Iron Uptake Proteins Research Articles

Transferrin receptor 1 upregulation in primary tumor and downregulation in benign kidney is associated with progression and mortality in renal cell carcinoma patients.

The central dysregulated pathway of clear cell (cc) renal cell carcinoma (RCC), the von Hippel Lindau/hypoxia inducible factor-α axis, is a key regulator of intracellular iron levels, however the role of iron uptake in human RCC tumorigenesis and progression remains unknown. We conducted a thorough, large-scale investigation of the expression and prognostic significance of the primary iron uptake protein, transferrin receptor 1 (TfR1/CD71/TFRC), in RCC patients. TfR1 immunohistochemistry was performed in over 1500 cores from 574 renal cell tumor patient tissues (primary tumors, matched benign kidneys, metastases) and non-neoplastic tissues from 36 different body sites. TfR1 levels in RCC tumors, particularly ccRCC, were significantly associated with adverse clinical prognostic features (anemia, lower body mass index, smoking), worse tumor pathology (size, stage, grade, multifocality, sarcomatoid dedifferentiation) and worse survival outcomes, including after adjustments for tumor pathology. Highest TfR1 tissue levels in the non-gravid body were detected in benign renal tubule epithelium. Opposite to TfR1 changes in the primary tumor, TfR1 levels in benign kidney dropped during tumor progression and were inversely associated with worse survival outcomes, independent of tumor pathology. Quantitative measurement of TfR1 subcellular localization in cell lines demonstrated mixed cytoplasmic and membranous expression with increased TfR1 in clusters in ccRCC versus benign renal cell lines. Results of this study support an important role for TfR1 in RCC progression and identify TfR1 as a novel RCC biomarker and therapeutic target.

Intraspinal TLR4 activation promotes iron storage but does not protect neurons or oligodendrocytes from progressive iron-mediated damage

Iron is essential for basic cellular functions but in excess is highly toxic. For this reason, free iron and iron storage are controlled in the periphery by elaborate regulatory mechanisms. In contrast, iron regulation in the central nervous system (CNS) is not well defined. Given that excess iron is present after trauma, hemorrhagic stroke and neurodegeneration, understanding normal iron regulation and promoting iron uptake in CNS pathology is crucial. Peripherally, toll-like receptor 4 (TLR4) activation promotes iron sequestration by macrophages. Notably, iron-rich sites of CNS pathology typically contain TLR4 agonists, which may promote iron uptake. Indeed, our recent work showed impaired iron storage after acute spinal cord injury in mice with TLR4 deficiency. Here we used a reductionist model to ask if TLR4 activation in the CNS stimulates iron uptake and promotes neuroprotection from iron-induced toxicity. For this, we measured the ability of microglia/macrophages to sequester exogenous iron and prevent pathology with and without concomitant intraspinal TLR4 activation. Results show that, similar to the periphery, activating intraspinal TLR4 via focal LPS injection increased mRNA encoding iron uptake and storage proteins and promoted iron sequestration into ferritin-expressing macrophages. However, this did not prevent oligodendrocyte and neuron loss. Moreover, replacement of oligodendrocytes by progenitor cells – a normally robust response to in vivo macrophage TLR4 activation – was significantly reduced if iron was present concomitant with TLR4 activation. Thus, while TLR4 signaling promotes CNS iron uptake, future work needs to determine ways to enhance iron removal without blocking the reparative effects of innate immune receptor signaling.

