Conditions Of Iron Deprivation Research Articles

Paracoccidioides spp. ferrous and ferric iron assimilation pathways.

Iron is an essential micronutrient for almost all organisms, including fungi. Usually, fungi can uptake iron through receptor-mediated internalization of a siderophore or heme, and/or reductive iron assimilation (RIA). Traditionally, the RIA pathway consists of ferric reductases (Fres), ferroxidase (Fet3) and a high-affinity iron permease (Ftr1). Paracoccidioides spp. genomes do not present an Ftr1 homolog. However, this fungus expresses zinc regulated transporter homologs (Zrts), members of the ZIP family of membrane transporters that are able in some organisms to transport zinc and iron. A 2,3,5-triphenyltetrazolium chloride (TTC)-overlay assay indicates that both Pb01 and Pb18 express a ferric reductase activity; however, 59Fe uptake assays indicate that only in Pb18 is this activity coupled to a reductase-dependent iron uptake pathway. In addition, Zrts are up-regulated in iron deprivation, as indicated by RNAseq and qRT-PCR using Pb01 transcripts. RNAseq strategy also demonstrated that transcripts related to siderophore uptake and biosynthesis are up-regulated in iron-deprived condition. The data suggest that the fungus could use both a non-classical RIA, comprising ferric reductases and Fe/Zn permeases (Zrts), and siderophore uptake pathways under iron-limited conditions. The study of iron metabolism reveals novel surface molecules that could function as accessible targets for drugs to block iron uptake and, consequently, inhibit pathogen's proliferation.

Salinity Influences the Response of Halomonas hydrothermalis to Artificial Fossilization by Evaporative Silicification

ABSTRACTEvaporative silicification can drive microbial fossilization within diverse natural habitats. Research into this process is pivotal to understanding the terrestrial fossil record and the preservation of biomarkers within extreme environments. We employed laboratory experiments to silicify the polyextremotolerant bacterium Halomonas hydrothermalis cultured at low, intermediate and high salinities (1, 3.5 or 11.8% w/v of NaCl) under iron-rich or iron-deprived conditions. Silicification was achieved by adding sodium silicate solution (30 or 150 ppm of Si) onto cultures, followed by evaporation. Scanning electron microscopy demonstrated the presence of mineralized bacteria with intact morphology across all culture conditions. However, multivariate analysis of the attenuated total reflectance Fourier-transform infrared (ATR-FT-IR) spectra of silicified cultures showed significant differences between the examined salinities, most notably between cultures silicified after incubation at high salinity and those at lower salinities. Although the spectra of mineralized low- and intermediate-salinity cultures appeared distinct from their nonsilicified counterparts, these differences were less pronounced at high salinity. By showing that differences in salinity can influence microbial responses to mineralization at the molecular level, these data indicate that the potential for evaporative silicification to contribute to microbial fossilization may differ between freshwater and hypersaline environments.

Role of manganese in protection against oxidative stress under iron starvation in cyanobacterium Anabaena 7120.

The cyanobacterium Anabaena sp. PCC 7120 was grown in presence and absence of iron to decipher the role of manganese in protection against the oxidative stress under iron starvation and growth, manganese uptake kinetics, antioxidative enzymes, lipid peroxidation, electrolyte leakage, thiol content, total peroxide, proline and NADH content was investigated. Manganese supported the growth of cyanobacterium Anabaena 7120 under iron deprived conditions where maximum uptake rate of manganese was observed with lower K(m) and higher V(max) values. Antioxidative enzymes were also found to be elevated in iron-starved conditions. Estimation of lipid peroxidation and electrolyte leakage depicted the role of manganese in stabilizing the integrity of the membrane which was considered as the prime target of oxygen free radicals in oxidative stress. The levels of total peroxide, thiol, proline and NADH content, which are the representative of oxidative stress response in Anabaena 7120, were also showed increasing trends in iron starvation. Hence, the results discerned, clearly suggested the role of manganese in protection against the oxidative stress in cyanobacterium Anabaena 7120 under iron starvation either due to its antioxidative properties or involvement as cofactor in a number of antioxidative enzymes.

Alterations of protein expression in conditions of copper-deprivation for Paracoccidioides lutzii in the presence of extracellular matrix components.

