Presence Of Iron Chelator Research Articles

Identification of the iron-limitation stimulon in Staphylococcus lugdunensis.

During the infectious process, pathogens such as Staphylococcus lugdunensis have to cope with the condition of host-induced iron-limitation. Using the RNAseq approach, we performed the first global transcriptomic analysis of S. lugdunensis cells incubated in the absence and presence of iron chelator. One hundred and seventy-five genes were identified as members of the iron-limitation stimulon (127 up- and 48 downregulated). Six gene clusters known or likely required for the acquisition of iron have been identified. Among them, a novel Energy-Coupling Factor type transporter (ECF), homologous to the lhaSTA operon, has been found into a 13-gene putative operon and strongly overexpressed under iron-limitation condition. Moreover, the transcription of genes involved in resistance to oxidative stress (including catalase), virulence, transcriptional regulation, and hemin detoxification were also modified. These data provide some answers on the cellular response to the iron-limitation stress that is important for the opportunistic behavior of this pathogen.

Inhibitory effects of iron depletion plus eribulin on the breast cancer microenvironment

BackgroundIron is required for the proliferation of cancer cells, and its depletion suppresses tumor growth. Eribulin mesylate (eribulin), a non-taxane microtubule inhibitor, disrupts the tumor microenvironment via vascular remodeling and obstruction of the epithelial-mesenchymal transition (EMT). Herein, we investigated the effects of the iron chelator on tumor-related properties of breast cancer cells and the effects of iron chelator plus eribulin on tumor growth in vivo.MethodsTwo triple-negative breast cancer (TNBC) cell lines, MDA-MB-231 and BT-549, and one hormone-receptor positive breast cancer cell line, MCF-7, were used in our study. Cell proliferation, cell migration, cell cycle position, and gene expression were analyzed via MTT assays, wound-healing assays, flow cytometry, and quantitative real-time-polymerase chain reaction, respectively. For the in vivo experiments, mice with breast cancer xenografts were treated with the inhibitors, alone or together, and tumor volume was determined.ResultsIron chelator inhibited breast cancer cell proliferation and decreased the proportion of S-phase cells. Conversely, it induced hypoxia, angiogenesis, EMT, and immune checkpoints, as determined by quantifying the expression of marker mRNAs in MDA-MB-231 and MCF-7 cells. Eribulin suppressed the expression of the hypoxia and EMT related marker mRNAs in the presence of iron chelator. Iron chelator plus eribulin inhibited tumor growth in vivo to a greater extent than did either inhibitor alone.ConclusionsAlthough iron chelator induces oncogenic events (hypoxia, angiogenesis, EMT, and immune checkpoints), it may be an effective treatment for breast cancer when administered in combination with eribulin.

Iron-Chelating Potential of Novel Phytochemicals in Poplar and Cedar Trees

Introduction: Iron overload is not only a consequence of diseases such as thalassemia and hereditary hemochromatosis (HFE), but also of neurodegeneration with brain iron accumulation (NBIA). In HFE, iron increases the risk of developing hepatocellular and colorectal cancers. Excess iron resulting from disruptions in normal iron homeostasis can accumulate in major organs including liver, heart and brain, and has devastating effects if left untreated. Currently, treatment includes using iron chelators, which at higher concentrations can have significant adverse effects and require constant medical supervision. Therefore, the search for alternative unique or adjuvant iron chelators that have reduced toxicity could be of significant benefit. Plants that grow in alkaline soils may be a good source of chelators for this purpose. Since iron is generally unavailable in such soils, plant roots have evolved mechanisms to solubilize iron for uptake, such as soil acidification, but need additional strategies to overcome high alkalinity. This may include producing secondary metabolites that are exuded into the soil and can chelate iron directly, including phenolic acids that may chelate iron at physiological pH in humans. This project is focused on finding, isolating, and testing bioactivity of compounds from western red cedar (alkaline tolerant) and poplar (rich in phenolics). Methods: Plants are grown in iron-normal and -deficient conditions in an innovative aeroponic system to stimulate the production of secondary metabolites related to Fe deficiency. Plant tissue extracts and root washings are collected and concentrated with solid phase extraction chromatography to form plant-derived concentrates (PDC) that are analyzed by UPLC-MS and colourimetric assays to isolate, identify, and characterize compounds induced by iron-deficiency. For bio-activity testing, PDCs are introduced to cultures of THP-1 cells, a model human monocytic cell line, to study their effect on Fe homeostasis. Prior to treatment with chelators, cells are cultured under normal (Con) and Fe-overload (CrFe) conditions (produced by treatment with 10 and 20 µM Fe-citrate) for one week to model human chronic iron overload. Deferoxamine (DFO), a well-known clinical iron chelator, model phenolics like caffeic (CafA) and chlorogenic acid (CGA), and PDCs have been applied to cultures as potential chelators. Results: Leaf compared to root tissues from poplar vary greatly in their CGA and phenolic content. Leaf extracts contained 5 times more phenolics than root extracts, and root extracts from iron-deficient plants produced 66% more phenolic compounds than those from iron-normal plants. Compared to leaf extracts, root extracts showed a 4-fold increase in iron-binding activity in vitro. PDCs including these extracts were found to contain compounds responsive to iron deficiency, which are semi-polar and low in molecular weight (140 - 340 m/z). Distinct iron-responsive compounds were also identified from cedar. Following acute dosage with Fe-citrate, THP-1 cells showed a moderate reduction in iron content after treatment with CafA, CGA, and PDCs from roots, with no influence on cell viability. On-going work includes dose-dependency with CafA and PDCs and co-operative effects of PDCs with DFO. Iron-loading in THP-1 cells is time sensitive, with maximum iron uptake measured at 8 hours following delivery of 20 µM Fe-citrate. Detailed kinetics of cellular iron-loading in the presence of iron-chelators is currently being investigated. Conclusions: We found that low-molecular weight and water-soluble PDCs from iron-deficient plants had excellent iron-binding capacity in vitro, and inhibited iron uptake in THP-1 cells. Effects of chelators on cellular iron uptake is both dose and time dependent. Screening plants for novel chelators provides an abundance of opportunity to search for new chelators for human medicinal use. Disclosures No relevant conflicts of interest to declare.

