Concentrations Of Ferric Citrate Research Articles

Investigation the mechanism of iron overload-induced colonic inflammation following ferric citrate exposure

Iron overload occurs due to excessive iron intake compared to the body’s demand, leading to iron deposition and impairment of multiple organ functions. Our previous study demonstrated that chronic oral administration of ferric citrate (FC) caused colonic inflammatory injury. However, the precise mechanism underlying this inflammatory response remains unclear. The current study aims to investigate the mechanism by which iron overload induced by FC exposure leads to colonic inflammation. To accomplish this, mice were orally exposed to three different concentrations of FC (71 mg/kg/bw (L), 143 mg/kg/bw (M) and 286 mg/kg/bw (H)) for continuous 16 weeks, with the control group receiving ultrapure water (C). Exposure to FC caused disturbances in the excretory system, altered colonic flora alpha diversity, and enriched pathogenic bacteria, such as Mucispirillum, Helicobacter, Desulfovibrio, and Shigella. These changes led to structural disorders of the colonic flora and an inflammatory response phenotype characterized by inflammatory cells infiltration, atrophy of intestinal glands, and irregular thickening of the intestinal wall. Mechanistic studies revealed that FC-exposure activated the NF-κB signaling pathway by up-regulating TLR4, MyD88, and NF-κB mRNA levels and protein expression. This activation resulted in increased production of pro-inflammatory cytokines, further contributing to the colonic inflammation. Additionally, in vitro experiments in SW480 cells confirmed the activation of NF-κB signaling pathway by FC exposure, consistent with the in vivo findings. The significance of this study lies in its elucidation of the mechanism by which iron overload caused by FC exposure leads to colonic inflammation. By identifying the role of pathogenic bacteria and the NF-κB signaling pathway, this study could potentially offer a crucial theoretical foundation for the research on iron overload, as well as provide valuable insights for clinical iron supplementation.

Optimization of photofermentative biohydrogen production in a mixed volatile fatty acid medium by Rhodobacter sp. MAY2: A response surface methodology (RSM) approach

Biohydrogen production through photofermentation has gained significant attention as it provides a sustainable solution for clean energy supply and addresses wastewater concerns by utilizing volatile fatty acids (VFA) which are produced in dark fermentation. In this study, response surface methodology via a Face-centered Central Composite Design (FCCD) was employed to optimize buffer solution pH, ferric citrate concentration, sodium glutamate concentration to maximize biohydrogen production by Rhodobacter sp. MAY2 with mixed VFA (acetate, propionate, butyrate) as substrates. Results showed that the concentrations of ferric citrate, sodium glutamate, and buffer solution pH positively impact the biohydrogen production of Rhodobacter sp. MAY2. The optimal conditions were shown to be 0.36 mM ferric citrate, 11 mM sodium glutamate, and buffer solution of pH 7. The predicted hydrogen production potential (HPP) at these conditions (661.1 mL/L) was within 2 % of the actual HPP (647.7 mL/L). Kinetic parameters using the modified Gompertz equation showed that the predicted values for maximum potential cumulative H2 production (P), maximum hydrogen production rate (Rm), and lag time (λ) are comparable to those reported in literature. Overall, the study highlighted the potential of a local isolate, Rhodobacter sp. MAY2, for biohydrogen production and the suitable fermentation conditions for its growth.

Iron deposition in ovarian endometriosis evaluated by magnetic resonance imaging R2* correlates with ovarian function

Research questionDoes iron overload in patients with endometriosis affect ovarian function? Can a method be developed to visually reflect this? DesignMagnetic resonance imaging (MRI) R2* was used to evaluate the correlation between iron deposition of ovarian and anti-Müllerian hormone (AMH) in patients with endometriosis. All patients underwent T2* MRI scanning. Serum AMH levels were measured preoperatively. The area of focal iron deposition, iron content of the cystic fluid and AMH levels between the endometriosis and control groups were compared using non-parametric tests. The effects of iron overload on AMH secretion in mouse ovarian granulosa cells were investigated by adding different concentrations of ferric citrate to the medium. ResultsA significant difference was found between endometriosis and control groups in area of iron deposition (P < 0.0001), cystic fluid iron content (P < 0.0001), R2* of lesions (P < 0.0001) and R2* of the cystic fluid (P < 0.0001). Negative correlations were found between serum AMH levels and R2* of cystic lesions in patients with endometriosis aged 18–35 years (rs = –0.6484, P < 0.0001), and between serum AMH levels and R2* of cystic fluid (rs = –0.5074, P = 0.0050). Transcription level (P < 0.0005) and secretion level (P < 0.005) of AMH significantly decreased with the increase in iron exposure. ConclusionIron deposits can impair ovarian function, which is reflected in MRI R2*. Serum AMH levels and R2* of cystic lesions or fluid in patients aged 18–35 years had a negative correlation with endometriosis. R2* can be used to reflect the changes of ovarian function caused by iron deposition.

