters derived from the photosynthesis versus irradiance curves indicated affinity to low irradiances for all algae tested. Some degree of photoinhibition, [β = -(0.30-0.13) mg 02 g-1 dry weight h-1 (μmol photons m-2 s-1)-1], low light compensation points (lc = 4-20 μmol photons m-2 s-1) were found for all species analyzed, as well as low values of light saturation parameter (lk) and saturation (ls) 29-130 and 92-169 μmol photons m-2 S-1, respectively. Photoacclimation was observed in two populations of N. subglomerata collected from sites with different irradiances, consisting of variations in photosynthetic parameters (higher values of α, and lower of lk and maximum photosynthetic rate, Pmax, in the population under lower irradiance). The highest photosynthetic rates for Chara species were observed at 10-15°C, while for Nitella the highest photosynthetic rate was observed at 20-25°C, despite the lack of significant differences among most levels tested. Rates of dark respiration significantly increase with temperature, with the highest values at 25°C. The results from pH experiments showed highest photosynthetic rates under pH 4.0 for all algae, suggesting higher affinity for inorganic carbon in the form of carbon dioxide, except in one population of N. subglomerata, with similar rates under the three levels, suggesting indistinct use of bicarbonate and carbon dioxide. Diurnal changes in photosynthetic rates revealed a general pattern for most algae tested, which was characterized by two peaks: the first (higher) during the morning (07.00-11.00) and the second (lower) in the afternoon (14.00-17.00). This suggests an endogenous rhythm determining the daily variations in photosynthetic rates.

Blue-greenish acrochaetioid algae in freshwater habitats are Chantransia stages of Batrachospermales sensu lato (Rhodophyta)

Fourteen culture isolates of freshwater acrochaetioid algae from distinct regions around the world were analysed, including the reddish species Audouinella hermannii, the dubious blue-greenish species A. pygmaea, and Chantransia stages from distinct taxonomic origins in the Batrachospermales sensu lato (Batrachospermaceae, Lemaneaceae and Thoreaceae). Four isolates (two 'Chantransia' stages and two species of Audouinella, A. hermannii and A. pygmaea) were tested under experimental conditions of temperature (10-25°C), irradiance (65 and 300 μmol photons m-2 s-1) and photoperiod (16:8 h and 8:16 h light/dark cycles). Plant colour is proposed as the only vegetative character that can be unequivocally applied to distinguish Audouinella from 'Chantransia', blue-greenish representing Chantransia stages and reddish applying to true Audouinella species (also forming reproductive structures other than monosporangia, e.g. tetrasporangia). Some isolates of A. pygmaea were proven to be unequivocally 'Chantransia stages owing either to production of juvenile gametophytes or to derivation from carpospores. No association of the morphology of A. pygmaea was found with any particular species, thus it should be regarded as a complex involving many species of the Batrachospermales sensu lato, as is also the case with A. macrospora. We therefore recommend that all blue-greenish acrochaetioid algae in freshwater habitats be considered as Chantransia stages of members of the Batrachospermales, and that the informal descriptors pygmaea and macrospora be used to distinguish the two discernable morphologies. Induction of gametophytes occurred under much wider conditions than previously reported, reinforcing the conclusion that requirements are probably species-specific. Although phenotypic plasticity was in evidence, with temperature, irradiance and photoperiod affecting morphology, no alga showed variation outside the limits based on traditional taxonomic studies. No overall trend was observed for vegetative or reproductive characters in response to temperature, irradiance and photoperiod for all the algae tested, only for specific algae or characters. Effects of temperature and irradiance on morphological characters were more evident, as well as strong interactions between these variables, whereas few differences were generally found in response to photoperiod and irradiance.

