Iron acquisition in Arabidopsis depends mainly on AtIRT1, a Fe 2؉ 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. 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.Iron deficiency in plants is visually expressed as chlorosis, first appearing in the youngest developing leaves and accompanied by a reduction in growth rate, dry matter production, and in most cases by a decrease of iron concentration (1). At the same time, root uptake capacities and leaf concentrations of other divalent metal cations increase (2, 3). In soils or nutrient solutions with unbalanced microelement supply, iron deficiency can then promote the toxicity of other transition metals (4, 5). Such toxicity seems also to be the case with nickel, supported by the observation that phytotoxicity of nickel decreased with increasing iron:nickel ratios in the leaf tissue (6, 7). Thus, an increased sensitivity to heavy metals under iron deficiency might represent a secondary, growth-limiting factor besides the lack of iron itself.Molecular studies in yeast showed that an iron deficiencyinduced accumulation of transition metals other than iron was explained by a higher activity of non-selective low affinity iron transport. Deletion of FET3, an essential component for high affinity iron uptake in yeast, leads to a constitutive iron-deficient phenotype and a concomitant up-regulation of FET4, which encodes a low affinity Fe(II) transporter with poor substrate selectivity (8). As a consequence, sensitivity to elevated concentrations of the transition metals cobalt, copper, zinc, and manganese was higher in fet3 mutants than in the corresponding wild type, consistent with increased metal accumulation (8).In Arabidopsis, iron-dependent overaccumulation of divalent metal cations was found to be mediated by the Fe(II) transporter AtIRT1, which in fact transports a broad range of transition metals (9). Atirt1 T-DNA insertion lines no longer accumulated manganese, zinc, and cobalt under iron deficiency and even showed an increased tolerance to toxic levels of cadmium (10). Thus, accumulation of certain transition metals in iron-deficient Arabidopsis plants directly depends on AtIRT1 and appears as an unavoidable side effect of iron deficiencyinduced iron acquisition.In the search for genes that might be involved in metal transport in Arabidopsis roots, homology to ...

We have previously reported that the CD14+ monocytic subpopulation of human PBMC induces programmed cell death (apoptosis) in cocultured endothelial cells (EC) when stimulated by bacterial endotoxin (LPS). Apoptosis is mediated by two routes, first via transmembrane TNF-α (mTNF) expressed on PBMC and, in addition, by TNF-independent soluble factors that trigger apoptosis in EC. Neutralizing anti-TNF mAb completely blocked coculture-mediated apoptosis, despite the fact that there should have been additional soluble cell death factors. This led to the hypothesis that a reverse signal is transmitted from the TNF receptor on EC to monocytes (MO) via mTNF that prevents the production of soluble apoptotic factors. Here we have tested this hypothesis. The results support the idea of a bidirectional cross-talk between MO and EC. Peripheral blood MO, MO-derived macrophages (MΦ), or the monocytic cell line Mono Mac 6 were preincubated with human microvascular EC that constitutively express TNF receptor type I (TNF-R1) and subsequently stimulated with LPS. Cell-free supernatants of these preparations no longer induced EC apoptosis. The preincubation of MO/MΦ with TNF-reactive agents, such as mAb and soluble receptors, also blocked the production of death factors, providing further evidence for reverse signaling via mTNF. Finally, we show that reverse signaling through mTNF mediated LPS resistance in MO/MΦ as indicated by the down-regulation of LPS-induced soluble TNF and IL-6 as well as IL-1 and IL-10.

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-␤-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 g/mL-1 g/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 g/ 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-Arainduced 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. (Blood. 2002;100:334-340)

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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.tRNA molecules are essential for protein synthesis, providing the amino acids during translation. They are not directly transcribed as functional molecules but as precursor RNAs, which require several processing steps to generate the functional tRNA molecule. Two of these processing steps are the removal of the additional 5Ј and 3Ј sequences of the tRNA. Although the removal of the additional 5Ј sequence (the 5Ј leader) is well understood (1), maturation of the tRNA 3Ј end is not as well studied, although a correctly generated tRNA 3Ј end is essential for the addition of the CCA triplet and thus for aminoacylation (2).It has been shown that in Escherichia coli, tRNA 3Ј maturation is a multistep process involving endo-as well as exonucleases, the final steps being performed by an exonuclease (3). In contrast, Bacillus subtilis employs an endonuclease, called tRNase 6 Z (EC 3.1.26.11), which cleaves CCA-less tRNA precursors directly 3Ј to the discriminator (4).Precursors, which do contain the CCA, are not processed by tRNase Z. Archaea and eukaryotes also use tRNase Z enzymes to process the tRNA 3Ј trailer in a single-step mechanism (5-8).The first tRNase Z, TRZ1, was isolated from Arabidopsis thaliana (5). Data base analyses showed that TRZ1 homologues are present in organisms from all three kingdoms, bacteria, archaea, and eukarya (Fig. 1). The tRNase Z family of proteins (also called Elac1/Elac2) can be divided into two subgroups: the short tRNase Z proteins (being 250 -350 amino acids long), tRNase Z S enzymes, and the long tRNase Z proteins (with 700 -950 amino acids), the tRNase Z L enzymes. Although the tRNase Z S proteins are present in all kingdoms, the tRNase Z L enzymes can only be found in eukarya. Both subgroups are part of the same protein family since the C-terminal part of the tRNase Z L proteins has high sequence similarity to the tRNase Z S enzymes. TRZ1 belongs to the family of metal-dependent ␤-lactamases (9), a group of met...