Control of heme synthesis during Friend cell differentiation: role of iron and transferrin

International Nuclear Information System (INIS)

Laskey, J.D.; Ponka, P.; Schulman, H.M.

1986-01-01

In many types of cells the synthesis of σ-aminolevulinic acid (ALA) limits the rate of heme formation. However, results from this laboratory with reticulocytes suggest that the rate of iron uptake from 125 I-transferrin (Tf), rather than ALA synthase activity, limits the rate of heme synthesis in erythroid cells. To determine whether changes occur in iron metabolism and the control of heme synthesis during erythroid cell development Friend erythroleukemia cells induced to erythroid differentiation by dimethylsulfoxide (DMSO) were studied. While added ALA stimulated heme synthesis in uninduced Friend cells (suggesting ALA synthase is limiting) it did not do so in induced cells. Therefore the possibility was investigated that, in induced cells, iron uptake from Tf limits and controls heme synthesis. Several aspects of iron metabolism were investigated using the synthetic iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH). Both induced and uninduced Friend cells take up and utilize Fe for heme synthesis directly from Fe-SIH without the involvement of transferrin and transferrin receptors and to a much greater extent than from saturating levels or 59 Fe-Tf (20 μM). Furthermore, in induced Friend cells 100 μM Fe-SIH stimulated 2- 14 C-glycine incorporation into heme up to 3.6-fold as compared to the incorporation observed with saturating concentrations of Fe-Tf. These results indicate that some step(s) in the pathway of iron from extracellular Tf to protoporphyrin, rather than the activity of ALA synthase, limits and controls the overall rate of heme and possibly hemoglobin synthesis in differentiating Friend erythroleukemia cells

Studying disorders of vertebrate iron and heme metabolism using zebrafish.

Science.gov (United States)

van der Vorm, Lisa N; Paw, Barry H

2017-01-01

Iron is a crucial component of heme- and iron-sulfur clusters, involved in vital cellular functions such as oxygen transport, DNA synthesis, and respiration. Both excess and insufficient levels of iron and heme-precursors cause human disease, such as iron-deficiency anemia, hemochromatosis, and porphyrias. Hence, their levels must be tightly regulated, requiring a complex network of transporters and feedback mechanisms. The use of zebrafish to study these pathways and the underlying genetics offers many advantages, among others their optical transparency, ex-vivo development and high genetic and physiological conservations. This chapter first reviews well-established methods, such as large-scale mutagenesis screens that have led to the initial identification of a series of iron and heme transporters and the generation of a variety of mutant lines. Other widely used techniques are based on injection of RNA, including complementary morpholino knockdown and gene overexpression. In addition, we highlight several recently developed approaches, most notably endonuclease-based gene knockouts such as TALENs or the CRISPR/Cas9 system that have been used to study how loss of function can induce human disease phenocopies in zebrafish. Rescue by chemical complementation with iron-based compounds or small molecules can subsequently be used to confirm causality of the genetic defect for the observed phenotype. All together, zebrafish have proven to be - and will continue to serve as an ideal model to advance our understanding of the pathogenesis of human iron and heme-related diseases and to develop novel therapies to treat these conditions. Copyright © 2017 Elsevier Inc. All rights reserved.

A relay network of extracellular heme-binding proteins drives C. albicans iron acquisition from hemoglobin.

Science.gov (United States)

Kuznets, Galit; Vigonsky, Elena; Weissman, Ziva; Lalli, Daniela; Gildor, Tsvia; Kauffman, Sarah J; Turano, Paola; Becker, Jeffrey; Lewinson, Oded; Kornitzer, Daniel

2014-10-01

Iron scavenging constitutes a crucial challenge for survival of pathogenic microorganisms in the iron-poor host environment. Candida albicans, like many microbial pathogens, is able to utilize iron from hemoglobin, the largest iron pool in the host's body. Rbt5 is an extracellular glycosylphosphatidylinositol (GPI)-anchored heme-binding protein of the CFEM family that facilitates heme-iron uptake by an unknown mechanism. Here, we characterize an additional C. albicans CFEM protein gene, PGA7, deletion of which elicits a more severe heme-iron utilization phenotype than deletion of RBT5. The virulence of the pga7-/- mutant is reduced in a mouse model of systemic infection, consistent with a requirement for heme-iron utilization for C. albicans pathogenicity. The Pga7 and Rbt5 proteins exhibit distinct cell wall attachment, and discrete localization within the cell envelope, with Rbt5 being more exposed than Pga7. Both proteins are shown here to efficiently extract heme from hemoglobin. Surprisingly, while Pga7 has a higher affinity for heme in vitro, we find that heme transfer can occur bi-directionally between Pga7 and Rbt5, supporting a model in which they cooperate in a heme-acquisition relay. Together, our data delineate the roles of Pga7 and Rbt5 in a cell surface protein network that transfers heme from extracellular hemoglobin to the endocytic pathway, and provide a paradigm for how receptors embedded in the cell wall matrix can mediate nutrient uptake across the fungal cell envelope.

PCBP1 and NCOA4 regulate erythroid iron storage and heme biosynthesis.

Science.gov (United States)

Ryu, Moon-Suhn; Zhang, Deliang; Protchenko, Olga; Shakoury-Elizeh, Minoo; Philpott, Caroline C

2017-05-01

Developing erythrocytes take up exceptionally large amounts of iron, which must be transferred to mitochondria for incorporation into heme. This massive iron flux must be precisely controlled to permit the coordinated synthesis of heme and hemoglobin while avoiding the toxic effects of chemically reactive iron. In cultured animal cells, iron chaperones poly rC-binding protein 1 (PCBP1) and PCBP2 deliver iron to ferritin, the sole cytosolic iron storage protein, and nuclear receptor coactivator 4 (NCOA4) mediates the autophagic turnover of ferritin. The roles of PCBP, ferritin, and NCOA4 in erythroid development remain unclear. Here, we show that PCBP1, NCOA4, and ferritin are critical for murine red cell development. Using a cultured cell model of erythroid differentiation, depletion of PCBP1 or NCOA4 impaired iron trafficking through ferritin, which resulted in reduced heme synthesis, reduced hemoglobin formation, and perturbation of erythroid regulatory systems. Mice lacking Pcbp1 exhibited microcytic anemia and activation of compensatory erythropoiesis via the regulators erythropoietin and erythroferrone. Ex vivo differentiation of erythroid precursors from Pcbp1-deficient mice confirmed defects in ferritin iron flux and heme synthesis. These studies demonstrate the importance of ferritin for the vectorial transfer of imported iron to mitochondria in developing red cells and of PCBP1 and NCOA4 in mediating iron flux through ferritin.

Acyl transfer from membrane lipids to peptides is a generic process.

Science.gov (United States)

Dods, Robert H; Bechinger, Burkhard; Mosely, Jackie A; Sanderson, John M

2013-11-15

The generality of acyl transfer from phospholipids to membrane-active peptides has been probed using liquid chromatography-mass spectrometry analysis of peptide-lipid mixtures. The peptides examined include melittin, magainin II, PGLa, LAK1, LAK3 and penetratin. Peptides were added to liposomes with membrane lipid compositions ranging from pure phosphatidylcholine (PC) to mixtures of PC with phosphatidylethanolamine, phosphatidylserine or phosphatidylglycerol. Experiments were typically conducted at pH7.4 at modest salt concentrations (90 mM NaCl). In favorable cases, lipidated peptides were further characterized by tandem mass spectrometry methods to determine the sites of acylation. Melittin and magainin II were the most reactive peptides, with significant acyl transfer detected under all conditions and membrane compositions. Both peptides were lipidated at the N-terminus by transfer from PC, phosphatidylethanolamine, phosphatidylserine or phosphatidylglycerol, as well as at internal sites: lysine for melittin; serine and lysine for magainin II. Acyl transfer could be detected within 3h of melittin addition to negatively charged membranes. The other peptides were less reactive, but for each peptide, acylation was found to occur in at least one of the conditions examined. The data demonstrate that acyl transfer is a generic process for peptides bound to membranes composed of diacylglycerophospholipids. Phospholipid membranes cannot therefore be considered as chemically inert toward peptides and by extension proteins. © 2013. Published by Elsevier Ltd. All rights reserved.

Control of intracellular heme levels: Heme transporters and Heme oxygenases

Science.gov (United States)

Khan, Anwar A.; Quigley, John G.

2011-01-01

Heme serves as a co-factor in proteins involved in fundamental biological processes including oxidative metabolism, oxygen storage and transport, signal transduction and drug metabolism. In addition, heme is important for systemic iron homeostasis in mammals. Heme has important regulatory roles in cell biology, yet excessive levels of intracellular heme are toxic; thus, mechanisms have evolved to control the acquisition, synthesis, catabolism and expulsion of cellular heme. Recently, a number of transporters of heme and heme synthesis intermediates have been described. Here we review aspects of heme metabolism and discuss our current understanding of heme transporters, with emphasis on the function of the cell-surface heme exporter, FLVCR. Knockdown of Flvcr in mice leads to both defective erythropoiesis and disturbed systemic iron homeostasis, underscoring the critical role of heme transporters in mammalian physiology. PMID:21238504

Heme oxygenase activity correlates with serum indices of iron homeostasis in healthy nonsmokers

Science.gov (United States)

Heme oxygenase (HO) catalyzes the breakdown of heme to carbon monoxide, iron, and biliverdin. While the use of genetically altered animal models in investigation has established distinct associations between HO activity and systemic iron availability, studies have not yet confirm...

Mononuclear non-heme iron(III) complexes of linear and tripodal ...

Indian Academy of Sciences (India)

The rate of oxygenation depends on the solvent and the. Lewis acidity of iron(III) ... has been achieved by non-heme iron enzymes and their ..... oxygen atoms of nitrate ion (figure 3). ... enhanced covalency of iron-catecholate interaction and.

Effects of dietary heme iron and exercise training on abdominal fat accumulation and lipid metabolism in high-fat diet-fed mice.

Science.gov (United States)

Katsumura, Masanori; Takagi, Shoko; Oya, Hana; Tamura, Shohei; Saneyasu, Takaoki; Honda, Kazuhisa; Kamisoyama, Hiroshi

2017-08-01

Animal by-products can be recycled and used as sources of essential nutrients. Water-soluble heme iron (WSHI), a functional food additive for supplementing iron, is produced by processing animal blood. In this study, we investigated the effects of dietary supplementation of 3% WSHI and exercise training for 4 weeks on the accumulation of abdominal fat and lipid metabolism in mice fed high-fat diet. Exercise-trained mice had significantly less perirenal adipose tissue, whereas WSHI-fed mice tended to have less epididymal adipose tissue. In addition, total weight of abdominal adipose tissues was significantly decreased in the Exercise + WSHI group. Dietary WSHI significantly increased the messenger RNA (mRNA) levels of lipoprotein lipase and hormone-sensitive lipase. WSHI-fed mice also tended to show increased mRNA levels of adipose triglyceride lipase in their epididymal adipose tissue. Dietary WSHI also significantly decreased the mRNA levels of fatty acid oxidation-related enzymes in the liver, but did not influence levels in the Gastrocnemius muscle. Exercise training did not influence the mRNA levels of lipid metabolism-related enzymes in the epididymal adipose tissue, liver or the Gastrocnemius muscle. These findings suggest that the accumulation of abdominal fat can be efficiently decreased by the combination of dietary WSHI and exercise training in mice fed high-fat diet. © 2016 Japanese Society of Animal Science.

Irradiation of bovine meat: effect of heme-iron concentration

International Nuclear Information System (INIS)

Mistura, Liliana Perazzini Furtado

2002-01-01

The irradiation is often used, nowadays, for meat conservation and it is important to know how much this process interferes with the nutritional quality of the meat. In this study round cut meat, ground and steaks (from a local supermarket) was irradiated with doses of O; 1; 2; 3; 4; 5; 7,5 and 10 kGy (JS-7500 Nordium Inc -Canada) and the interference of irradiation and the process of food preparation on heme-iron (H Fe) content was determined. Half of the sample was kept raw and the other half was grilled in a pre-warmed oven at 250 deg C for 9 min and a controlled humidity of 70%. The chemical composition, the total iron (T Fe) (EM) and the heme iron concentration were determined (Hornsey,1956) and the sensorial quality evaluated. The average T Fe concentration of raw and ground , ground and grilled, raw steaks and grilled steak meat, on dry and degreased basis was 113 mug/g, 121 mug/g , 91 mug/g and 77 mug/g; and the H Fe concentration 105 mug/g (93% of T Fe) , 88 mug/g (73% of T Fe), 90 mug/g (99% of T Fe) and 52 mug/g (68% of T Fe) respectively. Data were evaluated by ANOVA with fixed effects and multiple comparisons. The irradiation neither altered the chemical composition nor the proportion of heme iron of meat. The preparation conditions (temperature, cooking time, environment humidity, meat presentation) of the sample interfered more with the heme iron content than the irradiation. With the sensorial analysis we verified that meats irradiated with doses of 3 kGy were better evaluated in softness and succulency attributes than the others. Meat submitted to irradiation doses up to 3 kGy were accepted by the specialists' panel. (author)

Alteration by irradiation and storage at amount of heme iron in poultry meat; Alteracoes provocadas pela irradiacao e armazenamento nos teores de ferro heme em carne de frango

Energy Technology Data Exchange (ETDEWEB)

Souza, Adriana Regia Marques de; Arthur, Valter Arthur [Centro de Energia Nuclear na Agricultura (CENA), Piracicaba, SP (Brazil). Lab. de Irradiacao de Alimentos e Radioentomologia; Canniatti-Brazaca, Solange Guidolin [Escola Superior de Agricultura Luiz de Queiroz (ESALQ/USP), Piracicaba, SP (Brazil). Dept. de Agroindustria, Alimentos e Nutricao]. E-mail: sgcbraza@esalq.usp.br

2007-04-15

Studies of irradiation and storage effects in chicken were carried out to discover the influence in iron heme, non-heme amount, color and total pigments. Chicken thighs and chicken breast were studied. These were irradiated to 0, 1 and 2 kGy stored by 14 days to 4 deg C in refrigerator. Determining the heme content and non-heme of meat was done using the colorimeter method and the Ferrozine reagent. The values of iron heme were influenced both by the irradiation and the storage, reducing the amount throughout the course of time. The iron non-heme was also influenced by the doses and the storage time, however the values increased throughout the course of time, because of the conversion of iron heme in non-heme. The color did not show that it was influenced by the studied doses, except for the storage, and the total number of pigments was affected by the irradiation and the time, reducing the values with the increase of storage. Irradiation was shown to be a good method to conserve iron. (author)

X-ray absorption spectroscopic studies of mononuclear non-heme iron enzymes

Energy Technology Data Exchange (ETDEWEB)

Westre, Tami E. [Stanford Univ., CA (United States)

1996-01-01

Fe-K-edge X-ray absorption spectroscopy (XAS) has been used to investigate the electronic and geometric structure of the iron active site in non-heme iron enzymes. A new theoretical extended X-ray absorption fine structure (EXAFS) analysis approach, called GNXAS, has been tested on data for iron model complexes to evaluate the utility and reliability of this new technique, especially with respect to the effects of multiple-scattering. In addition, a detailed analysis of the 1s→3d pre-edge feature has been developed as a tool for investigating the oxidation state, spin state, and geometry of iron sites. Edge and EXAFS analyses have then been applied to the study of non-heme iron enzyme active sites.

Characterization of Lipid A Variants by Energy-Resolved Mass Spectrometry: Impact of Acyl Chains

Science.gov (United States)

Crittenden, Christopher M.; Akin, Lucas D.; Morrison, Lindsay J.; Trent, M. Stephen; Brodbelt, Jennifer S.

2017-06-01

Lipid A molecules consist of a diglucosamine sugar core with a number of appended acyl chains that vary in their length and connectivity. Because of the challenging nature of characterizing these molecules and differentiating between isomeric species, an energy-resolved MS/MS strategy was undertaken to track the fragmentation trends and map genealogies of product ions originating from consecutive cleavages of acyl chains. Generalizations were developed based on the number and locations of the primary and secondary acyl chains as well as variations in preferential cleavages arising from the location of the phosphate groups. Secondary acyl chain cleavage occurs most readily for lipid A species at the 3' position, followed by primary acyl chain fragmentation at both the 3' and 3 positions. In the instances of bisphosphorylated lipid A variants, phosphate loss occurs readily in conjunction with the most favorable primary and secondary acyl chain cleavages. [Figure not available: see fulltext.

Heme iron content in lamb meat is differentially altered upon boiling, grilling, or frying as assessed by four distinct analytical methods.

Science.gov (United States)

Pourkhalili, Azin; Mirlohi, Maryam; Rahimi, Ebrahim

2013-01-01

Lamb meat is regarded as an important source of highly bioavailable iron (heme iron) in the Iranians diet. The main objective of this study is to evaluate the effect of traditional cooking methods on the iron changes in lamb meat. Four published experimental methods for the determination of heme iron were assessed analytically and statistically. Samples were selected from lambs' loin. Standard methods (AOAC) were used for proximate analysis. For measuring heme iron, the results of four experimental methods were compared regarding their compliance to Ferrozine method which was used for the determination of nonheme iron. Among three cooking methods, the lowest total iron and heme iron were found in boiling method. The heme iron proportions to the total iron in raw, boiled lamb meat and grilled, were counted as 65.70%, 67.75%, and 76.01%, receptively. Measuring the heme iron, the comparison of the methods in use showed that the method in which heme extraction solution was composed of 90% acetone, 18% water, and 2% hydrochloric acid was more appropriate and more correlated with the heme iron content calculated by the difference between total iron and nonheme iron.

Heme Iron Content in Lamb Meat Is Differentially Altered upon Boiling, Grilling, or Frying as Assessed by Four Distinct Analytical Methods

Directory of Open Access Journals (Sweden)

Azin Pourkhalili

2013-01-01

Full Text Available Lamb meat is regarded as an important source of highly bioavailable iron (heme iron in the Iranians diet. The main objective of this study is to evaluate the effect of traditional cooking methods on the iron changes in lamb meat. Four published experimental methods for the determination of heme iron were assessed analytically and statistically. Samples were selected from lambs' loin. Standard methods (AOAC were used for proximate analysis. For measuring heme iron, the results of four experimental methods were compared regarding their compliance to Ferrozine method which was used for the determination of nonheme iron. Among three cooking methods, the lowest total iron and heme iron were found in boiling method. The heme iron proportions to the total iron in raw, boiled lamb meat and grilled, were counted as 65.70%, 67.75%, and 76.01%, receptively. Measuring the heme iron, the comparison of the methods in use showed that the method in which heme extraction solution was composed of 90% acetone, 18% water, and 2% hydrochloric acid was more appropriate and more correlated with the heme iron content calculated by the difference between total iron and nonheme iron.

Silencing of Iron and Heme-Related Genes Revealed a Paramount Role of Iron in the Physiology of the Hematophagous Vector Rhodnius prolixus

Directory of Open Access Journals (Sweden)

Ana B. Walter-Nuno

2018-02-01

Full Text Available Iron is an essential element for most organisms However, free iron and heme, its complex with protoporphyrin IX, can be extremely cytotoxic, due to the production of reactive oxygen species, eventually leading to oxidative stress. Thus, eukaryotic cells control iron availability by regulating its transport, storage and excretion as well as the biosynthesis and degradation of heme. In the genome of Rhodnius prolixus, the vector of Chagas disease, we identified 36 genes related to iron and heme metabolism We performed a comprehensive analysis of these genes, including identification of homologous genes described in other insect genomes. We observed that blood-meal modulates the expression of ferritin, Iron Responsive protein (IRP, Heme Oxygenase (HO and the heme exporter Feline Leukemia Virus C Receptor (FLVCR, components of major pathways involved in the regulation of iron and heme metabolism, particularly in the posterior midgut (PM, where an intense release of free heme occurs during the course of digestion. Knockdown of these genes impacted the survival of nymphs and adults, as well as molting, oogenesis and embryogenesis at different rates and time-courses. The silencing of FLVCR caused the highest levels of mortality in nymphs and adults and reduced nymph molting. The oogenesis was mildly affected by the diminished expression of all of the genes whereas embryogenesis was dramatically impaired by the knockdown of ferritin expression. Furthermore, an intense production of ROS in the midgut of blood-fed insects occurs when the expression of ferritin, but not HO, was inhibited. In this manner, the degradation of dietary heme inside the enterocytes may represent an oxidative challenge that is counteracted by ferritins, conferring to this protein a major antioxidant role. Taken together these results demonstrate that the regulation of iron and heme metabolism is of paramount importance for R. prolixus physiology and imbalances in the levels of

Heme in pathophysiology: a matter of scavenging, metabolism and trafficking across cell membranes

OpenAIRE

Chiabrando, Deborah; Vinchi, Francesca; Fiorito, Veronica; Mercurio, Sonia; Tolosano, Emanuela

2014-01-01

Heme (iron-protoporphyrin IX) is an essential co-factor involved in multiple biological processes: oxygen transport and storage, electron transfer, drug and steroid metabolism, signal transduction, and micro RNA processing. However, excess free-heme is highly toxic due to its ability to promote oxidative stress and lipid peroxidation, thus leading to membrane injury and, ultimately, apoptosis. Thus, heme metabolism needs to be finely regulated. Intracellular heme amount is controlled at multi...

Covalent heme attachment to the protein in human heme oxygenase-1 with selenocysteine replacing the His25 proximal iron ligand.

Science.gov (United States)

Jiang, Yongying; Trnka, Michael J; Medzihradszky, Katalin F; Ouellet, Hugues; Wang, Yongqiang; Ortiz de Montellano, Paul R

2009-03-01

To characterize heme oxygenase with a selenocysteine (SeCys) as the proximal iron ligand, we have expressed truncated human heme oxygenase-1 (hHO-1) His25Cys, in which Cys-25 is the only cysteine, in the Escherichia coli cysteine auxotroph strain BL21(DE3)cys. Selenocysteine incorporation into the protein was demonstrated by both intact protein mass measurement and mass spectrometric identification of the selenocysteine-containing tryptic peptide. One selenocysteine was incorporated into approximately 95% of the expressed protein. Formation of an adduct with Ellman's reagent (DTNB) indicated that the selenocysteine in the expressed protein was in the reduced state. The heme-His25SeCys hHO-1 complex could be prepared by either (a) supplementing the overexpression medium with heme, or (b) reconstituting the purified apoprotein with heme. Under reducing conditions in the presence of imidazole, a covalent bond is formed by addition of the selenocysteine residue to one of the heme vinyl groups. No covalent bond is formed when the heme is replaced by mesoheme, in which the vinyls are replaced by ethyl groups. These results, together with our earlier demonstration that external selenolate ligands can transfer an electron to the iron [Y. Jiang, P.R. Ortiz de Montellano, Inorg. Chem. 47 (2008) 3480-3482 ], indicate that a selenyl radical is formed in the hHO-1 His25SeCys mutant that adds to a heme vinyl group.

Heme Mobilization in Animals: A Metallolipid's Journey.

Science.gov (United States)

Reddi, Amit R; Hamza, Iqbal

2016-06-21

Heme is universally recognized as an essential and ubiquitous prosthetic group that enables proteins to carry out a diverse array of functions. All heme-dependent processes, from protein hemylation to heme signaling, require the dynamic and rapid mobilization of heme to hemoproteins present in virtually every subcellular compartment. The cytotoxicity and hydrophobicity of heme necessitates that heme mobilization is carefully controlled at the cellular and systemic level. However, the molecules and mechanisms that mediate heme homeostasis are poorly understood. In this Account, we provide a heuristic paradigm with which to conceptualize heme trafficking and highlight the most recent developments in the mechanisms underlying heme trafficking. As an iron-containing tetrapyrrole, heme exhibits properties of both transition metals and lipids. Accordingly, we propose its transport and trafficking will reflect principles gleaned from the trafficking of both metals and lipids. Using this conceptual framework, we follow the flow of heme from the final step of heme synthesis in the mitochondria to hemoproteins present in various subcellular organelles. Further, given that many cells and animals that cannot make heme can assimilate it intact from nutritional sources, we propose that intercellular heme trafficking pathways must exist. This necessitates that heme be able to be imported and exported from cells, escorted between cells and organs, and regulated at the organismal level via a coordinated systemic process. In this Account, we highlight recently discovered heme transport and trafficking factors and provide the biochemical foundation for the cell and systems biology of heme. Altogether, we seek to reconceptualize heme from an exchange inert cofactor buried in hemoprotein active sites to an exchange labile and mobile metallonutrient.

Irradiation of bovine meat: effect of heme-iron concentration.; Irradiacao de carne bovina: efeito na concentracao de ferro heme

Energy Technology Data Exchange (ETDEWEB)

Mistura, Liliana Perazzini Furtado

2002-07-01

The irradiation is often used, nowadays, for meat conservation and it is important to know how much this process interferes with the nutritional quality of the meat. In this study round cut meat, ground and steaks (from a local supermarket) was irradiated with doses of O; 1; 2; 3; 4; 5; 7,5 and 10 kGy (JS-7500 Nordium Inc -Canada) and the interference of irradiation and the process of food preparation on heme-iron (H Fe) content was determined. Half of the sample was kept raw and the other half was grilled in a pre-warmed oven at 250 deg C for 9 min and a controlled humidity of 70%. The chemical composition, the total iron (T Fe) (EM) and the heme iron concentration were determined (Hornsey,1956) and the sensorial quality evaluated. The average T Fe concentration of raw and ground , ground and grilled, raw steaks and grilled steak meat, on dry and degreased basis was 113 mug/g, 121 mug/g , 91 mug/g and 77 mug/g; and the H Fe concentration 105 mug/g (93% of T Fe) , 88 mug/g (73% of T Fe), 90 mug/g (99% of T Fe) and 52 mug/g (68% of T Fe) respectively. Data were evaluated by ANOVA with fixed effects and multiple comparisons. The irradiation neither altered the chemical composition nor the proportion of heme iron of meat. The preparation conditions (temperature, cooking time, environment humidity, meat presentation) of the sample interfered more with the heme iron content than the irradiation. With the sensorial analysis we verified that meats irradiated with doses of 3 kGy were better evaluated in softness and succulency attributes than the others. Meat submitted to irradiation doses up to 3 kGy were accepted by the specialists' panel. (author)

Control of intracellular heme levels: Heme transporters and heme oxygenases

OpenAIRE

Khan, Anwar A.; Quigley, John G.

2011-01-01

Heme serves as a co-factor in proteins involved in fundamental biological processes including oxidative metabolism, oxygen storage and transport, signal transduction and drug metabolism. In addition, heme is important for systemic iron homeostasis in mammals. Heme has important regulatory roles in cell biology, yet excessive levels of intracellular heme are toxic; thus, mechanisms have evolved to control the acquisition, synthesis, catabolism and expulsion of cellular heme. Recently, a number...

A dual component heme biosensor that integrates heme transport and synthesis in bacteria.

Science.gov (United States)

Nobles, Christopher L; Clark, Justin R; Green, Sabrina I; Maresso, Anthony W

2015-11-01

Bacterial pathogens acquire host iron to power cellular processes and replication. Heme, an iron-containing cofactor bound to hemoglobin, is scavenged by bacterial proteins to attain iron. Methods to measure intracellular heme are laborious, involve complex chemistry, or require radioactivity. Such drawbacks limit the study of the mechanistic steps of heme transport and breakdown. Hypothesizing heme homeostasis could be measured with fluorescent methods, we coupled the conversion of heme to biliverdin IXα (a product of heme catabolism) by heme oxygenase 1 (HO1) with the production of near-infrared light upon binding this verdin by infrared fluorescent protein (IFP1.4). The resultant heme sensor, IFP-HO1, was fluorescent in pathogenic E. coli exposed to heme but not in the absence of the heme transporter ChuA and membrane coupling protein TonB, thereby validating their long-standing proposed role in heme uptake. Fluorescence was abolished in a strain lacking hemE, the central gene in the heme biosynthetic pathway, but stimulated by iron, signifying the sensor reports on intracellular heme production. Finally, an invasive strain of E. coli harboring the sensor was fluorescent during an active infection. This work will allow researchers to expand the molecular toolbox used to study heme and iron acquisition in culture and during infection. Copyright © 2015 Elsevier B.V. All rights reserved.

The effect of proteins from animal source foods on heme iron bioavailability in humans.

Science.gov (United States)

Pizarro, Fernando; Olivares, Manuel; Valenzuela, Carolina; Brito, Alex; Weinborn, Valerie; Flores, Sebastián; Arredondo, Miguel

2016-04-01

Forty-five women (35-45 year) were randomly assigned to three iron (Fe) absorption sub-studies, which measured the effects of dietary animal proteins on the absorption of heme Fe. Study 1 was focused on heme, red blood cell concentrate (RBCC), hemoglobin (Hb), RBCC+beef meat; study 2 on heme, heme+fish, chicken, and beef; and study 3 on heme and heme+purified animal protein (casein, collagen, albumin). Study 1: the bioavailability of heme Fe from Hb was similar to heme only (∼13.0%). RBCC (25.0%) and RBCC+beef (21.3%) were found to be increased 2- and 1.6-fold, respectively, when compared with heme alone (pProteins from animal source foods and their digestion products did not enhance heme Fe absorption. Copyright © 2015. Published by Elsevier Ltd.

Heme Iron Content in Lamb Meat Is Differentially Altered upon Boiling, Grilling, or Frying as Assessed by Four Distinct Analytical Methods

OpenAIRE

Pourkhalili, Azin; Mirlohi, Maryam; Rahimi, Ebrahim

2013-01-01

Lamb meat is regarded as an important source of highly bioavailable iron (heme iron) in the Iranians diet. The main objective of this study is to evaluate the effect of traditional cooking methods on the iron changes in lamb meat. Four published experimental methods for the determination of heme iron were assessed analytically and statistically. Samples were selected from lambs' loin. Standard methods (AOAC) were used for proximate analysis. For measuring heme iron, the results of four experi...

Heme Iron Concentrate and Iron Sulfate Added to Chocolate Biscuits: Effects on Hematological Indices of Mexican Schoolchildren.

Science.gov (United States)

Quintero-Gutiérrez, Adrián Guillermo; González-Rosendo, Guillermina; Pozo, Javier Polo; Villanueva-Sánchez, Javier

2016-08-01

Food fortification is one of the most effective strategies for increasing iron intake in the population. A simple blind trial was conducted to compare the effect of 2 forms of iron fortification and assess the changes in hemoglobin and iron status indices among preschool children from rural communities. Hemoglobin was evaluated in 47 children aged 3-6 years old. For 72 days (10-week period), children ate Nito biscuits. Thirteen pupils with elevated hemoglobin levels were assigned to the biscuit control group, and pupils with hemoglobin equal to 13.5 mg/dL or less were randomly allocated to consume fortified biscuits with a heme iron concentrate (n = 15) or iron sulfate (n = 19). Changes in hemoglobin, plasma ferritin, and other hematological indices were evaluated with analysis of variance (ANOVA) for repeated measurements. Except mean corpuscular hemoglobin concentrations (+1.27 ± 2.25 g/dL), hematological indices increased significantly across the study: Mean corpuscular volume (+2.2 ± 1.0 f/dL), red blood cells (+0.30 ± 0.37 M/μL), mean corpuscular hemoglobin (+1.8 ± 1.74 pg), hemoglobin (+1.68 ± 0.91 g/dL), hematocrit (+3.43% ± 3.03%), and plasma ferritin (+18.38 ± 22.1 μg/L) were all p effect of the iron-fortified chocolate biscuits in the hemoglobin levels was higher than the control group (+1.1 ± 0.2 g/dL) but no difference was found between consumers of fortified biscuits with heme iron concentrate or iron sulfate (+1.9 ± 0.2 g/dL and +2.0 ± 0.2 g/dL, respectively). Heme iron concentrate and iron sulfate were equally effective in increasing Hb levels and hematological indices. Processed foods were shown to be an effective, valuable, and admissible intervention to prevent anemia in preschool children.

Suppression of acyl migration in enzymatic production of structured lipids through temperature programming

DEFF Research Database (Denmark)

Yang, Tiankui; Fruekilde, Maj-Britt; Xu, Xuebing

2005-01-01

Acyl migration in the glycerol backbone often leads to the increase of by-products in the enzymatic production of specific structured lipids. Acyl migration is a thermodynamic process and is very difficult to stop fully in actual reactions. The objective of this study was to investigate...

Deciphering the acylation pattern of Yersinia enterocolitica lipid A.

Science.gov (United States)

Reinés, Mar; Llobet, Enrique; Dahlström, Käthe M; Pérez-Gutiérrez, Camino; Llompart, Catalina M; Torrecabota, Nuria; Salminen, Tiina A; Bengoechea, José A

2012-01-01

Pathogenic bacteria may modify their surface to evade the host innate immune response. Yersinia enterocolitica modulates its lipopolysaccharide (LPS) lipid A structure, and the key regulatory signal is temperature. At 21°C, lipid A is hexa-acylated and may be modified with aminoarabinose or palmitate. At 37°C, Y. enterocolitica expresses a tetra-acylated lipid A consistent with the 3'-O-deacylation of the molecule. In this work, by combining genetic and mass spectrometric analysis, we establish that Y. enterocolitica encodes a lipid A deacylase, LpxR, responsible for the lipid A structure observed at 37°C. Western blot analyses indicate that LpxR exhibits latency at 21°C, deacylation of lipid A is not observed despite the expression of LpxR in the membrane. Aminoarabinose-modified lipid A is involved in the latency. 3-D modelling, docking and site-directed mutagenesis experiments showed that LpxR D31 reduces the active site cavity volume so that aminoarabinose containing Kdo(2)-lipid A cannot be accommodated and, therefore, not deacylated. Our data revealed that the expression of lpxR is negatively controlled by RovA and PhoPQ which are necessary for the lipid A modification with aminoarabinose. Next, we investigated the role of lipid A structural plasticity conferred by LpxR on the expression/function of Y. enterocolitica virulence factors. We present evidence that motility and invasion of eukaryotic cells were reduced in the lpxR mutant grown at 21°C. Mechanistically, our data revealed that the expressions of flhDC and rovA, regulators controlling the flagellar regulon and invasin respectively, were down-regulated in the mutant. In contrast, the levels of the virulence plasmid (pYV)-encoded virulence factors Yops and YadA were not affected in the lpxR mutant. Finally, we establish that the low inflammatory response associated to Y. enterocolitica infections is the sum of the anti-inflammatory action exerted by pYV-encoded YopP and the reduced activation of

Asymmetric Chemoenzymatic Reductive Acylation of Ketones by a Combined Iron-Catalyzed Hydrogenation-Racemization and Enzymatic Resolution Cascade

KAUST Repository

El-Sepelgy, Osama

2017-02-28

A general and practical process for the conversion of prochiral ketones into the corresponding chiral acetates has been realized. An iron carbonyl complex is reported to catalyze the hydrogenation-dehydrogenation-hydrogenation of prochiral ketones. By merging the iron-catalyzed redox reactions with enantioselective enzymatic acylations a wide range of benzylic, aliphatic and (hetero)aromatic ketones, as well as diketones, were reductively acylated. The corresponding products were isolated with high yields and enantioselectivities. The use of an iron catalyst together with molecular hydrogen as the hydrogen donor and readily available ethyl acetate as acyl donor make this cascade process highly interesting in terms of both economic value and environmental credentials.

Association of acylated cationic decapeptides with dipalmitoylphosphatidylserine-dipalmitoyl- phosphatidylcholine lipid membranes

DEFF Research Database (Denmark)

Pedersen, T. B.; Sabra, Mads Christian; Frokjaer, Sven

2001-01-01

decapeptides that are N-terminally linked with C-2, C-8, and C-14 acyl chains contain four basic histidine residues in their identical amino acid sequence. A binding model, based on changes in the intrinsic fluorescent properties of the peptides upon association with the DPPC-DPPS membranes, is used...... DPPC-DPPS lipid mixture. The extent of peptide association deduced from the heat capacity measurements suggests a strong binding and membrane insertion of the C-14 acylated peptide in accordance with the fluorescence measurements....

Deciphering the acylation pattern of Yersinia enterocolitica lipid A.

Directory of Open Access Journals (Sweden)

Mar Reinés

Full Text Available Pathogenic bacteria may modify their surface to evade the host innate immune response. Yersinia enterocolitica modulates its lipopolysaccharide (LPS lipid A structure, and the key regulatory signal is temperature. At 21°C, lipid A is hexa-acylated and may be modified with aminoarabinose or palmitate. At 37°C, Y. enterocolitica expresses a tetra-acylated lipid A consistent with the 3'-O-deacylation of the molecule. In this work, by combining genetic and mass spectrometric analysis, we establish that Y. enterocolitica encodes a lipid A deacylase, LpxR, responsible for the lipid A structure observed at 37°C. Western blot analyses indicate that LpxR exhibits latency at 21°C, deacylation of lipid A is not observed despite the expression of LpxR in the membrane. Aminoarabinose-modified lipid A is involved in the latency. 3-D modelling, docking and site-directed mutagenesis experiments showed that LpxR D31 reduces the active site cavity volume so that aminoarabinose containing Kdo(2-lipid A cannot be accommodated and, therefore, not deacylated. Our data revealed that the expression of lpxR is negatively controlled by RovA and PhoPQ which are necessary for the lipid A modification with aminoarabinose. Next, we investigated the role of lipid A structural plasticity conferred by LpxR on the expression/function of Y. enterocolitica virulence factors. We present evidence that motility and invasion of eukaryotic cells were reduced in the lpxR mutant grown at 21°C. Mechanistically, our data revealed that the expressions of flhDC and rovA, regulators controlling the flagellar regulon and invasin respectively, were down-regulated in the mutant. In contrast, the levels of the virulence plasmid (pYV-encoded virulence factors Yops and YadA were not affected in the lpxR mutant. Finally, we establish that the low inflammatory response associated to Y. enterocolitica infections is the sum of the anti-inflammatory action exerted by pYV-encoded YopP and the

Mechanisms of heme utilization by Francisella tularensis.

Directory of Open Access Journals (Sweden)

Helena Lindgren

Full Text Available Francisella tularensis is a highly virulent facultative intracellular pathogen causing the severe disease tularemia in mammals. As for other bacteria, iron is essential for its growth but very few mechanisms for iron acquisition have been identified. Here, we analyzed if and how F. tularensis can utilize heme, a major source of iron in vivo. This is by no means obvious since the bacterium lacks components of traditional heme-uptake systems. We show that SCHU S4, the prototypic strain of subspecies tularensis, grew in vitro with heme as the sole iron source. By screening a SCHU S4 transposon insertion library, 16 genes were identified as important to efficiently utilize heme, two of which were required to avoid heme toxicity. None of the identified genes appeared to encode components of a potential heme-uptake apparatus. Analysis of SCHU S4 deletion mutants revealed that each of the components FeoB, the siderophore system, and FupA, contributed to the heme-dependent growth. In the case of the former two systems, iron acquisition was impaired, whereas the absence of FupA did not affect iron uptake but led to abnormally high binding of iron to macromolecules. Overall, the present study demonstrates that heme supports growth of F. tularensis and that the requirements for the utilization are highly complex and to some extent novel.

Production of specific-structured lipids by enzymatic interesterification: elucidation of acyl migration by response surface design

DEFF Research Database (Denmark)

Xu, Xuebing; Skands, Anja; Høy, Carl-Erik

1998-01-01

Production of specific-structured lipids (SSL) by lipase-catalyzed interesterification has been attracting more and more attention recently. However, it was found that acyl migration occurs during the reaction and causes the production of by-products. In this paper, the elucidation of acyl...

Heme Recognition By a Staphylococcus Aureus IsdE

Energy Technology Data Exchange (ETDEWEB)

Grigg, J.C.; Vermeiren, C.L.; Heinrichs, D.E.; Murphy, M.E.P.

2009-06-03

Staphylococcus aureus is a Gram-positive bacterial pathogen and a leading cause of hospital acquired infections. Because the free iron concentration in the human body is too low to support growth, S. aureus must acquire iron from host sources. Heme iron is the most prevalent iron reservoir in the human body and a predominant source of iron for S. aureus. The iron-regulated surface determinant (Isd) system removes heme from host heme proteins and transfers it to IsdE, the cognate substrate-binding lipoprotein of an ATP-binding cassette transporter, for import and subsequent degradation. Herein, we report the crystal structure of the soluble portion of the IsdE lipoprotein in complex with heme. The structure reveals a bi-lobed topology formed by an N- and C-terminal domain bridged by a single {alpha}-helix. The structure places IsdE as a member of the helical backbone metal receptor superfamily. A six-coordinate heme molecule is bound in the groove established at the domain interface, and the heme iron is coordinated in a novel fashion for heme transporters by Met{sup 78} and His{sup 229}. Both heme propionate groups are secured by H-bonds to IsdE main chain and side chain groups. Of these residues, His{sup 299} is essential for IsdE-mediated heme uptake by S. aureus when growth on heme as a sole iron source is measured. Multiple sequence alignments of homologues from several other Gram-positive bacteria, including the human pathogens pyogenes, Bacillus anthracis, and Listeria monocytogenes, suggest that these other systems function equivalently to S. aureus IsdE with respect to heme binding and transport.

Asymmetric Chemoenzymatic Reductive Acylation of Ketones by a Combined Iron-Catalyzed Hydrogenation-Racemization and Enzymatic Resolution Cascade

KAUST Repository

El-Sepelgy, Osama; Brzozowska, Aleksandra; Rueping, Magnus

2017-01-01

. By merging the iron-catalyzed redox reactions with enantioselective enzymatic acylations a wide range of benzylic, aliphatic and (hetero)aromatic ketones, as well as diketones, were reductively acylated. The corresponding products were isolated with high

Biosynthesis of heme in immature erythroid cells. The regulatory step for heme formation in the human erythron

International Nuclear Information System (INIS)

Gardner, L.C.; Cox, T.M.

1988-01-01

Heme formation in reticulocytes from rabbits and rodents is subject to end product negative feedback regulation: intracellular free heme has been shown to control acquisition of transferrin iron for heme synthesis. To identify the site of control of heme biosynthesis in the human erythron, immature erythroid cells were obtained from peripheral blood and aspirated bone marrow. After incubation with human 59Fe transferrin, 2-[14C]glycine, or 4-[14C]delta-aminolevulinate, isotopic incorporation into extracted heme was determined. Addition of cycloheximide to increase endogenous free heme, reduced incorporation of labeled glycine and iron but not delta-aminolevulinate into cell heme. Incorporation of glycine and iron was also sensitive to inhibition by exogenous hematin (Ki, 30 and 45 microM, respectively) i.e. at concentrations in the range which affect cell-free protein synthesis in reticulocyte lysates. Hematin treatment rapidly diminished incorporation of intracellular 59Fe into heme by human erythroid cells but assimilation of 4-[14C]delta-aminolevulinate into heme was insensitive to inhibition by hematin (Ki greater than 100 microM). In human reticulocytes (unlike those from rabbits), addition of ferric salicylaldehyde isonicotinoylhydrazone, to increase the pre-heme iron pool independently of the transferrin cycle, failed to promote heme synthesis or modify feedback inhibition induced by hematin. In human erythroid cells (but not rabbit reticulocytes) pre-incubation with unlabeled delta-aminolevulinate or protoporphyrin IX greatly stimulated utilization of cell 59Fe for heme synthesis and also attenuated end product inhibition. In human erythroid cells heme biosynthesis is thus primarily regulated by feedback inhibition at one or more steps which lead to delta-aminolevulinate formation

Structural and Functional Models of Non-Heme Iron Enzymes : A Study of the 2-His-1-Carboxylate Facial Triad Structural Motif

NARCIS (Netherlands)

Bruijnincx, P.C.A.