Variation of genetic expression during development, revealed by esterase patterns in Aedes aegypti (Diptera, Culicidae)

Polyacrylamide gel electrophoresis was used to analyze esterase patterns during development of Aedes aegypti from the cities of Marília and São José do Rio Preto (SJRP), Brazil. The zymograms showed a total of 23 esterase bands, 22 of which were in the specimens from Marília and 19 in those from SJRP. These esterase bands were considered to be the product of 23 alleles distributed tentatively in eight genetic loci. Most of the alleles were developmentally regulated. The larval stage expressed the greatest number of them (19 alleles, from the eight loci, in Marília; and 17 alleles, from seven loci, in SJRP). The pupal stage expressed 10 alleles from seven loci, in both populations, and the adult stage expressed 8 alleles from five and six loci in SJRP and Marília, respectively. Some alleles that were active in every stage were developmentally controlled at the level of expression (amount of product). A single allele was constitutively and highly expressed, in larvae, pupae, and adults, in both populations. Differences in esterase synthesis among stages are probably due to regulatory mechanisms acting in agreement with the requirements of a variable number of processes in which esterases are involved. The larval stage is the most active in developmental processes and shows very intense intake of food and very high mobility. These features may demand increased esterase production at that stage. Comparison of the two populations examined showed (besides the existence of alleles that they do not share) that they exhibit differences in the control of expression of other alleles. Such findings may reflect genetic differences between founders in each population, but the possibility of involvement of the intensive use of insecticides in SJRP is also discussed.

Photosynthetic responses to temperature in tropical lotic macroalgae

A comparative analysis of the photosynthetic responses to temperature (10-30°C) was carried out under short-term laboratory conditions by chlorophyll fluorescence and oxygen (O2) evolution. Ten lotic macroalgal species from southeastern Brazil (20°11-20°48′S, 49°18-49°41′W) were tested, including Bacillariophyta, Chlorophyta, Cyanophyta, Rhodophyta and Xanthophyta. Temperature had significant effects on electron transport rate (ETR) only for three species (Terpsinoe musica, Bacillariophyta; Cladophora glomerata, Chlorophyta; and C. coeruleus, Rhodophyta), with highest values at 25-30°C, whereas the remaining species had no significant responses. It also had similar effects on non-photochemical quenching and ETR. Differences in net photosynthesis/dark respiration ratios at distinct temperatures were found, with an increasing trend of respiration with higher temperatures. This implies in a decreasing balance between net primary production and temperature, representing more critical conditions toward higher temperatures for most species. In contrast, high net photosynthesis and photosynthesis/dark respiration ratios at high and wide ranges of temperature were found in three species of green algae, suggesting that these algae can be important primary producers in lotic ecosystems, particularly in tropical regions. Optimal photosynthetic rates were observed under similar environmental temperatures for five species (two rhodophytes, two chlorophytes and one diatom) considering both techniques, suggesting acclimation to their respective ambient temperatures. C. coeruleus was the only species with peaks of ETR and O 2 evolution under similar field-measured temperatures. All species kept values of ETR and net photosynthesis close to the optimum under a broad range of temperatures. Increased non-photochemical quenching, as a measure of thermal dissipation of excess energy, toward higher temperatures was observed in some species, as well as positive correlation of non-photochemical quenching with ETR, and were interpreted as two mechanisms of adaptation of the photosynthetic apparatus to temperature changes. Different optimal temperatures were found for individual species by each technique, generally under lower temperatures by O2 evolution, indicating dependence on distinct factors: increases in temperature generally induced higher ETR due to increased enzymatic activity, whereas increments of enzymatic activity were compensated by increased respiration and photorespiration leading to decreases in net photosynthesis.

Identification and frequency of transposable elements in Eucalyptus

Transposable elements (TE) are major components of eukaryotic genomes and involved in cell regulation and organism evolution. We have analyzed 123,889 expressed sequence tags of the Eucalyptus Genome Project database and found 124 sequences representing 76 TE in 9 groups, of which copia, MuDR and FAR1 groups were the most abundant. The low amount of sequences of TE may reflect the high efficiency of repression of these elements, a process that is called TE silencing. Frequency of groups of TE in Eucalyptus libraries which were prepared with different tissues or physiologic conditions from seedlings or adult plants indicated that developing plants experience the expression of a much wider spectrum of TE groups than that seen in adult plants. These are preliminary results that identify the most relevant TE groups involved with Eucalyptus development, which is important for industrial wood production. Copyright by the Brazilian Society of Genetics.