Iron Enhances Hepatic Fibrogenesis and Activates Transforming Growth Factor-β Signaling in Murine Hepatic Stellate Cells

BackgroundAlthough excess iron induces oxidative stress in the liver, it is unclear whether it directly activates the hepatic stellate cells (HSC). Materials and MethodsWe evaluated the effects of excess iron on fibrogenesis and transforming growth factor beta (TGF-β) signaling in murine HSC. Cells were treated with holotransferrin (0.005-5g/L) for 24 hours, with or without the iron chelator deferoxamine (10µM). Gene expressions (α-SMA, Col1-α1, Serpine-1, TGF-β, Hif1-α, Tfrc and Slc40a1) were analyzed by quantitative real time-polymerase chain reaction, whereas TfR1, ferroportin, ferritin, vimentin, collagen, TGF-β RII and phospho-Smad2 proteins were evaluated by immunofluorescence, Western blot and enzyme-linked immunosorbent assay. ResultsHSC expressed the iron-uptake protein transferrin receptor 1 (TfR1) and the iron-export protein ferroportin. Holotransferrin upregulated TfR1 expression by 1.8-fold (P < 0.03) and ferritin accumulation (iron storage) by 2-fold (P < 0.01), and activated HSC with 2-fold elevations (P < 0.03) in α-SMA messenger RNA and collagen secretion, and a 1.6-fold increase (P < 0.01) in vimentin protein. Moreover, holotransferrin activated the TGF-β pathway with TGF-β messenger RNA elevated 1.6-fold (P = 0.05), and protein levels of TGF-β RII and phospho-Smad2 increased by 1.8-fold (P < 0.01) and 1.6-fold (P < 0.01), respectively. In contrast, iron chelation decreased ferritin levels by 30% (P < 0.03), inhibited collagen secretion by 60% (P < 0.01), repressed fibrogenic genes α-SMA (0.2-fold; P < 0.05) and TGF-β (0.4-fold; P < 0.01) and reduced levels of TGF-β RII and phospho-Smad2 proteins. ConclusionsHSC express iron-transport proteins. Holotransferrin (iron) activates HSC fibrogenesis and the TGF-β pathway, whereas iron depletion by chelation reverses this, suggesting that this could be a useful adjunct therapy for patients with fibrosis. Further studies in primary human HSC and animal models are necessary to confirm this.

Iron metabolism and drug resistance in cancer.

Iron is an essential inorganic element for various cellular events. It is directly associated with cell proliferation and growth; therefore, it is expected that iron metabolism is altered in tumor cells which usually have rapid growth rates. The studies on iron metabolism of tumor cells have shown that tumor cells necessitated higher concentrations of iron and the genes of iron uptake proteins were highly over-expressed. However, there are limited number of studies on overall iron metabolism in drug-resistant tumor cells. In this article, we evaluated the studies reporting the relationship between drug resistance and iron metabolism and the utilization of this knowledge for the reversal of drug resistance. Also, the studies on iron-related cell death mechanism, ferroptosis, and its relation to drug resistance were reviewed. We focus on the importance of iron metabolism in drug-resistant cancer cells and how alterations in iron metabolism participate in drug-resistant phenotype.

Phylogenetic Analysis of Antibiotic Resistance Genes and Virulence Genes of Klebsiella species in silico

A total of twelve isolates were screened for virulence and antibiotic resistance genes associated with Klebsiella pneumoniae infections. Virulence and antibiotic resistance genes were detected by in silico PCR amplification. Iron uptake protein entB was detected in 66.67% (n=8) of the isolates while no isolate was found to harbour chelating agent irp2. Iron uptake system kfu, involved in purulent tissue infections and capsule formation, was identified in 25% (n=3) of the isolates. Regulator of mucoid phenotype A, rmpA was not found in any of the isolates. The wabG gene, responsible for urinary tract infections was found in seven K. pneumoniae strains. Five uge positive strains might play role in the pathogenicity of K. pneumoniae infections. About 83.33% of the isolates were positive for type 1 fimbriae fimH1 while no type 3 fimbriae mrkD gene was found. Complement reaction blocked by plasmid traT gene was not observed in Klebsiella species while eight isolates harboured outer membrane lipoprotein, ycfM which protects Klebsiella species from antibiotics. Antibiotic resistance genes blaTEM and blaSHV were detected in 33.33% (n=4) and 66.67% (n=8) of the isolates while 25% isolates carried both blaTEM and blaSHV genes. Genotype 1 carried fimH1 and ycfM genes while all the virulence genes studied were present in genotype 2 and 3. The blaSHV gene was detected in all the genotypes while blaTEM gene was found in only genotype 1 and 3. The findings of this study would be helpful to predict virulence gene associated with Klebsiella infections. This data also helps us to choose antibiotics for treating Klebsiella infections. By assessing the genotypic distribution of antibiotic resistance gene, correct antibiotic can be used to treat the infection. This could help reduce emergence of antibiotic resistance since it is known that incorrect choice of antibiotics contributes to antibiotic resistance.Dhaka Univ. J. Pharm. Sci. 16(1): 119-127, 2017 (June)

