Cell cycle effects of iron depletion on T-47D human breast cancer cells

T-47D human breast cancer cells grown in culture medium containing low concentrations of fetal calf serum (FCS) proliferated very slowly, with an accumulation of cells in the G2 phase of the cell cycle, increased polyploid cells, and increased expression of transferrin receptors. Cell proliferation was stimulated by the addition of human transferrin or ammonium ferric citrate to the medium. Growth inhibition and accumulation of G2-phase cells could also be produced in T-47D cells grown in medium containing 10% FCS by the addition of the iron chelator, desferrioxamine. It is concluded that cellular deprivation of iron and/or transferrin is the major cause of reduced proliferation rates and G2-phase arrest which accompany the culture of these cells in medium supplemented with low concentrations of FCS.     

Modulation of cell proliferation and polyamine metabolism in rat liver cell cultures by the iron chelator O-trensox

The antiproliferative effects of the iron chelator O-trensox and the ornithine-decarboxylase (ODC) inhibitor alpha-difluoromethylornithine (DFMO) were characterized in the rat hepatoma cell line FAO, the rat liver epithelial cell line (RLEC) and the primary rat hepatocyte cultures stimulated by EGF. We observed that O-trensox and DFMO decreased cell viabilty and DNA replication in the three culture models. The cytostatic effect of O-trensox was correlated to a cytotoxicity, higher than for DFMO, and to a cell cycle arrest in G0/G1 or S phases. Moreover, O-trensox and DFMO decreased the intracellular concentration of spermidine in the three models without changing significantly the spermine level. We concluded that iron, but also polyamine depletion, decrease cell growth. However, the drop in cell proliferation obtained with O-trensox was stronger compared to DFMO effect. Altogether, our data provide insights that, in the three rat liver cell culture models, the cytostatic effect of the iron chelator O-trensox may be the addition of two mechanisms: iron and polyamine depletion.     

Effects of an external magnetic field on the sedimentation of activated sludge

By applying an external magnetic field (800–3000 G, 0.08–0.30 T) using permanent magnets to the aeration vessel of an activated sludge culture, the sedimentation of activated sludge was enhanced and chemical oxygen demand (COD) removal was also improved in an indoor continuous culture system. Adding a small amount of iron(III) chloride (FeCl₃, less than 0.1%, w/v) stimulated these enhancements. The possibility was suggested that the small amount of molecular iron incorporated into the activated sludge stimulated the flocculation and sedimentation by external magnetization.     

Targeted inhibition of transferrin-mediated iron uptake in Hep G2 hepatoma cells

We have used a model system consisting of two human hepatoma cell lines, Hep G2, representing well differentiated normal hepatocytes, and PLC/PRF/5, representing poorly differentiated malignant hepatocytes, to demonstrate that the differential presence of asialoglycoprotein receptor activity in these cell lines can be used to influence transferrin-mediated iron uptake. We based our experiments on the following facts: Hep G2 cells possess receptors that bind, internalize, and degrade galactose-terminal (asialo-)glycoproteins; PLC/PRF/5 cells have barely detectable asialoglycoprotein receptor activity; both cell lines possess active transferrin-mediated iron uptake; transferrin releases iron during acidification of intracellular vesicular compartments; primary amines, e.g. primaquine, inhibit acidification and iron release from transferrin. When added to culture medium, [55Fe]transferrin delivered 55Fe well to both cell lines. As expected, in the presence of [55Fe]transferrin, free primaquine caused a concentration-dependent decrease in 55Fe uptake in both cell lines. To create a targetable conjugate, primaquine was covalently coupled to asialofetuin to form asialofetuin-primaquine. When PLC/PRF/5 (asialoglycoprotein receptor (-)) cells were preincubated with this conjugate, transferrin-mediated 55Fe uptake was unaffected. However, transferrin-mediated 55Fe uptake by Hep G2 (asialoglycoprotein receptor (+)) cells under identical conditions was specifically decreased by 55% compared to control cells incubated without the conjugate.     

