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.     

Efficacy of <Emphasis Type="Italic">Neoseiulus californicus</Emphasis> and <Emphasis Type="Italic">Phytoseiulus persimilis</Emphasis> in suppression of <Emphasis Type="Italic">Tetranychus urticae</Emphasis> in young clementine plants

Tetranychus urticae is one of the most damaging tetranychid mites affecting clementine orchards in Spain, where natural control is insufficient. Furthermore, in clementine nurseries, tender foliage is highly susceptible to attack and natural enemies are almost always absent. Therefore, acaricides are often used indiscriminately. Alternative control measures are necessary, both in commercial orchards and clementine nurseries. In order to assess the efficacy of inoculative releases of N. californicus and P. persimilis to reduce T. urticae populations in young Spanish clementine plants, a semi-field experiment was conducted and repeated in three seasons (spring, summer and autumn). Phytoseiulus persimilis was highly effective in reducing both T. urticae infestations and the damage level inflicted on plants at both release rates evaluated (40 and 80 phytoseiids/plant) and all three periods considered. By contrast, N. californicus demonstrated low performance under certain conditions. The results of this study could be adapted and transferred to nurseries and young citrus plantations.     

Anisotropic elastic network modeling of entire microtubules

Microtubules are supramolecular structures that make up the cytoskeleton and strongly affect the mechanical properties of the cell. Within the cytoskeleton filaments, the microtubule (MT) exhibits by far the highest bending stiffness. Bending stiffness depends on the mechanical properties and intermolecular interactions of the tubulin dimers (the MT building blocks). Computational molecular modeling has the potential for obtaining quantitative insights into this area. However, to our knowledge, standard molecular modeling techniques, such as molecular dynamics (MD) and normal mode analysis (NMA), are not yet able to simulate large molecular structures like the MTs; in fact, their possibilities are normally limited to much smaller protein complexes. In this work, we developed a multiscale approach by merging the modeling contribution from MD and NMA. In particular, MD simulations were used to refine the molecular conformation and arrangement of the tubulin dimers inside the MT lattice. Subsequently, NMA was used to investigate the vibrational properties of MTs modeled as an elastic network. The coarse-grain model here developed can describe systems of hundreds of interacting tubulin monomers (corresponding to up to 1,000,000 atoms). In particular, we were able to simulate coarse-grain models of entire MTs, with lengths up to 350 nm. A quantitative mechanical investigation was performed; from the bending and stretching modes, we estimated MT macroscopic properties such as bending stiffness, Young modulus, and persistence length, thus allowing a direct comparison with experimental data.     

Toward a resolution of a taxonomic enigma: first genetic analyses of Paradoxornis webbianus and Paradoxornis alphonsianus (Aves: Paradoxornithidae) from China and Italy

Paradoxornis webbianus and Paradoxornis alphonsianus naturally occur in South-East Asia. Due to a recent introduction, a mixed population currently occurs in northern Italy. A preliminary phylogeographic analysis using samples from Italy and China found little genetic differentiation between the two taxa and revealed the existence of two molecular lineages, sympatric in some part of China, that do not correspond to the morphological classification. Possible taxonomic changes and preliminary inferences on the relationships between Chinese and the Italian populations and on the likely provenance of the founders introduced in Italy are also discussed.     

Characterization of ��/��- and ��-Gliadins in Commercial Varieties and Breeding Lines of Durum Wheat Using MALDI-TOF and A-PAGE Gels

In this work, gliadin composition has been analyzed in 33 accessions of durum wheat using MALDI-TOF MS and compared with A-PAGE results. The MALDI-TOF MS spectra were 29,900-42,500 Da, which corresponds to the α/β- and γ-gliadin regions in A-PAGE. The average of gliadin peaks per line was 23 for MALDI-TOF MS and only 14.8 bands for A-PAGE. MALDI-TOF MS identified 33 gliadin peaks in the durum wheat collection, 20 of which were unique peaks present in 7 lines. A-PAGE analysis identified 30 bands, of which only 4 were unique. Thus, the MALDI-TOF MS method was more sensitive than A-PAGE for identifying α/β- and γ-gliadins in the 33 durum wheat lines studied. Phylogenetic analyses performed using MALDI-TOF MS data assigned the durum wheat lines to two groups. The utility of MALDI-TOF MS to determine relationships among genotypes and for identification of durum wheat accessions is discussed.     