MRNA Levels of Placental Iron and Zinc Transporter Genes Are Upregulated in Gambian Women with Low Iron and Zinc Status

Background: The role of the placenta in regulating micronutrient transport in response to maternal status is poorly understood.Objective: We investigated the effect of prenatal nutritional supplementation on the regulation of placental iron and zinc transport.Methods: In a randomized trial in rural Gambia [ENID (Early Nutrition and Immune Development)], pregnant women were allocated to 1 of 4 nutritional intervention arms: 1) iron and folic acid (FeFol) tablets (FeFol group); 2) multiple micronutrient (MMN) tablets (MMN group); 3) protein energy (PE) as a lipid-based nutrient supplement (LNS; PE group); and 4) PE and MMN (PE+MMN group) as LNS. All arms included iron (60 mg/d) and folic acid (400 μg/d). The MMN and PE+MMN arms included 30 mg supplemental Zn/d. In a subgroup of ∼300 mother-infant pairs, we measured maternal iron status, mRNA levels of genes encoding for placental iron and zinc transport proteins, and cord blood iron levels.Results: Maternal plasma iron concentration in late pregnancy was 45% and 78% lower in the PE and PE+MMN groups compared to the FeFol and MMN groups, respectively (P < 0.001). The mRNA levels of the placental iron uptake protein transferrin receptor 1 were 30–49% higher in the PE and PE+MMN arms than in the FeFol arm (P < 0.031), and also higher in the PE+MMN arm (29%; P = 0.042) than in the MMN arm. Ferritin in infant cord blood was 18–22% lower in the LNS groups (P < 0.024). Zinc supplementation in the MMN arm was associated with higher maternal plasma zinc concentrations (10% increase; P < 0.001) than in other intervention arms. mRNA levels for intracellular zinc-uptake proteins, in this case zrt, irt-like protein (ZIP) 4 and ZIP8, were 96–205% lower in the PE+MMN arm than in the intervention arms without added zinc (P < 0.025). Furthermore, mRNA expression of ZIP1 was 85% lower in the PE+MMN group than in the PE group (P = 0.003).Conclusion: In conditions of low maternal iron and in the absence of supplemental zinc, the placenta upregulates the gene expression of iron and zinc uptake proteins, presumably in order to meet fetal demands in the face of low maternal supply. The ENID trial was registered at www.controlled-trials.com as ISRCTN49285450.

(Z)-ligustilide increases ferroportin1 expression and ferritin content in ischemic SH-SY5Y cells

The mechanisms involved in the antioxidant and anti-apoptotic properties of (Z)-ligustilide (LIG) are not fully elucidated. Based on the accumulated data, we hypothesized that LIG might be able to reduce ischemia/reperfusion-induced increase in brain iron by regulating expression of iron transport proteins. We therefore investigated the effects of LIG on iron uptake protein transferrin receptor 1, iron exporter protein ferroportin 1, iron storage protein ferritin light chain and also hypoxia inducible factor-1 alpha (HIF-1 alpha) in oxygen-glucose deprivation/reoxygenation (OGD/R)-treated SH-SY5Y cells, using Western blot analysis. We demonstrated that LIG completely reversed the OGD/R-induced reduction of ferroportin 1, increased ferritin light chain content, and also suppressed the OGD-induced increase in HIF-1 alpha in SH-SY5Y cells. These findings imply that LIG might reduce the OGD/R-induced increase in brain iron by promoting cell iron release and iron corporation into ferritin, and also by inhibiting the HIF-1 alpha-induced increase in transferrin-bound iron uptake and iron accumulation in the brain, consequently attenuating iron-mediated free radical formation, oxidative stress and apoptosis.