Synergistic inhibition of tumor cell proliferation by metformin and mito-metformin in the presence of iron chelators.

ABSTRACTWe demonstrate that combined treatment with metformin (Met) or its mitochondria-targeted analog, mito-metformin (Mito-Met), and various iron chelators synergistically inhibits proliferation of pancreatic and triple-negative breast cancer cells. Previously, we reported that Met (at millimolar levels) and Mito-Met (at micromolar levels) inhibited pancreatic cancer cell proliferation. Inhibition of mitochondrial complex I and mitochondrial oxidative phosphorylation (OXPHOS) through stimulation of mitochondrial redox signaling was proposed as a key mechanism for decreased cancer cell proliferation. Because of its poor bioavailability, the use of Met as a “stand-alone” antitumor drug has been questioned. Iron chelators such as deferasirox (DFX) and dexrazoxane (DXR) exhibit antiproliferative effects in cancer cells. The potency of Met and Mito-Met was synergistically enhanced in the presence of iron chelators, DFX, N,N'-bis(2-hydroxyphenyl)ethylendiamine-N,N'-diacetic acid (HBED), and deferoxamine (DFO). Met, DXR (also ICRF-187), and DFO are FDA-approved drugs for treating diabetes, decreasing the incidence and severity of cardiotoxicity following chemotherapy, and mitigating iron toxicity, respectively. Thus, the synergistic antiproliferative effects of Met and Met analogs and iron chelators may have significant clinical relevance in cancer treatment. These findings may have implications in other OXPHOS-mediated cancer therapies.

Interaction of ascaridole, carvacrol, and caryophyllene oxide from essential oil of Chenopodium ambrosioides L. with mitochondria in Leishmania and other eukaryotes.

The antileishmanial activity of the essential oil (EO) from Chenopodium ambrosioides L. has been demonstrated in vitro and in animal models, attributed to the major components of the EO. This study focused on the effects of the three major EO compounds carvacrol, caryophyllene oxide (Caryo), and the antileishmanial endoperoxide ascaridole (Asc) on mitochondrial functions in Leishmania tarentolae promastigotes (LtP). EO and Caryo were able to partially inhibit the leishmanial electron transport chain, whereas other components failed to demonstrate a direct immediate effect. Caryo demonstrated inhibition of complex III activity in LtP and in isolated complex III from other species. The formation of superoxide radicals was studied in Leishmania by electron spin resonance spectroscopy in the presence of iron chelators wherein selected compounds failed to trigger a significant immediate additional superoxide production in LtP. However, upon prolonged incubation of Leishmania with Asc and especially in the absence of iron chelators (allowing the activation of Asc), an increased superoxide radical production and significant impairment of mitochondrial coupling in Leishmania was observed. Prolonged incubation with all EO components resulted in thiol depletion. Taken together, the major components of EO mediate their leishmanicidal activity via different mitochondrial targets and time profiles. Further studies are required to elucidate possible synergistic effects of carvacrol and Asc and the influence of minor compounds.