2007-01-01

The structural and functional modeling of a specific group of non-heme iron enzymes by the synthesis of small synthetic analogues is the topic of this thesis. The group of non-heme iron enzymes with the 2-His-1-carboxylate facial triad has recently been established as a common platform for the

Production of structured lipids: acyl migration during enzymatic interesterification and downstream processing

DEFF Research Database (Denmark)

Xu, Xuebing

1997-01-01

Production of structured lipids by lipase-catalyzed interesterification attracts great interests recently. Structured lipids are defined, in this article, as triacylglycerols which contain both medium or short chain fatty acids and long chain fatty acids, each groups locating specifically in the sn......-2 position or sn-1,3 positions of glycerol backbone. These kinds of lipids are reported to be promising for both enteral and parenteral nutrition. However, acyl migration occurs in the reaction stage and downstream purification process. This side-reaction causes by-products which are harmful...

Single or functionalized fullerenes interacting with heme group

Energy Technology Data Exchange (ETDEWEB)

Costa, Wallison Chaves; Diniz, Eduardo Moraes, E-mail: eduardo.diniz@ufma.br [Departamento de Física, Universidade Federal do Maranhão, Avenida dos Portugueses, 1966, CEP 65080-805, São Luís - MA (Brazil)

2014-09-15

The heme group is responsible for iron transportation through the bloodstream, where iron participates in redox reactions, electron transfer, gases detection etc. The efficiency of such processes can be reduced if the whole heme molecule or even the iron is somehow altered from its original oxidation state, which can be caused by interactions with nanoparticles as fullerenes. To verify how such particles alter the geometry and electronic structure of heme molecule, here we report first principles calculations based on density functional theory of heme group interacting with single C{sub 60} fullerene or with C{sub 60} functionalized with small functional groups (−CH{sub 3}, −COOH, −NH{sub 2}, −OH). The calculations shown that the system heme + nanoparticle has a different spin state in comparison with heme group if the fullerene is functionalized. Also a functional group can provide a stronger binding between nanoparticle and heme molecule or inhibit the chemical bonding in comparison with single fullerene results. In addition heme molecule loses electrons to the nanoparticles and some systems exhibited a geometry distortion in heme group, depending on the binding energy. Furthermore, one find that such nanoparticles induce a formation of spin up states in heme group. Moreover, there exist modifications in density of states near the Fermi energy. Although of such changes in heme electronic structure and geometry, the iron atom remains in the heme group with the same oxidation state, so that processes that involve the iron might not be affected, only those that depend on the whole heme molecule.

Heme Gazing: Illuminating Eukaryotic Heme Trafficking, Dynamics, and Signaling with Fluorescent Heme Sensors.

Science.gov (United States)

Hanna, David A; Martinez-Guzman, Osiris; Reddi, Amit R

2017-04-04

Heme (iron protoporphyrin IX) is an essential protein prosthetic group and signaling molecule required for most life on Earth. All heme-dependent processes require the dynamic and rapid mobilization of heme from sites of synthesis or uptake to hemoproteins present in virtually every subcellular compartment. The cytotoxicity and hydrophobicity of heme necessitate that heme mobilization be carefully controlled to mitigate the deleterious effects of this essential toxin. Indeed, a number of disorders, including certain cancers, cardiovascular diseases, and aging and age-related neurodegenerative diseases, are tied to defects in heme homeostasis. However, the molecules and mechanisms that mediate heme transport and trafficking, and the dynamics of these processes, are poorly understood. This is in large part due to the lack of physical tools for probing cellular heme. Herein, we discuss the recent development of fluorescent probes that can monitor and image kinetically labile heme with respect to its mobilization and role in signaling. In particular, we will highlight how heme gazing with these tools can uncover new heme trafficking factors upon being integrated with genetic screens and illuminate the concentration, subcellular distribution, and dynamics of labile heme in various physiological contexts. Altogether, the monitoring of labile heme, along with recent biochemical and cell biological studies demonstrating the reversible regulation of certain cellular processes by heme, is challenging us to reconceptualize heme from being a static cofactor buried in protein active sites to a dynamic and mobile signaling molecule.

X-ray absorption spectroscopy of soybean lipoxygenase-1 : Influence of lipid hydroperoxide activation and lyophilization on the structure of the non-heme iron active site

NARCIS (Netherlands)

Vliegenthart, J.F.G.; Heijdt, L.M. van der; Feiters, M.C.; Navaratnam, S.; Nolting, H.-F.; Hermes, C.; Veldink, G.A.

1992-01-01

X-ray absorption spectra at the Fe K-edge of the non-heme iron site in Fe(II) as well as Fe(III) soybean lipoxygenase-1, in frozen solution or lyophilized, are presented; the latter spectra were obtained by incubation of the Fe(II) enzyme with its product hydroperoxide. An edge shift of about 23 eV

Structures of the multicomponent Rieske non-heme iron toluene 2, 3-dioxygenase enzyme system

Energy Technology Data Exchange (ETDEWEB)

Friemann, Rosmarie [Department of Molecular Biology, Swedish University of Agricultural Sciences, Box 590, 751 24 Uppsala (Sweden); Lee, Kyoung [Department of Microbiology, Changwon National University, Changwon, Kyoungnam 641-773 (Korea, Republic of); Department of Microbiology, The University of Iowa, Iowa City, Iowa 52242 (United States); Brown, Eric N. [Department of Biochemistry, The University of Iowa, Iowa City, Iowa 52242 (United States); Gibson, David T. [Department of Microbiology, The University of Iowa, Iowa City, Iowa 52242 (United States); Eklund, Hans [Department of Molecular Biology, Swedish University of Agricultural Sciences, Box 590, 751 24 Uppsala (Sweden); Ramaswamy, S., E-mail: s-ramaswamy@uiowa.edu [Department of Biochemistry, The University of Iowa, Iowa City, Iowa 52242 (United States); Department of Molecular Biology, Swedish University of Agricultural Sciences, Box 590, 751 24 Uppsala (Sweden)

2009-01-01

The crystal structures of the three-component toluene 2, 3-dioxygenase system provide a model for electron transfer among bacterial Rieske non-heme iron dioxygenases. Bacterial Rieske non-heme iron oxygenases catalyze the initial hydroxylation of aromatic hydrocarbon substrates. The structures of all three components of one such system, the toluene 2, 3-dioxygenase system, have now been determined. This system consists of a reductase, a ferredoxin and a terminal dioxygenase. The dioxygenase, which was cocrystallized with toluene, is a heterohexamer containing a catalytic and a structural subunit. The catalytic subunit contains a Rieske [2Fe–2S] cluster and mononuclear iron at the active site. This iron is not strongly bound and is easily removed during enzyme purification. The structures of the enzyme with and without mononuclear iron demonstrate that part of the structure is flexible in the absence of iron. The orientation of the toluene substrate in the active site is consistent with the regiospecificity of oxygen incorporation seen in the product formed. The ferredoxin is Rieske type and contains a [2Fe–2S] cluster close to the protein surface. The reductase belongs to the glutathione reductase family of flavoenzymes and consists of three domains: an FAD-binding domain, an NADH-binding domain and a C-terminal domain. A model for electron transfer from NADH via FAD in the reductase and the ferredoxin to the terminal active-site mononuclear iron of the dioxygenase is proposed.

Hal Is a Bacillus anthracis Heme Acquisition Protein

Science.gov (United States)

Balderas, Miriam A.; Nobles, Christopher L.; Honsa, Erin S.; Alicki, Embriette R.

2012-01-01

The metal iron is a limiting nutrient for bacteria during infection. Bacillus anthracis, the causative agent of anthrax and a potential weapon of bioterrorism, grows rapidly in mammalian hosts, which suggests that it efficiently attains iron during infection. Recent studies have uncovered both heme (isd) and siderophore-mediated (asb) iron transport pathways in this pathogen. Whereas deletion of the asb genes results in reduced virulence, the loss of three surface components from isd had no effect, thereby leaving open the question of what additional factors in B. anthracis are responsible for iron uptake from the most abundant iron source for mammals, heme. Here, we describe the first functional characterization of bas0520, a gene recently implicated in anthrax disease progression. bas0520 encodes a single near-iron transporter (NEAT) domain and several leucine-rich repeats. The NEAT domain binds heme, despite lacking a stabilizing tyrosine common to the NEAT superfamily of hemoproteins. The NEAT domain also binds hemoglobin and can acquire heme from hemoglobin in solution. Finally, deletion of bas0520 resulted in bacilli unable to grow efficiently on heme or hemoglobin as an iron source and yielded the most significant phenotype relative to that for other putative heme uptake systems, a result that suggests that this protein plays a prominent role in the replication of B. anthracis in hematogenous environments. Thus, we have assigned the name of Hal (heme-acquisition leucine-rich repeat protein) to BAS0520. These studies advance our understanding of heme acquisition by this dangerous pathogen and justify efforts to determine the mechanistic function of this novel protein for vaccine or inhibitor development. PMID:22865843

Over-expression of heme oxygenase-1 promotes oxidative mitochondrial damage in rat astroglia.

Science.gov (United States)

Song, Wei; Su, Haixiang; Song, Sisi; Paudel, Hemant K; Schipper, Hyman M

2006-03-01

Glial heme oxygenase-1 is over-expressed in the CNS of subjects with Alzheimer disease (AD), Parkinson disease (PD) and multiple sclerosis (MS). Up-regulation of HO-1 in rat astroglia has been shown to facilitate iron sequestration by the mitochondrial compartment. To determine whether HO-1 induction promotes mitochondrial oxidative stress, assays for 8-epiPGF(2alpha) (ELISA), protein carbonyls (ELISA) and 8-OHdG (HPLC-EC) were used to quantify oxidative damage to lipids, proteins, and nucleic acids, respectively, in mitochondrial fractions and whole-cell compartments derived from cultured rat astroglia engineered to over-express human (h) HO-1 by transient transfection. Cell viability was assessed by trypan blue exclusion and the MTT assay, and cell proliferation was determined by [3H] thymidine incorporation and total cell counts. In rat astrocytes, hHO-1 over-expression (x 3 days) resulted in significant oxidative damage to mitochondrial lipids, proteins, and nucleic acids, partial growth arrest, and increased cell death. These effects were attenuated by incubation with 1 microM tin mesoporphyrin, a competitive HO inhibitor, or the iron chelator, deferoxamine. Up-regulation of HO-1 engenders oxidative mitochondrial injury in cultured rat astroglia. Heme-derived ferrous iron and carbon monoxide (CO) may mediate the oxidative modification of mitochondrial lipids, proteins and nucleic acids in these cells. Glial HO-1 hyperactivity may contribute to cellular oxidative stress, pathological iron deposition, and bioenergetic failure characteristic of degenerating and inflamed neural tissues and may constitute a rational target for therapeutic intervention in these conditions. Copyright 2005 Wiley-Liss, Inc.

A role for heme in Alzheimer's disease: Heme binds amyloid β and has altered metabolism

OpenAIRE

Atamna, Hani; Frey, William H.

2004-01-01

Heme is a common factor linking several metabolic perturbations in Alzheimer's disease (AD), including iron metabolism, mitochondrial complex IV, heme oxygenase, and bilirubin. Therefore, we determined whether heme metabolism was altered in temporal lobes obtained at autopsy from AD patients and age-matched nondemented subjects. AD brain demonstrated 2.5-fold more heme-b (P < 0.01) and 26% less heme-a (P = 0.16) compared with controls, resulting in a highly significant 2.9-fold decrease in he...

Mono- and binuclear non-heme iron chemistry from a theoretical perspective

Czech Academy of Sciences Publication Activity Database

Rokob, T. A.; Chalupský, Jakub; Bím, Daniel; Andrikopoulos, Prokopis C.; Srnec, Martin; Rulíšek, Lubomír

2016-01-01

Roč. 21, 5/6 (2016), s. 619-644 ISSN 0949-8257 R&D Projects: GA ČR(CZ) GJ15-10279Y; GA ČR(CZ) GA14-31419S; GA ČR GA15-19143S Grant - others:COST(XE) CM1305 Institutional support: RVO:61388963 ; RVO:61388955 Keywords : non-heme iron * density functional theory * multireference methods * dioxygen activation * reactivity Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.894, year: 2016

Peroxide Activation for Electrophilic Reactivity by the Binuclear Non-heme Iron Enzyme AurF

International Nuclear Information System (INIS)

Park, Kiyoung; Li, Ning; Kwak, Yeonju; Srnec, Martin

2017-01-01

Binuclear non-heme iron enzymes activate O 2 for diverse chemistries that include oxygenation of organic substrates and hydrogen atom abstraction. This process often involves the formation of peroxo-bridged biferric intermediates, only some of which can perform electrophilic reactions. To elucidate the geometric and electronic structural requirements to activate peroxo reactivity, the active peroxo intermediate in 4-aminobenzoate N-oxygenase (AurF) has been characterized spectroscopically and computationally. A magnetic circular dichroism study of reduced AurF shows that its electronic and geometric structures are poised to react rapidly with O 2 . Nuclear resonance vibrational spectroscopic definition of the peroxo intermediate formed in this reaction shows that the active intermediate has a protonated peroxo bridge. Density functional theory computations on the structure established here show that the protonation activates peroxide for electrophilic/single-electron-transfer reactivity. As a result, this activation of peroxide by protonation is likely also relevant to the reactive peroxo intermediates in other binuclear non-heme iron enzymes.

Peroxo-Type Intermediates in Class I Ribonucleotide Reductase and Related Binuclear Non-Heme Iron Enzymes

DEFF Research Database (Denmark)

Kepp, Kasper Planeta; Bell, Caleb B.; Clay, MIchael D.

2009-01-01

We have performed a systematic study of chemically possible peroxo-type intermediates occurring in the non-heme di-iron enzyme class la ribonucleotide reductase, using spectroscopically calibrated computational chemistry. Density functional computations of equilibrium structures, Fe-O and O-O str...

Binding analysis of ferritin with heme using α-casein and biotinylated-hemin: detection of heme-binding capacity of Dpr derived from heme synthesis-deficient Streptococcus mutans.

Science.gov (United States)

Mieno, Ayako; Yamamoto, Yuji; Yoshikawa, Yasunaga; Watanabe, Kiyotaka; Mukai, Takao; Orino, Koichi

2013-01-01

Bacterial and mammalian ferritins are known to bind heme. The use of α-casein and biotinylated hemin could be applicable to detection of protein-bound heme and of proteins with heme-binding capacity, respectively. Although commercial horse spleen ferritin and purified horse spleen ferritin (L:H subunit ratio=4) bound to an α-casein-coated plate, and this binding could be inhibited by hemin, recombinant iron-binding protein (rDpr), derived from heme-deficient Streptococcus mutans and expressed in Escherichia coli, did not bind to an α-casein-coated plate. Both horse spleen ferritins bound to α-casein-immobilized beads. Commercial horse spleen ferritin and rDpr showed direct binding to hemin-agarose beads. After preincubation of commercial horse spleen ferritin or rDpr with biotinylated hemin, they showed indirect binding to avidin-immobilized beads through biotinylated hemin. These results demonstrate that α-casein is useful for detection of heme-binding ferritin and that both hemin-agarose and the combination of biotinylated hemin and avidin-beads are useful for detection of the heme-binding capacity of ferritin. In addition, this study also revealed that Dpr, a decameric iron-binding protein, from heme-deficient cells binds heme.

Dietary heme-mediated PPARα activation does not affect the heme-induced epithelial hyperproliferation and hyperplasia in mouse colon.

Directory of Open Access Journals (Sweden)

Noortje Ijssennagger

Full Text Available Red meat consumption is associated with an increased colon cancer risk. Heme, present in red meat, injures the colon surface epithelium by luminal cytotoxicity and reactive oxygen species. This surface injury is overcompensated by hyperproliferation and hyperplasia of crypt cells. Transcriptome analysis of mucosa of heme-fed mice showed, besides stress- and proliferation-related genes, many upregulated lipid metabolism-related PPARα target genes. The aim of this study was to investigate the role of PPARα in heme-induced hyperproliferation and hyperplasia. Male PPARα KO and WT mice received a purified diet with or without heme. As PPARα is proposed to protect against oxidative stress and lipid peroxidation, we hypothesized that the absence of PPARα leads to more surface injury and crypt hyperproliferation in the colon upon heme-feeding. Heme induced luminal cytotoxicity and lipid peroxidation and colonic hyperproliferation and hyperplasia to the same extent in WT and KO mice. Transcriptome analysis of colonic mucosa confirmed similar heme-induced hyperproliferation in WT and KO mice. Stainings for alkaline phosphatase activity and expression levels of Vanin-1 and Nrf2-targets indicated a compromised antioxidant defense in heme-fed KO mice. Our results suggest that the protective role of PPARα in antioxidant defense involves the Nrf2-inhibitor Fosl1, which is upregulated by heme in PPARα KO mice. We conclude that PPARα plays a protective role in colon against oxidative stress, but PPARα does not mediate heme-induced hyperproliferation. This implies that oxidative stress of surface cells is not the main determinant of heme-induced hyperproliferation and hyperplasia.

Polymorphism in 'L' shaped lipids: structure of N-, O-diacylethanolamines with mixed acyl chains.

Science.gov (United States)

Tarafdar, Pradip K; Swamy, Musti J

2009-11-01

Although solid state polymorphism in lipids has been established by spectroscopic and calorimetric studies long ago, only in a few cases crystal structures of different polymorphs of the same compound have been reported, possibly due to difficulties in obtaining high quality single crystals of individual polymorphs. Recent studies show that N-, O-diacylethanolamines (DAEs) can be derived by the O-acylation of the stress-related lipids, the N-acylethanolamines under physiological conditions. In this study, two DAEs with mixed acyl chains, namely N-palmitoyl, O-octanoylethanolamine and N-palmitoyl, O-decanoylethanolamine have been synthesized and their three-dimensional structures were determined. Both the compounds were found to adopt 'L' shaped structures and exist in two polymorphic forms, alpha and beta. In the alpha form a mixed-type chain packing has been observed whereas in the beta form the chain packing is symmetric. Similar polymorphic forms are likely to exist in other 'L' shaped lipids such as 1,3-diacylglycerols and ceramides, where polymorphism has been detected earlier, but three-dimensional structures - which can give precise information about the packing at atomic resolution - have not been reported.

Experimental and Computational Evidence for the Mechanism of Intradiol Catechol Dioxygenation by Non-Heme Iron(III) Complexes

Science.gov (United States)

Jastrzebski, Robin; Quesne, Matthew G; Weckhuysen, Bert M; de Visser, Sam P; Bruijnincx, Pieter C A

2014-01-01

Catechol intradiol dioxygenation is a unique reaction catalyzed by iron-dependent enzymes and non-heme iron(III) complexes. The mechanism by which these systems activate dioxygen in this important metabolic process remains controversial. Using a combination of kinetic measurements and computational modelling of multiple iron(III) catecholato complexes, we have elucidated the catechol cleavage mechanism and show that oxygen binds the iron center by partial dissociation of the substrate from the iron complex. The iron(III) superoxide complex that is formed subsequently attacks the carbon atom of the substrate by a rate-determining C=O bond formation step. PMID:25322920

Heme Synthesis and Acquisition in Bacterial Pathogens

OpenAIRE

Choby, Jacob E.; Skaar, Eric P.

2016-01-01

Bacterial pathogens require the iron-containing cofactor heme to cause disease. Heme is essential to the function of hemoproteins, which are involved in energy generation by the electron transport chain, detoxification of host immune effectors, and other processes. During infection, bacterial pathogens must synthesize heme or acquire heme from the host; however, host heme is sequestered in high-affinity hemoproteins. Pathogens have evolved elaborate strategies to acquire heme from host source...

Fulminant lipid storage myopathy due to multiple acyl-coenzyme a dehydrogenase deficiency.

Science.gov (United States)

Whitaker, Charles H; Felice, Kevin J; Silvers, David; Wu, Qian

2015-08-01

The lipid storage myopathies, primary carnitine deficiency, neutral lipid storage disease, and multiple acyl coenzyme A dehydrogenase deficiency (MADD), are progressive disorders that cause permanent weakness. These disorders of fatty acid metabolism and intracellular triglyceride degradation cause marked fat deposition and damage to muscle cells. We describe a rapidly progressive myopathy in a previously healthy 33-year-old woman. Over 4 months, she developed a proximal and axial myopathy associated with diffuse myalgia and dysphagia, ultimately leading to respiratory failure and death. Muscle biopsy showed massive accumulation of lipid. Plasma acylcarnitine and urine organic acid analysis was consistent with MADD. This was confirmed by molecular genetic testing, which revealed 2 pathogenic mutations in the ETFDH gene. This report illustrates a late-onset case of MADD and reviews the differential diagnosis and evaluation of patients with proximal myopathy and excessive accumulation of lipid on muscle biopsy. © 2014 Wiley Periodicals, Inc.

Iron, Oxidative Stress and Gestational Diabetes

Directory of Open Access Journals (Sweden)

Taifeng Zhuang

2014-09-01

Full Text Available Both iron deficiency and hyperglycemia are highly prevalent globally for pregnant women. Iron supplementation is recommended during pregnancy to control iron deficiency. The purposes of the review are to assess the oxidative effects of iron supplementation and the potential relationship between iron nutrition and gestational diabetes. High doses of iron (~relative to 60 mg or more daily for adult humans can induce lipid peroxidation in vitro and in animal studies. Pharmaceutical doses of iron supplements (e.g., 10× RDA or more for oral supplements or direct iron supplementation via injection or addition to the cell culture medium for a short or long duration will induce DNA damage. Higher heme-iron intake or iron status measured by various biomarkers, especially serum ferritin, might contribute to greater risk of gestational diabetes, which may be mediated by iron oxidative stress though lipid oxidation and/or DNA damage. However, information is lacking about the effect of low dose iron supplementation (≤60 mg daily on lipid peroxidation, DNA damage and gestational diabetes. Randomized trials of low-dose iron supplementation (≤60 mg daily for pregnant women are warranted to test the relationship between iron oxidative stress and insulin resistance/gestational diabetes, especially for iron-replete women.

Effects of illumination and packaging on non-heme iron and color attributes of sliced ham.

Science.gov (United States)

Li, H; Li, C B; Xu, X L; Zhou, G H

2012-08-01

This study was designed to investigate effects of illumination and packaging on color of cooked cured sliced ham during refrigeration, and the possibility of decomposition of nitrosylheme under light and oxygen exposure. Three illumination levels and three packaging films with different oxygen transmission rates (OTRs) were used in two separate experiments during 35 days storage, and pH value, a* value, nitrosylheme, residual nitrite and non-heme iron were evaluated. Packaging OTRs had significant effects (P0.05) nitrosylheme concentration during storage. For both groups, storage time had a significant effect (P<0.01) on a* value and nitrosylheme. Negative relationships between nitrosylheme and nitrite in the illumination group, and between nitrosylheme and non-heme iron in the packaging group were observed. Therefore, illumination level and packaging OTR had limited effects on overall pigment stability, but more discoloration and loss of redness occurred on the surface of products. Copyright © 2012 Elsevier Ltd. All rights reserved.

Reversible formation of high-valent-iron-oxo-porphyrin intermediate in heme-based catalysis: revisiting the kinetic model for horseradish peroxidase.

NARCIS (Netherlands)

Haandel, van M.J.H.; Primus, J.L.; Teunis, C.; Boersma, M.G.; Osman, A.M.; Veeger, C.; Rietjens, I.M.C.M.

1998-01-01

Many heme-containing biocatalysts exert their catalytic action through the initial formation of so-called high-valent-iron-oxo porphyrin intermediates. For horseradish peroxidase the initial intermediate formed has been identified as a high-valent-iron-oxo porphyrin π-radical cation, called compound

Tick iron and heme metabolism – New target for an anti-tick intervention

Czech Academy of Sciences Publication Activity Database

Hajdušek, Ondřej; Šíma, Radek; Perner, Jan; Loosová, Gabriela; Harcubová, Adéla; Kopáček, Petr

2016-01-01

Roč. 7, č. 4 (2016), s. 565-572 ISSN 1877-959X R&D Projects: GA ČR GA13-11043S; GA ČR GP13-27630P; GA ČR GP13-12816P EU Projects: European Commission(XE) 316304 - MODBIOLIN Institutional support: RVO:60077344 Keywords : tick * iron * heme * RNAi * vaccine Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.230, year: 2016

Antioxidant Enzyme Activity, Iron Content and Lipid Oxidation of Raw and Cooked Meat of Korean Native Chickens and Other Poultry

Directory of Open Access Journals (Sweden)

Muhlisin

2016-05-01

Full Text Available This study was conducted to observe antioxidant enzyme activity, iron content and lipid oxidation of Korean native chickens and other poultry. The breast and thigh meat of three Korean native chicken breeds including Woorimatdak, Hyunin black and Yeonsan ogye, and three commercial poultry breeds including the broiler, White Leghorn and Pekin duck (Anasplatyrhyncos domesticus were studied. The analyses of the antioxidant enzymes activity, iron content and lipid oxidation were performed in raw and cooked samples. The activity of catalase (CAT in the thigh meat was higher than that of the breast meat of three Korean native chickens and the broiler, respectively. The activity of glutathione peroxidase (GPx in the uncooked thigh meat of three Korean native chickens was higher than that of the breasts. The breast meat of Woorimatdak and Pekin duck had higher superoxide dismutase (SOD activity than the others, while only the thigh meat of Pekin duck had the highest activity. Cooking inactivated CAT and decreased the activity of GPx and SOD. The thigh meat of Woorimatdak, White Leghorn, Yeonsan ogye and Hyunin black contained more total iron than the breast meat of those breeds. The heme-iron lost during cooking ranged from 3.2% to 14.8%. It is noted that the thigh meat had higher thiobarbituric acid reactive substances values than the breast in all chicken breeds. Though Woorimatdak showed higher antioxidant enzyme activity and lower released-iron percentage among Korean native chickens, no differences were found on lipid oxidation. We confirm that the dark meat of poultry exhibited higher antioxidant enzyme activity and contained more iron than the white meat.

Sugars increase non-heme iron bioavailability in human epithelial intestinal and liver cells.

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Tatiana Christides

Full Text Available Previous studies have suggested that sugars enhance iron bioavailability, possibly through either chelation or altering the oxidation state of the metal, however, results have been inconclusive. Sugar intake in the last 20 years has increased dramatically, and iron status disorders are significant public health problems worldwide; therefore understanding the nutritional implications of iron-sugar interactions is particularly relevant. In this study we measured the effects of sugars on non-heme iron bioavailability in human intestinal Caco-2 cells and HepG2 hepatoma cells using ferritin formation as a surrogate marker for iron uptake. The effect of sugars on iron oxidation state was examined by measuring ferrous iron formation in different sugar-iron solutions with a ferrozine-based assay. Fructose significantly increased iron-induced ferritin formation in both Caco-2 and HepG2 cells. In addition, high-fructose corn syrup (HFCS-55 increased Caco-2 cell iron-induced ferritin; these effects were negated by the addition of either tannic acid or phytic acid. Fructose combined with FeCl3 increased ferrozine-chelatable ferrous iron levels by approximately 300%. In conclusion, fructose increases iron bioavailability in human intestinal Caco-2 and HepG2 cells. Given the large amount of simple and rapidly digestible sugars in the modern diet their effects on iron bioavailability may have important patho-physiological consequences. Further studies are warranted to characterize these interactions.

Heme and erythropoieis: more than a structural role.

Science.gov (United States)

Chiabrando, Deborah; Mercurio, Sonia; Tolosano, Emanuela

2014-06-01

Erythropoiesis is the biological process that consumes the highest amount of body iron for heme synthesis. Heme synthesis in erythroid cells is finely coordinated with that of alpha (α) and beta (β)-globin, resulting in the production of hemoglobin, a tetramer of 2α- and 2β-globin chains, and heme as the prosthetic group. Heme is not only the structural component of hemoglobin, but it plays multiple regulatory roles during the differentiation of erythroid precursors since it controls its own synthesis and regulates the expression of several erythroid-specific genes. Heme is synthesized in developing erythroid progenitors by the stage of proerythroblast, through a series of eight enzymatic reactions divided between mitochondria and cytosol. Defects of heme synthesis in the erythroid lineage result in sideroblastic anemias, characterized by microcytic anemia associated to mitochondrial iron overload, or in erythropoietic porphyrias, characterized by porphyrin deposition in erythroid cells. Here, we focus on the heme biosynthetic pathway and on human erythroid disorders due to defective heme synthesis. The regulatory role of heme during erythroid differentiation is discussed as well as the heme-mediated regulatory mechanisms that allow the orchestration of the adaptive cell response to heme deficiency. Copyright© Ferrata Storti Foundation.

Rieske non-heme iron-dependent oxygenases catalyse diverse reactions in natural product biosynthesis.

Science.gov (United States)

Perry, Christopher; de Los Santos, Emmanuel L C; Alkhalaf, Lona M; Challis, Gregory L

2018-04-13

Covering: up to the end of 2017The roles played by Rieske non-heme iron-dependent oxygenases in natural product biosynthesis are reviewed, with particular focus on experimentally characterised examples. Enzymes belonging to this class are known to catalyse a range of transformations, including oxidative carbocyclisation, N-oxygenation, C-hydroxylation and C-C desaturation. Examples of such enzymes that have yet to be experimentally investigated are also briefly described and their likely functions are discussed.

Meat, Dietary Heme Iron, and Risk of Type 2 Diabetes Mellitus: The Singapore Chinese Health Study.

Science.gov (United States)

Talaei, Mohammad; Wang, Ye-Li; Yuan, Jian-Min; Pan, An; Koh, Woon-Puay

2017-10-01

We evaluated the relationships of red meat, poultry, fish, and shellfish intakes, as well as heme iron intake, with the risk of type 2 diabetes mellitus (T2D).The Singapore Chinese Health Study is a population-based cohort study that recruited 63,257 Chinese adults aged 45-74 years from 1993 to 1998. Usual diet was evaluated using a validated 165-item semiquantitative food frequency questionnaire at recruitment. Physician-diagnosed T2D was self-reported during 2 follow-up interviews in 1999-2004 and 2006-2010. During a mean follow-up of 10.9 years, 5,207 incident cases of T2D were reported. When comparing persons in the highest intake quartiles with those in the lowest, the multivariate-adjusted hazard ratio for T2D was 1.23 (95% confidence interval (CI): 1.14, 1.33) for red meat intake (P for trend meat intake remained significantly associated with T2D risk (multivariate-adjusted hazard ratio = 1.13, 95% CI: 1.01, 1.25; P for trend = 0.02). Heme iron was associated with a higher risk of T2D even after additional adjustment for red meat intake (multivariate-adjusted hazard ratio = 1.14, 95% CI: 1.02, 1.28; P for trend = 0.03). In conclusion, red meat and poultry intakes were associated with a higher risk of T2D. These associations were mediated completely for poultry and partially for red meat by heme iron intake. © The Author(s) 2017. Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Heme degrading protein HemS is involved in oxidative stress response of Bartonella henselae.

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MaFeng Liu

Full Text Available Bartonellae are hemotropic bacteria, agents of emerging zoonoses. These bacteria are heme auxotroph Alphaproteobacteria which must import heme for supporting their growth, as they cannot synthesize it. Therefore, Bartonella genome encodes for a complete heme uptake system allowing the transportation of this compound across the outer membrane, the periplasm and the inner membranes. Heme has been proposed to be used as an iron source for Bartonella since these bacteria do not synthesize a complete system required for iron Fe³⁺ uptake. Similarly to other bacteria which use heme as an iron source, Bartonellae must transport this compound into the cytoplasm and degrade it to allow the release of iron from the tetrapyrrole ring. For Bartonella, the gene cluster devoted to the synthesis of the complete heme uptake system also contains a gene encoding for a polypeptide that shares homologies with heme trafficking or degrading enzymes. Using complementation of an E. coli mutant strain impaired in heme degradation, we demonstrated that HemS from Bartonella henselae expressed in E. coli allows the release of iron from heme. Purified HemS from B. henselae binds heme and can degrade it in the presence of a suitable electron donor, ascorbate or NADPH-cytochrome P450 reductase. Knocking down the expression of HemS in B. henselae reduces its ability to face H₂O₂ induced oxidative stress.

Heme Synthesis and Acquisition in Bacterial Pathogens.

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Choby, Jacob E; Skaar, Eric P

2016-08-28

Bacterial pathogens require the iron-containing cofactor heme to cause disease. Heme is essential to the function of hemoproteins, which are involved in energy generation by the electron transport chain, detoxification of host immune effectors, and other processes. During infection, bacterial pathogens must synthesize heme or acquire heme from the host; however, host heme is sequestered in high-affinity hemoproteins. Pathogens have evolved elaborate strategies to acquire heme from host sources, particularly hemoglobin, and both heme acquisition and synthesis are important for pathogenesis. Paradoxically, excess heme is toxic to bacteria and pathogens must rely on heme detoxification strategies. Heme is a key nutrient in the struggle for survival between host and pathogen, and its study has offered significant insight into the molecular mechanisms of bacterial pathogenesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Are meat and heme iron intake associated with pancreatic cancer? Results from the NIH-AARP Diet and Health Cohort

Science.gov (United States)

Taunk, Pulkit; Hecht, Eric; Stolzenberg-Solomon, Rachael

2015-01-01

Several studies on pancreatic cancer have reported significant positive associations for intake of red meat but null associations for heme iron. We assessed total, red, white, and processed meat intake, meat cooking methods and doneness, and heme iron and mutagen intake in relation to pancreatic cancer in the NIH-AARP Diet and Health Study cohort. 322,846 participants (187,265 men; 135,581 women) successfully completed and returned the food frequency questionnaire between 1995–1996. After a mean follow-up of 9.2 years (up to 10.17 years), 1,417 individuals (895 men, 522 women) developed exocrine pancreatic cancer. Cox proportional hazard models were used to calculate hazard ratios (HR) and 95% confidence intervals (CI), and trends were calculated using the median value of each quantile. Models incorporated age as the time metric and were adjusted for smoking history, BMI, self-reported diabetes, and energy-adjusted saturated fat. Pancreatic cancer risk significantly increased with intake of total meat (Q5 vs. Q1 HR=1.20, 95% CI 1.02–1.42, p-trend=0.03), red meat (HR=1.22, 95% CI 1.01–1.48, p-trend=0.02), high-temperature cooked meat (HR=1.21, 95% CI 1.00–1.45, p-trend=0.02), grilled/barbequed meat (HR=1.24, 95% CI 1.03–1.50, p-trend=0.007), well/very well done meat (HR=1.32, 95% CI 1.10–1.58, p-trend = 0.005), and heme iron from red meat (Q4 vs. Q1 HR=1.21, 95% CI 1.01–1.45, p-trend=0.04). When stratified by sex, these associations remained significant in men but not women except for white meat intake in women (HR = 1.33, 95% CI 1.02–1.74, p-trend = 0.04). Additional studies should confirm our findings that consuming heme iron from red meat increases pancreatic cancer risk. PMID:26666579

Stanniocalcin 1 binds hemin through a partially conserved heme regulatory motif

International Nuclear Information System (INIS)

Westberg, Johan A.; Jiang, Ji; Andersson, Leif C.

2011-01-01

Highlights: → Stanniocalcin 1 (STC1) binds heme through novel heme binding motif. → Central iron atom of heme and cysteine-114 of STC1 are essential for binding. → STC1 binds Fe 2+ and Fe 3+ heme. → STC1 peptide prevents oxidative decay of heme. -- Abstract: Hemin (iron protoporphyrin IX) is a necessary component of many proteins, functioning either as a cofactor or an intracellular messenger. Hemoproteins have diverse functions, such as transportation of gases, gas detection, chemical catalysis and electron transfer. Stanniocalcin 1 (STC1) is a protein involved in respiratory responses of the cell but whose mechanism of action is still undetermined. We examined the ability of STC1 to bind hemin in both its reduced and oxidized states and located Cys 114 as the axial ligand of the central iron atom of hemin. The amino acid sequence differs from the established (Cys-Pro) heme regulatory motif (HRM) and therefore presents a novel heme binding motif (Cys-Ser). A STC1 peptide containing the heme binding sequence was able to inhibit both spontaneous and H 2 O 2 induced decay of hemin. Binding of hemin does not affect the mitochondrial localization of STC1.

Heme oxygenase-1: a metabolic nike.

Science.gov (United States)

Wegiel, Barbara; Nemeth, Zsuzsanna; Correa-Costa, Matheus; Bulmer, Andrew C; Otterbein, Leo E

2014-04-10

Heme degradation, which was described more than 30 years ago, is still very actively explored with many novel discoveries on its role in various disease models every year. The heme oxygenases (HO) are metabolic enzymes that utilize NADPH and oxygen to break apart the heme moiety liberating biliverdin (BV), carbon monoxide (CO), and iron. Heme that is derived from hemoproteins can be toxic to the cells and if not removed immediately, it causes cell apoptosis and local inflammation. Elimination of heme from the milieu enables generation of three products that influences numerous metabolic changes in the cell. CO has profound effects on mitochondria and cellular respiration and other hemoproteins to which it can bind and affect their function, while BV and bilirubin (BR), the substrate and product of BV, reductase, respectively, are potent antioxidants. Sequestration of iron into ferritin and its recycling in the tissues is a part of the homeodynamic processes that control oxidation-reduction in cellular metabolism. Further, heme is an important component of a number of metabolic enzymes, and, therefore, HO-1 plays an important role in the modulation of cellular bioenergetics. In this review, we describe the cross-talk between heme oxygenase-1 (HO-1) and its products with other metabolic pathways. HO-1, which we have labeled Nike, the goddess who personified victory, dictates triumph over pathophysiologic conditions, including diabetes, ischemia, and cancer.

Heme oxygenase activity increases after exercise in healthy volunteers

Science.gov (United States)

AbstractHeme oxygenase (HO) is an essential, rate-limiting protein which participates in the catabolism of heme to iron, carbon monoxide (CO), and biliverdin. The alpha methene bridge carbon of the heme is eliminated as CO which can be measured as blood carboxyhemoglobin (COHb)....

Mechanism governing heme synthesis reveals a GATA factor/heme circuit that controls differentiation.

Science.gov (United States)

Tanimura, Nobuyuki; Miller, Eli; Igarashi, Kazuhiko; Yang, David; Burstyn, Judith N; Dewey, Colin N; Bresnick, Emery H

2016-02-01

Metal ion-containing macromolecules have fundamental roles in essentially all biological processes throughout the evolutionary tree. For example, iron-containing heme is a cofactor in enzyme catalysis and electron transfer and an essential hemoglobin constituent. To meet the intense demand for hemoglobin assembly in red blood cells, the cell type-specific factor GATA-1 activates transcription of Alas2, encoding the rate-limiting enzyme in heme biosynthesis, 5-aminolevulinic acid synthase-2 (ALAS-2). Using genetic editing to unravel mechanisms governing heme biosynthesis, we discovered a GATA factor- and heme-dependent circuit that establishes the erythroid cell transcriptome. CRISPR/Cas9-mediated ablation of two Alas2 intronic cis elements strongly reduces GATA-1-induced Alas2 transcription, heme biosynthesis, and surprisingly, GATA-1 regulation of other vital constituents of the erythroid cell transcriptome. Bypassing ALAS-2 function in Alas2 cis element-mutant cells by providing its catalytic product 5-aminolevulinic acid rescues heme biosynthesis and the GATA-1-dependent genetic network. Heme amplifies GATA-1 function by downregulating the heme-sensing transcriptional repressor Bach1 and via a Bach1-insensitive mechanism. Through this dual mechanism, heme and a master regulator collaborate to orchestrate a cell type-specific transcriptional program that promotes cellular differentiation. © 2015 The Authors.

Visualization of the role of host heme on the virulence of the heme auxotroph Streptococcus agalactiae.

Science.gov (United States)

Joubert, Laetitia; Dagieu, Jean-Baptiste; Fernandez, Annabelle; Derré-Bobillot, Aurélie; Borezée-Durant, Elise; Fleurot, Isabelle; Gruss, Alexandra; Lechardeur, Delphine

2017-01-16

Heme is essential for several cellular key functions but is also toxic. Whereas most bacterial pathogens utilize heme as a metabolic cofactor and iron source, the impact of host heme during bacterial infection remains elusive. The opportunist pathogen Streptococcus agalactiae does not synthesize heme but still uses it to activate a respiration metabolism. Concomitantly, heme toxicity is mainly controlled by the HrtBA efflux transporter. Here we investigate how S. agalactiae manages heme toxicity versus benefits in the living host. Using bioluminescent bacteria and heme-responsive reporters for in vivo imaging, we show that the capacity of S. agalactiae to overcome heme toxicity is required for successful infection, particularly in blood-rich organs. Host heme is simultaneously required, as visualized by a generalized infection defect of a respiration-negative mutant. In S. agalactiae, HrtBA expression responds to an intracellular heme signal via activation of the two-component system HssRS. A hssRS promoter-driven intracellular luminescent heme sensor was designed to identify host compartments that supply S. agalactiae with heme. S. agalactiae acquires heme in heart, kidneys, and liver, but not in the brain. We conclude that S. agalactiae response to heme is organ-dependent, and its efflux may be particularly relevant in late stages of infection.

Mononuclear non-heme iron enzymes with the 2-His-1-carboxylate facial triad: recent developments in enzymology and modeling studies.

Science.gov (United States)

Bruijnincx, Pieter C A; van Koten, Gerard; Klein Gebbink, Robertus J M

2008-12-01

Iron-containing enzymes are one of Nature's main means of effecting key biological transformations. The mononuclear non-heme iron oxygenases and oxidases have received the most attention recently, primarily because of the recent availability of crystal structures of many different enzymes and the stunningly diverse oxidative transformations that these enzymes catalyze. The wealth of available structural data has furthermore established the so-called 2-His-1-carboxylate facial triad as a new common structural motif for the activation of dioxygen. This superfamily of mononuclear iron(ii) enzymes catalyzes a wide range of oxidative transformations, ranging from the cis-dihydroxylation of arenes to the biosynthesis of antibiotics such as isopenicillin and fosfomycin. The remarkable scope of oxidative transformations seems to be even broader than that associated with oxidative heme enzymes. Not only are many of these oxidative transformations of key biological importance, many of these selective oxidations are also unprecedented in synthetic organic chemistry. In this critical review, we wish to provide a concise background on the chemistry of the mononuclear non-heme iron enzymes characterized by the 2-His-1-carboxylate facial triad and to discuss the many recent developments in the field. New examples of enzymes with unique reactivities belonging to the superfamily have been reported. Furthermore, key insights into the intricate mechanistic details and reactive intermediates have been obtained from both enzyme and modeling studies. Sections of this review are devoted to each of these subjects, i.e. the enzymes, biomimetic models, and reactive intermediates (225 references).

Interactions between Carotenoids from Marine Bacteria and Other Micronutrients: Impact on Stability and Antioxidant Activity.

Science.gov (United States)

Sy, Charlotte; Dangles, Olivier; Borel, Patrick; Caris-Veyrat, Catherine

2015-11-19

Recently isolated spore-forming pigmented marine bacteria Bacillus indicus HU36 are sources of oxygenated carotenoids with original structures (about fifteen distinct yellow and orange pigments with acylated d-glucosyl groups). In this study, we evaluated the stability (sensitivity to iron-induced autoxidation) and antioxidant activity (inhibition of iron-induced lipid peroxidation) of combinations of bacterial HU36 carotenoids with the bacterial vitamin menaquinone MQ-7 and with phenolic antioxidants (vitamin E, chlorogenic acid, rutin). Unexpectedly, MQ-7 strongly improves the ability of HU36 carotenoids to inhibit Fe(II)-induced lipid peroxidation, although MQ-7 was not consumed in the medium. We propose that their interaction modifies the carotenoid antioxidant mechanism(s), possibly by allowing carotenoids to scavenge the initiating radicals. For comparison, β-carotene and lycopene in combination were shown to exhibit a slightly higher stability toward iron-induced autoxidation, as well as an additive antioxidant activity as compared to the carotenoids, individually. HU36 carotenoids and phenolic antioxidants displayed synergistic activities in the inhibition of linoleic acid peroxidation induced by heme iron, but not by free iron. Synergism could arise from antioxidants interacting via electron transfer through the porphyrin nucleus of heme iron. Overall, combining antioxidants acting via complementary mechanisms could be the key for optimizing the activity of this bacterial carotenoid cocktail.

