Cellular redox state and activating protein-1 are involved in ascorbate effect on calcitriol-induced differentiation

Summary Ascorbate has been related to the differentiation of several mesenchymal cells including haematopoietic cells. We have previously demonstrated that ascorbate enhances the activity of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) on monocytic differentiation of HL-60 cells. Here, we show that ascorbate-mediated modification of cellular redox state and AP-1 (activating protein-1) DNA binding during early phases are related to the enhancing effect of ascorbate on differentiation. Ascorbate, but not its fully oxidized form, dehydroascorbate, or an ascorbate analogue with a low rate of oxidation, ascorbate-2-phosphate, enhanced the differentiation induced by 1,25(OH)₂D₃, modified cytosolic reactive oxygen species levels and mitochondrial redox potential (_m), and modulated AP-1 DNA binding in HL-60 cells. Ascorbate itself increased AP-1 binding to DNA in noninduced cells, whereas it inhibited AP-1 binding in 1,25(OH)₂D₃-induced cells. However, ascorbate increased the mRNA levels ofc-jun, junB. andc-fos in 1,25(OH)₂D₃-induced cells. Taken together, these results suggest that the enhancing effect of ascorbate on HL-60 differentiation induced by 1,25(OH)₂D₃ is related to its effect on the cellular redox state and the modulation of AP-1 activity.Abbreviations 1,25-(OH)₂D₃ 1,25-dihydroxyvitamin D₃ - _m mitochondrial transmembrane potential - AP-1 activating protein-1 - Asc-2-P ascorbate-2-phosphate - DHA dehydroascorbate - DiOC₆(3) 3,3-dihexyloxacarboxyanine iodide - EMSA electromobility shift assay - NBT nitroblue tetrazolium - ROS reactive oxygen species     

Terguride attenuates prolactin levels and ameliorates insulin sensitivity and insulin binding in obese spontaneously hypertensive rats

Glucose tolerance, serum insulin, insulin receptors in epididymal fat tissue, circulating total cholesterol and triglyceride concentrations as well as serum prolactin were studied in obese and lean spontaneously hypertensive rats (SHR) of both sexes. Obese animals displayed insulin resistance and elevated insulin and triglyceride concentrations. Moreover, in obese rats the increased mass of epididymal fat tissue was accompanied with decreased capacity of high affinity binding sites of insulin receptors in the tissue plasma membranes. Terguride treatment lowered prolactin serum levels which was accompanied by ameliorated insulin sensitivity in obese animals of both sexes. In addition, terguride treatment decreased serum insulin and triglyceride concentrations in obese females and at the same time enhanced the affinity of high affinity insulin binding sites. Our results show that obesity in SHR is associated with a decreased capacity of insulin receptors and that prolactin may play a role in obesity-induced insulin resistance, particularly in female rats.     