Impact of Iron Availability on <i>Bacillus amyloliquefaciens</i> Growth

Bacillus amyloliquefaciens is a biocontrol agent whose genome has been sequenced. Within the genome of B. amyloliquefaciens are genes associated with iron chelation, but these genes are not found within all sequenced strains. The impact of iron availability on the B. amyloliquefaciens physiology was examined in this study. B. amyloliquefaciens ATCC 23843 was cultured under ironreplete and iron-deplete conditions for 48 hours, at 37°C. Final growth yields were dependent on iron concentration. Cultures grown in the absence of detectable iron were restricted in growth, but reached their highest yields at 48 hours. Iron restriction was confirmed by the presence of iron chelators in the filtrates. In contrast, B. amyloliquefaciens ATCC 23843 cultures grown with ferric ammonium citrate as the iron source research reached the highest yields at 24 hours. Iron chelator production was not detected in the ferric ammonium samples. A significant decrease in turbidity was observed for these cultures, which coincided with elevated spore production in B. amyloliquefaciens ATCC 23843. A decrease in turbidity was also observed on blood agar, where hemolysis was readily evident. We propose that iron impacts numerous physiological responses and further studies will elucidate the complex regulatory mechanisms governed by iron availability.

Constitutive expression of RyhB regulates the heme biosynthesis pathway and increases the 5-aminolevulinic acid accumulation inEscherichia coli

In the current study, the small RNA ryhB, which regulates the metabolism of iron in Escherichia coli, was constitutively expressed in engineered E. coli DALA. The resulting strain E. coli DALRA produced 16% more 5-aminolevulinic acid (ALA) than the parent strain E. coli DALA in batch fermentation. Meanwhile, we found that addition of iron in the medium increased heme formation and reduced ALA yield, whereas the presence of iron chelator in the medium decreased heme concentration and increased the ALA production efficiency (ALA yield per OD600). The qRT-PCR analysis showed that the mRNA levels of hemB and hemH were also decreased as well as the known RyhB target genes of acnAB, sdhAB, fumA, and cydAB in E. coli DALRA. These results indicated that small RNA can be used as a tool for regulating ALA accumulation in E. coli.

An Anti-Apoptotic Molecule, Anamorsin, Is Essential for Erythropoiesis Through the Regulation of Cellular Labile Iron Pool

Abstract 610 Introduction:Iron has crucial roles in many cellular biological processes. Cellular iron uptake and export must be tightly regulated. Insufficient iron concentrations impair the function of numerous iron proteins, whereas excess free iron can oxidize and damage the contents of cells.Anamorsin (AM, also called CIAPIN-1) is an anti-apoptotic factor, which we originally isolated as a molecule that confers factor-independent survival of hematopoietic cells. AM-deficient mice are embryonic lethal at late gestation due to the defect of definitive hematopoiesis. It is thought that AM plays a crucial role in hematopoiesis, however its precise biological mechanisms remain unclear. Recently, it was reported that the yeast AM homolog, Dre2, was implicated in cytosolic iron-sulfur (Fe/S) cluster assembly (Zhang Y., et al. Mol.Cell.Biol. 28:5569–5582, 2008). The AM carries conserved cysteine motifs (CX2CXC and twin CX2C) at its C termini, which may form iron binding sites.In this study, we have focused on the possibility that AM may be involved in the maturation of Fe/S cluster and the cellular iron homeostasis, especially, the regulation of labile iron pool (LIP) and that AM may affect the accumulation of reactive oxygen species (ROS), leading to impaired erythropoiesis. Methods and Results:To analyze the function of Fe/S protein, we established wild-type cell lines (AMWT) and AM-deficient cell lines (AMKO) from wild-type and AM-deficient fetal liver (14.5dpc) respectively by using SV40 large T antigen.Iron regulatory protein 1 (IRP1) is a well-known Fe/S protein with dual functions. In the presence of Fe/S cluster, IRP1 functions as a cytosolic aconitase. While, in the absence of Fe/S cluster, IRP1 stabilizes the transferrin receptor (TfR) mRNA by binding to the iron responsive element (IRE). We compared the aconitase activity and the IRE binding activity of IRP1 between AMWT and AMKO. The results showed that the cytosolic aconitase activity in AMKO decreased approximately 30% compared to AMWT and the IRE binding activity of IRP1 in AMKO increased 3-fold compared to AMWT. Furthermore, we compared the iron homeostasis. In the presence of iron chelator, desferrioxamine, the expression of TfR in AMWT was markedly elevated, while it was hardly elevated in AMKO.The LIP is a pool of chelatable and redox-active iron, which serves as a crossroad of cell iron metabolism. The measurement of LIP with the metal-sensitive sensor calcein acetoxymethyl ester showed that AMKO had 5-fold higher cellular LIP than AMWT. Moreover we evaluated the accumulation of ROS and the induction of apoptosis by extracellular iron uptake between AMWT and AMKO. The results showed the accumulation of ROS and the induction of apoptosis in AMKO were enhanced about twice as much as in AMWT. These enhancements could be restored by transduction of AM expressing retrovirus vector to AMKO.We also evaluated the effects of AM-deficiency on erythroid differentiation. Fetal liver cells from wild-type or AM-deficient embryos (14.5dpc) were divided into primitive and more matured erythroid populations based on their expression of CD71 and Ter119 by FACS analysis. AM-deficient fetal liver cells had a significant increase in the CD71low TER119low population, containing primitive erythroid progenitors, compared to wild-type (9.4±2.1% vs. 5.2±1.1%, P<0.05). Conversely, the CD71lowTER119highpopulation, comprised of late orthochromatophilic erythroblasts and reticulocytes, decreased in AM-deficient fetal liver cells compared to wild-type cells (2.3±0.8% vs. 7.4±1.3%, P < 0.05).Moreover we studied LIP in wild-type or AM-deficient embryo fetal liver cells. In accordance with the cell lines, the LIP in AM-deficient fetal liver cells increased 3 to 5-fold more than in wild-type fetal liver cells. The accumulation of ROS and the number of apoptotic cells also increased 2 to 5-fold in AM- deficient fetal liver cells compared to wild-type fetal liver cells.Thus, it was showed that AM deficiency impaired the iron homeostasis and conferred low sensitivity for iron concentration, resulting in the increase of LIP, the accumulation of ROS and the induction of apoptosis. Furthermore, dysregulation of cellular iron homeostasis was thought to be the cause of the embryonic lethal due to AM deficiency. Conclusion:Our current findings indicate that AM functions in cytosolic Fe/S cluster biogenesis and iron homeostasis and is essential for erythropoiesis. Disclosures:Kanakura:Shire: Consultancy.