Stanniocalcin 1 binds hemin through a partially conserved heme regulatory motif

Energy Technology Data Exchange (ETDEWEB)

Westberg, Johan A., E-mail: johan.westberg@helsinki.fi [Department of Pathology, Haartman Institute, University of Helsinki and HUSLAB, P.O. Box 21, Haartmaninkatu 3, FI-00014 Helsinki (Finland); Jiang, Ji, E-mail: ji.jiang@helsinki.fi [Department of Pathology, Haartman Institute, University of Helsinki and HUSLAB, P.O. Box 21, Haartmaninkatu 3, FI-00014 Helsinki (Finland); Andersson, Leif C., E-mail: leif.andersson@helsinki.fi [Department of Pathology, Haartman Institute, University of Helsinki and HUSLAB, P.O. Box 21, Haartmaninkatu 3, FI-00014 Helsinki (Finland)

2011-06-03

Highlights: {yields} Stanniocalcin 1 (STC1) binds heme through novel heme binding motif. {yields} Central iron atom of heme and cysteine-114 of STC1 are essential for binding. {yields} STC1 binds Fe{sup 2+} and Fe{sup 3+} heme. {yields} STC1 peptide prevents oxidative decay of heme. -- Abstract: Hemin (iron protoporphyrin IX) is a necessary component of many proteins, functioning either as a cofactor or an intracellular messenger. Hemoproteins have diverse functions, such as transportation of gases, gas detection, chemical catalysis and electron transfer. Stanniocalcin 1 (STC1) is a protein involved in respiratory responses of the cell but whose mechanism of action is still undetermined. We examined the ability of STC1 to bind hemin in both its reduced and oxidized states and located Cys{sup 114} as the axial ligand of the central iron atom of hemin. The amino acid sequence differs from the established (Cys-Pro) heme regulatory motif (HRM) and therefore presents a novel heme binding motif (Cys-Ser). A STC1 peptide containing the heme binding sequence was able to inhibit both spontaneous and H{sub 2}O{sub 2} induced decay of hemin. Binding of hemin does not affect the mitochondrial localization of STC1.

Introduction of a covalent histidine-heme linkage in a hemoglobin: a promising tool for heme protein engineering.

Science.gov (United States)

Rice, Selena L; Preimesberger, Matthew R; Johnson, Eric A; Lecomte, Juliette T J

2014-12-01

The hemoglobins of the cyanobacteria Synechococcus and Synechocystis (GlbNs) are capable of spontaneous and irreversible attachment of the b heme to the protein matrix. The reaction, which saturates the heme 2-vinyl by addition of a histidine residue, is reproduced in vitro by preparing the recombinant apoprotein, adding ferric heme, and reducing the iron to the ferrous state. Spontaneous covalent attachment of the heme is potentially useful for protein engineering purposes. Thus, to explore whether the histidine-heme linkage can serve in such applications, we attempted to introduce it in a test protein. We selected as our target the heme domain of Chlamydomonas eugametos LI637 (CtrHb), a eukaryotic globin that exhibits less than 50% sequence identity with the cyanobacterial GlbNs. We chose two positions, 75 in the FG corner and 111 in the H helix, to situate a histidine near a vinyl group. We characterized the proteins with gel electrophoresis, absorbance spectroscopy, and NMR analysis. Both T111H and L75H CtrHbs reacted upon reduction of the ferric starting material containing cyanide as the distal ligand to the iron. With L75H CtrHb, nearly complete (>90%) crosslinking was observed to the 4-vinyl as expected from the X-ray structure of wild-type CtrHb. Reaction of T111H CtrHb also occurred at the 4-vinyl, in a 60% yield indicating a preference for the flipped heme orientation in the starting material. The work suggests that the His-heme modification will be applicable to the design of proteins with a non-dissociable heme group. Copyright © 2014 Elsevier Inc. All rights reserved.

A functional mimic of natural peroxidases : synthesis and catalytic activity of a non-heme iron peptide hydroperoxide complex

NARCIS (Netherlands)

Choma, CT; Schudde, EP; Kellogg, RM; Robillard, GT; Feringa, BL

1998-01-01

Site-selective attachment of unprotected peptides to a non-heme iron complex is achieved by displacing two halides on the catalyst by peptide caesium thiolates, This coupling approach should be compatible with any peptide sequence provided there is only a single reduced cysteine. The oxidation

Heme oxygenase-1, oxidation, inflammation and atherosclerosis

Directory of Open Access Journals (Sweden)

Jesus A Araujo

2012-07-01

Full Text Available Atherosclerosis is an inflammatory process of the vascular wall characterized by the infiltration of lipids and inflammatory cells. Oxidative modifications of infiltrating low density lipoproteins and induction of oxidative stress play a major role in lipid retention in the vascular wall, uptake by macrophages and generation of foam cells, a hallmark of this disorder. The vasculature has a plethora of protective resources against oxidation and inflammation, many of them regulated by the Nrf2 transcription factor. Heme oxygenase-1 (HO-1 is a Nrf2-regulated gene that plays a critical role in the prevention of vascular inflammation. It is the inducible isoform of heme oxygenase, responsible for the oxidative cleavage of heme groups leading to the generation of biliverdin, carbon monoxide and release of ferrous iron. HO-1 has important antioxidant, antiinflammatory, antiapoptotic, antiproliferative and immunomodulatory effects in vascular cells, most of which play a significant role in the protection against atherogenesis. HO-1 may also be an important feature in macrophage differentiation and polarization to certain subtypes. The biological effects of HO-1 are largely attributable to its enzymatic activity, which can be conceived as a system with three arms of action, corresponding to its three enzymatic byproducts. HO-1 mediated vascular protection may be due to a combination of systemic and vascular local effects. It is usually expressed at low levels but can be highly upregulated in the presence of several proatherogenic stimuli. The HO-1 system is amenable for use in the development of new therapies, some of them currently under experimental and clinical trials. Interestingly, in contrast to the HO-1 antiatherogenic actions, the expression of its transcriptional regulator Nrf2 leads to proatherogenic effects instead. This article reviews the evidence that supports the antiatherogenic role of HO-1, potential pathways and mechanisms mediating

The Bacillus subtilis Acyl Lipid Desaturase Is a Δ5 Desaturase

Science.gov (United States)

Altabe, Silvia G.; Aguilar, Pablo; Caballero, Gerardo M.; de Mendoza, Diego

2003-01-01

Bacillus subtilis was recently reported to synthesize unsaturated fatty acids (UFAs) with a double bond at positions Δ5, Δ7, and Δ9 (M. H. Weber, W. Klein, L. Muller, U. M. Niess, and M. A. Marahiel, Mol. Microbiol. 39:1321-1329, 2001). Since this finding would have considerable importance in the double-bond positional specificity displayed by the B. subtilis acyl lipid desaturase, we have attempted to confirm this observation. We report that the double bond of UFAs synthesized by B. subtilis is located exclusively at the Δ5 position, regardless of the growth temperature and the length chain of the fatty acids. PMID:12730185

LipidPedia: a comprehensive lipid knowledgebase.

Science.gov (United States)

Kuo, Tien-Chueh; Tseng, Yufeng Jane

2018-04-10

Lipids are divided into fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, sterols, prenol lipids and polyketides. Fatty acyls and glycerolipids are commonly used as energy storage, whereas glycerophospholipids, sphingolipids, sterols and saccharolipids are common used as components of cell membranes. Lipids in fatty acyls, glycerophospholipids, sphingolipids and sterols classes play important roles in signaling. Although more than 36 million lipids can be identified or computationally generated, no single lipid database provides comprehensive information on lipids. Furthermore, the complex systematic or common names of lipids make the discovery of related information challenging. Here, we present LipidPedia, a comprehensive lipid knowledgebase. The content of this database is derived from integrating annotation data with full-text mining of 3,923 lipids and more than 400,000 annotations of associated diseases, pathways, functions, and locations that are essential for interpreting lipid functions and mechanisms from over 1,400,000 scientific publications. Each lipid in LipidPedia also has its own entry containing a text summary curated from the most frequently cited diseases, pathways, genes, locations, functions, lipids and experimental models in the biomedical literature. LipidPedia aims to provide an overall synopsis of lipids to summarize lipid annotations and provide a detailed listing of references for understanding complex lipid functions and mechanisms. LipidPedia is available at http://lipidpedia.cmdm.tw. yjtseng@csie.ntu.edu.tw. Supplementary data are available at Bioinformatics online.

Heme and erythropoieis: more than a structural role

OpenAIRE

Chiabrando, Deborah; Mercurio, Sonia; Tolosano, Emanuela

2014-01-01

Erythropoiesis is the biological process that consumes the highest amount of body iron for heme synthesis. Heme synthesis in erythroid cells is finely coordinated with that of alpha (α) and beta (β)-globin, resulting in the production of hemoglobin, a tetramer of 2α- and 2β-globin chains, and heme as the prosthetic group. Heme is not only the structural component of hemoglobin, but it plays multiple regulatory roles during the differentiation of erythroid precursors since it controls its own ...

Myoglobins: the link between discoloration and lipid oxidation in muscle and meat

Directory of Open Access Journals (Sweden)

Jens K. S. Møller

2006-12-01

Full Text Available Aerobic metabolism changes rapidly to glycolysis post-mortem resulting in a pH-decrease during the transformation of muscle in to meat affecting ligand binding and redox potential of the heme iron in myoglobin, the meat pigment. The "inorganic chemistry" of meat involves (i redox-cycling between iron(II, iron(III, and iron(IV/protein radicals; (ii ligand exchange processes; and (iii spin-equilibra with a change in coordination number for the heme iron. In addition to the function of myoglobin for oxygen storage, new physiological roles of myoglobin are currently being discovered, which notably find close parallels in the processes in fresh meat and nitrite-cured meat products. Myoglobin may be characterized as a bioreactor for small molecules like O2, NO, CO, CO2, H2O, and HNO with importance in bio-regulation and in protection against oxidative stress in vivo otherwise affecting lipids in membranes. Many of these processes may be recognised as colour changes in fresh meat and cured meat products under different atmospheric conditions, and could also be instructive for teaching purposes.

Practice guidelines for the diagnosis and management of microcytic anemias due to genetic disorders of iron metabolism or heme synthesis

NARCIS (Netherlands)

Donker, A.E.; Raymakers, R.A.P.; Vlasveld, L.T.; Barneveld, T. van; Terink, R.; Dors, N.; Brons, P.P.T.; Knoers, N.V.A.M.; Swinkels, D.W.

2014-01-01

During recent years, our understanding of the pathogenesis of inherited microcytic anemias has gained from the identification of several genes and proteins involved in systemic and cellular iron metabolism and heme syntheses. Numerous case reports illustrate that the implementation of these novel

Nitric oxide and iron modulate heme oxygenase activity as a long distance signaling response to salt stress in sunflower seedling cotyledons.

Science.gov (United States)

Singh, Neha; Bhatla, Satish C

2016-02-29

Nitric oxide is a significant component of iron signaling in plants. Heme is one of the iron sensors in plants. Free heme is highly toxic and can cause cell damage as it catalyzes the formation of reactive oxygen species (ROS). Its catabolism is carried out by heme oxygenase (HOs; EC 1.14.99.3) which uses heme both as a prosthetic group and as a substrate. Two significant events, which accompany adaptation to salt stress in sunflower seedlings, are accumulation of ROS and enhanced production of nitric oxide (NO) in roots and cotyledons. Present investigations on the immunolocalization of heme oxygenase distribution in sunflower seedling cotyledons by confocal laser scanning microscopic (CLSM) imaging provide new information on the differential spatial distribution of the inducible form of HO (HO-1) as a long distance in response to NaCl stress. The enzyme is abundantly distributed in the specialized cells around the secretory canals (SCs) in seedling cotyledons. Abundance of tyrosine nitrated proteins has also been observed in the specialized cells around the secretory canals in cotyledons derived from salt stressed seedlings. The spatial distribution of tyrosine nitrated proteins and HO-1 expression further correlates with the abundance of mitochondria in these cells. Present findings, thus, highlight a link among distribution of HO-1 expression, abundance of tyrosine nitrated proteins and mitochondria in specialized cells around the secretory canal as a long distance mechanism of salt stress tolerance in sunflower seedlings. Enhanced spatial distribution of HO-1 in response to NaCl stress in seedling cotyledons is in congruence with the observed increase in specific activity of HO-1 in NaCl stressed conditions. The enzyme activity is further enhanced by hemin (HO-1 inducer) both in the absence or presence of NaCl stress and inhibited by zinc protoporphyrin. Western blot analysis of cotyledon homogenates using anti-HO-1 polyclonal antibody shows one major band (29

Targeted Lipidomics in Drosophila melanogaster Identifies Novel 2-Monoacylglycerols and N-acyl Amides

Science.gov (United States)

Takacs, Sara M.; Stuart, Jordyn M.; Basnet, Arjun; Raboune, Siham; Widlanski, Theodore S.; Doherty, Patrick; Bradshaw, Heather B.

2013-01-01

Lipid metabolism is critical to coordinate organ development and physiology in response to tissue-autonomous signals and environmental cues. Changes to the availability and signaling of lipid mediators can limit competitiveness, adaptation to environmental stressors, and augment pathological processes. Two classes of lipids, the N-acyl amides and the 2-acyl glycerols, have emerged as important signaling molecules in a wide range of species with important signaling properties, though most of what is known about their cellular functions is from mammalian models. Therefore, expanding available knowledge on the repertoire of these lipids in invertebrates will provide additional avenues of research aimed at elucidating biosynthetic, metabolic, and signaling properties of these molecules. Drosophila melanogaster is a commonly used organism to study intercellular communication, including the functions of bioactive lipids. However, limited information is available on the molecular identity of lipids with putative biological activities in Drosophila. Here, we used a targeted lipidomics approach to identify putative signaling lipids in third instar Drosophila larvae, possessing particularly large lipid mass in their fat body. We identified 2-linoleoyl glycerol, 2-oleoyl glycerol, and 45 N-acyl amides in larval tissues, and validated our findings by the comparative analysis of Oregon-RS, Canton-S and w1118 strains. Data here suggest that Drosophila represent another model system to use for the study of 2-acyl glycerol and N-acyl amide signaling. PMID:23874457

4-Hydroxy-2(E)-nonenal metabolism differs in Apc(+/+) cells and in Apc(Min/+) cells: it may explain colon cancer promotion by heme iron.

Science.gov (United States)

Baradat, Maryse; Jouanin, Isabelle; Dalleau, Sabine; Taché, Sylviane; Gieules, Mathilde; Debrauwer, Laurent; Canlet, Cécile; Huc, Laurence; Dupuy, Jacques; Pierre, Fabrice H F; Guéraud, Françoise

2011-11-21

Animal and epidemiological studies suggest that dietary heme iron would promote colorectal cancer. Oxidative properties of heme could lead to the formation of cytotoxic and genotoxic secondary lipid oxidation products, such as 4-hydroxy-2(E)-nonenal (HNE). This compound is more cytotoxic to mouse wild-type colon cells than to isogenic cells with a mutation on the adenomatous polyposis coli (APC) gene. The latter thus have a selective advantage, possibly leading to cancer promotion. This mutation is an early and frequent event in human colorectal cancer. To explain this difference, the HNE biotransformation capacities of the two cell types have been studied using radiolabeled and stable isotope-labeled HNE. Apc-mutated cells showed better biotransformation capacities than nonmutated cells did. Thiol compound conjugation capacities were higher for mutated cells, with an important advantage for the extracellular conjugation to cysteine. Both cells types were able to reduce HNE to 4-hydroxynonanal, a biotransformation pathway that has not been reported for other intestinal cells. Mutated cells showed higher capacities to oxidize 4-hydroxynonanal into 4-hydroxynonanoic acid. The mRNA expression of different enzymes involved in HNE metabolism such as aldehyde dehydrogenase 1A1, 2 and 3A1, glutathione transferase A4-4, or cystine transporter xCT was upregulated in mutated cells compared with wild-type cells. In conclusion, this study suggests that Apc-mutated cells are more efficient than wild-type cells in metabolizing HNE into thiol conjugates and 4-hydroxynonanoic acid due to the higher expression of key biotransformation enzymes. These differential biotransformation capacities would explain the differences of susceptibility between normal and Apc-mutated cells regarding secondary lipid oxidation products.

Characterization of Heme Proteins Involved in Microbial Exoelectric Activity and Small Molecule-Sensing

KAUST Repository

Vogler, Malvina M.

2018-01-01

Heme proteins, also termed cytochromes, are a widespread class of metalloproteins containing an Fe-protoporphyrin IX cofactor. They perform numerous functions in nature such as oxygen-transport by hemoglobin, monooxygenation reactions catalyzed by Cytochrome P-450, and electron transfer reactions during photosynthesis. The differences between proteincofactor binding characteristics and the cofactor environment greatly influence the extensive range of functions. In this dissertation, proteins from the Mtr pathway of Shewanella oneidensis are characterized. These c-type cytochromes contain multiple heme cofactors per protein molecule that covalently attach to the protein amino acid sequence and are involved in electron transfer to extracellular metal oxides during anaerobic conditions. Successful recombinant expression of pathway components MtrC and MtrA is achieved in Escherichia coli. Heme-dependent gel staining and UV/Vis spectroscopy show characteristic c-type cytochrome characteristics. Mass spectrometry confirms that the correct extensive post-translational modifications were performed and the ten heme groups were incorporated per protein of MtrC and MtrA and the correct lipid-anchor was attached to extracellular MtrC. Raman spectroscopy measurements of MtrA provide intriguing structural information and highlight the strong influence of the heme cofactors within the protein structure. Next, an Arabidopsis thaliana protein is analyzed. It was previously identified via a motif search of the plant genome, based on conserved residues in the H4 NOX pocket. Here, the incorporation of a heme b cofactor is confirmed. UV/Vis spectroscopy under anaerobic conditions demonstrates reversible binding of nitric oxide to the heme iron and depicts the previously published characteristic absorption maxima for other H-NOX proteins.

A Heme-responsive Regulator Controls Synthesis of Staphyloferrin B in Staphylococcus aureus.

Science.gov (United States)

Laakso, Holly A; Marolda, Cristina L; Pinter, Tyler B; Stillman, Martin J; Heinrichs, David E

2016-01-01

Staphylococcus aureus possesses a multitude of mechanisms by which it can obtain iron during growth under iron starvation conditions. It expresses an effective heme acquisition system (the iron-regulated surface determinant system), it produces two carboxylate-type siderophores staphyloferrin A and staphyloferrin B (SB), and it expresses transporters for many other siderophores that it does not synthesize. The ferric uptake regulator protein regulates expression of genes encoding all of these systems. Mechanisms of fine-tuning expression of iron-regulated genes, beyond simple iron regulation via ferric uptake regulator, have not been uncovered in this organism. Here, we identify the ninth gene of the sbn operon, sbnI, as encoding a ParB/Spo0J-like protein that is required for expression of genes in the sbn operon from sbnD onward. Expression of sbnD-I is drastically decreased in an sbnI mutant, and the mutant does not synthesize detectable SB during early phases of growth. Thus, SB-mediated iron acquisition is impaired in an sbnI mutant strain. We show that the protein forms dimers and tetramers in solution and binds to DNA within the sbnC coding region. Moreover, we show that SbnI binds heme and that heme-bound SbnI does not bind DNA. Finally, we show that providing exogenous heme to S. aureus growing in an iron-free medium results in delayed synthesis of SB. This is the first study in S. aureus that identifies a DNA-binding regulatory protein that senses heme to control gene expression for siderophore synthesis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of the heme acquisition system in Vibrio vulnificus M2799.

Science.gov (United States)

Kawano, Hiroaki; Miyamoto, Katsushiro; Yasunobe, Megumi; Murata, Masahiro; Yamahata, Eri; Yamaguchi, Ryo; Miyaki, Yuta; Tsuchiya, Takahiro; Tanabe, Tomotaka; Funahashi, Tatsuya; Tsujibo, Hiroshi

2018-04-01

Vibrio vulnificus, the causative agent of serious, often fatal, infections in humans, requires iron for its pathogenesis. As such, it obtains iron via both vulnibactin and heme-mediated iron-uptake systems. In this study, we identified the heme acquisition system in V. vulnificus M2799. The nucleotide sequences of the genes encoding heme receptors HupA and HvtA and the ATP-binding cassette (ABC) transport system proteins HupB, HupC, and HupD were determined, and then used in the construction of deletion mutants developed from a Δics strain, which could not synthesize vulnibactin. Growth experiments using these mutants indicated that HupA and HvtA are major and minor heme receptors, respectively. The expressions of two proteins were analyzed by the quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Furthermore, complementation analyses confirmed that the HupBCD proteins are the only ABC transport system shared by both the HupA and HvtA receptors. This is the first genetic evidence that the HupBCD proteins are essential for heme acquisition by V. vulnificus. Further investigation showed that hupA, hvtA, and hupBCD are regulated by Fur. The qRT-PCR analysis of the heme receptor genes revealed that HupR, a LysR-family positive transcriptional activator, upregulates the expression of hupA, but not hvtA. In addition, ptrB was co-transcribed with hvtA, and PtrB had no influence on growth in low-iron CM9 medium supplemented with hemin, hemoglobin, or cytochrome C. Copyright © 2018 Elsevier Ltd. All rights reserved.

The detection of iron protoporphyrin (heme b) in phytoplankton and marine particulate material by electrospray ionisation mass spectrometry – comparison with diode array detection

Energy Technology Data Exchange (ETDEWEB)

Gledhill, Martha, E-mail: m.gledhill@geomar.de

2014-09-02

Highlights: • Mass spectrometry was applied to the analysis of heme b in biological material. • Optimal conditions involved selective reactant monitoring of the heme b product ion. • The isotopic signature for this iron tetrapyrrole further improved selectivity. • Mass spectrometry and spectrophotometry were compared for heme b analysis. • Combining techniques made a powerful tool for analysis of heme in marine microbes. - Abstract: A mass spectrometric (MS) method for the identification of iron protoporphyrin (IX) (FePTP, heme b) in marine particulate material and phytoplankton is described. Electrospray ionisation of FePTP produced the molecular Fe(III)PTP{sup +} ion (m/z = 616) or the pseudomolecular [Fe(II)PTP + H]{sup +} ion (m/z = 617), depending on the oxidation state of the central iron ion. Collision induced dissociation (CID) in the ion trap mass spectrometer resulted in a single detected product ion (m/z = 557) indicative of loss of ethanoic acid from a carboxylic acid side chain. Widening the isolation width to 616 ± 3 resulted in production of a mass spectrum demonstrating the distinctive isotopic ratio of the iron containing fragment, further increasing the specificity of the analysis. Selective reactant monitoring (SRM) of the fragment ion (m/z = 557) was applied to the detection of FePTP after chromatography of ammoniacal OGP extracts of marine samples. The detection limit for FePTP analysed by SRM after chromatography was 1.2 ± 0.5 fmol. For phytoplankton samples, reasonably good agreement was achieved between results obtained with SRM and those obtained by monitoring absorbance at λ = 400 nm using a diode array detector (DAD). Use of SRM for analysis of particulate material obtained from the high latitude North Atlantic allowed for the analysis of FePTP in the presence of a co-eluting compound that interfered with detection by DAD. Simultaneous collection of mass spectra from m/z = 300 to 1500 resulted in identification of the

Moessbauer spectroscopic study of polymer-bound heme complexes

International Nuclear Information System (INIS)

Tsuchida, Eishun; Nishide, Hiroyuki; Yokoyama, Hiroyuki; Inoue, Hidenari; Shirai, Tsuneo.

1984-01-01

Moessbauer spectra were measured on the heme complexes of poly(1-vinyl- and 1-vinyl-2-methylimidazole)(PVI and PMI) and heme derivatives with covalently bound imidazoleligand (IH) and 2-methylimidazole-ligand (MIH) embedded in poly(1-vinyl-2-pyrrolidone) film. Quadrupole splitting (ΔE sub(Q)) for the carbon monoxide adduct of PMI-heme indicated large electronic field gradient at the iron nucleus, probably due to steric hindrance of the polymer chain, and this behavior agreed with its low affinity with carbon monoxide. PMI-heme formed an oxygen adduct and its isomer shift and ΔE sub(Q) values were obtained. (author)

Identification of the Mitochondrial Heme Metabolism Complex.

Science.gov (United States)

Medlock, Amy E; Shiferaw, Mesafint T; Marcero, Jason R; Vashisht, Ajay A; Wohlschlegel, James A; Phillips, John D; Dailey, Harry A

2015-01-01

Heme is an essential cofactor for most organisms and all metazoans. While the individual enzymes involved in synthesis and utilization of heme are fairly well known, less is known about the intracellular trafficking of porphyrins and heme, or regulation of heme biosynthesis via protein complexes. To better understand this process we have undertaken a study of macromolecular assemblies associated with heme synthesis. Herein we have utilized mass spectrometry with coimmunoprecipitation of tagged enzymes of the heme biosynthetic pathway in a developing erythroid cell culture model to identify putative protein partners. The validity of these data obtained in the tagged protein system is confirmed by normal porphyrin/heme production by the engineered cells. Data obtained are consistent with the presence of a mitochondrial heme metabolism complex which minimally consists of ferrochelatase, protoporphyrinogen oxidase and aminolevulinic acid synthase-2. Additional proteins involved in iron and intermediary metabolism as well as mitochondrial transporters were identified as potential partners in this complex. The data are consistent with the known location of protein components and support a model of transient protein-protein interactions within a dynamic protein complex.

A Heme-responsive Regulator Controls Synthesis of Staphyloferrin B in Staphylococcus aureus*♦

Science.gov (United States)

Laakso, Holly A.; Marolda, Cristina L.; Pinter, Tyler B.; Stillman, Martin J.; Heinrichs, David E.

2016-01-01

Staphylococcus aureus possesses a multitude of mechanisms by which it can obtain iron during growth under iron starvation conditions. It expresses an effective heme acquisition system (the iron-regulated surface determinant system), it produces two carboxylate-type siderophores staphyloferrin A and staphyloferrin B (SB), and it expresses transporters for many other siderophores that it does not synthesize. The ferric uptake regulator protein regulates expression of genes encoding all of these systems. Mechanisms of fine-tuning expression of iron-regulated genes, beyond simple iron regulation via ferric uptake regulator, have not been uncovered in this organism. Here, we identify the ninth gene of the sbn operon, sbnI, as encoding a ParB/Spo0J-like protein that is required for expression of genes in the sbn operon from sbnD onward. Expression of sbnD–I is drastically decreased in an sbnI mutant, and the mutant does not synthesize detectable SB during early phases of growth. Thus, SB-mediated iron acquisition is impaired in an sbnI mutant strain. We show that the protein forms dimers and tetramers in solution and binds to DNA within the sbnC coding region. Moreover, we show that SbnI binds heme and that heme-bound SbnI does not bind DNA. Finally, we show that providing exogenous heme to S. aureus growing in an iron-free medium results in delayed synthesis of SB. This is the first study in S. aureus that identifies a DNA-binding regulatory protein that senses heme to control gene expression for siderophore synthesis. PMID:26534960

In vivo heme scavenging by Staphylococcus aureus IsdC and IsdE proteins

International Nuclear Information System (INIS)

Mack, John; Vermeiren, Christie; Heinrichs, David E.; Stillman, Martin J.

2004-01-01

We report the first characterization of the in vivo porphyrin scavenging abilities of two components of a newly discovered heme scavenging system involving iron-regulated surface determinant (Isd) proteins. These proteins are present within the cell envelope of the Gram-positive human pathogen Staphylococcus aureus. IsdC and IsdE, when expressed heterologously in Escherichia coli, efficiently scavenged intracellular heme and resulted in de novo heme synthesis in excess of 100-fold above background. Magnetic circular dichroism analyses showed that the heme-binding properties of the two proteins differ significantly from one another. IsdC bound almost exclusively free-base protoporphyrin IX, whereas the IsdE protein was associated with low spin Fe(III) and Fe(II) heme. These properties provide important insight into the possible mechanisms of iron scavenging from bound heme by Isd proteins

One ring to rule them all: trafficking of heme and heme synthesis intermediates in the metazoans.

Science.gov (United States)

Hamza, Iqbal; Dailey, Harry A

2012-09-01

The appearance of heme, an organic ring surrounding an iron atom, in evolution forever changed the efficiency with which organisms were able to generate energy, utilize gasses and catalyze numerous reactions. Because of this, heme has become a near ubiquitous compound among living organisms. In this review we have attempted to assess the current state of heme synthesis and trafficking with a goal of identifying crucial missing information, and propose hypotheses related to trafficking that may generate discussion and research. The possibilities of spatially organized supramolecular enzyme complexes and organelle structures that facilitate efficient heme synthesis and subsequent trafficking are discussed and evaluated. Recently identified players in heme transport and trafficking are reviewed and placed in an organismal context. Additionally, older, well established data are reexamined in light of more recent studies on cellular organization and data available from newer model organisms. This article is part of a Special Issue entitled: Cell Biology of Metals. Copyright © 2012 Elsevier B.V. All rights reserved.

Pilot batch production of specific-structured lipids by lipase-catalyzed interesterification: preliminary study on incorporation and acyl migration

DEFF Research Database (Denmark)

Xu, Xuebing; Balchen, Steen; Høy, Carl-Erik

1998-01-01

Effects of water content, reaction time and their relationships in the production of two types of specific-structured lipids (sn-MLM- and sn-LML-types: L-long chain fatty acids; M-medium chain fatty acids) by lipase-catalyzed interesterification in a solvent-free system were studied...... of two totally position-opposed lipids can be observed. Presumably these are caused by the different chain length of the fatty acids. The relationships between reaction time and water content are inverse and give a quantitative prediction of incorporation and acyl migration in selected reaction...

Dinitrosyl non-heme iron complexes at the gamma radiation treatment of animals

International Nuclear Information System (INIS)

Aliev, D.I.; Alieva, I.N.; Abilov, Z.G.; Gurbanov, I.S.

2003-01-01

Full text: At the present time there are a great number investigations dedicated to revealing of mechanism formation of 2,03 complexes at the some pathologies in an organism. These complexes are represented weakly bounded form of non-heme iron, including into beside iron two nitrogen oxide molecules (NO) and two paired RS- groups of proteins or low-molecular compounds. 2,03 complexes are characterized by an axial symmetrical tensory of the g-factor with g=2,037, g=2,012 and g=2,03. In this study the data testifying 2,03 complexes formation into liver of animal treated by the fatal dose of gamma-radiation are reported. The changing of the ESR signal form was observed. It was shown that the form and intensity of the 2,03 signal in healthy and irradiated animals are differ from each other. The analysis of the 2,03 signal parameters is confirm this fact, too. The conclusion was made that 2,03 complexes ESR signal may be considered as an indicator of integrity of intracellular membranes of the gamma-irradiated animals

Understanding Acyl Chain and Glycerolipid Metabolism in Plants

Energy Technology Data Exchange (ETDEWEB)

Ohlrogge, John B.

2013-11-05

Progress is reported in these areas: acyl-editing in initial eukaryotic lipid assembly in soybean seeds; identification and characterization of two Arabidopsis thaliana lysophosphatidyl acyltransferases with preference for lysophosphatidylethanolamine; and characterization and subcellular distribution of lysolipid acyl transferase activity of pea leaves.

In vivo genotoxic effects of dietary heme iron on rat colon mucosa and ex vivo effects on colon cells monitored by an optimized alkaline comet assay.

Directory of Open Access Journals (Sweden)

Océane, C Martin

2015-04-01

In conclusion, our results offer a suitable protocol to evaluate genotoxicity on in vivo cryopreserved colon mucosa and on in vitro murine colonic cells, with a middle throughput capacity. This protocol confirms the increase of genotoxicity in rat colon mucosa after an heme-iron diet. Moreover, this protocol enables the demonstration that aldehydes from heme-induced lipoperoxidation are responsible for this increase of genotoxicity.

Antibiotic suppression of intestinal microbiota reduces heme-induced lipoperoxidation associated with colon carcinogenesis in rats.

Science.gov (United States)

Martin, O C B; Lin, C; Naud, N; Tache, S; Raymond-Letron, I; Corpet, D E; Pierre, F H

2015-01-01

Epidemiological studies show that heme iron from red meat is associated with increased colorectal cancer risk. In carcinogen-induced-rats, a heme iron-rich diet increases the number of precancerous lesions and raises associated fecal biomarkers. Heme-induced lipoperoxidation measured by fecal thiobarbituric acid reagents (TBARs) could explain the promotion of colon carcinogenesis by heme. Using a factorial design we studied if microbiota could be involved in heme-induced carcinogenesis, by modulating peroxidation. Rats treated or not with an antibiotic cocktail were given a control or a hemoglobin-diet. Fecal bacteria were counted on agar and TBARs concentration assayed in fecal water. The suppression of microbiota by antibiotics was associated with a reduction of crypt height and proliferation and with a cecum enlargement, which are characteristics of germ-free rats. Rats given hemoglobin diets had increased fecal TBARs, which were suppressed by the antibiotic treatment. A duplicate experiment in rats given dietary hemin yielded similar results. These data show that the intestinal microbiota is involved in enhancement of lipoperoxidation by heme iron. We thus suggest that microbiota could play a role in the heme-induced promotion of colorectal carcinogenesis.

Heme exporter FLVCR1a regulates heme synthesis and degradation and controls activity of cytochromes P450.

Science.gov (United States)

Vinchi, Francesca; Ingoglia, Giada; Chiabrando, Deborah; Mercurio, Sonia; Turco, Emilia; Silengo, Lorenzo; Altruda, Fiorella; Tolosano, Emanuela

2014-05-01

The liver has one of the highest rates of heme synthesis of any organ. More than 50% of the heme synthesized in the liver is used for synthesis of P450 enzymes, which metabolize exogenous and endogenous compounds that include natural products, hormones, drugs, and carcinogens. Feline leukemia virus subgroup C cellular receptor 1a (FLVCR1a) is plasma membrane heme exporter that is ubiquitously expressed and controls intracellular heme content in hematopoietic lineages. We investigated the role of Flvcr1a in liver function in mice. We created mice with conditional disruption of Mfsd7b, which encodes Flvcr1a, in hepatocytes (Flvcr1a(fl/fl);alb-cre mice). Mice were analyzed under basal conditions, after phenylhydrazine-induced hemolysis, and after induction of cytochromes P450 synthesis. Livers were collected and analyzed by histologic, quantitative real-time polymerase chain reaction, and immunoblot analyses. Hepatic P450 enzymatic activities were measured. Flvcr1a(fl/fl);alb-cre mice accumulated heme and iron in liver despite up-regulation of heme oxygenase 1, ferroportin, and ferritins. Hepatic heme export activity of Flvcr1a was closely associated with heme biosynthesis, which is required to sustain cytochrome induction. Upon cytochromes P450 stimulation, Flvcr1a(fl/fl);alb-cre mice had reduced cytochrome activity, associated with accumulation of heme in hepatocytes. The expansion of the cytosolic heme pool in these mice was likely responsible for the early inhibition of heme synthesis and increased degradation of heme, which reduced expression and activity of cytochromes P450. In livers of mice, Flvcr1a maintains a free heme pool that regulates heme synthesis and degradation as well as cytochromes P450 expression and activity. These findings have important implications for drug metabolism. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

Practice guidelines for the diagnosis and management of microcytic anemias due to genetic disorders of iron metabolism or heme synthesis.

Science.gov (United States)

Donker, Albertine E; Raymakers, Reinier A P; Vlasveld, L Thom; van Barneveld, Teus; Terink, Rieneke; Dors, Natasja; Brons, Paul P T; Knoers, Nine V A M; Swinkels, Dorine W

2014-06-19

During recent years, our understanding of the pathogenesis of inherited microcytic anemias has gained from the identification of several genes and proteins involved in systemic and cellular iron metabolism and heme syntheses. Numerous case reports illustrate that the implementation of these novel molecular discoveries in clinical practice has increased our understanding of the presentation, diagnosis, and management of these diseases. Integration of these insights into daily clinical practice will reduce delays in establishing a proper diagnosis, invasive and/or costly diagnostic tests, and unnecessary or even detrimental treatments. To assist the clinician, we developed evidence-based multidisciplinary guidelines on the management of rare microcytic anemias due to genetic disorders of iron metabolism and heme synthesis. These genetic disorders may present at all ages, and therefore these guidelines are relevant for pediatricians as well as clinicians who treat adults. This article summarizes these clinical practice guidelines and includes background on pathogenesis, conclusions, and recommendations and a diagnostic flowchart to facilitate using these guidelines in the clinical setting. © 2014 by The American Society of Hematology.

Genetic Variability of the Heme Uptake System among Different Strains of the Fish Pathogen Vibrio anguillarum: Identification of a New Heme Receptor

Science.gov (United States)

Mouriño, Susana; Rodríguez-Ares, Isabel; Osorio, Carlos R.; Lemos, Manuel L.

2005-01-01

The ability to utilize heme compounds as iron sources was investigated in Vibrio anguillarum strains belonging to serotypes O1 to O10. All strains, regardless of their serotype or isolation origin could utilize hemin and hemoglobin as sole iron sources. Similarly, all of the isolates could bind hemin and Congo red, and this binding was mediated by cell envelope proteins. PCR and Southern hybridization were used to assay the occurrence of heme transport genes huvABCD, which have been previously described in serotype O1. Of 23 strains studied, two serotype O3 isolates proved negative for all huvABCD genes, whereas nine strains included in serotypes O2, O3, O4, O6, O7, and O10 tested negative for the outer membrane heme receptor gene huvA. A gene coding for a novel outer membrane heme receptor was cloned and characterized in a V. anguillarum serotype O3 strain lacking huvA. The new heme receptor, named HuvS, showed significant similarity to other outer membrane heme receptors described in Vibrionaceae, but little homology (39%) to HuvA. This heme receptor was present in 9 out of 11 of the V. anguillarum strains that tested negative for HuvA. Furthermore, complementation experiments demonstrated that HuvS could substitute for the HuvA function in Escherichia coli and V. anguillarum mutants. The huvS and huvA sequences alignment, as well as the analysis of their respective upstream and downstream DNA sequences, suggest that horizontal transfer and recombination might be responsible for generating this genetic diversity. PMID:16332832

No changes in heme synthesis in human Friedreich´s ataxia erythroid progenitor cells.

Science.gov (United States)

Steinkellner, Hannes; Singh, Himanshu Narayan; Muckenthaler, Martina U; Goldenberg, Hans; Moganty, Rajeswari R; Scheiber-Mojdehkar, Barbara; Sturm, Brigitte

2017-07-20

Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by reduced expression of the protein frataxin. Frataxin is thought to play a role in iron-sulfur cluster biogenesis and heme synthesis. In this study, we used erythroid progenitor stem cells obtained from FRDA patients and healthy donors to investigate the putative role, if any, of frataxin deficiency in heme synthesis. We used electrochemiluminescence and qRT-PCR for frataxin protein and mRNA quantification. We used atomic absorption spectrophotometry for iron levels and a photometric assay for hemoglobin levels. Protoporphyrin IX and Ferrochelatase were analyzed using auto-fluorescence. An "IronChip" microarray analysis followed by a protein-protein interaction analysis was performed. FRDA patient cells showed no significant changes in iron levels, hemoglobin synthesis, protoporphyrin IX levels, and ferrochelatase activity. Microarray analysis presented 11 genes that were significantly changed in all patients compared to controls. The genes are especially involved in oxidative stress, iron homeostasis and angiogenesis. The mystery about the involvement of frataxin on iron metabolism raises the question why frataxin deficiency in primary FRDA cells did not lead to changes in biochemical parameters of heme synthesis. It seems that alternative pathways can circumvent the impact of frataxin deficiency on heme synthesis. We show for the first time in primary FRDA patient cells that reduced frataxin levels are still sufficient for heme synthesis and possibly other mechanisms can overcome reduced frataxin levels in this process. Our data strongly support the fact that so far no anemia in FRDA patients was reported. Copyright © 2017 Elsevier B.V. All rights reserved.

Heme Exporter FLVCR1a Regulates Heme Synthesis and Degradation and Controls Activity of Cytochromes P450

Science.gov (United States)

Vinchi, Francesca; Ingoglia, Giada; Chiabrando, Deborah; Mercurio, Sonia; Turco, Emilia; Silengo, Lorenzo; Altruda, Fiorella; Tolosano, Emanuela

2014-01-01

Background & Aims The liver has one of the highest rates of heme synthesis of any organ. More than 50% of the heme synthesized in the liver is used for synthesis of P450 enzymes, which metabolize exogenous and endogenous compounds that include natural products, hormones, drugs, and carcinogens. Feline leukemia virus subgroup C cellular receptor 1a (FLVCR1a) is plasma membrane heme exporter that is ubiquitously expressed and controls intracellular heme content in hematopoietic lineages. We investigated the role of Flvcr1a in liver function in mice. Methods We created mice with conditional disruption of Mfsd7b, which encodes Flvcr1a, in hepatocytes (Flvcr1afl/fl;alb-cre mice). Mice were analyzed under basal conditions, after phenylhydrazine-induced hemolysis, and after induction of cytochromes P450 synthesis. Livers were collected and analyzed by histologic, quantitative real-time polymerase chain reaction, and immunoblot analyses. Hepatic P450 enzymatic activities were measured. Results Flvcr1afl/fl;alb-cre mice accumulated heme and iron in liver despite up-regulation of heme oxygenase 1, ferroportin, and ferritins. Hepatic heme export activity of Flvcr1a was closely associated with heme biosynthesis, which is required to sustain cytochrome induction. Upon cytochromes P450 stimulation, Flvcr1afl/fl;alb-cre mice had reduced cytochrome activity, associated with accumulation of heme in hepatocytes. The expansion of the cytosolic heme pool in these mice was likely responsible for the early inhibition of heme synthesis and increased degradation of heme, which reduced expression and activity of cytochromes P450. Conclusions In livers of mice, Flvcr1a maintains a free heme pool that regulates heme synthesis and degradation as well as cytochromes P450 expression and activity. These findings have important implications for drug metabolism. PMID:24486949

Solid lipid nanoparticles loaded with iron to overcome barriers for treatment of iron deficiency anemia

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Hosny KM

2015-01-01

Full Text Available Khaled Mohamed Hosny,1,2 Zainy Mohammed Banjar,3 Amani H Hariri,4 Ali Habiballah Hassan5 1Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia; 2Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Beni Suef University, Beni Suef, Egypt; 3Department of Clinical Biochemistry, Faculty of medicine, King Abdulaziz University, Jeddah, Saudi Arabia; 4Consultant Obstetrics and Gynecology, Hera Genaral Hospital, Makkah, Saudi Arabia; 5Department of Orthodontics, Faculty of Dentistry, King Abdulaziz University, Jeddah, Saudi Arabia Abstract: According to the World Health Organization, 46% of the world’s children suffer from anemia, which is usually treated with iron supplements such as ferrous sulfate. The aim of this study was to prepare iron as solid lipid nanoparticles, in order to find an innovative way for alleviating the disadvantages associated with commercially available tablets. These limitations include adverse effects on the digestive system resulting in constipation and blood in the stool. The second drawback is the high variability in the absorption of iron and thus in its bioavailability. Iron solid lipid nanoparticles (Fe-SLNs were prepared by hot homogenization/ultrasonication. Solubility of ferrous sulfate in different solid lipids was measured, and effects of process variables such as the surfactant type and concentration, homogenization and ultrasonication times, and charge-inducing agent on the particle size, zeta potential, and encapsulation efficiency were determined. Furthermore, in vitro drug release and in vivo pharmacokinetics were studied in rabbits. Results indicated that Fe-SLNs consisted of 3% Compritol 888 ATO, 1% Lecithin, 3% Poloxamer 188, and 0.2% dicetylphosphate, with an average particle size of 25 nm with 92.3% entrapment efficiency. In vivo pharmacokinetic study revealed more than fourfold enhanced bioavailability. In

Red and processed meat, nitrite, and heme iron intakes and postmenopausal breast cancer risk in the NIH-AARP Diet and Health Study

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Inoue-Choi, Maki; Sinha, Rashmi; Gierach, Gretchen L.; Ward, Mary H.

