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.     

Intravesicular Factors Controlling Exocytosis in Chromaffin Cells

Chromaffin granules are similar organelles to the large dense core vesicles (LDCV) present in many secretory cell types including neurons. LDCV accumulate solutes at high concentrations (catecholamines, 0.5–1 M; ATP, 120–300 mM; or Ca²⁺, 40 mM (Bulenda and Gratzl Biochemistry 24:7760–7765, 1985). Solutes seem to aggregate to a condensed matrix to elude osmotic lysis. The affinity of solutes for LDCV matrix is responsible for the delayed release of catecholamines during exocytosis. The aggregation of solutes occurs due to a specific H⁺ pump denominated V-ATPase that maintains an inner acidic media (pH ≈5.5). This pH gradient against cytosol is also responsible for the vesicular accumulation of amines and Ca²⁺. When this gradient is reduced by modulation of the V-ATPase activity, catecholamines and Ca²⁺ are moved toward the cytosol. In addition, some drugs largely accumulate inside LDCV and not only impair the accumulation of natural solutes, but also act as false neurotransmitters when they are co-released with catecholamines. There is much experimental evidence to conclude that the physiological modulation of vesicle pH and the manipulation of intravesicular media with drugs affect the LDCV cargo and change the kinetics of exocytosis. Here, we will present some experimental data demonstrating the participation of drugs in the kinetics of exocytosis through changes in the composition of vesicular media. We also offer a model to explain the regulation of exocytosis by the intravesicular media that conciliate the experimentally obtained data.     

Over-expression of peptide deformylase in chloroplasts confers actinonin resistance,but is not a suitable selective marker system for plastid transformation

Arabidopsis thaliana peptide deformylase PDF1B was expressed in tobacco chloroplasts using spectinomycin as the selective agent. The foreign protein accumulated in chloroplasts (6% of the total soluble protein) and was enzymatically active. Transplastomic plants were evaluated for resistance to the peptide deformylase inhibitor actinonin. In vitro seed germination in the presence of actinonin and in planta application of the inhibitor demonstrated the resistance of the transformed plants. In addition, transgenic leaf explants were able to develop shoots via organogenesis in the presence of actinonin. However, when the combination of the PDF1B gene and actinonin was used as the primary selective marker system for chloroplast transformation of tobacco, all developed shoots were escapes. Therefore, under the experimental conditions tested, the use of this system for plastid transformation would be limited to function as a secondary selective marker.     

Systematic characterization of the ionic basis of rabbit cellular electrophysiology using two ventricular models

Several mathematical models of rabbit ventricular action potential (AP) have been proposed to investigate mechanisms of arrhythmias and excitation-contraction coupling. Our study aims at systematically characterizing how ionic current properties modulate the main cellular biomarkers of arrhythmic risk using two widely-used rabbit ventricular models, and comparing simulation results using the two models with experimental data available for rabbit. A sensitivity analysis of AP properties, Ca²⁺ and Na⁺ dynamics, and their rate dependence to variations (±15% and ±30%) in the main transmembrane current conductances and kinetics was performed using the Shannon et al. (2004) and the [Mahajan et?al., 2008a] and [Mahajan et?al., 2008b] AP rabbit models. The effects of severe transmembrane current blocks (up to 100%) on steady-state AP and calcium transients, and AP duration (APD) restitution curves were also simulated using both models. Our simulations show that, in both virtual rabbit cardiomyocytes, APD is significantly modified by most repolarization currents, AP triangulation is regulated mostly by the inward rectifier K⁺ current (I_K1) whereas APD rate adaptation as well as [Na⁺]_i rate dependence is influenced by the Na⁺/K⁺ pump current (I_NaK). In addition, steady-state [Ca²⁺]_i levels, APD restitution properties and [Ca²⁺]_i rate dependence are strongly dependent on I_NaK, the L-Type Ca²⁺ current (I_CaL) and the Na⁺/Ca²⁺ exchanger current (I_NaCa), although the relative role of these currents is markedly model dependent. Furthermore, our results show that simulations using both models agree with many experimentally-reported electrophysiological characteristics. However, our study shows that the Shannon et al. model mimics rabbit electrophysiology more accurately at normal pacing rates, whereas Mahajan et al. model behaves more appropriately at faster rates. Our results reinforce the usefulness of sensitivity analysis for further understanding of cellular electrophysiology and validation of cardiac AP models.     

Seasonal and wastewater stream variation of trace organic compounds in a dairy processing plant aerobic bioreactor

Bioreactors are often an integral part of dairy factory efforts to reduce the biological oxygen demand of their wastewater. In this study, infeed, mixed liquor and supernatant samples of an aerobic bioreactor used by a dairy factory in South-Eastern Australia were analyzed for nutrients and organic compounds using gas chromatography-mass spectrometry and physicochemical analyses. Despite different concentrations of organic inputs into the bioreactor, nutrients and trace organic compounds were reduced significantly (i.e. average concentration of trace organic compounds: infeed = 1681 μg/L; mixed liquor = 257 μg/L; supernatant = 23 μg/L). However, during one sampling period the bioreactor was adversely affected by the organic loading. Trace organic compounds in the samples were predominantly fatty acids associated with animal products. The analyses suggest that it is possible to trace a disruptive input (i.e. infeed with high organic carbon concentrations) into an aerobic bioreactor by measuring concentrations of fatty acids or ammonia.     

