Physiological functions of TNF family receptor/ligand interactions in hematopoiesis and transplantation.

Secretion of ligands of the tumor necrosis factor (TNF) superfamily is a conserved response of parenchymal tissues to injury and inflammation that commonly perpetuates elimination of dysfunctional cellular components by apoptosis. The same signals of tissue injury that induce apoptosis in somatic cells activate stem cells and initiate the process of tissue regeneration as a coupling mechanism of injury and recovery. Hematopoietic stem and progenitor cells upregulate the TNF family receptors under stress conditions and are transduced with trophic signals. The progeny gradually acquires sensitivity to receptor-mediated apoptosis along the differentiation process, which becomes the major mechanism of negative regulation of mature proliferating hematopoietic lineages and immune homeostasis. Receptor/ligand interactions of the TNF family are physiological mechanisms transducing the need for repair, which may be harnessed in pathological conditions and transplantation. Because these interactions are physiological mechanisms of injury, neutralization of these pathways has to be carefully considered in disorders that do not involve intrinsic aberrations of excessive susceptibility to apoptosis.

Stable activity of diabetogenic cells with age in NOD mice: dynamics of reconstitution and adoptive diabetes transfer in immunocompromised mice.

The non-obese diabetic (NOD) mouse is a prevalent disease model of type 1 diabetes. Immune aberrations that cause and propagate autoimmune insulitis in these mice are being continually debated, with evidence supporting both dominance of effector cells and insufficiency of suppressor mechanisms. In this study we assessed the behaviour of NOD lymphocytes under extreme expansion conditions using adoptive transfer into immunocompromised NOD.SCID (severe combined immunodeficiency) mice. CD4(+)  CD25(+) T cells do not cause islet inflammation, whereas splenocytes and CD4(+)  CD25(-) T cells induce pancreatic inflammation and hyperglycaemia in 80-100% of the NOD.SCID recipients. Adoptively transferred effector T cells migrate to the lymphoid organs and pancreas, proliferate, are activated in the target organ in situ and initiate inflammatory insulitis. Reconstitution of all components of the CD4(+) subset emphasizes the plastic capacity of different cell types to adopt effector and suppressor phenotypes. Furthermore, similar immune profiles of diabetic and euglycaemic NOD.SCID recipients demonstrate dissociation between fractional expression of CD25 and FoxP3 and the severity of insulitis. There were no evident and consistent differences in diabetogenic activity and immune reconstituting activity of T cells from pre-diabetic (11 weeks) and new onset diabetic NOD females. Similarities in immune phenotypes and variable distribution of effector and suppressor subsets in various stages of inflammation commend caution in interpretation of quantitative and qualitative aberrations as markers of disease severity in adoptive transfer experiments.

Resistance of hematopoietic progenitors to Fas-mediated apoptosis is actively sustained by NFκB with a characteristic transcriptional signature.

Umbilical cord blood (UCB) is a good source of hematopoietic progenitors with increasing implementation in the clinical transplant setting. This study evaluates the molecular mechanisms of progenitor resistance to apoptosis triggered by Fas cross-linking. CD34(+) and lineage-negative progenitors survive short-term ex vivo incubation and are not induced into apoptosis by Fas cross-linking. Furthermore, brief exposure of UCB cells to Fas-ligand for 24-48 h does not impair quantitative severe combine immune deficiency (SCID) reconstitution activity and appears to foster myelomonocyte reconstitution. The transcriptome of Fas receptor-positive CD34(+) cells that survived an apoptotic challenge showed significant transcriptional upregulation of caspase-8, mucosa-associated lymphoid tissue lymphoma translocation gene-1 (MALT1), HtrA2, and GSK3ß in addition to higher levels of c-FLICE inhibitory protein (FLIP), Bcl-2, and cytosolic inhibitor of apoptosis protein (cIAP) in all Fas-positive cells. Most prominent is the transcriptional upregulation of several key components the NFκB1 pathway including the membrane receptors TGF-ß, interleukin-1 (IL-1), and TCR, the associated factor TNF receptor-associated factor-6 (TRAF6), and the converting enzymes TGF-ß-activated kinase-1 (TAK1), double-stranded RNA-activated protein kinase (PKR), and α-catalytic subunit of IκB kinase (IKKα), that promote activation and nuclear translocation of this transcription factor. These data indicate that hematopoietic progenitors are not insensitive to apoptosis but are actively shielded from the extrinsic and intrinsic apoptotic pathways. This may occur through inherent transcriptional upregulation of the entire NFκB pathway in the presence of competent apoptotic signaling.

