Electron paramagnetic resonance studies and effects of vanadium in Saccharomyces cerevisiae

Vanadium uptake by whole cells and isolated cell walls of the yeast Saccharomyces cerevisiae was studied. When orthovanadate was added to wild-type S. cerevisiae cells growing in rich medium, growth was inhibited as a function of the VO₄ ^3- concentration and the growth was completely arrested at a concentration of 20 mM of VO₄ ^3- in YEPD. Electron paramagnetic resonance (EPR) spectroscopy was used to obtain structural and dynamic information about the cell-associated paramagnetic vanadyl ion. The presence of EPR signals indicated that vanadate was reduced by whole cells to the vanadyl ion. On the contrary, no EPR signals were detected after interaction of vanadate with isolated cell walls. A mobile and an immobile species associated in cells with small chelates and with macromolecular sites, respectively, were identified. The value of rotational correlation time _r indicated the relative motional freedom at the macromolecular site. A strongly immobilized vanadyl species bound to polar sites mainly through coulombic attractions was detected after interaction of VO²⁺ ions with isolated cell walls.     

The beneficial effect of Trichoderma spp. on tomato is modulated by the plant genotype

Rhizosphere-competent fungi of the genus Trichoderma are widely used as biofertilizers and biopesticides in commercial formulates because of the multiple beneficial effects on plant growth and disease resistance. In this work, we demonstrate that genetic variability among wild and cultivated tomato lines affects the outcome of the interaction with two 'elite' biocontrol strains of T. atroviride and T. harzianum. The beneficial response, which included enhanced growth and systemic resistance against Botrytis cinerea, was clearly evident for some, but not all, the tested lines. At least in one case (line M82), treatment with the biocontrol agents had no effect or was even detrimental. Expression studies on defence-related genes suggested that the fungus is able to trigger, in the responsive lines, a long-lasting up-regulation of the salicylic acid pathway in the absence of a pathogen, possibly activating a priming mechanism in the plant. Consequently, infection with B. cinerea on plants pretreated with Trichoderma is followed by enhanced activation of jasmonate-responsive genes, eventually boosting systemic resistance to the pathogen in a plant genotype-dependent manner. Our data indicate that, at least in tomato, the Trichoderma induced systemic resistance mechanism is much more complex than considered so far, and the ability of the plant to benefit from this symbiotic-like interaction can be genetically improved.     

A JAK2 Interdomain Linker Relays Epo Receptor Engagement Signals to Kinase Activation