BackgroundParacoccidioides spp is a fungi genus and the agent of paracoccidioidomycosis. The strategies of infection used by these pathogens involve the expression of proteins related to adaptation to the host, particularly regarding the uptake of micronutrients. This study analyzed the adhesion of Paracoccidioides lutzii during conditions of copper (Cu) and iron (Fe) deprivation, while also evaluating the proteins expressed in conditions of Cu depletion in the presence of four extracellular matrix (ECM) components (laminin, fibronectin and types I and IV collagen).ResultsWe cultured the P. lutzii in a chemically defined media without Cu and Fe. The fungus was then placed in contact with different ECM components and adhesion was evaluated. A significant increase in binding to all ECM components was observed when the fungus was cultured without Cu; which might be related to some adhesins expression. A proteomic assay was developed and revealed 39 proteins expressed that are involved in processes such as virulence, protein synthesis, metabolism, energy, transcription, transport, stress response and the cell cycle when the fungus was interacting with the ECM components. The up-regulated expression of two important adhesins, enolase and 14-3-3, was observed at the fungal cell wall during the interaction with the ECM components, indicating the role of these proteins in the Paracoccidioides–host interaction.ConclusionsThis study is important for determining prospective proteins that may be involved in the interaction of Paracoccidioides with a host. Understanding the adaptive response to different growth conditions, elucidating the processes of adhesion and cell invasion, and identifying the proteins that are differentially expressed during the fungus-host interaction may help elucidate mechanisms used for survival and growth of Paracoccidioides in various human tissues.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0302-7) contains supplementary material, which is available to authorized users.

Identification of genes expressed by Cryptococcus gattii during iron deprivation.

Cryptococcus neoformans and C. gattii are pathogenic yeasts that cause life-threatening diseases in humans and animals. Iron is an essential nutrient for virtually every organism as it functions as a cofactor in numerous essential enzymatic reactions. In the literature, the competition for iron between microbes and mammalian hosts during infection is well documented. In this study, we used representational difference analysis (RDA) in order to gain a better understanding of how C. gattii responds to iron starvation. A total of 15 and 29 genes were identified as having altered expression levels due to iron depletion after 3 h and 12 h, respectively. Of these, eight genes were identified in both libraries. The transcripts were related to many biological processes, such as cell cycle, ergosterol metabolism, cell wall organization, transportation, translation, cell respiration and the stress response. These data suggest a remodeling of C. gattii metabolism during conditions of iron deprivation.

Protein expression and transcription profiles of three strains of Aeromonas salmonicida ssp. salmonicida under normal and iron-limited culture conditions.

BackgroundAeromonas salmonicida is an important fish pathogen that produces a wide and varied array of virulence factors. Here we used iron deprivation by addition of the chelator 2’2-dipyridyl to induce the expression of several such virulence factors in three isolates of Aeromonas salmonicida (one avirulent and two virulent). By using SDS-PAGE followed by mass spectrometry, we identified proteins that appeared differentially expressed under these conditions. The differential transcription of the identified gene products were subsequently measured by reverse transcription quantitative real-time PCR (RT-qPCR).ResultsOur initial screening using SDS-PAGE identified five proteins that appeared differentially expressed in virulent and avirulent isolates or, within the same isolates, between bacteria cultivated under iron-rich or iron-deprived conditions. The transcription of the genes coding for these proteins were subsequently quantified by RT-qPCR. Results of this analysis demonstrated that the gene coding for alkyl hydroperoxide reductase (AhpC), a protein involved in oxidative stress response, was transcribed at a higher rate in the virulent strain as compared to the avirulent strain. Additionally, it was observed that addition of an iron chelator to the culture medium lead to a reduction of the transcription levels of the regulatory histone-like nucleoid structuring protein (H-NS). This was consistent in all three isolates. On the other hand, the transcription levels of the virulence array protein (VapA) and the protein ATP-synthetase F (ATPF) displayed only limited changes, despite being the dominant component of a protein fraction that displayed changes during the preliminary SDS-PAGE screening. This was true regardless of the culture conditions and of the isolates considered. Finally, transcription of the enzyme enolase was upregulated in the iron-deprived broths in all isolates.ConclusionsWe identified several genes differentially expressed under culture conditions known to lead to the overexpression of virulence factors. In addition, we identified alkyl hydroperoxide as being overexpressed in the virulent isolates compared to the avirulent isolates. The results from this study will contribute to enhance our understanding of the virulence of A. salmonicida and may suggest new directions for further research.