2015-01-01

Previous studies have shown inconsistent associations between red and processed meat intake and breast cancer risk. N-nitroso compounds and heme iron have been hypothesized as contributing factors. We followed 193,742 postmenopausal women in the NIH-AARP Diet and Health Study and identified 9,305 incident breast cancers (1995–2006). Dietary intake was assessed using a food frequency questionnaire at baseline. We adjusted daily intakes of meat, nitrite, and heme iron for energy intake using the nutrient density method. We estimated multivariable-adjusted hazard ratios (HR) and 95% confidence intervals (CI) by quintiles of dietary exposures for all breast cancer, by stage (in-situ, localized, regional/distant), and by estrogen/progesterone receptor (ER/PR) status using Cox proportional hazards regression. Total red meat intake was positively associated with risk of regional/distant cancer (p-trend=0.02). The risk was 25% higher in the highest vs. lowest intake quintile (95%CI=1.03–1.52). Higher processed red meat intake (Q5 vs. Q1) was associated with 27% higher risk of localized breast cancer (95%CI=1.01–1.27, p-trend=0.03) and a 19% higher risk of regional/distant cancer (95%CI=0.98–1.44, p-trend=0.10). In addition, higher nitrite intake from processed red meat was positively associated with localized cancer (HR for Q5 vs. Q1=1.23, 95%CI=1.09–1.39, p-trendmeat and processed meat may increase risk of postmenopausal breast cancer. Added nitrite and heme iron may partly contribute to these observed associations. PMID:26505173

Expression of Vibrio harveyi acyl-ACP synthetase allows efficient entry of exogenous fatty acids into the Escherichia coli fatty acid and lipid A synthetic pathways.

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Jiang, Yanfang; Morgan-Kiss, Rachael M; Campbell, John W; Chan, Chi Ho; Cronan, John E

2010-02-02

Although the Escherichia coli fatty acid synthesis (FAS) pathway is the best studied type II fatty acid synthesis system, a major experimental limitation has been the inability to feed intermediates into the pathway in vivo because exogenously supplied free fatty acids are not efficiently converted to the acyl-acyl carrier protein (ACP) thioesters required by the pathway. We report that expression of Vibrio harveyi acyl-ACP synthetase (AasS), a soluble cytosolic enzyme that ligates free fatty acids to ACP to form acyl-ACPs, allows exogenous fatty acids to enter the E. coli fatty acid synthesis pathway. The free fatty acids are incorporated intact and can be elongated or directly incorporated into complex lipids by acyltransferases specific for acyl-ACPs. Moreover, expression of AasS strains and supplementation with the appropriate fatty acid restored growth to E. coli mutant strains that lack essential fatty acid synthesis enzymes. Thus, this strategy provides a new tool for circumventing the loss of enzymes essential for FAS function.

Endotoxin Structures in the Psychrophiles Psychromonas marina and Psychrobacter cryohalolentis Contain Distinctive Acyl Features

Directory of Open Access Journals (Sweden)

Charles R. Sweet

2014-07-01

Full Text Available Lipid A is the essential component of endotoxin (Gram-negative lipopolysaccharide, a potent immunostimulatory compound. As the outer surface of the outer membrane, the details of lipid A structure are crucial not only to bacterial pathogenesis but also to membrane integrity. This work characterizes the structure of lipid A in two psychrophiles, Psychromonas marina and Psychrobacter cryohalolentis, and also two mesophiles to which they are related using MALDI-TOF MS and fatty acid methyl ester (FAME GC-MS. P. marina lipid A is strikingly similar to that of Escherichia coli in organization and total acyl size, but incorporates an unusual doubly unsaturated tetradecadienoyl acyl residue. P. cryohalolentis also shows structural organization similar to a closely related mesophile, Acinetobacter baumannii, however it has generally shorter acyl constituents and shows many acyl variants differing by single methylene (-CH2- units, a characteristic it shares with the one previously reported psychrotolerant lipid A structure. This work is the first detailed structural characterization of lipid A from an obligate psychrophile and the second from a psychrotolerant species. It reveals distinctive structural features of psychrophilic lipid A in comparison to that of related mesophiles which suggest constitutive adaptations to maintain outer membrane fluidity in cold environments.

Isocyanides inhibit human heme oxygenases at the verdoheme stage.

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Evans, John P; Kandel, Sylvie; Ortiz de Montellano, Paul R

2009-09-22

Heme oxygenases (HO) catalyze the oxidative cleavage of heme to generate biliverdin, CO, and free iron. In humans, heme oxygenase-1 (hHO-1) is overexpressed in tumor tissues, where it helps to protect cancer cells from anticancer agents, while HOs in fungal pathogens, such as Candida albicans, function as the primary means of iron acquisition. Thus, HO can be considered a potential therapeutic target for certain diseases. In this study, we have examined the equilibrium binding of three isocyanides, isopropyl, n-butyl, and benzyl, to the two major human HO isoforms (hHO-1 and hHO-2), Candida albicans HO (CaHmx1), and human cytochrome P450 CYP3A4 using electronic absorption spectroscopy. Isocyanides coordinate to both ferric and ferrous HO-bound heme, with tighter binding by the more hydrophobic isocyanides and 200-300-fold tighter binding to the ferrous form. Benzyl isocyanide was the strongest ligand to ferrous heme in all the enzymes. Because the dissociation constants (KD) of the ligands for ferrous heme-hHO-1 were below the limit of accuracy for equilibrium titrations, stopped-flow kinetic experiments were used to measure the binding parameters of the isocyanides to ferrous hHO-1. Steady-state activity assays showed that benzyl isocyanide was the most potent uncompetitive inhibitor with respect to heme with a KI = 0.15 microM for hHO-1. Importantly, single turnover assays revealed that the reaction was completely stopped by coordination of the isocyanide to the verdoheme intermediate rather than to the ferric heme complex. Much tighter binding of the inhibitor to the verdoheme intermediate differentiates it from inhibition of, for example, CYP3A4 and offers a possible route to more selective inhibitor design.

Isocyanides Inhibit Human Heme Oxygenases at the Verdoheme Stage†

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Evans, John P.; Kandel, Sylvie; Ortiz de Montellano, Paul R.

2010-01-01

Heme oxygenases (HO) catalyze the oxidative cleavage of heme to generate biliverdin, CO, and free iron. In humans, heme oxygenase-1 (hHO-1) is overexpressed in tumor tissues, where it helps to protect cancer cells from anticancer agents, while HOs in fungal pathogens, such as Candida albicans, function as the primary means of iron acquisition. Thus, HO can be considered a potential therapeutic target for certain diseases. In this study, we have examined the equilibrium binding of three isocyanides; isopropyl, n-butyl, and benzyl, to the two major human HO isoforms (hHO-1 and hHO-2), Candida albicans HO (CaHmx1), and human cytochrome P450 CYP3A4 using electronic absorption spectroscopy. Isocyanides coordinate to both ferric and ferrous HO-bound heme, with tighter binding by the more hydrophobic isocyanides, and 200-300-fold tighter binding to the ferrous form. Benzyl isocyanide was the strongest ligand to ferrous heme in all the enzymes. Because the dissociation constants (KD) of the ligands for ferrous heme-hHO-1 were below the limit of accuracy for equilibrium titrations, stopped-flow kinetic experiments were used to measure the binding parameters of the isocyanides to ferrous hHO-1. Steady-state activity assays showed that benzyl isocyanide was the most potent uncompetitive inhibitor with respect to heme with a KI = 0.15 μM for hHO-1. Importantly, single turnover assays revealed that the reaction was completely stopped by coordination of the isocyanide to the verdoheme intermediate rather than to the ferric heme complex. Much tighter binding of the inhibitor to the verdoheme intermediate differentiates it from inhibition of, for example, CYP3A4 and offers a possible route to more selective inhibitor design. PMID:19694439

Lack of Plasma Protein Hemopexin Results in Increased Duodenal Iron Uptake.

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Fiorito, Veronica; Geninatti Crich, Simonetta; Silengo, Lorenzo; Aime, Silvio; Altruda, Fiorella; Tolosano, Emanuela

2013-01-01

The body concentration of iron is regulated by a fine equilibrium between absorption and losses of iron. Iron can be absorbed from diet as inorganic iron or as heme. Hemopexin is an acute phase protein that limits iron access to microorganisms. Moreover, it is the plasma protein with the highest binding affinity for heme and thus it mediates heme-iron recycling. Considering its involvement in iron homeostasis, it was postulated that hemopexin may play a role in the physiological absorption of inorganic iron. Hemopexin-null mice showed elevated iron deposits in enterocytes, associated with higher duodenal H-Ferritin levels and a significant increase in duodenal expression and activity of heme oxygenase. The expression of heme-iron and inorganic iron transporters was normal. The rate of iron absorption was assessed by measuring the amount of (57)Fe retained in tissues from hemopexin-null and wild-type animals after administration of an oral dose of (57)FeSO4 or of (57)Fe-labelled heme. Higher iron retention in the duodenum of hemopexin-null mice was observed as compared with normal mice. Conversely, iron transfer from enterocytes to liver and bone marrow was unaffected in hemopexin-null mice. The increased iron level in hemopexin-null duodenum can be accounted for by an increased iron uptake by enterocytes and storage in ferritins. These data indicate that the lack of hemopexin under physiological conditions leads to an enhanced duodenal iron uptake thus providing new insights to our understanding of body iron homeostasis.

Synthesis, delivery and regulation of eukaryotic heme and Fe-S cluster cofactors.

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Barupala, Dulmini P; Dzul, Stephen P; Riggs-Gelasco, Pamela Jo; Stemmler, Timothy L

2016-02-15

In humans, the bulk of iron in the body (over 75%) is directed towards heme- or Fe-S cluster cofactor synthesis, and the complex, highly regulated pathways in place to accomplish biosynthesis have evolved to safely assemble and load these cofactors into apoprotein partners. In eukaryotes, heme biosynthesis is both initiated and finalized within the mitochondria, while cellular Fe-S cluster assembly is controlled by correlated pathways both within the mitochondria and within the cytosol. Iron plays a vital role in a wide array of metabolic processes and defects in iron cofactor assembly leads to human diseases. This review describes progress towards our molecular-level understanding of cellular heme and Fe-S cluster biosynthesis, focusing on the regulation and mechanistic details that are essential for understanding human disorders related to the breakdown in these essential pathways. Copyright © 2016 Elsevier Inc. All rights reserved.

Heme acquisition mechanisms of Porphyromonas gingivalis - strategies used in a polymicrobial community in a heme-limited host environment.

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Smalley, J W; Olczak, T

2017-02-01

Porphyromonas gingivalis, a main etiologic agent and key pathogen responsible for initiation and progression of chronic periodontitis requires heme as a source of iron and protoporphyrin IX for its survival and the ability to establish an infection. Porphyromonas gingivalis is able to accumulate a defensive cell-surface heme-containing pigment in the form of μ-oxo bisheme. The main sources of heme for P. gingivalis in vivo are hemoproteins present in saliva, gingival crevicular fluid, and erythrocytes. To acquire heme, P. gingivalis uses several mechanisms. Among them, the best characterized are those employing hemagglutinins, hemolysins, and gingipains (Kgp, RgpA, RgpB), TonB-dependent outer-membrane receptors (HmuR, HusB, IhtA), and hemophore-like proteins (HmuY, HusA). Proteins involved in intracellular heme transport, storage, and processing are less well characterized (e.g. PgDps). Importantly, P. gingivalis may also use the heme acquisition systems of other bacteria to fulfill its own heme requirements. Porphyromonas gingivalis displays a novel paradigm for heme acquisition from hemoglobin, whereby the Fe(II)-containing oxyhemoglobin molecule must first be oxidized to methemoglobin to facilitate heme release. This process not only involves P. gingivalis arginine- and lysine-specific gingipains, but other proteases (e.g. interpain A from Prevotella intermedia) or pyocyanin produced by Pseudomonas aeruginosa. Porphyromonas gingivalis is then able to fully proteolyze the more susceptible methemoglobin substrate to release free heme or to wrest heme from it directly through the use of the HmuY hemophore. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Using porphyrin-amino acid pairs to model the electrochemistry of heme proteins: experimental and theoretical investigations.

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Samajdar, Rudra N; Manogaran, Dhivya; Yashonath, S; Bhattacharyya, Aninda J

2018-04-18

Quasi reversibility in electrochemical cycling between different oxidation states of iron is an often seen characteristic of iron containing heme proteins that bind dioxygen. Surprisingly, the system becomes fully reversible in the bare iron-porphyrin complex: hemin. This leads to the speculation that the polypeptide bulk (globin) around the iron-porphyrin active site in these heme proteins is probably responsible for the electrochemical quasi reversibility. To understand the effect of such polypeptide bulk on iron-porphyrin, we study the interaction of specific amino acids with the hemin center in solution. We choose three representative amino acids-histidine (a well-known iron coordinator in bio-inorganic systems), tryptophan (a well-known fluoroprobe for proteins), and cysteine (a redox-active organic molecule). The interactions of these amino acids with hemin are studied using electrochemistry, spectroscopy, and density functional theory. The results indicate that among these three, the interaction of histidine with the iron center is strongest. Further, histidine maintains the electrochemical reversibility of iron. On the other hand, tryptophan and cysteine interact weakly with the iron center but disturb the electrochemical reversibility by contributing their own redox active processes to the system. Put together, this study attempts to understand the molecular interactions that can control electrochemical reversibility in heme proteins. The results obtained here from the three representative amino acids can be scaled up to build a heme-amino acid interaction database that may predict the electrochemical properties of any protein with a defined polypeptide sequence.

The Staphylococcus aureus Protein IsdH Inhibits Host Hemoglobin Scavenging to Promote Heme Acquisition by the Pathogen

DEFF Research Database (Denmark)

Saederup, Kirstine Lindhardt; Stødkilde-Jørgensen, Kristian; Graversen, Jonas Heilskov

2016-01-01

Hemolysis is a complication in septic infections with Staphylococcus aureus, which utilizes the released Hb as an iron source. S. aureus can acquire heme in vitro from hemoglobin (Hb) by a heme-sequestering mechanism that involves proteins from the S. aureus iron-regulated surface determinant (Isd...

Mortality from different causes associated with meat, heme iron, nitrates, and nitrites in the NIH-AARP Diet and Health Study: population based cohort study

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Sinha, Rashmi; Ward, Mary H; Graubard, Barry I; Inoue-Choi, Maki; Dawsey, Sanford M; Abnet, Christian C

2017-01-01

Objective To determine the association of different types of meat intake and meat associated compounds with overall and cause specific mortality. Design Population based cohort study. Setting Baseline dietary data of the NIH-AARP Diet and Health Study (prospective cohort of the general population from six states and two metropolitan areas in the US) and 16 year follow-up data until 31 December 2011. Participants 536 969 AARP members aged 50-71 at baseline. Exposures Intake of total meat, processed and unprocessed red meat (beef, lamb, and pork) and white meat (poultry and fish), heme iron, and nitrate/nitrite from processed meat based on dietary questionnaire. Adjusted Cox proportional hazards regression models were used with the lowest fifth of calorie adjusted intakes as reference categories. Main outcome measure Mortality from any cause during follow-up. Results An increased risk of all cause mortality (hazard ratio for highest versus lowest fifth 1.26, 95% confidence interval 1.23 to 1.29) and death due to nine different causes associated with red meat intake was observed. Both processed and unprocessed red meat intakes were associated with all cause and cause specific mortality. Heme iron and processed meat nitrate/nitrite were independently associated with increased risk of all cause and cause specific mortality. Mediation models estimated that the increased mortality associated with processed red meat was influenced by nitrate intake (37.0-72.0%) and to a lesser degree by heme iron (20.9-24.1%). When the total meat intake was constant, the highest fifth of white meat intake was associated with a 25% reduction in risk of all cause mortality compared with the lowest intake level. Almost all causes of death showed an inverse association with white meat intake. Conclusions The results show increased risks of all cause mortality and death due to nine different causes associated with both processed and unprocessed red meat, accounted for, in part, by

Synthesis and Evaluation of Amyloid β Derived and Amyloid β Independent Enhancers of the Peroxidase-like Activity of Heme.

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Wißbrock, Amelie; Kühl, Toni; Silbermann, Katja; Becker, Albert J; Ohlenschläger, Oliver; Imhof, Diana

2017-01-12

Labile heme has been suggested to have an impact in several severe diseases. In the context of Alzheimer's disease (AD), however, decreased levels of free heme have been reported. Therefore, we were looking for an assay system that can be used for heme concentration determination. From a biochemical point of view the peroxidase activity of the Aβ-heme complex seemed quite attractive to pursue this goal. As a consequence, a peptide that is able to increase the readout even in the case of a low heme concentration is favorable. The examination of Aβ- and non-Aβ-derived peptides in complex with heme revealed that the peroxidase-like activity significantly depends on the peptide sequence and length. A 23mer His-based peptide derived from human fatty acyl-CoA reductase 1 in complex with heme exhibited a significantly higher peroxidase activity than Aβ(40)-heme. Structural modeling of both complexes demonstrated that heme binding via a histidine can be supported by hydrogen bond interactions of a basic residue near the propionate carboxyl function of protoporphyrin IX. Furthermore, the interplay of Aβ-heme and the lipoprotein LDL as a potential physiological effector of Aβ was examined.

Dual acylation and lipid raft association of Src-family protein tyrosine kinases are required for SDF-1/CXCL12-mediated chemotaxis in the Jurkat human T cell lymphoma cell line.

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Zaman, Sabiha N; Resek, Mary E; Robbins, Stephen M

2008-10-01

Chemokines play pivotal roles in regulating a wide variety of biological processes by modulating cell migration and recruitment. Deregulation of chemokine signaling can alter cell recruitment, contributing to the pathogenic states associated with autoimmune disease, inflammatory disorders, and sepsis. During chemotaxis, lipid rafts and their resident signaling molecules have been demonstrated to partition to different parts of the cell. Herein, we investigated the role of lipid raft resident Src-family kinases (SFK) in stromal cell-derived factor 1/CXCL12-mediated chemotaxis. We have shown that Lck-deficient J.CaM 1.6 cells are defective in CXCL12-mediated chemotaxis in contrast to their parental counterpart, Jurkat cells. Ectopic expression of the SFK hematopoietic cell kinase (Hck) in J.CaM 1.6 cells reconstituted CXCL12 responsiveness. The requirement of lipid raft association of SFK was assessed using both isoforms of Hck: the dually acylated p59(Hck) isoform that is targeted to lipid rafts and the monoacylated p61(Hck) isoform that is nonraft-associated. We have shown using several gain and loss of acylation alleles that dual acylation of Hck was required for CXCL12-mediated chemotaxis in J.CaM 1.6 cells. These results highlight the importance of the unique microenvironment provided by lipid rafts and their specific contribution in providing specificity to CXCL12 signaling.

Fatty Acyl Chains of Mycobacterium marinum Lipooligosaccharides

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Rombouts, Yoann; Alibaud, Laeticia; Carrère-Kremer, Séverine; Maes, Emmanuel; Tokarski, Caroline; Elass, Elisabeth; Kremer, Laurent; Guérardel, Yann

2011-01-01

We have recently established the fine structure of the glycan backbone of lipooligosaccharides (LOS-I to LOS-IV) isolated from Mycobacterium marinum, a close relative of Mycobacterium tuberculosis. These studies culminated with the description of an unusual terminal N-acylated monosaccharide that confers important biological functions to LOS-IV, such as macrophage activation, that may be relevant to granuloma formation. It was, however, also suggested that the lipid moiety was required for LOSs to exert their immunomodulatory activity. Herein, using highly purified LOSs from M. marinum, we have determined through a combination of mass spectrometric and NMR techniques, the structure and localization of the fatty acids composing the lipid moiety. The occurrence of two distinct polymethyl-branched fatty acids presenting specific localizations is consistent with the presence of two highly related polyketide synthases (Pks5 and Pks5.1) in M. marinum and presumably involved in the synthesis of these fatty acyl chains. In addition, a bioinformatic search permitted us to identify a set of enzymes potentially involved in the biosynthesis or transfer of these lipids to the LOS trehalose unit. These include MMAR_2343, a member of the Pap (polyketide-associated protein) family, that acylates trehalose-based glycolipids in M. marinum. The participation of MMAR_2343 to LOS assembly was demonstrated using a M. marinum mutant carrying a transposon insertion in the MMAR_2343 gene. Disruption of MMAR_2343 resulted in a severe LOS breakdown, indicating that MMAR_2343, hereafter designated PapA4, fulfills the requirements for LOS acylation and assembly. PMID:21803773

Phase behavior and nanoscale structure of phospholipid membranes incorporated with acylated C-14-peptides

DEFF Research Database (Denmark)

Pedersen, T.B.; Kaasgaard, Thomas; Jensen, M.O.

2005-01-01

The thermotropic phase behavior and lateral structure of dipalmitoylphosphatidylcholine (DPPC) lipid bilayers containing an acylated peptide has been characterized by differential scanning calorimetry (DSC) on vesicles and atomic force microscopy (AFM) on mica-supported bilayers. The acylated...... peptide, which is a synthetic decapeptide N-terminally linked to a C-14 acyl chain (C-14-peptide), is incorporated into DPPC bilayers in amounts ranging from 0-20 mol %. The calorimetric scans of the two-component system demonstrate a distinct influence of the C-14-peptide on the lipid bilayer...... gel phase DPPC bilayers, inserts preferentially into preexisting defect regions and has a noticeable influence on the organization of the surrounding lipids. The presence of the C-14-peptide gives rise to a laterally heterogeneous bilayer structure with coexisting lipid domains characterized by a 10...

The Structure of the Complex between Yeast Frataxin and Ferrochelatase: CHARACTERIZATION AND PRE-STEADY STATE REACTION OF FERROUS IRON DELIVERY AND HEME SYNTHESIS.

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Söderberg, Christopher; Gillam, Mallory E; Ahlgren, Eva-Christina; Hunter, Gregory A; Gakh, Oleksandr; Isaya, Grazia; Ferreira, Gloria C; Al-Karadaghi, Salam

2016-05-27

Frataxin is a mitochondrial iron-binding protein involved in iron storage, detoxification, and delivery for iron sulfur-cluster assembly and heme biosynthesis. The ability of frataxin from different organisms to populate multiple oligomeric states in the presence of metal ions, e.g. Fe(2+) and Co(2+), led to the suggestion that different oligomers contribute to the functions of frataxin. Here we report on the complex between yeast frataxin and ferrochelatase, the terminal enzyme of heme biosynthesis. Protein-protein docking and cross-linking in combination with mass spectroscopic analysis and single-particle reconstruction from negatively stained electron microscopic images were used to verify the Yfh1-ferrochelatase interactions. The model of the complex indicates that at the 2:1 Fe(2+)-to-protein ratio, when Yfh1 populates a trimeric state, there are two interaction interfaces between frataxin and the ferrochelatase dimer. Each interaction site involves one ferrochelatase monomer and one frataxin trimer, with conserved polar and charged amino acids of the two proteins positioned at hydrogen-bonding distances from each other. One of the subunits of the Yfh1 trimer interacts extensively with one subunit of the ferrochelatase dimer, contributing to the stability of the complex, whereas another trimer subunit is positioned for Fe(2+) delivery. Single-turnover stopped-flow kinetics experiments demonstrate that increased rates of heme production result from monomers, dimers, and trimers, indicating that these forms are most efficient in delivering Fe(2+) to ferrochelatase and sustaining porphyrin metalation. Furthermore, they support the proposal that frataxin-mediated delivery of this potentially toxic substrate overcomes formation of reactive oxygen species. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Mimicking heme enzymes in the solid state: metal-organic materials with selectively encapsulated heme.

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Larsen, Randy W; Wojtas, Lukasz; Perman, Jason; Musselman, Ronald L; Zaworotko, Michael J; Vetromile, Carissa M

2011-07-13

To carry out essential life processes, nature has had to evolve heme enzymes capable of synthesizing and manipulating complex molecules. These proteins perform a plethora of chemical reactions utilizing a single iron porphyrin active site embedded within an evolutionarily designed protein pocket. We herein report the first class of metal-organic materials (MOMs) that mimic heme enzymes in terms of both structure and reactivity. The MOMzyme-1 class is based upon a prototypal MOM, HKUST-1, into which catalytically active metalloporphyrins are selectively encapsulated in a "ship-in-a-bottle" fashion within one of the three nanoscale cages that exist in HKUST-1. MOMs offer unparalleled levels of permanent porosity and their modular nature affords enormous diversity of structures and properties. The MOMzyme-1 class could therefore represent a new paradigm for heme biomimetic catalysis since it combines the activity of a homogeneous catalyst with the stability and recyclability of heterogeneous catalytic systems within a single material.

Deuterium-labelled N-acyl-l-homoserine lactones (AHLs) - inter-kingdom signalling molecules - synthesis, structural studies, and interactions with model lipid membranes

International Nuclear Information System (INIS)

Jakubczyk, Dorota; Barth, Christoph; Anastassacos, Frances; Koelsch, Patrick; Schepers, Ute; Kubas, Adam; Fink, Karin; Brenner-Weiss, Gerald; Braese, Stefan

2012-01-01

N-Acyl-l-homoserine lactones (AHLs) are synthesized by Gram-negative bacteria. These quorum-sensing molecules play an important role in the context of bacterial infection and biofilm formation. They also allow communication between microorganisms and eukaryotic cells (inter-kingdom signalling). However, very little is known about the entire mechanism of those interactions. Precise structural studies are required to analyse the different AHL isomers as only one form is biologically most active. Theoretical studies combined with experimental infrared and Raman spectroscopic data are therefore undertaken to characterise the obtained compounds. To mimic interactions between AHL and cell membranes, we studied the insertion of AHL in supported lipid bilayers, using vibrational sum-frequency-generation spectroscopy. Deuterium-labelled AHLs were thus synthesized. Starting from readily available deuterated fatty acids, a two-step procedure towards deuterated N-acyl-l-homoserine lactones with varying chain lengths is described. This included the acylation of Meldrum's acid followed by amidation. Additionally, the detailed analytical evaluation of the products is presented herein. (orig.)

Production and Early Preservation of Lipid Biomarkers in Iron Hot Springs

Energy Technology Data Exchange (ETDEWEB)

Parenteau, Mary N.; Jahnke, Linda L.; Farmer, Jack D.; Cady, Sherry L.

2014-06-01

The bicarbonate-buffered anoxic vent waters at Chocolate Pots hot springs in Yellowstone National Park are 51–54°C, pH 5.5–6.0, and are very high in dissolved Fe(II) at 5.8–5.9 mg/L. The aqueous Fe(II) is oxidized by a combination of biotic and abiotic mechanisms and precipitated as primary siliceous nanophase iron oxyhydroxides (ferrihydrite). Four distinct prokaryotic photosynthetic microbial mat types grow on top of these iron deposits. Lipids were used to characterize the community composition of the microbial mats, link source organisms to geologically significant biomarkers, and investigate how iron mineralization degrades the lipid signature of the community. The phospholipid and glycolipid fatty acid profiles of the highest-temperature mats indicate that they are dominated by cyanobacteria and green nonsulfur filamentous anoxygenic phototrophs (FAPs). Diagnostic lipid biomarkers of the cyanobacteria include midchain branched mono- and dimethylalkanes and, most notably, 2-methylbacteriohopanepolyol. Diagnostic lipid biomarkers of the FAPs (Chloroflexus and Roseiflexus spp.) include wax esters and a long-chain tri-unsaturated alkene. Surprisingly, the lipid biomarkers resisted the earliest stages of microbial degradation and diagenesis to survive in the iron oxides beneath the mats. Understanding the potential of particular sedimentary environments to capture and preserve fossil biosignatures is of vital importance in the selection of the best landing sites for future astrobiological missions to Mars. Finally, this study explores the nature of organic degradation processes in moderately thermal Fe(II)-rich groundwater springs—environmental conditions that have been previously identified as highly relevant for Mars exploration.

Acyl Chain Preference in Foam Cell Formation from Mouse Peritoneal Macrophages.

Science.gov (United States)

Fujiwara, Yuko; Hama, Kotaro; Tsukahara, Makoto; Izumi-Tsuzuki, Ryosuke; Nagai, Toru; Ohe-Yamada, Mihoko; Inoue, Keizo; Yokoyama, Kazuaki

2018-01-01

Macrophage foam cells play critical roles in the initiation and development of atherosclerosis by synthesizing and accumulating cholesteryl ester (CE) in lipid droplets. However, in analyzing lipid metabolism in foam cell formation, studies have focused on the sterol group, and little research has been done on the acyl chains. Therefore, we adapted a model system using liposomes containing particular acyl chains and examined the effect of various acyl chains on foam cell formation. Of the phosphatidylserine (PS) liposomes tested containing PS, phosphatidylcholine, and cholesterol, we found that unsaturated (C18:1), but not saturated (C16:0 and C18:0), PS liposomes induced lipid droplet formation, indicating that foam cell formation depends on the nature of the acyl chain of the PS liposomes. Experiments on the uptake and accumulation of cholesterol from liposomes by adding [ 14 C]cholesterol suggested that foam cell formation could be induced only when cholesterol was converted to CE in the case of C18:1 PS liposomes. Both microscopic observations and metabolic analysis suggest that cholesterol incorporated into either C16:0 or C18:0 PS liposomes may stay intact after being taken in by endosomes. The [ 14 C]C18:1 fatty acyl chain in the C18:1 PS liposome was used to synthesize CE and triacylglycerol (TG). Interestingly, the [ 14 C]C16:0 in the C18:1 PS liposome was metabolized to sphingomyelin rather than being incorporated into either CE or TG, which could be because of enzymatic acyl chain selectivity. In conclusion, our results indicate that the acyl chain preference of macrophages could have some impact on their progression to foam cells.

Improvement of bioavailability for iron from vegetarian meals by ascorbic acid

International Nuclear Information System (INIS)

Sritongkul, N.; Tuntawiroon, M.; Pleehachinda, R.; Suwanik, R.

1996-01-01

There are two kinds of iron in the diet with respect to the mechanism of absorption, heme-iron which is present as haemoglobin or myoglobin in meat and blood products, and, non-heme iron which is the main source of dietary iron. The bioavailability of the non-heme food iron is much lower than heme-iron. Vegetarian diets contain only non-heme iron. Iron intake from vegetarian meals are generally satisfied with the requirements, however, the bioavailabilities for non-heme iron is determined not only by iron content byt also the balance between different dietary factors enhancing and inhibiting iron absorption. The main enhancing factor in vegetarian meals is ascorbic acid in fruits and vegetables, inhibitors are phytate in cereals and grains, and tannins in some spices and vegetables. It has been reported that iron deficiency is one of the common micronutrient problems associated with unplanned vegetarian diets. In the present study the absorption of non-heme iron was measured from 2 vegetarian meals containing considerable amounts of phytate and tannin. The extrinsic tay method ( 59 Fe/ 55 Fe) was used to labelled the non-heme iron. The mean percentage absorption of non-heme iron from both meals was slightly different due to differences in their dietary contents. Their initial percentages iron absorption were apparent low (3.5% and 4.1%), however, the absorption progressively increased with increase in the level of ascorbic acid, 2-3 times with 100 mg and 4-5 times with 200 mg of ascorbic acid. The average amount of iron absorbed per 2000 kcal increased from 0.37 mg to 0.86 mg and 1.45 mg with the addition of 100 mg and 200 mg ascorbic acid respectively (p < 0.001). Considering the limited caloric intakes and the iron content in the meals, the amount of iron absorbed from vegetarian meals without ascorbic acid was not able to meet certain requirements for children, adolescents and menstruating women. The minimal requirement for dietary iron needed to be absorbed is

Improvement of bioavailability for iron from vegetarian meals by ascorbic acid

Energy Technology Data Exchange (ETDEWEB)

Sritongkul, N; Tuntawiroon, M; Pleehachinda, R; Suwanik, R [Siriraj Hospital Medical School, Bangkok (Thailand). Section of Nuclear Medicine

1996-12-01

There are two kinds of iron in the diet with respect to the mechanism of absorption, heme-iron which is present as haemoglobin or myoglobin in meat and blood products, and, non-heme iron which is the main source of dietary iron. The bioavailability of the non-heme food iron is much lower than heme-iron. Vegetarian diets contain only non-heme iron. Iron intake from vegetarian meals are generally satisfied with the requirements, however, the bioavailabilities for non-heme iron is determined not only by iron content byt also the balance between different dietary factors enhancing and inhibiting iron absorption. The main enhancing factor in vegetarian meals is ascorbic acid in fruits and vegetables, inhibitors are phytate in cereals and grains, and tannins in some spices and vegetables. It has been reported that iron deficiency is one of the common micronutrient problems associated with unplanned vegetarian diets. In the present study the absorption of non-heme iron was measured from 2 vegetarian meals containing considerable amounts of phytate and tannin. The extrinsic tay method ({sup 59}Fe/ {sup 55}Fe) was used to labelled the non-heme iron. The mean percentage absorption of non-heme iron from both meals was slightly different due to differences in their dietary contents. Their initial percentages iron absorption were apparent low (3.5% and 4.1%), however, the absorption progressively increased with increase in the level of ascorbic acid, 2-3 times with 100 mg and 4-5 times with 200 mg of ascorbic acid. The average amount of iron absorbed per 2000 kcal increased from 0.37 mg to 0.86 mg and 1.45 mg with the addition of 100 mg and 200 mg ascorbic acid respectively (p < 0.001). Considering the limited caloric intakes and the iron content in the meals, the amount of iron absorbed from vegetarian meals without ascorbic acid was not able to meet certain requirements for children, adolescents and menstruating women. The minimal requirement for dietary iron needed to be

Controlling lipid oxidation via a biomimetic iron chelating active packaging material.

Science.gov (United States)

Tian, Fang; Decker, Eric A; Goddard, Julie M

2013-12-18

Previously, a siderophore-mimetic metal chelating active packaging film was developed by grafting poly(hydroxamic acid) (PHA) from the surface of polypropylene (PP) films. The objective of the current work was to demonstrate the potential applicability of this PP-g-PHA film to control iron-promoted lipid oxidation in food emulsions. The iron chelating activity of this film was investigated, and the surface chemistry and color intensity of films were also analyzed after iron chelation. In comparison to the iron chelating activity in the free Fe(3+) solution, the PP-g-PHA film retained approximately 50 and 30% of its activity in nitrilotriacetic acid (NTA)/Fe(3+) and citric acid/Fe(3+) solutions, respectively (pH 5.0), indicating a strong chelating strength for iron. The ability of PP-g-PHA films to control lipid oxidation was demonstrated in a model emulsion system (pH 3.0). PP-g-PHA films performed even better than ethylenediaminetetraacetic acid (EDTA) in preventing the formation of volatile oxidation products. The particle size and ζ potential results of emulsions indicated that PP-g-PHA films had no adverse effects on the stability of the emulsion system. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) analysis suggested a non-migratory nature of the PP-g-PHA film surface. These results suggest that such biomimetic, non-migratory metal chelating active packaging films have commercial potential in protecting foods against iron-promoted lipid oxidation.

Salivary proline-rich protein may reduce tannin-iron chelation: a systematic narrative review

OpenAIRE

Delimont, Nicole M.; Rosenkranz, Sara K.; Haub, Mark D.; Lindshield, Brian L.

2017-01-01

Background Tannins are often cited for antinutritional effects, including chelation of non-heme iron. Despite this, studies exploring non-heme iron bioavailability inhibition with long-term consumption have reported mixed results. Salivary proline-rich proteins (PRPs) may mediate tannin-antinutritional effects on non-heme iron bioavailability. Aim To review evidence regarding biochemical binding mechanisms and affinity states between PRPs and tannins, as well as effects of PRPs on non-heme ir...

Kidney injury and heme oxygenase-1

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Hai-xing MAI

2012-02-01

Full Text Available     Heme oxygenase-1 (HO-1 is one of the main pathways to degrade heme in mammals, and the main degradation products are free iron (Fe2+, carbon monoxide (CO, and bilirubin. Heme plays an important role in promoting cell survival, circulation of intracellular substrates, and immune regulation. Previous studies suggest that HO-1 pathway is an important internal factor in determining the susceptibility and severity of acute kidney injury (AKI. The induction of HO-1 expression can attenuate the severity of renal ischemia-reperfusion injury (IRI, and the inhibition of HO-1 expression will aggravate IRI. The present article summarizes the latest advances in research abroad and at home on protective mechanism by which HO-1 prevents AKI to further deepen our understanding of the role of HO-1 in the treatment of AKI.   

Staphylococcus aureus HemX Modulates Glutamyl-tRNA Reductase Abundance To Regulate Heme Biosynthesis

OpenAIRE

Jacob E. Choby; Caroline M. Grunenwald; Arianna I. Celis; Svetlana Y. Gerdes; Jennifer L. DuBois; Eric P. Skaar; Kimberly A. Kline

2018-01-01

Staphylococcus aureus is responsible for a significant amount of devastating disease. Its ability to colonize the host and cause infection is supported by a variety of proteins that are dependent on the cofactor heme. Heme is a porphyrin used broadly across kingdoms and is synthesized de novo from common cellular precursors and iron. While heme is critical to bacterial physiology, it is also toxic in high concentrations, requiring that organisms encode regulatory processes to control heme hom...

Radioisotope investigation of iron absorption in humans. Part of a WHO/IAEA coordinated programme on iron nutrition

International Nuclear Information System (INIS)

Bothwell, T.H.

1975-08-01

The gastrointestinal absorption of iron was investigated in a number of multiparous women living under low socio-economic conditions which are known to lead commonly to iron deficiency. The percentage absorption was deduced by comparing the concentration of 55 Fe and/or 59 Fe in blood with the activity of the corresponding isotope(s) administered orally about two weeks earlier. It was confirmed or established that: (1) food or supplemental iron, if available at all, tends to be absorbed from the intestines as if present there in one of two alternative pools: heme and non-heme, (2) vitamin C substantially increases absorption of food or supplemental non-heme iron, (3) it is technically possible to use common salt or sugar as a carrier for supplemental iron and vitamin C, but practical difficulties of using such systems in a public health prophylactic programme are severe, and (4) tea, presumably because of its tannin content, inhibits absorption of accompanying iron

Genome-based analysis of heme biosynthesis and uptake in prokaryotic systems.

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Cavallaro, Gabriele; Decaria, Leonardo; Rosato, Antonio

2008-11-01

Heme is the prosthetic group of many proteins that carry out a variety of key biological functions. In addition, for many pathogenic organisms, heme (acquired from the host) may constitute a very important source of iron. Organisms can meet their heme demands by taking it up from external sources, by producing the cofactor through a dedicated biosynthetic pathway, or both. Here we analyzed the distribution of proteins specifically involved in the processes of heme biosynthesis and heme uptake in 474 prokaryotic organisms. These data allowed us to identify which organisms are capable of performing none, one, or both processes, based on the similarity to known systems. Some specific instances where one or more proteins along the pathways had unusual modifications were singled out. For two key protein domains involved in heme uptake, we could build a series of structural models, which suggested possible alternative modes of heme binding. Future directions for experimental work are given.

Cellular Studies with UVA Radiation: A Role for Iron (invited paper)

International Nuclear Information System (INIS)

Tyrrell, R.M.; Pourzand, C.A.; Brown, J.; Hejmadi, V.; Kvam, V.; Ryter, S.; Watkin, R.D.

2000-01-01

The UVA (320-380 nm) component of sunlight or sunbeds acts as an oxidising carcinogen and has been clearly implicated in skin cancer. Since UVA radiation interacts with cells by generating active oxygen species, the damaging effects of this radiation will be exacerbated by the presence of catalytically reactive iron in cells. It has now been shown by two independent techniques (dequenching of metal-quenched calcein fluorescence in cells and changes in the binding activity of the iron responsive protein IRPI) that UVA radiation causes an immediate release of 'free' iron in human skin fibroblasts via the proteolysis of ferritin (Ft). Within minutes of exposure to a range of doses of UVA at natural exposure levels, the binding activity of IRP-1, as well as Ft levels, decrease in a dose-dependent manner. It is proposed that the oxidative damage to lysosomes that leads to Ft degradation and the consequent release of potentially harmful 'free' iron to the cytosol might be a major factor in UVA-induced damage to the skin. UVA radiation also breaks down heme-containing proteins in the microsomal membrane to release free heme as an additional photosensitising component. This will provide another source of enhanced free iron in skin cells since constitutive heme oxygenase 2 (in keratinocytes) and UVA-inducible heme oxygenase-1 (in fibroblasts) are likely to break down any free heme to biliverdin and release iron and carbon monoxide in the process. It is postulated that, in skin fibroblasts, this free heme release and the enhanced free iron pools will lead to an adaptive response involving heme oxygenase (with a maximum about 10 h) and ferritin (in 24-48 h) that will scavenge the heme and iron released by subsequent oxidising (UVA) treatments. (author)

Heme synthesis in normal mouse liver and mouse liver tumors

International Nuclear Information System (INIS)

Stout, D.L.; Becker, F.F.

1990-01-01

Hepatic cancers from mice and rats demonstrate decreased levels of delta-aminolevulinic acid synthase, the rate-limiting enzyme in the heme synthetic pathway, and increased heme oxygenase, the heme-catabolizing enzyme. These findings suggest that diminution of P-450, b5, and catalase in these lesions may result from a heme supply that is limited by decreased heme synthesis and increased heme catabolism. Heme synthesis was measured in mouse liver tumors (MLT) and adjacent tumor-free lobes (BKG) by administering the radiolabeled heme precursors 55 FeCl3 and [2- 14 C]glycine and subsequently extracting the heme for determination of specific activity. Despite reduced delta-aminolevulinic acid synthase activity in MLT, both tissues incorporated [2-14C]glycine into heme at similar rates. At early time points, heme extracted from MLT contained less 55Fe than that from BKG. This was attributed to the findings that MLT took up 55Fe at a slower rate than BKG and had larger iron stores than BKG. The amount of heme per milligram of protein was also similar in both tissues. These findings militate against the hypothesis that diminished hemoprotein levels in MLT result from limited availability of heme. It is probable, therefore, that decreased hemoprotein levels in hepatic tumors are linked to a general program of dedifferentiation associated with the cancer phenotype. Diminution of hemoprotein in MLT may result in a relatively increased intracellular heme pool. delta-Aminolevulinic acid synthase and heme oxygenase are, respectively, negatively and positively regulated by heme. Thus, their alteration in MLT may be due to the regulatory influences of the heme pool

Increase on the initial soluble heme levels in acidic conditions is an important mechanism for spontaneous heme crystallization in vitro.

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Renata Stiebler

Full Text Available BACKGROUND: Hemozoin (Hz is a heme crystal that represents a vital pathway for heme disposal in several blood-feeding organisms. Recent evidence demonstrated that β-hematin (βH (the synthetic counterpart of Hz formation occurs under physiological conditions near synthetic or biological hydrophilic-hydrophobic interfaces. This seems to require a heme dimer acting as a precursor of Hz crystals that would be formed spontaneously in the absence of the competing water molecules bound to the heme iron. Here, we aimed to investigate the role of medium polarity on spontaneous βH formation in vitro. METHODOLOGY/PRINCIPAL FINDINGS: We assessed the effect of water content on spontaneous βH formation by using the aprotic solvent dimethylsulfoxide (DMSO and a series of polyethyleneglycols (PEGs. We observed that both DMSO and PEGs (3.350, 6.000, 8.000, and 22.000 increased the levels of soluble heme under acidic conditions. These compounds were able to stimulate the production of βH crystals in the absence of any biological sample. Interestingly, the effects of DMSO and PEGs on βH formation were positively correlated with their capacity to promote previous heme solubilization in acidic conditions. Curiously, a short chain polyethyleneglycol (PEG 300 caused a significant reduction in both soluble heme levels and βH formation. Finally, both heme solubilization and βH formation strongly correlated with reduced medium water activity provided by increased DMSO concentrations. CONCLUSIONS: The data presented here support the notion that reduction of the water activity is an important mechanism to support spontaneous heme crystallization, which depends on the previous increase of soluble heme levels.

The effect of lipids, a lipid-rich ready-to-use therapeutic food, or a phytase on iron absorption from maize-based meals fortified with micronutrient powders.