Converse modulation of toxic α-synuclein oligomers in living cells by N′-benzylidene-benzohydrazide derivates and ferric iron

Intracellular α-synuclein (α-syn) aggregates are the pathological hallmark in several neurodegenerative diseases including Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy. Recent evidence suggests that small oligomeric aggregates rather than large amyloid fibrils represent the main toxic particle species in these diseases. We recently characterized iron-dependent toxic α-syn oligomer species by confocal single molecule fluorescence techniques and used this aggregation model to identify several N′-benzylidene-benzohydrazide (NBB) derivatives inhibiting oligomer formation in vitro. In our current work, we used the bioluminescent protein-fragment complementation assay (BPCA) to directly analyze the formation of toxic α-syn oligomers in cell culture and to investigate the effect of iron and potential drug-like compounds in living cells. Similar to our previous findings in vitro, we found a converse modulation of toxic α-syn oligomers by NBB derivates and ferric iron, which was characterized by an increase in aggregate formation by iron and an inhibitory effect of certain NBB compounds. Inhibition of α-syn oligomer formation by the NBB compound 293G02 was paralleled by a reduction in cytotoxicity indicating that toxic α-syn oligomers are present in the BPCA cell culture model and that pharmacological inhibition of oligomer formation can reduce toxicity. Thus, this approach provides a suitable model system for the development of new disease-modifying drugs targeting toxic oligomer species. Moreover, NBB compounds such as 293G02 may provide useful tool compounds to dissect the functional role of toxic oligomer species in cell culture models and in vivo.     

Reduction of synthetic ferrihydrite by a binary anaerobic culture of <Emphasis Type="Italic">Anaerobacillus alkalilacustris</Emphasis> and <Emphasis Type="Italic">Geoalkalibacter ferrihydriticus</Emphasis> grown on mannitol at pH 9.5

In the course of an investigation of alkaliphilic iron reduction, metabiotic interactions in a binary culture reducing synthetic ferrihydrite (SF) have been studied. The binary culture contained two anaerobic bacteria: the alkaliphilic organotrophic bacillus Anaerobacillus alkalilacustris, which ferments sugars and sugar alcohols and is incapable of iron reduction, and the dissimilatory iron-reducing bacterium Geoalkalibacter ferrihydriticus, which is able to grow on acetate at the expense of anaerobic respiration. The experiments were performed under conditions of SF excess and deficiency. It was expected that G. ferrihydriticus would oxidize the acetate formed in the course of mannitol fermentation by A. alkalilacustris. The results were different from the expected ones: in the binary culture, fermentation products other than acetate were used for iron reduction; these were primarily formate and ethanol, which led to acetate accumulation rather than consumption. The reduction of SF to magnetite and/or siderite followed the earlier established regularities. The preferential order of donor utilization by G. ferrihydriticus did not conform to the energy yields of the corresponding reactions. Thus, it has been shown that there may be interactions in microbial communities that cannot be predicted from the characteristics of pure cultures. The degradation pathways of organic matter in communities may differ considerably from those observed in pure cultures, even in pure cultures of highly specialized organisms.     

Degradation of 2-fluorophenol by the brown-rot fungus<Emphasis Type="Italic"> Gloeophyllum striatum</Emphasis>: evidence for the involvement of extracellular Fenton chemistry

Iron-containing liquid cultures of the brown-rot basidiomycete Gloeophyllum striatum degraded 2-fluorophenol. Two simultaneously appearing degradation products, 3-fluorocatechol and catechol, were identified by gas chromatography and mass spectrometry (GC-MS). Concomitantly, fluoride was produced at approximately 50% of the amount that theoretically could be achieved upon complete dehalogenation. Defluorination was strongly inhibited in the presence of either the hydroxyl radical scavenger mannitol or superoxide dismutase, as well as in the absence of iron. The addition of the natural iron chelator oxalate caused a clear but less extensive inhibition, whereas supplementation with the artificial iron chelator nitrilotriacetic acid increased fluoride production. Extracellular 2-fluorophenol degradation was evidenced by defluorination, observed upon addition of 2-fluorophenol to cell-free culture supernatants derived from iron-containing fungal cultures. Ultrafiltered culture supernatants oxidized methanol to formaldehyde, known as a product of the reaction of methanol with hydroxyl radical. In addition, G. striatum was found to produce metabolites extractable with ethyl acetate that are capable of reducing Fe³⁺. GC-MS analysis of such extracts revealed the presence of several compounds. The mass spectrum of a prominent peak matched those previously reported for 2,5-dimethoxyhydroquinone and 4,5-dimethoxycatechol, fungal metabolites implicated to drive hydroxyl radical production in Gloeophyllum. Taken together, these findings further support an extracellular Fenton-type mechanism operative during halophenol degradation by G. striatum.     

