Cell wall proteomics of sugarcane cell suspension cultures

The use of cell walls to produce cellulosic ethanol from sugarcane bagasse is a new challenge. A better knowledge of proteins involved in cell wall remodelling is essential to improve the saccharification processes. Cell suspension cultures were used for this first cell wall proteomics study of sugarcane. Proteins extracted from cell walls were identified using an adapted protocol. They were extracted using 0.2 M CaCl₂ and 2 M LiCl after purification of cell walls. The proteins were then identified by the innovative nanoACQUITY UPLC MS/MS technology and bioinformatics using the translated SUCEST EST cluster database of sugarcane. The experiments were reproduced three times. Since Sorghum bicolor is the closest plant with a fully sequenced genome, homologous proteins were searched for to complete the annotation of proteins, that is, prediction of subcellular localization and functional domains. Altogether, 69 different proteins predicted to be secreted were identified among 377 proteins. The reproducibility of the experiments is discussed. These proteins were distributed into eight functional classes. Oxidoreductases such as peroxidases were well represented, whereas glycoside hydrolases were scarce. This work provides information about the proteins that could be manipulated through genetic transformation, to increase second‐generation ethanol production.     

Microsatellite loci from the northern house mosquito (Culex pipiens), a principal vector of West Nile virus in North America

Microsatellites were isolated and characterized in the northern house mosquito, Culex pipiens, a widespread pest species and important vector of diseases such as West Nile virus. An enrichment protocol yielded 150 positive clones. We designed primers to amplify 17 unique (GT)_n microsatellites, eight of which amplified cleanly and were polymorphic. A survey of 29 individuals showed that these loci are highly variable with the number of alleles ranging from seven to 19 and expected heterozygosity ranging from 0.66 to 0.93. These markers will be useful for studies of population structure and intraspecific variation in epidemiological characteristics of Cx. pipiens.     

Habitat specific growth rates and condition indices for the sympatric soles Solea solea (Linnaeus, 1758) and Solea senegalensis Kaup 1858, in the Tagus estuary, Portugal, based on otolith daily increments and RNA-DNA ratio

Habitat specific growth rates and condition indices were estimated for Solea solea and Solea senegalensis, in two nursery areas within the Tagus estuary, at the end of the estuarine colonization process, in 2005. While in the uppermost nursery area the two species of sole live in sympatry, in the lower nursery only S. senegalensis is present. Daily increments of left lapillar otoliths were used to estimate age (in days) and determine growth rates (mm per day). Condition indices were assessed through RNA‐DNA ratio in muscle samples. Growth rates were higher for S. senegalensis (0.970and 1.180 mm per day in nursery A and B, respectively) than for S. solea (0.767 mm per day in nursery A). Growth rates of S. senegalensis from the uppermost nursery area were lower when compared to those obtained for the other nursery. The RNA/DNA condition index followed the general trend given by the growth rate estimates, i.e. values were higher for S. senegalensis than for S. solea. However, no significant differences were detected in S. senegalensis from the two nurseries. Larger variations in salinity (10‰ amplitude in the uppermost nursery vs 0.2‰ in the lower nursery) and highest pollution loads may be important factors lowering the habitat quality of the uppermost nursery in comparison to the lower nursery. The use of growth rate estimates based on otolith readings and the RNA/DNA index as tools for habitat quality assessment was discussed.     

Ouabain-insensitive Na+-ATPase of proximal tubules is an effector for urodilatin and atrial natriuretic peptide

In the present paper we studied the effect of urodilatin and atrial natriuretic peptide (ANP) on the proximal tubule Na+-ATPase and (Na+K+)ATPase activities. Urodilatin and ANP inhibit the Na+-ATPase activity but not the (Na+K+)ATPase activity. Maximal effect was observed at a concentration of 10(-11) M for both peptides. In this condition, the enzyme activity decreases from 10.8 +/- 1.6 (control) to 5.7 +/- 0.9 or 6.1 +/- 0.7 nmol Pi mg(-1) min(-1) in the presence of urodilatin or ANP, respectively. This effect was completely reversed by 10(-6) M LY83583, a guanylyl cyclase inhibitor, and mimicked by 10 nM cGMP. Furthermore, both ANP and urodilatin increase cGMP production by 33% and 49%, respectively. This is the first demonstration that it was shown that urodilatin and ANP directly modulate primary active sodium transport in the proximal tubule. The data obtained indicate that this effect is mediated by the activation of the NPR-A/guanylate cyclase/cGMP pathway.     