JAK2 (Janus kinase 2) is essential for cytokine receptor signaling, and several lines of evidence support a causal role of an activating JAK2 mutation in myeloproliferative disorders. JAK2 activity is autoinhibited by its pseudokinase domain in the basal state, and the inhibition is released by cytokine stimulation; how engagement of the cognate receptor triggers this release is unknown. From a functional screen for gain-of-function JAK2 mutations, we discovered 13 missense mutations, nine in the pseudokinase domain and four in the Src homology 2 (SH2)-pseudokinase domain linker. These mutations identified determinants for autoinhibition and inducible activation in JAK2. Two of the mutants, K539I and N622I, resulted in erythrocytosis in mice. Scanning mutagenesis of the SH2-pseudokinase domain linker indicated that its N-terminal part was essential for interaction of JAK2 with the Epo receptor, whereas certain mutations in the C-terminal region conferred constitutive activation. We further showed that substitutions for Glu⁵⁴³-Asp⁵⁴⁴ in this linker or Leu⁶¹¹, Arg⁶⁸³, or Phe⁶⁹⁴ in the hinge proximal region of the pseudokinase domain resulted in activated JAK2 mutants that could not be further stimulated by Epo. These results suggest that the SH2-pseudokinase domain linker acts as a switch that relays cytokine engagement to JAK2 activation by flexing the pseudokinase domain hinge.The Janus family of tyrosine kinases (JAKs)² are key regulators of cytokine receptor signaling in hematopoiesis and immune responses (1). Of the four mammalian JAK kinases, JAK2 transmits signals for a variety of cytokine receptors, including the erythropoietin receptor (EpoR) that is essential for red blood cell production (2). Upon Epo stimulation, JAK2 activates downstream signaling, such as STAT5, Ras/mitogen-activated protein kinase, and phosphatidylinositol 3-kinase/AKT pathways (2). Mice deficient in Epo, EpoR, or JAK2 die embryonically due to the absence of definitive erythropoiesis (3–5).In addition to regulation by phosphatases and suppressors of cytokine signaling (6, 7), JAK2 kinase activity is critically controlled by an autoinhibitory mechanism. Like other JAK members, JAK2 contains an N-terminal segment followed by a pseudokinase domain and a C-terminal tyrosine kinase domain. The N-terminal segment, consisting of a FERM (protein 4.1, ezrin, moezin, radixin homologous) domain and an atypical SH2 domain (1), mediates association with the membrane-proximal region of the cytokine receptors (8). Binding of JAK2 through its N-terminal segment to the EpoR is essential for EpoR surface expression (9). The pseudokinase domain is predicted to adopt a kinase fold but lacks residues essential for catalysis (10). Deletion of the pseudokinase domain leads to a marked increase in JAK2 kinase activity and loss of response to cytokine stimulation (11–13). Therefore, this domain is essential for JAK2 autoinhibition and is essential for JAK2 activation upon cytokine stimulation. Consistent with this notion, a point mutation in the JAK2 pseudokinase domain was identified in the majority of myeloproliferative disorder patients, including 90% of polycythemia vera (PV) patients (14–18). This mutation, V617F, in the presence of a dimerized receptor scaffold, such as the EpoR, resulted in the constitutive activation of JAK2 and downstream signaling effectors (19, 20) and caused erythrocytosis in a murine bone marrow transplant model (14, 21–23). Recently, mutations immediately adjacent to the JAK2 pseudokinase domain in the SH2-pseudokinase domain linker were identified in PV patients and shown to cause constitutive activation of JAK2 and a PV-like phenotype in mice (24–26). The molecular mechanisms underlying the control of JAK2 activity (i.e. the swift augmentation of its activity upon receptor activation) are poorly understood. The residues involved in the autoinhibition in JAK2 are unknown.In this work, we sought to characterize the regulatory mechanisms controlling JAK2 kinase activity. Using a functional screen for activating JAK2 mutations that signal constitutively, we discovered 13 mutations in the pseudokinase domain and in the SH2-pseudokinase domain linker. These mutations identified specific residues that are important for the inhibition of basal JAK2 kinase activity and for cytokine-induced JAK2 activation. In addition, we showed that the SH2-pseudokinase domain linker is essential for interaction with the EpoR, autoinhibitory regulation, and Epo-inducible JAK2 activation and may act as a switch in relaying cytokine receptor engagement to JAK2 activation by flexing the pseudokinase domain hinge.     

Exposure to 50 Hz electromagnetic field raises the levels of the anti-apoptotic protein BAG3 in melanoma cells

The expression of the anti‐apoptotic protein BAG3 is induced in several cell types by exposure to high temperature, oxidants, and other stressful agents. We investigated whether exposure to 50 Hz electromagnetic fields raised BAG3 levels in the human melanoma cell line M14, in vitro and in orthotopic xenografts. Exposure of cultured cells or xenografts for 6 h or 4 weeks, respectively, produced a significant (P < 0.01) increase in BAG3 protein amounts. Interestingly, at the same times, we could not detect any significant variation in the levels of HSP70/72 protein or cell apoptosis. These results confirm the stressful effect of exposure to ELF in human cells, by identifying BAG3 protein as a marker of ELF‐induced stress. Furthermore, they suggest that BAG3 induction by ELF may contribute to melanoma cell survival and/or resistance to therapy. J. Cell. Physiol. 226: 2901–2907, 2011. © 2011 Wiley‐Liss, Inc.     