P5-05-06: Dysregulation in the Transport of Iron and Haem Is Implicated in the Aetiopathogenesis of Breast Cancer.

Abstract Backround: Mounting evidence implicates iron in the development of a number of cancers, including breast. The responsible mechanism is poorly understood in breast cancer, although recent work has demonstrated irregular expression of various molecular iron transporters. Materials and Methods: Immunohistochemistry on archived material was used to evaluate differences in expression of iron and haem exporters, importers and storage proteins between breast cancer, DCIS and normal breast tissue. The results were corroborated by Western blotting and RT-qPCR on protein and RNA samples generated from matched normal and cancer specimens collected at mastectomy. The ability of benign and malignant breast cells in vitro to accumulate excess iron in the presence of exogenous iron/haem was evaluated with a ferrozine assay. In addition the effect of exogenous iron and haem on cell phenotype in vitro was assessed in benign and malignant cell lines. Assays utilised included viability (MTT), proliferation (BrdU), migration, anchorage-independent growth (colony formation) and invasive capacity. These assays were then repeated in the presence of iron chelators to investigate their potential utility as a therapeutic modality. Results: Statistically significant increases (p&amp;lt;0.05) were observed in expression of the iron importers DCytB, DMT1 and TfR in breast cancer relative to normal tissue. Levels of the storage protein ferritin were also significantly raised. The iron exporters ferroportin and hephaestin were significantly under-expressed in breast cancer, while hepcidin (which induces exporter degradation) was over-expressed. Levels of the haem importer HCP1 and the exporters BCRP and FLVCR were all significantly down-regulated in cancer relative to normal. Both benign and malignant breast cells were capable of importing excess iron, although only malignant cells could import excess haem. Iron induced increased viability and proliferation in benign cells but had no effect on their migratory or invasive capability, or their ability to form colonies. Haem had no effect on benign cell phenotype. Iron and haem both had significantly positive effects on all these aspects of cell phenotype when introduced to malignant breast cells in vitro. Iron chelators were capable of abrogating these positive effects. Discussion: Archived material and prospectively collected matched tissue pairs demonstrate that breast cancers exhibit altered expression of the proteins involved in transporting iron and haem through the cell. In vitro experiments reveal malignant cells to be capable of taking up both substances, which both appear to drive a more malignant phenotype. These in vitro changes can be reversed through the introduction of iron chelators, implying possible efficacy as a novel therapy in breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-05-06.

An Anti-Apoptotic Molecule, Anamorsin, Functions in Both Iron-Sulfur Protein Assembly and Cellular Iron Homeostasis