Allergen1 regulates polysaccharide structure in Cryptococcus neoformans

Cryptococcus neoformans is an important human, fungal pathogen that sheds polysaccharide (exo-PS) into host tissues. While shed exo-PS mediates numerous untoward effects (including promoting increased intracranial pressure), little is known about the regulation of this phenomenon. Since downregulation of the Allergen 1 (ALL1) gene is associated with high ICP, we investigated the relationship between ALL1 expression and exo-PS structure using a variety of biophysical techniques. The Δall1 mutants of two serotypes produced a shorter exo-PS with less branching and structural complexity than the parental strains. Consistent with lower branching, these exo-PSs manifested higher intrinsic viscosity than the parental strains. The Δall1 mutant strains manifested differences in epitope expression and significant resistance to phagocytosis. Exo-PS of Δall1 mutant exhibited anti-phagocytic properties. Comparative transcriptome analysis of mutant and parental strain under iron-deprived conditions indicated a role of ALL1 in iron homeostasis, characterized by differential regulation of genes that mediate iron reduction and transport. Together, our results demonstrate a role of ALL1 in regulating conformational aspects of PS structure and iron homeostasis. These findings provide a mechanism to explain how changes in ALL1 expression influence virulence of switch variants and suggest that structural changes and polymer length are epigenetically regulated.

Systematic validation of candidate reference genes for qRT-PCR normalization under iron deficiency in Arabidopsis

A reliable result obtained by qRT-PCR highly depends on accurate transcript normalization using stably expressed reference genes. However, the transcript levels of traditional reference genes are not always stable. Also, the inaccurate normalization could easily lead to the false conclusions. In this report, by using geNorm and NormFinder algorithms, 12 candidate reference genes were evaluated in Arabidopsis under iron deficiency. Our results revealed that three novel reference genes (SAND, YLS8 and TIP41-like) were identified and validated as suitable reference genes for qRT-PCR normalization in both iron deprivation (the addition of Ferrozine to the medium) and starvation (withdrawal of iron from the medium) conditions. This conclusion was also confirmed by publicly available microarray data. In addition, when using SAND, YLS8 and TIP41-like as multiple reference genes, the expression patterns of FIT1 and IRT1, two iron deficiency marker genes, were approximately similar with that reported previously. However, a weaker inducible response was obtained from qRT-PCR by normalizating EF-1α alone. Together, we proposed that the combination of SAND, YLS8 and TIP41-like can be used for accurate normalization of gene expression in iron deficiency research. These results provide a valuable evidence for the importance of adequate reference genes in qRT-PCR normalization, insisting on the use of appropriate reference gene validation in all transcriptional analyses.

Oversynthesis of Riboflavin in the Yeast Pichia guilliermondii is Accompanied by Reduced Catalase and Superoxide Dismutases Activities

Iron deficiency causes oversynthesis of riboflavin in several yeast species, known as flavinogenic yeasts. Under iron deprivation conditions, Pichia guilliermondii cells increase production of riboflavin and malondialdehyde and the formation of protein carbonyl groups, which reflect increased intracellular content of reactive oxygen species. In this study, we found that P. guilliermondii iron deprived cells showed dramatically decreased catalase and superoxide dismutase activities. Previously reported mutations rib80, rib81, and hit1, which affect repression of riboflavin synthesis and iron accumulation by iron ions, caused similar drops in activities of the mentioned enzymes. These findings could explain the previously described development of oxidative stress in iron deprived or mutated P. guilliermondii cells that overproduce riboflavin. Similar decrease in superoxide dismutase activities was observed in iron deprived cells in the non-flavinogenic yeast Saccharomyces cerevisiae.

The Yersinia High-Pathogenicity Island in Escherichia coli and Klebsiella pneumoniae Isolated from Polymicrobial Infections

We examined 12 pairs of strains of Escherichia coli and Klebsiella pneumoniae isolated from mixed infections in human for the presence of the Yersinia high-pathogenicity island (HPI). In one case both isolates carried the HPI, whereas in 11 cases one strain of the pair was HPI-positive. Although there were differences in the organization of the Yersinia HPI, all HPI-positive isolates were able to produce yersiniabactin. The presence of the Yersinia HPI may enhance the capability of strains involved in mixed infections to replicate in iron-deprived conditions in the host.