Two separate pathways for d-lactate oxidation by Saccharomyces cerevisiae mitochondria which differ in energy production and carrier involvement

In this work we looked at whether and how mitochondria isolated from Saccharomyces cerevisiae (SCM) oxidize d-lactate. We found that: (1). externally added d-lactate causes oxygen uptake by SCM with P/O ratio equal to 1.5; in the presence of antimycin A (AA), P/O ratio was 1.8, differently in the presence of the non-penetrant alpha-cyanocinnamate (alpha-CCN-) no P/O ratio could be measured. Consistently, mitochondrial electrical membrane potential (deltapsi) generation was found, due to externally added d-lactate in the presence of antimycin A, but not of alpha-CCN-. (2). SCM oxidize d-lactate in two different manners: (i). via inner membrane d-lactate dehydrogenase which leads to d-lactate oxidation without driving deltapsi generation and ATP synthesis and (ii). via the matrix d-lactate dehydrogenase, which drives deltapsi generation and ATP synthesis by using taken up d-lactate. (3). Pyruvate newly synthesised in the mitochondrial matrix is exported via the novel d-lactate/pyruvate antiporter. d-Lactate/pyruvate antiport proved to regulate the rate of pyruvate efflux in vitro. (4). The existence of the d-lactate/H+ symporter is also proposed as shown by mitochondrial swelling. The d-lactate carriers and d-lactate dehydrogenases could account for the removal of the toxic methylglyoxal from cytosol, as well as for the d-lactate-dependent gluconeogenesis.     

Impaired effector function of hepatitis C virus-specific CD8+ T cells in chronic hepatitis C virus infection

The cellular immune response contributes to clearance of hepatitis C virus (HCV) and persists for decades after recovery from infection. The immunological basis for the inefficiency of the cellular immune response in chronically infected persons is not known. Here, we used four HLA-A2 tetramers, specific for two HCV core and two HCV NS3 epitopes, to investigate at the single-cell level effector function and phenotype of HCV-specific CD8+ T cells in 20 chronically infected and 12 long-term recovered patients. Overall, HCV-specific, tetramer+ T cells were more frequently found in PBMCs of chronically infected patients than in those of recovered patients. However, when compared with HCV-tetramer+ T cells of recovered patients, they displayed an impaired proliferative capacity. As a result of the impaired proliferative capacity, HCV-specific T cell lines derived from chronically infected patients displayed less peptide-specific cytotoxicity than those from recovered patients. In addition, proliferation and ex vivo IFN-gamma production of HCV-tetramer+ cells, but not influenza-virus-specific T cells, were defective in chronically infected patients and could not be restored by in vitro stimulation with peptide and IL-2. At least three distinct phenotypes of HCV-specific CD8+ T cells were identified and associated with certain functional characteristics. In addition, impairment of proliferative, cytokine, and cytotoxic effector functions of tetramer+ T cells in viremic patients was associated with weak ex vivo HCV-specific CD4+ T cell responses. Thus, the defective functions of HCV-specific CD8+ T cells might contribute to viral persistence in chronically infected patients, and knowledge on their reversibility may facilitate the development of immunotherapeutic vaccines.     

Molecular and immunological significance of chimpanzee major histocompatibility complex haplotypes for hepatitis C virus immune response and vaccination studies

The chimpanzee is a critical animal model for studying cellular immune responses to infectious pathogens such as hepatitis B and C viruses, human immunodeficiency virus, and malaria. Several candidate vaccines and immunotherapies for these infections aim at the induction or enhancement of cellular immune responses against viral epitopes presented by common human major histocompatibility complex (MHC) alleles. To identify and characterize chimpanzee MHC class I molecules that are functionally related to human alleles, we sequenced 18 different Pan troglodytes (Patr) alleles of 14 chimpanzees, 2 of them previously unknown and 3 with only partially reported sequences. Comparative analysis of Patr binding pockets and binding assays with biotinylated peptides demonstrated a molecular homology between the binding grooves of individual Patr alleles and the common human alleles HLA-A1, -A2, -A3, and -B7. Using cytotoxic T cells isolated from the blood of hepatitis C virus (HCV)-infected chimpanzees, we then mapped the Patr restriction of these HCV peptides and demonstrated functional homology between the Patr-HLA orthologues in cytotoxicity and gamma interferon (IFN-gamma) release assays. Based on these results, 21 HCV epitopes were selected to characterize the chimpanzees' cellular immune response to HCV. In each case, IFN-gamma-producing T cells were detectable in the blood after but not prior to HCV infection and were specifically targeted against those HCV peptides predicted by Patr-HLA homology. This study demonstrates a close functional homology between individual Patr and HLA alleles and shows that HCV infection generates HCV peptides that are recognized by both chimpanzees and humans with Patr and HLA orthologues. These results are relevant for the design and evaluation of vaccines in chimpanzees that can now be selected according to the most frequent human MHC haplotypes.