Surge in regulatory T cells does not prevent onset of hyperglycemia in NOD mice: immune profiles do not correlate with disease severity.

Immune profiling of non-obese diabetic (NOD) is a widely employed tool to assess the mechanisms of inflammatory insulitis. Our analysis of the female NOD colony revealed similar distribution of lymphoid lineages to wild type mice, and at various ages of prediabetic and diabetic mice. The profiles of mesenteric and pancreatic lymph nodes differ and often change reciprocally due to directed migration of T cells towards the site of inflammation. Significant events in our colony include early decline in CD4(+)CD25(+)CD62L(+) Treg, accompanied by gradual increase in CD4(+)CD25(+)FoxP3(+) Treg in peripheral lymphoid organs and pancreatic infiltrates. Impressively, aged euglycemic mice display significant transient rise in CD4(+)CD25(-)FoxP3(+) Treg in the thymus, pancreas and draining lymph nodes. A significant difference was superior viability of effector and suppressor cells from new onset diabetics in the presence of high interleukin-2 (IL-2) concentrations in vitro as compared to cells of prediabetic mice. Overall, we found no correlation between FoxP3(+) Treg in the pancreatic lymph nodes and the inflammatory scores of individual NOD mice. CD25(-)FoxP3(+) Treg are markedly increased in the pancreatic infiltrates in late stages of inflammation, possibly an effort to counteract destructive insulitis. Considering extensive evidence that Treg in aged NOD mice are functionally sufficient, quantitative profiling evolves as an unreliable tool to assess mechanism and causes of inflammation under baseline conditions. Immune profiles are modulated by thymic output, cell migration, shedding of markers, proliferation, survival and in-situ evolution of regulatory cells.

Age dependent course of EAE in Aire-/- mice.

This study explores the consequences of deficiency in the autoimmune regulator (Aire) on the susceptibility to experimental autoimmune encephalomyelitis (EAE). Increased susceptibility to EAE was found in Aire knockout (KO) compared to wild type (WT) in 6month old mice. In contrast, 2month old Aire KO mice were less susceptible to EAE than WT mice, and this age-related resistance correlated with elevated proportions of T regulatory (Treg) cells in their spleen and brain. Combined with our previous findings in experimental autoimmune myasthenia gravis, we suggest an age-related association between Aire and Treg cells in the susceptibility to autoimmunity.

Immunomodulation with donor regulatory T cells armed with Fas-ligand alleviates graft-versus-host disease.

Infusion of large numbers of donor regulatory T cells (Tregs) is an effective approach to suppress graft-versus-host disease (GvHD). We have reported previously that enhancing the killing activity of CD25(+) Tregs by decoration with short-lived Fas-ligand (FasL) protein (killer Tregs) is effective in abrogation of autoimmunity. In this study, we assessed the therapeutic efficacy of killer Tregs in murine models of lethal GvHD. In a model in which disease-associated mortality was not prevented by infusion of naive donor Tregs (3 days after transplant) at an effector:suppressor ratio of 10:1, killer Tregs rescued 70% of the mice and improved the clinical and histologic scores. We found that both effector lymphocytes and therapeutic Tregs migrate to and proliferate in the mesenteric lymph nodes of irradiated recipients; however, only killer Tregs increased fractional apoptosis of effector lymphocytes. Although the lymphoid organs were primarily reconstituted from the bone marrow with little contribution of the infused effector and suppressor subsets, immunomodulation with FasL caused a durable rise in fractions of CD4(+)FoxP3(+) Tregs. Our findings demonstrate that a short-lived apoptotic protein increases the suppressive activity of Tregs and ameliorates GvHD severity.

Killer Treg cells ameliorate inflammatory insulitis in non-obese diabetic mice through local and systemic immunomodulation.

