Plant Growth‐promoting Fungus Penicillium oxalicum Enhances Plant Growth and Induces Resistance in Pearl Millet Against Downy Mildew Disease

Plant Growth‐promoting Fungus (PGPF) Penicillium oxalicum was isolated from rhizosphere soil of pearl millet and was tested for its ability to promote growth and induce systemic resistance in pearl millet against downy mildew disease. The fungal isolate P. oxalicum UOM PGPF 16 was identified as P. oxalicum using ITS sequencing and morphological analysis and sequence was deposited at NCBI with accession number KF150220. Pearl millet susceptible seeds were treated with three different inducers (CS, CF and LCF) of PGPF P. oxalicum and all the inducers significantly reduced the downy mildew disease and enhanced plant growth. Among the inducers tested, CS treatment recorded highest seed germination of 91% and 1427 seedling vigour followed by LCF and CF treatments. The vegetative growth parameter and NPK uptake studies under greenhouse conditions revealed that the CS treatment of P. oxalicum remarkably enhanced the parameters tested when compared to control plants. A significant disease protection of 62% and 58% against downy mildew disease was observed in plants pretreated with CS of P. oxalicum under greenhouse and field conditions, respectively. The spatio‐temporal studies revealed that inducers P. oxalicum required a minimum of 3 days for developing maximum disease resistance which was maintained thereafter. The maximum Peroxidase (POX) activity (62.7 U) was observed at 24 h in seedlings treated with CS of PGPF P. oxalicum and the activity gradually reduced at later time points after pathogen inoculation. Chitinase (CHT) activity was significantly higher in inducer treated seedlings when compared to control seedlings inoculated with pathogen after 48 h and remained constant at all time points.     

Engineering of a linear inactive analog of human β‐defensin 4 to generate peptides with potent antimicrobial activity

Human β‐defensins (HBDs) are cationic antimicrobial peptides constrained by three disulfide bridges. They have diverse range of functions in the innate immune response. It is of interest to investigate whether linear analogs of defensins can be generated, which possess antimicrobial activity. In this study, we have designed linear peptides with potent antimicrobial activity from an inactive peptide spanning the N‐terminus of HBD4. Our results show that l ‐arginine to d ‐arginine substitution imparts considerable antimicrobial activity against both bacteria and Candida albicans. Increase in hydrophobicity by fatty acylation of the peptides with myristic acid further enhances their potency. In the presence of high concentrations of salt, antimicrobial activity of the myristoylated peptide with l ‐arginine is attenuated relatively to a lesser extent as compared with the linear active peptide with d ‐arginine. Substitution of cysteine with the hydrophobic helix‐promoting amino acid α‐aminoisobutyric acid favors candidacidal activity but not antibacterial activity. The mechanism of killing by d ‐arginine substituted unacylated analog involves transient interaction with the bacterial membrane followed by translocation into the cytoplasm without membrane permeabilization. Accumulation of peptides in the cytoplasm can affect various cellular processes that lead to cell death. However, the peptide causes membrane permeabilization in case of C. albicans. Myristoylation results in greater interaction of the peptide chain with the microbial cell surface and causes membrane permeabilization. Results described in the study demonstrate that it is possible to generate highly active linear analogs of defensins by selective introduction of d ‐amino acids and fatty acids, which could be attractive candidates for development as therapeutic agents. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Single muscle fiber proteomics reveals unexpected mitochondrial specialization

Mammalian skeletal muscles are composed of multinucleated cells termed slow or fast fibers according to their contractile and metabolic properties. Here, we developed a high‐sensitivity workflow to characterize the proteome of single fibers. Analysis of segments of the same fiber by traditional and unbiased proteomics methods yielded the same subtype assignment. We discovered novel subtype‐specific features, most prominently mitochondrial specialization of fiber types in substrate utilization. The fiber type‐resolved proteomes can be applied to a variety of physiological and pathological conditions and illustrate the utility of single cell type analysis for dissecting proteomic heterogeneity.     

The nucleosome (histone-DNA complex) is the TLR9-specific immunostimulatory component of Plasmodium falciparum that activates DCs

The systemic clinical symptoms of Plasmodium falciparum infection such as fever and chills correspond to the proinflammatory cytokines produced in response to the parasite components released during the synchronized rupture of schizonts. We recently demonstrated that, among the schizont-released products, merozoites are the predominant components that activate dendritic cells (DCs) by TLR9-specific recognition to induce the maturation of cells and to produce proinflammatory cytokines. We also demonstrated that DNA is the active constituent and that formation of a DNA-protein complex is essential for the entry of parasite DNA into cells for recognition by TLR9. However, the nature of endogenous protein-DNA complex in the parasite is not known. In this study, we show that parasite nucleosome constitute the major protein-DNA complex involved in the activation of DCs by parasite nuclear material. The parasite components were fractionated into the nuclear and non-nuclear materials. The nuclear material was further fractionated into chromatin and the proteins loosely bound to chromatin. Polynucleosomes and oligonucleosomes were prepared from the chromatin. These were tested for their ability to activate DCs obtained by the FLT3 ligand differentiation of bone marrow cells from the wild type, and TLR2(-/-), TLR9(-/-) and MyD88(-/-) mice. DCs stimulated with the nuclear material and polynucleosomes as well as mono- and oligonucleosomes efficiently induced the production of proinflammatory cytokines in a TLR9-dependent manner, demonstrating that nucleosomes (histone-DNA complex) represent the major TLR9-specific DC-immunostimulatory component of the malaria parasite nuclear material. Thus, our data provide a significant insight into the activation of DCs by malaria parasites and have important implications for malaria vaccine development.     