Promoting effect of Fe3+ on gentamicin resistance in Escherichia coli

Kinds of antibiotics are used to prevent and control bacteria infections, unfortunately, the overuse and misuse of antibiotic have promoted the emergence and spread of antibiotic-resistant bacteria. Therefore, understanding the mechanism of antibiotic resistance is very important. This study explores the combined effection of metal ions and antibiotic to the drug resistance of Escherichia coli. Our results found that the minimum inhibitory concentration (MIC) increased as the ammonium ferric citrate concentration increased, especially for gentamicin antibiotic. When the Fe3+ concentration reached 300 μM, the survival of E. coli was stably restored with the increased gentamicin concentration. Exogenous Fe3+ could decrease intracellular gentamicin concentration. On the other hand, Fe3+ treatment together with gentamicin could reduce reactive oxygen species (ROS) production, characterized by decreased levels of NADH and ATP. Furthermore, ROS-scavenging enzymes of superoxide dismutase (SOD) and catalase (CAT) were up-regulated and H2O2 plus gentamicin-mediated killing was restored by Fe3+. These results may have significant implications in understanding bacterial antibiotic-resistant mechanisms based on the external Fe3+ concentration.

Influence of cobalt chloride and ferric citrate on purple non-sulfur bacteria Rhodopseudomonas yavorovii

Heavy metals that enter the environment due to natural processes or industrial activities, when accumulated, have a negative impact on organisms, including microorganisms. Microorganisms have developed various adaptations to heavy metal compounds. The aim of our work was to investigate the influence of ferric citrate and cobalt (II) chloride on biomass accumulation, indicators of free radical damage and activity of enzymes of the antioxidant defense system of bacteria Rhodopseudomonas yavorovii IMV B-7620, that were isolated from the water of Yavorivske Lake (Ukraine, Lviv region), which was formed as a result of flooding of a sulfur quarry. We used cultural, photometric methods, and statistical processing of the results was performed using two-way ANOVA and factor analysis. It was found that ferric citrate at a concentration of 1–12 mM causes inhibition of the accumulation of biomass of bacteria Rh. yavorovii IMV B-7620 up to 44.7%, and cobalt (II) chloride at a concentration of 1–15 mM – up to 70.4%, compared with the control. The studied concentrations of ferric citrate and cobalt (II) chloride cause free radical damage to lipids and proteins of Rh. yavorovii IMV B-7620. As a result of two-way ANOVA we found that under the influence of ferric citrate statistically significant changes in biomass accumulation, lipid hydroperoxides and thiobarbiturate reactive species content, superoxide dismutase activity were predetermined by increasing the concentration of metal salts as well as increasing the duration of cultivation of bacteria, while the content of diene conjugates and catalase activity changed with increasing duration of cultivation. Under the influence of cobalt (II) chloride, statistically significant changes in all studied indicators were found both due to the increase in the concentration of metal salts and with increasing duration of bacterial cultivation. The studied parameters of Rh. yavorovii IMV B-7620 cells under the influence of ferric citrate and cobalt (II) chloride are combined into two factors, that explain 95.4% and 99.2% of the total data variance, respectively. Under the influence of ferric citrate, the first latent factor included diene conjugates, thiobarbiturate reactive species, carbonyl groups in proteins, which are closely linked by a direct bond and inversely related to the content of lipid hydroperoxides and catalase activity. The second latent factor included duration of cultivation of bacteria, biomass accumulation, and superoxide dismutase activity, which are inversely related to lipid hydroperoxide content and catalase activity. Under the influence of cobalt (II) chloride, the first latent factor included the content of lipid hydroperoxides, carbonyl groups in proteins, as well as catalase and superoxide dismutase activities, which are inversely related to bacterial biomass.

