A barley mutant with improved salt tolerance through ion homeostasis and ROS scavenging under salt stress

To investigate key regulatory components and genes with great impact on salt tolerance, near isogenic or mutant lines with distinct salinity tolerance are suitable genetic materials to simplify and dissect the complex genes networks. In this study, we evaluated responses of a barley mutant genotype (73-M4-30), in comparison with its wild-type background (Zarjou) under salt stress. Although the root growth of both genotypes was significantly decreased by exposure to sodium chloride (NaCl), the effect was greater in the wild type. The chlorophyll content decreased under salt stress for the wild type, but no change occurred in the mutant. The mutant maintained the steady-state level of [K⁺] and significantly lower [Na⁺] concentrations in roots and higher [K⁺]/[Na⁺] ratio in shoots under salt conditions. The catalase (CAT), peroxidase (POD) activity, and proline content were higher in the mutant than those in the wild type under controlled conditions. The soluble proline was higher after 24 h of salt stress in roots of the mutant but was higher after 96 h of salt stress in the wild type. The CAT and POD activity of the mutant increased under salt stress which was as a coincidence to lower levels of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) contents. The ratio of dry-to-fresh weight of the roots increased for the mutant under salt stress which was as a result of the higher phenylalanine ammonia-lyase (PAL) gene expression and peroxidase activity and involved in cell wall lignification. Consequently, it seems that ion homeostasis and increased peroxidase activity have led to salt tolerance in the mutant’s genotype.     

Epidermal bladder cells confer salinity stress tolerance in the halophyte quinoa and Atriplex species

Epidermal bladder cells (EBCs) have been postulated to assist halophytes in coping with saline environments. However, little direct supporting evidence is available. Here, Chenopodium quinoa plants were grown under saline conditions for 5 weeks. One day prior to salinity treatment, EBCs from all leaves and petioles were gently removed by using a soft cosmetic brush and physiological, ionic and metabolic changes in brushed and non‐brushed leaves were compared. Gentle removal of EBC neither initiated wound metabolism nor affected the physiology and biochemistry of control‐grown plants but did have a pronounced effect on salt‐grown plants, resulting in a salt‐sensitive phenotype. Of 91 detected metabolites, more than half were significantly affected by salinity. Removal of EBC dramatically modified these metabolic changes, with the biggest differences reported for gamma‐aminobutyric acid (GABA), proline, sucrose and inositol, affecting ion transport across cellular membranes (as shown in electrophysiological experiments). This work provides the first direct evidence for a role of EBC in salt tolerance in halophytes and attributes this to (1) a key role of EBC as a salt dump for external sequestration of sodium; (2) improved K⁺ retention in leaf mesophyll and (3) EBC as a storage space for several metabolites known to modulate plant ionic relations.     

A semi-empirical model of apparent blood viscosity as a function of vessel diameter and discharge hematocrit

A semi-empirical model is developed to describe the dependence of apparent viscosity of blood on vessel diameter (2.7 to 500 microns) and vessel discharge hematocrit (5% to 60%). The blood flow is modeled as a cell-rich core and a cell-free marginal layer in the larger vessels and an axial-train in the smaller vessels. Laminar (Poiseuille) flow is assumed in all cases. An equation is derived in which apparent viscosity is a function of vessel diameter, core viscosity, and width of marginal layer. This is then complemented by empirical equations in which core viscosity varies exponentially with discharge hematocrit while the width of marginal layer varies linearly with discharge hematocrit. The model correlates well with several sets of experimental data and behaves according to the Fahraeus-Lindqvist effect. Predicted apparent viscosity tends to the expected finite value for large vessel diameters. Dependence of apparent viscosity on vessel diameter is realistically smooth in the whole diameter range.     

Prediction of T Cell Epitopes from Leishmania major Potentially Excreted/Secreted Proteins Inducing Granzyme B Production

Leishmania-specific cytotoxic T cell response is part of the acquired immune response developed against the parasite and contributes to resistance to reinfection. Herein, we have used an immune-informatic approach for the identification, among Leishmania major potentially excreted/secreted proteins previously described, those generating peptides that could be targeted by the cytotoxic immune response. Seventy-eight nonameric peptides that are predicted to be loaded by HLA-A*0201 molecule were generated and their binding capacity to HLA-A2 was evaluated. These peptides were grouped into 20 pools and their immunogenicity was evaluated by in vitro stimulation of peripheral blood mononuclear cells from HLA-A2⁺-immune individuals with a history of zoonotic cutaneous leishmaniasis. Six peptides were identified according to their ability to elicit production of granzyme B. Furthermore, among these peptides 3 showed highest affinity to HLA-A*0201, one derived from an elongation factor 1-alpha and two from an unknown protein. These proteins could constitute potential vaccine candidates against leishmaniasis.     