Quinolobactin, a new siderophore of Pseudomonas fluorescens ATCC 17400, the production of which is repressed by the cognate pyoverdine

Transposon mutant strain 3G6 of Pseudomonas fluorescens ATCC 17400 which was deficient in pyoverdine production, was found to produce another iron-chelating molecule; this molecule was identified as 8-hydroxy-4-methoxy-quinaldic acid (designated quinolobactin). The pyoverdine-deficient mutant produced a supplementary 75-kDa iron-repressed outer membrane protein (IROMP) in addition to the 85-kDa IROMP present in the wild type. The mutant was also characterized by substantially increased uptake of (59)Fe-quinolobactin. The 75-kDa IROMP was produced by the wild type after induction by quinolobactin-containing culture supernatants obtained from the pyoverdine-negative mutant or by purified quinolobactin. Conversely, adding purified wild-type pyoverdine to the growth medium resulted in suppression of the 75-kDa IROMP in the pyoverdine-deficient mutant; however, suppression was not observed when Pseudomonas aeruginosa PAO1 pyoverdine, a siderophore utilized by strain 3G6, was added to the culture. Therefore, we assume that the quinolobactin receptor is the 75-kDa IROMP and that the quinolobactin-mediated iron uptake system is repressed by the cognate pyoverdine.     

Production of a Pseudomonas lipase in n-alkane substrate and its isolation using an improved ammonium sulfate precipitation technique

Among the various lipidic and non-lipidic substances, normal alkanes within the chain lengths of C-12 to C-20 served as the best carbon substrates for the production of extracellular lipase by Pseudomonas species G6. Maximum lipase production of 25 U/ml of the culture broth was obtained by using n-hexadecane as the sole carbon substrate. The optimum pH of 8 and temperature of 34 + 1 degrees C were demonstrated for the production of lipase in n-hexadecane substrate. The optimum concentration of iron, which played a critical role on the lipase production, was found to be 0.25 mg/l. Lipase production could be enhanced to nearly 2.4-fold by using tributyrin at a concentration of 0.05% (v/v) in the culture medium. High recovery of the lipase protein (83%) from the culture broth was achieved by treating the culture supernatant with Silicone 21 Defoamer followed by ammonium sulfate (60% saturation) fractionation.     

缺铁和过量重碳酸盐胁迫下丛枝菌根真菌对枳活性氧代谢的影响

采用盆栽沙培试验,研究了缺铁处理及重碳酸盐胁迫下丛枝菌根真菌地表球囊霉对枳实生苗抗活性氧系统的影响。试验结果表明,在缺铁以及重碳酸盐胁迫处理下,丛枝菌根真菌显著提高了枳叶片和根系中可溶性蛋白含量、超氧化物岐化酶活性、过氧化物酶活性和过氧化氢酶活性,明显增强了叶片类胡萝卜素含量,显著降低了枳叶片和根系中的丙二醛含量,增强了枳自身防御能力,减少了胁迫对枳细胞膜的伤害。     

Quinolobactin, a New Siderophore of Pseudomonas fluorescens ATCC 17400, the Production of Which Is Repressed by the Cognate Pyoverdine

Transposon mutant strain 3G6 of Pseudomonas fluorescens ATCC 17400 which was deficient in pyoverdine production, was found to produce another iron-chelating molecule; this molecule was identified as 8-hydroxy-4-methoxy-quinaldic acid (designated quinolobactin). The pyoverdine-deficient mutant produced a supplementary 75-kDa iron-repressed outer membrane protein (IROMP) in addition to the 85-kDa IROMP present in the wild type. The mutant was also characterized by substantially increased uptake of ⁵⁹Fe-quinolobactin. The 75-kDa IROMP was produced by the wild type after induction by quinolobactin-containing culture supernatants obtained from the pyoverdine-negative mutant or by purified quinolobactin. Conversely, adding purified wild-type pyoverdine to the growth medium resulted in suppression of the 75-kDa IROMP in the pyoverdine-deficient mutant; however, suppression was not observed when Pseudomonas aeruginosa PAO1 pyoverdine, a siderophore utilized by strain 3G6, was added to the culture. Therefore, we assume that the quinolobactin receptor is the 75-kDa IROMP and that the quinolobactin-mediated iron uptake system is repressed by the cognate pyoverdine.     

