Ferroxidase Activity Research Articles

Multi-Copper Oxidases and Human Iron Metabolism

Multi-copper oxidases (MCOs) are a small group of enzymes that oxidize their substrate with the concomitant reduction of dioxygen to two water molecules. Generally, multi-copper oxidases are promiscuous with regards to their reducing substrates and are capable of performing various functions in different species. To date, three multi-copper oxidases have been detected in humans—ceruloplasmin, hephaestin and zyklopen. Each of these enzymes has a high specificity towards iron with the resulting ferroxidase activity being associated with ferroportin, the only known iron exporter protein in humans. Ferroportin exports iron as Fe2+, but transferrin, the major iron transporter protein of blood, can bind only Fe3+ effectively. Iron oxidation in enterocytes is mediated mainly by hephaestin thus allowing dietary iron to enter the bloodstream. Zyklopen is involved in iron efflux from placental trophoblasts during iron transfer from mother to fetus. Release of iron from the liver relies on ferroportin and the ferroxidase activity of ceruloplasmin which is found in blood in a soluble form. Ceruloplasmin, hephaestin and zyklopen show distinctive expression patterns and have unique mechanisms for regulating their expression. These features of human multi-copper ferroxidases can serve as a basis for the precise control of iron efflux in different tissues. In this manuscript, we review the biochemical and biological properties of the three human MCOs and discuss their potential roles in human iron homeostasis.

Investigation of Iron Metabolism in Mice Expressing a Mutant Menke’s Copper Transporting ATPase (Atp7a) Protein with Diminished Activity (Brindled; MoBr/y)

During iron deficiency, perturbations in copper homeostasis have frequently been documented. Previous studies in iron-deprived rats demonstrated that enterocyte and hepatic copper levels increase and a copper transporter (the Menkes Copper ATPase; Atp7a) is induced in the duodenal epithelium in parallel to iron transport-related genes (e.g. Dmt1, Dcytb, Fpn1). Moreover, two ferroxidase proteins involved in iron homeostasis, hephaestin expressed in enterocytes and ceruloplasmin, produced and secreted into blood by the liver, are copper-dependent enzymes. We thus aimed to test the hypothesis that Atp7a function is important for the copper-related compensatory response of the intestinal epithelium to iron deficiency. Accordingly, iron homeostasis was studied for the first time in mice expressing a mutant Atp7a protein with minimal activity (Brindled [MoBr /y]). Mutant mice were rescued by perinatal copper injections, and, after a 7–8 week recovery period, were deprived of dietary iron for 3 weeks (along with WT littermates). Adult MoBr /y mice displayed copper-deficiency anemia but had normal iron status; in contrast, iron-deprived MoBr /y mice were iron deficient and more severely anemic with partial amelioration of the copper-deficient phenotype. Intestinal iron absorption in both genotypes (WT and MoBr /y) increased ∼3-fold when mice consumed a low-iron diet and ∼6-fold when mice were concurrently bled. WT mice exhibited no alterations in copper homeostasis in response to iron deprivation or phlebotomy. Conversely, upregulation of iron absorption was associated with increased enterocyte and liver copper levels and serum ferroxidase (ceruloplasmin) activity in MoBr /y mice, typifying the response to iron deprivation in many mammalian species. We thus speculate that a copper threshold exists that is necessary to allow appropriate regulate of iron absorption. In summary, MoBr /y mice were able to adequately regulate iron absorption, but unlike in WT mice, concurrent increases in enterocyte and liver copper levels and serum ferroxidase activity may have contributed to maintenance of iron homeostasis.