Mechanism of desensitization of the Ca2(+)-mobilizing system to bombesin by Ha-ras. Independence from down-modulation of agonist-stimulated inositol phosphate production.

Expression of a transforming Ha-ras gene in NIH 3T3 cells transfected with an inducible Ha-ras construct leads to a rapid desensitization of the intracellular Ca2(+)-mobilizing system to bombesin and serum growth factors. Half-maximal depression of the Ca2+ response is observed 2 h after induction of p21ras. A maximum is obtained after 6 h. Bombesin-induced elevation of inositol 1,4,5-trisphosphate formation is also depressed in cells expressing Ha-ras. This, however, is a relatively late phenomenon and not yet detectable when maximal depression of the Ca2+ signal is observed. We conclude that the rapid densensitization of the Ca2(+)-releasing system to bombesin by Ha-ras is not caused by down-modulation or uncoupling of phospholipase C-coupled bombesin receptors. The inositol 1,4,5-trisphosphate-mediated release of intracellular Ca2+ is reduced in permeabilized cells expressing the Ha-ras oncogene. A depletion of intracellular Ca2+ stores by Ha-ras is unlikely since (i) the Ha-ras-induced growth factor-independent stimulation of inositol phosphate formation occurs several hours after reduction of the Ca2+ response and (ii) the Ca2+ load of intracellular nonmitochondrial Ca2+ stores was found to be unaffected by Ha-ras. We conclude that the desensitization of the Ca2(+)-mobilizing system is caused either by partial inhibition of inositol 1,4,5-trisphosphate-regulated Ca2+ channels or by interference of Ha-ras with Ca2+ translocation between intracellular Ca2+ compartments.     

Dendritic cells loaded with exosomes derived from cancer stem cell-enriched spheroids as a potential immunotherapeutic option

Cancer stem cells (CSCs) are responsible for therapeutic resistance and recurrence in colorectal cancer. Despite advances in immunotherapy, the inability to specifically eradicate CSCs has led to treatment failure. Hence, identification of appropriate antigen sources is a major challenge in designing dendritic cell (DC)-based therapeutic strategies against CSCs. Here, in an in vitro model using the HT-29 colon cancer cell line, we explored the efficacy of DCs loaded with exosomes derived from CSC-enriched colonospheres (CSC_enr-EXOs) as an antigen source in activating CSC-specific T-cell responses. HT-29 lysate, HT-29-EXOs and CSC_enr lysate were independently assessed as separate antigen sources. Having confirmed CSCs enrichment in spheroids, CSC_enr-EXOs were purified and characterized, and their impact on DC maturation was investigated. Finally, the impact of the antigen-pulsed DCs on the proliferation rate and also spheroid destructive capacity of autologous T cells was assessed. CSC_enr-EXOs similar to other antigen groups had no suppressive/negative impacts on phenotypic maturation of DCs as judged by the expression level of costimulatory molecules. Notably, similar to CSC_enr lysate, CSC_enr-EXOs significantly increased the IL-12/IL-10 ratio in supernatants of mature DCs. CSC_enr-EXO-loaded DCs effectively promoted T-cell proliferation. Importantly, T cells stimulated with CSC_enr-EXOs disrupted spheroids' structure. Thus, CSC_enr-EXOs present a novel and promising antigen source that in combination with conventional tumour bulk-derived antigens should be further explored in pre-clinical immunotherapeutic settings for the efficacy in hampering recurrence and metastatic spread.     

The C‐terminal linker of Escherichia coli FtsZ functions as an intrinsically disordered peptide

The tubulin homologue FtsZ provides the cytoskeletal framework and constriction force for bacterial cell division. FtsZ has an ～ 50‐amino‐acid (aa) linker between the protofilament‐forming globular domain and the C‐terminal (Ct) peptide that binds FtsA and ZipA, tethering FtsZ to the membrane. This Ct‐linker is widely divergent across bacterial species and thought to be an intrinsically disordered peptide (IDP). We confirmed that the Ct‐linkers from three bacterial species behaved as IDPs in vitro by circular dichroism and trypsin proteolysis. We made chimeras, swapping the Escherichia coli linker for Ct‐linkers from other bacteria, and even for an unrelated IDP from human α‐adducin. Most substitutions allowed for normal cell division, suggesting that sequence of the IDP did not matter. With few exceptions, almost any sequence appears to work. Length, however, was important: IDPs shorter than 43 or longer than 95 aa had compromised or no function. We conclude that the Ct‐linker functions as a flexible tether between the globular domain of FtsZ in the protofilament, and its attachment to FtsA/ZipA at the membrane. Modelling the Ct‐linker as a worm‐like chain, we predict that it functions as a stiff entropic spring linking the bending protofilaments to the membrane.     