Reduction of ferric iron by L-lactate and DL-glycerol-3-phosphate in membrane preparations from Staphylococcus aureus and interactions with the nitrate reductase system.

Membrane fractions with L-lactate dehydrogenase, sn-glycerol-3-phosphate (G3P) dehydrogenase, and nitrate reductase activities were prepared from Staphylococcus aureus wild-type and hem mutant strains. These preparations reduced ferric to ferrous iron with L-lactate or G3P as the source of reductant, using ferrozine to trap the ferrous iron. Reduction of ferric iron was insensitive to 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) with either L-lactate or G3P as reductant, but oxalate and dicumarol inhibited reduction with L-lactate as substrate. The membranes had L-lactate- and G3P-nitrate reductase activities, which were inhibited by azide and by HQNO. Reduction of ferric iron under anaerobic conditions was inhibited by nitrate with preparations from the wild-type strain. This effect of nitrate was abolished by blocking electron transport to the nitrate reductase system with azide or HQNO. Nitrate did not inhibit reduction of ferric iron in heme-depleted membranes from the hem mutant unless hemin was added to restore L-lactate- and G3P-nitrate reductase activity. We conclude that reduced components of the electron transport chain that precede cytochrome b serve as the source of reductant for ferric iron and that these components are oxidized preferentially by a functional nitrate reductase system.     

Globulins enhance in vitro iron but not zinc dialysability: a study on six legume species.

The study was addressed to evaluate the in vitro iron and zinc dialysability from the globulin fraction of six legumes. Five legume species including white bean, mottled bean (Taylor bean), chickpea, lentil, lupin, and a modified mottled bean variety, selected by back-crossing to obtain seeds with globulins composed by G1 fraction only, were used. Globulins (G1 + G2) were extracted from the seeds and analysed for their in vitro iron and zinc dialysability. The highest globulin concentration was detected in lentil (89%). The percentage of globulins in the modified variety of Taylor bean (G1 only) was higher than that of the commercial variety (G1 + G2). The highest concentration of iron was found in Taylor bean globulins. The modified variety of Taylor bean contained 2.6-fold higher iron concentration than the whole seed, and the commercial variety had 1.8-fold higher iron only. The highest zinc concentration was found in lentil globulins. Also iron dialysability from globulins was markedly higher than that of the respective whole seed. The highest value of iron dialysability was found in lentil (10.8%). Zinc dialysability was generally high (above 20%), but no significant differences between whole seed and globulins were detected. The results showed that globulins enhanced iron but not zinc dialysability. Lupin and the modified variety of Taylor bean showed a different behaviour in terms of mineral dialysability compared to the other legumes. The amino acid composition of the digestion products of whole seeds and globulins failed to evidence any direct influence on iron and zinc availability.     

Iron in Neisseria meningitidis: minimum requirements, effects of limitation, and characteristics of uptake.

A simple defined medium (neisseria defined medium) was devised that does not require iron extraction to produce iron-limited growth of Neisseria meningitidis (SDIC). Comparison of this medium to Mueller-Hinton broth and agar showed nearly identical growth rates and yields. The defined medium was used in batch cultures to determine the disappearance of iron from the medium and its uptake by cells. To avoid a number of problems inherent in batch culture, continuous culture, in which iron and dissolved oxygen were varied independently, was used. Most of the cellular iron was found to be nonheme and associated with the particulate fraction in sonically disrupted cells. Nonheme and catalase-heme iron were reduced by iron starvation far more than cytochromes b and c and N,N,N',N'-tetramethylphenylenediamine-oxidase. The respiration rate and efficiency also decreased under iron limitation, whereas generation times increased. The iron-starved meningococcus took up iron by an energy-independent system operating in the first minute after an iron pulse and a slower energy-dependent system inhibited by respiratory poisons and an uncoupler. The energy-dependent system showed saturation kinetics and was stimulated nearly fourfold by iron privation. In addition, to determine the availability to the meningococcus of the iron in selected compounds, a sensitive assay was devised in which an iron-limited continuous culture was pulsed with the iron-containing compound.     

