Analysis of the grasspea proteome and identification of stress-responsive proteins upon exposure to high salinity, low temperature, and abscisic acid treatment

Abiotic stress causes diverse biochemical and physiological changes in plants and limits crop productivity. Plants respond and adapt to such stress by altering their cellular metabolism and activating various defense machineries. To understand the molecular basis of stress tolerance in plants, we have developed differential proteomes in a hardy legume, grasspea (Lathyrus sativus L.). Five-week-old grasspea seedlings were subjected independently to high salinity, low temperature and abscisic acid treatment for duration of 36 h. The physiological changes of stressed seedlings were monitored, and correlated with the temporal changes of proteome using two-dimensional gel electrophoresis. Approximately, 400 protein spots were detected in each of the stress proteome with one-fourth showing more than 2-fold differences in expression values. Eighty such proteins were subjected to LC-tandem MS/MS analyses that led to the identification of 48 stress-responsive proteins (SRPs) presumably involved in a variety of functions, including metabolism, signal transduction, protein biogenesis and degradation, and cell defense and rescue. While 33 proteins were responsive to all three treatments, 15 proteins were expressed in stress-specific manner. Further, we explored the possible role of ROS in triggering the stress-induced degradation of large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase (Rubisco). These results might help in understanding the spectrum of stress-regulated proteins and the biological processes they control as well as having implications for strategies to improve stress adaptation in plants.     

Diet Composition and Intensity of Feeding of Tenualosa ilisha (Hamilton, 1822) Occurring in the Northern Bay of Bengal,India

Feeding intensity and diet composition of Hilsa Shad (Tenualosa ilisha) from Northern Bay of Bengal were studied between June 2010 and March 2011. The stomach contents of 320 fishes were analyzed from the northern part of the Bay of Bengal to understand the food items of this species. The major constituents of food are organic debris (26.06 ± 5.19 % SD), diatoms (31.22 ± 11.97 % SD), other algae (12.41 ± 2.62 % SD), and crustaceans (3.50 ± 1.28 % SD). The most abundant species of diatoms were Coscinodiscus, Pleurosigma, Bacillaria, Nitzschia, Biddulphia, Diatoma and Asterionella. The stomach of Hilsa was found to be almost empty during June to October while it was almost full during November to March. Significant positive correlation among feeding intensity, chlorophyll-A concentration and salinity of the ambient water indicated that feeding in T. ilisha is influenced by a number of factors. Strong positive correlation between percentage occurrence of diatoms and intensity of feeding indicated their preference for diatom–food.     

Cell interaction knowledgebase: an online database for innate immune cells, cytokines and chemokines

The innate immune system is fundamental to the recognition of pathogens, triggering of immune-inflammatory response and host defense. Recent advance in this area has resulted in enormous amount of data, which are stored across different databases. Integrating relevant information from these different data sources is difficult because of their heterogeneous nature and dispersed physical location. We present here a single portal system, Cell Interaction Knowledgebase, with focus on the innate immunity. In particular, the knowledgebase houses comprehensive information on innate immune cells and cytokines/chemokines which are the principal mediators of communication among the immune cells. Currently the knowledgebase consists of extensive information on 2 major innate immune cell types (Macrophages and Dendritic cells) and 7 6 cytokines/chemokines for both human and mouse. In addition, intra-cellular molecular interactions and inter-cellular interactions involved in the innate immunity are curated and presented in an interactive and dynamic manner by animated pathways and query-driven cell-interaction map respectively. This is one of the first databases that houses extensive phenotypic, signaling, genomic, proteomic and knockout data on both the innate immune cells and their attendant cytokines/chemokines, and is aimed to evolve as a one-stop-shop for immunologists. The first version of database is available at http://cell-interaction.bii.a-star.edu.sg/.     

Role for MyD88-independent, TRIF pathway in lipid A/TLR4-induced endotoxin tolerance

Repeated exposure to low doses of endotoxin results in progressive hyporesponsiveness to subsequent endotoxin challenge, a phenomenon known as endotoxin tolerance. In spite of its clinical significance in sepsis and characterization of the TLR4 signaling pathway as the principal endotoxin detection mechanism, the molecular determinants that induce tolerance remain obscure. We investigated the role of the TRIF/IFN-beta pathway in TLR4-induced endotoxin tolerance. Lipid A-induced homotolerance was characterized by the down-regulation of MyD88-dependent proinflammatory cytokines TNF-alpha and CCL3, but up-regulation of TRIF-dependent cytokine IFN-beta. This correlated with a molecular phenotype of defective NF-kappaB activation but a functional TRIF-dependent STAT1 signaling. Tolerance-induced suppression of TNF-alpha and CCL3 expression was significantly relieved by TRIF and IFN regulatory factor 3 deficiency, suggesting the involvement of the TRIF pathway in tolerance. Alternatively, selective activation of TRIF by poly(I:C)-induced tolerance to lipid A. Furthermore, pretreatment with rIFN-beta also induced tolerance, whereas addition of IFN-beta-neutralizing Ab during the tolerization partially alleviated tolerance to lipid A but not TLR2-induced endotoxin homo- or heterotolerance. Furthermore, IFNAR1-/- murine embryonal fibroblast and bone-marrow derived macrophages failed to induce tolerance. Together, these observations constitute evidence for a role of the TRIF/IFN-beta pathway in the regulation of lipid A/TLR4-mediated endotoxin homotolerance.     