Optimization of anthocyanidins conversion using chokeberry pomace rich in polymeric proanthocyanidins and cellular antioxidant activity analysis

Chokeberry pomace contains abundant polymeric proanthocyanidins with low antioxidant activity and bioaccessibility. In this study, polymeric proanthocyanidins from chokeberry pomace were converted into anthocyanidins with higher antioxidant activity. Optimal conversion conditions were determined by full factorial design. The composition of anthocyanidin was analyzed by TOF-MS and UPLC. In addition, the antioxidant activity of the conversion products was evaluated by PSC and CAA assays. The results showed that the yield of anthocyanidins was 11.59 mg/g when the pomace was treated with 95% ethanol concentration, 50:1 liquid-solid ratio, 4% hydrochloric acid and 0.2 mg/mL ammonium ferric citrate concentration at 80 °C for 30 min. The newly generated anthocyanidins were identified as cyanidin, which accounting for 51.3% of the total anthocyanidins. The antioxidant activity values of PSC and CAA increased 2.81 and 4.77 folds respectively. The proposed method provides a new strategy for preparation of antioxidants from naturally abundant polymeric ones.

Iron Level and Monocyte Morphology Predict TLR4 Expression and Reactive Oxygen Species Production Which Influences Chronic Inflammation in β-Thalassemia

Introduction: β-thalassemia major requires a lifetime of transfusion and chelation therapy and despite improved chelation therapies, patients endure organ iron overload and increased hemolysis leading to organ injury and release of damage associated bio-markers. The structure of these markers are recognized by pattern recognition receptors (PRR) of the innate immune system as damage associated molecular patterns (DAMPs), which trigger inflammation and increased reactive oxygen species (ROS). The specificity of this recognition depends on PRRs, such as toll-like receptor 4 (TLR4), to provide the first line of defense against pathogens and DAMPS to initiate inflammation. Therefore, chronic organ iron loading is implicated in the chronic inflammation and morbidity of β-thalassemia major; however, the mechanism of how monocytes respond to iron loading in β-thalassemia remains largely unknown. This study investigated how chronic iron and heme overload affected innate immune cell morphology and expression of TLR4 and ROS by modeling THP-1 cellscompared to β-thalassemia monocytes.Methods: Cultured THP-1 monocytic cells were exposed to increasing concentrations of ferric citrate (0-250 μM) or heme (0-50 μM) for 24 hours or to chronic exposures of 40 μM ferric citrate for up to 3 weeks. Anti-human TLR4 phycoerythrin conjugated antibodies (TLR4-PE) were used to quantify TLR4 expression and 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) was used to measure ROS. Flow cytometry was done using a BD FACSCalibur. Peripheral-blood samples were obtained from 13 consenting β-thalassemia patients as part of the Novartis sponsored study investigating the effects of combined chelation therapy (CICL670AUS42T). Samples were also obtained from ethnically and age matched healthy controls. Peripheral-blood monocytes and THP-1 cells were gated on the parameters of forward and side scatter to stratify cells based on size and granularity, separating cells into two populations: larger more granular (LG) and smaller less granular (SG) cells. Results are reported as the MFI ratios of LG versus SG cells for TLR4-PE or DCFH-DA as a functional measure of the difference in TLR4 or ROS expression between these populations.Results:In vitro, LG THP-1 cells expressed 4-fold more TLR4 and 10-fold more ROS than SG cells (p < 0.001). Treatment of THP-1 cells with increasing concentrations of iron or heme for 1 day or 3 weeks, increased the ROS and TLR4 heterogeneity between LG and SG monocytes. The LG versus SG ratio of ROS production significantly correlated to the concentration of ferric citrate or heme added to the culture media (r = 0.64, p = 0.019 and r = 0.58 and p = 0.048, respectively). The mean ratio for iron but not heme treated THP-1 cells was significantly greater than for untreated cells (p = 0.008 and p = 0.406, respectively). Mean LG versus SG ratios of TLR4-PE MFI were 5.20 ± 1.56 for ferric citrate, 4.11 ± 0.90 for heme, and 3.78 ± 1.41 for untreated cells. We observed a similar TLR4 morphological heterogenic pattern in peripheral-blood monocytes from iron overloaded β-thalassemia patients. β-thalassemia LG monocytes had significantly greater TLR4 MFI than SG monocytes (LG mean = 11.91 ± 1.17, SG mean = 6.56 ± 1.02, p < 0.001). Long-term treatment of THP-1 monocytic cells for 3 weeks with 40 μM ferric citrate also resulted in the same phenomena regarding TLR4. No difference was observed in ROS production for control LG cells and SG cells; however, THP-1 cells treated for 3 weeks with 40uM ferric citrate revealed populations of LG cells that expressed 3 fold more ROS than SG cells.Conclusion: THP-1 monocytic cells and β-thalassemic peripheral-blood monocytes exhibit a morphological heterogeneity where LG cells express more TLR4 and in vitro, produce more ROS than SG cells. As TLR4 is one of the receptors for the initiation of inflammation, LG cells are likely greater contributors to the chronic inflammation experienced by β-thalassemia patients. Thus, we hypothesize that iron overload may give rise to monocytes that are larger and more granular and express more TLR4 and ROS. DisclosuresWalter:Novartis: Research Funding. Porter:Shire: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy.