Science.gov (United States)

Monnard, Arnaud; Moretti, Diego; Zeder, Christophe; Steingötter, Andreas; Zimmermann, Michael B

2017-06-01

Background: Ready-to-use-therapeutic foods (RUTFs) high in lipid, protein, and iron are used to treat malnutrition. Lipids increase gastric residence time, which could increase iron absorption, particularly from poorly soluble iron compounds and in combination with phytase. Objectives: The objectives were to 1 ) assess the effect on iron absorption of a lipid emulsion given 20 min before or together with an iron-fortified maize meal and 2 ) assess iron absorption from a micronutrient powder (MNP) given with a nutrient-dense RUTF and/or a microbial phytase. Design: A total of 41 women participated in 3 studies. They consumed a maize meal fortified with isotopically labeled ferrous sulfate (FeSO 4 ; study 1) or ferric pyrophosphate (FePP; study 2). In studies 1 and 2, a lipid emulsion was given with or 20 min before the meal. In study 3, with the use of a 2 × 2 factorial design, subjects consumed a maize meal fortified with an MNP containing labeled FeSO 4 (MNP) given with an RUTF (MNP+RUTF), with a phytase (MNP+phytase), or both (MNP+RUTF+phytase). Iron absorption was assessed by isotope incorporation in erythrocytes 14 d after the test meals. Results: The lipid emulsion given either before or with the meal significantly increased iron absorption from FePP by 2.55-fold (95% CI: 1.48-, 4.37-fold; P = 0.001) but not from FeSO 4 There was a trend to increase iron absorption with the MNP+RUTF meal, which did not reach significance (1.21-fold; 95% CI: 0.92-, 1.61-fold; P = 0.060). The addition of phytase to MNP and MNP+RUTF significantly increased iron absorption by 1.85-fold (95% CI: 1.49-, 2.29-fold; P phytase and RUTF. Conclusions: In iron-fortified maize-based meals, the addition of lipids more than doubles iron absorption from FePP. Our results suggest the possibility of an enhancing effect on iron absorption of lipid-rich RUTFs, but more research is needed to determine this. This trial was registered at clinicaltrials.gov as NCT01991626. © 2017 American Society

Deuterium-labelled N-acyl-l-homoserine lactones (AHLs) - inter-kingdom signalling molecules - synthesis, structural studies, and interactions with model lipid membranes

Energy Technology Data Exchange (ETDEWEB)

Jakubczyk, Dorota [Institute of Organic Chemistry, Karlsruhe Institute of Technology, Karlsruhe (Germany); Institute of Functional Interfaces, Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen (Germany); Barth, Christoph; Anastassacos, Frances; Koelsch, Patrick; Schepers, Ute [Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen (Germany); Kubas, Adam; Fink, Karin [Institute of Nanotechnology, Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen (Germany); Brenner-Weiss, Gerald [Institute of Functional Interfaces, Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen (Germany); Braese, Stefan [Institute of Organic Chemistry, Karlsruhe Institute of Technology, Karlsruhe (Germany)

2012-04-15

N-Acyl-l-homoserine lactones (AHLs) are synthesized by Gram-negative bacteria. These quorum-sensing molecules play an important role in the context of bacterial infection and biofilm formation. They also allow communication between microorganisms and eukaryotic cells (inter-kingdom signalling). However, very little is known about the entire mechanism of those interactions. Precise structural studies are required to analyse the different AHL isomers as only one form is biologically most active. Theoretical studies combined with experimental infrared and Raman spectroscopic data are therefore undertaken to characterise the obtained compounds. To mimic interactions between AHL and cell membranes, we studied the insertion of AHL in supported lipid bilayers, using vibrational sum-frequency-generation spectroscopy. Deuterium-labelled AHLs were thus synthesized. Starting from readily available deuterated fatty acids, a two-step procedure towards deuterated N-acyl-l-homoserine lactones with varying chain lengths is described. This included the acylation of Meldrum's acid followed by amidation. Additionally, the detailed analytical evaluation of the products is presented herein. (orig.)

Heme-coordinated histidine residues form non-specific functional "ferritin-heme" peroxidase system: Possible and partial mechanistic relevance to oxidative stress-mediated pathology in neurodegenerative diseases.

Science.gov (United States)

Esmaeili, Sajjad; Kooshk, Mohammad Reza Ashrafi; Asghari, Seyyed Mohsen; Khodarahmi, Reza

2016-10-01

Ferritin is a giant protein composed of 24 subunits which is able to sequester up to 4500 atoms of iron. We proposed two kinds of heme binding sites in mammalian ferritins and provided direct evidence for peroxidase activity of heme-ferritin, since there is the possibility that "ferritin-heme" systems display unexpected catalytic behavior like heme-containing enzymes. In the current study, peroxidase activity of heme-bound ferritin was studied using TMB(1), l-DOPA, serotonin, and dopamine, in the presence of H2O2, as oxidant substrate. The catalytic oxidation of TMB was consistent with first-order kinetics with respect to ferritin concentration. Perturbation of the binding affinity and catalytic behavior of heme-bound His-modified ferritin were also documented. We also discuss the importance of the peroxidase-/nitrative-mediated oxidation of vital molecules as well as ferritin-induced catalase inhibition using in vitro experimental system. Uncontrollable "heme-ferritin"-based enzyme activity as well as up-regulation of heme and ferritin may inspire that some oxidative stress-mediated cytotoxic effects in AD-affected cells could be correlated to ferritin-heme interaction and/or ferritin-induced catalase inhibition and describe its contribution as an important causative pathogenesis mechanism in some neurodegenerative disorders. Copyright © 2016 Elsevier B.V. All rights reserved.

Heme-Induced Biomarkers Associated with Red Meat Promotion of colon Cancer Are Not Modulated by the Intake of Nitrite

Science.gov (United States)

Chenni, Fatima Z; Taché, Sylviane; Naud, Nathalie; Guéraud, Françoise; Hobbs, Ditte A; Kunhle, Gunter G C; Pierre, Fabrice H; Corpet, Denis E

2013-01-01

Red and processed meat consumption is associated with the risk of colorectal cancer. Three hypotheses are proposed to explain this association, via heme/alcenal, heterocyclic amines or N-nitroso compounds. Rats have often been used to study these hypotheses, but the lack of enterosalivary cycle of nitrate in rats casts doubt on the relevance of this animal model to predict nitroso- and heme-associated human colon carcinogenesis. The present study was thus designed to clarify whether a nitrite intake that mimics the enterosalivary cycle can modulate heme-induced nitrosation and fat peroxidation. This study shows that, in contrast with the starting hypothesis, salivary nitrite did not change the effect of hemoglobin on biochemical markers linked to colon carcinogenesis, notably lipid peroxidation and cytotoxic activity in the colon of rat. However, ingested sodium nitrite increased fecal nitroso-compounds level, but their fecal concentration and their nature (iron-nitrosyl) would not be associated with an increased risk of cancer. The rat model could thus be relevant to study the effect of red meat on colon carcinogenesis in spite of the lack of nitrite recycling in rat’s saliva. PMID:23441609

Unique structure and stability of HmuY, a novel heme-binding protein of Porphyromonas gingivalis.

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Halina Wójtowicz

2009-05-01

Full Text Available Infection, survival, and proliferation of pathogenic bacteria in humans depend on their capacity to impair host responses and acquire nutrients in a hostile environment. Among such nutrients is heme, a co-factor for oxygen storage, electron transport, photosynthesis, and redox biochemistry, which is indispensable for life. Porphyromonas gingivalis is the major human bacterial pathogen responsible for severe periodontitis. It recruits heme through HmuY, which sequesters heme from host carriers and delivers it to its cognate outer-membrane transporter, the TonB-dependent receptor HmuR. Here we report that heme binding does not significantly affect the secondary structure of HmuY. The crystal structure of heme-bound HmuY reveals a new all-beta fold mimicking a right hand. The thumb and fingers pinch heme iron through two apical histidine residues, giving rise to highly symmetric octahedral iron co-ordination. The tetrameric quaternary arrangement of the protein found in the crystal structure is consistent with experiments in solution. It shows that thumbs and fingertips, and, by extension, the bound heme groups, are shielded from competing heme-binding proteins from the host. This may also facilitate heme transport to HmuR for internalization. HmuY, both in its apo- and in its heme-bound forms, is resistant to proteolytic digestion by trypsin and the major secreted proteases of P. gingivalis, gingipains K and R. It is also stable against thermal and chemical denaturation. In conclusion, these studies reveal novel molecular properties of HmuY that are consistent with its role as a putative virulence factor during bacterial infection.

Iron release from ferritin and lipid peroxidation by radiolytically generated reducing radicals

International Nuclear Information System (INIS)

Reif, D.W.; Schubert, J.; Aust, S.D.

1988-01-01

Iron is involved in the formation of oxidants capable of damaging membranes, protein, and DNA. Using 137 Cs gamma radiation, we investigated the release of iron from ferritin and concomitant lipid peroxidation by radiolytically generated reducing radicals, superoxide and the carbon dioxide anion radical. Both radicals released iron from ferritin with similar efficiencies and iron mobilization from ferritin required an iron chelator. Radiolytically generated superoxide anion resulted in peroxidation of phospholipid liposomes as measured by malondialdehyde formation only when ferritin was included as an iron source and the released iron was found to be chelated by the phospholipid liposomes

Chloroquine Interference with Hemoglobin Endocytic Trafficking Suppresses Adaptive Heme and Iron Homeostasis in Macrophages: The Paradox of an Antimalarial Agent

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Christian A. Schaer

2013-01-01

Full Text Available The CD163 scavenger receptor pathway for Hb:Hp complexes is an essential mechanism of protection against the toxicity of extracellular hemoglobin (Hb, which can accumulate in the vasculature and within tissues during hemolysis. Chloroquine is a lysosomotropic agent, which has been extensively used as an antimalarial drug in the past, before parasite resistance started to limit its efficacy in most parts of the world. More recent use of chloroquine is related to its immunomodulatory activity in patients with autoimmune diseases, which may also involve hemolytic disease components. In this study we examined the effects of chloroquine on the human Hb clearance pathway. For this purpose we developed a new mass-spectrometry-based method to specifically quantify intracellular Hb peptides within the endosomal-lysosomal compartment by single reaction monitoring (SRM. We found that chloroquine exposure impairs trafficking of Hb:Hp complexes through the endosomal-lysosomal compartment after internalization by CD163. Relative quantification of intracellular Hb peptides by SRM confirmed that chloroquine blocked cellular Hb:Hp catabolism. This effect suppressed the cellular heme-oxygenase-1 (HO-1 response and shifted macrophage iron homeostasis towards inappropriately high expression of the transferrin receptor with concurrent inhibition of ferroportin expression. A functional deficiency of Hb detoxification and heme-iron recycling may therefore be an adverse consequence of chloroquine treatment during hemolysis.

Coordinate expression of heme and globin is essential for effective erythropoiesis.

Science.gov (United States)

Doty, Raymond T; Phelps, Susan R; Shadle, Christina; Sanchez-Bonilla, Marilyn; Keel, Siobán B; Abkowitz, Janis L

2015-12-01

Erythropoiesis requires rapid and extensive hemoglobin production. Heme activates globin transcription and translation; therefore, heme synthesis must precede globin synthesis. As free heme is a potent inducer of oxidative damage, its levels within cellular compartments require stringent regulation. Mice lacking the heme exporter FLVCR1 have a severe macrocytic anemia; however, the mechanisms that underlie erythropoiesis dysfunction in these animals are unclear. Here, we determined that erythropoiesis failure occurs in these animals at the CFU-E/proerythroblast stage, a point at which the transferrin receptor (CD71) is upregulated, iron is imported, and heme is synthesized--before ample globin is produced. From the CFU-E/proerythroblast (CD71(+) Ter119(-) cells) stage onward, erythroid progenitors exhibited excess heme content, increased cytoplasmic ROS, and increased apoptosis. Reducing heme synthesis in FLVCR1-defient animals via genetic and biochemical approaches improved the anemia, implying that heme excess causes, and is not just associated with, the erythroid marrow failure. Expression of the cell surface FLVCR1 isoform, but not the mitochondrial FLVCR1 isoform, restored normal rbc production, demonstrating that cellular heme export is essential. Together, these studies provide insight into how heme is regulated to allow effective erythropoiesis, show that erythropoiesis fails when heme is excessive, and emphasize the importance of evaluating Ter119(-) erythroid cells when studying erythroid marrow failure in murine models.

Synthesis of 5-hydroxyectoine from ectoine: crystal structure of the non-heme iron(II and 2-oxoglutarate-dependent dioxygenase EctD.

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Klaus Reuter

2010-05-01

Full Text Available As a response to high osmolality, many microorganisms synthesize various types of compatible solutes. These organic osmolytes aid in offsetting the detrimental effects of low water activity on cell physiology. One of these compatible solutes is ectoine. A sub-group of the ectoine producer's enzymatically convert this tetrahydropyrimidine into a hydroxylated derivative, 5-hydroxyectoine. This compound also functions as an effective osmostress protectant and compatible solute but it possesses properties that differ in several aspects from those of ectoine. The enzyme responsible for ectoine hydroxylation (EctD is a member of the non-heme iron(II-containing and 2-oxoglutarate-dependent dioxygenases (EC 1.14.11. These enzymes couple the decarboxylation of 2-oxoglutarate with the formation of a high-energy ferryl-oxo intermediate to catalyze the oxidation of the bound organic substrate. We report here the crystal structure of the ectoine hydroxylase EctD from the moderate halophile Virgibacillus salexigens in complex with Fe(3+ at a resolution of 1.85 A. Like other non-heme iron(II and 2-oxoglutarate dependent dioxygenases, the core of the EctD structure consists of a double-stranded beta-helix forming the main portion of the active-site of the enzyme. The positioning of the iron ligand in the active-site of EctD is mediated by an evolutionarily conserved 2-His-1-carboxylate iron-binding motif. The side chains of the three residues forming this iron-binding site protrude into a deep cavity in the EctD structure that also harbours the 2-oxoglutarate co-substrate-binding site. Database searches revealed a widespread occurrence of EctD-type proteins in members of the Bacteria but only in a single representative of the Archaea, the marine crenarchaeon Nitrosopumilus maritimus. The EctD crystal structure reported here can serve as a template to guide further biochemical and structural studies of this biotechnologically interesting enzyme family.

Increased lipid peroxidation in pregnant women after iron and vitamin C supplementation.

Science.gov (United States)

Lachili, B; Hininger, I; Faure, H; Arnaud, J; Richard, M J; Favier, A; Roussel, A M

2001-11-01

Iron overload could promote the generation of free radicals and result in deleterious cellular damages. A physiological increase of oxidative stress has been observed in pregnancy. A routine iron supplement, especially a combined iron and vitamin C supplementation, without biological justifications (low hemoglobin [Hb] and iron stores) could therefore aggravate this oxidative risk. We investigated the effect of a daily combined iron supplementation (100 mg/d as fumarate) and vitamin C (500 mg/d as ascorbate) for the third trimester of pregnancy on lipid peroxidation (plasma TBARS), antioxidant micronutriments (Zn, Se, retinol, vitamin E, (beta-carotene) and antioxidant metalloenzymes (RBC Cu-Zn SOD and Se-GPX). The iron-supplemented group (n = 27) was compared to a control group (n = 27), age and number of pregnancies matched. At delivery, all the women exhibited normal Hb and ferritin values. In the supplemented group, plasma iron level was higher than in the control group (26.90 +/- 5.52 mmol/L) and TBARs plasma levels were significantly enhanced (p cell antioxidant metalloenzymes. Furthermore, the alpha-tocopherol plasma level was lowered in the iron-supplemented groups, suggesting an increased utilization of vitamin E. These data show that pharmalogical doses of iron, associated with high vitamin C intakes, can result in uncontrolled lipid peroxidation. This is predictive of adverse effects for the mother and the fetus. This study illustrates the potential harmful effects of iron supplementation when prescribed only on the assumption of anemia and not on the bases of biological criteria.

Iron depletion in HCT116 cells diminishes the upregulatory effect of phenethyl isothiocyanate on heme oxygenase-1

International Nuclear Information System (INIS)

Bolloskis, Michael P.; Carvalho, Fabiana P.; Loo, George

2016-01-01

Some of the health-promoting properties of cruciferous vegetables are thought to be partly attributed to isothiocyanates. These phytochemicals can upregulate the expression of certain cytoprotective stress genes, but it is unknown if a particular nutrient is involved. Herein, the objective was to ascertain if adequate iron is needed for enabling HCT116 cells to optimally express heme oxygenase-1 (HO-1) when induced by phenethyl isothiocyanate (PEITC). PEITC increased HO-1 expression and also nuclear translocation of Nrf2, which is a transcription factor known to activate the HO-1 gene. However, in HCT116 cells that were made iron-deficient by depleting intracellular iron with deferoxamine (DFO), PEITC was less able to increase HO-1 expression and nuclear translocation of Nrf2. These suppressive effects of DFO were overcome by replenishing the iron-deficient cells with the missing iron. To elucidate these findings, it was found that PEITC-induced HO-1 upregulation can be inhibited with thiol antioxidants (glutathione and N-acetylcysteine). Furthermore, NADPH oxidase inhibitors (diphenyleneiodonium and apocynin) and a superoxide scavenger (Tiron) each inhibited PEITC-induced HO-1 upregulation. In doing so, diphenyleneiodonium was the most potent and also inhibited nuclear translocation of redox-sensitive Nrf2. Collectively, the results imply that the HO-1 upregulation by PEITC involves an iron-dependent, oxidant signaling pathway. Therefore, it is concluded that ample iron is required to enable PEITC to fully upregulate HO-1 expression in HCT116 cells. As such, it is conceivable that iron-deficient individuals may not reap the full health benefits of eating PEITC-containing cruciferous vegetables that via HO-1 may help protect against multiple chronic diseases. - Highlights: • PEITC increased HO-1 expression in HCT116 cells. • PEITC-induced HO-1 upregulation was impaired in iron-depleted HCT116 cells. • Impairment of PEITC-induced HO-1 upregulation was

Regiospecificity determinants of human heme oxygenase: differential NADPH- and ascorbate-dependent heme cleavage by the R183E mutant.

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Wang, Jinling; Lad, Latesh; Poulos, Thomas L; Ortiz de Montellano, Paul R

2005-01-28

The ability of the human heme oxygenase-1 (hHO-1) R183E mutant to oxidize heme in reactions supported by either NADPH-cytochrome P450 reductase or ascorbic acid has been compared. The NADPH-dependent reaction, like that of wild-type hHO-1, yields exclusively biliverdin IXalpha. In contrast, the R183E mutant with ascorbic acid as the reductant produces biliverdin IXalpha (79 +/- 4%), IXdelta (19 +/- 3%), and a trace of IXbeta. In the presence of superoxide dismutase and catalase, the yield of biliverdin IXdelta is decreased to 8 +/- 1% with a corresponding increase in biliverdin IXalpha. Spectroscopic analysis of the NADPH-dependent reaction shows that the R183E ferric biliverdin complex accumulates, because reduction of the iron, which is required for sequential iron and biliverdin release, is impaired. Reversal of the charge at position 183 makes reduction of the iron more difficult. The crystal structure of the R183E mutant, determined in the ferric and ferrous-NO bound forms, shows that the heme primarily adopts the same orientation as in wild-type hHO-1. The structure of the Fe(II).NO complex suggests that an altered active site hydrogen bonding network supports catalysis in the R183E mutant. Furthermore, Arg-183 contributes to the regiospecificity of the wild-type enzyme, but its contribution is not critical. The results indicate that the ascorbate-dependent reaction is subject to a lower degree of regiochemical control than the NADPH-dependent reaction. Ascorbate may be able to reduce the R183E ferric and ferrous dioxygen complexes in active site conformations that cannot be reduced by NADPH-cytochrome P450 reductase.

Alteration of the Regiospecificity of Human Heme Oxygenase-1 by Unseating of the Heme but not Disruption of the Distal Hydrogen Bonding Network†

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Wang, Jinling; Evans, John P.; Ogura, Hiroshi; La Mar, Gerd N.; Ortiz de Montellano, Paul R.

2008-01-01

Heme oxygenase regiospecifically oxidizes heme at the α-meso position to give biliverdin IXα, CO, and iron. The heme orientation within the active site, which is thought to determine the oxidation regiospecificity, is shown here for the human enzyme (hHO1) to be largely determined by interactions between the heme carboxylic acid groups and residues Arg183 and Lys18 but not Tyr134. Mutation of either Arg183 or Lys18 individually does not significantly alter the NADPH-cytochrome P450 reductase-dependent reaction regiochemistry, but partially shifts the oxidation to the β/δ-meso positions in the reaction supported by ascorbic acid. Mutation of Glu29 to a lysine, which places a positive charge where it can interact with a heme carboxyl if the heme rotates by ~90°, causes a slight loss of regiospecificity, but combined with the R183E and K18E mutations results primarily in β/δ-meso oxidation of the heme under all conditions. NMR analysis of heme binding to the triple K18E/E29K/R183E mutant confirms rotation of the heme in the active site. Kinetic studies demonstrate that mutations of Arg183 greatly impair the rate of the P450 reductase-dependent reaction, in accord with the earlier finding that Arg183 is involved in binding of the reductase to hHO1, but have little effect on the ascorbate reaction. Mutations of Asp140 and Tyr58 that disrupt the active site hydrogen bonding network, impair catalytic rates but do not influence the oxidation regiochemistry. The results indicate both that the oxidation regiochemistry is largely controlled by ionic interactions of the heme propionic acid groups with the protein and that shifts in regiospecificity involve rotation of the heme about an axis perpendicular to the heme plane. PMID:16388581

Plasma protein haptoglobin modulates renal iron loading

DEFF Research Database (Denmark)

Fagoonee, Sharmila; Gburek, Jakub; Hirsch, Emilio

2005-01-01

Haptoglobin is the plasma protein with the highest binding affinity for hemoglobin. The strength of hemoglobin binding and the existence of a specific receptor for the haptoglobin-hemoglobin complex in the monocyte/macrophage system clearly suggest that haptoglobin may have a crucial role in heme...... distribution of hemoglobin in haptoglobin-deficient mice resulted in abnormal iron deposits in proximal tubules during aging. Moreover, iron also accumulated in proximal tubules after renal ischemia-reperfusion injury or after an acute plasma heme-protein overload caused by muscle injury, without affecting...... morphological and functional parameters of renal damage. These data demonstrate that haptoglobin crucially prevents glomerular filtration of hemoglobin and, consequently, renal iron loading during aging and following acute plasma heme-protein overload....

Species- and age-dependent sensitivity to ozone in young plants of pea, wheat and spinach. Effects on acyl lipid and pigment content and metabolism

Energy Technology Data Exchange (ETDEWEB)

Carlsson, A.S.; Wallin, G.; Sandelius, A.S.

1996-11-01

Acyl lipids and pigments were analyzed in young plants of garden pea, spring wheat and spinach exposed to < 5 or 65 nl l{sup -1} ozone 12 h per day for 5 days, in one set of experiments, the plants were exposed to {sup 14}CO{sub 2} for 2 h 3 days prior to ozone exposure. The plants responded differently to the moderately enhanced level of ozone used. Spinach was not at all sensitive while in both pea and wheat, leaves of different ages differed in ozone sensitivity. In pea, ozone sensitivity increased with leaf age. In the second and third oldest leaves, the amounts of galactolipids per leaf area and the proportions of 18:3 of the total lipid extract and of phosphatidylglycerol decreased. In the second oldest leaf, ozone also caused a decreased proportion of 18:3 of monogalactosyldiacylglycerol. In the fourth oldest leaf, lipid composition and galactolipid unsaturation was unaffected, but ozone caused decreased leaf expansion resulting in increased acyl lipid content per leaf area. In both the first and second leaves of wheat, ozone fumigation caused a marked decrease in the content of monogalactosyldiacylglycerol and in the first leaf, the contents of phosphatidylcholine and phosphatidylethanolamine increased. The proportion of 18:3 in phosphatidylcholine was larger in ozone-fumigated than in control plants, while the reverse applied for phosphatidylglycerol. In the oldest sampled leaves of pea and wheat, ozone caused an increase in the radioactivity associated with {beta}-carotene, indicting increased turnover. Thus, while spinach was unaffected, in both pea and whet ozone caused a decrease in the proportion of chloroplast membrane lipids to non-chloroplast membrane lipids in older leaves while younger leaves were less sensitive. (au) 21 refs.

Modulation of cellulase activity by charged lipid bilayers with different acyl chain properties for efficient hydrolysis of ionic liquid-pretreated cellulose.

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Mihono, Kai; Ohtsu, Takeshi; Ohtani, Mai; Yoshimoto, Makoto; Kamimura, Akio

2016-10-01

The stability of cellulase activity in the presence of ionic liquids (ILs) is critical for the enzymatic hydrolysis of insoluble cellulose pretreated with ILs. In this work, cellulase was incorporated in the liposomes composed of negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and zwitterionic phosphatidylcholines (PCs) with different length and degree of unsaturation of the acyl chains. The liposomal cellulase-catalyzed reaction was performed at 45°C in the acetate buffer solution (pH 4.8) with 2.0g/L CC31 as cellulosic substrate. The crystallinity of CC31 was reduced by treating with 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) at 120°C for 30min. The liposomal cellulase continuously catalyzed hydrolysis of the pretreated CC31 for 48h producing glucose in the presence of 15wt% [Bmim]Cl. The charged lipid membranes were interactive with [Bmim](+), as elucidated by the [Bmim]Cl-induced alterations in fluorescence polarization of the membrane-embedded 1,6-diphenyl-1,3,5-hexatriene (DPH) molecules. The charged membranes offered the microenvironment where inhibitory effects of [Bmim]Cl on the cellulase activity was relieved. The maximum glucose productivity GP of 10.8 mmol-glucose/(hmol-lipid) was obtained at the reaction time of 48h with the cellulase incorporated in the liposomes ([lipid]=5.0mM) composed of 50mol% POPG and 1,2-dilauroyl-sn-glycero-3-phosohocholine (DLPC) with relatively short and saturated acyl chains. Copyright © 2016 Elsevier B.V. All rights reserved.

A Heme Oxygenase-1 Transducer Model of Degenerative and Developmental Brain Disorders

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Hyman M. Schipper

2015-03-01

Full Text Available Heme oxygenase-1 (HO-1 is a 32 kDa protein which catalyzes the breakdown of heme to free iron, carbon monoxide and biliverdin. The Hmox1 promoter contains numerous consensus sequences that render the gene exquisitely sensitive to induction by diverse pro-oxidant and inflammatory stimuli. In “stressed” astroglia, HO-1 hyperactivity promotes mitochondrial iron sequestration and macroautophagy and may thereby contribute to the pathological iron deposition and bioenergetic failure documented in Alzheimer disease, Parkinson disease and certain neurodevelopmental conditions. Glial HO-1 expression may also impact neuroplasticity and cell survival by modulating brain sterol metabolism and the proteasomal degradation of neurotoxic proteins. The glial HO-1 response may represent a pivotal transducer of noxious environmental and endogenous stressors into patterns of neural damage and repair characteristic of many human degenerative and developmental CNS disorders.

Effects of moderately enhanced levels of ozone on the acyl lipid composition and dynamical properties of plasma membranes isolated from garden pea (Pisum sativum)

DEFF Research Database (Denmark)

Hellgren, Lars; Sellden, G.; Sandelius, A.S.

2001-01-01

Plasma membranes were isolated from leaves of 16-day-old garden pea, Pisum sativum L., that had been grown in the absence or presence of 65 nl l(-1) ozone for 4 days prior to membrane isolation, Plasma membranes from ozone-fumigated plants contained significantly more acyl lipids per protein than....../stigmasterol and lipid/protein ratios, and suggesting that ozone-fumigated pea plants may be more susceptible to freezing injuries....... lipids, as well as in PC and PE, The amount of free sterols per protein was unaltered, but the percentage of campesterol increased, concomitant with a decrease in stigmasterol, The dynamical properties of the isolated plasma membranes were assessed using Laurdan fluorescence spectroscopy, which monitors...

Mini Heme-Proteins: Designability of Structure and Diversity of Functions.

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Rai, Jagdish

2017-08-30

Natural heme proteins may have heme bound to poly-peptide chain as a cofactor via noncovalent forces or heme as a prosthetic group may be covalently bound to the proteins. Nature has used porphyrins in diverse functions like electron transfer, oxidation, reduction, ligand binding, photosynthesis, signaling, etc. by modulating its properties through diverse protein matrices. Synthetic chemists have tried to utilize these molecules in equally diverse industrial and medical applications due to their versatile electro-chemical and optical properties. The heme iron has catalytic activity which can be modulated and enhanced for specific applications by protein matrix around it. Heme proteins can be designed into novel enzymes for sterio specific catalysis ranging from oxidation to reduction. These designed heme-proteins can have applications in industrial catalysis and biosensing. A peptide folds around heme easily due to hydrophobic effect of the large aromatic ring of heme. The directional property of co-ordinate bonding between peptide and metal ion in heme further specifies the structure. Therefore heme proteins can be easily designed for targeted structure and catalytic activity. The central aromatic chemical entity in heme viz. porphyrin is a very ancient molecule. Its presence in the prebiotic soup and in all forms of life suggests that it has played a vital role in the origin and progressive evolution of living organisms. Porphyrin macrocycles are highly conjugated systems composed of four modified pyrrole subunits interconnected at their α -carbon atoms via methine (=CH-) bridges. Initial minimalist models of hemoproteins focused on effect of heme-ligand co-ordinate bonding on chemical reactivity, spectroscopy, electrochemistry and magnetic properties of heme. The great sensitivity of these spectroscopic features of heme to its surrounding makes them extremely useful in structural elucidation of designed heme-peptide complexes. Therefore heme proteins are

Staphylococcus aureus redirects central metabolism to increase iron availability.

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David B Friedman

2006-08-01

Full Text Available Staphylococcus aureus pathogenesis is significantly influenced by the iron status of the host. However, the regulatory impact of host iron sources on S. aureus gene expression remains unknown. In this study, we combine multivariable difference gel electrophoresis and mass spectrometry with multivariate statistical analyses to systematically cluster cellular protein response across distinct iron-exposure conditions. Quadruplicate samples were simultaneously analyzed for alterations in protein abundance and/or post-translational modification state in response to environmental (iron chelation, hemin treatment or genetic (Deltafur alterations in bacterial iron exposure. We identified 120 proteins representing several coordinated biochemical pathways that are affected by changes in iron-exposure status. Highlighted in these experiments is the identification of the heme-regulated transport system (HrtAB, a novel transport system which plays a critical role in staphylococcal heme metabolism. Further, we show that regulated overproduction of acidic end-products brought on by iron starvation decreases local pH resulting in the release of iron from the host iron-sequestering protein transferrin. These findings reveal novel strategies used by S. aureus to acquire scarce nutrients in the hostile host environment and begin to define the iron and heme-dependent regulons of S. aureus.

Novel endogenous N-acyl amides activate TRPV1-4 receptors, BV-2 microglia, and are regulated in brain in an acute model of inflammation

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Raboune, Siham; Stuart, Jordyn M.; Leishman, Emma; Takacs, Sara M.; Rhodes, Brandon; Basnet, Arjun; Jameyfield, Evan; McHugh, Douglas; Widlanski, Theodore; Bradshaw, Heather B.

2014-01-01

A family of endogenous lipids, structurally analogous to the endogenous cannabinoid, N-arachidonoyl ethanolamine (Anandamide), and called N-acyl amides have emerged as a family of biologically active compounds at TRP receptors. N-acyl amides are constructed from an acyl group and an amine via an amide bond. This same structure can be modified by changing either the fatty acid or the amide to form potentially hundreds of lipids. More than 70 N-acyl amides have been identified in nature. We have ongoing studies aimed at isolating and characterizing additional members of the family of N-acyl amides in both central and peripheral tissues in mammalian systems. Here, using a unique in-house library of over 70 N-acyl amides we tested the following three hypotheses: (1) Additional N-acyl amides will have activity at TRPV1-4, (2) Acute peripheral injury will drive changes in CNS levels of N-acyl amides, and (3) N-acyl amides will regulate calcium in CNS-derived microglia. Through these studies, we have identified 20 novel N-acyl amides that collectively activate (stimulating or inhibiting) TRPV1-4. Using lipid extraction and HPLC coupled to tandem mass spectrometry we showed that levels of at least 10 of these N-acyl amides that activate TRPVs are regulated in brain after intraplantar carrageenan injection. We then screened the BV2 microglial cell line for activity with this N-acyl amide library and found overlap with TRPV receptor activity as well as additional activators of calcium mobilization from these lipids. Together these data provide new insight into the family of N-acyl amides and their roles as signaling molecules at ion channels, in microglia, and in the brain in the context of inflammation. PMID:25136293

Duodenal Cytochrome b (DCYTB in Iron Metabolism: An Update on Function and Regulation

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Darius J. R. Lane

2015-03-01

Full Text Available Iron and ascorbate are vital cellular constituents in mammalian systems. The bulk-requirement for iron is during erythropoiesis leading to the generation of hemoglobin-containing erythrocytes. Additionally; both iron and ascorbate are required as co-factors in numerous metabolic reactions. Iron homeostasis is controlled at the level of uptake; rather than excretion. Accumulating evidence strongly suggests that in addition to the known ability of dietary ascorbate to enhance non-heme iron absorption in the gut; ascorbate regulates iron homeostasis. The involvement of ascorbate in dietary iron absorption extends beyond the direct chemical reduction of non-heme iron by dietary ascorbate. Among other activities; intra-enterocyte ascorbate appears to be involved in the provision of electrons to a family of trans-membrane redox enzymes; namely those of the cytochrome b561 class. These hemoproteins oxidize a pool of ascorbate on one side of the membrane in order to reduce an electron acceptor (e.g., non-heme iron on the opposite side of the membrane. One member of this family; duodenal cytochrome b (DCYTB; may play an important role in ascorbate-dependent reduction of non-heme iron in the gut prior to uptake by ferrous-iron transporters. This review discusses the emerging relationship between cellular iron homeostasis; the emergent “IRP1-HIF2α axis”; DCYTB and ascorbate in relation to iron metabolism.

MR imaging of abscess by use of lipid-coated iron oxide particles

International Nuclear Information System (INIS)

Chan, T.W.; Eley, C.G.S.; Kressel, H.Y.

1990-01-01

The authors of this paper investigate the potential application of lipid-coated iron oxide particles as an MR contrast agent for imaging inflammatory process by using a rat subcutaneous abscess model induced by turpentine. Ten male Sprague-Dawley rats received subcutaneous injections of 0.1 mL of turpentine in the flank. At 24-36 hours later, the rats developed a subcutaneous abscess of 1-1.8 cm. An intravenous injection of lipid-coated iron oxide particles, Ferrosome (Vestar) at doses of 25, 40, 100, 200 μg/kg was administered. The animals were imaged at 12-24 hours later on a 1.5-T magnet using a 3-inch (7.62-cm) surface coil. Two animals were also imaged 5 days later. T1-weighted, T2-weighted, and multiplanar gradient-recalled (MPGR) sequences were obtained. The abscess was then excised and examined with routine H-E and iron staining

Iron accumulation with age, oxidative stress and functional decline.

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Jinze Xu

2008-08-01

Full Text Available Identification of biological mediators in sarcopenia is pertinent to the development of targeted interventions to alleviate this condition. Iron is recognized as a potent pro-oxidant and a catalyst for the formation of reactive oxygen species in biological systems. It is well accepted that iron accumulates with senescence in several organs, but little is known about iron accumulation in muscle and how it may affect muscle function. In addition, it is unclear if interventions which reduced age-related loss of muscle quality, such as calorie restriction, impact iron accumulation. We investigated non-heme iron concentration, oxidative stress to nucleic acids in gastrocnemius muscle and key indices of sarcopenia (muscle mass and grip strength in male Fischer 344 X Brown Norway rats fed ad libitum (AL or a calorie restricted diet (60% of ad libitum food intake starting at 4 months of age at 8, 18, 29 and 37 months of age. Total non-heme iron levels in the gastrocnemius muscle of AL rats increased progressively with age. Between 29 and 37 months of age, the non-heme iron concentration increased by approximately 200% in AL-fed rats. Most importantly, the levels of oxidized RNA in gastrocnemius muscle of AL rats were significantly increased as well. The striking age-associated increase in non-heme iron and oxidized RNA levels and decrease in sarcopenia indices were all attenuated in the calorie restriction (CR rats. These findings strongly suggest that the age-related iron accumulation in muscle contributes to increased oxidative damage and sarcopenia, and that CR effectively attenuates these negative effects.

Increased Heme Levels in the Heart Lead to Exacerbated Ischemic Injury.

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Sawicki, Konrad Teodor; Shang, Meng; Wu, Rongxue; Chang, Hsiang-Chun; Khechaduri, Arineh; Sato, Tatsuya; Kamide, Christine; Liu, Ting; Naga Prasad, Sathyamangla V; Ardehali, Hossein

2015-07-31

Heme is an essential iron-containing molecule for cardiovascular physiology, but in excess it may increase oxidative stress. Failing human hearts have increased heme levels, with upregulation of the rate-limiting enzyme in heme synthesis, δ-aminolevulinic acid synthase 2 (ALAS2), which is normally not expressed in cardiomyocytes. We hypothesized that increased heme accumulation (through cardiac overexpression of ALAS2) leads to increased oxidative stress and cell death in the heart. We first showed that ALAS2 and heme levels are increased in the hearts of mice subjected to coronary ligation. To determine the causative role of increased heme in the development of heart failure, we generated transgenic mice with cardiac-specific overexpression of ALAS2. While ALAS2 transgenic mice have normal cardiac function at baseline, their hearts display increased heme content, higher oxidative stress, exacerbated cell death, and worsened cardiac function after coronary ligation compared to nontransgenic littermates. We confirmed in cultured cardiomyoblasts that the increased oxidative stress and cell death observed with ALAS2 overexpression is mediated by increased heme accumulation. Furthermore, knockdown of ALAS2 in cultured cardiomyoblasts exposed to hypoxia reversed the increases in heme content and cell death. Administration of the mitochondrial antioxidant MitoTempo to ALAS2-overexpressing cardiomyoblasts normalized the elevated oxidative stress and cell death levels to baseline, indicating that the effects of increased ALAS2 and heme are through elevated mitochondrial oxidative stress. The clinical relevance of these findings was supported by the finding of increased ALAS2 induction and heme accumulation in failing human hearts from patients with ischemic cardiomyopathy compared to nonischemic cardiomyopathy. Heme accumulation is detrimental to cardiac function under ischemic conditions, and reducing heme in the heart may be a novel approach for protection against the

Association of dietary and supplemental iron and colorectal cancer in a population-based study.

Science.gov (United States)

Ashmore, Joseph H; Lesko, Samuel M; Miller, Paige E; Cross, Amanda J; Muscat, Joshua E; Zhu, Junjia; Liao, Jason; Harper, Gregory; Lazarus, Philip; Hartman, Terryl J

2013-11-01

We evaluated the role of dietary iron, heme iron, and supplemental iron on colorectal cancer (CRC) risk in a population-based case-control study in Pennsylvania, including 1005 incident cases and 1062 controls. Diet was assessed through a modified food frequency questionnaire that included supplement use and a meat-specific module. Cases reported intakes for the year before diagnosis, whereas controls reported intakes for the year before interview. Heme iron intake was calculated using a new heme database developed by the US National Cancer Institute. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using unconditional logistic regression. After multivariate adjustment, there were no significant associations between heme iron or total iron intake and CRC incidence. Dietary iron intake was inversely associated with CRC among women (OR Q5 vs. Q1=0.45; 95% CI=0.22-0.92), but not among men. Supplemental iron intake of more than 18 mg/day versus none was positively associated with CRC incidence (OR=2.31; 95% CI=1.48-3.59; P-trendconsumption of more than 18 mg/day of supplemental iron may increase risk for CRC.

Enhancement of nitrite on heme-induced oxidative reactions: A potential toxicological implication.

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Lu, Naihao; Chen, Wei; Zhu, Jingjie; Peng, Yi-Yuan

2012-02-01

Evidence to support the role of heme as major inducers of oxidative damage is increasingly present. Nitrite (NO(2)(-)) is one of the major end products of NO metabolism. Although the biological significance of heme/NO(2)(-)-mediated protein tyrosine nitration is a subject of great interest, the important roles of NO(2)(-) on heme-dependent redox reaction have been greatly underestimated. In this study, we investigated the influence of NO(2)(-) on heme -dependent oxidative reactions. It was found that NO(2)(-) had the capacity to act as a reducing agent to remove high oxidation states of heme iron. In the reduction of ferryl heme to ferric heme, NO(2)(-) was oxidized to a nitrating agent NO(2), and subsequently, tyrosine residues in bovine serum albumin (BSA) were nitrated. However, the presence of NO(2)(-) surprisingly exerted pro-oxidant effect on heme-H(2)O(2)-induced formation of BSA carbonyls at lower concentrations and enhanced the loss of HepG2 cell viability dose-dependently, which was probably due to the ability of this inorganic compound to efficiently enhance the peroxidase activity and oxidative degradation of heme. These data provide novel evidence that the dietary intake and experimental use of NO(2)(-) in vivo and in vitro would possess the pro-oxidant activity through interfering in heme-dependent oxidative reactions. Besides the classic role in protein tyrosine nitration, the deleterious effects on heme redox reactions may provide new insights into the toxicological implications of NO(2)(-) with cellular heme proteins. Copyright © 2011 Elsevier Ltd. All rights reserved.

Subpicosecond oxygen trapping in the heme pocket of the oxygen sensor FixL observed by time-resolved resonance Raman spectroscopy.

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Kruglik, Sergei G; Jasaitis, Audrius; Hola, Klara; Yamashita, Taku; Liebl, Ursula; Martin, Jean-Louis; Vos, Marten H

2007-05-01

Dissociation of oxygen from the heme domain of the bacterial oxygen sensor protein FixL constitutes the first step in hypoxia-induced signaling. In the present study, the photodissociation of the heme-O2 bond was used to synchronize this event, and time-resolved resonance Raman (TR(3)) spectroscopy with subpicosecond time resolution was implemented to characterize the heme configuration of the primary photoproduct. TR(3) measurements on heme-oxycomplexes are highly challenging and have not yet been reported. Whereas in all other known six-coordinated heme protein complexes with diatomic ligands, including the oxymyoglobin reported here, heme iron out-of-plane motion (doming) occurs faster than 1 ps after iron-ligand bond breaking; surprisingly, no sizeable doming is observed in the oxycomplex of the Bradyrhizobium japonicum FixL sensor domain (FixLH). This assessment is deduced from the absence of the iron-histidine band around 217 cm(-1) as early as 0.5 ps. We suggest that efficient ultrafast oxygen rebinding to the heme occurs on the femtosecond time scale, thus hindering heme doming. Comparing WT oxy-FixLH, mutant proteins FixLH-R220H and FixLH-R220Q, the respective carbonmonoxy-complexes, and oxymyoglobin, we show that a hydrogen bond of the terminal oxygen atom with the residue in position 220 is responsible for the observed behavior; in WT FixL this residue is arginine, crucially implicated in signal transmission. We propose that the rigid O2 configuration imposed by this residue, in combination with the hydrophobic and constrained properties of the distal cavity, keep dissociated oxygen in place. These results uncover the origin of the "oxygen cage" properties of this oxygen sensor protein.