Abstract 2106 Introduction:Anamorsin (AM, also called CIAPIN-1) is an anti-apoptotic factor, which we originally isolated as a molecule that confers factor-independent survival of hematopoietic cells (J.Exp.Med. 199:581–592, 2004). AM has no structural homology to any known anti-apoptosis molecules such as Bcl-2 and IAP family members. AM-deficient (AM−/−) mice are embryonic lethal at late gestation. Its embryos are anemic and the size of embryos is very small. It is thought that AM plays a crucial role in hematopoiesis, however the precise biological mechanisms of AM remain unclear. Recently, it was reported that the yeast AM homolog, Dre2, was implicated in cytosolic iron-sulfur (Fe/S) cluster assembly (Zhang Y., et al. Mol.Cell.Biol. 28:5569-5582, 2008). The AM carries conserved cysteine motifs (CX2CXC and twin CX2C) at its C termini, which may form iron binding sites. In this study, we have focused on the possibility that AM may be involved in the cellular iron regulation. Methods and Results:At first, in order to analyze molecular and cellular events, we established cell lines from wild-type or AM-deficient embryonic fetal liver (14.5dpc) by using SV40 large T antigen. We isolated 5 wild-type cell lines (AM WTs) and 2 AM-deficient cell lines (AM KOs) respectively. Next, we compared the cell growth and apoptosis in both cell lines and found that the growth rate of AM KOs were slightly lower than that of AM WTs although these cell lines were immortalized. AM KOs showed more significant apoptosis induced by oxidative stress; the percents of Annexin V positive fraction were 12 ± 4 and 36 ± 6 in AM WTs and AM KOs respectively under the condition of 0.1mM H2O2 for 16hr. In addition to oxidative stress, AM KOs were more sensitive to UV irradiation. These differences were cancelled by transduction of AM-expression retrovirus vector in AM KOs.It was reported that Dre2 functions in cytosolic Fe/S protein biogenesis. We examined whether AM might be involved in the maturation of cytosolic Fe/S proteins. Iron regulatory protein 1 (IRP1) is a well-known cytosolic Fe/S protein with dual functions; in the presence of an [4Fe-4S] cluster it functions as a cytosolic aconitase, while IRP1 binds to mRNA stem-loop structures called iron responsive elements (IREs) and confer the mRNA stability when the [4Fe-4S] cluster is missing. In the iron-deficient cells, IRP1 binds to IREs located at the mRNA of iron transferrin receptor (TfR), ferritin and other iron metabolism transcripts, thereby enhancing iron uptake. In this way, it is thought that IRP1 plays important roles in iron homeostasis. We therefore compared the aconitase activity and IRE binding activities of IRP1 between AM WTs and AM KOs and found that AM deficiency resulted in the decrease of cytosolic aconitase activity (approximately 30% compared to AM WTs). In contrast to cytosolic aconitase activity, the mitochondrial aconitase activity showed little change regardless of AM deficiency. In order to analyze whether AM deficiency might increase IRE binding activity of IRP1, cytoplasmic extracts of AM WTs and AM KOs were compared by RNA precipitation assay. In AM KOs, the expression level of IRP1 decreased approximately one third compared to AM WTs. However, the binding activity of IRP1 to biotin-labeled IRE increased in the extract of AM KOs approximately three-fold in comparison to AM WTs. These differences was cancelled by transduction of AM-expression retrovirus vector to AM KOs. All these findings demonstrated the involvement of AM in the maturation of the cytosolic Fe/S protein, IRP1.Furthermore, we examined the expression of TfR, which is known to be modulated by IRP1-mediated posttranscriptional regulation. In the presence of iron chelator, desferrioxamine, the expression of TfR in AM WTs was markedly elevated. On the other hand, in AM KOs, the expression of TfR was hardly elevated. Thus, it was showed that AM deficiency impaired the iron homeostasis and conferred low sensitivity for iron concentration due to the decreased function of IRP1. Conclusion:Our current findings indicate that AM is essential for cytosolic Fe/S cluster biogenesis and iron homeostasis. Now the influence of the AM-mediated iron homeostasis on hematopoiesis is under investigation. Disclosures:No relevant conflicts of interest to declare.

Characteristics and modifying factors of asbestos‐induced oxidative DNA damage

Respiratory exposure to asbestos has been linked with mesothelioma in humans. However, its carcinogenic mechanism is still unclear. Here we studied the ability of chrysotile, crocidolite and amosite fibers to induce oxidative DNA damage and the modifying factors using four distinct approaches. Electron spin resonance analyses revealed that crocidolite and amosite containing high amounts of iron, but not chrysotile, catalyzed hydroxyl radical generation in the presence of H(2)O(2), which was enhanced by an iron chelator, nitrilotriacetic acid, and suppressed by desferal. Natural iron chelators, such as citrate, adenosine 5'-triphosphate and guanosine 5'-triphosphate, did not inhibit this reaction. Second, we used time-lapse video microscopy to evaluate how cells cope with asbestos fibers. RAW264.7 cells, MeT-5 A and HeLa cells engulfed asbestos fibers, which reached not only cytoplasm but also the nucleus. Third, we utilized supercoiled plasmid DNA to evaluate the ability of each asbestos to induce DNA double strand breaks (DSB). Crocidolite and amosite, but not chrysotile, induced DNA DSB in the presence of iron chelators. We cloned the fragments to identify break sites. DSB occurred preferentially within repeat sequences and between two G:C sequences. Finally, i.p. administration of each asbestos to rats induced not only formation of nuclear 8-hydroxy-2'-deoxyguanosine in the mesothelia, spleen, liver and kidney but also significant iron deposits in the spleen. Together with the established carcinogenicity of i.p. chrysotile, our data suggest that asbestos-associated catalytic iron, whether constitutional or induced by other mechanisms, plays an important role in asbestos-induced carcinogenesis and that chemoprevention may be possible through targeting the catalytic iron.