Influence of Iron on Production of the Antibacterial Compound Tropodithietic Acid and Its Noninhibitory Analog in Phaeobacter inhibens.

Tropodithietic acid (TDA) is an antibacterial compound produced by some Phaeobacter and Ruegeria spp. of the Roseobacter clade. TDA production is studied in marine broth or agar since antibacterial activity in other media is not observed. The purpose of this study was to determine how TDA production is influenced by substrate components. High concentrations of ferric citrate, as present in marine broth, or other iron sources were required for production of antibacterially active TDA. However, when supernatants of noninhibitory, low-iron cultures of Phaeobacter inhibens were acidified, antibacterial activity was detected in a bioassay. The absence of TDA in nonacidified cultures and the presence of TDA in acidified cultures were verified by liquid chromatography-high-resolution mass spectrometry. A noninhibitory TDA analog (pre-TDA) was produced by P. inhibens, Ruegeria mobilis F1926, and Phaeobacter sp. strain 27-4 under low-iron concentrations and was instantaneously converted to TDA when pH was lowered. Production of TDA in the presence of Fe(3+) coincides with formation of a dark brown substance, which could be precipitated by acid addition. From this brown pigment TDA could be liberated slowly with aqueous ammonia, and both direct-infusion mass spectrometry and elemental analysis indicated a [Fe(III)(TDA)2]x complex. The pigment could also be produced by precipitation of pure TDA with FeCl3. Our results raise questions about how biologically active TDA is produced in natural marine settings where iron is typically limited and whether the affinity of TDA to iron points to a physiological or ecological function of TDA other than as an antibacterial compound.

OXIDATIVE MODIFICATION OF PROTEINS AND SPECIFIC SUPEROXIDE DISMUASE ACTIVITY OF DESULFUROMONAS ACETOXIDANS ІМV В-7384 BACTERIA UNDER THE INFLUENCE OF FERRIC CITRATE

Aim. Investigation of oxidative modification of proteins and specific superoxide dismutase activity of Desulfuromonas acetoxidans ІМV В-7384 bacteria under the influence of ferric citrate. Methods. Generation of protein carbonyl groups (CG) were determined in reaction with 2,4- dinitrophenylhydrazine. Specific superoxide dismutase activity was measured by the ability of enzyme to inhibit autoxidation of quercetin at рН 10 in the presence of tetramethylethylenediamine. Results. The content of protein CG under the influence of ferric citrate changed in dependence of duration of bacterial cultivation. Under the influence of all investigated concentrations of ferric citrate on second and third days of cultivation no significant increase of protein CG in comparison with control sample was observed. The increase of content of protein CG by 2–4 times under the influence of metal salt was reached on the fourth day of cultivation. Addition of ferric citrate caused substantial enhancing of specific SOD activity. Under the influence of all investigated concentrations of ferric citrate specific SOD activity increased with enhancing of duration of cultivation. Conclusions. Accumulation of significant concentrations of CG in  acetoxidans ІМ -В7384  proteins under the influence of ferric citrate indicates free radical mechanism of damage of cell proteins. Under these conditions neutralization of reactive oxygen species in D. acetoxidans ІМV-В7384 bacteria is evidently made by the enzymes of antioxidative defense system, including SOD.