Acetylcholinesterase-independent protective effects of huperzine A against iron overload-induced oxidative damage and aberrant iron metabolism signaling in rat cortical neurons.

Iron dyshomeostasis is one of the primary causes of neuronal death in Alzheimer's disease (AD). Huperzine A (HupA), a natural inhibitor of acetylcholinesterase (AChE), is a licensed anti-AD drug in China and a nutraceutical in the United Sates. Here, we investigated the protective effects of HupA against iron overload-induced injury in neurons. Rat cortical neurons were treated with ferric ammonium citrate (FAC), and cell viability was assessed with MTT assays. Reactive oxygen species (ROS) assays and adenosine triphosphate (ATP) assays were performed to assess mitochondrial function. The labile iron pool (LIP) level, cytosolic-aconitase (c-aconitase) activity and iron uptake protein expression were measured to determine iron metabolism changes. The modified Ellman's method was used to evaluate AChE activity. HupA significantly attenuated the iron overload-induced decrease in neuronal cell viability. This neuroprotective effect of HupA occurred concurrently with a decrease in ROS and an increase in ATP. Moreover, HupA treatment significantly blocked the upregulation of the LIP level and other aberrant iron metabolism changes induced by iron overload. Additionally, another specific AChE inhibitor, donepezil (Don), at a concentration that caused AChE inhibition equivalent to that of HupA negatively, influenced the aberrant changes in ROS, ATP or LIP that were induced by excessive iron. We provide the first demonstration of the protective effects of HupA against iron overload-induced neuronal damage. This beneficial role of HupA may be attributed to its attenuation of oxidative stress and mitochondrial dysfunction and elevation of LIP, and these effects are not associated with its AChE-inhibiting effect.

The Bradyrhizobium japonicum Ferrous Iron Transporter FeoAB Is Required for Ferric Iron Utilization in Free Living Aerobic Cells and for Symbiosis

The bacterium Bradyrhizobium japonicum USDA110 does not synthesize siderophores for iron utilization in aerobic environments, and the mechanism of iron uptake within symbiotic soybean root nodules is unknown. An mbfA bfr double mutant defective in iron export and storage activities cannot grow aerobically in very high iron medium. Here, we found that this phenotype was suppressed by loss of function mutations in the feoAB operon encoding ferrous (Fe(2+)) iron uptake proteins. Expression of the feoAB operon genes was elevated under iron limitation, but mutants defective in either gene were unable to grow aerobically over a wide external ferric (Fe(3+)) iron (FeCl3) concentration range. Thus, FeoAB accommodates iron acquisition under iron limited and iron replete conditions. Incorporation of radiolabel from either (55)Fe(2+) or (59)Fe(3+) into cells was severely defective in the feoA and feoB strains, suggesting Fe(3+) reduction to Fe(2+) prior to traversal across the cytoplasmic membrane by FeoAB. The feoA or feoB deletion strains elicited small, ineffective nodules on soybean roots, containing few bacteria and lacking nitrogen fixation activity. A feoA(E40K) mutant contained partial iron uptake activity in culture that supported normal growth and established an effective symbiosis. The feoA(E40K) strain had partial iron uptake activity in situ within nodules and in isolated cells, indicating that FeoAB is the iron transporter in symbiosis. We conclude that FeoAB supports iron acquisition under limited conditions of soil and in the iron-rich environment of a symbiotic nodule.