The Relationship between Haptoglobin Polymorphism and Oxidative Stress in Hemodialysis Patients

Summary Background: The relationship between haptoglobin polymorphism and oxidative stress in hemodialysis patients is not fully understood. In this study, total antioxidant capacity and ce ru - loplasmin ferroxidase activity were evaluated in relation to haptoglobin phenotype distribution in hemodialysis patients. Methods: Serum samples collected from 161 patients and 84 healthy controls were haptoglobin-typed by electrophoresis. Ceruloplasmin ferroxidase activity and total antioxidant capacity were assayed using colorimetric methods. Results: Irrespective of the haptoglobin phenotype, patients exhibited significantly lower total antioxidant capacity (1.42± 0.29 vs. 1.55±0.28 mmol/L, P=0.002) and higher ferroxidase activity than controls. Frequency of Hp1-1 and Hp2-1 in patients was 15.5% and 36% as compared with 9.5% and 41.7% in controls. While ferroxidase activity was lower in Hp2-2 patients than in controls (142±61 vs. 179±47 U/L, P=0.002), it was higher in Hp2-1 (173±56 U/L) and Hp1-1 (170±54 U/L) patients than in controls (141±43 and 99±30 U/L respectively) (P=0.002 and 0.009). Ferroxidase activity in Hp2-2 patients was significantly lower than that of Hp2-1 or Hp1-1 patients (P=0.004 and 0.034). Total antioxidant capacity was significantly lower only in Hp2-2 patients (1.44±0.25) compared to that in Hp2-2 controls (1.65±0.22) (P=0.000). Conclusions:These findings suggest that haptoglobin polymorphism can differentially impact oxidative stress levels in hemodialysis patients.

Iron Absorption in Drosophila melanogaster

The way in which Drosophila melanogaster acquires iron from the diet remains poorly understood despite iron absorption being of vital significance for larval growth. To describe the process of organismal iron absorption, consideration needs to be given to cellular iron import, storage, export and how intestinal epithelial cells sense and respond to iron availability. Here we review studies on the Divalent Metal Transporter-1 homolog Malvolio (iron import), the recent discovery that Multicopper Oxidase-1 has ferroxidase activity (iron export) and the role of ferritin in the process of iron acquisition (iron storage). We also describe what is known about iron regulation in insect cells. We then draw upon knowledge from mammalian iron homeostasis to identify candidate genes in flies. Questions arise from the lack of conservation in Drosophila for key mammalian players, such as ferroportin, hepcidin and all the components of the hemochromatosis-related pathway. Drosophila and other insects also lack erythropoiesis. Thus, systemic iron regulation is likely to be conveyed by different signaling pathways and tissue requirements. The significance of regulating intestinal iron uptake is inferred from reports linking Drosophila developmental, immune, heat-shock and behavioral responses to iron sequestration.

A low-molecular-weight ferroxidase is increased in the CSF of sCJD cases: CSF ferroxidase and transferrin as diagnostic biomarkers for sCJD.

Most biomarkers used for the premortem diagnosis of sporadic Creutzfeldt-Jakob disease (CJD) are surrogate in nature, and provide suboptimal sensitivity and specificity. We report that CJD-associated brain iron dyshomeostasis is reflected in the cerebrospinal fluid (CSF), providing disease-specific diagnostic biomarkers. Analysis of 290 premortem CSF samples from confirmed cases of CJD, Alzheimer's disease, and other dementias (DMs), and 52 non-DM (ND) controls revealed a significant difference in ferroxidase (Frx) activity and transferrin (Tf) levels in sporadic Creutzfeldt-Jakob disease (sCJD) relative to other DM and ND controls. A combination of CSF Frx and Tf discriminated sCJD from other DMs with a sensitivity of 86.8%, specificity of 92.5%, accuracy of 88.9%, and area-under-the receiver-operating-characteristic (ROC) curve of 0.94. This combination provided a similar diagnostic accuracy in discriminating CJD from rapidly progressing cases who died within 6 months of sample collection. Surprisingly, ceruloplasmin and amyloid precursor protein, the major brain Frxs, displayed minimal activity in the CSF. Most of the Frx activity was concentrated in the <3-kDa fraction in normal and diseased CSF, and resisted heat and proteinase-K treatment. (i) A combination of CSF Frx and Tf provides disease-specific premortem diagnostic biomarkers for sCJD. (ii) A novel, nonenzymatic, nonprotein Frx predominates in human CSF that is distinct from the currently known CSF Frxs. The underlying cause of iron imbalance is distinct in sCJD relative to other DMs associated with the brain iron imbalance. Thus, change in the CSF levels of iron-management proteins can provide disease-specific biomarkers and insight into the cause of iron imbalance in neurodegenerative conditions.