Structural Basis for Evolution of Product Diversity in Soybean Glutathione Biosynthesis

The redox active peptide glutathione is ubiquitous in nature, but some plants also synthesize glutathione analogs in response to environmental stresses. To understand the evolution of chemical diversity in the closely related enzymes homoglutathione synthetase (hGS) and glutathione synthetase (GS), we determined the structures of soybean (Glycine max) hGS in three states: apoenzyme, bound to γ-glutamylcysteine (γEC), and with hGSH, ADP, and a sulfate ion bound in the active site. Domain movements and rearrangement of active site loops change the structure from an open active site form (apoenzyme and γEC complex) to a closed active site form (hGSH•ADP•SO₄²⁻ complex). The structure of hGS shows that two amino acid differences in an active site loop provide extra space to accommodate the longer β-Ala moiety of hGSH in comparison to the glycinyl group of glutathione. Mutation of either Leu-487 or Pro-488 to an Ala improves catalytic efficiency using Gly, but a double mutation (L487A/P488A) is required to convert the substrate preference of hGS from β-Ala to Gly. These structures, combined with site-directed mutagenesis, reveal the molecular changes that define the substrate preference of hGS, explain the product diversity within evolutionarily related GS-like enzymes, and reinforce the critical role of active site loops in the adaptation and diversification of enzyme function.     

Novel method to obtain highly enriched cultures of adult rat Schwann cells

Schwann cells (SCs) can be used to repair both the peripheral and central nervous systems. Therefore, establishment of a procedure to obtain activated, highly proliferative SCs, in an appropriate time for clinical applications, is a prerequisite. Purification is complicated by contamination with fibroblasts which often become the predominant cell type in an in vitro SC culture. This study describes a novel and efficient method to enrich SCs by utilizing the differential detachment properties of the two cell types. In culture, cells were treated with two different media and the chelator, EGTA, which detached SCs faster than fibroblasts and allowed for easy isolation of SCs. Within seven days, high yields of SCs with a purity of greater than 99% were achieved. This was confirmed by immunostaining characterization and flow-cytometric analyses using an antibody against the p75 low affinity nerve growth factor receptor (p75LNGFR). The entire procedure was completed in approximately 21 days. This method has the advantage of being technically easier, faster, and more efficient than other previously described methods. An SC culture that was about 99% homogenous was achieved.     

A tumor vasculature targeted liposome delivery system for combretastatin A4: Design, characterization, and in vitro evaluation

The objective of this study was to develop an efficient tumor vasculature targeted liposome delivery system for combretastatin A4, a novel antivascular agent. Liposomes composed of hydrogenated soybean phosphatidylcholine (HSPC), cholesterol, distearoyl phosphoethanolamine-polyethylene-glycol-2000 conjugate (DSPE-PEG), and DSPE-PEG-maleimide were prepared by the lipid film hydration and extrusion process. Cyclic RGD (Arg-Gly-Asp) peptides with affinity for αvβ3-integrins expressed on tumor vascular endothelial cells were coupled to the distal end of PEG on the liposomes sterically stabilized with PEG (long circulating liposomes, LCL). The liposome delivery system was characterized in terms of size, lamellarity, ligand density, drug loading, and leakage properties. Targeting nature of the delivery system was evaluated in vitro using cultured human umbilical vein endothelial cells (HUVEC). Electron microscopic observations of the formulations revealed presence of small unilamellar liposomes of ∼120 nm in diameter. High performance liquid chromatography determination of ligand coupling to the liposome surface indicated that more than 99% of the RGD peptides were reacted with maleimide groups on the liposome surface. Up to 3 mg/mL of stable liposomal combretastatin A4 loading was achieved with ∼80% of this being entrapped within the liposomes. In the in vitro cell culture studies, targeted liposomes showed significantly higher binding to their target cells than non-targeted liposomes, presumably through specific interaction of the RGD with its receptors on the cell surface. It was concluded that the targeting properties of the prepared delivery system would potentially improve the therapeutic benefits of combretastatin A4 compared with nontargeted liposomes or solution dosage forms.