Effects of Calcification Inhibitors on the Viability of the Coralline Algae Lithophyllum yessoense and Corallina pilulifera

Coralline algae, the algal whitening phenomenon-causing seaweeds, are characterized by calcareous deposits in the cell wall. The viability of the coralline algae Lithophyllum yessoense and Corallina pilulifera was quantitated using a triphenyltetrazolium chloride assay and eight calcification inhibitors. Among these inhibitors, ferric citrate showed the strongest inhibition of coralline algae viability. The concentrations of ferric citrate conferring 50% inhibition were 1.7 and 3.8 mM for L. yessoense and C. pilulifera, respectively. Thus, at a specific concentration and in a localized area, ferric citrate may be used to prevent the blooming of coralline algae.

Iron Availability Increases the Pathogenic Potential of Salmonella Typhimurium and Other Enteric Pathogens at the Intestinal Epithelial Interface

Recent trials have questioned the safety of untargeted oral iron supplementation in developing regions. Excess of luminal iron could select for enteric pathogens at the expense of beneficial commensals in the human gut microflora, thereby increasing the incidence of infectious diseases. The objective of the current study was to determine the effect of high iron availability on virulence traits of prevalent enteric pathogens at the host-microbe interface. A panel of enteric bacteria was cultured under iron-limiting conditions and in the presence of increasing concentrations of ferric citrate to assess the effect on bacterial growth, epithelial adhesion, invasion, translocation and epithelial damage in vitro. Translocation and epithelial integrity experiments were performed using a transwell system in which Caco-2 cells were allowed to differentiate to a tight epithelial monolayer mimicking the intestinal epithelial barrier. Growth of Salmonella typhimurium and other enteric pathogens was increased in response to iron. Adhesion of S. typhimurium to epithelial cells markedly increased when these bacteria were pre-incubated with increasing iron concentration (P = 0.0001), whereas this was not the case for the non-pathogenic Lactobacillus plantarum (P = 0.42). Cellular invasion and epithelial translocation of S. typhimurium followed the trend of increased adhesion. Epithelial damage was increased upon incubation with S. typhimurium or Citrobacter freundii that were pre-incubated under iron-rich conditions. In conclusion, our data fit with the consensus that oral iron supplementation is not without risk as iron could, in addition to inducing pathogenic overgrowth, also increase the virulence of prevalent enteric pathogens.

A Cyclic AMP Receptor Protein-Regulated Cell-Cell Communication System Mediates Expression of a FecA Homologue in Stenotrophomonas maltophilia

Stenotrophomonas maltophilia WR-C possesses an rpf/diffusible signal factor (DSF) cell-cell communication system. It produces cis-Delta2-11-methyl-dodecenoic acid, a DSF, and seven structural derivatives, which require rpfF and rpfB for synthesis. Acquisition of iron from the environment is important for bacterial growth as well as the expression of virulence genes. We identified a gene homologous to fecA, which encodes a ferric citrate receptor that transports exogenous siderophore ferric citrate from the environment into the bacterial periplasm. Western blot analysis with anti-FecA-His(6) antibody showed that the FecA homologue was induced in the iron-depleted medium supplemented with a low concentration of ferric citrate. Deletion of rpfF or rpfB resulted in reduced FecA expression compared to the wild type. Synthetic DSF restored FecA expression by the DeltarpfF mutant to the wild-type level. Reverse transcription-PCR showed that the fecA transcript was decreased in the DeltarpfF mutant compared to the wild type. These data suggest that DSF affected the level of fecA mRNA. Transposon inactivation of crp, which encodes cyclic AMP (cAMP) receptor protein (CRP) resulted in reduced FecA expression and rpfF transcript level. Putative CRP binding sites were located upstream of the rpfF promoter, indicating that the effect of CRP on FecA is through the rpf/DSF pathway and by directly controlling rpfF. We propose that CRP may serve as a checkpoint for iron uptake, protease activity, and hemolysis in response to environmental changes such as changes in concentrations of glucose, cAMP, iron, or DSF.

