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.     

HIV-1 Tat protein induces glial cell autophagy through enhancement of BAG3 protein levels

BAG3 protein has been described as an anti-apoptotic and pro-autophagic factor in several neoplastic and normal cells. We previously demonstrated that BAG3 expression is elevated upon HIV-1 infection of glial and T lymphocyte cells. Among HIV-1 proteins, Tat is highly involved in regulating host cell response to viral infection. Therefore, we investigated the possible role of Tat protein in modulating BAG3 protein levels and the autophagic process itself. In this report, we show that transfection with Tat raises BAG3 levels in glioblastoma cells. Moreover, BAG3 silencing results in highly reducing Tat- induced levels of LC3-II and increasing the appearance of sub G0/G1 apoptotic cells, in keeping with the reported role of BAG3 in modulating the autophagy/apoptosis balance. These results demonstrate for the first time that Tat protein is able to stimulate autophagy through increasing BAG3 levels in human glial cells.     

On/off TLR signaling decides proinflammatory or tolerogenic dendritic cell maturation upon CD1d-mediated interaction with invariant NKT cells

Invariant NKT (iNKT) cells play an effector/adjuvant function during antimicrobial and antitumoral immunity and a regulatory role to induce immune tolerance and prevent autoimmunity. iNKT cells that differentially modulate adaptive immunity do not bear a unique phenotype and/or specific cytokine secretion profile, thus opening questions on how a single T cell subset can exert opposite immunological tasks. In this study, we show that iNKT cells perform their dual roles through a single mechanism of action relying on the cognate interaction with myeloid dendritic cells (DCs) and leading to opposite effects depending on the presence of other maturation stimuli simultaneously acting on DCs. The contact of murine purified iNKT cells with immature autologous DCs directly triggers the tolerogenic maturation of DCs, rendering them able to induce regulatory T cell differentiation and prevent autoimmune diabetes in vivo. Conversely, the interaction of the same purified iNKT cells with DCs, in the presence of simultaneous TLR4 stimulation, significantly enhances proinflammatory DC maturation and IL-12 secretion. The different iNKT cell effects are mediated through distinct mechanisms and activation of different molecular pathways within the DC: CD1d signaling and activation of the ERK1/2 pathway for the tolerogenic action, and CD40-CD40L interaction and NF-κB activation for the adjuvant effect. Our data suggest that the DC decision to undergo proinflammatory or tolerogenic maturation results from the integration of different signals received at the time of iNKT cell contact and could have important therapeutic implications for exploiting iNKT cell adjuvant/regulatory properties in autoimmune diseases, infections, and cancer.     

On the general dynamic model of oceanic island biogeography

Aim To investigate the biological meaning of equations used to apply the general dynamic model (GDM) of oceanic island biogeography proposed by R. J. Whittaker, K. A. Triantis and R. J. Ladle. Location Analyses are presented for 17 animal groups living on the Aeolian Islands, a volcanic archipelago in the central Mediterranean, near Sicily. Methods In addition to the mathematical implementation of the GDM proposed by Whittaker, Triantis and Ladle, and termed here logATT² (, where S is species number or any other diversity metric, t is island age, A is island area, and a, b, c and d are fitted parameters), a new implementation based on the Arrhenius equation of the species–area relationship (SAR) is investigated. The new model (termed powerATT²) is: . For logATT² and powerATT² models, equations were developed to calculate (1) the expected number of species at equilibrium (i.e. when the island has reached maturity) per unit area (S_eq), and (2) the time required to obtain this value (t_eq). Whereas the intercept in the Gleason model (S = C + z log A) or the coefficient of the Arrhenius power model (S = CA^z) of the SAR can be considered measures of the expected number of species per unit area, this is not the case for the parameter a of the ATT² models. However, values of S_eq can be used for this purpose. The index of ‘colonization ability’ (CAB), calculated as the ratio , may provide a measure of the mean number of species added per unit area per unit time. Results Both ATT² models fitted most of the data well, but the powerATT² model was in most cases superior. Equilibrial values of species richness (S_eq) varied from c. 3 species km^?2 (reptiles) to 100 species km^?2 (mites). The fitted curves for the powerATT² model showed large variations in d, from 0.03 to 3. However, most groups had values of d around 0.2–0.4, as commonly observed for the z‐values of SARs modelled by a power function. Equilibration times ranged from about 170,000 years to 400,000 years. Mites and springtails had very high values of CAB, thus adding many more species per unit area per unit time than others. Reptiles and phytophagous scarabs showed very low values, being the groups that added fewest species per unit area per unit time. Main conclusions Values of equilibrial species richness per unit area are influenced by species biology (e.g. body size and ecological specialization). Theoretical and empirical evidence suggests that higher immigration rates should increase the z‐values of the Arrhenius model. Thus, in the same archipelago, groups with larger z‐values should be characterized by higher dispersal ability. Results obtained here for the parameter d conform to this prediction.     