Novel vaccine antigen combinations elicit protective immune responses against Escherichia coli sepsis

Systemic infections caused by extraintestinal pathogenic Escherichia coli (ExPEC) have emerged as the most common community-onset bacterial infections and are major causes of nosocomial infections worldwide. The management of ExPEC infections has been complicated by the heterogeneity of ExPEC strains and the emergence of antibiotic resistance, thus their prevention through vaccination would be beneficial. The protective efficacy of four common ExPEC antigen candidates composed of common pilus antigens EcpA and EcpD and iron uptake proteins IutA and IroN, were tested by both active and passive immunization in lethal and non-lethal murine models of sepsis. Additionally, antibody raised to a synthetic form of a conserved surface polysaccharide, β-(1-6)-linked poly-N-acetylglucosamine (dPNAG) containing 9 monomers of (non-acetylated) glucosamine (9GlcNH2) conjugated to tetanus toxoid TT (9GlcNH2-TT) was tested in passive immunization protocols. Active immunization of mice with recombinant antigens EcpA, EcpD, IutA, or IroN elicited high levels of total IgG antibody of IgG1/IgG2a isotypes, and were determined to be highly protective against E. coli infection in lethal and non-lethal sepsis challenges. Moreover, passive immunization against these four antigens resulted in significant reductions of bacteria in internal organs and blood of the mice, especially when the challenge strain was grown in iron-restricted media. Inclusion of antibodies to PNAG increased the efficacy of the passive immunization under conditions where the challenge bacteria were grown in LB medium but not in iron-restricted media. The information and data presented are the first step toward the development of a broadly protective vaccine against sepsis-causing E. coli strains.

Effects of aspirin on expression of iron transport and storage proteins in BV-2 microglial cells

In the light of recent studies, we hypothesized that aspirin might have the functions to regulate the expression of iron transport proteins and then affect cellular iron levels. To test this hypothesis, we investigated the effects of aspirin on expression of iron uptake protein transferrin receptor 1 (TfR1), iron release protein ferroportin 1 (Fpn1) and iron storage protein ferritin using Western blot analysis and on tumor necrosis factor (TNF)-αlpha, interleukin (IL)-6, interleukin (IL)-10 and hepcidin using quantitative real-time PCR in BV-2 microglial cells treated with lipopolysaccharides (LPS). We found that aspirin significantly down-regulated TfR1, while also up-regulated Fpn1 and ferritin expressions in BV-2 microglial cells in vitro. We also showed that TfR1 and Fpn1 expressions were significantly higher, while ferritin contents, IL-6, TNF-alpha and hepcidin mRNA levels were lower in cells treated with aspirin plus LPS than those in cells treated with LPS only. We concluded that aspirin has a negative effect on cell iron contents under ‘normal’ conditions and could partly reverse LPS-induced-disruption in cell iron balance under in vitro inflammatory conditions. Our findings also suggested that hepcidin might play a dominant role in the control of TfR1 expression by aspirin in the cells treated with LPS.

Analysis of Salmonella PhoP/PhoQ regulation by dimethyl-SRM-based quantitative proteomics

SRM (selected reaction monitoring), a tandem mass spectrometry-based method characterized by high repeatability and accuracy, is an effective tool for the quantification of predetermined proteins. In this study, we built a time-scheduled dimethyl-SRM method that can provide the precise relative quantification of 92 proteins in one run. By applying this method to the Salmonella PhoP/PhoQ two-component system, we found that the expression of selected PhoP/PhoQ-activated proteins in response to Mg2+ concentrations could be divided into two distinct patterns. For the time-course SRM experiment, we found that the dynamics of the selected PhoP/PhoQ-activated proteins could be divided into three distinct patterns, providing a new clue regarding PhoP/PhoQ activation and regulation. Moreover, the results for iron homeostasis proteins in response to Mg2+ concentrations revealed that the PhoP/PhoQ two-component system may serve as a repressor for iron uptake proteins. And ribosomal protein levels clearly showed a response to different Mg2+ concentrations and to time.

Iron Regulatory Protein 1 Suppresses Hypoxia-Induced Iron Uptake Proteins Expression and Decreases Iron Levels in HepG2 Cells.

