AtIREG2 encodes a tonoplast transport protein involved in iron-dependent nickel detoxification in Arabidopsis thaliana roots.

Iron acquisition in Arabidopsis depends mainly on AtIRT1, a Fe2+ transporter in the plasma membrane of root cells. However, substrate specificity of AtIRT1 is low, leading to an excess accumulation of other transition metals in iron-deficient plants. In the present study we describe AtIREG2 as a nickel transporter at the vacuolar membrane that counterbalances the low substrate specificity of AtIRT1 and possibly other iron transport systems in iron-deficient root cells. AtIREG2 is co-regulated with AtIRT1 by the transcription factor FRU/FIT1, encodes a membrane protein, which has 10 putative transmembrane domains and shares homology with vertebrate Fe2+ exporters. Heterologous expression of AtIREG2 in various yeast mutants, however, did not demonstrate an iron transport function. Instead, expression in wild-type and nickel-sensitive cot1 yeast cells conferred enhanced tolerance to elevated concentrations of nickel at acidic pH. A role in vacuolar substrate transport was further supported by localization of AtIREG2-GFP fusion proteins to the tonoplast in Arabidopsis suspension cells and root cells of intact plants. Transgenic plants overexpressing AtIREG2 showed an increased tolerance to elevated concentrations of nickel, whereas T-DNA insertion lines lacking AtIREG2 expression were more sensitive to nickel, particularly under iron deficiency, and accumulated less nickel in roots. We therefore propose a role of AtIREG2 in vacuolar loading of nickel under iron deficiency and thus identify it as a novel component in the iron deficiency stress response.

Analysis of the functional modules of the tRNA 3' endonuclease (tRNase Z).

tRNA 3' processing is one of the essential steps during tRNA maturation. The tRNA 3'-processing endonuclease tRNase Z was only recently isolated, and its functional domains have not been identified so far. We performed an extensive mutational study to identify amino acids and regions involved in dimerization, tRNA binding, and catalytic activity. 29 deletion and point variants of the tRNase Z enzyme were generated. According to the results obtained, variants can be sorted into five different classes. The first class still had wild type activity in all three respects. Members of the second and third class still formed dimers and bound tRNAs but had reduced catalytic activity (class two) or no catalytic activity (class three). The fourth class still formed dimers but did not bind the tRNA and did not process precursors. Since this class still formed dimers, it seems that the amino acids mutated in these variants are important for RNA binding. The fifth class did not have any activity anymore. Several conserved amino acids could be mutated without or with little loss of activity.

Reverse signalling of membrane-integrated tumour necrosis factor differentially regulates alloresponses of CD4+ and CD8+ T cells against human microvascular endothelial cells.

Reverse signalling of membrane-integrated ligands is a common phenomenon in the tumour necrosis factor (TNF) family and contributes to the pleiotropy of this pro-inflammatory cytokine and to the plasticity of the immune system in general. Transmembrane TNF (mTNF) itself can induce resistance to bacterial endotoxin in monocytes and can stimulate the immune activity of mitogen-activated, as well as of virus-infected, T cells. The aim of the present study was to investigate the influence of reverse signalling of mTNF on the allogeneic activity of CD4+ and CD8+ T cells against human microvascular endothelial cells (HMEC), as targets of various inflammatory responses. The proliferative potential of CD4+ T cells towards HMEC was attenuated by mTNF signalling, whereas stimulation of mTNF on CD8+ T cells increased their cytotoxic potential against HMEC. These effects were specific for reverse signalling of mTNF, as a blockade of the classical TNF-TNF receptor interaction by a neutralizing TNF receptor antibody had no effect. Cytokine profiling of the effector cells revealed that the anti-endothelial CD4+ T cells were of a T helper 2 (Th2) phenotype, whereas CD8+ T cells mainly produced cytotox. T cell 1 (Tc1) cytokines. From the results obtained in this study, we conclude that reverse signalling of mTNF differentially modulates CD4+ and CD8+ T-cell activity against allogeneic endothelial cells, which should be taken into account in settings of therapeutic cytokine antagonisms.