Inhibition of eukaryotic translation initiation factor 5A (eIF5A) hypusination impairs melanoma growth

The eukaryotic translation initiation factor 5A (eIF5A) undergoes a specific post-translational modification called hypusination. This modification is required for the functionality of this protein. The compound N1-guanyl-1,7-diaminoheptane (GC7) is a potent and selective inhibitor of deoxyhypusine synthase, which catalyses the first step of eIF5A hypusination process. In the present study, the effects of GC7 on cell death were investigated using two cell lines: melan-a murine melanocytes and Tm5 marine melanoma. In vitro treatment with GC7 increased by 3-fold the number of cells presenting DNA fragmentation in Tm5 cells. Exposure to GC7 also decreased viability to both cell lines. This study also describes, for the first time, the in vivo antitumour effect of GC7, as indicated by impaired melanoma growth in C57BL/6 mice. Copyright © 2006 John Wiley & Sons, Ltd.

Use of a synthetic lethal screen to identify genes related to TIF51A in Saccharomyces cerevisiae

The putative eukaryotic translation initiation factor 5A (eIF5A) is an essential protein for cell viability and the only cellular protein known to contain the unusual amino acid residue hypusine. eIF5A has been implicated in translation initiation, cell proliferation, nucleocytoplasmic transport, mRNA decay, and actin polarization, but the precise biological function of this protein is not clear. However, eIF5A was recently shown to be directly involved with the translational machinery. A screen for synthetic lethal mutations was carried out with one of the temperature-sensitive alleles of TIF51A (tif51A-3) to identify factors that functionally interact with eIF5A and revealed the essential gene YPT1. This gene encodes a small GTPase, a member of the rab family involved with secretion, acting in the vesicular trafficking between endoplasmatic reticulum and the Golgi. Thus, the synthetic lethality between TIF51A and YPT1 may reveal the connection between translation and the polarized distribution of membrane components, suggesting that these proteins work together in the cell to guarantee proper protein synthesis and secretion necessary for correct bud formation during G1/ S transition. Future studies will investigate the functional interaction between eIF5A and Ypt1 in order to clarify this involvement of eIF5A with vesicular trafficking. ©FUNPEC-RP.

UMP kinase from Mycobacterium tuberculosis: Mode of action and allosteric interactions, and their likely role in pyrimidine metabolism regulation

The pyrH-encoded uridine 5′-monophosphate kinase (UMPK) is involved in both de novo and salvage synthesis of DNA and RNA precursors. Here we describe Mycobacterium tuberculosis UMPK (MtUMPK) cloning and expression in Escherichia coli. N-terminal amino acid sequencing and electrospray ionization mass spectrometry analyses confirmed the identity of homogeneous MtUMPK. MtUMPK catalyzed the phosphorylation of UMP to UDP, using ATP-Mg 2+ as phosphate donor. Size exclusion chromatography showed that the protein is a homotetramer. Kinetic studies revealed that MtUMPK exhibits cooperative kinetics towards ATP and undergoes allosteric regulation. GTP and UTP are, respectively, positive and negative effectors, maintaining the balance of purine versus pyrimidine synthesis. Initial velocity studies and substrate(s) binding measured by isothermal titration calorimetry suggested that catalysis proceeds by a sequential ordered mechanism, in which ATP binds first followed by UMP binding, and release of products is random. As MtUMPK does not resemble its eukaryotic counterparts, specific inhibitors could be designed to be tested as antitubercular agents. © 2010 Elsevier Inc. All rights reserved.