Treg cells endowed with enhanced killing activity through decoration with Fas-ligand (FasL) protein (killer Treg) have been effective in delay of hyperglycemia in prediabetic non-obese diabetic (NOD) mice. In this study, we assessed the therapeutic efficacy of these cells, harvested from age-matched euglycemic NOD donors, on the course of disease in new-onset diabetics. One dose of 4 × 10(6) killer Treg cells stabilized blood glucose associated with increased insulin levels in 5 of 9 mice and partially reversed the severity of islet inflammation, whereas naive Treg cells did not modulate the course of disease significantly. Killer Treg cells were shown to operate through induction of cell apoptosis within the pancreatic lymph nodes, resulting in reduced efficiency of adoptive disease transfer to NOD/SCID recipients. A second mechanism of action consisted of increased fractions of CD4(+)CD25(-)FoxP3(+) T cells in the pancreas and all lymphoid organs. Immunomodulation with FasL rather than Treg cells enhanced the expression of CD25 and FoxP3 in the thymus, suggesting a possible contribution of thymic output to prolonged stabilization of the glucose levels. Autologous Treg cells evolve as excellent vehicles for targeted delivery of FasL as an immunomodulatory protein, which delete pathogenic cells at the site of inflammation and induce systemic dominance of suppressor subsets.

Phylogeny and a structural model of plant MHX transporters.

BACKGROUND: The Arabidopsis thaliana MHX gene (AtMHX) encodes a Mg²âº/H⁺ exchanger. Among non-plant proteins, AtMHX showed the highest similarity to mammalian Na⁺/Ca²âº exchanger (NCX) transporters, which are part of the Ca²âº/cation (CaCA) exchanger superfamily. RESULTS: Sequences showing similarity to AtMHX were searched in the databases or sequenced from cDNA clones. Phylogenetic analysis showed that the MHX family is limited to plants, and constitutes a sixth family within the CaCA superfamily. Some plants include, besides a full MHX gene, partial MHX-related sequences. More than one full MHX gene was currently identified only in Oryza sativa and Mimulus guttatus, but an EST for more than one MHX was identified only in M. guttatus. MHX genes are not present in the currently available chlorophyte genomes. The prevalence of upstream ORFs in MHX genes is much higher than in most plant genes, and can limit their expression. A structural model of the MHXs, based on the resolved structure of NCX1, implies that the MHXs include nine transmembrane segments. The MHXs and NCXs share 32 conserved residues, including a GXG motif implicated in the formation of a tight-turn in a reentrant-loop. Three residues differ between all MHX and NCX proteins. Altered mobility under reducing and non-reducing conditions suggests the presence of an intramolecular disulfide-bond in AtMHX. CONCLUSIONS: The absence of MHX genes in non-plant genomes and in the currently available chlorophyte genomes, and the presence of an NCX in Chlamydomonas, are consistent with the suggestion that the MHXs evolved from the NCXs after the split of the chlorophyte and streptophyte lineages of the plant kingdom. The MHXs underwent functional diploidization in most plant species. De novo duplication of MHX occurred in O. sativa before the split between the Indica and Japonica subspecies, and was apparently followed by translocation of one MHX paralog from chromosome 2 to chromosome 11 in Japonica. The structural analysis presented and the identification of elements that differ between the MHXs and the NCXs, or between the MHXs of specific plant groups, can contribute to clarification of the structural basis of the function and ion selectivity of MHX transporters.

Depletion of naïve lymphocytes with fas ligand ex vivo prevents graft-versus-host disease without impairing T cell support of engraftment or graft-versus-tumor activity.

Graft-versus-host disease (GVHD) can be prevented by Fas-mediated selective depletion of host-sensitized donor lymphocytes ex vivo. We tested the hypothesis that Fas-mediated depletion of lymphocytes in the absence of host-specific antigenic stimulation can alleviate GVHD. Brief exposure (24 hours) of unstimulated donor lymphocytes to Fas ligand (FasL) ex vivo results in balanced apoptosis of CD8(+) and CD4(+) subsets with preferential depletion of CD62L and CD69, increased T regulatory fractions, and sustained responses to stimulation. This procedure ameliorates weight loss and improves the clinical and histologic score of skin and gastrointestinal GVHD with and without concurrent transplantation of hematopoietic progenitors and irrespective of conditioning-induced tissue injury. Although FasL-resistant donor T cells are less potent effectors of GVHD, they facilitate hematopoietic progenitor engraftment when infused with or after the graft and retain the potential to elaborate graft-versus-tumor reactions. These findings in a preclinical model together with the known trophic effects of FasL on primitive hematopoietic progenitors suggest that brief ex vivo incubation of hematopoietic grafts with FasL may improve the outcome and safety of clinical T cell-replete allogeneic and haploidentical transplants.

TNF-α has tropic rather than apoptotic activity in human hematopoietic progenitors: involvement of TNF receptor-1 and caspase-8.