Transferrin receptor (TfR1) and divalent metal transporter 1 (DMT1) are important proteins for cellular iron uptake, and both are regulated transcriptionally through the binding of hypoxia-inducible factor 1 (HIF-1) to hypoxia-responsive elements (HREs) under hypoxic conditions. These proteins are also regulated post-transcriptionally through the binding of iron regulatory protein 1 (IRP1) to iron-responsive elements (IREs) located in the mRNA untranslated region (UTR) to control cellular iron homeostasis. In iron-deficient cells, IRP1-IRE interactions stabilize TfR1 and DMT1 mRNAs, enhancing iron uptake. However, little is known about the impact of IRP1 on the regulation of cellular iron homeostasis under hypoxia. Thus, to investigate the role of IRP1 in hypoxic condition, overexpression and knockdown assays were performed using HepG2 cells. The overexpression of IRP1 suppressed the hypoxia-induced increase in TfR1 and DMT1 (+IRE) expression and reduced the stability of TfR1 and DMT1 (+IRE) mRNAs under hypoxia, whereas IRP1 knockdown further increased the hypoxia-induced expression of both proteins, preventing the decrease in IRE-dependent luciferase activity induced by hypoxia. Under hypoxic conditions, ferrous iron uptake, the labile iron pool (LIP), and total intracellular iron reduced when IRP1 was overexpressed and further increased when IRP1 was knocked down. IRP1 expression declined and TfR1/DMT1 (+IRE) expression increased with the time of hypoxia prolonged, whereas the binding of IRP1 to the IRE of TfR1/DMT1 mRNA maintained. In summary, IRP1 suppressed TfR1/DMT1 (+IRE) expression, limited the cellular iron content and decreased lactate dehydrogenase (LDH) release induced by hypoxia.

Mechanisms and regulation of iron trafficking across the capillary endothelial cells of the blood-brain barrier.

The transcellular trafficking of iron from the blood into the brain interstitium depends on iron uptake proteins in the apical membrane of brain microvascular capillary endothelial cells and efflux proteins at the basolateral, abluminal membrane. In this review, we discuss the three mechanisms by which these cells take-up iron from the blood and the sole mechanism by which they efflux this iron into the abluminal space. We then focus on the regulation of this efflux pathway by exocrine factors that are released from neighboring astrocytes. Also discussed are the cytokines secreted by capillary cells that regulate the expression of these glial cell signals. Among the interstitial factors that regulate iron efflux into the brain is the Amyloid precursor protein (APP). The role of this amyliodogenic species in brain iron metabolism is discussed. Last, we speculate on the potential relationship between iron transport at the blood-brain barrier and neurological disorders associated with iron mismanagement.

Role of extracellular Hydrogen peroxide in regulation of iron homeostasis genes in neuronal cells: Implication in iron accumulation

Iron accumulation and oxidative stress are associated with neurodegenerative disease. Labile iron is known to catalyze free radical generation and subsequent neuronal damage, whereas the role of oxidative stress in neuronal iron accumulation is less well understood. Here, we examined the effect of hydrogen peroxide (H2O2) treatment on cellular iron-uptake, -storage, and -release proteins in the neuroblastoma cell line SH-SY5Y. We found no detectable change in the iron-uptake proteins transferrin receptor-1 and divalent metal ion transporter. In contrast, H2O2 treatment resulted in significant degradation of the iron-exporter ferroportin (Fpn). A decrease in Fpn is expected to increase the labile iron pool (LIP), reducing the iron-regulatory protein (IRP)–iron-responsive element interaction and increasing the expression of ferritin-H (Ft-H) for iron storage. Instead, we detected IRP1 activation, presumably due to oxidative stress, and a decrease in Ft-H translation. A reduction in Ft-H mRNA was also observed, probably dependent on an antioxidant-response element present in the Ft-H enhancer. The decrease in Fpn and Ft-H upon H2O2 treatment led to a time-dependent increase in the cellular LIP. Our study reveals a complex regulation of neuronal iron-release and iron-storage components in response to H2O2 that may explain iron accumulation detected in neurodegenerative diseases associated with oxidative stress.

High fat diet subverts hepatocellular iron uptake determining dysmetabolic iron overload.