Identification of clinically relevant protein targets in prostate cancer with 2D-DIGE coupled mass spectrometry and systems biology network platform

Prostate cancer (PCa) is the most common type of cancer found in men and among the leading causes of cancer death in the western world. In the present study, we compared the individual protein expression patterns from histologically characterized PCa and the surrounding benign tissue obtained by manual micro dissection using highly sensitive two-dimensional differential gel electrophoresis (2D-DIGE) coupled with mass spectrometry. Proteomic data revealed 118 protein spots to be differentially expressed in cancer (n = 24) compared to benign (n = 21) prostate tissue. These spots were analysed by MALDI-TOF-MS/MS and 79 different proteins were identified. Using principal component analysis we could clearly separate tumor and normal tissue and two distinct tumor groups based on the protein expression pattern. By using a systems biology approach, we could map many of these proteins both into major pathways involved in PCa progression as well as into a group of potential diagnostic and/or prognostic markers. Due to complexity of the highly interconnected shortest pathway network, the functional sub networks revealed some of the potential candidate biomarker proteins for further validation. By using a systems biology approach, our study revealed novel proteins and molecular networks with altered expression in PCa. Further functional validation of individual proteins is ongoing and might provide new insights in PCa progression potentially leading to the design of novel diagnostic and therapeutic strategies.     

Endogenous phosphotyrosine signaling in zebrafish embryos

In the developing embryo, cell growth, differentiation, and migration are strictly regulated by complex signaling pathways. One of the most important cell signaling mechanisms is protein phosphorylation on tyrosine residues, which is tightly controlled by protein-tyrosine kinases and protein-tyrosine phosphatases. Here we investigated endogenous phosphotyrosine signaling in developing zebrafish embryos. Tyrosine phosphorylated proteins were immunoaffinity-purified from zebrafish embryos at 3 and 5 days postfertilization and identified by multidimensional LC-MS. Among the identified proteins were tyrosine kinases, including Src family kinases, Eph receptor kinases, and focal adhesion kinases, as well as the adaptor proteins paxillin, p130Cas, and Crk. We identified several known and some unknown in vivo tyrosine phosphorylation sites in these proteins. Whereas most immunoaffinity-purified proteins were detected at both developmental stages, significant differences in abundance and/or phosphorylation state were also observed. In addition, multiplex in vitro kinase assays were performed by incubating a microarray of peptide substrates with the lysates of the two developmental stages. Many of the in vivo observations were confirmed by this on-chip in vitro kinase assay. Our experiments are the first to show that global tyrosine phosphorylation-mediated signaling can be studied at endogenous levels in complex multicellular organisms.     

Molecular characterization of Inonotus rickii /Ptychogaster cubensis isolates from different geographic provenances

Thirteen isolates of Inonotus rickii/Ptychogaster cubensis, from different geographic provenances, were analyzed by sequencing ITS1, ITS 2 and 5,8S ribosomal RNA region. A phylogenetic tree, also including sequences available in Genbank database, showed that the strains enclosed in this study fall into two well-separated groups, one formed by isolates from Florida (USA) and the other one by isolates from Europe, Argentina and China. Differences were also highlighted on the growth rate of mycelial cultures at different temperatures. In fact, although the tested isolates generally attained the best growth at 30°C, isolates from Europe seem well adapted to higher temperatures and went on growing at 40°C whilst the growth of isolates from Florida significantly decreased at 35°C. Since the teleomorph I. rickii was never detected in Florida, and in this study noticeable differences were detected by analysis of ITS region, the existence of two possible distinct species, not discriminated solely on the basis of morphological characters, could be suggested.     