Role of Fur on cyanide tolerance of Pseudomonas pseudoalcaligenes CECT5344

Pseudomonas pseudoalcaligenes CECT5344 can be used in cyanide bioremediation processes because it grows at pH 9.5 using 2.0 mM cyanide at the sole nitrogen source. Cyanide strongly binds to metals creating iron-deprivation conditions. The bacterium responds to the presence of cyanide by inducing several processes such as siderophore synthesis for iron capture, cyanide-insensitive respiration system and defence mechanisms against oxidative stress. Since high concentrations of cyanide cause iron deficiency and because iron is an essential nutrient, bacterial growth in the presence of cyanide requires an efficient iron uptake. Fur is a global transcription factor that regulates a diversity of biological processes such as iron homoeostasis, TCA (tricarboxylic acid) cycle metabolism and oxidative stress response. Fur's regulation of iron uptake and storage genes should play a significant role in the lives of these bacteria. In the present review, current knowledge of Fur is summarized.

The Iron-Responsive Regulator Irr Is Required for Wild-Type Expression of the Gene Encoding the Heme Transporter BhuA in Brucella abortus 2308

Irr and RirA, rather than Fur, serve as the major iron-responsive regulators in the alphaproteobacteria. With only a few exceptions, however, the relative contributions of these transcriptional regulators to the differential expression of specific iron metabolism genes in Brucella strains are unclear. The gene encoding the outer membrane heme transporter BhuA exhibits maximum expression in Brucella abortus 2308 during growth under iron-deprived conditions, and mutational studies indicate that this pattern of bhuA expression is mediated by the iron-responsive regulator Irr. Specifically, a bhuA-lacZ transcriptional fusion does not produce elevated levels of β-galactosidase in response to iron deprivation in the isogenic irr mutant BEA5, and, unlike the parental strain, B. abortus BEA5 cannot utilize heme as an iron source in vitro and is attenuated in mice. A derivative of the bhuA-lacZ transcriptional fusion lacking the predicted Irr binding site upstream of the bhuA promoter does not produce elevated levels of β-galactosidase in response to iron deprivation in the parental B. abortus 2308 strain, and a direct and specific interaction between a recombinant version of the Brucella Irr and the bhuA promoter region was observed in an electrophoretic mobility shift assay. Despite the fact that it lacks the heme regulatory element linked to the iron-responsive degradation of its counterpart in Bradyrhizobium japonicum, readily detectable levels of Irr were found only in B. abortus 2308 cells by Western blot analysis following growth under iron-deprived conditions.

Cap2-HAP Complex Is a Critical Transcriptional Regulator That Has Dual but Contrasting Roles in Regulation of Iron Homeostasis in Candida albicans

Iron homeostasis is highly regulated in organisms across evolutionary time scale as iron is essential for various cellular processes. In a computational screen, we identified the Yap/bZIP domain family in Candida clade genomes. Cap2/Hap43 is essential for C. albicans growth under iron-deprivation conditions and for virulence in mouse. Cap2 has an amino-terminal bipartite domain comprising a fungal-specific Hap4-like domain and a bZIP domain. Our mutational analyses showed that both the bZIP and Hap4-like domains perform critical and independent functions for growth under iron-deprivation conditions. Transcriptome analysis conducted under iron-deprivation conditions identified about 16% of the C. albicans ORFs that were differentially regulated in a Cap2-dependent manner. Microarray data also suggested that Cap2 is required to mobilize iron through multiple mechanisms; chiefly by activation of genes in three iron uptake pathways and repression of iron utilizing and iron storage genes. The expression of HAP2, HAP32, and HAP5, core components of the HAP regulatory complex was induced in a Cap2-dependent manner indicating a feed-forward loop. In a feed-back loop, Cap2 repressed the expression of Sfu1, a negative regulator of iron uptake genes. Cap2 was coimmunoprecipitated with Hap5 from cell extracts prepared from iron-deprivation conditions indicating an in vivo association. ChIP assays demonstrated Hap32-dependent recruitment of Hap5 to the promoters of FRP1 (Cap2-induced) and ACO1 (Cap2-repressed). Together our data indicates that the Cap2-HAP complex functions both as a positive and a negative regulator to maintain iron homeostasis in C. albicans.