Levels of Serum Ceruloplasmin Associate With Pediatric Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) in adolescents and children is rapidly becoming one of the most common causes of chronic liver disease worldwide. NAFLD varies from simple fatty liver to nonalcoholic steatohepatitis (NASH) with possible fibrosis. Several studies suggest that oxidative stress plays a central role in several metabolic abnormalities and cellular damage that characterize NAFLD. We investigated whether transition metals and their related proteins were related to NAFLD symptoms and their underlying processes. We measured copper, iron, ceruloplasmin (Cp) concentration and activity, transferrin (Tf), ferroxidase activity, and ferritin, and we calculated Tf saturation and Cp to Tf ratio (Cp/Tf) as an index of the activity of the antioxidant Cp-Tf system in 100 children with biopsy-proven NAFLD. Pediatric patients were grouped by nonalcoholic fatty liver disease score (NAS) ≥ 5 (30 subjects) and NAS < 5 (70). Cp distinguished children with NAS ≥ 5 from those with NAS < 5 with an accuracy of 82%. Specifically, a receiver operator characteristics curve showed that a cutoff of 28.6 mg/dL separated NAS ≥ 5 from NAS < 5 with a specificity of 92% and a sensitivity of 76%. The Cp/Tf ratio, as well as copper concentration and Cp activity, decreased in the NAS ≥ 5 group, pointing out an imbalance in metal regulation. Either copper or Cp concentrations were lower in subjects having ballooning. Serum antioxidant capacity owing to Cp failure is strongly associated with NAFLD-related damage. Further studies are, however, required to clarify the role of Cp in NAFLD pathogenesis and to evaluate its potential application as diagnostic marker.

Iron Metabolism in a Rodent Model of Menkes Disease (Brindled Mice)

The Menkes copper‐transporting ATPase (Atp7a) is induced in the rodent duodenum during iron deficiency. To understand its role in intestinal iron metabolism, we thus initiated an investigation in Atp7a mutant mice (Brindled). As mutant mice die perinatally of severe copper deficiency, IP copper injections were administered at 7 and 9 days‐of‐age. Mice developed normally thereafter and were similar in size and activity level to wt littermates. Subsequently, mice were deprived of dietary iron for 3 wks and iron and copper homeostasis was studied. Several important observations arose from these analyses, including: 1) mutant mice on control diet were copper deficient and anemic with no changes in iron status; 2) mutant mice on iron‐deficient diet were iron and copper deficient and more significantly anemic; 3) the transcriptional response to iron deficiency in enterocytes was seen only in mutant mice on low Fe diet, suggesting that the HIF2α‐mediated induction of iron and copper homeostasis‐related genes is triggered by iron levels and not hypoxia per se; and 4) Brindled mice had increased hepatic copper levels during iron deprivation, leading to a concomitant increase in serum ferroxidase activity, while no such changes were noted in control or low Fe wt mice. This study has thus revealed novel aspects of the iron‐copper connection and is the first to investigate iron homeostasis in a model of genetic copper deficiency.Grant Funding Source: NIH

The Molecular Basis of Iron-induced Oligomerization of Frataxin and the Role of the Ferroxidation Reaction in Oligomerization

The role of the mitochondrial protein frataxin in iron storage and detoxification, iron delivery to iron-sulfur cluster biosynthesis, heme biosynthesis, and aconitase repair has been extensively studied during the last decade. However, still no general consensus exists on the details of the mechanism of frataxin function and oligomerization. Here, using small-angle x-ray scattering and x-ray crystallography, we describe the solution structure of the oligomers formed during the iron-dependent assembly of yeast (Yfh1) and Escherichia coli (CyaY) frataxin. At an iron-to-protein ratio of 2, the initially monomeric Yfh1 is converted to a trimeric form in solution. The trimer in turn serves as the assembly unit for higher order oligomers induced at higher iron-to-protein ratios. The x-ray crystallographic structure obtained from iron-soaked crystals demonstrates that iron binds at the trimer-trimer interaction sites, presumably contributing to oligomer stabilization. For the ferroxidation-deficient D79A/D82A variant of Yfh1, iron-dependent oligomerization may still take place, although >50% of the protein is found in the monomeric state at the highest iron-to-protein ratio used. This demonstrates that the ferroxidation reaction controls frataxin assembly and presumably the iron chaperone function of frataxin and its interactions with target proteins. For E. coli CyaY, the assembly unit of higher order oligomers is a tetramer, which could be an effect of the much shorter N-terminal region of this protein. The results show that understanding of the mechanistic features of frataxin function requires detailed knowledge of the interplay between the ferroxidation reaction, iron-induced oligomerization, and the structure of oligomers formed during assembly.