Structural Characterization of Heme Environmental Mutants of CgHmuT that Shuttles Heme Molecules to Heme Transporters

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Norifumi Muraki

2016-05-01

Full Text Available Corynebacteria contain a heme uptake system encoded in hmuTUV genes, in which HmuT protein acts as a heme binding protein to transport heme to the cognate transporter HmuUV. The crystal structure of HmuT from Corynebacterium glutamicum (CgHmuT reveals that heme is accommodated in the central cleft with His141 and Tyr240 as the axial ligands and that Tyr240 forms a hydrogen bond with Arg242. In this work, the crystal structures of H141A, Y240A, and R242A mutants were determined to understand the role of these residues for the heme binding of CgHmuT. Overall and heme environmental structures of these mutants were similar to those of the wild type, suggesting that there is little conformational change in the heme-binding cleft during heme transport reaction with binding and the dissociation of heme. A loss of one axial ligand or the hydrogen bonding interaction with Tyr240 resulted in an increase in the redox potential of the heme for CgHmuT to be reduced by dithionite, though the wild type was not reduced under physiological conditions. These results suggest that the heme environmental structure stabilizes the ferric heme binding in CgHmuT, which will be responsible for efficient heme uptake under aerobic conditions where Corynebacteria grow.

Introduction of water into the heme distal side by Leu65 mutations of an oxygen sensor, YddV, generates verdoheme and carbon monoxide, exerting the heme oxygenase reaction.

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Stranava, Martin; Martínková, Markéta; Stiborová, Marie; Man, Petr; Kitanishi, Kenichi; Muchová, Lucie; Vítek, Libor; Martínek, Václav; Shimizu, Toru

2014-11-01

The globin-coupled oxygen sensor, YddV, is a heme-based oxygen sensor diguanylate cyclase. Oxygen binding to the heme Fe(II) complex in the N-terminal sensor domain of this enzyme substantially enhances its diguanylate cyclase activity which is conducted in the C-terminal functional domain. Leu65 is located on the heme distal side and is important for keeping the stability of the heme Fe(II)-O2 complex by preventing the entry of the water molecule to the heme complex. In the present study, it was found that (i) Escherichia coli-overexpressed and purified L65N mutant of the isolated heme-bound domain of YddV (YddV-heme) contained the verdoheme iron complex and other modified heme complexes as determined by optical absorption spectroscopy and mass spectrometry; (ii) CO was generated in the reconstituted system composed of heme-bound L65N and NADPH:cytochrome P450 reductase as confirmed by gas chromatography; (iii) CO generation of heme-bound L65N in the reconstituted system was inhibited by superoxide dismutase and catalase. In a concordance with the result, the reactive oxygen species increased the CO generation; (iv) the E. coli cells overexpressing the L65N protein of YddV-heme also formed significant amounts of CO compared to the cells overexpressing the wild type protein; (v) generation of verdoheme and CO was also observed for other mutants at Leu65 as well, but to a lesser extent. Since Leu65 mutations are assumed to introduce the water molecule into the heme distal side of YddV-heme, it is suggested that the water molecule would significantly contribute to facilitating heme oxygenase reactions for the Leu65 mutants. Copyright © 2014 Elsevier Inc. All rights reserved.

Effect of irradiation and storage in the iron availability in lamb meat treated with different diets

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Souza, Adriana Regia Marques de; Arthur, Valter

2008-01-01

Irradiation is an efficient method to increase the microbiological safety and to maintain the nutrients such as iron in the meat. The best absorption form, heme iron, should be preserved in order to increase the nutritional quality of stored meat. The diet can alter the nutrients contents and form in the meat. The iron is provided from the diet and it is an essential element for the metabolic processes such as oxygen transport, oxidative metabolism, and cellular growth. Meat lamb samples treated with different diets (it controls, TAC1, TAC2 and sorghum) were wrapped to vacuous, and irradiated in the doses 0, 2 and 4 kGy and stored at 4 deg C during 15 days. The values of total iron and heme iron were measured at 0 and 15 days of storage. The storage reduced the content of total iron (18.36 for 14.28 mg.100 g -1 ) and heme iron (13.78 for 10.52 mg.100 g -1 ). The diets affected the levels of total and heme iron of the meat, and the sorghum diet was the one that presented the larger content. The dose of 2 kGy was the one that affected the iron the most independently of the storage time. It was verified that the amounts of total and heme iron varied according to the storage time, irradiation doses, and lamb diets. (author)

N-acylated peptides derived from human lactoferricin perturb organization of cardiolipin and phosphatidylethanolamine in cell membranes and induce defects in Escherichia coli cell division.

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Dagmar Zweytick

Full Text Available Two types of recently described antibacterial peptides derived from human lactoferricin, either nonacylated or N-acylated, were studied for their different interaction with membranes of Escherichia coli in vivo and in model systems. Electron microscopy revealed striking effects on the bacterial membrane as both peptide types induced formation of large membrane blebs. Electron and fluorescence microscopy, however demonstrated that only the N-acylated peptides partially induced the generation of oversized cells, which might reflect defects in cell-division. Further a different distribution of cardiolipin domains on the E. coli membrane was shown only in the presence of the N-acylated peptides. The lipid was distributed over the whole bacterial cell surface, whereas cardiolipin in untreated and nonacylated peptide-treated cells was mainly located at the septum and poles. Studies with bacterial membrane mimics, such as cardiolipin or phosphatidylethanolamine revealed that both types of peptides interacted with the negatively charged lipid cardiolipin. The nonacylated peptides however induced segregation of cardiolipin into peptide-enriched and peptide-poor lipid domains, while the N-acylated peptides promoted formation of many small heterogeneous domains. Only N-acylated peptides caused additional severe effects on the main phase transition of liposomes composed of pure phosphatidylethanolamine, while both peptide types inhibited the lamellar to hexagonal phase transition. Lipid mixtures of phosphatidylethanolamine and cardiolipin revealed anionic clustering by all peptide types. However additional strong perturbation of the neutral lipids was only seen with the N-acylated peptides. Nuclear magnetic resonance demonstrated different conformational arrangement of the N-acylated peptide in anionic and zwitterionic micelles revealing possible mechanistic differences in their action on different membrane lipids. We hypothesized that both peptides kill

Influence of iron solubility and charged surface-active compounds on lipid oxidation in fatty acid ethyl esters containing association colloids.

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Homma, Rika; Johnson, David R; McClements, D Julian; Decker, Eric A

2016-05-15

The impact of iron compounds with different solubilities on lipid oxidation was studied in the presence and absence of association colloids. Iron (III) sulfate only accelerated lipid oxidation in the presence of association colloids while iron (III) oleate accelerated oxidation in the presence and absence of association colloids. Further, iron (III) oxide retarded lipid oxidation both with and without association colloids. The impact of charged association colloids on lipid oxidation in ethyl oleate was also investigated. Association colloids consisting of the anionic surface-active compound dodecyl sulphosuccinate sodium salt (AOT), cationic surface-active compound hexadecyltrimethylammonium bromide (CTAB), and nonionic surface-active compound 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol (Triton X-100) retarded, promoted, and had no effect on lipid oxidation rates, respectively. These results indicate that the polarity of metal compounds and the charge of association colloids play a big role in lipid oxidation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Clinically Important Features of Porphyrin and Heme Metabolism and the Porphyrias

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Siddesh Besur

2014-11-01

Full Text Available Heme, like chlorophyll, is a primordial molecule and is one of the fundamental pigments of life. Disorders of normal heme synthesis may cause human diseases, including certain anemias (X-linked sideroblastic anemias and porphyrias. Porphyrias are classified as hepatic and erythropoietic porphyrias based on the organ system in which heme precursors (5-aminolevulinic acid (ALA, porphobilinogen and porphyrins are chiefly overproduced. The hepatic porphyrias are further subdivided into acute porphyrias and chronic hepatic porphyrias. The acute porphyrias include acute intermittent, hereditary copro-, variegate and ALA dehydratase deficiency porphyria. Chronic hepatic porphyrias include porphyria cutanea tarda and hepatoerythropoietic porphyria. The erythropoietic porphyrias include congenital erythropoietic porphyria (Gűnther’s disease and erythropoietic protoporphyria. In this review, we summarize the key features of normal heme synthesis and its differing regulation in liver versus bone marrow. In both organs, principal regulation is exerted at the level of the first and rate-controlling enzyme, but by different molecules (heme in the liver and iron in the bone marrow. We also describe salient clinical, laboratory and genetic features of the eight types of porphyria.

Lipid accumulation in human breast cancer cells injured by iron depletors.

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De Bortoli, Maida; Taverna, Elena; Maffioli, Elisa; Casalini, Patrizia; Crisafi, Francesco; Kumar, Vikas; Caccia, Claudio; Polli, Dario; Tedeschi, Gabriella; Bongarzone, Italia

2018-04-03

Current insights into the effects of iron deficiency in tumour cells are not commensurate with the importance of iron in cell metabolism. Studies have predominantly focused on the effects of oxygen or glucose scarcity in tumour cells, while attributing insufficient emphasis to the inadequate supply of iron in hypoxic regions. Cellular responses to iron deficiency and hypoxia are interlinked and may strongly affect tumour metabolism. We examined the morphological, proteomic, and metabolic effects induced by two iron chelators-deferoxamine (DFO) and di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT)-on MDA-MB-231 and MDA-MB-157 breast cancer cells. These chelators induced a cytoplasmic massive vacuolation and accumulation of lipid droplets (LDs), eventually followed by implosive, non-autophagic, and non-apoptotic death similar to methuosis. Vacuoles and LDs are generated by expansion of the endoplasmic reticulum (ER) based on extracellular fluid import, which includes unsaturated fatty acids that accumulate in LDs. Typical physiological phenomena associated with hypoxia are observed, such as inhibition of translation, mitochondrial dysfunction, and metabolic remodelling. These survival-oriented changes are associated with a greater expression of epithelial/mesenchymal transcription markers. Iron starvation induces a hypoxia-like program able to scavenge nutrients from the extracellular environment, and cells assume a hypertrophic phenotype. Such survival strategy is accompanied by the ER-dependent massive cytoplasmic vacuolization, mitochondrial dysfunctions, and LD accumulation and then evolves into cell death. LDs containing a greater proportion of unsaturated lipids are released as a consequence of cell death. The consequence of the disruption of iron metabolism in tumour tissue and the effects of LDs on intercellular communication, cancer-inflammation axis, and immunity remain to be explored. Considering the potential benefits, these are crucial

The induction of two biosynthetic enzymes helps Escherichia coli sustain heme synthesis and activate catalase during hydrogen peroxide stress.

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Mancini, Stefano; Imlay, James A

2015-05-01

Hydrogen peroxide pervades many natural environments, including the phagosomes that mediate cell-based immunity. Transcriptomic analysis showed that during protracted low-grade H(2)O(2) stress, Escherichia coli responds by activating both the OxyR defensive regulon and the Fur iron-starvation response. OxyR induced synthesis of two members of the nine-step heme biosynthetic pathway: ferrochelatase (HemH) and an isozyme of coproporphyrinogen III oxidase (HemF). Mutations that blocked either adaptation caused the accumulation of porphyrin intermediates, inadequate activation of heme enzymes, low catalase activity, defective clearance of H(2)O(2) and a failure to grow. Genetic analysis indicated that HemH induction is needed to compensate for iron sequestration by the mini-ferritin Dps. Dps activity protects DNA and proteins by limiting Fenton chemistry, but it interferes with the ability of HemH to acquire the iron that it needs to complete heme synthesis. HemF is a manganoprotein that displaces HemN, an iron-sulfur enzyme whose synthesis and/or stability is apparently problematic during H(2)O(2) stress. Thus, the primary responses to H(2)O(2), including the sequestration of iron, require compensatory adjustments in the mechanisms of iron-cofactor synthesis. The results support the growing evidence that oxidative stress is primarily an iron pathology. © 2015 John Wiley & Sons Ltd.

Iron Acquisition in Bacillus cereus: The Roles of IlsA and Bacillibactin in Exogenous Ferritin Iron Mobilization

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Buisson, Christophe; Daou, Nadine; Kallassy, Mireille; Lereclus, Didier; Arosio, Paolo; Bou-Abdallah, Fadi; Nielsen Le Roux, Christina

2014-01-01

In host-pathogen interactions, the struggle for iron may have major consequences on the outcome of the disease. To overcome the low solubility and bio-availability of iron, bacteria have evolved multiple systems to acquire iron from various sources such as heme, hemoglobin and ferritin. The molecular basis of iron acquisition from heme and hemoglobin have been extensively studied; however, very little is known about iron acquisition from host ferritin, a 24-mer nanocage protein able to store thousands of iron atoms within its cavity. In the human opportunistic pathogen Bacillus cereus, a surface protein named IlsA (Iron-regulated leucine rich surface protein type A) binds heme, hemoglobin and ferritin in vitro and is involved in virulence. Here, we demonstrate that IlsA acts as a ferritin receptor causing ferritin aggregation on the bacterial surface. Isothermal titration calorimetry data indicate that IlsA binds several types of ferritins through direct interaction with the shell subunits. UV-vis kinetic data show a significant enhancement of iron release from ferritin in the presence of IlsA indicating for the first time that a bacterial protein might alter the stability of the ferritin iron core. Disruption of the siderophore bacillibactin production drastically reduces the ability of B. cereus to utilize ferritin for growth and results in attenuated bacterial virulence in insects. We propose a new model of iron acquisition in B. cereus that involves the binding of IlsA to host ferritin followed by siderophore assisted iron uptake. Our results highlight a possible interplay between a surface protein and a siderophore and provide new insights into host adaptation of B. cereus and general bacterial pathogenesis. PMID:24550730

Iron acquisition in Bacillus cereus: the roles of IlsA and bacillibactin in exogenous ferritin iron mobilization.

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Diego Segond

2014-02-01

Full Text Available In host-pathogen interactions, the struggle for iron may have major consequences on the outcome of the disease. To overcome the low solubility and bio-availability of iron, bacteria have evolved multiple systems to acquire iron from various sources such as heme, hemoglobin and ferritin. The molecular basis of iron acquisition from heme and hemoglobin have been extensively studied; however, very little is known about iron acquisition from host ferritin, a 24-mer nanocage protein able to store thousands of iron atoms within its cavity. In the human opportunistic pathogen Bacillus cereus, a surface protein named IlsA (Iron-regulated leucine rich surface protein type A binds heme, hemoglobin and ferritin in vitro and is involved in virulence. Here, we demonstrate that IlsA acts as a ferritin receptor causing ferritin aggregation on the bacterial surface. Isothermal titration calorimetry data indicate that IlsA binds several types of ferritins through direct interaction with the shell subunits. UV-vis kinetic data show a significant enhancement of iron release from ferritin in the presence of IlsA indicating for the first time that a bacterial protein might alter the stability of the ferritin iron core. Disruption of the siderophore bacillibactin production drastically reduces the ability of B. cereus to utilize ferritin for growth and results in attenuated bacterial virulence in insects. We propose a new model of iron acquisition in B. cereus that involves the binding of IlsA to host ferritin followed by siderophore assisted iron uptake. Our results highlight a possible interplay between a surface protein and a siderophore and provide new insights into host adaptation of B. cereus and general bacterial pathogenesis.

Comparative analysis of the heme iron electronic structure and stereochemistry in tetrameric rabbit hemoglobin and monomeric soybean leghemoglobin a using Mössbauer spectroscopy with a high velocity resolution

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Alenkina, I. V.; Kumar, A.; Berkovsky, A. L.; Oshtrakh, M. I.

2018-02-01

A comparative study of tetrameric rabbit hemoglobin and monomeric soybean leghemoglobin a in the oxy- and deoxy-forms was carried out using 57Fe Mössbauer spectroscopy with a high velocity resolution in order to analyze the heme iron electronic structure and stereochemistry in relation to the Mössbauer hyperfine parameters. The Mössbauer spectra of tetrameric rabbit hemoglobin in both forms were fitted using two quadrupole doublets related to the 57Fe in ɑ- and β-subunits. In contrast, the Mössbauer spectra of monomeric soybean leghemoglobin a were fitted using: (i) two quadrupole doublets for the oxy-form related to two conformational states of the distal His E7 imidazole ring and different hydrogen bonding of oxygen molecule in the oxy-form and (ii) using three quadrupole doublets for deoxy-form related to three conformational states of the proximal His F8 imidazole ring. Small variations of Mössbauer hyperfine parameters related to small differences in the heme iron electronic structure and stereochemistry in tetrameric rabbit hemoglobin and monomeric soybean leghemoglobin a are discussed.

Conserved residues of the human mitochondrial holocytochrome c synthase mediate interactions with heme.

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Babbitt, Shalon E; San Francisco, Brian; Bretsnyder, Eric C; Kranz, Robert G

2014-08-19

C-type cytochromes are distinguished by the covalent attachment of a heme cofactor, a modification that is typically required for its subsequent folding, stability, and function. Heme attachment takes place in the mitochondrial intermembrane space and, in most eukaryotes, is mediated by holocytochrome c synthase (HCCS). HCCS is the primary component of the eukaryotic cytochrome c biogenesis pathway, known as System III. The catalytic function of HCCS depends on its ability to coordinate interactions between its substrates: heme and cytochrome c. Recent advancements in the recombinant expression and purification of HCCS have facilitated comprehensive analyses of the roles of conserved residues in HCCS, as demonstrated in this study. Previously, we proposed a four-step model describing HCCS-mediated cytochrome c assembly, identifying a conserved histidine residue (His154) as an axial ligand to the heme iron. In this study, we performed a systematic mutational analysis of 17 conserved residues in HCCS, and we provide evidence that the enzyme contains two heme-binding domains. Our data indicate that heme contacts mediated by residues within these domains modulate the dynamics of heme binding and contribute to the stability of the HCCS-heme-cytochrome c steady state ternary complex. While some residues are essential for initial heme binding (step 1), others impact the subsequent release of the holocytochrome c product (step 4). Certain HCCS mutants that were defective in heme binding were corrected for function by exogenous aminolevulinic acid (ALA, the precursor to heme). This chemical "correction" supports the proposed role of heme binding for the corresponding residues.

Bacterial Nitric Oxide Synthase Is Required for the Staphylococcus aureus Response to Heme Stress.

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Surdel, Matthew C; Dutter, Brendan F; Sulikowski, Gary A; Skaar, Eric P

2016-08-12

Staphylococcus aureus is a pathogen that causes significant morbidity and mortality worldwide. Within the vertebrate host, S. aureus requires heme as a nutrient iron source and as a cofactor for multiple cellular processes. Although required for pathogenesis, excess heme is toxic. S. aureus employs a two-component system, the heme sensor system (HssRS), to sense and protect against heme toxicity. Upon activation, HssRS induces the expression of the heme-regulated transporter (HrtAB), an efflux pump that alleviates heme toxicity. The ability to sense and respond to heme is critical for the pathogenesis of numerous Gram-positive organisms, yet the mechanism of heme sensing remains unknown. Compound '3981 was identified in a high-throughput screen as an activator of staphylococcal HssRS that triggers HssRS independently of heme accumulation. '3981 is toxic to S. aureus; however, derivatives of '3981 were synthesized that lack toxicity while retaining HssRS activation, enabling the interrogation of the heme stress response without confounding toxic effects of the parent molecule. Using '3981 derivatives as probes of the heme stress response, numerous genes required for '3981-induced activation of HssRS were uncovered. Specifically, multiple genes involved in the production of nitric oxide were identified, including the gene encoding bacterial nitric oxide synthase (bNOS). bNOS protects S. aureus from oxidative stress imposed by heme. Taken together, this work identifies bNOS as crucial for the S. aureus heme stress response, providing evidence that nitric oxide synthesis and heme sensing are intertwined.

Liposomes Loaded with Hydrophobic Iron Oxide Nanoparticles: Suitable T2 Contrast Agents for MRI

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Raquel Martínez-González

2016-07-01

Full Text Available There has been a recent surge of interest in the use of superparamagnetic iron oxide nanoparticles (SPIONs as contrast agents (CAs for magnetic resonance imaging (MRI, due to their tunable properties and their low toxicity compared with other CAs such as gadolinium. SPIONs exert a strong influence on spin-spin T2 relaxation times by decreasing the MR signal in the regions to which they are delivered, consequently yielding darker images or negative contrast. Given the potential of these nanoparticles to enhance detection of alterations in soft tissues, we studied the MRI response of hydrophobic or hydrophilic SPIONs loaded into liposomes (magnetoliposomes of different lipid composition obtained by sonication. These hybrid nanostructures were characterized by measuring several parameters such as size and polydispersity, and number of SPIONs encapsulated or embedded into the lipid systems. We then studied the influence of acyl chain length as well as its unsaturation, charge, and presence of cholesterol in the lipid bilayer at high field strength (7 T to mimic the conditions used in preclinical assays. Our results showed a high variability depending on the nature of the magnetic particles. Focusing on the hydrophobic SPIONs, the cholesterol-containing samples showed a slight reduction in r2, while unsaturation of the lipid acyl chain and inclusion of a negatively charged lipid into the bilayer appeared to yield a marked increase in negative contrast, thus rendering these magnetoliposomes suitable candidates as CAs, especially as a liver CA.

The DFT-DVM theoretical study of the differences of quadrupole splitting and the iron electronic structure for the rough heme models for {alpha}- and {beta}-subunits in deoxyhemoglobin and for deoxymyoglobin

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Yuryeva, E. I. [Institute of Solid State Chemistry of the Ural Branch of the Russian Academy of Sciences (Russian Federation); Oshtrakh, M. I., E-mail: oshtrakh@mail.utnet.ru [Ural State Technical University-UPI, Faculty of Physical Techniques and Devices for Quality Control (Russian Federation)

2008-01-15

Quantum chemical calculations of the iron electron structure and {sup 57}Fe quadrupole splitting were made by density functional theory and X{alpha} discrete variation method for the rough heme models for {alpha}- and {beta}-subunits in deoxyhemoglobin and for deoxymyoglobin accounting stereochemical differences of the active sites in native proteins. The calculations revealed differences of quadrupole splitting temperature dependences for three models indicating sensitivity of quadrupole splitting and Fe(II) electronic structure to small variations of iron stereochemistry.

The heme-heme oxygenase system: a molecular switch in wound healing.

NARCIS (Netherlands)

Wagener, F.A.D.T.G.; Beurden, H.E. van; Hoff, J.W. Von den; Adema, G.J.; Figdor, C.G.

2003-01-01

When cells are injured they release their contents, resulting in a local accumulation of free heme proteins and heme. Here, we investigated the involvement of heme and its degrading enzyme heme oxygenase (HO) in the inflammatory process during wound healing. We observed that heme directly

Studies on lactoferricin-derived Escherichia coli membrane-active peptides reveal differences in the mechanism of N-acylated versus nonacylated peptides.

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Zweytick, Dagmar; Deutsch, Günter; Andrä, Jörg; Blondelle, Sylvie E; Vollmer, Ekkehard; Jerala, Roman; Lohner, Karl

2011-06-17

To improve the low antimicrobial activity of LF11, an 11-mer peptide derived from human lactoferricin, mutant sequences were designed based on the defined structure of LF11 in the lipidic environment. Thus, deletion of noncharged polar residues and strengthening of the hydrophobic N-terminal part upon adding a bulky hydrophobic amino acid or N-acylation resulted in enhanced antimicrobial activity against Escherichia coli, which correlated with the peptides' degree of perturbation of bacterial membrane mimics. Nonacylated and N-acylated peptides exhibited different effects at a molecular level. Nonacylated peptides induced segregation of peptide-enriched and peptide-poor lipid domains in negatively charged bilayers, although N-acylated peptides formed small heterogeneous domains resulting in a higher degree of packing defects. Additionally, only N-acylated peptides perturbed the lateral packing of neutral lipids and exhibited increased permeability of E. coli lipid vesicles. The latter did not correlate with the extent of improvement of the antimicrobial activity, which could be explained by the fact that elevated binding of N-acylated peptides to lipopolysaccharides of the outer membrane of gram-negative bacteria seems to counteract the elevated membrane permeabilization, reflected in the respective minimal inhibitory concentration for E. coli. The antimicrobial activity of the peptides correlated with an increase of membrane curvature stress and hence bilayer instability. Transmission electron microscopy revealed that only the N-acylated peptides induced tubular protrusions from the outer membrane, whereas all peptides caused detachment of the outer and inner membrane of E. coli bacteria. Viability tests demonstrated that these bacteria were dead before onset of visible cell lysis.

Studies on Lactoferricin-derived Escherichia coli Membrane-active Peptides Reveal Differences in the Mechanism of N-Acylated Versus Nonacylated Peptides*

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Zweytick, Dagmar; Deutsch, Günter; Andrä, Jörg; Blondelle, Sylvie E.; Vollmer, Ekkehard; Jerala, Roman; Lohner, Karl

2011-01-01

To improve the low antimicrobial activity of LF11, an 11-mer peptide derived from human lactoferricin, mutant sequences were designed based on the defined structure of LF11 in the lipidic environment. Thus, deletion of noncharged polar residues and strengthening of the hydrophobic N-terminal part upon adding a bulky hydrophobic amino acid or N-acylation resulted in enhanced antimicrobial activity against Escherichia coli, which correlated with the peptides' degree of perturbation of bacterial membrane mimics. Nonacylated and N-acylated peptides exhibited different effects at a molecular level. Nonacylated peptides induced segregation of peptide-enriched and peptide-poor lipid domains in negatively charged bilayers, although N-acylated peptides formed small heterogeneous domains resulting in a higher degree of packing defects. Additionally, only N-acylated peptides perturbed the lateral packing of neutral lipids and exhibited increased permeability of E. coli lipid vesicles. The latter did not correlate with the extent of improvement of the antimicrobial activity, which could be explained by the fact that elevated binding of N-acylated peptides to lipopolysaccharides of the outer membrane of Gram-negative bacteria seems to counteract the elevated membrane permeabilization, reflected in the respective minimal inhibitory concentration for E. coli. The antimicrobial activity of the peptides correlated with an increase of membrane curvature stress and hence bilayer instability. Transmission electron microscopy revealed that only the N-acylated peptides induced tubular protrusions from the outer membrane, whereas all peptides caused detachment of the outer and inner membrane of E. coli bacteria. Viability tests demonstrated that these bacteria were dead before onset of visible cell lysis. PMID:21515687

Effects of Iron Overload on the Activity of Na,K-ATPase and Lipid Profile of the Human Erythrocyte Membrane.

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Leilismara Sousa

Full Text Available Iron is an essential chemical element for human life. However, in some pathological conditions, such as hereditary hemochromatosis type 1 (HH1, iron overload induces the production of reactive oxygen species that may lead to lipid peroxidation and a change in the plasma-membrane lipid profile. In this study, we investigated whether iron overload interferes with the Na,K-ATPase activity of the plasma membrane by studying erythrocytes that were obtained from the whole blood of patients suffering from iron overload. Additionally, we treated erythrocytes of normal subjects with 0.8 mM H2O2 and 1 μM FeCl3 for 24 h. We then analyzed the lipid profile, lipid peroxidation and Na,K-ATPase activity of plasma membranes derived from these cells. Iron overload was more frequent in men (87.5% than in women and was associated with an increase (446% in lipid peroxidation, as indicated by the amount of the thiobarbituric acid reactive substances (TBARS and an increase (327% in the Na,K-ATPase activity in the plasma membrane of erythrocytes. Erythrocytes treated with 1 μM FeCl3 for 24 h showed an increase (132% in the Na,K-ATPase activity but no change in the TBARS levels. Iron treatment also decreased the cholesterol and phospholipid content of the erythrocyte membranes and similar decreases were observed in iron overload patients. In contrast, erythrocytes treated with 0.8 mM H2O2 for 24 h showed no change in the measured parameters. These results indicate that erythrocytes from patients with iron overload exhibit higher Na,K-ATPase activity compared with normal subjects and that this effect is specifically associated with altered iron levels.

Design and synthesis of a tetradentate '3-amine-1-carboxylate' ligand to mimic the metal binding environment at the non-heme iron(II) oxidase active site.

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Dungan, Victoria J; Ortin, Yannick; Mueller-Bunz, Helge; Rutledge, Peter J

2010-04-07

Non-heme iron(II) oxidases (NHIOs) catalyse a diverse array of oxidative chemistry in Nature. As part of ongoing efforts to realize biomimetic, iron-mediated C-H activation, we report the synthesis of a new 'three-amine-one-carboxylate' ligand designed to complex with iron(II) and mimic the NHIO active site. The tetradentate ligand has been prepared as a single enantiomer in nine synthetic steps from N-Cbz-L-alanine, pyridine-2,6-dimethanol and diphenylamine, using Seebach oxazolidinone chemistry to control the stereochemistry. X-Ray crystal structures are reported for two important intermediates, along with variable temperature NMR experiments to probe the hindered interconversion of conformational isomers of several key intermediates, 2,6-disubstituted pyridine derivatives. The target ligand and an N-Cbz-protected precursor were each then complexed with iron(II) and tested for their ability to promote alkene dihydroxylation, using hydrogen peroxide as the oxidant.

Binding of Pseudomonas aeruginosa Apo-Bacterioferritin Associated Ferredoxin to Bacterioferritin B Promotes Heme Mediation of Electron Delivery and Mobilization of Core Mineral Iron†

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Weeratunga, Saroja K.; Gee, Casey E.; Lovell, Scott; Zeng, Yuhong; Woodin, Carrie L.; Rivera, Mario

2009-01-01

The bfrB gene from Pseudomonas aeruginosa was cloned and expressed in E. coli. The resultant protein (BfrB), which assembles into a 445.3 kDa complex0020from 24 identical subunits, binds 12 molecules of heme axially coordinated by two Met residues. BfrB, isolated with 5–10 iron atoms per protein molecule, was reconstituted with ferrous ions to prepare samples with a core mineral containing 600 ± 40 ferric ions per BfrB molecule and approximately one phosphate molecule per iron atom. In the presence of sodium dithionite or in the presence of P. aeruginosa ferredoxin NADP reductase (FPR) and NADPH the heme in BfrB remains oxidized and the core iron mineral is mobilized sluggishly. In stark contrast, addition of NADPH to a solution containing BfrB, FPR and the apo-form of P. aeruginosa bacterioferritin associated ferredoxin (apo-Bfd) results in rapid reduction of the heme in BfrB and in the efficient mobilization of the core iron mineral. Results from additional experimentation indicate that Bfd must bind to BfrB to promote heme mediation of electrons from the surface to the core to support the efficient mobilization of ferrous ions from BfrB. In this context, the thus far mysterious role of heme in bacterioferritins has been brought to the front by reconstituting BfrB with its physiological partner, apo-Bfd. These findings are discussed in the context of a model for the utilization of stored iron in which the significant upregulation of the bfd gene under low-iron conditions [Ochsner, U.A., Wilderman, P.J., Vasil, A.I., and Vasil, M.L. (2002) Mol. Microbiol. 45, 1277–1287] ensures sufficient concentrations of apo-Bfd to bind BfrB and unlock the iron stored in its core. Although these findings are in contrast to previous speculations suggesting redox mediation of electron transfer by holo-Bfd, the ability of apo-Bfd to promote iron mobilization is an economical strategy used by the cell because it obviates the need to further deplete cellular iron levels to

Lipopolysaccharides with acylation defects potentiate TLR4 signaling and shape T cell responses.

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Anna Martirosyan

Full Text Available Lipopolysaccharides or endotoxins are components of Gram-negative enterobacteria that cause septic shock in mammals. However, a LPS carrying hexa-acyl lipid A moieties is highly endotoxic compared to a tetra-acyl LPS and the latter has been considered as an antagonist of hexa-acyl LPS-mediated TLR4 signaling. We investigated the relationship between the structure and the function of bacterial LPS in the context of human and mouse dendritic cell activation. Strikingly, LPS with acylation defects were capable of triggering a strong and early TLR4-dependent DC activation, which in turn led to the activation of the proteasome machinery dampening the pro-inflammatory cytokine secretion. Upon activation with tetra-acyl LPS both mouse and human dendritic cells triggered CD4(+ T and CD8(+ T cell responses and, importantly, human myeloid dendritic cells favored the induction of regulatory T cells. Altogether, our data suggest that LPS acylation controlled by pathogenic bacteria might be an important strategy to subvert adaptive immunity.

Acyl coenzyme A thioesterase 7 regulates neuronal fatty acid metabolism to prevent neurotoxicity.

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Ellis, Jessica M; Wong, G William; Wolfgang, Michael J

2013-05-01

Numerous neurological diseases are associated with dysregulated lipid metabolism; however, the basic metabolic control of fatty acid metabolism in neurons remains enigmatic. Here we have shown that neurons have abundant expression and activity of the long-chain cytoplasmic acyl coenzyme A (acyl-CoA) thioesterase 7 (ACOT7) to regulate lipid retention and metabolism. Unbiased and targeted metabolomic analysis of fasted mice with a conditional knockout of ACOT7 in the nervous system, Acot7(N-/-), revealed increased fatty acid flux into multiple long-chain acyl-CoA-dependent pathways. The alterations in brain fatty acid metabolism were concomitant with a loss of lean mass, hypermetabolism, hepatic steatosis, dyslipidemia, and behavioral hyperexcitability in Acot7(N-/-) mice. These failures in adaptive energy metabolism are common in neurodegenerative diseases. In agreement, Acot7(N-/-) mice exhibit neurological dysfunction and neurodegeneration. These data show that ACOT7 counterregulates fatty acid metabolism in neurons and protects against neurotoxicity.

Intersections of pathways involving biotin and iron relative to therapeutic mechanisms for progressive multiple sclerosis.

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Heidker, Rebecca M; Emerson, Mitchell R; LeVine, Steven M

2016-12-01

While there are a variety of therapies for relapsing remitting multiple sclerosis (MS), there is a lack of treatments for progressive MS. An early study indicated that high dose biotin therapy has beneficial effects in approximately 12-15% of patients with progressive MS. The mechanisms behind the putative improvements seen with biotin therapy are not well understood, but have been postulated to include: 1) improving mitochondrial function which is impaired in MS, 2) increasing synthesis of lipids and cholesterol to facilitate remyelination, and 3) affecting gene expression. We suggest one reason that a greater percentage of patients with MS didn't respond to biotin therapy is the inaccessibility or lack of other nutrients, such as iron. In addition to biotin, iron (or heme) is necessary for energy production, biosynthesis of cholesterol and lipids, and for some protective mechanisms. Both biotin and iron are required for myelination during development, and by inference, remyelination. However, iron can also play a role in the pathology of MS. Increased deposition of iron can occur in some CNS structures possibly promoting oxidative damage while low iron levels can occur in other areas. Thus, the potential, detrimental effects of iron need to be considered together with the need for iron to support metabolic demands associated with repair and/or protective processes. We propose the optimal utilization of iron may be necessary to maximize the beneficial effects of biotin. This review will examine the interactions between biotin and iron in pathways that may have therapeutic or pathogenic implications for MS.

Rationale and design of the oral HEMe iron polypeptide Against Treatment with Oral Controlled Release Iron Tablets trial for the correction of anaemia in peritoneal dialysis patients (HEMATOCRIT trial

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Isbel Nicole M

2009-07-01

Full Text Available Abstract Background The main hypothesis of this study is that oral heme iron polypeptide (HIP; Proferrin® ES administration will more effectively augment iron stores in erythropoietic stimulatory agent (ESA-treated peritoneal dialysis (PD patients than conventional oral iron supplementation (Ferrogradumet®. Methods Inclusion criteria are peritoneal dialysis patients treated with darbepoietin alpha (DPO; Aranesp®, Amgen for ≥ 1 month. Patients will be randomized 1:1 to receive either slow-release ferrous sulphate (1 tablet twice daily; control or HIP (1 tablet twice daily for a period of 6 months. The study will follow an open-label design but outcome assessors will be blinded to study treatment. During the 6-month study period, haemoglobin levels will be measured monthly and iron studies (including transferring saturation [TSAT] measurements will be performed bi-monthly. The primary outcome measure will be the difference in TSAT levels between the 2 groups at the end of the 6 month study period, adjusted for baseline values using analysis of covariance (ANCOVA. Secondary outcome measures will include serum ferritin concentration, haemoglobin level, DPO dosage, Key's index (DPO dosage divided by haemoglobin concentration, and occurrence of adverse events (especially gastrointestinal adverse events. Discussion This investigator-initiated multicentre study has been designed to provide evidence to help nephrologists and their peritoneal dialysis patients determine whether HIP administration more effectively augments iron stores in ESP-treated PD patients than conventional oral iron supplementation. Trial Registration Australia New Zealand Clinical Trials Registry number ACTRN12609000432213.

Determination of lanthanum and rare earth elements in bovine whole blood reference material by ICP-MS after coprecipitation preconcentration with heme-iron as coprecipitant

International Nuclear Information System (INIS)

Fujimori, Eiji; Hayashi, Tatsuya; Inagaki, Kazumi; Haraguchi, H.

1999-01-01

An analytical method for the determination of lanthanide elements in the bovine whole blood reference material (IAEA A-13) has been investigated by inductively coupled plasma mass spectrometry (ICP-MS). The bovine whole blood reference material was digested with HNO 3 and HClO 4 , and then the pH of the digested solution was adjusted to 12 with 3 M sodium hydroxide aqueous solution. In this experimental procedure, lanthanide elements in the blood sample were coprecipitated with iron mainly derived from heme-iron in blood itself. In order to minimize matrix effects due to iron, excess iron in the analysis solution was removed by solvent extraction using methyl isobutyl ketone (MIBK) prior to the determination of lanthanide elements by ICP-MS. The recoveries of all lanthanide elements were almost quantitative in the recovery test. In consequence, it has been found that all lanthanide elements in bovine whole blood reference material are at the wide concentration range of 0.90 pg/g for Tm ∝1880 pg/g for Ce. (orig.)

Tyrosine B10 triggers a heme propionate hydrogen bonding network loop with glutamine E7 moiety

International Nuclear Information System (INIS)

Ramos-Santana, Brenda J.; López-Garriga, Juan

2012-01-01

Highlights: ► H-bonding network loop by PheB10Tyr mutation is proposed. ► The propionate group H-bonding network restricted the flexibility of the heme. ► The hydrogen bonding interaction modulates the electron density of the iron. ► Propionate H-bonding network loop explains the heme-ligand stabilization. -- Abstract: Propionates, as peripheral groups of the heme active center in hemeproteins have been described to contribute in the modulation of heme reactivity and ligand selection. These electronic characteristics prompted the question of whether the presence of hydrogen bonding networks between propionates and distal amino acids present in the heme ligand moiety can modulate physiological relevant events, like ligand binding association and dissociation activities. Here, the role of these networks was evaluated by NMR spectroscopy using the hemoglobin I PheB10Tyr mutant from Lucina pectinata as model for TyrB10 and GlnE7 hemeproteins. 1 H-NMR results for the rHbICN PheB10Tyr derivative showed chemical shifts of TyrB10 OHη at 31.00 ppm, GlnE7 N ε1 H/N ε2 H at 10.66 ppm/−3.27 ppm, and PheE11 C δ H at 11.75 ppm, indicating the presence of a crowded, collapsed, and constrained distal pocket. Strong dipolar contacts and inter-residues crosspeaks between GlnE7/6-propionate group, GlnE7/TyrB10 and TyrB10/CN suggest that this hydrogen bonding network loop between GlnE7, TyrB10, 6-propionate group, and the heme ligand contribute significantly to the modulation of the heme iron electron density as well as the ligand stabilization mechanism. Therefore, the network loop presented here support the fact that the electron withdrawing character of the hydrogen bonding is controlled by the interaction of the propionates and the nearby electronic environments contributing to the modulation of the heme electron density state. Thus, we hypothesize that in hemeproteins with similar electrostatic environment the flexibility of the heme-6-propionate promotes a hydrogen

Lxr-driven enterocyte lipid droplet formation delays transport of ingested lipids.

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Cruz-Garcia, Lourdes; Schlegel, Amnon

2014-09-01

Liver X receptors (Lxrs) are master regulators of cholesterol catabolism, driving the elimination of cholesterol from the periphery to the lumen of the intestine. Development of pharmacological agents to activate Lxrs has been hindered by synthetic Lxr agonists' induction of hepatic lipogenesis and hypertriglyceridemia. Elucidating the function of Lxrs in regulating enterocyte lipid handling might identify novel aspects of lipid metabolism that are pharmacologically amenable. We took a genetic approach centered on the single Lxr gene nr1h3 in zebrafish to study the role of Lxr in enterocyte lipid metabolism. Loss of nr1h3 function causes anticipated gene regulatory changes and cholesterol intolerance, collectively reflecting high evolutionary conservation of zebrafish Lxra function. Intestinal nr1h3 activation delays transport of absorbed neutral lipids, with accumulation of neutral lipids in enterocyte cytoplasmic droplets. This delay in transport of ingested neutral lipids protects animals from hypercholesterolemia and hepatic steatosis induced by a high-fat diet. On a gene regulatory level, Lxra induces expression of acsl3a, which encodes acyl-CoA synthetase long-chain family member 3a, a lipid droplet-anchored protein that directs fatty acyl chains into lipids. Forced overexpression of acls3a in enterocytes delays, in part, the appearance of neutral lipids in the vasculature of zebrafish larvae. Activation of Lxr in the intestine cell-autonomously regulates the rate of delivery of absorbed lipids by inducting a temporary lipid intestinal droplet storage depot. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

Heme oxygenase-1 mediates BAY 11-7085 induced ferroptosis.

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Chang, Ling-Chu; Chiang, Shih-Kai; Chen, Shuen-Ei; Yu, Yung-Luen; Chou, Ruey-Hwang; Chang, Wei-Chao

2018-03-01

Ferroptosis is a form of oxidative cell death and has become a chemotherapeutic target for cancer treatment. BAY 11-7085 (BAY), which is a well-known IκBα inhibitor, suppressed viability in cancer cells via induction of ferroptotic death in an NF-κB-independent manner. Reactive oxygen species scavenging, relief of lipid peroxidation, replenishment of glutathione and thiol-containing agents, as well as iron chelation, rescued BAY-induced cell death. BAY upregulated a variety of Nrf2 target genes related to redox regulation, particularly heme oxygenase-1 (HO-1). Studies with specific inhibitors and shRNA interventions suggested that the hierarchy of induction is Nrf2-SLC7A11-HO-1. SLC7A11 inhibition by erastin, sulfasalazine, or shRNA interference sensitizes BAY-induced cell death. Overexperession of SLC7A11 attenuated BAY-inhibited cell viability. The ferroptotic process induced by hHO-1 overexpression further indicated that HO-1 is a key mediator of BAY-induced ferroptosis that operates through cellular redox regulation and iron accumulation. BAY causes compartmentalization of HO-1 into the nucleus and mitochondrion, and followed mitochondrial dysfunctions, leading to lysosome targeting for mitophagy. In this study, we first discovered that BAY induced ferroptosis via Nrf2-SLC7A11-HO-1 pathway and HO-1 is a key mediator by responding to the cellular redox status. Copyright © 2017 Elsevier B.V. All rights reserved.

[Iron absorption of the habitual diet in a population of low socioeconomic level].

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Morón, C; Kremenchuzky, S; Passamai, M I; D'Andrea de Rivero, S; Pérez de Galíndez, G; Gerschcovich, C

1985-06-01

Iron absorption using the extrinsic double-tag method was determined in the habitual diet consumed by a group of 32 volunteers of both sexes, pertaining to the low socioeconomic strata. The diet was made up of bread, spaghetti, vegetables and meat, totalling 2,022 kcal, 65.0 g protein, 17.57 mg iron, and 28.75 mg ascorbic acid. According to our findings, men were found to be neither anemic nor iron-deficient. Among the women, however, 4.8% had anemia and 57.1% suffered from iron deficiency. The non-heme iron absorption was very low: 1.35% at breakfast, 3.29% at lunch, and 3.82% at dinner. Among those subjects found to be normal, the absorption was half the above figures, whereas among those with iron deficiency it was threefold, the differences being highly significant. The absorption of heme-iron for lunch and dinner was 17.53%. The iron deficient group had an absorption value four times greater than the normal group, the differences also being highly significant. The daily availability of non-heme, heme and total iron was 0.44, 1.13 and 1.57 mg, respectively. In the subjects who formed the normal group, total iron available was 1.14 mg, barely covering a man's daily requirements, but not those of a woman. In the iron-deficient group, it was 4.31 mg, that is, four times greater than in the normal group; while this value improves the balance, it does not prevent deficiency in women, with great blood losses. Bearing these results in mind, it is suggested that measures tending to improve dietary iron content and bio-availability, be enforced.

A Neisseria meningitidis fbpABC mutant is incapable of using nonheme iron for growth.