Increased serum ferritin associated with mild hepatic iron accumulation, despite preserved upregulation of the iron hormone hepcidin, is frequently observed in patients with dysmetabolic overload syndrome (DIOS). Genetic factors and Western diet represent predisposing conditions, but the mechanisms favoring iron accumulation in DIOS are still unclear. Aims of this study were to assess the effect a high-fat diet (HFD) on hepatic iron metabolism in an experimental model in rats, to further characterize the effect of free fatty acids on iron metabolism in HepG2 hepatocytes in vitro, and to assess the translational relevance in patients with fatty liver with and without iron accumulation. Despite decreased uptake of dietary iron, rats fed HFD accumulated more hepatic iron than those fed regular diet, which was associated with steatosis development. Hepatic iron accumulation was paralleled by induction of ferritin, in the presence of preserved upregulation of hepcidin, recapitulating the features of DIOS. HFD was associated with increased expression of the major iron uptake protein Transferrin receptor-1 (TfR-1), consistently with upregulation of the intracellular iron sensor Iron regulated protein-1 (IRP1). Supplementation with fatty acids induced TfR-1 and IRP1 in HepG2 hepatocytes, favoring intracellular iron accumulation following exposure to iron salts. IRP1 silencing completely abrogated TfR-1 induction and the facilitation of intracellular iron accumulation induced by fatty acids. Hepatic TfR-1 mRNA levels were upregulated in patients with fatty liver and DIOS, whereas they were not associated with liver fat nor with inflammation. In conclusion, increased exposure to fatty acids subverts hepatic iron metabolism, favoring the induction of an iron uptake program despite hepatocellular iron accumulation.

Effect of Glutamate on Brain Iron Metabolism and the Regulation Mechanism

Glutamate is an excitatory transmitter and can induce neurotoxicity, it can also increase the iron concentrations in the brain, but little is known about the detailed molecular regulation mechanism of iron metabolism by Glu. Based on our previous data, iron metabolism related proteins might be associated with the increase of brain iron contents induced by neurotransmitter. To investigate the issues, the iron contents, non-transferrin-bound iron (NTBI) uptake and the expression of iron uptake and iron release proteins were firstly examined in vivo and in vitro with iron histochemistry, inductively coupled plasma mass spectroscopy (ICP-MS), 55Fe radioactive liquid scintillation counting and western blot methods. Data showed that glutamate induced the increase of total iron contents, storage iron contents and NTBI uptake activity. Moreover, only divalent metal transporter 1, one of iron uptake proteins, was increased in rat brain and PC12 cells treated with glutamate. Further investigations revealed that nuclear factor ÐoB (NF-ÐoB) and protein kinase C (PKC) were involved in the regulation of DMT1 in PC12 cells treated with glutamate. These findings demonstrate that glutamate increases iron contents in the brain through increased NTBI, and that DMT1 is the key molecule underlying regulation of iron metabolism by glutamate, Furthermore, NF-ÐoB and PKC play important roles in the regulatory pathway of DMT1 expression by glutamate. Thus, it implicates that inhibiting the expression of DMT1 and disruption of its regulation pathway might be effective strategies in attenuating glutamate neurotoxicity through decreased iron contents.

Iron Liposome: A more Effective Iron Supplement for Sports Anemia and Anemia of Inflammation

Anemia is such a serious global health problem that it has aroused the focus of the world. Anemia is characterized by decreased number of red blood cells (RBC), lower haemoglobin concentrations or even altered RBC morphology. Due to the critical role of iron in the production of haemoglobin and RBC, iron-deficiency is often accompanied with anemia. In general, it is recommended to supplement iron formulations when iron is deficiency in anemia patients. However, sports anemia and anemia of inflammation (AI) cannot be treated with traditional iron supplements because the iron cannot be absorbed adequately resulting from the decreased expression of iron uptake and exported proteins in duodenum. Therefore, new types of iron supplements should be designed to prevent or treat iron deficiency in sports anemia and AI. Iron liposome is a new type of iron supplement that can be absorbed without the limitation of iron uptake and export proteins. Iron liposome has the superiority of liposome that can cross the duodenum through membrane fusion, membrane diffusion or phagocytosis. This review mainly covers the advantage and usage of iron liposomes in the prevention and treatment of sports anemia and AI. This will shed light on the application of iron liposomes in clinic in future.