The role of the synergistic phosphate anion in iron transport by the periplasmic iron-binding protein from Haemophilus influenzae

The acquisition of iron from transferrin by Gram-negative bacterial pathogens is dependent on a periplasmic ferric-ion-binding protein, FbpA. FbpA shuttles iron from the outer membrane to an inner membrane transport complex. A bound phosphate anion completes the iron co-ordination shell of FbpA and kinetic studies demonstrate that the anion plays a critical role in iron binding and release in vitro. The present study was initiated to directly address the hypothesis that the synergistic anion is required for transport of iron in intact cells. A series of site-directed mutants in the anion-binding amino acids of the Haemophilus influenzae FbpA (Gln-58, Asn-175 and Asn-193) were prepared to provide proteins defective in binding of the phosphate anion. Crystal structures of various mutants have revealed that alteration of the C-terminal domain ligands (Asn-175 or Asn-193) but not the N-terminal domain ligand (Gln-58) abrogated binding of the phosphate anion. The mutant proteins were introduced into H. influenzae to evaluate their ability to mediate iron transport. All of the single site-directed mutants (Q58L, N175L and N193L) were capable of mediating iron acquisition from transferrin and from limiting concentrations of ferric citrate. The results suggest that the transport of iron by FbpA is not dependent on binding of phosphate in the synergistic anion-binding site.

Utilization of heterologous siderophores enhances levels of iron available to Pseudomonas putida in the rhizosphere.

Pseudomonas spp. have the capacity to utilize siderophores produced by diverse species of bacteria and fungi, and the present study was initiated to determine if siderophores produced by rhizosphere microorganisms enhance the levels of iron available to a strain of Pseudomonas putida in this natural habitat. We used a previously described transcriptional fusion (pvd-inaZ) between an iron-regulated promoter (pvd) and the ice nucleation reporter gene (inaZ) to detect alterations in iron availability to P. putida. Ice nucleation activity (INA) expressed from the pvd-inaZ fusion by P. putida N1R or N1R Pvd(-), a derivative deficient in the production of a pyoverdine siderophore, was inversely related to the concentration of ferric citrate in a culture medium. In culture, INA expressed by N1R Pvd(-) (pvd-inaZ) was reduced in the presence of the ferric complex of pseudobactin-358, a pyoverdine siderophore produced by P. putida WCS358 that can be utilized as a source of iron by N1R Pvd(-). In the rhizosphere of cucumbers grown in sterilized soil, N1R Pvd(-) (pvd-inaZ) expressed INA, indicating that iron availability was sufficiently low in that habitat to allow transcription of the iron-regulated pvd promoter. Coinoculation with WCS358 or N1R significantly decreased INA expressed by N1R Pvd(-) (pvd-inaZ) in the rhizosphere, whereas coinoculation with a pyoverdine-deficient mutant of WCS358 did not reduce INA expressed by N1R Pvd(-) (pvd-inaZ). These results indicate that iron availability to N1R Pvd(-) (pvd-inaZ) in the rhizosphere was enhanced by the presence of another strain of P. putida that produces a pyoverdine that N1R Pvd(-) (pvd-inaZ) was able to utilize as a source of iron. In culture, strain N1R Pvd(-) also utilized ferric complexes of the siderophores enterobactin and aerobactin as sources of iron. In the rhizosphere of cucumbers grown in sterilized soil, INA expressed by N1R Pvd(-) (pvd-inaZ) was reduced in the presence of strains of Enterobacter cloacae that produced enterobactin, aerobactin, or both siderophores, but INA expressed by N1R Pvd(-) (pvd-inaZ) was not altered in the presence of a mutant of E. cloacae deficient in both enterobactin and aerobactin production. Therefore, the iron status of P. putida was altered by siderophores produced by an unrelated bacterium coinhabiting the rhizosphere. Finally, we demonstrated that INA expressed by N1R containing pvd-inaZ in the rhizosphere differed between plants grown in sterilized versus nonsterilized field soil. The results of this study demonstrate that (i) P. putida expresses genes for pyoverdine production and uptake in the rhizosphere, but the level of gene expression is influenced by other bacteria that coexist with P. putida in this habitat, and (ii) diverse groups of microorganisms can alter the availability of chemical resources in microbial habitats on root surfaces.