Roscovitine inhibits differentiation and invasion in a three-dimensional skin reconstruction model of metastatic melanoma

The aim of this study was to investigate the therapeutic potential of a cyclin-dependent kinase inhibitor, roscovitine, in cultured melanoma cells and a three-dimensional skin reconstruction model of metastatic melanoma. The modulatory effects of roscovitine on the growth and survival of normal melanocytes and cultured melanoma cell lines were tested. Additionally, we investigated the potential of roscovitine to regulate the growth and differentiation of a metastatic melanoma cell line (A375) in a three-dimensional skin reconstruction culture consisting of A375 cells admixed with normal human keratinocytes embedded within a collagen-constricted fibroblast matrix. We show that roscovitine is able to induce apoptosis in the melanoma cell lines A375, 888, and 624 but not in normal human cultured epithelial melanocytes. The degree of apoptosis within these cell lines correlated with the accumulation of p53 protein and concomitant reduction of X-linked inhibitor of apoptosis protein, with no change in the proteins Bcl-2 and survivin. We also found that roscovitine inhibited the growth and differentiation of A375 melanoma cells within the dermal layer of the skin. The results of this study show that roscovitine has the potential to inhibit the differentiation and invasion of metastatic melanoma and may be useful as a therapy for the treatment of patients with metastatic melanoma.     

Developing hybrid approaches to predict pKa values of ionizable groups

Accurate predictions of pKa values of titratable groups require taking into account all relevant processes associated with the ionization/deionization. Frequently, however, the ionization does not involve significant structural changes and the dominating effects are purely electrostatic in origin allowing accurate predictions to be made based on the electrostatic energy difference between ionized and neutral forms alone using a static structure and the subtle structural changes be accounted by using dielectric constant larger than two. On another hand, if the change of the charge state is accompanied by a large structural reorganization of the target protein, then the relevant conformational changes have to be explicitly taken into account in the pKa calculations. Here we report a hybrid approach that first predicts the titratable groups whose ionization is expected to cause large conformational changes, termed "problematic" residues, and then applies a special protocol on them, while the rest of the pK(a)s are predicted with rigid backbone approach as implemented in multi-conformation continuum electrostatics (MCCE) method. The backbone representative conformations for "problematic" groups are generated with either molecular dynamics simulations with charged and uncharged amino acid or with ab-initio local segment modeling. The corresponding ensembles are then used to calculate the titration curves of the "problematic" residues and then the results are averaged to obtain the corresponding pKa.     

Characterization of the secretome of chickpea suspension culture reveals pathway abundance and the expected and unexpected secreted proteins

The secretome of an organism is defined as a set of secreted proteins that encompasses all proteins exported to the extracellular space. To better understand the chickpea secretome, we used callus culture to isolate and identify secreted proteins as a step toward determining their functions. Proteins in the extracellular media of the suspension culture were examined using SDS-PAGE and mass spectrometry (LC-MS/MS). Proteomic analysis led to the identification of 773 proteins, presumably involved in a variety of functions including metabolism, signal transduction, transport, and cell defense, in addition to maintaining redox status of extracellular space. Bioinformatic analysis confirmed 724 proteins, accounting for 94% of the identified proteins, as constituents of the secretome. Analysis of the secretome revealed the presence of several proteins of unknown function and a large number of classical and nonclassical secreted proteins. This represents the first comprehensive secretome of a legume genome, which is yet to be sequenced. Comparative analysis of the chickpea secretome with those of Medicago, Arabidopsis, and rice revealed that the majority of identified proteins are seemingly species-specific. This study demonstrates that characterization of the chickpea secretome in vitro can be used to identify secreted proteins, which has implications for systems biology research.     

Dehydration-responsive reversible and irreversible changes in the extracellular matrix: comparative proteomics of chickpea genotypes with contrasting tolerance

Dehydration is the most crucial environmental factor that limits plant growth, development, and productivity affecting agriculture throughout the world. Studies on genetic variations for dehydration tolerance in plants is crucial because divergent cultivars with contrasting traits aid the identification of key cellular components that confer better adaptability. The extracellular matrix (ECM) is a dynamic structure that serves as the repository for important signaling components and acts as a front-line defense. To better understand dehydration adaptation, a proteomic study was performed on the extracellular matrix of ICCV-2, a dehydration-susceptible genotype of chickpea. The proteome was generated with ECM-enriched fractions using two-dimensional gel electrophoresis. The LC-ESI-MS/MS analysis led to the identification of 81 dehydration-responsive proteins. The proteome was then compared with that of JG-62, a tolerant genotype. Comparative proteomics revealed genotype-specific expression of many proteins involved in a variety of cellular functions. Further, the reversible and irreversible changes in the proteomes revealed their differing ability to recover from dehydration-induced damage. We propose that cell wall restructuring and superior homeostasis, particularly the management of reactive oxygen species, may render better dehydration-adaptation. To our knowledge, this is the first report on the comprehensive comparison of dehydration-responsive organellar proteome of two genotypes with contrasting tolerance.     

Biochemical characterization of compatible plant-viral interaction: A case study with a begomovirus-kenaf host-pathosystem

Yellow vein mosaic disease of mesta, a compatible plant virus interaction, poses a serious threat to mesta cultivation in India. Plants respond to invasion by pathogens with multi-component defense responses particularly in incompatible interaction. With the aim of understanding, a biochemical approach was attempted to study the cellular redox status in early stages of yellow vein mosaic virus infection associated with different ages plants of Hibiscus cannabinus. Comparative analysis of GSH and GSSG content in infected and control plants of different ages indicated that infected plants are under oxidative or nitrosative stress condition. A significant change was observed in Glutathione Reductase, Catalase and Ascorbate Peroxidase level in early stage of infection. We also showed microscopic evidence of nitrosylated thiols in infected leaves, stems and roots of H. cannabinus. Furthermore, we identified few defense related proteins in infected plant using MALDI TOF mass spectrometric analysis.Key words: mesta yellow vein mosaic virus, cellular redox status, redox active enzymes, nitrosylated thiol, defense proteins