Conformation and activity of delta-lysin and its analogs

Delta-Lysin is a 26-residue hemolytic peptide secreted by Staphylococcus aureus. Unlike the bee venom peptide melittin, delta-lysin does not exhibit antibacterial activity. We have synthesized delta-lysin and several analogs wherein the N-terminal residues of the toxin were sequentially deleted. The toxin has three aspartic acids, four lysines and no prolines. Analogs were also generated in which all the aspartic acids were replaced with lysines. A proline residue was introduced in the native sequences as well as in the analogs where aspartic acids were replaced with lysines. We observed that 20- and 22-residue peptides corresponding to residues 7-26 and 5-26 of delta-lysin, respectively, had greater hemolytic activity than the parent peptide. These shorter peptides, unlike delta-lysin, did not self-associate to adopt alpha-helical conformation in water, at lytic concentrations. Introduction of proline or substitution of aspartic acids by lysines resulted in loss in propensity to adopt helical conformation in water. When proline was introduced in the peptides corresponding to the native toxin sequence, loss of hemolytic activity was observed. Substitution of all the aspartic acids with lysines resulted in enhanced hemolytic activity in all the analogs. However, when both proline and aspartic acid to lysine changes were made, only antibacterial activity was observed in the shorter peptides. Our investigations on delta-lysin and its analogs provide insights into the positioning of anionic, cationic residues and proline in determining hemolytic and antibacterial activities.     

Phospho-caveolin-1 mediates integrin-regulated membrane domain internalization

Growth of normal cells is anchorage dependent because signalling through multiple pathways including Erk, phosphatidylinositol-3-OH kinase (PI(3)K) and Rac requires integrin-mediated cell adhesion. Components of these pathways localize to low-density, cholesterol-rich domains in the plasma membrane named 'lipid rafts' or 'cholesterol-enriched membrane microdomains' (CEMM). We previously reported that integrin-mediated adhesion regulates CEMM transport such that cell detachment from the extracellular matrix triggers CEMM internalization and clearance from the plasma membrane. We now report that this internalization is mediated by dynamin-2 and caveolin-1. Internalization requires phosphorylation of caveolin-1 on Tyr 14. A shift in localization of phospho-caveolin-1 from focal adhesions to caveolae induces CEMM internalization upon cell detachment, which mediates inhibition of Erk, PI(3)K and Rac. These data define a novel molecular mechanism for growth and tumour suppression by caveolin-1.     

Effect of methylglyoxal modification and phosphorylation on the chaperone and anti-apoptotic properties of heat shock protein 27

Heat shock protein 27 (Hsp27) is a stress-inducible protein in cells that functions as a molecular chaperone and also as an anti-apoptotic protein. Methylglyoxal (MGO) is a reactive dicarbonyl compound produced from cellular glycolytic intermediates that reacts non-enzymatically with proteins to form products such as argpyrimidine. We found considerable amount of Hsp27 in phosphorylated form (pHsp27) in human cataractous lenses. pHsp27 was the major argpyrimidine-modified protein in brunescent cataractous lenses. Modification by MGO enhanced the chaperone function of both pHsp27 and native Hsp27, but the effect on Hsp27 was at least three-times greater than on pHsp27. Phosphorylation of Hsp27 abolished its chaperone function. Transfer of Hsp27 using a cationic lipid inhibited staurosporine (SP)-induced apoptotic cell death by 53% in a human lens epithelial cell line (HLE B-3). MGO-modified Hsp27 had an even greater effect (62% inhibition). SP-induced reactive oxygen species in HLE-B3 cells was significantly lower in cells transferred with MGO-modified Hsp27 when compared to native Hsp27. In vitro incubation experiments showed that MGO-modified Hsp27 reduced the activity of caspase-9, and MGO-modified pHsp27 reduced activities of both caspase-9 and caspase-3. Based on these results, we propose that Hsp27 becomes a better anti-apoptotic protein after modification by MGO, which may be due to multiple mechanisms that include enhancement of chaperone function, reduction in oxidative stress, and inhibition of activity of caspases. Our results suggest that MGO modification and phosphorylation of Hsp27 may have important consequences for lens transparency and cataract development.     

Integrin-mediated adhesion regulates membrane order

The properties of cholesterol-dependent domains (lipid rafts) in cell membranes have been controversial. Because integrin-mediated cell adhesion and caveolin both regulate trafficking of raft components, we investigated the effects of adhesion and caveolin on membrane order. The fluorescent probe Laurdan and two-photon microscopy revealed that focal adhesions are highly ordered; in fact, they are more ordered than caveolae or domains that stain with cholera toxin subunit B (CtxB). Membrane order at focal adhesion depends partly on phosphorylation of caveolin1 at Tyr14, which localizes to focal adhesions. Detachment of cells from the substratum triggers a rapid, caveolin-independent decrease in membrane order, followed by a slower, caveolin-dependent decrease that correlates with internalization of CtxB-stained domains. Endocytosed CtxB domains also become more fluid. Thus, membrane order is highly dependent on caveolae and focal adhesions. These results show that lipid raft properties are conferred by assembly of specific protein complexes. The ordered state within focal adhesions may have important consequences for signaling at these sites.