Effect of different tumor necrosis factor (TNF) reactive agents on reverse signaling of membrane integrated TNF in monocytes.

Reverse signaling of transmembrane TNF (mTNF) contributes to the versatility of this cytokine superfamily. Previously, we could demonstrate that mTNF acting as receptor confers resistance to bacterial lipopolysaccharide in monocytes and macrophages (MO/MPhi). Reverse signaling can be induced by incubation with the monoclonal anti-TNF antibody 195F and other TNF antagonists, such as the humanized monoclonal antibody infliximab and the humanized soluble TNF receptor construct etanercept, respectively, all in former or present clinical use. Here, we addressed the question whether there are differences in modulating the LPS response in MO/MPhi among these three antagonists. Whereas 195F and infliximab suppress both, the release of an LPS-induced endothelial cell apoptotic factor and proinflammatory cytokines, etanercept only protected against the LPS-triggered apoptosis activity, but left the LPS-induced cytokine release unchanged. These data could have clinical impact with regard to TNF neutralization strategies.

LPS resistance in monocytic cells caused by reverse signaling through transmembrane TNF (mTNF) is mediated by the MAPK/ERK pathway.

The transmembrane form of tumor necrosis factor (mTNF), expressed on activated monocytes (MO) and macrophages (MPhi), is able to induce apoptosis in human endothelial cells (EC). Apoptosis is mediated by two distinct mechanisms: direct cell contact and a yet-unidentified soluble protein, death factor X. In addition, mTNF acts as a receptor that transduces a "reverse signal" into MO/MPhi when bound to the TNF receptor on EC. Reverse signaling by mTNF confers resistance to bacterial lipopolysaccharide (LPS). Stimulation of reverse signaling by mTNF blocks the ability of MO/MPhi to produce death factor X and proinflammatory cytokines. We have investigated which signaling pathways are used by mTNF acting as receptor. Reverse signaling triggers two independent pathways that can be distinguished by protein kinase C (PKC) inhibitors. The suppression of LPS-induced death factor X is dependent on PKC, whereas the suppression of LPS-mediated cytokine release is not. LPS and reverse signaling stimulate the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway. It is interesting that the activation of reverse signaling by mTNF renders MO/MPhi refractory to a subsequent activation of the MAPK/ERK pathway by LPS. Thus, reverse signaling achieves LPS resistance in monocytic cells through interference with key signal-transduction pathways.

Fludarabine induces apoptosis, activation, and allogenicity in human endothelial and epithelial cells: protective effect of defibrotide.

Fludarabine is a nonmyeloablative immunosuppressant increasingly used as a component of alternative conditioning regimens before allogeneic bone marrow transplantation. It is expected to reduce conditioning-related toxicity and proinflammatory activation of the host tissues. However, in our in vitro study, we provide evidence that 2-fluoroadenine 9-beta-D-arabinofuranoside (F-Ara) as the active metabolized form of fludarabine damages human microvascular endothelial cells (HMECs) and dermal and alveolar epithelial cell lines after 48 hours of culture when it is used in pharmacologically relevant concentrations (range, 10 microg/mL-1 microg/mL). In addition, flow cytometric analyses revealed a significant up-regulation of intercellular adhesion molecule 1 and major histocompatibility complex (MHC) class I molecules by F-Ara, suggesting a proinflammatory activation of HMECs. Cytotoxicity assays demonstrated that target HMECs pretreated with F-Ara (10 microg/mL) showed increased lysis by allogeneic MHC class I-restricted cytotoxic T lymphocytes from healthy human donors. We conclude that, beside its immunosuppressive activities, F-Ara can be harmful for target tissues of transplantation-related complications and can even stimulate allogeneic immune responses. We identified the pharmaceutical compound defibrotide as protective against F-Ara- induced apoptosis and alloactivation, importantly, without affecting the antileukemic effect of F-Ara. This observation argues for a potential clinical usage of defibrotide in pretransplantation conditioning.