Iron Chelation in Mantle Cell Lymphoma.

Cell proliferation is dependent upon iron, and numerous studies have shown that iron limitation arrests cells in the G1 phase of the cell cycle. A recent study of the molecular basis of these observations (Richardson, et al. Blood 2007; 109:4045) examined the ability of iron chelators to inhibit cell proliferation and to induce apoptosis, focusing on the role of iron chelation on cyclin D1. Cyclin D1 assembles with cdk-4 or cdk-6, generating an active holo-enzyme that catalyzes a rate limiting step in G1/S progression. This complex phosphorylates substrates, including the retinoblastoma protein, which regulate S phase entrance. Richardson's group demonstrated that the G1/S arrest after Fe depletion is mediated, in part, by a decrease in cyclin D1 via ubiquitin-independent proteasomal degradation. Studies looking specifically at mantle cell lymphoma cell lines, however, have not yet been reported. Mantle Cell lymphoma is an interesting target for potential iron chelation as it is associated with a balanced translocation (t11;14) which leads to upregulation of BCL1 and to the constitutive overproduction of cyclin D1. We studied five different cell lines - JeKo (Mantle Cell Lymphoma), BL-41 (Burkitt Cell Lymphoma), DG-75 (Burkitt Cell Lymphoma), SUDHL-6 (Diffuse Large B cell Lymphoma) and EBV-immortalized lymphocytes from normal controls - and incubated them with four different iron chelators - deferoxamine (DFO), 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311), Pyridoxal Isonicotinoyl Hydrazone (PIH), and Salicylaldehyde Isocotinoyl Hydrazone (SIH). We then measured and compared cell cycle proliferation (using the Cellometer Auto T4, an instrument that measures cell count, cell viability, and cell size) and the rate of apoptosis (via propidium iodide FACS analysis). At 24 hours incubation, the mantle cell lymphoma lines showed significantly increased rates of apoptosis compared with non-chelated mantle cell controls (5% vs. 48%, p=0.04). The diffuse large B cell lymphoma line showed a lesser increase in apoptosis that did not reach statistical significant (6.5% vs. 14%, p=0.07), while the Burkitt's lymphoma lines and the EBV immortalized lymphocytes showed no significant difference (BL-41, 3.4% vs. 4.1%, p=0.50; DG-75, 6% vs. 5.9%, p=0.99; EBV lymphocytes, 12.5% vs. 12.7%, p=0.96). At 72 hours of incubation with chelators, the EBV lymphocytes showed increased apoptosis compared to untreated controls (2.5% vs. 44.5%, p=0.002), while the apoptotic rate increased in the diffuse large B cell lymphoma line (3.8% vs. 48%, p=0.001) and even more dramatically in the mantle cell lymphoma line (1.5% vs. 64%, p=0.0006). The two Burkitt's lymphoma lines were affected to a lesser degree at 72 hours by the presence of iron chelators (BL-41, 0.9% vs, 3.9%, p=0.02; DG-75, 5.5% vs. 8.9%, p=0.11). Although iron chelation, especially at longer incubation times, did affect all cell lines to various degrees, the chelator-mediated effects do appear to be specific for cell type, with mantle cell lymphoma cells displaying higher rates of apoptosis compared with other lymphomas and normal lymphocytes. These initial results will now be followed by examination of cyclin D1 expression after iron chelation. If overexpression of cyclin D1 in mantle cell lymphoma releases cells from their normal controls and acts as an oncogene, then a decrease in cyclin D1 levels via iron chelation could be added to the therapeutic armamentarium of mantle cell lymphoma.

Iron-dependent vs. iron- independent cold-induced injury to cultured rat hepatocytes: A comparative study in physiological media and organ preservation solutions

We previously described the entity of cold-induced apoptosis to rat hepatocytes and characterized its major, iron-dependent pathway. However, after cold incubation in some solutions, e.g. cell culture medium, hepatocytes show an additional, yet uncharacterized component of cold-induced injury. We here assessed the effects of organ preservation solutions on both components of cold-induced injury and tried to further characterize the iron- independent component. None of the preservation solutions (University of Wisconsin, histidine–tryptophan–ketoglutarate, Euro-Collins, histidine–lactobionate, sodium–lactobionate–sucrose and Celsior solutions) provided significant protection against cold-induced cell injury (LDH release after 24-h cold incubation/3 h rewarming >65% for all solutions); three solutions even enhanced cold-induced injury. However, when the predominant iron-dependent mechanism was eliminated by the addition of iron chelators, all preservation solutions yielded hepatocyte protection that was clearly superior to the one obtainable in cell culture medium or Krebs–Henseleit buffer with iron chelators (LDH release after 24-h cold incubation/3 h rewarming ⩽35% in all preservation solutions and 65 ± 10% in culture medium). The iron-dependent and the weaker iron- independent component of cold-induced injury showed a different temperature dependence, and in experiments with modified Krebs–Henseleit buffer the principle of the preservation solutions that inhibits the iron- independent component was identified as the low chloride concentration of these solutions (LDH release after cold incubation/rewarming in the presence of iron chelators: 66 ± 6% in regular and 22 ± 8% in chloride-poor Krebs–Henseleit buffer). Taken together, these results suggest that solutions for cold storage of hepatocytes should be chloride-poor and contain an iron chelator.