Moderate exercise training provides left ventricular tolerance to acute pressure overload

The present study evaluated the impact of moderate exercise training on the cardiac tolerance to acute pressure overload. Male Wistar rats were randomly submitted to exercise training or sedentary lifestyle for 14 wk. At the end of this period, the animals were anaesthetized, mechanically ventilated, and submitted to hemodynamic evaluation with biventricular tip pressure manometers. Acute pressure overload was induced by banding the descending aorta to induce a 60% increase of peak systolic left ventricular pressure during 120 min. This resulted in the following experimental groups: 1) sedentary without banding (SED + Sham), 2) sedentary with banding (SED + Band), and 3) exercise trained with banding (EX + Band). In response to aortic banding, SED + Band animals could not sustain the 60% increase of peak systolic pressure for 120 min, even with additional narrowing of the banding. This was accompanied by a reduction of dP/dt(max) and dP/dt(min) and a prolongation of the time constant tau, indicating impaired systolic and diastolic function. This impairment was not observed in EX + Band (P < 0.05 vs. SED + Band). Additionally, compared with SED + Band, EX + Band presented less myocardial damage, exhibited attenuated protein expression of active caspase-3 and NF-κB (P < 0.016), and showed less protein carbonylation and nitration (P < 0.05). These findings support our hypothesis that exercise training has a protective role in the modulation of the early cardiac response to pressure overload.     

Optimization of a pseudo-affinity process for penicillin acylase purification

A pseudo-affinity process for penicillin acylase (EC 3.5.1.11) purification using an affinity ligand (Ampicillin) attached on Sepharose 4B-CNBr was optimized. The enzyme adsorption on this affiant (Amp-Seph) is independent of pH between 5.5 and 8.8, in 100?mM phosphate containing 22% (w/v) ammonium sulphate. The desorption of the penicillin acylase from the affinity gels was carried out, the best desorption results being obtained through a non specific eluent, 100?mM phosphate pH 4.6 with 15% (w/v) ammonium sulphate. The best purification results were obtained with an enzymatic extract, produced through osmotic shock of Escherichia coli cells (3.7?IU/mg prot). With this extract and an affinity gel of Sepharose 4B-CNBr derivatized with ampicillin (3.8?μmol/cm³?gel), a maximum activity capacity adsorbed of 20?IU/cm³?gel was obtained for initial values of activity and protein concentration of 1.7?IU/cm³ and 0.4?mg prot/cm³, respectively. With the optimized eluent it was possible to obtain penicillin acylase in only one purification step with a desorption yield of enzyme activity higher than 90%. The penicillin acylase produced with this process was characterized by a maximum purity of 34?IU/mg prot, corresponding to a purification degree higher than 150 in relation to the lowest pure enzymatic extract. The enzyme purity of the eluted fractions was certified by SDS gel electrophoresis and liquid chromatography through a Mono Q column in a FPLC apparatus. The gel electrophoresis presented 4 main stained bands with 2 corresponding to α and β subunits of the penicillin acylase with equivalent molecular weights of 27 and 63?kDa. No external diffusion resistance on penicillin acylase and total protein adsorption on this affiant (Amp-Seph 3.8?μmol/cm³?gel) were observed for continuous adsorption processes performed at two different agitation speeds (120 and 400?rpm).     