Modulation of platelet responsiveness through selective phosphorylation of plasma membrane proteins: Antagonistic eeffects of cyclic AMP and cyclic GMP

³²P phosphorylation of plasma membranes from human blood platelets, under conditions that closely resemble physiological ones (endogeneous phosphate donors and intact platelets in homologous plasma), result in the incorporation of the label mainly in a membrane glycoprotein of apparently high molecular weight (greater than 400 000). Dibutyryl cyclic AMP, an inhibitor of platelet aggregation, specifically increases the degree of phosphorylation of this glycoprotein. Moreover, it has been found that prostaglandin E₁ one of the most potent inhibitors of platelet aggregation which also increases phosphorylation of the same glycoprotein, is significantly more effective than cyclic AMP.Cyclic GMP does not have any apparent effect on platelet aggregation. However, incubation of platelet-rich plasma with both cyclic GMP and cyclic AMP results in a partial recovery of the platelet responsiveness towards ADP-induced aggregation. Coincidently, the degree of phosphorylation of the high molecular weight glycoprotein under these conditions, although still higher than in controls (no nucleotides added), is significantly decreased as compared with cyclic AMP-treated cells. Furthermore, cyclic GMP inhibits the cyclic AMP-dependent protein kinase activity in isolated platelet plasma membranes.These results suggest a central role for this membrane phosphoglycoprotein in the triggering of platelet aggregation and, furthermore, suggest that modulation of its degree of phosphorylation may be exerted through some cyclic AMP/cyclic GMP relationship, which in the basal state might be critical for platelet responsiveness.     

Modeling cancer: integration of "omics" information in dynamic systems

The last 10 years have seen the rise of many technologies that produce an unprecedented amount of genome-scale data from many organisms. Although the research community has been successful in exploring these data, many challenges still persist. One of them is the effective integration of such data sets directly into approaches based on mathematical modeling of biological systems. Applications in cancer are a good example. The bridge between information and modeling in cancer can be achieved by two major types of complementary strategies. First, there is a bottom-up approach, in which data generates information about structure and relationship between components of a given system. In addition, there is a top-down approach, where cybernetic and systems-theoretical knowledge are used to create models that describe mechanisms and dynamics of the system. These approaches can also be linked to yield multi-scale models combining detailed mechanism and wide biological scope. Here we give an overall picture of this field and discuss possible strategies to approach the major challenges ahead.     

KIAA0101 interacts with BRCA1 and regulates centrosome number

To find genes and proteins that collaborate with BRCA1 or BRCA2 in the pathogenesis of breast cancer, we used an informatics approach and found a candidate BRCA interactor, KIAA0101, to function like BRCA1 in exerting a powerful control over centrosome number. The effect of KIAA0101 on centrosomes is likely direct, as its depletion does not affect the cell cycle, KIAA0101 localizes to regions coincident with the centrosomes, and KIAA0101 binds to BRCA1. We analyzed whether KIAA0101 protein is overexpressed in breast cancer tumor samples in tissue microarrays, and we found that overexpression of KIAA0101 correlated with positive Ki67 staining, a biomarker associated with increased disease severity. Furthermore, overexpression of the KIAA0101 gene in breast tumors was found to be associated with significantly decreased survival time. This study identifies KIAA0101 as a protein important for breast tumorigenesis, and as this factor has been reported as a UV repair factor, it may link the UV damage response to centrosome control.     

Structures of wall heterogalactomannans isolated from three genera of entomopathogenic fungi

O-linked heterogalactomannans with similar structural features have been purified from the fungal walls of the entomopathogenic fungi Lecanicillium muscarium, Beauveria bassiana, Beauveriabrongniartii, and Cordyceps sphingum. Their composition and structure have been determined using acid hydrolysis, methylation analysis, gas-liquid chromatography-mass spectrometry (GC-MS) and Nuclear magnetic resonance spectroscopy (NMR). All structures have an α-(1→6)-mannose backbone, but one of the two strains of L. muscarium included in this study contained an acidic heterogalactomannan instead of the neutral polysaccharide isolated in the rest of the species analyzed. Sequence analysis of the internal transcribed spacer (ITS) region of this strain indicated that it belongs to the related genus Simplicillium, displaying low identity (83%) with the closest Lecanicillium species. This is a new demonstration of the structural diversity of fungal wall heteromannans and validates their interest as chemotaxonomic markers. The production of a pullulan-like extracellular polysaccharide in strain CBS 413.70C of L. muscarium is also reported.     

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.