Structural insights into selectivity and cofactor binding in snake venom l-amino acid oxidases

l-Amino acid oxidases (LAAOs) are flavoenzymes that catalytically deaminate l-amino acids to corresponding α-keto acids with the concomitant production of ammonia (NH 3) and hydrogen peroxide (H 2O 2). Particularly, snake venom LAAOs have been attracted much attention due to their diverse clinical and biological effects, interfering on human coagulation factors and being cytotoxic against some pathogenic bacteria and Leishmania ssp. In this work, a new LAAO from Bothrops jararacussu venom (BjsuLAAO) was purified, functionally characterized and its structure determined by X-ray crystallography at 3.1å resolution. BjsuLAAO showed high catalytic specificity for aromatic and aliphatic large side-chain amino acids. Comparative structural analysis with prokaryotic LAAOs, which exhibit low specificity, indicates the importance of the active-site volume in modulating enzyme selectivity. Surprisingly, the flavin adenine dinucleotide (FAD) cofactor was found in a different orientation canonically described for both prokaryotic and eukaryotic LAAOs. In this new conformational state, the adenosyl group is flipped towards the 62-71 loop, being stabilized by several hydrogen-bond interactions, which is equally stable to the classical binding mode. © 2012 Elsevier Inc.

EIF5A dimerizes not only in vitro but also in vivo and its molecular envelope is similar to the EF-P monomer

The protein eukaryotic initiation factor 5A (eIF5A) is highly conserved among archaea and eukaryotes, but not in bacteria. Bacteria have the elongation factor P (EF-P), which is structurally and functionally related to eIF5A. eIF5A is essential for cell viability and the only protein known to contain the amino acid residue hypusine, formed by post-translational modification of a specific lysine residue. Although eIF5A was initially identified as a translation initiation factor, recent studies strongly support a function for eIF5A in the elongation step of translation. However, the mode of action of eIF5A is still unknown. Here, we analyzed the oligomeric state of yeast eIF5A. First, by using size-exclusion chromatography, we showed that this protein exists as a dimer in vitro, independent of the hypusine residue or electrostatic interactions. Protein-protein interaction assays demonstrated that eIF5A can form oligomers in vitro and in vivo, in an RNA-dependent manner, but independent of the hypusine residue or the ribosome. Finally, small-angle X-ray scattering (SAXS) experiments confirmed that eIF5A behaves as a stable dimer in solution. Moreover, the molecular envelope determined from the SAXS data shows that the eIF5A dimer is L-shaped and superimposable on the tRNAPhe tertiary structure, analogously to the EF-P monomer. © 2012 Springer-Verlag.

The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication

Background: Yellow fever virus (YFV) belongs to the Flavivirus genus and causes an important disease. An alarming resurgence of viral circulation and the expansion of YFV-endemic zones have been detected in Africa and South America in recent years. NS5 is a viral protein that contains methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, which are essential for viral replication, and the interactions between NS5 and cellular proteins have been studied to better understand viral replication. The aim of this study was to characterize the interaction of the NS5 protein with eukaryotic translation initiation factor 3 subunit L (eIF3L) and to evaluate the role of eIF3L in yellow fever replication. Methods. To identify interactions of YFV NS5 with cellular proteins, we performed a two-hybrid screen using the YFV NS5 RdRp domain as bait with a human cDNA library, and RNApol deletion mutants were generated and analyzed using the two-hybrid system for mapping the interactions. The RNApol region involved was segmented into three fragments and analyzed using an eIF3L-expressing yeast strain. To map the NS5 residues that are critical for the interactions, we performed site-direct mutagenesis in segment 3 of the interaction domain (ID) and confirmed the interaction using in vitro assays and in vivo coimmunoprecipitation. The significance of eIF3L for YFV replication was investigated using eIF3L overexpression and RNA interference. Results: In this work, we describe and characterize the interaction of NS5 with the translation factor eIF3L. The interaction between NS5 and eIF3L was confirmed using in vitro binding and in vivo coimmunoprecipitation assays. This interaction occurs at a region (the interaction domain of the RNApol domain) that is conserved in several flaviviruses and that is, therefore, likely to be relevant to the genus. eIF3L overexpression and plaque reduction assays showed a slight effect on YFV replication, indicating that the interaction of eIF3L with YFV NS5 may play a role in YFV replication. Conclusions: Although the precise function of eIF3L on interactions with viral proteins is not entirely understood, these results indicate an interaction of eIF3L with YF NS5 and that eIF3L overexpression facilitates translation, which has potential implications for virus replication. © 2013 Morais et al.; licensee BioMed Central Ltd.