Comparative analysis of the uropathogenic Escherichia coli surface proteome by tandem mass-spectrometry of artificially induced outer membrane vesicles

Uropathogenic Escherichia coli (UPEC) are the major cause of urinary tract infections. For successful colonisation of the urinary tract, UPEC employ multiple surface-exposed or secreted virulence factors, including adhesins and iron uptake systems. Whilst individual UPEC strains and their virulence factors have been the focus of extensive research, there have been no outer membrane (OM) proteomic studies based on large clinical UPEC collections, primarily due to limitations of traditional methods. In this study, a high-throughput method based on tandem mass-spectrometry of EDTA heat-induced outer membrane vesicles (OMVs) was developed for the characterisation of the UPEC surface-associated proteome. The method was applied to compare the OM proteome of fifty-four UPEC isolates, resulting in the identification of 8789 proteins, consisting of 619 unique proteins, which were subsequently interrogated for their subcellular origin, prevalence and homology to characterised virulence factors. Multiple distinct virulence-associated proteins were identified, including two novel putative iron uptake proteins, an uncharacterised type of chaperone-usher fimbriae and various highly prevalent hypothetical proteins. Our results give fundamental insight into the physiology of UPEC and provide a framework for understanding the composition of the UPEC OM proteome. In this study a high-throughput method based on tandem mass-spectrometry of EDTA heat-induced outer membrane vesicles was used to define the outer membrane proteome of a large uropathogenic E. coli (UPEC) collection. Our results provide an inventory of proteins expressed on the surface of UPEC, and provide a framework for understanding the composition of the UPEC OM proteome. The method enables the rapid characterisation of the E. coli surface proteome and could easily be applied to the large-scale outer membrane protein profiling of other Gram-negative bacteria.

Hypoxia induced changes in expression of proteins involved in iron uptake and storage in cultured lens epithelial cells

Hypoxia inducible factor (HIF) regulates expression of over 60 genes by binding to hypoxia response elements (HRE) located upstream of the transcriptional start sites. Many genes encoding proteins involved in iron transport and homeostasis are regulated by HIF. Expression of iron handling proteins can also be translationally regulated by binding of iron regulatory protein (IRP) to iron responsive elements (IREs) on the mRNA of ferritin chains and transferrin receptor (TfR). Lens epithelial cells (LEC) function in a low oxygen environment. This increases the risk of iron catalyzed formation of reactive oxygen species (ROS) and oxidative cell damage. We examined changes in expression of ferritin (iron storage protein) and Tf/TfR1 (iron uptake proteins) in LEC cultured under hypoxic conditions. Ferritin consists of 24 subunits of two types, heavy (H-chain) and light (L-chain) assembled in a cell specific ratio. Real-time PCR showed that 24 h exposure to hypoxia lowered transcription of both ferritin chains by over 50% when compared with normoxic LEC. However it increased the level of ferritin chain proteins (20% average). We previously found that 6 h exposure of LEC to hypoxia increased the concentration of cytosolic iron which would stimulate translation of ferritin chains. This elevated ferritin concentration increased the iron storage capacity of LEC. Hypoxic LEC labeled with 59FeTf incorporated 70% more iron into ferritin after 6 h as compared to normoxic LEC. Exposure of LEC to hypoxia for 24 h reduced the concentration of TfR1 in cell lysates. As a result, hypoxic LEC internalized less Tf at this later time point. Incorporation of 59Fe into ferritin of hypoxic LEC after 24 h did not differ from that of normoxic LEC due to lower 59FeTf uptake. This study showed that hypoxia acutely increased iron storage capacity and lowered iron uptake due to changes in expression of iron handling proteins. These changes may better protect LEC against oxidative stress by limiting iron-catalyzed ROS formation in the low oxygen environment in which the lens resides.