Genetic characterization of pcpS, encoding the multifunctional phosphopantetheinyl transferase of Pseudomonas aeruginosa.

Fatty acid synthases (primary metabolism), non-ribosomal peptide synthases and polyketide synthases (secondary metabolism) contain phosphopantetheinyl (Ppant)-dependent carrier proteins that must be made functionally active by transfer of the 4'-Ppant moiety from coenzyme A. These reactions are usually catalysed by dedicated Ppant transferases. Although rich in Ppant-dependent carrier proteins, it was previously shown that Pseudomonas aeruginosa possesses only one Ppant transferase, encoded by pcpS, which functions in both primary and secondary metabolism. Consistent with this notion are our findings that pcpS can genetically complement mutations in the Escherichia coli acpS and entD genes, encoding the apo-acyl carrier protein (ACP) synthase of fatty acid synthesis and a Ppant transferase of enterobactin synthesis, respectively. It also complements a Bacillus subtilis sfp mutation affecting a gene encoding a Ppant transferase essential for surfactin synthesis. A pcpS insertion mutant could only be constructed in a strain carrying the E. coli acpS gene on a chromosomally integrated element in trans, implying that the in vitro essentiality of pcpS is due to its requirement for activation of apo-ACP of fatty acid synthesis. The conditional pcpS mutant is non-fluorescent, does not produce pyoverdine and pyochelin, and does not grow in the presence of iron chelators. The data presented here for the first time confirm that PcpS plays an essential role in both fatty acid and siderophore metabolism.

Impact of cultivation conditions on haemolytic activity of Pseudoalteromonas issachenkonii KMM 3549T.

The present work aimed to study the effects of cultivation conditions on the haemolytic activities of Pseudoalteromonas issachenkonii. The kinetics of growth and haemolytic activities was investigated on sea-salts and NaCl-based nutrient media supplemented with either starch, or KBr over a period of 140 h. The first haemolytic activity occurred when bacterial cells reached the late stationary phase. The second haemolytic activity was observed in marine broth (MB) after 110 h of incubation. Addition of Fe to the culture medium neither affected bacterial growth nor reduced the haemolytic activity. However, the activity was enhanced in the presence of iron chelator. The second haemolytic activity was not affected by Ca2+, or inhibited by chymotrypsin or EDTA. The production of haemolysins by P. issachenkonii was greater on MB and was dependent on both the medium composition and time of incubation. The second haemolytic activity was heat stable, nonproteinaceous, calcium-independent and was regulated by Fe. The results demonstrated the importance of optimization of both the media composition and monitoring the haemolytic activity over a prolonged cultivation time to detect different types of haemolysins.

Role of the photo-Fenton reaction in the production of hydroxyl radicals and photobleaching of colored dissolved organic matter in a coastal river of the southeastern United States

Photochemical reactions involving colored dissolved organic matter (CDOM) in natural waters are important determinants of nutrient cycling, trace gas production and control of light penetration into the water column. In this study the role of the hydroxyl radical (•OH) in CDOM photodegradation was explored as well as the contribution of photo-Fenton chemistry to •OH formation. Photochemically produced •OH was observed under aerobic and dioxygen-depleted conditions in highly colored, acidic natural water samples obtained from a freshwater reach of the Satilla River, a river in the southeastern United States. Net aerobic •OH formation along with the production of hydrogen peroxide and Fe(II) provided evidence of photo-Fenton produced •OH. A reduction in •OH production in the presence of iron chelators further suggests the importance of iron and the photo-Fenton reaction in this water. Apparent quantum yield values for the photochemical production of •OH were determined from 300–320 nm. In addition, the relationship between •OH photoproduction and effects of irradiation on the optical properties of CDOM was examined. Changes in the light absorption and fluorescence properties of water samples from the Satilla River and other natural waters were compared to •OH production rates. The ability of constituents of Satilla River water, principally the dissolved organic matter, to scavenge •OH was also considered. Results indicate that the photo-Fenton reaction accounts for more than 70% of total photochemical •OH production in Satilla River water. Given the significant levels of •OH produced in this water, it is possible that •OH influences CDOM photobleaching.