Opioids protect against substantia nigra cell degeneration under conditions of iron deprivation: A mechanism of possible relevance to the Restless Legs Syndrome (RLS) and Parkinson's Disease

Hypofunction of the endogenous opioid, dopamine and iron systems are implicated in the pathogenesis of Restless Legs Syndrome (RLS). Therefore, we probed the interrelationship of these 3 systems in an in vitro model. Cell cultures of the substantia nigra (SN) of Sprague–Dawley rats were established and the cells were determined to be primarily dopaminergic. The numbers of cells surviving under different concentrations of the iron chelator desferoxamine were reduced in a concentration and time dependent manner (p < 0.01 at day 10, n = 19). The cell death was determined to be apoptotic and DNA analysis revealed that 48-hour 100 μM desferoxamine exposure caused DNA fragmentation of the cells. Pre-administration of the δ-opioid peptide [ d -Ala2, d -Leu5]Enkephalin (DADLE) significantly protected the SN cells from damage by iron deficiency (n = 6, p < 0.01). Our previous studies indicate that the DNA-damage induced apoptosis family gene P53 is activated in this model and that pre-exposure to DADLE prevents this activation. The implications of this model are that in RLS patients with iron deficiency, dopaminergic system dysfunction may result and an intact endogenous opioid system or opioid treatment may protect the dopamine system from dysfunction. Implications of this model for Parkinson's Disease are also briefly discussed.

Bacterial cyanide degradation is under review: Pseudomonas pseudoalcaligenes CECT5344, a case of an alkaliphilic cyanotroph

There are thousands of areas in the U.S.A. and Europe contaminated with cyanide-containing wastes as a consequence of a large number of industrial activities such as gold mining, steel and aluminium manufacturing, electroplating and nitrile pesticides used in agriculture. Chemical treatments to remove cyanide are expensive and generate other toxic products. By contrast, cyanide biodegradation constitutes an appropriate alternative treatment. In the present review we provide an overview of how cells deal in the presence of the poison cyanide that irreversible binds to metals causing, among other things, iron-deprivation conditions outside the cell and metalloenzymes inhibition inside the cell. In this sense, several systems must be present in a cyanotrophic organism, including a siderophore-based acquisition mechanism, a cyanide-insensitive respiratory system and a cyanide degradation/assimilation pathway. The alkaliphilic autochthonous bacterium Pseudomonas pseudocaligenes CECT5344 presents all these requirements with the production of siderophores, a cyanide-insensitive bd-related cytochrome [Cio (cyanide-insensitive oxidase)] and a cyanide assimilation pathway that generates ammonium, which is further incorporated into organic nitrogen.

Iron-Dependent Remodeling of Fungal Metabolic Pathways Associated with Ferrichrome Biosynthesis

The fission yeast Schizosaccharomyces pombe excretes and accumulates the hydroxamate-type siderophore ferrichrome. The sib1(+) and sib2(+) genes encode, respectively, a siderophore synthetase and an l-ornithine N(5)-oxygenase that participate in ferrichrome biosynthesis. In the present report, we demonstrate that sib1(+) and sib2(+) are repressed by the GATA-type transcriptional repressor Fep1 in response to high levels of iron. We further found that the loss of Fep1 results in increased ferrichrome production. We showed that a sib1Delta sib2Delta mutant strain exhibits a severe growth defect on iron-poor media. We determined that two metabolic pathways are involved in biosynthesis of ornithine, an obligatory precursor of ferrichrome. Ornithine is produced by hydrolysis of arginine by the Car1 and Car3 proteins. Although car3(+) was constitutively expressed, car1(+) transcription levels were repressed upon exposure to iron, with a concomitant decrease of Car1 arginase activity. Ornithine is also generated by transformation of glutamate, which itself is produced by two separate biosynthetic pathways which are transcriptionally regulated by iron in an opposite fashion. In one pathway, the glutamate dehydrogenase Gdh1, which produces glutamate from 2-ketoglutarate, was repressed under iron-replete conditions in a Fep1-dependent manner. The other pathway involves two coupled enzymes, glutamine synthetase Gln1 and Fe-S cluster-containing glutamate synthase Glt1, which were both repressed under iron-limiting conditions but were expressed under iron-replete conditions. Collectively, these results indicate that under conditions of iron deprivation, yeast remodels metabolic pathways linked to ferrichrome synthesis in order to limit iron utilization without compromising siderophore production and its ability to sequester iron from the environment.