Unraveling the plant diversity of the Amazonian canga through DNA barcoding

The canga of the Serra dos Carajás, in Eastern Amazon, is home to a unique open plant community, harboring several endemic and rare species. Although a complete flora survey has been recently published, scarce to no genetic information is available for most plant species of the ironstone outcrops of the Serra dos Carajás. In this scenario, DNA barcoding appears as a fast and effective approach to assess the genetic diversity of the Serra dos Carajás flora, considering the growing need for robust biodiversity conservation planning in such an area with industrial mining activities. Thus, after testing eight different DNA barcode markers (matK, rbcL, rpoB, rpoC1, atpF‐atpH, psbK‐psbI, trnH‐psbA, and ITS2), we chose rbcL and ITS2 as the most suitable markers for a broad application in the regional flora. Here we describe DNA barcodes for 1,130 specimens of 538 species, 323 genera, and 115 families of vascular plants from a highly diverse flora in the Amazon basin, with a total of 344 species being barcoded for the first time. In addition, we assessed the potential of using DNA metabarcoding of bulk samples for surveying plant diversity in the canga. Upon achieving the first comprehensive DNA barcoding effort directed to a complete flora in the Brazilian Amazon, we discuss the relevance of our results to guide future conservation measures in the Serra dos Carajás.     

Pharmacological and genetic evaluation of proposed roles of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), extracellular signal-regulated kinase (ERK), and p90(RSK) in the control of mTORC1 protein signaling by phorbol esters

The mammalian target of rapamycin complex 1 (mTORC1) links the control of mRNA translation, cell growth, and metabolism to diverse stimuli. Inappropriate activation of mTORC1 can lead to cancer. Phorbol esters are naturally occurring products that act as potent tumor promoters. They activate isoforms of protein kinase C (PKCs) and stimulate the oncogenic MEK/ERK signaling cascade. They also activate mTORC1 signaling. Previous work indicated that mTORC1 activation by the phorbol ester PMA (phorbol 12-myristate 13-acetate) depends upon PKCs and may involve MEK. However, the precise mechanism(s) through which they activate mTORC1 remains unclear. Recent studies have implicated both the ERKs and the ERK-activated 90-kDa ribosomal S6 kinases (p90(RSK)) in activating mTORC1 signaling via phosphorylation of TSC2 (a regulator of mTORC1) and/or the mTORC1 component raptor. However, the relative importance of each of these kinases and phosphorylation events for the activation of mTORC1 signaling is unknown. The recent availability of MEK (PD184352) and p90(RSK) (BI-D1870) inhibitors of improved specificity allowed us to address the roles of these protein kinases in controlling mTORC1 in a variety of human and rodent cell types. In parallel, we used specific shRNAs against p90(RSK1) and p90(RSK2) to further test their roles in regulating mTORC1 signaling. Our data indicate that p90(RSKs) are dispensable for the activation of mTORC1 signaling by phorbol esters in all cell types tested. Our data also reveal striking diversity in the requirements for MEK/ERK in the control of mTORC1 between different cell types, pointing to additional signaling connections between phorbol esters and mTORC1, which do not involve MEK/ERK. This study provides important information for the design of efficient strategies to combat the hyperactivation of mTORC1 signaling by oncogenic pathways.     

Behavioral phenotype of maLPA1-null mice: increased anxiety-like behavior and spatial memory deficits

Lysophosphatidic acid (LPA) has emerged as a new regulatory molecule in the brain. Recently, some studies have shown a role for this molecule and its LPA₁ receptor in the regulation of plasticity and neurogenesis in the adult brain. However, no systematic studies have been conducted to investigate whether the LPA₁ receptor is involved in behavior. In this study, we studied the phenotype of maLPA₁-null mice, which bear a targeted deletion at the lpa ₁ locus, in a battery of tests examining neurologic performance, habituation in exploratory behavior in response to low and mild anxiety environments and spatial memory. MaLPA₁-null mutants showed deficits in both olfaction and somesthesis, but not in retinal or auditory functions. Sensorimotor co-ordination was impaired only in the equilibrium and grasping reflexes. The mice also showed impairments in neuromuscular strength and analgesic response. No additional differences were observed in the rest of the tests used to study sensoriomotor orientation, limb reflexes and co-ordinated limb use. At behavioral level, maLPA₁-null mice showed an impaired exploration in the open field and increased anxiety-like response when exposed to the elevated plus maze. Furthermore, the mice exhibit impaired spatial memory retention and reduced use of spatial strategies in the Morris water maze. We propose that the LPA₁ receptor may play a major role in both spatial memory and response to anxiety-like conditions.