The structural characterization of bacterioferritin, BfrA, from Mycobacterium tuberculosis

Tuberculosis is a deadly disease caused by Mycobacterium tuberculosis. Like most bacterial pathogens, iron acquisition, regulation, and storage are critical for its survival. Due to the poor solubility of iron under physiological conditions, both eukaryotes and prokaryotes possess ferritins, large protein complexes that store iron and keep it bioavailable. Mtb encodes for two ferritin homologs: a heme-containing bacterioferritin (Mtb-BfrA) and a non-heme eukaryotic-like ferritin (Mtb-BfrB). A conserved feature of bacterioferritins is the presence of a heme group at the interface between two subunits of each dimer that is related by a non-crystallographic two-fold axis. The structure of a selenomethionine derivative of Mtb-BfrA was previously reported (PDB ID: 2WTL); however, a proposed heme degradation product was modeled into the heme-binding site, as electron density for intact heme was not observed. Here, the purification and structure determination of recombinant Mtb-BfrA is reported. As-isolated Mtb-BfrA from Escherichia coli is not fully heme loaded. However, the absorption spectrum features suggest binding of intact heme. In an attempt to fully complement Mtb-BfrA with heme, two different methodologies are described. Electronic spectroscopy and structure determination were used to confirm varying amounts of intact bis-methionine coordinated heme to Mtb-BfrA. We also report that increased heme incorporation only slightly increases Mtb-BfrA ferroxidase activity. Finally, the cognate partner of Mtb-BfrA is proposed to be a putative encoded gene which is located approximately 300 bps upstream of Mycobacterium tuberculosisbfrA, homologous to the cognate partner of Pseudomonas aeruginosa bacterioferritin, a 7 kDa ferrodoxin Bfd.

Ceruloplasmin dysfunction and therapeutic potential for Parkinson disease

Ceruloplasmin is an iron-export ferroxidase that is abundant in plasma and also expressed in glia. We found a ∼80% loss of ceruloplasmin ferroxidase activity in the substantia nigra of idiopathic Parkinson disease (PD) cases, which could contribute to the pro-oxidant iron accumulation that characterizes the pathology. Consistent with a role for ceruloplasmin in PD etiopathogenesis, ceruloplasmin knockout mice developed parkinsonism that was rescued by iron chelation. Additionally, peripheral infusion of ceruloplasmin attenuated neurodegeneration and nigral iron elevation in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model for PD. These findings show, in principle, that intravenous ceruloplasmin may have therapeutic potential in PD.

Identification and characterization of four ferritin subunits involved in immune defense of the Yesso scallop (Patinopecten yessoensis)

As a primary iron storage protein, ferritin plays a vital role in iron homeostasis and innate immunity. In this study, four ferritin subunits (PyFer1, PyFer2, PyFer3, and PyFer4) were cloned from the Yesso scallop, Patinopecten yessoensis, by rapid amplification of cDNA ends (RACE) following in silico transcriptome analysis. The full-length cDNAs of the four ferritins are 895, 920, 891, and 1400bp in length, respectively, and each contains a putative iron response element (IRE) in its 5′ UTR. Meanwhile, multiple A+U-destabilizing elements (TATT or ATTTA) are present in the 3′ UTRs of PyFer2 and PyFer4. The open reading frames of the four ferritins are 522, 516, 516, and 519bp, encoding 173, 171, 171, and 172 amino acids, respectively. These proteins have typical ferritin structures, with four long α-helices, one short α-helix and an L-loop. All of the predicted proteins possess both the ferroxidase center of mammalian H ferritins (E25, Y32, E59, E60, H63, E105, and Q139) and the iron nucleation site of mammalian L ferritins (H116, D129, and E132), and the recombinant proteins possess apparent ferroxidase activity. Quantitative real-time PCR analysis revealed that the expression of the four PyFers was significantly elevated at the D-shaped stage and was relatively high in the adult mantle and hepatopancreas. Furthermore, the four PyFers were significantly up-regulated by iron or bacterial challenge, and all four purified recombinant PyFers were able to inhibit the growth of the scallop pathogen Vibrio anguillarum. These results suggest that these PyFers are likely to play important roles in many fundamental biological processes in P. yessoensis, including immune defense, iron homeostasis, and shell development.