Growth of human tumor cell lines in transferrin-free, low-iron medium.

Iron is essential for tumor cell growth. Previous studies have demonstrated that apart from transferrin-bound iron uptake, mammalian cells also possess a transport system capable of efficiently obtaining iron from small molecular weight iron chelates (Sturrock et al., 1990). In the present study, we have examined the ability of tumor cells to grow in the presence of low molecular weight iron chelates of citrate. In chemically defined serum-free medium, most human tumor cell lines required either transferrin (5 micrograms/ml) or a higher concentration of ferric citrate (500 microM) as an iron source. However, we have also found that from 13 human cell lines tested, 4 were capable of long-term growth in transferrin-free medium with a substantially lower concentration of ferric citrate (5 microM). When grown in medium containing transferrin, both regular and low-iron dependent cell lines use transferrin-bound iron. Growth of both cell types in transferrin medium was inhibited to a certain degree by monoclonal antibody 42/6, which specifically blocks the binding of transferrin to the transferrin receptor. On the contrary, growth of low-iron dependent cell lines in transferrin-free, low-iron medium (5 microM ferric citrate) could not be inhibited by monoclonal antibody 42/6. Furthermore, no autocrine production of transferrin was observed. Low-iron dependent cell lines still remain sensitive to iron depletion as the iron(III) chelator, desferrioxamine, inhibited their growth. We conclude that low-iron dependent tumor cells in transferrin-free, low-iron medium may employ a previously unknown mechanism for uptake of non-transferrin-bound iron that allows them to efficiently use low concentrations of ferric citrate as an iron source. The results are discussed in the context of alternative iron uptake mechanisms to the well-characterized receptor-mediated endocytosis process.

Purification and characterization of an iron-induced ferritin from soybean (Glycine max) cell suspensions

Ferric citrate induces ferritin synthesis and accumulation in soybean (Glycine max) cell suspension cultures [Proudhon, Briat & Lescure (1989) Plant Physiol. 90, 586-590]. This iron-induced ferritin has been purified from cells grown for 72 h in the presence of either 100 microM- or 500 microM-ferric citrate. It has a molecular mass of about 600 kDa and is built up from a 28 kDa subunit which is recognized by antibodies raised against pea (Pisum sativum) seed ferritin and it has the same N-terminal sequence as this latter, except for residue number 3, which is alanine in pea seed ferritin instead of valine in iron-induced soybean cell ferritin. It contains an average of 1800 atoms of iron per molecule whatever the ferric citrate concentration used to induce its synthesis. It is shown that the presence of 100 microM- or 500 microM-ferric citrate in the culture medium leads respectively to an 11- and 28-fold increase in the total intracellular iron concentration and to a 30- and 60-fold increase in the ferritin concentration. However, the percentage of iron stored in the mineral core of ferritin remains constant whatever the ferric citrate concentration used and represents only 5-6% of cellular iron.

Floral Induction in a Photoperiodically Neutral Duckweed, Lemna paucicostata Strain LP6: Role of Chelating Agents and Iron

Lemna paucicostata, strain LP6, does not ordinarily flower under any photoperiodic schedule, when grown in the modified Bonner-Devirian medium supplemented with 10−4 M EDTA and 1% sucrose (this is a medium which is otherwise satisfactory for short day induction of flowering of other strains of this duckweed). However, when the ferric citrate concentration in the culture medium containing EDTA was raised 10-fold over that in the normal medium, a low but significant flowering could be initiated in this duckweed, irrespective of the length of the photoperiod. A similar flowering response was obtained when ferric citrate was replaced by ferrous sulphate or ferric chloride, thereby indicating that the inductive effect of higher level of ferric citrate on flowering in strain LP6 is due to its iron component. Some flowering in this strain could be induced even in medium containing normal level of iron, provided the level of EDTA, itself, was raised to 5×10−4 (5% flowering) or to 10−3 M (12% flowering), but replacement of EDTA by EDDHA led to truly remarkable effects. With EDDHA, flowering could be induced at very high levels (90%) under all photoperiodic regimes tried. Floral initiation could be obtained even with 10−6 M EDDHA, though the optimal level ranged from 5×10−6 to 10−5 M.