Mineral and iron oxidation at low temperatures by pure and mixed cultures of acidophilic microorganisms

An enrichment culture from a boreal sulfide mine environment containing a low-grade polymetallic ore was tested in column bioreactors for simulation of low temperature heap leaching. PCR-denaturing gradient gel electrophoresis and 16S rRNA gene sequencing revealed the enrichment culture contained an Acidithiobacillus ferrooxidans strain with high 16S rRNA gene similarity to the psychrotolerant strain SS3 and a mesophilic Leptospirillum ferrooxidans strain. As the mixed culture contained a strain that was within a clade with SS3, we used the SS3 pure culture to compare leaching rates with the At. ferrooxidans type strain in stirred tank reactors for mineral sulfide dissolution at various temperatures. The psychrotolerant strain SS3 catalyzed pyrite, pyrite/arsenopyrite, and chalcopyrite concentrate leaching. The rates were lower at 5 degrees C than at 30 degrees C, despite that all the available iron was in the oxidized form in the presence of At. ferrooxidans SS3. This suggests that although efficient At. ferrooxidans SS3 mediated biological oxidation of ferrous iron occurred, chemical oxidation of the sulfide minerals by ferric iron was rate limiting. In the column reactors, the leaching rates were much less affected by low temperatures than in the stirred tank reactors. A factor for the relatively high rates of mineral oxidation at 7 degrees C is that ferric iron remained in the soluble phase whereas, at 21 degrees C the ferric iron precipitated. Temperature gradient analysis of ferrous iron oxidation by this enrichment culture demonstrated two temperature optima for ferrous iron oxidation and that the mixed culture was capable of ferrous iron oxidation at 5 degrees C.     

Identification of glucose 6-phosphate dehydrogenase deficiency in a population with a high frequency of thalassemia

High frequencies of both thalassemia trait (5.2%) and glucose 6-phosphate dehydrogenase (G6PD) deficiency for only males (1.3%) have been observed in the Calabrian population. The G6PD activity measurement was carried out on 1239 samples of whole blood from Calabrian subjects of both sexes (age range 10-55) by a differential pH-metry technique which was quite suitable to determine the G6PD deficiency in mass screenings. The analyzed subjects showed: only the thalassemia trait; or only the G6PD deficiency; or only the total iron serum deficiency; or G6PD deficiency associated with the thalassemia trait or with the total iron serum deficiency. The G6PD heterozygous subjects have an enzymatic activity which is masked by both the thalassemia trait and the total iron serum deficiency. In a population showing high frequencies of both thalassemia trait and G6PD deficiency, the comparison of G6PD activity of heterozygous subjects also affected with the thalassemia trait is more reliable if referred to the enzymatic activity of the carriers of the latter inherited anomaly rather than to G6PD activity of normal subjects.     

Utilization and cell-surface binding of hemin by Histoplasma capsulatum

Histoplasma capsulatum, a dimorphic fungus capable of causing severe respiratory illness in immunocompromised individuals, resides in macrophages during mammalian infection. Previous studies suggest that siderophore-mediated iron transport may be important for the acquisition of iron from transferrin while the organism resides in macrophages. However, iron is also present as hemin in the intracellular environment of the macrophage and may serve as a major source of iron during infection. Thus the ability of H. capsulatum to use hemin and heme-containing compounds was examined. Histoplasma capsulatum G217B was iron-starved by adding the iron chelator deferoxamine mesylate to the culture. The addition of 10 microM hemin in the presence of deferoxamine mesylate restored growth to the levels seen in the absence of the chelator. Histoplasma capsulatum was also cultivated in an iron-limited, chemically defined medium without the addition of chelators and it was determined that the organism could also use hemoglobin as a sole source of iron. The method of iron internalization from heme was examined by measuring hemin binding to the yeast-cell surface. The ability of H. capsulatum to bind hemin was related to the nutritional status of the cells. Cells grown under iron-limited conditions bound more heme to the cell surface than did cells grown in medium without chelator. Pretreatment of iron-starved cells with proteinase K eliminated the ability of the organism to bind hemin. Additionally, the pre-incubation of iron-starved H. capsulatum with hemin eliminated the ability of these cells to remove hemin from the solution, although pre-incubation of cells with the iron-free form of hemin, protoporphyrin IX, only modestly affected the ability of the organism to bind hemin. These results suggest that H. capsulatum uses hemin as a sole source of iron and that one mechanism of iron acquisition involves a cell-surface receptor for hemin.     