Up-regulation of neutrophil activating protein in Helicobacter pylori under high-salt stress: Structural and phylogenetic comparison with bacterial iron-binding ferritins

It is generally accepted that most gastrointestinal diseases are probably caused by the bacterial pathogen Helicobacter pylori (H. pylori). In this study we have focused on the comparison of protein expression profiles of H. pylori grown under normal and high-salt conditions by a proteomics approach. We have identified about 190 proteins whose expression levels changed after growth at high salt concentration. Among these proteins, neutrophil-activating protein (NapA) was found to be consistently up-regulated under osmotic stress brought by high salts. We have investigated the effect of high salt on secondary and tertiary structures of NapA by circular dichroism spectroscopy followed by analytical ultracentrifugation to monitor the change of quaternary structure of recombinant NapA with increasing salt concentration. The loss of iron-binding activity of NapA coupled with noticeable energetic variation in protein association of NapA as revealed by isothermal titration calorimetry was found under high salt condition. The phylogenetic tree analysis based on sequence comparison of 16 protein sequences encompassing NapA proteins and ferritin of H. pylori and other prokaryotic organisms pointed to the fact that all H. pylori NapA proteins of human origin are more homologous to NapA of Helicobacter genus than to other bacterial NapA. Based on computer modeling, NapA proteins from H. pylori of human isolates are found more similar to ferritin from H. pylori than to NapA from other species of bacteria. Taken together, these results suggested that divergent evolution of NapA and ferritin possessing dissimilar and diverse sequences follows a path distinct from that of convergent evolution of NapA and ferritin with similar dual functionality of iron-binding and ferroxidase activities.

Induction of ferroxidase enzymatic activity by copper reduces MPP+-evoked neurotoxicity in rats

Parkinson's disease (PD) is a neurodegenerative disorder characterized by decreased dopamine, intracellular inclusions (Lewy bodies) and brain iron deposits. PD has also been related with reduced ferroxidase activity, diminished antioxidant defenses and lipid peroxidation. Striatal injection of 1-methyl-4-phenylpyridinium (MPP+) into rodents reproduces the major biochemical characteristics of PD, including oxidative stress. Copper (Cu) plays an important role as prosthetic group of several proteins involved in iron metabolism and antioxidant responses, such as ceruloplasmin (Cp). In the present study, intraperitoneal CuSO4 injection (10μmol/kg) produced an insignificant increase of Cu content in striatum and midbrain (17.5% and 7%, respectively). After 10 and 11h, Cu induced 6- and 4-fold increase Cp mRNA in midbrain and striatum, respectively. Cu-supplement also produced a time-dependent increase ferroxidase activity in striatal tissue, reaching a maximum 16h after Cu treatment in midbrain; while, ferrous iron content diminished 18% in striatum and 8% in midbrain. In regard the PD model, we found that MPP+ (10μg/8μL, intrastriatal), induced a significant (P<0.05) reduction of striatal ferroxidase activity; this effect was reverted by Cu pre-treatment 16h before MPP+. Likewise, Cu-supplement prevented lipid fluorescent products formation in striatum, evaluated (P<0.01) 6h after MPP+. In the long term, apomorphine-evoked circling behavior was evaluated 6 days after MPP+ injury; Cu pre-treatment significantly reduced (P<0.05) the apomorphine-induced ipsilateral turns in MPP+-lesioned rats. These results suggest that Cu-induced expression of Cp could be an interesting scope against the deleterious effects of iron deposits in PD.