Tumor necrosis factor-α (TNF-α) has been suggested to exert detrimental effects on hematopoietic progenitor function that might limit the success of transplants. In this study, we assessed the influences of TNF-α and its two cognate receptors on the function of fresh umbilical cord blood (UCB) and cryopreserved mobilized peripheral blood (mPB). CD34(+) progenitors from both sources are less susceptible to spontaneous apoptosis than lineage-committed cells and are not induced into apoptosis by TNF-α. Consequently, the activity of UCB-derived severe combined immune deficiency (SCID) reconstituting cells and long-term culture-initiating cells is unaffected by this cytokine. On the contrary, transient exposure of cells from both sources to TNF-α stimulates the activity of myeloid progenitors, which persists in vivo in UCB cell transplants. Progenitor stimulation is selectively mediated by TNF-R1 and involves activation of caspase-8, without redundant activity of TNF-R2. Despite significant differences between fresh UCB cells and cryopreserved mPB cells in susceptibility to apoptosis and time to activation, TNF-α is primarily involved in tropic signaling in hematopoietic progenitors from both sources. Cytokine-mediated tropism cautions against TNF-α neutralization under conditions of stress hematopoiesis and may be particularly beneficial in overcoming the limitations of UCB cell transplants.

Activation and crosstalk between TNF family receptors in umbilical cord blood cells is not responsible for loss of engraftment capacity following culture.

Umbilical cord blood (UCB) is a rich source of hematopoietic progenitors for transplantation. Murine and human progenitors are insensitive to apoptotic signaling mediated by the TNF family receptors, however extension of culture over 48 hours is accompanied by severe deterioration in engraftment and hematopoietic reconstituting capacity. In this study we assessed crosstalk between the Fas, TNF and TRAIL receptors, and questioned whether it contributes to increased mortality and decreased activity of UCB progenitors following extended ex vivo culture for 72 hours. The well-characterized TNF-induced expression of Fas is mediated by both TNF receptors, yet the TNF receptors determine survival rather than Fas: superior viability of TNF-R1 progenitors. Additional cross talk includes upregulation of TRAIL-R1 by Fas-ligand, mediated both by fast cycling and inductive crosstalk. These inductive interactions are not accompanied by concomitant sensitization of progenitors to receptor-mediated apoptosis during extended culture, but rather decreased fractional apoptosis in expanded progenitor subsets expressing the receptors. TRAIL upregulates both TRAIL-R1 and TRAIL-R2, accompanied by commensurate susceptibility to spontaneous apoptosis. The current data reveal inductive crosstalk between TNF family receptors, which are largely dissociated from the sensitivity of hematopoietic progenitors to apoptosis. Activation of Fas, TNF and TRAIL receptors and excessive apoptosis are not responsible for loss of engraftment and impaired reconstituting activity of UCB progenitors following extended culture.

Immunosuppressive therapy exacerbates autoimmunity in NOD mice and diminishes the protective activity of regulatory T cells.

Mounting evidence indicates that immunosuppressive therapy and autologous bone marrow transplantation are relatively inefficient approaches to treat autoimmune diabetes. In this study we assessed the impact of immunosuppression on inflammatory insulitis in NOD mice, and the effect of radiation on immunomodulation mediated by adoptive transfer of various cell subsets. Sublethal radiation of NOD females at the age of 14 weeks (onset of hyperglycemia) delayed the onset of hyperglycemia, however two thirds of the mice became diabetic. Adoptive transfer of splenocytes into irradiated NON and NOD mice precipitated disease onset despite increased contents of CD25(+)FoxP3(+) T cells in the pancreas and regional lymphatics. Similar phenotypic changes were observed when CD25(+) T cells were infused after radiation, which also delayed disease onset without affecting its incidence. Importantly, irradiation increased the susceptibility to diabetes in NOD and NON mice (71-84%) as compared to immunomodulation with splenocytes and CD25(+) T cells in naïve recipients (44-50%). Although irradiation had significant and durable influence on pancreatic infiltrates and the fractions of functional CD25(+)FoxP3(+) Treg cells were elevated by adoptive cell transfer, this approach conferred no protection from disease progression. Irradiation was ineffective both in debulking of pathogenic clones and in restoring immune homeostasis, and the consequent homeostatic expansion evolves as an unfavorable factor in attempts to restore self-tolerance and might even provoke uncontrolled proliferation of pathogenic clones. The obstacles imposed by immunosuppression on abrogation of autoimmune insulitis require replacement of non-specific immunosuppressive therapy by selective immunomodulation that does not cause lymphopenia.