Iron Chelators Inhibit VCAM-1 Expression in Human Dermal Microvascular Endothelial Cells

Vascular cell adhesion molecule (VCAM)-1 expression may be coupled to redox-sensitive regulatory pathways, and iron may play a role in generation of reactive oxygen species that participate in these signaling pathways. To investigate the role of iron in TNF alpha-induced VCAM-1 gene expression, human dermal microvascular endothelial cells (HDMEC) were stimulated with TNF alpha in the presence of iron chelators and examined for expression of VCAM-1. The iron chelators dipyridyl (DP) and desferoxamine (DFO) inhibited VCAM-1 protein and mRNA induction in a concentration- and time-dependent manner. The induction of VCAM-1 was not inhibited by nonmetal binding reactive oxygen species (ROS) scavengers, implying a direct effect of iron in the expression of these adhesion molecules. The effect of iron was mediated at the level of gene transcription since pretreatment with DP abrogated the TNF alpha-mediated up-regulation of VCAM-1 heterogeneous nuclear RNA. Pretreatment of HDMEC with DP prior to TNFalpha treatment had no effect on p65 nuclear localization, DNA binding, or serine phosphorylation. DP pretreatment inhibited TNF alpha- and IFN gamma-mediated interferon regulatory factor 1 (IRF-1) protein expression, although restoration of IRF-1 expression failed to reconstitute VCAM-1 expression. DP treatment also blocked VCAM-1 induction in human umbilical vein endothelium and blocked induction of a host of NF-kB activated genes in HDMEC including ICAM-1, IL-8, and tissue factor. I kappa B alpha, an NF-kappa B inducible and constitutively accessible gene not requiring chromatin remodeling for transcription, was not affected by DP treatment. These data suggest that iron plays a critical role in TNF alpha mediated VCAM-1 induction in HDMEC, and the target for iron effects may be IRF-1, NF-kappa B, and potentially chromatin remodeling.

Lack of a role for iron in the Lyme disease pathogen.

A fundamental tenet of microbial pathogenesis is that bacterial pathogens must overcome host iron limitation to establish a successful infection. Surprisingly, the Lyme disease pathogen Borrelia burgdorferi has bypassed this host defense by eliminating the need for iron. B. burgdorferi grew normally and did not alter gene expression in the presence of iron chelators. Furthermore, typical bacterial iron-containing proteins were not detected in cell lysates, nor were the genes encoding such proteins identified in the genome sequence. The intracellular concentration of iron in B. burgdorferi was estimated to be less than 10 atoms per cell, well below a physiologically relevant concentration.

Nuclear DNA damage during NAD(P)H oxidation by membrane redox chains

Nuclear DNA damage, as the result of active oxygen formation by NAD(P)H-dependent redox chains, was studied. Isolated rat liver nuclei were incubated in the presence of NAD(P)H and iron chelators. Nuclear DNA damage was analyzed by electrophoresis in alkaline agarose. DNA damage after the addition of electron donors alone or with FeCl 3 or DFO-Fe 3+ was not visualized. Dramatic decay of high molecular weight DNA was observed with EDTA-Fe 3+ or DTPA-Fe 3+ in the presence of NAD(P)H. SOD did not prevent DNA damage, whereas catalase was protective. DNA damage was revealed after the addition of cumene hydroperoxide with EDTA-Fe 3+, and it was sharply increased in the presence of NADPH. It is suggested that alkoxyl radicals in addition to hydroxyl radicals are involved in DNA damage during NAD(P)H oxidation in the presence of iron chelators, which can be reduced by membrane redox chains.

Methylene Blue and Rose Bengal Photoinactivation of RNA Bacteriophages: Comparative Studies of 8-Oxoguanine Formation in Isolated RNA

Several reactive oxygen species, including singlet oxygen ( 1O 2) and hydroxyl free radical ( . OH), may potentially be involved in the photoinactivation of viruses by agents such as methylene blue (MB) and rose bengal (RB). Both 1O 2 and . OH also mediate the formation of 8-oxoguanine (8-oxoGua) in DNA and RNA. Evidence that MB-or RB-induced bacteriophage (R17 or Qβ) inactivation and 8-oxoGua formation in UNA result from 1O 2 rather than . OH was obtained utilizing complementary experimental approaches which show that: (i) the rate of phage photoinactivation by MB was unchanged by the presence of iron chelators or by different temperatures in the 13-37°C range; (ii) MB- and RB-mediated rates of 8-oxoGua formation in isolated RNA have very little, if any, temperature dependence, in contrast to a significant temperature dependence of 8-oxoGua formation by a . OH generating system, the ultraviolet light irradiation of H 2O 2 and (iii) deuterium oxide (D 2O) enhanced the RB-mediated rate of phage photoinactivation and 8-oxoGua formation in isolated RNA. The presence of superoxide dismutase in the RB photoinactivation reaction did not alter the rate of phage inactivation. The data suggest that 8-oxoGua serves as a marker that correlates qualitatively with 1O 2-mediated lethal lesions in RNA bacteriophages.