Effect of chelating agents on bud induction and accumulation of iron and copper by the moss Bartramidula bartramioides

The chelating agents, EDDHA, its iron salt, EDTA, and salicylic acid enhance bud formation in Bartramidula bartramioides (Griff.) Wijk &amp; Marg. Salicylic acid elicits optimal response at 10–4M, whereas the other substances do so at 10–7M. Increased concentration of ferric citrate and cupric sulphate also stimulate bud induction. The accumulation of Fe3+ and Cu2+ is facilitated by chelators. The endogenous iron content is maximum at 10–7M EDDHA or EDTA, which is also the concentration optimal for bud induction.

Differential inhibition of the MLR by iron: association with HLA phenotype.

The effect of iron on the MLR was examined by pretreating peripheral blood mononuclear cells from 77 unrelated Caucasians with five concentrations of Ferric-citrate (10.0 mM, 1.0 mM, 0.1 mM, 0.01 mM and 0.005 mM). After incubation with the metal, the cells were washed and cultured in a one-way MLR with a pool of stimulator cells. Cell viability remained unchanged (greater than 90 percent) during the 6-day culture period. Citrate per se had no effect on either the responder or the stimulator population. Iron treatment influenced the MLR in the following ways: (1) a variable degree of inhibition was observed which related to the dose of Ferric-citrate used and to HLA phenotype, (2) the responder but not the stimulator cells were affected, (3) no statistically significant differences were seen between female and male donor cells and (4) the mean percent response of cells from HLA-A2 donors were significantly (0.005 less than P less than 0.01) less susceptible to iron exposure than those from non-HLA-A2 individuals. The present results indicate that iron can interact with lymphoid cells and influence some immunological functions in vitro. The possibility is discussed that similar interactions take place in vivo which could contribute to the prognosis of certain diseases associated with particular HLA phenotypes.

&lt;i&gt;Petunia hybrida&lt;/i&gt; のはい珠培養における鉄, ホウ酸, および糖が発芽種子数におよぼす影響

The present investigation deals with in vitro growth of ovules on a half of the placenta of Petunia hybrida, W166H×K146BH (each clone is self-incompatible), with special reference to the influence of iron, boric acid, and sugars. The culture medium of NITSCH (1951), modified by the addition of 500mg/l KNO3 and 80mg/l NH4NO3, was used as a basal medium.The optimal initial hydrogen-ion concentration was at pH of 4.5-5.0 in the basal medium and at pH of 6.0 in the basal medium with Fe-EDTA at an iron concentration of ca. 2.5ppm instead of ferric citrate.The number of viable seeds per ovary increased with an increasing concentration of ferric citrate from 10 to 40mg/l in the basal medium. Iron complex Fe-EDTA composed of 50mg/l FeSO4•7H2O and 40mg/l disodium ethylenediaminetetraacetate, dihydrate (Na2-EDTA) was also an effective compound and was favorable for the viable seed production.10mg/l or above boric acid should be added to the basal medium containing either ferric citrate or Fe-EDTA. Medium containing 50mg/l boric acid was superior for the viable seed production to those with any other concentration. The efficancy of boron was influenced by the organic forms of iron. The medium with Fe-EDTA at an iron concentration of ca. 2.5ppm was superior to that with 10mg/l ferric citrate.Basal medium containing 7.5 per cent sucrose or 5 per cent fructose produced the maximal viable seeds and that containing a mixture of sucrose and fructose or sucrose and glucose was inferior to the basal medium.From these results, experiments were conducted to clarify the interaction among three components, iron, boric acid, and sugars. About 5-6 normal seedlings with two cotyledons and roots per ovary were obtained in the medium containing ca. 10 ppm Fe as Fe-EDTA, 25mg/l boric acid, and 7.5 per cent sucrose. This was about six times as the number of seedlings produced in the basal medium.In the basal medium containing fructose instead of sucrose, however, the higher concentrations of boric acid and/or iron failed to bring about the noticeable effect.