Science.gov (United States)

Khun, H H; Kirby, S D; Lee, B C

1998-05-01

The neisserial fbpABC locus has been proposed to act as an iron-specific ABC transporter system. To confirm this assigned function, we constructed an fbpABC mutant in Neisseria meningitidis by insertional inactivation of fbpABC with a selectable antibiotic marker. The mutant was unable to use iron supplied from human transferrin, human lactoferrin, or iron chelates. However, the use of iron from heme and human hemoglobin was unimpaired. These results support the obligatory participation of fbpABC in neisserial periplasmic iron transport and do not indicate a role for this genetic locus in the heme iron pathway.

Heme oxygenase-2 gene deletion attenuates oxidative stress in neurons exposed to extracellular hemin

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Benvenisti-Zarom Luna

2004-09-01

Full Text Available Abstract Background Hemin, the oxidized form of heme, accumulates in intracranial hematomas and is a potent oxidant. Growing evidence suggests that it contributes to delayed injury to surrounding tissue, and that this process is affected by the heme oxygenase enzymes. In a prior study, heme oxygenase-2 gene deletion increased the vulnerability of cultured cortical astrocytes to hemin. The present study tested the effect of HO-2 gene deletion on protein oxidation, reactive oxygen species formation, and cell viability after mixed cortical neuron/astrocyte cultures were incubated with neurotoxic concentrations of hemin. Results Continuous exposure of wild-type cultures to 1–10 μM hemin for 14 h produced concentration-dependent neuronal death, as detected by both LDH release and fluorescence intensity after propidium iodide staining, with an EC50 of 1–2 μM; astrocytes were not injured by these low hemin concentrations. Cell death was consistently reduced by at least 60% in knockout cultures. Exposure to hemin for 4 hours, a time point that preceded cell lysis, increased protein oxidation in wild-type cultures, as detected by staining of immunoblots for protein carbonyl groups. At 10 μM hemin, carbonylation was increased 2.3-fold compared with control sister cultures subjected to medium exchanges only; this effect was reduced by about two-thirds in knockout cultures. Cellular reactive oxygen species, detected by fluorescence intensity after dihydrorhodamine 123 (DHR staining, was markedly increased by hemin in wild-type cultures and was localized to neuronal cell bodies and processes. In contrast, DHR fluorescence intensity in knockout cultures did not differ from that of sham-washed controls. Neuronal death in wild-type cultures was almost completely prevented by the lipid-soluble iron chelator phenanthroline; deferoxamine had a weaker but significant effect. Conclusions These results suggest that HO-2 gene deletion protects neurons in mixed

Heme Sensor Proteins*

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Girvan, Hazel M.; Munro, Andrew W.

2013-01-01

Heme is a prosthetic group best known for roles in oxygen transport, oxidative catalysis, and respiratory electron transport. Recent years have seen the roles of heme extended to sensors of gases such as O2 and NO and cell redox state, and as mediators of cellular responses to changes in intracellular levels of these gases. The importance of heme is further evident from identification of proteins that bind heme reversibly, using it as a signal, e.g. to regulate gene expression in circadian rhythm pathways and control heme synthesis itself. In this minireview, we explore the current knowledge of the diverse roles of heme sensor proteins. PMID:23539616

Monomeric Yeast Frataxin is an Iron-Binding Protein

International Nuclear Information System (INIS)

Cook, J.; Bencze, K.; Jankovic, A.; Crater, A.; Busch, C.; Bradley, P.; Stemmler, A.; Spaller, M.; Stemmler, T.

2006-01-01

Friedreich's ataxia, an autosomal cardio- and neurodegenerative disorder that affects 1 in 50 000 humans, is caused by decreased levels of the protein frataxin. Although frataxin is nuclear-encoded, it is targeted to the mitochondrial matrix and necessary for proper regulation of cellular iron homeostasis. Frataxin is required for the cellular production of both heme and iron-sulfur (Fe-S) clusters. Monomeric frataxin binds with high affinity to ferrochelatase, the enzyme involved in iron insertion into porphyrin during heme production. Monomeric frataxin also binds to Isu, the scaffold protein required for assembly of Fe-S cluster intermediates. These processes (heme and Fe-S cluster assembly) share requirements for iron, suggesting that monomeric frataxin might function as the common iron donor. To provide a molecular basis to better understand frataxin's function, we have characterized the binding properties and metal-site structure of ferrous iron bound to monomeric yeast frataxin. Yeast frataxin is stable as an iron-loaded monomer, and the protein can bind two ferrous iron atoms with micromolar binding affinity. Frataxin amino acids affected by the presence of iron are localized within conserved acidic patches located on the surfaces of both helix-1 and strand-1. Under anaerobic conditions, bound metal is stable in the high-spin ferrous state. The metal-ligand coordination geometry of both metal-binding sites is consistent with a six-coordinate iron-(oxygen/nitrogen) based ligand geometry, surely constructed in part from carboxylate and possibly imidazole side chains coming from residues within these conserved acidic patches on the protein. On the basis of our results, we have developed a model for how we believe yeast frataxin interacts with iron

Heme transport and erythropoiesis

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Yuan, Xiaojing; Fleming, Mark D.; Hamza, Iqbal

2013-01-01

In humans, systemic heme homeostasis is achieved via coordinated regulation of heme synthesis, transport and degradation. Although the heme biosynthesis and degradation pathways have been well characterized, the pathways for heme trafficking and incorporation into hemoproteins remains poorly understood. In the past few years, researchers have exploited genetic, cellular and biochemical tools, to identify heme transporters and, in the process, reveal unexpected functions for this elusive group of proteins. However, given the complexity of heme trafficking pathways, current knowledge of heme transporters is fragmented and sometimes contradictory. This review seeks to focus on recent studies on heme transporters with specific emphasis on their functions during erythropoiesis. PMID:23415705

Phenol degradation catalyzed by a peroxidase mimic constructed through the grafting of heme onto metal-organic frameworks.

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Jiang, Wei; Yang, Jiebing; Wang, Xinghuo; Han, Haobo; Yang, Yan; Tang, Jun; Li, Quanshun

2018-01-01

The aim of this work was to construct a peroxidase mimic for achieving the phenol degradation through Fenton reaction. The enzyme mimic was synthesized through the conjugation of heme with the amino group of 2-amino-1,4-benzene dicarboxylate in UiO-66-NH 2 (ZrMOF), namely Heme-ZrMOF. Compared to free heme, the composite Heme-ZrMOF exhibited an obviously enhanced ability for phenol degradation with up to 97.3% of phenol removal after 2h. Meanwhile, it could achieve the easy separation of catalyst from the system and the elimination of iron residues in the process of phenol degradation. Finally, the catalyst Heme-ZrMOF was observed to possess good recyclability in the phenol degradation with still 76.2% of phenol removal after 4 cycles. Copyright © 2017 Elsevier Ltd. All rights reserved.

Nitric oxide heme interactions in nitrophorin from Cimex lectularius

Energy Technology Data Exchange (ETDEWEB)

Christmann, R.; Auerbach, H., E-mail: auerbach@physik.uni-kl.de [University of Kaiserslautern, Department of Physics (Germany); Berry, R. E.; Walker, F. A. [The University of Arizona, Department of Chemistry and Biochemistry (United States); Schünemann, V. [University of Kaiserslautern, Department of Physics (Germany)

2016-12-15

The nitrophorin from the bedbug Cimex lectularius (cNP) is a nitric oxide (NO) carrying protein. Like the nitrophorins (rNPs) from the kissing bug Rhodnius prolixus, cNP forms a stable heme Fe(III)-NO complex, where the NO can be stored reversibly for a long period of time. In both cases, the NPs are found in the salivary glands of blood-sucking bugs. The insects use the nitrophorins to transport the NO to the victim’s tissues, resulting in vasodilation and reduced blood coagulation. However, the structure of cNP is significantly different to those of the rNPs from Rhodnius prolixus. Furthermore, the cNP can bind a second NO molecule to the proximal heme cysteine when present at higher concentrations. High field Mössbauer spectroscopy on {sup 57}Fe enriched cNP complexed with NO shows reduction of the heme iron and formation of a ferrous nitric oxide (Fe(II)-NO) complex. Density functional theory calculations reproduce the experimental Mössbauer parameters and confirm this observation.

Current understanding of iron homeostasis.

Science.gov (United States)

Anderson, Gregory J; Frazer, David M

2017-12-01

Iron is an essential trace element, but it is also toxic in excess, and thus mammals have developed elegant mechanisms for keeping both cellular and whole-body iron concentrations within the optimal physiologic range. In the diet, iron is either sequestered within heme or in various nonheme forms. Although the absorption of heme iron is poorly understood, nonheme iron is transported across the apical membrane of the intestinal enterocyte by divalent metal-ion transporter 1 (DMT1) and is exported into the circulation via ferroportin 1 (FPN1). Newly absorbed iron binds to plasma transferrin and is distributed around the body to sites of utilization with the erythroid marrow having particularly high iron requirements. Iron-loaded transferrin binds to transferrin receptor 1 on the surface of most body cells, and after endocytosis of the complex, iron enters the cytoplasm via DMT1 in the endosomal membrane. This iron can be used for metabolic functions, stored within cytosolic ferritin, or exported from the cell via FPN1. Cellular iron concentrations are modulated by the iron regulatory proteins (IRPs) IRP1 and IRP2. At the whole-body level, dietary iron absorption and iron export from the tissues into the plasma are regulated by the liver-derived peptide hepcidin. When tissue iron demands are high, hepcidin concentrations are low and vice versa. Too little or too much iron can have important clinical consequences. Most iron deficiency reflects an inadequate supply of iron in the diet, whereas iron excess is usually associated with hereditary disorders. These disorders include various forms of hemochromatosis, which are characterized by inadequate hepcidin production and, thus, increased dietary iron intake, and iron-loading anemias whereby both increased iron absorption and transfusion therapy contribute to the iron overload. Despite major recent advances, much remains to be learned about iron physiology and pathophysiology. © 2017 American Society for Nutrition.

Stearoyl-acyl carrier protein and unusual acyl-acyl carrier protein desaturase activities are differentially influenced by ferredoxin.

Science.gov (United States)

Schultz, D J; Suh, M C; Ohlrogge, J B

2000-10-01

Acyl-acyl carrier protein (ACP) desaturases function to position a single double bond into an acyl-ACP substrate and are best represented by the ubiquitous Delta9 18:0-ACP desaturase. Several variant acyl-ACP desaturases have also been identified from species that produce unusual monoenoic fatty acids. All known acyl-ACP desaturase enzymes use ferredoxin as the electron-donating cofactor, and in almost all previous studies the photosynthetic form of ferredoxin rather than the non-photosynthetic form has been used to assess activity. We have examined the influence of different forms of ferredoxin on acyl-ACP desaturases. Using combinations of in vitro acyl-ACP desaturase assays and [(14)C]malonyl-coenzyme A labeling studies, we have determined that heterotrophic ferredoxin isoforms support up to 20-fold higher unusual acyl-ACP desaturase activity in coriander (Coriandrum sativum), Thunbergia alata, and garden geranium (Pelargonium x hortorum) when compared with photosynthetic ferredoxin isoforms. Heterotrophic ferredoxin also increases activity of the ubiquitous Delta9 18:0-ACP desaturase 1.5- to 3.0-fold in both seed and leaf extracts. These results suggest that ferredoxin isoforms may specifically interact with acyl-ACP desaturases to achieve optimal enzyme activity and that heterotrophic isoforms of ferredoxin may be the in vivo electron donor for this reaction.

Characterization of Heme Proteins Involved in Microbial Exoelectric Activity and Small Molecule-Sensing

KAUST Repository

Vogler, Malvina M.

2018-01-01

spectrometry confirms that the correct extensive post-translational modifications were performed and the ten heme groups were incorporated per protein of MtrC and MtrA and the correct lipid-anchor was attached to extracellular MtrC. Raman spectroscopy

Role of Serum Iron in the Activation of Lipid Peroxidation in Critical Conditions

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Yu. P. Orlov

2006-01-01

Full Text Available Twenty-four critically ill patients due to generalized purulent peritonitis, pancreatonecrosis, thermal skin injuries, and severe poisoning by acetic acid were examined. The general regularities of the effect of high serum iron concentrations on the health status of patients, on the activity of antioxidative enzymes, and on the initiation of lipid peroxidation (LPO processes, as supported by the values of Fe2+-induced chemiluminescence, were revealed. In critically ill patients, iron metabolism occurs with the overload of a transport protein, such as transferrin, which is caused by intravascular hemolysis and hemoglobin metabolism to ionized iron. The overload of proteins responsible for iron transport leads to the tissue accumulation of free (ferrous and ferric iron that is actively involved in the processes of LPO initiation with excess synthesis of cytotoxic radicals, which in turn accounts for the severity of endotoxicosis.

Calcineurin signaling and membrane lipid homeostasis regulates iron mediated multidrug resistance mechanisms in Candida albicans.

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Saif Hameed

2011-04-01

Full Text Available We previously demonstrated that iron deprivation enhances drug susceptibility of Candida albicans by increasing membrane fluidity which correlated with the lower expression of ERG11 transcript and ergosterol levels. The iron restriction dependent membrane perturbations led to an increase in passive diffusion and drug susceptibility. The mechanisms underlying iron homeostasis and multidrug resistance (MDR, however, are not yet resolved. To evaluate the potential mechanisms, we used whole genome transcriptome and electrospray ionization tandem mass spectrometry (ESI-MS/MS based lipidome analyses of iron deprived Candida cells to examine the new cellular circuitry of the MDR of this pathogen. Our transcriptome data revealed a link between calcineurin signaling and iron homeostasis. Among the several categories of iron deprivation responsive genes, the down regulation of calcineurin signaling genes including HSP90, CMP1 and CRZ1 was noteworthy. Interestingly, iron deprived Candida cells as well as iron acquisition defective mutants phenocopied molecular chaperone HSP90 and calcineurin mutants and thus were sensitive to alkaline pH, salinity and membrane perturbations. In contrast, sensitivity to above stresses did not change in iron deprived DSY2146 strain with a hyperactive allele of calcineurin. Although, iron deprivation phenocopied compromised HSP90 and calcineurin, it was independent of protein kinase C signaling cascade. Notably, the phenotypes associated with iron deprivation in genetically impaired calcineurin and HSP90 could be reversed with iron supplementation. The observed down regulation of ergosterol (ERG1, ERG2, ERG11 and ERG25 and sphingolipid biosynthesis (AUR1 and SCS7 genes followed by lipidome analysis confirmed that iron deprivation not only disrupted ergosterol biosynthesis, but it also affected sphingolipid homeostasis in Candida cells. These lipid compositional changes suggested extensive remodeling of the membranes in iron

Disparate patterns of age-related changes in lipid peroxidation in long-lived naked mole-rats and shorter-lived mice.

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Andziak, Blazej; Buffenstein, Rochelle

2006-12-01

A key tenet of the oxidative stress theory of aging is that levels of accrued oxidative damage increase with age. Differences in damage generation and accumulation therefore may underlie the natural variation in species longevity. We compared age-related profiles of whole-organism lipid peroxidation (urinary isoprostanes) and liver lipid damage (malondialdehyde) in long living naked mole-rats [maximum lifespan (MLS) > 28.3 years] and shorter-living CB6F1 hybrid mice (MLS approximately 3.5 years). In addition, we compared age-associated changes in liver non-heme iron to assess how intracellular conditions, which may modulate oxidative processes, are affected by aging. Surprisingly, even at a young age, concentrations of both markers of lipid peroxidation, as well as of iron, were at least twofold (P naked mole tats than in mice. This refutes the hypothesis that prolonged naked mole-rat longevity is due to superior protection against oxidative stress. The age-related profiles of all three parameters were distinctly species specific. Rates of lipid damage generation in mice were maintained throughout adulthood, while accrued damage in old animals was twice that of young mice. In naked mole-rats, urinary isoprostane excretion declined by half with age (P naked mole-rats is independent of oxidative stress parameters.

Human heme oxygenase oxidation of 5- and 15-phenylhemes.

Science.gov (United States)

Wang, Jinling; Niemevz, Fernando; Lad, Latesh; Huang, Liusheng; Alvarez, Diego E; Buldain, Graciela; Poulos, Thomas L; de Montellano, Paul R Ortiz

2004-10-08

Human heme oxygenase-1 (hHO-1) catalyzes the O2-dependent oxidation of heme to biliverdin, CO, and free iron. Previous work indicated that electrophilic addition of the terminal oxygen of the ferric hydroperoxo complex to the alpha-meso-carbon gives 5-hydroxyheme. Earlier efforts to block this reaction with a 5-methyl substituent failed, as the reaction still gave biliverdin IXalpha. Surprisingly, a 15-methyl substituent caused exclusive cleavage at the gamma-meso-rather than at the normal, unsubstituted alpha-meso-carbon. No CO was formed in these reactions, but the fragment cleaved from the porphyrin eluded identification. We report here that hHO-1 cleaves 5-phenylheme to biliverdin IXalpha and oxidizes 15-phenylheme at the alpha-meso position to give 10-phenylbiliverdin IXalpha. The fragment extruded in the oxidation of 5-phenylheme is benzoic acid, one oxygen of which comes from O2 and the other from water. The 2.29- and 2.11-A crystal structures of the hHO-1 complexes with 1- and 15-phenylheme, respectively, show clear electron density for both the 5- and 15-phenyl rings in both molecules of the asymmetric unit. The overall structure of 15-phenylheme-hHO-1 is similar to that of heme-hHO-1 except for small changes in distal residues 141-150 and in the proximal Lys18 and Lys22. In the 5-phenylheme-hHO-1 structure, the phenyl-substituted heme occupies the same position as heme in the heme-HO-1 complex but the 5-phenyl substituent disrupts the rigid hydrophobic wall of residues Met34, Phe214, and residues 26-42 near the alpha-meso carbon. The results provide independent support for an electrophilic oxidation mechanism and support a role for stereochemical control of the reaction regiospecificity.

Selenolate complexes of CYP101 and the heme-bound hHO-1/H25A proximal cavity mutant.

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Jiang, Yongying; Ortiz de Montellano, Paul R

2008-05-05

Thiolate and selenolate complexes of CYP101 (P450cam) and the H25A proximal cavity mutant of heme-bound human heme oxygenase-1 (hHO-1) have been examined by UV-vis spectroscopy. Both thiolate and selenolate ligands bound to the heme distal side in CYP101 and gave rise to characteristic hyperporphyrin spectra. Thiolate ligands also bound to the proximal side of the heme in the cavity created by the H25A mutation in hHO-1, giving a Soret absorption similar to that of the H25C hHO-1 mutant. Selenolate ligands also bound to this cavity mutant under anaerobic conditions but reduced the heme iron to the ferrous state, as shown by the formation of a ferrous CO complex. Under aerobic conditions, the selenolate ligand but not the thiolate ligand was rapidly oxidized. These results indicate that selenocysteine-coordinated heme proteins will not be stable species in the absence of a redox potential stabilizing effect.

The Haptoglobin-CD163-Heme Oxygenase-1 Pathway for Hemoglobin Scavenging

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Jens Haugbølle Thomsen

2013-01-01

Full Text Available The haptoglobin- (Hp- CD163-heme oxygenase-1 (HO-1 pathway is an efficient captor-receptor-enzyme system to circumvent the hemoglobin (Hb/heme-induced toxicity during physiological and pathological hemolyses. In this pathway, Hb tightly binds to Hp leading to CD163-mediated uptake of the complex in macrophages followed by lysosomal Hp-Hb breakdown and HO-1-catalyzed conversion of heme into the metabolites carbon monoxide (CO, biliverdin, and iron. The plasma concentration of Hp is a limiting factor as evident during accelerated hemolysis, where the Hp depletion may cause serious Hb-induced toxicity and put pressure on backup protecting systems such as the hemopexin-CD91-HO pathway. The Hp-CD163-HO-1 pathway proteins are regulated by the acute phase mediator interleukin-6 (IL-6, but other regulatory factors indicate that this upregulation is a counteracting anti-inflammatory response during inflammation. The heme metabolites including bilirubin converted from biliverdin have overall an anti-inflammatory effect and thus reinforce the anti-inflammatory efficacy of the Hp-CD163-HO-1 pathway. Future studies of animal models of inflammation should further define the importance of the pathway in the anti-inflammatory response.

Differences and Comparisons of the Properties and Reactivities of Iron(III)–hydroperoxo Complexes with Saturated Coordination Sphere

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Faponle, Abayomi S; Quesne, Matthew G; Sastri, Chivukula V; Banse, Frédéric; de Visser, Sam P

2015-01-01

Heme and nonheme monoxygenases and dioxygenases catalyze important oxygen atom transfer reactions to substrates in the body. It is now well established that the cytochrome P450 enzymes react through the formation of a high-valent iron(IV)–oxo heme cation radical. Its precursor in the catalytic cycle, the iron(III)–hydroperoxo complex, was tested for catalytic activity and found to be a sluggish oxidant of hydroxylation, epoxidation and sulfoxidation reactions. In a recent twist of events, evidence has emerged of several nonheme iron(III)–hydroperoxo complexes that appear to react with substrates via oxygen atom transfer processes. Although it was not clear from these studies whether the iron(III)–hydroperoxo reacted directly with substrates or that an initial O–O bond cleavage preceded the reaction. Clearly, the catalytic activity of heme and nonheme iron(III)–hydroperoxo complexes is substantially different, but the origins of this are still poorly understood and warrant a detailed analysis. In this work, an extensive computational analysis of aromatic hydroxylation by biomimetic nonheme and heme iron systems is presented, starting from an iron(III)–hydroperoxo complex with pentadentate ligand system (L52). Direct C–O bond formation by an iron(III)–hydroperoxo complex is investigated, as well as the initial heterolytic and homolytic bond cleavage of the hydroperoxo group. The calculations show that [(L52)FeIII(OOH)]2+ should be able to initiate an aromatic hydroxylation process, although a low-energy homolytic cleavage pathway is only slightly higher in energy. A detailed valence bond and thermochemical analysis rationalizes the differences in chemical reactivity of heme and nonheme iron(III)–hydroperoxo and show that the main reason for this particular nonheme complex to be reactive comes from the fact that they homolytically split the O–O bond, whereas a heterolytic O–O bond breaking in heme iron(III)–hydroperoxo is found. PMID:25399782

Enhancement of total lipid yield by nitrogen, carbon, and iron supplementation in isolated microalgae.

Science.gov (United States)

Sivaramakrishnan, Ramachandran; Incharoensakdi, Aran

2017-08-01

The biochemical contents and biodiesel production ability of three microalgal strains grown under different sodium nitrate, sodium carbonate, and ferric ammonium citrate (iron) levels were investigated. The highest biomass and lipid contents were found in Scenedesmus sp., Chlorella sp., and Chlamydomonas sp. when grown in normal BG-11 containing sodium carbonate concentration at 0.03 g · L -1 , and in normal BG-11 containing iron concentration (IC) at 0.009 or 0.012 g · L -1 . Increasing the sodium nitrate level increased the biomass content, but decreased the lipid content in all three microalgae. Among the three microalgae, Scenedesmus sp. showed the highest total lipid yield of 0.69 g · L -1 under the IC of 0.012 g · L -1 . Palmitic and oleic acids were the major fatty acids of Scenedesmus sp. and Chlamydomonas sp. lipids. On the other hand, Chlorella sp. lipids were rich in palmitic, oleic, and linolenic acids, and henceforth contributing to poor biodiesel properties below the standard limits. The three isolated strains had a potential for biodiesel production. Nevertheless, Scenedesmus sp. from stone quarry pond water was the most suitable source for biodiesel production with tolerance toward the high concentration of sodium carbonate without the loss of its biodiesel properties. © 2017 Phycological Society of America.

Influence of food tannins on certain aspects of iron metabolism : Part 3 -- Heme synthesis and haematopoiesis in normal and anemic rats

International Nuclear Information System (INIS)

Roy, S.N.; Mukherjee, S.

1979-01-01

Tannin from various fruits and vegetables at a dose level of 0.5 mg/kg wt/day helps approximately 65% recovery of the blood hemoglobin concentration in hemolytic anemic rats within 7 days resulting in normal levels of haematological parameters. While in vitro tannin at low doses (5-10 μg/mg protein) stimulates iron incorporation into protoporphyrin IX by rat liver subcellular fractions, at higher doses (15-40 μg/mg protein) it inhibits the heme synthesis in liver, the inhibition being complete at 40 μg/mg protein. In vivo studies indicate that the administration of tannin (0.5 mg/kg) exhibits significant increase in incorporation of label into hemin of anemic rats compared to that of anemic control and tannin-fed normal groups. In rats receiving supplements of tannin (0.5 mg/k.o.), incorporation of the label into hemin of anemic ones is comparatively greater when 59 Fe is given by intravenous route instead of oral administration of radio-iron. The total labelling of 59 Fe in red blood cells is significantly greater in tannin-fed anemic rats than anemic control. These results suggest that tannin (0.5 mg/kg) from fruits and vegetables may help iron utilization more effectively for greater haematopoiesis in hemolytic anemia. (auth.)

Influence of food tannins on certain aspects of iron metabolism : Part 3 -- Heme synthesis and haematopoiesis in normal and anemic rats

Energy Technology Data Exchange (ETDEWEB)

Roy, S N [Albert Einstein Coll. of Medicine, Bronx, NY (USA); Mukherjee, S [Calcutta Univ. (India). Dept. of Applied Chemistry

1979-06-01

Tannin from various fruits and vegetables at a dose level of 0.5 mg/kg wt/day helps approximately 65% recovery of the blood hemoglobin concentration in hemolytic anemic rats within 7 days resulting in normal levels of haematological parameters. While in vitro tannin at low doses (5-10 ..mu..g/mg protein) stimulates iron incorporation into protoporphyrin IX by rat liver subcellular fractions, at higher doses (15-40 ..mu..g/mg protein) it inhibits the heme synthesis in liver, the inhibition being complete at 40 ..mu..g/mg protein. In vivo studies indicate that the administration of tannin (0.5 mg/kg) exhibits significant increase in incorporation of label into hemin of anemic rats compared to that of anemic control and tannin-fed normal groups. In rats receiving supplements of tannin (0.5 mg/k.o.), incorporation of the label into hemin of anemic ones is comparatively greater when /sup 59/Fe is given by intravenous route instead of oral administration of radio-iron. The total labelling of /sup 59/Fe in red blood cells is significantly greater in tannin-fed anemic rats than anemic control. These results suggest that tannin (0.5 mg/kg) from fruits and vegetables may help iron utilization more effectively for greater haematopoiesis in hemolytic anemia.

Nutrient intake, serum lipids and iron status of colligiate rugby players.

Science.gov (United States)

Imamura, Hiroyuki; Iide, Kazuhide; Yoshimura, Yoshitaka; Kumagai, Kenya; Oshikata, Reika; Miyahara, Keiko; Oda, Kazuto; Miyamoto, Noriko; Nakazawa, Anthony

2013-02-13

There are two main playing positions in rugby (backs and forwards), which demonstrate different exercise patterns, roles, and physical characteristics. The purpose of this study was: 1) to collect baseline data on nutrient intake in order to advise the athletes about nutrition practices that might enhance performance, and 2) to compare serum lipids, lipoproteins, apolipoproteins (apo), lecithin:cholesterol acyltransferase (LCAT) activity, and iron status of forwards and backs. The sporting group was divided into 18 forwards and 16 backs and were compared with 26 sedentary controls. Dietary information was obtained with a food frequency questionnaire. There were significant differences among the three groups. The forwards had the highest body weight, body mass index, percentage of body fat (calculated by sum of four skinfold thicknesses), as well as the highest lean body mass, followed by the backs and the control group. The mean carbohydrate intake was marginal and protein intake was lower than the respective recommended targets in all three groups. The mean intakes of calcium, magnesium, and vitamins A, B1, B2, and C were lower than the respective Japanese recommended dietary allowances or adequate dietary intakes for the rugby players. The forwards had significantly lower high-density lipoprotein cholesterol (HDL-C) and HDL2-C than the backs and had significantly higher apo B and LCAT activity than the controls. The backs showed significantly higher HDL-C, HDL3-C, low-density lipoprotein cholesterol, and apo A-I, and LCAT activity than the controls. Four forwards (22%), five backs (31%), and three controls (12%) had hemolysis. None of the rugby players had anemia or iron depletion. The findings of our study indicate that as the athletes increased their carbohydrate and protein intake, their performance and lean body mass increased. Further, to increase mineral and vitamin intakes, we recommended athletes increase their consumption of green and other vegetables

Homeoviscous adaptation and the regulation of membrane lipids

DEFF Research Database (Denmark)

Ernst, Robert; Ejsing, Christer S; Antonny, Bruno

2016-01-01

Biological membranes are complex and dynamic assemblies of lipids and proteins. Poikilothermic organisms including bacteria, fungi, reptiles, and fish do not control their body temperature and must adapt their membrane lipid composition in order to maintain membrane fluidity in the cold. This ada......Biological membranes are complex and dynamic assemblies of lipids and proteins. Poikilothermic organisms including bacteria, fungi, reptiles, and fish do not control their body temperature and must adapt their membrane lipid composition in order to maintain membrane fluidity in the cold....... This adaptive response was termed homeoviscous adaptation and has been frequently studied with a specific focus on the acyl chain composition of membrane lipids. Massspectrometry-based lipidomics can nowadays provide more comprehensive insights into the complexity of lipid remodeling during adaptive responses...... such as neurons maintain unique lipid compositions with specific physicochemical properties. To date little is known about the sensory mechanisms regulating the acyl chain profile in such specialized cells or during adaptive responses. Here we summarize our current understanding of lipid metabolic networks...

Selective Acylation Enhances Membrane Charge Sensitivity of the Antimicrobial Peptide Mastoparan-X

DEFF Research Database (Denmark)

Etzerodt, Thomas Povl; Henriksen, Jonas Rosager; Rasmussen, Palle

2011-01-01

and positioning of the peptide in the membrane caused by either PA or OA acylation play a critical role in the fine-tuning of the effective charge of the peptide and thereby the fine-tuning of the peptide's selectivity between neutral and negatively charged lipid membranes. This finding is unique compared...... to previous reports where peptide acylation enhanced membrane affinity but also resulted in impaired selectivity. Our result may provide a method of enhancing selectivity of antimicrobial peptides toward bacterial membranes due to their high negative charge—a finding that should be investigated for other...

Cloning and Characterization of an Outer Membrane Protein of Vibrio vulnificus Required for Heme Utilization: Regulation of Expression and Determination of the Gene Sequence

Science.gov (United States)

Litwin, Christine M.; Byrne, Burke L.

1998-01-01

Vibrio vulnificus is a halophilic, marine pathogen that has been associated with septicemia and serious wound infections in patients with iron overload and preexisting liver disease. For V. vulnificus, the ability to acquire iron from the host has been shown to correlate with virulence. V. vulnificus is able to use host iron sources such as hemoglobin and heme. We previously constructed a fur mutant of V. vulnificus which constitutively expresses at least two iron-regulated outer membrane proteins, of 72 and 77 kDa. The N-terminal amino acid sequence of the 77-kDa protein purified from the V. vulnificus fur mutant had 67% homology with the first 15 amino acids of the mature protein of the Vibrio cholerae heme receptor, HutA. In this report, we describe the cloning, DNA sequence, mutagenesis, and analysis of transcriptional regulation of the structural gene for HupA, the heme receptor of V. vulnificus. DNA sequencing of hupA demonstrated a single open reading frame of 712 amino acids that was 50% identical and 66% similar to the sequence of V. cholerae HutA and similar to those of other TonB-dependent outer membrane receptors. Primer extension analysis localized one promoter for the V. vulnificus hupA gene. Analysis of the promoter region of V. vulnificus hupA showed a sequence homologous to the consensus Fur box. Northern blot analysis showed that the transcript was strongly regulated by iron. An internal deletion in the V. vulnificus hupA gene, done by using marker exchange, resulted in the loss of expression of the 77-kDa protein and the loss of the ability to use hemin or hemoglobin as a source of iron. The hupA deletion mutant of V. vulnificus will be helpful in future studies of the role of heme iron in V. vulnificus pathogenesis. PMID:9632577

Analysis of lipid profile in lipid storage myopathy.

Science.gov (United States)

Aguennouz, M'hammed; Beccaria, Marco; Purcaro, Giorgia; Oteri, Marianna; Micalizzi, Giuseppe; Musumesci, Olimpia; Ciranni, Annmaria; Di Giorgio, Rosa Maria; Toscano, Antonio; Dugo, Paola; Mondello, Luigi

2016-09-01

Lipid dysmetabolism disease is a condition in which lipids are stored abnormally in organs and tissues throughout the body, causing muscle weakness (myopathy). Usually, the diagnosis of this disease and its characterization goes through dosage of Acyl CoA in plasma accompanied with evidence of droplets of intra-fibrils lipids in the patient muscle biopsy. However, to understand the pathophysiological mechanisms of lipid storage diseases, it is useful to identify the nature of lipids deposited in muscle fiber. In this work fatty acids and triglycerides profile of lipid accumulated in the muscle of people suffering from myopathies syndromes was characterized. In particular, the analyses were carried out on the muscle biopsy of people afflicted by lipid storage myopathy, such as multiple acyl-coenzyme A dehydrogenase deficiency, and neutral lipid storage disease with myopathy, and by the intramitochondrial lipid storage dysfunctions, such as deficiencies of carnitine palmitoyltransferase II enzyme. A single step extraction and derivatization procedure was applied to analyze fatty acids from muscle tissues by gas chromatography with a flame ionization detector and with an electronic impact mass spectrometer. Triglycerides, extracted by using n-hexane, were analyzed by high performance liquid chromatography coupled to mass spectrometer equipped with an atmospheric pressure chemical ionization interface. The most representative fatty acids in all samples were: C16:0 in the 13-24% range, C18:1n9 in the 20-52% range, and C18:2n6 in the 10-25% range. These fatty acids were part of the most representative triglycerides in all samples. The data obtained was statistically elaborated performing a principal component analysis. A satisfactory discrimination was obtained among the different diseases. Using component 1 vs component 3 a 43.3% of total variance was explained. Such results suggest the important role that lipid profile characterization can have in supporting a correct

Effect of lead on heme synthesis

Energy Technology Data Exchange (ETDEWEB)

Neuberger, A.

1975-01-01

Recently, a fair amount of work has been done on the effect of lead on porphobilinogen dehydratase, which has been used as a sensitive indicator of lead poisoning. How far this is in itself harmful depends on the Michaelis constants of both the aminolaevulinic synthetase and of the dehydratase, and in addition on the relative activities of the two enzymes in a cell and also on the tissue concentration of glycine. Information on some of these points is still fragmentary, and a reliable judgement is at the present not very easy. Another step in the heme synthesis, which is sensitive to low concentrations of lead, is the incorporation of iron into protoporphyrin. Inhibition of this step may be important in accounting to a large extent for the anaemia found in individuals with lead poisoning. Reduction in the tissue concentration of heme or of heme-like compounds may also explain, through the mechanism of de-repression, the excretion of increased amounts of aminolaevulinic acid in the urine observed in cases of lead poisoning. A third step in heme synthesis, which might be sensitive to lead, is the oxidative decarboxylation of coproporphyrin to protoporphyrin, and this may explain why the former derivative is excreted in the urine. Recent work of the Harvard Medical School has indicated that greatly reduced levels of ALA dehydratase may be found in most cases of severe liver damage due to alcoholism. In most of these cases the level of lead in the blood is within normal limits, and there is no history of exposure to toxic amounts of lead. We therefore have to assume that a reduction in the blood level of this enzyme is not necessarily an indication of lead poisoning.

Stearoyl-Acyl Carrier Protein and Unusual Acyl-Acyl Carrier Protein Desaturase Activities Are Differentially Influenced by Ferredoxin1

Science.gov (United States)

Schultz, David J.; Suh, Mi Chung; Ohlrogge, John B.

2000-01-01

Acyl-acyl carrier protein (ACP) desaturases function to position a single double bond into an acyl-ACP substrate and are best represented by the ubiquitous Δ9 18:0-ACP desaturase. Several variant acyl-ACP desaturases have also been identified from species that produce unusual monoenoic fatty acids. All known acyl-ACP desaturase enzymes use ferredoxin as the electron-donating cofactor, and in almost all previous studies the photosynthetic form of ferredoxin rather than the non-photosynthetic form has been used to assess activity. We have examined the influence of different forms of ferredoxin on acyl-ACP desaturases. Using combinations of in vitro acyl-ACP desaturase assays and [14C]malonyl-coenzyme A labeling studies, we have determined that heterotrophic ferredoxin isoforms support up to 20-fold higher unusual acyl-ACP desaturase activity in coriander (Coriandrum sativum), Thunbergia alata, and garden geranium (Pelargonium × hortorum) when compared with photosynthetic ferredoxin isoforms. Heterotrophic ferredoxin also increases activity of the ubiquitous Δ9 18:0-ACP desaturase 1.5- to 3.0-fold in both seed and leaf extracts. These results suggest that ferredoxin isoforms may specifically interact with acyl-ACP desaturases to achieve optimal enzyme activity and that heterotrophic isoforms of ferredoxin may be the in vivo electron donor for this reaction. PMID:11027717

Regulation of C. elegans fat uptake and storage by acyl-CoA synthase-3 is dependent on NR5A family nuclear hormone receptor nhr-25

DEFF Research Database (Denmark)

Mullaney, Brendan C; Blind, Raymond D; Lemieux, George A

2010-01-01

Acyl-CoA synthases are important for lipid synthesis and breakdown, generation of signaling molecules, and lipid modification of proteins, highlighting the challenge of understanding metabolic pathways within intact organisms. From a C. elegans mutagenesis screen, we found that loss of ACS-3...... mutant phenotypes require the nuclear hormone receptor NHR-25, a key regulator of C. elegans molting. Our findings suggest that ACS-3-derived long-chain fatty acyl-CoAs, perhaps incorporated into complex ligands such as phosphoinositides, modulate NHR-25 function, which in turn regulates an endocrine...... program of lipid uptake and synthesis. These results reveal a link between acyl-CoA synthase function and an NR5A family nuclear receptor in C. elegans....

The Acylation State of Surface Lipoproteins of Mollicute Acholeplasma laidlawii*

Science.gov (United States)

Serebryakova, Marina V.; Demina, Irina A.; Galyamina, Maria A.; Kondratov, Ilya G.; Ladygina, Valentina G.; Govorun, Vadim M.

2011-01-01

Acylation of the N-terminal Cys residue is an essential, ubiquitous, and uniquely bacterial posttranslational modification that allows anchoring of proteins to the lipid membrane. In Gram-negative bacteria, acylation proceeds through three sequential steps requiring lipoprotein diacylglyceryltransferase, lipoprotein signal peptidase, and finally lipoprotein N-acyltransferase. The apparent lack of genes coding for recognizable homologs of lipoprotein N-acyltransferase in Gram-positive bacteria and Mollicutes suggests that the final step of the protein acylation process may be absent in these organisms. In this work, we monitored the acylation state of eight major lipoproteins of the mollicute Acholeplasma laidlawii using a combination of standard two-dimensional gel electrophoresis protein separation, blotting to nitrocellulose membranes, and MALDI-MS identification of modified N-terminal tryptic peptides. We show that for each A. laidlawii lipoprotein studied a third fatty acid in an amide linkage on the N-terminal Cys residue is present, whereas diacylated species were not detected. The result thus proves that A. laidlawii encodes a lipoprotein N-acyltransferase activity. We hypothesize that N-acyltransferases encoded by genes non-homologous to N-acyltransferases of Gram-negative bacteria are also present in other mollicutes and Gram-positive bacteria. PMID:21540185

Effect of Andrographolide‭ Extract on Blood Glucose and Lipid Profile in Rats with Secondary Iron Overload

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َArash Mehri Pirayvatlo

2017-01-01

Full Text Available Background & objectives: Iron overload is involved in the pathophysiology of many diseases including diabetes. In fact, the excess iron by creating free radicals makes damage to pancreas and leads to insulin resistance and diabetes. Andrographolide extract has hypoglycemic and antioxidant properties. This study has surveyed the effects of andrographolide on blood glucose and lipid profile in rats with secondary iron overload. Methods: In this experimental study, 36 male Wistar rats were randomly divided into 6 groups: the healthy control group, secondary iron overload group, secondary iron overload groups treated with a dose of 3.5 and 7 mg/kg of andrographolide extract, and andrographolide groups treated with a dose of 3.5 and 7 mg/kg of extract. Iron and extract were injected for 6 and 12 days, respectively. Blood samples were taken for measurement of blood glucose and lipid profiles. Data were analyzed using ANOVA test. Results: The pathological results of samples from liver of animals receiving iron showed that the iron was deposited in the liver tissues. Iron injection significantly increased blood glucose levels compared to healthy control group (p<0.05. In the iron overload group, andrographolide extract with a dose of 3.5 mg/kg or 7 mg/kg significantly decreased blood glucose levels (p<0.05. Iron injections did not increase the serum triglyceride and cholesterollevels. Injections of andrographolide extract with a dose of 3.5 mg/kg and 7 mg/kg, significantly decreased the cholesterol levels compared to iron receiving group (p<0.05. Conclusion: Results of this study showed that the andrographolide with different doses may be effective in the treatment of diabetes by reducing serum glucose and cholesterol levels.

Metabolic incorporation of unsaturated fatty acids into boar spermatozoa lipids and de novo formation of diacylglycerols

DEFF Research Database (Denmark)

Svetlichnyy, V.; Müller, P.; Günther-Pomorski, Thomas

2014-01-01

Lipids play an important role in the maturation, viability and function of sperm cells. In this study, we examined the neutral and polar lipid composition of boar spermatozoa by thin-layer chromatography/mass spectrometry. Main representatives of the neutral lipid classes were diacylglycerols...... containing saturated (myristoyl, palmitoyl and stearoyl) fatty acyl residues. Glycerophosphatidylcholine and glycerophosphatidylethanolamine with alk(en)yl ether residues in the sn-1 position and unsaturated long chained fatty acyl residues in sn-2 position were identified as the most prominent polar lipids....... The only glycoglycerolipid was sulfogalactosylglycerolipid carrying 16:0-alkyl- and 16:0-acyl chains. Using stable isotope-labelling, the metabolic incorporation of exogenously supplied fatty acids was analysed. Boar spermatozoa incorporated hexadecenoic (16:1), octadecenoic (18:1), octadecadienoic (18...

Gene expression in plant lipid metabolism in Arabidopsis seedlings.

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An-Shan Hsiao

Full Text Available Events in plant lipid metabolism are important during seedling establishment. As it has not been experimentally verified whether lipid metabolism in 2- and 5-day-old Arabidopsis thaliana seedlings is diurnally-controlled, quantitative real-time PCR analysis was used to investigate the expression of target genes in acyl-lipid transfer, β-oxidation and triacylglycerol (TAG synthesis and hydrolysis in wild-type Arabidopsis WS and Col-0. In both WS and Col-0, ACYL-COA-BINDING PROTEIN3 (ACBP3, DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1 and DGAT3 showed diurnal control in 2- and 5-day-old seedlings. Also, COMATOSE (CTS was diurnally regulated in 2-day-old seedlings and LONG-CHAIN ACYL-COA SYNTHETASE6 (LACS6 in 5-day-old seedlings in both WS and Col-0. Subsequently, the effect of CIRCADIAN CLOCK ASSOCIATED1 (CCA1 and LATE ELONGATED HYPOCOTYL (LHY from the core clock system was examined using the cca1lhy mutant and CCA1-overexpressing (CCA1-OX lines versus wild-type WS and Col-0, respectively. Results revealed differential gene expression in lipid metabolism between 2- and 5-day-old mutant and wild-type WS seedlings, as well as between CCA1-OX and wild-type Col-0. Of the ACBPs, ACBP3 displayed the most significant changes between cca1lhy and WS and between CCA1-OX and Col-0, consistent with previous reports that ACBP3 is greatly affected by light/dark cycling. Evidence of oil body retention in 4- and 5-day-old seedlings of the cca1lhy mutant in comparison to WS indicated the effect of cca1lhy on storage lipid reserve mobilization. Lipid profiling revealed differences in primary lipid metabolism, namely in TAG, fatty acid methyl ester and acyl-CoA contents amongst cca1lhy, CCA1-OX, and wild-type seedlings. Taken together, this study demonstrates that lipid metabolism is subject to diurnal regulation in the early stages of seedling development in Arabidopsis.