Iron deprivation inducesEFG1-mediated hyphal development inCandida albicanswithout affecting biofilm formation

In this study, we investigated the role of cellular iron status in hyphae and biofilm formation in Candida albicans. Iron deprivation by a chelator, bathophenanthrolene disulfonic acid, promoted hyphal development even in nonhyphal-inducing media without affecting growth of C. albicans cells. Iron-acquisition defective mutants, Deltaftr1 and Deltaccc2, also showed hyphal formation, which was prevented by iron supplementation. Notably, most of the tested morphological mutants Deltacph1, Deltaefh1 and Deltatpk1 continued to form hyphae under iron-deprived conditions, except the Deltaefg1 null mutant, which showed a complete block in hyphae formation. The role of EFG1 in filamentation under iron-deprived conditions was further confirmed by Northern analysis, which showed a considerable upregulation of the EFG1 transcript. Of notable importance, all the morphological mutants including Deltaefg1 mutant possessed enhanced membrane fluidity under iron-deprived conditions; however, this did not appear to contribute to hyphal development. Interestingly, iron deprivation did not affect the ability of C. albicans to form biofilms on the catheter surface and led to no gross defects in azole resistance phenotype of these biofilms of C. albicans cells. Our study, for the first time, establishes a link between cellular iron, Efg1p and hyphal development of C. albicans cells that is independent of biofilm formation.

Influence of iron and chelator on siderophore production in Frankia strains nodulating Hippophae salicifolia D. Don

Effect of iron and chelator on the growth and siderophore production in the ten newly Frankia strains isolated from the root nodules of Hippophae salicifolia D. Don and the two reference strains were studied. Growth of the strains was greatly affected when grown in the iron and EDTA deprived conditions. All the strains were capable of producing both the hydroxamate and catecholate type siderophore that was detected using the Csaky and Arnow assays. Production of siderophore was enhanced in the EDTA replenish condition in contrast to the iron supplemented medium suggesting that EDTA reduces the availability of other free metals and hence creates the stress condition for which the secretion of siderophore is enhanced. A decrease in siderophore production was observed with an increase in iron concentration. Strains HsIi2 and HsIi10 were found to be producing more siderophore than the other strains.

Hydrogen peroxide inhibits caspase-dependent apoptosis by inactivating procaspase-9 in an iron-dependent manner

Apoptosis represents a physiological form of cell death, the perturbation of which may contribute to the development of several diseases connected with accumulation of unwanted cells or excessive cell loss. We have previously shown that the continuous presence of low concentrations of H 2O 2 (generated by the action of glucose oxidase) was able to inhibit caspase-mediated apoptosis in Jurkat cells. The main purpose of the present study was to elucidate the exact molecular mechanism(s) underlying this inhibitory action of H 2O 2. The results presented show that events like outer mitochondrial membrane permeabilization, release of cytochrome c from mitochondria, oligomerization of Apaf-1, and recruitment of procaspase-9 to apoptosomes were taking place normally, but further advancement toward activation of the execution caspases was interrupted when H 2O 2 was present during the apoptotic process. From the results presented in this work, it emerges that the inhibition of procaspase-9 autoactivation was probably due to the reversible oxidation of sensitive cysteine residues in this molecule. Remarkably, caspase-9 activation and the ensuing caspase cascade proceeded normally in the presence of H 2O 2 under conditions of iron deprivation, indicating that the inhibition of procaspase-9 activation was an iron-dependent process. Collectively, these results highlighted the potential role of available intracellular iron ions in signaling mechanisms related to apoptotic cell death.

Iron deprivation blocks multilineage haematopoietic differentiation by inhibiting induction of p21(WAF1/CIP1)

Iron is required for the differentiation of HL-60 cells along the monocyte lineage in vitro, reflecting a requirement for iron in the transcriptional induction of the p21(WAF1/CIP1) gene. To determine if the same requirement holds true for differentiation in other cell lineages and for primary human CD34(+) bone marrow precursor cells, we induced granulocyte differentiation by treating HL-60 cells with dimethyl sulphoxide, and erythroid or megakaryocytic differentiation by treating K562 cells with butyrate or phorbol myristate acetate, respectively. Nitro blue tetrazolium reduction, expression of haem, or expression of CD41 was used to assess granulocytic, erythroid, or megakaryocytic differentiation respectively. Purified CD34(+) cells were cultured with granulocyte/macrophage-colony stimulating factor and stem cell factor to induce myelomonocytic differentiation. Iron deprivation was induced by desferrioxamine. p21(WAF1/CIP1) antisense oligonucleotides were used to inhibit p21 expression. Iron deprivation blocked p21 induction as judged by real-time polymerase chain reaction assays. In addition, both iron deprivation and p21 antisense blocked CD34(+) cell differentiation. These observations were not explained by induction of widespread apoptosis under conditions of iron deprivation. We concluded that both iron and functional p21(WAF1/CIP1) are required for in vitro differentiation of human haematopoietic precursors along multiple cell lineages.