Phase-Dependent Astroglial Alterations in Li–Pilocarpine-Induced <Emphasis Type="Italic">Status Epilepticus</Emphasis> in Young Rats

Epilepsy prevalence is high in infancy and in the elderly population. Lithium–pilocarpine is widely used to induce experimental animal models of epilepsy, leading to similar neurochemical and morphological alterations to those observed in temporal lobe epilepsy. As astrocytes have been implicated in epileptic disorders, we hypothesized that specific astroglial changes accompany and contribute to epileptogenesis. Herein, we evaluated time-dependent astroglial alterations in the hippocampus of young (27-day-old) rats at 1, 14 and 56 days after Li–pilocarpine-induced status epilepticus (SE), corresponding to different phases in this model of epilepsy. We determined specific markers of astroglial activation: GFAP, S100B, glutamine synthetase (GS), glutathione (GSH) content, aquaporin-4 (AQP-4) and potassium channel Kir 4.1; as well as epileptic behavioral, inflammatory and neurodegenerative changes. Phase-dependent signs of hippocampal astrogliosis were observed, as demonstrated by increments in GFAP, S100B and GS. Astrocyte dysfunction in the hippocampus was characterized, based on the decrease in GSH content, AQP-4 and Kir 4.1 channels. Degenerating neurons were identified by Fluoro-Jade C staining. We found a clear, early (at SE1) and persistent (at SE56) increase in cerebrospinal fluid (CSF) S100B levels. Additionally, serum S100B was found to decrease soon after SE induction, implicating a rapid-onset increase in the CSF/serum S100B ratio. However, serum S100B increased at SE14, possibly reflecting astroglial activation and/or long-term increase in cerebrovascular permeability. Moreover, we suggest that peripheral S100B levels may represent a useful marker for SE in young rats and for follow up during the chronic phases of this model of epilepsy. Together, results reinforce and extend the idea of astroglial involvement in epileptic disorders.     

Biological Effects of Trichoderma harzianum Peptaibols on Mammalian Cells

Trichoderma species isolated from water-damaged buildings were screened for toxicity by using boar sperm cells as indicator cells. The crude methanolic cell extract from Trichoderma harzianum strain ES39 inhibited the boar sperm cell motility at a low exposure concentration (50% effective concentration, 1 to 5 μg [dry weight] ml of extended boar semen⁻¹). The same exposure concentration depleted the boar sperm cells of NADH₂. Inspection of the exposed boar sperm cells by transmission electron microscopy revealed damage to the plasma membrane. By using the black lipid membrane technique, it was shown that the semipurified metabolites (eluted from a SepPak C₁₈ cartridge) of T. harzianum strain ES39 induced voltage-dependent conductivity. The high-performance liquid chromatography-purified metabolites of T. harzianum strain ES39 dissipated the mitochondrial membrane potential (Δψ_m) of human lung epithelial carcinoma cells (cell line A549). The semipurified metabolites (eluted from a SepPak C₁₈ cartridge) of T. harzianum strain ES39 were analyzed by mass spectrometry (MS). Matrix-assisted laser desorption ionization and nanoflow electrospray ionization MS revealed five major peptaibols, each of which contained 18 residues and had a mass ranging from 1,719 to 1,775 Da. Their partial amino acid sequences were determined by collision-induced dissociation tandem MS.     

Photooxidation of pterin in aqueous solutions: biological and biomedical implications

Studies of the photochemical reactivity of pterin (= 2-aminopteridin-4(3H)-one; PT) in acidic (pH 5.0-6.0) and alkaline (pH 10.2-10.8) aqueous solutions have been performed. The photochemical reactions were followed by UV/VIS spectrophotometry, thin layer chromatography (TLC), high-performance liquid chromatography (HPLC), and an enzymatic method for H2O2 determination. PT is not light-sensitive in the absence of molecular oxygen, but it undergoes photooxidation in the presence of O2, yielding several nonpteridinic products. The quantum yields for PT disappearance were found to be 8.2 (+/-0.6) x 10(-4) and 1.2 (+/-0.2) x 10(-3) in acidic and alkaline media, respectively. H2O2 was detected and quantified in irradiated solutions of PT; and its importance from a biomedical point of view is discussed. The rate constant of the chemical reaction between singlet oxygen ((1)O2) and PT was determined to be 2.5 (+/-0.2) x 10(5) l mol(-1) s(-1) in alkaline medium, and the role of (1)O2 in the photooxidation of pterin was evaluated.     

Towards a molecular dynamics consensus view of B-DNA flexibility

We present a systematic study of B-DNA flexibility in aqueous solution using long-scale molecular dynamics simulations with the two more recent versions of nucleic acids force fields (CHARMM27 and parmbsc0) using four long duplexes designed to contain several copies of each individual base pair step. Our study highlights some differences between pambsc0 and CHARMM27 families of simulations, but also extensive agreement in the representation of DNA flexibility. We also performed additional simulations with the older AMBER force fields parm94 and parm99, corrected for non-canonical backbone flips. Taken together, the results allow us to draw for the first time a consensus molecular dynamics picture of B-DNA flexibility.