Tumor necrosis factor receptors support murine hematopoietic progenitor function in the early stages of engraftment.

Tumor necrosis factor (TNF) family receptors/ligands are important participants in hematopoietic homeostasis, in particular as essential negative expansion regulators of differentiated clones. As a prominent injury cytokine, TNF-alpha has been traditionally considered to suppress donor hematopoietic stem and progenitor cell function after transplantation. We monitored the involvement of TNF receptors (TNF-R) 1 and 2 in murine hematopoietic cell engraftment and their inter-relationship with Fas. Transplantation of lineage-negative (lin(-)) bone marrow cells (BMC) from TNF receptor-deficient mice into wild-type recipients showed defective early engraftment and loss of durable hematopoietic contribution upon recovery of host hematopoiesis. Consistently, cells deficient in TNF receptors had reduced competitive capacity as compared to wild-type progenitors. The TNF receptors were acutely upregulated in bone marrow (BM)-homed donor cells (wild-type) early after transplantation, being expressed in 60%-75% of the donor cells after 6 days. Both TNF receptors were detected in fast cycling, early differentiating progenitors, and were ubiquitously expressed in the most primitive progenitors with long-term reconstituting potential (lin(-)c-kit(+) stem cell antigen (SCA)-1(+)). BM-homed donor cells were insensitive to apoptosis induced by TNF-alpha and Fas-ligand and their combination, despite reciprocal inductive cross talk between the TNF and Fas receptors. The engraftment supporting effect of TNF-alpha is attributed to stimulation of progenitors through TNF-R1, which involves activation of the caspase cascade. This stimulatory effect was not observed for TNF-R2, and this receptor did not assume redundant stimulatory function in TNFR1-deficient cells. It is concluded that TNF-alpha plays a tropic role early after transplantation, which is essential to successful progenitor engraftment.

Low levels of allogeneic but not syngeneic hematopoietic chimerism reverse autoimmune insulitis in prediabetic NOD mice.

The relative efficiencies of allogeneic and syngeneic bone marrow transplantation and the threshold levels of donor chimerism required to control autoimmune insulitis were evaluated in prediabetic NOD mice. Male and female NOD mice were conditioned by radiation and grafted with bone marrow cells from allogeneic and syngeneic sex-mismatched donors. Establishment of full allogeneic chimerism in peripheral blood reversed insulitis and restored glucose tolerance despite persistence of residual host immune cells. By contrast, sublethal total body irradiation (with or without syngeneic transplant) reduced the incidence and delayed the onset of diabetes. The latter pattern was also seen in mice that rejected the bone marrow allografts. Low levels of stable allogeneic hematopoietic chimerism (>1%) were sufficient to prevent the evolution of diabetes following allogeneic transplantation. The data indicate that immunomodulation attained at low levels of allogeneic, but not syngeneic, hematopoietic chimerism is effective in resolution of islet inflammation at even relatively late stages in the evolution of the prediabetic state in a preclinical model. However, our data question the efficacy and rationale behind syngeneic (autologous-like) immuno-hematopoietic reconstitution in type 1 diabetes.

Overexpression of AtMHX in tobacco causes increased sensitivity to Mg2+, Zn2+, and Cd2+ ions, induction of V-ATPase expression, and a reduction in plant size.

AtMHX is a vacuolar transporter encoded by a single gene in Arabidopsis. Electrophysiological analysis showed that it exchanges protons with Mg(2+), Zn(2+), and Fe(2+) ions. The physiological impact of AtMHX was examined so far only in tissue-culture grown seedlings of tobacco plants overexpressing this transporter. Here we investigated the impact of AtMHX on growth, response to different metals, and metal accumulation of mature tobacco plants, as well as Arabidopsis plants in which we overexpressed this transporter. The analyses were carried out in hydroponic growth-systems, in which the mineral composition could be effectively controlled, and the metal content of roots could be examined. Transformed tobacco plants showed necrotic lesions and apical burnings upon growth with increased levels of Mg(2+), Zn(2+), and Cd(2+) ions. This suggested that AtMHX can carry in planta not only Mg(2+) and Zn(2+) ions, as previously deduced based on observations in tissue-culture, but also Cd(2+) ions. Transformed plants of both tobacco and Arabidopsis showed a reduction in plant size. However, the overall response of Arabidopsis to AtMHX overexpression was minor. No change was detected in the mineral content of any organ of the transgenic tobacco or Arabidopsis plants. The necrotic lesions in tobacco resembled those seen in plants with perturbed proton balancing, raising the assumption that AtMHX can affect the proton homeostasis of cells. In agreement with this assumption, the transformed tobacco plants had increased expression and activity of the vacuolar H(+)-ATPase. The relative significance of AtMHX for metal and proton homeostasis still has to be elucidated.