Manganese and iron oxidation by fungi isolated from building stone

Acid and nonacid generating fungal strains isolated from weathered sandstone, limestone, and granite of Spanish cathedrals were assayed for their ability to oxidize iron and manganese. In general, the concentration of the different cations present in the mineral salt media directly affected Mn(IV) oxide formation, although in some cases, the addition of glucose and nitrate to the culture media was necessary. Mn(II) oxidation in acidogenic strains was greater in a medium containing the highest concentrations of glucose, nitrate, and manganese. High concentrations of Fe(II), glucose, and mineral salts were optimal for iron oxidation. Mn(IV) precipitated as oxides or hydroxides adhered to the mycelium. Most of the Fe(III) remained in solution by chelation with organic acids excreted by acidogenic strains. Other metabolites acted as Fe(III) chelators in nonacidogenic strains, although Fe(III) deposits around the mycelium were also detected. Both iron and manganese oxidation were shown to involve extracellular, hydrosoluble enzymes, with maximum specific activities during exponential growth. Strains able to oxidize manganese were also able to oxidize iron. It is concluded that iron and manganese oxidation reported in this work were biologically induced by filamentous fungi mainly by direct (enzymatic) mechanisms.Correspondence to: G. Gomez-Alarcon.     

Iron mobilization from cultured rat bone marrow macrophages

The reticuloendothelial system is responsible for removing old and damaged erythrocytes from the circulation, allowing iron to return to bone marrow for hemoglobin synthesis. Cultured bone marrow macrophages were loaded with ⁵⁹Fe-labelled erythroblasts and iron mobilization was studied. After erythroblast digestion, iron taken up by macrophages was found in ferritin as well as in a low-molecular-weight fraction. The analysis of iron mobilization from macrophages shows: (1) the iron was mobilized as ferritin. (2) A higher mobilization was observed when apotransferrin was present in the culture medium. (3) In the presence of apotransferrin in the culture medium, part of the iron was found as transferrin iron. (4) Iron transfer from ferritin to apotransferrin was observed in a cell-free culture medium and this process was temperature independent. The results indicate that after phagocytosis of ⁵⁹Fe-labelled erythroblasts by macrophages, iron is mobilized as ferritin. In the plasma, this iron can be transferred to apotransferrin.     

Theoretical investigations of the hydrolysis pathway of verdoheme to biliverdin

Conversion of iron(II) verdoheme to iron biliverdin in the presence of OH(-) was investigated using B3LYP method. Both 3-21G and 6-31G* basis sets were employed for geometry optimization calculation as well as energy stabilization estimation. Calculation at 6-31G* level was found necessary for a correct spin state estimation of the iron complexes. Two possible pathways for the conversion of iron verdoheme to iron biliverdin were considered. In one path the iron was six-coordinate while in the other it was considered to be five-coordinate. In the six-coordinated pathway, the ground state of bis imidazole iron verdoheme is singlet while that for open chain iron biliverdin it is triplet state with 4.86 kcal/mol more stable than the singlet state. The potential energy surface suggests that a spin inversion take place during the course of reaction after TS. The ring opening process in the six-coordinated pathway is in overall -2.26 kcal/mol exothermic with a kinetic barrier of 9.76 kcal/mol. In the five-coordinated pathway the reactant and product are in the ground triplet state. In this path, hydroxyl ion attacks the iron center to produce a complex, which is only 1.59 kcal/mol more stable than when OH(-) directly attacks the macrocycle. The activation barrier for the conversion of iron hydroxy species to the iron biliverdin complex by a rebound mechanism is estimated to be 32.68 kcal/mol. Large barrier for rebound mechanism, small barrier of 4.18 kcal/mol for ring opening process of the hydroxylated macrocycle, and relatively same stabilities for complexes resulted by the attack of nucleophile to the iron and macrocycle indicate that five-coordinated pathway with direct attack of nucleophile to the 5-oxo position of macrocycle might be the path for the conversion of verdoheme to biliverdin.