Insights into plant immunity signaling: The bacterial competitive index angle

The interaction between a bacterial pathogen and its potential plant host develops from a complex combination of bacterial and plant elements, which determines either the establishment of resistance or the development of disease. The use of virulence assays based on competitive index in mixed infections constitutes a powerful tool for the analysis of bacterial virulence factors. In this work, we describe how the use of competitive index assays also constitutes an alternative approach for the analysis of plant immunity, to determine the contribution of different elements to bacterial recognition or immunity signaling.Key words: competitive index, mixed infections, pathogen, plant immunity, defence response, effector-triggered immunityThe type III secretion system (T3SS) allows Gram negative bacterial pathogens to deliver a set of effector proteins into the host cell. The plant pathogen Pseudomonas syringae employs a large inventory of type III-secreted effectors (T3SEs) to suppress plant immunity. Individual mutation of effector genes has traditionally failed to provide a relevant virulence phenotype, a fact generally associated to a high degree of functional redundancy between T3SEs, which also hinders the characterisation of effector activities within the plant cell. This problem has led researchers to use alternative approaches to overcome functional redundancy, including the generation of polymutants lacking several effector genes, ectopic expression of effectors in heterologous strains lacking the corresponding homolog, as well as the generation of transgenic plants expressing a given effector.¹ We have previously established that the use of competitive index in mixed infections provides an accurate and sensitive manner of establishing virulence phenotypes for single effector mutants for which other assays have failed,² thus providing an alternative to previously used approaches for the analysis of effector function within the context of the infection. This increase in sensitivity and accuracy is due to the direct comparison between growth of the co-inoculated strains within the same infection (Fig. 1), which replicate as they would in individual infections under the appropriate experimental settings.²Open in a separate windowFigure 1Basis for the increased sensitivity and acuracy of CI assays. A mixed inoculum with equal amounts of wild type and query strain is inoculated within the same plant (A), allowing a direct comparison between the replication values of both strains within the same infection (B). On the contrary, in regular individual infections, the values obtained from different plants have to be pulled first and compared afterwards (B), thus accumulating experimental and plant-to-plant variation.Plant immunity can be triggered by a group of conserved microbial molecules known as PAMPs (pathogen-associated molecular patterns). PAMP-triggered immunity (PTI) can be suppressed by effectors which in turn can be recognized by nucleotide binding-leucine rich repeat (NB-LRR) proteins, encoded by resistance genes or R genes.^3,4 Detection of such effectors by NB-LRR proteins determines effector-triggered immunity (ETI),³ an amplified version of PTI, which usually crosses the threshold inducing the hypersensitive response (HR), a localized cell death response. Furthermore, bacteria have evolved effectors that suppress ETI, some of which can in turn be recognized by the plant, thus triggering a secondary ETI.⁴ Selection favors new plant NB-LRRs that can recognize such secondary, newly acquired effectors.R-gene-mediated defences are usually associated with the accumulation of salycilic acid (SA),⁵ although SA-independent pathways such as that dependent on EDS1 (enhanced disease susceptibility-1),⁶ can also mediate ETI and trigger an HR.⁷ Although regulating independent pathways, EDS1 and SA have been recently described to function redundantly to regulate R-gene-mediated signaling.⁸     

Negative control of BAK1 by protein phosphatase 2A during plant innate immunity

Recognition of pathogen‐associated molecular patterns (PAMPs) by surface‐localized pattern‐recognition receptors (PRRs) activates plant innate immunity, mainly through activation of numerous protein kinases. Appropriate induction of immune responses must be tightly regulated, as many of the kinases involved have an intrinsic high activity and are also regulated by other external and endogenous stimuli. Previous evidences suggest that PAMP‐triggered immunity (PTI) is under constant negative regulation by protein phosphatases but the underlying molecular mechanisms remain unknown. Here, we show that protein Ser/Thr phosphatase type 2A (PP2A) controls the activation of PRR complexes by modulating the phosphostatus of the co‐receptor and positive regulator BAK1. A potential PP2A holoenzyme composed of the subunits A1, C4, and B’η/ζ inhibits immune responses triggered by several PAMPs and anti‐bacterial immunity. PP2A constitutively associates with BAK1 in planta. Impairment in this PP2A‐based regulation leads to increased steady‐state BAK1 phosphorylation, which can poise enhanced immune responses. This work identifies PP2A as an important negative regulator of plant innate immunity that controls BAK1 activation in surface‐localized immune receptor complexes.     

SGT1 is not required for plant LRR-RLK-mediated immunity

Plant immune signalling activated by the perception of pathogen-associated molecular patterns (PAMPs) or effector proteins is mediated by pattern-recognition receptors (PRRs) and nucleotide-binding and leucine-rich repeat domain-containing receptors (NLRs), which often share cellular components and downstream responses. Many PRRs are leucine-rich repeat receptor-like kinases (LRR-RLKs), which mostly perceive proteinaceous PAMPs. The suppressor of the G2 allele of skp1 (SGT1) is a core immune regulator required for the activation of NLR-mediated immunity. In this work, we examined the requirement of SGT1 for immune responses mediated by several LRR-RLKs in both Nicotiana benthamiana and Arabidopsis. Using complementary genetic approaches, we found that SGT1 is not limiting for early PRR-dependent responses or antibacterial immunity. We therefore conclude that SGT1 does not play a significant role in bacterial PAMP-triggered immunity.