Overexpression of the vacuolar metal/proton exchanger AtMHX in tomato causes decreased cell expansion and modifications in the mineral content.

AtMHX is an Arabidopsis vacuolar transporter that exchanges protons with Mg2+, Zn2+ and Fe2+ ions. Tobacco (Nicotiana tabacum (L.)) plants that overexpressed AtMHX showed necrotic lesions, similar to those shown by plants having increased proton influx from the apoplast into the cytosol. This raised the assumption that AtMHX affects the proton homeostasis of cells. Here, we expressed AtMHX in tomato (Lycopersicon esculentum Mill.). The results clarified that the common response of all plant species in which AtMHX was overexpressed thus far was a reduction in plant mass. Transformed tomato plants, in which this reduction was greater compared with tobacco or Arabidopsis thaliana (L.), exhibited reduced cell expansion and a reduction in potassium content. Modifications were also seen in the content of other minerals, including not only metals that can be carried by AtMHX. These changes may thus reflect not only direct metal transport by AtMHX but also the consequences of reduction in cell size. Decreased cell expansion characterises plants with diminished expression of vacuolar proton pumps, presumably due to reduction in the proton-motive force (PMF) necessary to drive solute (mainly potassium) influx into vacuoles and consequently water uptake. This supported a model in which AtMHX-mediated proton efflux from vacuoles affects the PMF, potassium influx, and cell expansion.

Participation of adult bone marrow-derived stem cells in pancreatic regeneration: neogenesis versus endogenesis.

Regenerative medicine opens new avenues and promises towards more effective therapies for autoimmune disorders. Current therapeutic strategies for type I diabetes focus on three major directions, with distinct advantages and disadvantages: arrest of autoimmunity, islet transplantation and generation of neoislets. There is mounting evidence that candidate stem cells residing in the hematopoietic compartments participate in regeneration of pancreatic islets following chemical and autoimmune injury in vivo. The apparent major mechanisms include immunomodulation, revascularization, support of endogenous beta-cell regeneration and differentiation into insulin-producing cells. Review of the current evidence suggests that some divergent observations depend primarily on the experimental design, which both limits and accentuates developmental events. The flood of publications reporting negative results appears to reflect primarily suboptimal experimental conditions for differentiation of putative stem cells, rather than limited developmental plasticity. Stem cells modulate the course of autoimmune diabetes through multiple mechanisms, including de novo generation of units capable to sense, produce and secrete insulin. Therefore, the charged debate over controversies surrounding developmental plasticity should not impede attempts to design curative therapies for this disease.

High expression in leaves of the zinc hyperaccumulator Arabidopsis halleri of AhMHX, a homolog of an Arabidopsis thaliana vacuolar metal/proton exchanger.

Zn hyperaccumulator plants sequester Zn into their shoot vacuoles. To date, the only transporters implicated in Zn sequestration into the vacuoles of hyperaccumulator plants are cation diffusion facilitators (CDFs). We investigated the expression in Arabidopsis halleri of a homolog of AtMHX, an A. thaliana tonoplast transporter that exchanges protons with Mg, Zn and Fe ions. A. halleri has a single copy of a homologous gene, encoding a protein that shares 98% sequence identity with AtMHX. Western blot analysis with vacuolar-enriched membrane fractions suggests localization of AhMHX in the tonoplast. The levels of MHX proteins are much higher in leaves of A. halleri than in leaves of the non-accumulator plant A. thaliana. At the same time, the levels of MHX transcripts are similar in leaves of the two species. This suggests that the difference in MHX levels is regulated at the post-transcriptional level. In vitro translation studies indicated that the difference between AhMHX and AtMHX expression is not likely to result from the variations in the sequence of their 5' untranslated regions (5'UTRs). The high expression of AhMHX in A. halleri leaves is constitutive and not significantly affected by the metal status of the plants. In both species, MHX transcript levels are higher in leaves than in roots, but the difference is higher in A. halleri. Metal sequestration into root vacuoles was suggested to inhibit hyperaccumulation in the shoot. Our data implicate AhMHX as a candidate gene in metal accumulation or tolerance in A. halleri.