Haptoglobin polymorphism in relation to antioxidative enzymes activity in type 2 diabetes mellitus

BackgroundIncreased oxidative stress and impaired antioxidative capacity are common findings in diabetics. This study reports on the status of antioxidative enzymes in relation to haptoglobin (Hp) polymorphism in type 2 diabetes. MethodsThe study comprised 165 type 2 diabetic patients and 94 controls. Erythrocytic superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT), and plasmatic ceruloplasmin ferroxidase (Cp) were measured by spectrophotometry and Hp phenotypes were determined by gel electrophoresis. ResultsIrrespective of Hp phenotype, while the activities of Cp ferroxidase and GPx were significantly higher in patients than in controls, those of SOD were significantly lower. No significant differences observed for CAT. However, significant Hp-phenotype dependent differences were observed between patients and controls regarding the activity of these enzymes. While ferroxidase activity in Hp2-2 patients was significantly higher than that in Hp1-1 or Hp2-1 patients, that of SOD and GPx were significantly lower. When patients were analyzed as a single group, Spearman's univariate analysis has demonstrated that HbA1c positively correlates with ferroxidase activity and negatively correlates with levels of GPx and SOD. However, when patients were treated as separate Hp-dependent groups, similar but stronger correlations between these variable were noted only in the case of Hp2-2 patients. ConclusionsThese findings suggest that Hp polymorphism has some bearing on the activity of antioxidative enzymes in type 2 diabetes and that Hp2-2 diabetics are under increased oxidative stress as compared with those expressing Hp1-1 or Hp2-1.

H-Ferritin Ferroxidase Activity Induces Cytoprotective Pathways and Inhibits Microvascular Stasis in Transgenic Sickle Mice

AbstractAbstract 378Hemolysis, oxidative stress, inflammation, vaso-occlusion and organ infarction are hallmarks of sickle cell disease (SCD). We hypothesize that intravascular heme, liberated from hemoglobin S derived from hemolyzed sickle red blood cells, is fundamental to inflammation and vaso-occlusion in SCD. Heme can be degraded by heme-oxygenase-1 (HO-1), releasing Fe2+, carbon monoxide (CO), and biliverdin/bilirubin. We have previously shown that increases in HO-1 activity inhibit vascular inflammation and vaso-occlusion in transgenic mouse models of SCD. HO-1 products, CO and biliverdin/bilirubin, also are protective in SCD, but Fe2+ released from the heme ring requires further processing. The released Fe2+ from heme is oxidized by ferritin heavy chain (FHC) ferroxidase activity and safely stored as catalytically-inactive Fe3+ inside ferritin clusters. FHC overexpression has been shown to be cytoprotective in response to inflammation and oxidative stress in vivo and in vitro. In this study, we hypothesize that overexpression of FHC with its ferroxidase activity will inhibit inflammation and microvascular stasis in transgenic sickle mice in response to stroma-free hemoglobin. We utilized a Sleeping Beauty transposase plasmid to deliver a human wt-ferritin heavy chain (wt-FHC) transposable element by hydrodynamic tail vein injections to NY1DD SCD mice. Control mice were infused with the same volume of lactated Ringer’s solution LRS) or a triple missense (ms-) human FHC plasmid encoding no ferroxidase enzyme activity. Eight weeks after injection, the mice were implanted with dorsal skin-fold chambers to access microvascular blood flow. LRS-treated mice had 40% microvascular stasis (% non-flowing venules) when infused with stroma-free hemoglobin, while wt-FHC overexpressing mice had only 5% stasis (p<0.05), suggesting vascular protection by wt-FHC. Ferroxidase activity was critical in this protection as mice overexpressing ms-FHC were not protected from microvascular stasis (33%). The wt-FHC SCD mice had marked increases in FHC mRNA and protein, light chain ferritin, 5-aminolevulinic acid synthase (5-ALA-synthase), cellular heme content, ferroportin, nuclear factor erythroid 2-related factor 2 (Nrf2), nuclear FHC and microsomal HO-1 activity and protein, and a decrease in activated phosho- and total nuclear factor-kappa B (NF-κB) p65 in the nuclei of the livers. We conclude that wt-FHC ferroxidase activity enhances 5-ALA-synthase activity and cytoprotective Nrf2-regulated proteins including HO-1 resulting in decreased NF-κB-activation, inflammation and microvascular stasis in transgenic SCD mice. Therapies directed at increasing ferritin heavy chain production and decreasing reactive iron availability may be beneficial in limiting sickle cell crises and end organ damage. Disclosures:Vercellotti:Sangart Inc: Consultancy, Research Funding. Chen:Sangart Inc: Research Funding. Hebbel:Sangart Inc: Consultancy, Research Funding. Belcher:Sangart Inc: Research Funding.