Two modes of sulfite oxidation in the extremely thermophilic and acidophilic archaeon Acidianus ambivalens

Sulfite-oxidizing enzyme activities were analyzed in cell-free extracts of aerobically grown cells of Acidianus ambivalens, an extremely thermophilic and chemolithoautotrophic archaeon. In the membrane and cytoplasmic fractions, two distinct enzyme activities were found. In the membrane fraction, a sulfite:acceptor oxidoreductase activity was found [530 mU (mg protein)^–1; apparent K _m for sulfite, 3.6 mM]. In the cytoplasmic fraction the following enzyme activities were found and are indicative of an oxidative adenylylsulfate pathway: adenylylsulfate reductase [138 mU (mg protein)^–1], adenylylsulfate:phosphate adenyltransferase [“ADP sulfurylase”; 86 mU (mg protein)^–1], adenylate kinase [650 mU (mg protein)^–1], and rhodanese [thiosulfate sulfur transferase, 9.2 mU (mg protein)^–1]. In addition, 5′,5′′′-P¹,P⁴-di(adenosine-5′) tetraphosphate (Ap₄A) synthase and Ap₄A pyrophosphohydrolase activities were detected. Received: 17 August 1998 / Accepted: 29 April 1999     

TNF-α -308 genotypes are associated with TNF-α and TGF-β₁ mRNA expression in blood leucocytes of humans

AimTumor necrosis factor α (TNF-α) influences the pathogenesis of lung-fibrosis and carcinogenesis in normal cells. Polymorphisms of this gene are suggested to be associated with susceptibility to lung-diseases. Additionally TNF-α is postulated to play a significant role in regulating. Transforming growth factor (TGF-β₁) expression Therefore we investigated if the TNF-α or TGF-β₁ gene expression level is different within the ?308 TNF-α genotypes.MethodsQuantitative Real-time PCR of TNF-α and TGF-β₁ was performed in 178 Germans. Calculations of expression were made with the 2^?ΔΔCT method. Detection of the ?308 promoter polymorphism of the TNF-α gene was performed by rapid capillary PCR with melting curve analysis.ResultsThe relative TNF-α mRNA expression revealed significant differences between the TNF-α ?308 homozygote wild-type G/G (0.00079 ± 0.00011; n = 113) and the heterozygote genotype G/A (0.0005 ± 0.00008; n = 52; p = 0.030) as well as between homozygote wild-type G/G and the homozygote mutant A/A (0.00029 ± 0.00009; n = 5; p = 0.004). The relative TGF-β mRNA expression showed, similar to TNF-α, the highest mRNA expression was seen within the TNF-α ?308 homozygote wild-types, while the lowest mRNA expression lay within the homozygote mutant-types.ConclusionOur findings suggest that the G-allele of TNF-α ?308 is associated with a significantly higher TNF-α mRNA expression compared to the A-allele and that this also reflects in TGF-β expression. Therefore we support the thesis that TGF-β is regulated by TNF-α.     

Survival and growth of two heterotrophic hydrothermal vent archaea, <Emphasis Type="Italic">Pyrococcus</Emphasis> strain GB-D and <Emphasis Type="Italic">Thermococcus fumicolans</Emphasis>, under low pH and high sulfide concentrations in combination with high temperature and pressure regimes

Growth and survival of hyperthermophilic archaea in their extreme hydrothermal vent and subsurface environments are controlled by chemical and physical key parameters. This study examined the effects of elevated sulfide concentrations, temperature, and acidic pH on growth and survival of two hydrothermal vent archaea (Pyrococcus strain GB-D and Thermococcus fumicolans) under high temperature and pressure regimes. These two strains are members of the Thermococcales, a family of hyperthermophilic, heterotrophic, sulfur-reducing archaea that occur in high densities at vent sites. As actively growing cells, these two strains tolerated regimes of pH, pressure, and temperature that were in most cases not tolerated under severe substrate limitation. A moderate pH of 5.5–7 extends their survival and growth range over a wider range of sulfide concentrations, temperature and pressure, relative to lower pH conditions. T. fumicolans and Pyrococcus strain GB-D grew under very high pressures that exceeded in-situ pressures typical of hydrothermal vent depths, and included deep subsurface pressures. However, under the same conditions, but in the absence of carbon substrates and electron acceptors, survival was generally lower, and decreased rapidly when low pH stress was combined with high pressure and high temperature. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.