Metabolic Adaptation to Tissue Iron Overload Confers Tolerance to Malaria

Disease tolerance is a defense strategy that limits the fitness costs of infection irrespectively of pathogen burden. While restricting iron (Fe) availability to pathogens is perceived as a host defense strategy, the resulting tissue Fe overload can be cytotoxic and promote tissue damage to exacerbate disease severity. Examining this interplay during malaria, the disease caused by Plasmodium infection, we find that expression of the Fe sequestering protein ferritin H chain (FtH) in mice, and ferritin in humans, is associated with reduced tissue damage irrespectively of pathogen burden. FtH protection relies on its ferroxidase activity, which prevents labile Fe from sustaining proapoptotic c-Jun N-terminal kinase (JNK) activation. FtH expression is inhibited by JNK activation, promoting tissue Fe overload, tissue damage, and malaria severity. Mimicking FtH's antioxidant effect or inhibiting JNK activation pharmacologically confers therapeutic tolerance to malaria in mice. Thus, FtH provides metabolic adaptation to tissue Fe overload, conferring tolerance to malaria.

The Role of Oxidative Stress in Anti-tumor Necrosis Factor Antibody Treatment in Crohn´s Disease

Administration of anti-tumor necrosis factor alpha antibodies [anti-TNF]-alpha represents a therapeutic approach aimed to diminish the effects of tumor necrosis factor [TNF]- alpha in Crohn's disease [CD]. Blockade of its action should be related to various changes including those in immune and inflammatory response. There is a growing body of experimental data to suggest that the chronically inflamed intestine may be subjected to considerable oxidative stress. The aim of this study was to study the impact of therapy with anti-TNF [infliximab] on Crohn's disease activity index [CDAI], markers of inflammatory activity and oxidative stress. Fourteen patients with active CD received 5mg/kg of infliximab in a single intravenous infusion. CDAI, erythrocyte sedimentation rate [ESR], C-reactive protein [CRP], fibrinogen [FBG], alpha-1 antitrypsin [AAT], albumin [ALB], ceruloplasmin apoprotein [CP], ferroxidase activity of CP [FOACP], specific enzymatic activity of CP [SEACP] and conjugated dienes [DIE] were determined before treatment in month 0 [M 0], in 1st control period in month 1 [M 1] and in 2nd control period in month 5 [M 5] after treatment. In clinical activity a sustained significant decrease in CDAI was observed, with a significant drop in M 1, remaining in M 5. A significant decrease in ESR in M 1, accompanied by insignificantly reduced levels of CRP and FBG was present. During the further follow up in M 5 a slight increase of ESR, CRP and FBG was noticed. A significant decrease of AAT in M 1 was present; this decrease was followed by a significant increase in M 5. Ceruloplasmin apoprotein levels dropped in M 1, with further slight insignificant increase in M 5. A significant increase of ALB sustaining in M 5 was present. The levels of DIE slightly decreased in M 1 and significantly in M 5, together with the slight increase of the FOACP and SEACP in M 1 and significant increase in M 5. We conclude, that oxidative stress may be important in the pathogenesis and perpetuation of tissue injury in CD patients. The decreasing levels of DIE together with the increase of the FOCP and SEACP after infliximab treatment together with changes of markers of inflammatory activity, can participate in the improvement of clinical status of patients with CD.

Synthesis and interfacing of biocompatible iron oxide nanoparticles through the ferroxidase activity of Helicobacter Pylori ferritin

Ferritin is an iron storage protein that is often used to coat metallic nanoparticles, such as iron oxide nanoparticles (IONPs). However, the synthesis and biocompatibility of ferritin-coated IONPs remain unclear. Therefore, this study reports the synthesis of a ferritin gene cloned and expressed from Helicobacter pylori (HPFn). The ferroxidase activity of the synthase HPFn was used for the de novo synthesis of HPFn-coated IONPs under mild conditions. Gel filtration chromatography and transmission electron microscopy analyses demonstrated that the core–shell structure of both the 5.0 nm IONP nanocore and the 12.4 nm HPFn shell were correctly assembled. The cellular uptake of mouse macrophage cells (RAW 264.7 cells) has shown that only a few HPFn-coated IONPs (3%) were taken up after 24 h of incubation. This study compares the biocompatibility of HPFn-coated IONPs, superparamagnetic iron oxide nanoparticles (SPIOs) and ferric salt (ferric ammonium citrate) in respect to cell growth inhibition, reactive oxygen species generation and pro-inflammatory cytokine TNF-α release. Assessment results showed that the responses elicited by HPFn-coated IONPs were similar to those elicited by SPIO treatment but milder than those elicited by ferric salt treatment. This accounts for the notion that ferritin-coated IONPs are biocompatible iron agents. These findings show that the ferroxidase activity of ferritin can be used to synthesize biocompatible IONPs. The favorable properties of HPFn-coated IONPs suggest that they can be used as a non-macrophage contrast agent through further surface conjugation.

ANTIOXIDANT PROTECTION SYSTEM DURING CHRONIC INFLAMATION OF THE CONNECTIVE TISSUE IN COWS

As clinical test material we studied the blood serum taken from clinically healthy animals (the control group) and animals with chronic inflammation of the connective tissue in an ankle joint (the experimental group). To study the antioxidant protection system enzyme component condition in cows we examined the following: catalase and glutathione peroxidase activity (as a substrate we used hydrogen dioxide and glutathione reductase), ferroxidase activity of ceruloplasmin, superoxide dismutase activity. A spectrocolorimeter «Specol 210» was used to determine the solutions absorbancy. The results shows the breakdown of membrane-bounded enzymes activity (such as glutathione reductase and superoxide dismutase). These enzymes characterize the antioxidant protection system condition. It is essential to examine the antioxidant protection system condition in animals with chronic inflammation of the connective tissue in an ankle joint to enrich our knowledge about the mechanism of metabolic disorder at this type of pathology, as well as to treat and correct possible complications.

Salivary Ceruloplasmin Ferroxidase &amp; Oxidase Activities in Celiac Patients

The aim of the current study was to evaluate salivary ferroxidase ceruloplasmin activities in celiac patients with different histopathological severity. This study included 75 celiac patients with different mean age (18.68 ± 11.13) year, who had positive screen for celiac antibodies, and who had gastrointestinal symptoms. In order to simplify the comparison with the healthy control group, celiac patients were divided into two groups according to their histopathological severity: severe (marsh IIIa, b, c) &amp; less severe (marsh 0, I). All these patients have been evaluating for salivary ceruloplasmin (Cp) concentration and Cp ferroxidase activities. To confirm the presence of the enzymatic activity of this protein, polyacrylamide gel electrophoresis was carried out and then stained for Cp ferroxidase, as well as for Cp oxidase activity. Furthermore, the concentrations of salivary total protein, albumin, and globulin were measured in the studied groups. A significant increase (p&lt;0.05) in salivary concentration of ceruloplasmin was found in all above mentioned patients groups in comparison to that of the control group, except for total villous atrophy (marsh IIIc) patients subgroup. Salivary Cp ferroxidase activity revealed statistically significant decrease among the patient groups as well as between them and the control group. The result of salivary total protein and globulin showed presence a significant increase (p&lt;0.05) in comparison to that of the control group. Meanwhile albumin levels was found to increase non-significantly (p=0.186).