Fatty acyl-CoA reductases of birds

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Hellenbrand Janine

2011-12-01

Full Text Available Abstract Background Birds clean and lubricate their feathers with waxes that are produced in the uropygial gland, a holocrine gland located on their back above the tail. The type and the composition of the secreted wax esters are dependent on the bird species, for instance the wax ester secretion of goose contains branched-chain fatty acids and unbranched fatty alcohols, whereas that of barn owl contains fatty acids and alcohols both of which are branched. Alcohol-forming fatty acyl-CoA reductases (FAR catalyze the reduction of activated acyl groups to fatty alcohols that can be esterified with acyl-CoA thioesters forming wax esters. Results cDNA sequences encoding fatty acyl-CoA reductases were cloned from the uropygial glands of barn owl (Tyto alba, domestic chicken (Gallus gallus domesticus and domestic goose (Anser anser domesticus. Heterologous expression in Saccharomyces cerevisiae showed that they encode membrane associated enzymes which catalyze a NADPH dependent reduction of acyl-CoA thioesters to fatty alcohols. By feeding studies of transgenic yeast cultures and in vitro enzyme assays with membrane fractions of transgenic yeast cells two groups of isozymes with different properties were identified, termed FAR1 and FAR2. The FAR1 group mainly synthesized 1-hexadecanol and accepted substrates in the range between 14 and 18 carbon atoms, whereas the FAR2 group preferred stearoyl-CoA and accepted substrates between 16 and 20 carbon atoms. Expression studies with tissues of domestic chicken indicated that FAR transcripts were not restricted to the uropygial gland. Conclusion The data of our study suggest that the identified and characterized avian FAR isozymes, FAR1 and FAR2, can be involved in wax ester biosynthesis and in other pathways like ether lipid synthesis.

Fatty acyl-CoA reductases of birds

Science.gov (United States)

2011-01-01

Background Birds clean and lubricate their feathers with waxes that are produced in the uropygial gland, a holocrine gland located on their back above the tail. The type and the composition of the secreted wax esters are dependent on the bird species, for instance the wax ester secretion of goose contains branched-chain fatty acids and unbranched fatty alcohols, whereas that of barn owl contains fatty acids and alcohols both of which are branched. Alcohol-forming fatty acyl-CoA reductases (FAR) catalyze the reduction of activated acyl groups to fatty alcohols that can be esterified with acyl-CoA thioesters forming wax esters. Results cDNA sequences encoding fatty acyl-CoA reductases were cloned from the uropygial glands of barn owl (Tyto alba), domestic chicken (Gallus gallus domesticus) and domestic goose (Anser anser domesticus). Heterologous expression in Saccharomyces cerevisiae showed that they encode membrane associated enzymes which catalyze a NADPH dependent reduction of acyl-CoA thioesters to fatty alcohols. By feeding studies of transgenic yeast cultures and in vitro enzyme assays with membrane fractions of transgenic yeast cells two groups of isozymes with different properties were identified, termed FAR1 and FAR2. The FAR1 group mainly synthesized 1-hexadecanol and accepted substrates in the range between 14 and 18 carbon atoms, whereas the FAR2 group preferred stearoyl-CoA and accepted substrates between 16 and 20 carbon atoms. Expression studies with tissues of domestic chicken indicated that FAR transcripts were not restricted to the uropygial gland. Conclusion The data of our study suggest that the identified and characterized avian FAR isozymes, FAR1 and FAR2, can be involved in wax ester biosynthesis and in other pathways like ether lipid synthesis. PMID:22151413

Dietary Heme Induces Gut Dysbiosis, Aggravates Colitis, and Potentiates the Development of Adenomas in Mice

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Marco Constante

2017-09-01

Full Text Available Dietary heme can be used by colonic bacteria equipped with heme-uptake systems as a growth factor and thereby impact on the microbial community structure. The impact of heme on the gut microbiota composition may be particularly pertinent in chronic inflammation such as in inflammatory bowel disease (IBD, where a strong association with gut dysbiosis has been consistently reported. In this study we investigated the influence of dietary heme on the gut microbiota and inferred metagenomic composition, and on chemically induced colitis and colitis-associated adenoma development in mice. Using 16S rRNA gene sequencing, we found that mice fed a diet supplemented with heme significantly altered their microbiota composition, characterized by a decrease in α-diversity, a reduction of Firmicutes and an increase of Proteobacteria, particularly Enterobacteriaceae. These changes were similar to shifts seen in dextran sodium sulfate (DSS-treated mice to induce colitis. In addition, dietary heme, but not systemically delivered heme, contributed to the exacerbation of DSS-induced colitis and facilitated adenoma formation in the azoxymethane/DSS colorectal cancer (CRC mouse model. Using inferred metagenomics, we found that the microbiota alterations elicited by dietary heme resulted in non-beneficial functional shifts, which were also characteristic of DSS-induced colitis. Furthermore, a reduction in fecal butyrate levels was found in mice fed the heme supplemented diet compared to mice fed the control diet. Iron metabolism genes known to contribute to heme release from red blood cells, heme uptake, and heme exporter proteins, were significantly enriched, indicating a shift toward favoring the growth of bacteria able to uptake heme and protect against its toxicity. In conclusion, our data suggest that luminal heme, originating from dietary components or gastrointestinal bleeding in IBD and, to lesser extent in CRC, directly contributes to microbiota dysbiosis

Characterization of a gene family encoding SEA (sea-urchin sperm protein, enterokinase and agrin-domain proteins with lectin-like and heme-binding properties from Schistosoma japonicum.

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Evaristus Chibunna Mbanefo

Full Text Available BACKGROUND: We previously identified a novel gene family dispersed in the genome of Schistosoma japonicum by retrotransposon-mediated gene duplication mechanism. Although many transcripts were identified, no homolog was readily identifiable from sequence information. METHODOLOGY/PRINCIPAL FINDINGS: Here, we utilized structural homology modeling and biochemical methods to identify remote homologs, and characterized the gene products as SEA (sea-urchin sperm protein, enterokinase and agrin-domain containing proteins. A common extracellular domain in this family was structurally similar to SEA-domain. SEA-domain is primarily a structural domain, known to assist or regulate binding to glycans. Recombinant proteins from three members of this gene family specifically interacted with glycosaminoglycans with high affinity, with potential implication in ligand acquisition and immune evasion. Similar approach was used to identify a heme-binding site on the SEA-domain. The heme-binding mode showed heme molecule inserted into a hydrophobic pocket, with heme iron putatively coordinated to two histidine axial ligands. Heme-binding properties were confirmed using biochemical assays and UV-visible absorption spectroscopy, which showed high affinity heme-binding (K D = 1.605×10(-6 M and cognate spectroscopic attributes of hexa-coordinated heme iron. The native proteins were oligomers, antigenic, and are localized on adult worm teguments and gastrodermis; major host-parasite interfaces and site for heme detoxification and acquisition. CONCLUSIONS: The results suggest potential role, at least in the nucleation step of heme crystallization (hemozoin formation, and as receptors for heme uptake. Survival strategies exploited by parasites, including heme homeostasis mechanism in hemoparasites, are paramount for successful parasitism. Thus, assessing prospects for application in disease intervention is warranted.

Characterization of a Gene Family Encoding SEA (Sea-urchin Sperm Protein, Enterokinase and Agrin)-Domain Proteins with Lectin-Like and Heme-Binding Properties from Schistosoma japonicum

Science.gov (United States)

Mbanefo, Evaristus Chibunna; Kikuchi, Mihoko; Huy, Nguyen Tien; Shuaibu, Mohammed Nasir; Cherif, Mahamoud Sama; Yu, Chuanxin; Wakao, Masahiro; Suda, Yasuo; Hirayama, Kenji

2014-01-01

Background We previously identified a novel gene family dispersed in the genome of Schistosoma japonicum by retrotransposon-mediated gene duplication mechanism. Although many transcripts were identified, no homolog was readily identifiable from sequence information. Methodology/Principal Findings Here, we utilized structural homology modeling and biochemical methods to identify remote homologs, and characterized the gene products as SEA (sea-urchin sperm protein, enterokinase and agrin)-domain containing proteins. A common extracellular domain in this family was structurally similar to SEA-domain. SEA-domain is primarily a structural domain, known to assist or regulate binding to glycans. Recombinant proteins from three members of this gene family specifically interacted with glycosaminoglycans with high affinity, with potential implication in ligand acquisition and immune evasion. Similar approach was used to identify a heme-binding site on the SEA-domain. The heme-binding mode showed heme molecule inserted into a hydrophobic pocket, with heme iron putatively coordinated to two histidine axial ligands. Heme-binding properties were confirmed using biochemical assays and UV-visible absorption spectroscopy, which showed high affinity heme-binding (K D = 1.605×10−6 M) and cognate spectroscopic attributes of hexa-coordinated heme iron. The native proteins were oligomers, antigenic, and are localized on adult worm teguments and gastrodermis; major host-parasite interfaces and site for heme detoxification and acquisition. Conclusions The results suggest potential role, at least in the nucleation step of heme crystallization (hemozoin formation), and as receptors for heme uptake. Survival strategies exploited by parasites, including heme homeostasis mechanism in hemoparasites, are paramount for successful parasitism. Thus, assessing prospects for application in disease intervention is warranted. PMID:24416467

Enhanced lipid accumulation and biodiesel production by oleaginous Chlorella protothecoides under a structured heterotrophic-iron (II) induction strategy.

Science.gov (United States)

Li, Yuqin; Mu, Jinxiu; Chen, Di; Xu, Hua; Han, Fangxin

2015-05-01

A structured heterotrophic-iron (II) induction (HII) strategy was proposed to enhance lipid accumulation in oleaginous Chlorella protothecoides. C. protothecoides subjected to heterotrophic-iron (II) induction achieved a favorable lipid accumulation up to 62 % and a maximum lipid productivity of 820.17 mg/day, representing 2.78-fold and 3.64-fold increase respectively over heterotrophic cultivation alone. HII-induced cells produced significantly elevated levels of 16:0, 18:1(Δ9), and 18:2(Δ9,12) fatty acids (over 90 %). The lipid contents and plant lipid-like fatty acid compositions exhibit the potential of HII-induced C. protothecoides as biodiesel feedstock. Furthermore, 31 altered proteins in HII-induced algal cells were successfully identified. These differentially expressed proteins were assigned into nine molecular function categories, including carbohydrate metabolism, lipid biosynthesis, Calvin cycle, cellular respiration, photosynthesis, energy and transport, protein biosynthesis, regulate and defense, and unclassified. Analysis using the Kyoto encyclopedia of genes and genomes and gene ontology annotation showed that malic enzyme, acyltransferase, and ACP were key metabolic checkpoints found to modulate lipid accumulation in C. protothecoides. The results provided possible applications of HII cultivation strategy in other microalgal species and new possibilities in developing genetic and metabolic engineering microalgae for desirable lipid productivity.

Acyl carrier proteins from sunflower (Helianthus annuus L.) seeds and their influence on FatA and FatB acyl-ACP thioesterase activities.

Science.gov (United States)

Aznar-Moreno, Jose A; Venegas-Calerón, Mónica; Martínez-Force, Enrique; Garcés, Rafael; Salas, Joaquín J

2016-08-01

The kinetics of acyl-ACP thioesterases from sunflower importantly changed when endogenous ACPs were used. Sunflower FatB was much more specific towards saturated acyl-ACPs when assayed with them. Acyl carrier proteins (ACPs) are small (~9 kDa), soluble, acidic proteins involved in fatty acid synthesis in plants and bacteria. ACPs bind to fatty acids through a thioester bond, generating the acyl-ACP lipoproteins that are substrates for fatty acid synthase (FAS) complexes, and that are required for fatty acid chain elongation, acting as important intermediates in de novo fatty acid synthesis in plants. Plants, usually express several ACP isoforms with distinct functionalities. We report here the cloning of three ACPs from developing sunflower seeds: HaACP1, HaACP2, and HaACP3. These proteins were plastidial ACPs expressed strongly in seeds, and as such they are probably involved in the synthesis of sunflower oil. The recombinant sunflower ACPs were expressed in bacteria but they were lethal to the prokaryote host. Thus, they were finally produced using the GST gene fusion system, which allowed the apo-enzyme to be produced and later activated to the holo form. Radiolabelled acyl-ACPs from the newly cloned holo-ACP forms were also synthesized and used to characterize the activity of recombinant sunflower FatA and FatB thioesterases, important enzymes in plant fatty acids synthesis. The activity of these enzymes changed significantly when the endogenous ACPs were used. Thus, FatA importantly increased its activity levels, whereas FatB displayed a different specificity profile, with much high activity levels towards saturated acyl-CoA derivatives. All these data pointed to an important influence of the ACP moieties on the activity of enzymes involved in lipid synthesis.

Cytosolic iron chaperones: Proteins delivering iron cofactors in the cytosol of mammalian cells.

Science.gov (United States)

Philpott, Caroline C; Ryu, Moon-Suhn; Frey, Avery; Patel, Sarju

2017-08-04

Eukaryotic cells contain hundreds of metalloproteins that are supported by intracellular systems coordinating the uptake and distribution of metal cofactors. Iron cofactors include heme, iron-sulfur clusters, and simple iron ions. Poly(rC)-binding proteins are multifunctional adaptors that serve as iron ion chaperones in the cytosolic/nuclear compartment, binding iron at import and delivering it to enzymes, for storage (ferritin) and export (ferroportin). Ferritin iron is mobilized by autophagy through the cargo receptor, nuclear co-activator 4. The monothiol glutaredoxin Glrx3 and BolA2 function as a [2Fe-2S] chaperone complex. These proteins form a core system of cytosolic iron cofactor chaperones in mammalian cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Impairment of heme synthesis in myelin as potential trigger of multiple sclerosis.

Science.gov (United States)

Morelli, Alessandro; Ravera, Silvia; Calzia, Daniela; Panfoli, Isabella

2012-06-01

The pathogenesis of multiple sclerosis (MS), a disease characterized by demyelination and subsequent axonal degeneration, is as yet unknown. Also, the nature of the disease is as yet not established, since doubts have been cast on its autoimmune origin. Genetic and environmental factors have been implied in MS, leading to the idea of an overall multifactorial origin. An unexpected role in energizing the axon has been reported for myelin, supposed to be the site of consumption of most of oxygen in brain. Myelin would be able to perform oxidative phosphorylation to supply the axons with ATP, thanks to the expression therein of mitochondrial F(o)F(1)-ATP synthase, and respiratory chains. Interestingly, myelin expresses the pathway of heme synthesis, hence of cytochromes, that rely on heme group, in turn depending on Fe availability. Poisoning by these pollutants shares the common characteristic to bring about demyelination both in animal models and in man. Carbon monoxide (CO) and lead poisoning which cause functional imbalance of the heme group, as well as of heme synthesis, cause myelin damage. On the other hand, a lack of essential metals such as iron and copper, produces dramatic myelin decrease. Myelin is a primary target, of iron shortage, indicating that in myelin Fe-dependent processes are more active than in other tissues. The predominant spread of MS in industrialized countries where pollution by heavy metals, and CO poisoning is widespread, suggests a relationship among toxic action of metal pollutants and MS. According to the present hypothesis, MS can be primarily triggered by environmental factors acting on a genetic susceptibility, while the immune response may be a consequence of a primary oxidative damage due to reactive oxygen species produced consequently to an imbalance of cytochromes and respiratory chains in the sheath. Copyright © 2012 Elsevier Ltd. All rights reserved.

Plant Acyl-CoA:Lysophosphatidylcholine Acyltransferases (LPCATs) Have Different Specificities in Their Forward and Reverse Reactions*

Science.gov (United States)

Lager, Ida; Yilmaz, Jenny Lindberg; Zhou, Xue-Rong; Jasieniecka, Katarzyna; Kazachkov, Michael; Wang, Peng; Zou, Jitao; Weselake, Randall; Smith, Mark A.; Bayon, Shen; Dyer, John M.; Shockey, Jay M.; Heinz, Ernst; Green, Allan; Banas, Antoni; Stymne, Sten

2013-01-01

Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) enzymes have central roles in acyl editing of phosphatidylcholine (PC). Plant LPCAT genes were expressed in yeast and characterized biochemically in microsomal preparations of the cells. Specificities for different acyl-CoAs were similar for seven LPCATs from five different species, including species accumulating hydroxylated acyl groups in their seed oil, with a preference for C18-unsaturated acyl-CoA and low activity with palmitoyl-CoA and ricinoleoyl (12-hydroxyoctadec-9-enoyl)-CoA. We showed that Arabidopsis LPCAT1 and LPCAT2 enzymes catalyzed the acylation and de-acylation of both sn positions of PC, with a preference for the sn-2 position. When acyl specificities of the Arabidopsis LPCATs were measured in the reverse reaction, sn-2-bound oleoyl, linoleoyl, and linolenoyl groups from PC were transferred to acyl-CoA to a similar extent. However, a ricinoleoyl group at the sn-2-position of PC was removed 4–6-fold faster than an oleoyl group in the reverse reaction, despite poor utilization in the forward reaction. The data presented, taken together with earlier published reports on in vivo lipid metabolism, support the hypothesis that plant LPCAT enzymes play an important role in regulating the acyl-CoA composition in plant cells by transferring polyunsaturated and hydroxy fatty acids produced on PC directly to the acyl-CoA pool for further metabolism or catabolism. PMID:24189065

Heme environment in HmuY, the heme-binding protein of Porphyromonas gingivalis

Energy Technology Data Exchange (ETDEWEB)

Wojtowicz, Halina [Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, Tamka 2, 50-137 Wroclaw (Poland); Wojaczynski, Jacek [Department of Chemistry, University of Wroclaw, 50-383 Wroclaw (Poland); Olczak, Mariusz [Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, Tamka 2, 50-137 Wroclaw (Poland); Kroliczewski, Jaroslaw [Laboratory of Biophysics, Faculty of Biotechnology, University of Wroclaw, 50-148 Wroclaw (Poland); Latos-Grazynski, Lechoslaw [Department of Chemistry, University of Wroclaw, 50-383 Wroclaw (Poland); Olczak, Teresa [Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, Tamka 2, 50-137 Wroclaw (Poland)

2009-05-29

Porphyromonas gingivalis, a Gram-negative anaerobic bacterium implicated in the development and progression of chronic periodontitis, acquires heme for growth by a novel mechanism composed of HmuY and HmuR proteins. The aim of this study was to characterize the nature of heme binding to HmuY. The protein was expressed, purified and detailed investigations using UV-vis absorption, CD, MCD, and {sup 1}H NMR spectroscopy were carried out. Ferric heme bound to HmuY may be reduced by sodium dithionite and re-oxidized by potassium ferricyanide. Heme complexed to HmuY, with a midpoint potential of 136 mV, is in a low-spin Fe(III) hexa-coordinate environment. Analysis of heme binding to several single and double HmuY mutants with the methionine, histidine, cysteine, or tyrosine residues replaced by an alanine residue identified histidines 134 and 166 as potential heme ligands.

Heme environment in HmuY, the heme-binding protein of Porphyromonas gingivalis

International Nuclear Information System (INIS)

Wojtowicz, Halina; Wojaczynski, Jacek; Olczak, Mariusz; Kroliczewski, Jaroslaw; Latos-Grazynski, Lechoslaw; Olczak, Teresa

2009-01-01

Porphyromonas gingivalis, a Gram-negative anaerobic bacterium implicated in the development and progression of chronic periodontitis, acquires heme for growth by a novel mechanism composed of HmuY and HmuR proteins. The aim of this study was to characterize the nature of heme binding to HmuY. The protein was expressed, purified and detailed investigations using UV-vis absorption, CD, MCD, and 1 H NMR spectroscopy were carried out. Ferric heme bound to HmuY may be reduced by sodium dithionite and re-oxidized by potassium ferricyanide. Heme complexed to HmuY, with a midpoint potential of 136 mV, is in a low-spin Fe(III) hexa-coordinate environment. Analysis of heme binding to several single and double HmuY mutants with the methionine, histidine, cysteine, or tyrosine residues replaced by an alanine residue identified histidines 134 and 166 as potential heme ligands.

Lipid-protein interactions. The leucine transport system of Lactococcus lactis.

NARCIS (Netherlands)

Veld, Geertruida Elisabeth in 't

1992-01-01

In summary, it is concluded, that a functionally reconstituted leucine transport system of L. lactis is affected by bilayer features in the following order of importance: lipid headgroup (H+-bonding) › acyl chain carbon number (thickness) › cholesterol (fluidity) › acyl chain unsaturation (indirect

An ethane-bridged porphyrin dimer as a model of di-heme proteins: inorganic and bioinorganic perspectives and consequences of heme-heme interactions.

Science.gov (United States)

Sil, Debangsu; Rath, Sankar Prasad

2015-10-07

Interaction between heme centers has been cleverly implemented by Nature in order to regulate different properties of multiheme cytochromes, thereby allowing them to perform a wide variety of functions. Our broad interest lies in unmasking the roles played by heme-heme interactions in modulating different properties viz., metal spin state, redox potential etc., of the individual heme centers using an ethane-bridged porphyrin dimer as a synthetic model of dihemes. The large differences in the structure and properties of the diheme complexes, as compared to the monoheme analogs, provide unequivocal evidence of the role played by heme-heme interactions in the dihemes. This Perspective provides a brief account of our recent efforts to explore these interesting aspects and the subsequent outcomes.

Red meat and colon cancer : The cytotoxic and hyperproliferative effects of dietary heme

NARCIS (Netherlands)

Sesink, ALA; Termont, DSML; Kleibeuker, JH; Van der Meer, R

1999-01-01

The intake of a Western diet with a high amount of red meat is associated with a high risk for colon cancer. We hypothesize that heme, the iron carrier of red meat, is involved in diet-induced colonic epithelial damage, resulting in increased epithelial proliferation. Rats were fed purified control

Interaction of nitric oxide with human heme oxygenase-1.

Science.gov (United States)

Wang, Jinling; Lu, Shen; Moënne-Loccoz, Pierre; Ortiz de Montellano, Paul R

2003-01-24

NO and CO may complement each other as signaling molecules in some physiological situations. We have examined the binding of NO to human heme oxygenase-1 (hHO-1), an enzyme that oxidizes heme to biliverdin, CO, and free iron, to determine whether inhibition of hHO-1 by NO can contribute to the signaling interplay of NO and CO. An Fe(3+)-NO hHO-1-heme complex is formed with NO or the NO donors NOC9 or 2-(N,N-diethylamino)-diazenolate-2-oxide.sodium salt. Resonance Raman spectroscopy shows that ferric hHO-1-heme forms a 6-coordinated, low spin complex with NO. The nu(N-O) vibration of this complex detected by Fourier transform IR is only 4 cm(-1) lower than that of the corresponding metmyoglobin (met-Mb) complex but is broader, suggesting a greater degree of ligand conformational freedom. The Fe(3+)-NO complex of hHO-1 is much more stable than that of met-Mb. Stopped-flow studies indicate that k(on) for formation of the hHO-1-heme Fe(3+)-NO complex is approximately 50-times faster, and k(off) 10 times slower, than for met-Mb, resulting in K(d) = 1.4 microm for NO. NO thus binds 500-fold more tightly to ferric hHO-1-heme than to met-Mb. The hHO-1 mutations E29A, G139A, D140A, S142A, G143A, G143F, and K179A/R183A do not significantly diminish the tight binding of NO, indicating that NO binding is not highly sensitive to mutations of residues that normally stabilize the distal water ligand. As expected from the K(d) value, the enzyme is reversibly inhibited upon exposure to pathologically, and possibly physiologically, relevant concentrations of NO. Inhibition of hHO-1 by NO may contribute to the pleiotropic responses to NO and CO.

An ER Protein Functionally Couples Neutral Lipid Metabolism on Lipid Droplets to Membrane Lipid Synthesis in the ER

DEFF Research Database (Denmark)

Markgraf, Daniel F; Klemm, Robin W; Junker, Mirco

2014-01-01

Eukaryotic cells store neutral lipids such as triacylglycerol (TAG) in lipid droplets (LDs). Here, we have addressed how LDs are functionally linked to the endoplasmic reticulum (ER). We show that, in S. cerevisiae, LD growth is sustained by LD-localized enzymes. When LDs grow in early stationary...... phase, the diacylglycerol acyl-transferase Dga1p moves from the ER to LDs and is responsible for all TAG synthesis from diacylglycerol (DAG). During LD breakdown in early exponential phase, an ER membrane protein (Ice2p) facilitates TAG utilization for membrane-lipid synthesis. Ice2p has a cytosolic...... and explain how cells switch neutral lipid metabolism from storage to consumption....

Levels of bioactive lipids in cooking oils: olive oil is the richest source of oleoyl serine.

Science.gov (United States)

Bradshaw, Heather B; Leishman, Emma

2016-05-01

Rates of osteoporosis are significantly lower in regions of the world where olive oil consumption is a dietary cornerstone. Olive oil may represent a source of oleoyl serine (OS), which showed efficacy in animal models of osteoporosis. Here, we tested the hypothesis that OS as well as structurally analogous N-acyl amide and 2-acyl glycerol lipids are present in the following cooking oils: olive, walnut, canola, high heat canola, peanut, safflower, sesame, toasted sesame, grape seed, and smart balance omega. Methanolic lipid extracts from each of the cooking oils were partially purified on C-18 solid-phase extraction columns. Extracts were analyzed with high-performance liquid chromatography-tandem mass spectrometry, and 33 lipids were measured in each sample, including OS and bioactive analogs. Of the oils screened here, walnut oil had the highest number of lipids detected (22/33). Olive oil had the second highest number of lipids detected (20/33), whereas grape-seed and high-heat canola oil were tied for lowest number of detected lipids (6/33). OS was detected in 8 of the 10 oils tested and the levels were highest in olive oil, suggesting that there is something about the olive plant that enriches this lipid. Cooking oils contain varying levels of bioactive lipids from the N-acyl amide and 2-acyl glycerol families. Olive oil is a dietary source of OS, which may contribute to lowered prevalence of osteoporosis in countries with high consumption of this oil.

Disorders of muscle lipid metabolism: diagnostic and therapeutic challenges.

Science.gov (United States)

Laforêt, Pascal; Vianey-Saban, Christine

2010-11-01

Disorders of muscle lipid metabolism may involve intramyocellular triglyceride degradation, carnitine uptake, long-chain fatty acids mitochondrial transport, or fatty acid β-oxidation. Three main diseases leading to permanent muscle weakness are associated with severe increased muscle lipid content (lipid storage myopathies): primary carnitine deficiency, neutral lipid storage disease and multiple acyl-CoA dehydrogenase deficiency. A moderate lipidosis may be observed in fatty acid oxidation disorders revealed by rhabdomyolysis episodes such as carnitine palmitoyl transferase II, very-long-chain acyl-CoA dehydrogenase, mitochondrial trifunctional protein deficiencies, and in recently described phosphatidic acid phosphatase deficiency. Respiratory chain disorders and congenital myasthenic syndromes may also be misdiagnosed as fatty acid oxidation disorders due to the presence of secondary muscle lipidosis. The main biochemical tests giving clues for the diagnosis of these various disorders are measurements of blood carnitine and acylcarnitines, urinary organic acid profile, and search for intracytoplasmic lipid on peripheral blood smear (Jordan's anomaly). Genetic analysis orientated by the results of biochemical investigation allows establishing a firm diagnosis. Primary carnitine deficiency and multiple acyl-CoA dehydrogenase deficiency may be treated after supplementation with carnitine, riboflavine and coenzyme Q10. New therapeutic approaches for fatty acid oxidation disorders are currently developed, based on pharmacological treatment with bezafibrate, and specific diets enriched in medium-chain triglycerides or triheptanoin. Copyright © 2010 Elsevier B.V. All rights reserved.

Heme isomers substantially affect heme's electronic structure and function

DEFF Research Database (Denmark)

Kepp, Kasper Planeta

2017-01-01

Inspection of heme protein structures in the protein data bank reveals four isomers of heme characterized by different relative orientations of the vinyl side chains; remarkably, all these have been reported in multiple protein structures. Density functional theory computations explain this as du...

Phase separation in lipid bilayers triggered by low pH

International Nuclear Information System (INIS)

Suresh, Swetha; Edwardson, J. Michael

2010-01-01

Research highlights: → Lipid bilayers have been imaged by atomic force microscopy (AFM). → At pH 5 phase separation occurs in lipid bilayers containing mixed acyl chains. → Phase separation does not occur when lipids have only unsaturated chains. → Phase separation might drive protein clustering during endocytosis. -- Abstract: Endocytosis involves the capture of membrane from the cell surface in the form of vesicles, which become rapidly acidified to about pH 5. Here we show using atomic force microscopy (AFM) imaging that this degree of acidification triggers phase separation in lipid bilayers containing mixed acyl chains (e.g. palmitoyl/oleoyl) or complex mixtures (e.g. total brain extract) but not in bilayers containing only lipids with unsaturated chains (e.g. dioleoyl). Since mixed-chain lipids are major constituents of the outer leaflet of the plasma membrane, the type of phase separation reported here might support protein clustering and signaling during endocytosis.

Studies to further investigate the inhibition of human liver microsomal CYP2C8 by the acyl-β-glucuronide of gemfibrozil.

Science.gov (United States)

Jenkins, S M; Zvyaga, T; Johnson, S R; Hurley, J; Wagner, A; Burrell, R; Turley, W; Leet, J E; Philip, T; Rodrigues, A D

2011-12-01

In previous studies, gemfibrozil acyl-β-glucuronide, but not gemfibrozil, was found to be a mechanism-based inhibitor of cytochrome P450 2C8. To better understand whether this inhibition is specific for gemfibrozil acyl-β-glucuronide or whether other glucuronide conjugates are potential substrates for inhibition of this enzyme, we evaluated several pharmaceutical compounds (as their acyl glucuronides) as direct-acting and metabolism-dependent inhibitors of CYP2C8 in human liver microsomes. Of 11 compounds that were evaluated as their acyl glucuronide conjugates, only gemfibrozil acyl-β-glucuronide exhibited mechanism-based inhibition, indicating that CYP2C8 mechanism-based inhibition is very specific to certain glucuronide conjugates. Structural analogs of gemfibrozil were synthesized, and their glucuronide conjugates were prepared to further examine the mechanism of inhibition. When the aromatic methyl groups on the gemfibrozil moiety were substituted with trifluoromethyls, the resulting glucuronide conjugate was a weaker inhibitor of CYP2C8 and mechanism-based inhibition was abolished. However, the glucuronide conjugates of monomethyl gemfibrozil analogs were mechanism-based inhibitors of CYP2C8, although not as potent as gemfibrozil acyl-β-glucuronide itself. The ortho-monomethyl analog was a more potent inhibitor than the meta-monomethyl analog, indicating that CYP2C8 favors the ortho position for oxidation and potential inhibition. Molecular modeling of gemfibrozil acyl-β-glucuronide in the CYP2C8 active site is consistent with the ortho-methyl position being the favored site of covalent attachment to the heme. Moreover, hydrogen bonding to four residues (Ser100, Ser103, Gln214, and Asn217) is implicated.

Whey Peptide-Iron Complexes Increase the Oxidative Stability of Oil-in-Water Emulsions in Comparison to Iron Salts.

Science.gov (United States)

Caetano-Silva, Maria Elisa; Barros Mariutti, Lilian Regina; Bragagnolo, Neura; Bertoldo-Pacheco, Maria Teresa; Netto, Flavia Maria

2018-02-28

Food fortification with iron may favor lipid oxidation in both food matrices and the human body. This study aimed at evaluating the effect of peptide-iron complexation on lipid oxidation catalyzed by iron, using oil-in-water (O/W) emulsions as a model system. The extent of lipid oxidation of emulsions containing iron salts (FeSO 4 or FeCl 2 ) or iron complexes (peptide-iron complexes or ferrous bisglycinate) was evaluated during 7 days, measured as primary (peroxide value) and secondary products (TBARS and volatile compounds). Both salts catalyzed lipid oxidation, leading to peroxide values 2.6- to 4.6-fold higher than the values found for the peptide-iron complexes. The addition of the peptide-iron complexes resulted in the formation of lower amounts of secondary volatiles of lipid oxidation (up to 78-fold) than those of iron salts, possibly due to the antioxidant activity of the peptides and their capacity to keep iron apart from the lipid phase, since the iron atom is coordinated and takes part in a stable structure. The peptide-iron complexes showed potential to reduce the undesirable sensory changes in food products and to decrease the side effects related to free iron and the lipid damage of cell membranes in the organism, due to the lower reactivity of iron in the complexed form.

Heme Oxygenase-1 and breast cancer resistance protein protect against heme-induced toxicity

NARCIS (Netherlands)

Wagener, Frank A D T G; Dankers, Anita C A; van Summeren, Frank; Scharstuhl, Alwin; van den Heuvel, Jeroen J M W; Koenderink, Jan B; Pennings, Sebastiaan W C; Russel, Frans G M; Masereeuw, R.

2013-01-01

Heme is the functional group of diverse hemoproteins and crucial for many cellular processes. However, heme is increasingly recognized as a culprit for a wide variety of pathologies, including sepsis, malaria, and kidney failure. Excess of free heme can be detrimental to tissues by mediating

Isotope aided studies of the bioavailability of iron from human diets consumed in Peru

International Nuclear Information System (INIS)

Zavaleta, N.; Penny, M.; Berlanga, R.; Diaz, A.; Montoya, E.; Lonnerdal, B.

1994-01-01

Iron deficiency anaemia is an important health problem in Peru, which affects approximately 25% of the population. The most vulnerable groups are children below 5 years of age and pregnant women, of whom 64% and 53% respectively are anemic. The main reason for this deficiency is inadequate iron intake. Heme iron consumption is very low, and non-heme iron is virtually the only source of iron in the diet. Despite regional differences in food consumption, wheat, salt and sugar are widely consumed in all areas. Wheat is likely to be the most suitable food vehicle for iron fortification due to the processing required. Based on the recent food consumption surveys conducted in Lima by the IIN, we selected examples of typical main meals and measured iron bioavailability in the diet using an extrinsic tag method with 1.5 μCi of 59 Fe and 5 μCi of 55 Fe as markers. Coffee with bread and butter for breakfast, noodle soup with vegetables, rice with seasoned tripe (cow), bread and lemonade for lunch; and noodle soup with vegetables and bread for dinner were used to measure iron absorption. Thirteen adults in apparent good health, 5 male and 8 female, with normal hemoglobin levels participated in the study. The mean iron absorption from breakfast was 4.2% ± 4.1; from lunch 14.65% ± 10/95, and from dinner 5.1% ± 2.84. The presence of heme iron from tripe and ascorbic acid from lemonade improved iron absorption. (author). 17 refs, 3 tabs

Hemolytic anemia repressed hepcidin level without hepatocyte iron overload: lesson from Günther disease model.

Science.gov (United States)

Millot, Sarah; Delaby, Constance; Moulouel, Boualem; Lefebvre, Thibaud; Pilard, Nathalie; Ducrot, Nicolas; Ged, Cécile; Lettéron, Philippe; de Franceschi, Lucia; Deybach, Jean Charles; Beaumont, Carole; Gouya, Laurent; De Verneuil, Hubert; Lyoumi, Saïd; Puy, Hervé; Karim, Zoubida

2017-02-01

Hemolysis occurring in hematologic diseases is often associated with an iron loading anemia. This iron overload is the result of a massive outflow of hemoglobin into the bloodstream, but the mechanism of hemoglobin handling has not been fully elucidated. Here, in a congenital erythropoietic porphyria mouse model, we evaluate the impact of hemolysis and regenerative anemia on hepcidin synthesis and iron metabolism. Hemolysis was confirmed by a complete drop in haptoglobin, hemopexin and increased plasma lactate dehydrogenase, an increased red blood cell distribution width and osmotic fragility, a reduced half-life of red blood cells, and increased expression of heme oxygenase 1. The erythropoiesis-induced Fam132b was increased, hepcidin mRNA repressed, and transepithelial iron transport in isolated duodenal loops increased. Iron was mostly accumulated in liver and spleen macrophages but transferrin saturation remained within the normal range. The expression levels of hemoglobin-haptoglobin receptor CD163 and hemopexin receptor CD91 were drastically reduced in both liver and spleen, resulting in heme- and hemoglobin-derived iron elimination in urine. In the kidney, the megalin/cubilin endocytic complex, heme oxygenase 1 and the iron exporter ferroportin were induced, which is reminiscent of significant renal handling of hemoglobin-derived iron. Our results highlight ironbound hemoglobin urinary clearance mechanism and strongly suggest that, in addition to the sequestration of iron in macrophages, kidney may play a major role in protecting hepatocytes from iron overload in chronic hemolysis. Copyright© Ferrata Storti Foundation.

Checks and balances in membrane phospholipid class and acyl chain homeostasis, the yeast perspective

NARCIS (Netherlands)

de Kroon, A.I.P.M.|info:eu-repo/dai/nl/084765283; Rijken, P.J.|info:eu-repo/dai/nl/32716297X; De Smet, C.H.|info:eu-repo/dai/nl/304824224

2013-01-01

Glycerophospholipids are the most abundant membrane lipid constituents in most eukaryotic cells. As a consequence, phospholipid class and acyl chain homeostasis are crucial for maintaining optimal physical properties of membranes that in turn are crucial for membrane function. The topic of this

A Non-Heme Iron Photocatalyst for Light-Driven Aerobic Oxidation of Methanol

NARCIS (Netherlands)

Chen, Juan; Stepanovic, Stepan; Draksharapu, Apparao; Gruden, Maja; Browne, Wesley R

2018-01-01

Non-heme (L)FeIIIand (L)FeIII-O-FeIII(L) complexes (L=1,1-di(pyridin-2-yl)-N,N-bis(pyridin-2-ylmethyl)ethan-1-amine) underwent reduction under irradiation to the FeIIstate with concomitant oxidation of methanol to methanal, without the need for a secondary photosensitizer. Spectroscopic and DFT

Heme-induced ROS in Trypanosoma cruzi activates CaMKII-like that triggers epimastigote proliferation. One helpful effect of ROS.

Directory of Open Access Journals (Sweden)

Natália Pereira de Almeida Nogueira

Full Text Available Heme is a ubiquitous molecule that has a number of physiological roles. The toxic effects of this molecule have been demonstrated in various models, based on both its pro-oxidant nature and through a detergent mechanism. It is estimated that about 10 mM of heme is released during blood digestion in the blood-sucking bug's midgut. The parasite Trypanosoma cruzi, the agent of Chagas' disease, proliferates in the midgut of the insect vector; however, heme metabolism in trypanosomatids remains to be elucidated. Here we provide a mechanistic explanation for the proliferative effects of heme on trypanosomatids. Heme, but not other porphyrins, induced T. cruzi proliferation, and this phenomenon was accompanied by a marked increase in reactive oxygen species (ROS formation in epimastigotes when monitored by ROS-sensitive fluorescent probes. Heme-induced ROS production was time- and concentration-dependent. In addition, lipid peroxidation and the formation of 4-hydroxy-2-nonenal (4-HNE adducts with parasite proteins were increased in epimastigotes in the presence of heme. Conversely, the antioxidants urate and GSH reversed the heme-induced ROS. Urate also decreased parasite proliferation. Among several protein kinase inhibitors tested only specific inhibitors of CaMKII, KN93 and Myr-AIP, were able to abolish heme-induced ROS formation in epimastigotes leading to parasite growth impairment. Taken together, these data provide new insight into T. cruzi- insect vector interactions: heme, a molecule from the blood digestion, triggers epimastigote proliferation through a redox-sensitive signalling mechanism.

Characterization of acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) enzyme of human small intestine.

Science.gov (United States)

Hiramine, Yasushi; Tanabe, Toshizumi

2011-06-01

Acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) enzyme plays a significant role in dietary triacylglycerol (TAG) absorption in the small intestine. However, the characteristics of human intestinal DGAT enzyme have not been examined in detail. The aim of our study was to characterize the human intestinal DGAT enzyme by examining acyl-CoA specificity, temperature dependency, and selectivity for 1,2-diacylglycerol (DAG) or 1,3-DAG. We detected DGAT activity of human intestinal microsome and found that the acyl-CoA specificity and temperature dependency of intestinal DGAT coincided with those of recombinant human DGAT1. To elucidate the selectivity of human intestinal DGAT to 1,2-DAG or 1,3-DAG, we conducted acyl-coenzyme A:monoacylglycerol acyltransferase assays using 1- or 2-monoacylglycerol (MAG) as substrates. When 2-MAG was used as acyl acceptor, both 1,2-DAG and TAG were generated; however, when 1-MAG was used, 1,3-DAG was predominantly observed and little TAG was detected. These findings suggest that human small intestinal DGAT, which is mainly encoded by DGAT1, utilizes 1,2-DAG as the substrate to form TAG. This study will contribute to understand the lipid absorption profile in the small intestine.

Heme Oxygenase Induction Suppresses Hepatic Hepcidin and Rescues Ferroportin and Ferritin Expression in Obese Mice

Directory of Open Access Journals (Sweden)

Nitin Puri

2017-01-01

Full Text Available Hepcidin, a phase II reactant secreted by hepatocytes, regulates cellular iron levels by increasing internalization of ferroportin-a transmembrane protein facilitating egress of cellular iron. Chronic low-grade inflammatory states, such as obesity, have been shown to increase oxidative stress and enhance hepcidin secretion from hepatocytes and macrophages. Heme-heme oxygenase (HO is a stress response system which reduces oxidative stress. We investigated the effects of HO-1 induction on hepatic hepcidin levels and on iron homeostasis in hepatic tissues from lean and obese mice. Obese mice exhibited hyperglycemia (p<0.05; increased levels of proinflammatory cytokines (MCP-1, IL-6, p<0.05; oxidative stress (p<0.05; and increased hepatic hepcidin levels (p<0.05. Enhancement of hepcidin was reflected in the reduced expression of ferroportin in obese mice (p<0.05. However, this effect is accompanied by a significant decline in ferritin expression. Additionally, there are reduced insulin receptor phosphorylation and attenuation of metabolic regulators pAMPK, pAKT, and pLKB1. Cobalt protoporphyrin- (CoPP- induced HO-1 upregulation in obese mice reversed these alterations (p<0.05, while attenuating hepatic hepcidin levels. These effects of CoPP were prevented in obese mice concurrently exposed to an inhibitor of HO (SnMP (p<0.05. Our results highlight a modulatory effect of HO on iron homeostasis mediated through the suppression of hepatic hepcidin.

Identification of residues in the heme domain of soluble guanylyl cyclase that are important for basal and stimulated catalytic activity.

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Padmamalini Baskaran

Full Text Available Nitric oxide signals through activation of soluble guanylyl cyclase (sGC, a heme-containing heterodimer. NO binds to the heme domain located in the N-terminal part of the β subunit of sGC resulting in increased production of cGMP in the catalytic domain located at the C-terminal part of sGC. Little is known about the mechanism by which the NO signaling is propagated from the receptor domain (heme domain to the effector domain (catalytic domain, in particular events subsequent to the breakage of the bond between the heme iron and Histidine 105 (H105 of the β subunit. Our modeling of the heme-binding domain as well as previous homologous heme domain structures in different states point to two regions that could be critical for propagation of the NO activation signal. Structure-based mutational analysis of these regions revealed that residues T110 and R116 in the αF helix-β1 strand, and residues I41 and R40 in the αB-αC loop mediate propagation of activation between the heme domain and the catalytic domain. Biochemical analysis of these heme mutants allows refinement of the map of the residues that are critical for heme stability and propagation of the NO/YC-1 activation signal in sGC.

Molecular hijacking of siroheme for the synthesis of heme and d1 heme.

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Bali, Shilpa; Lawrence, Andrew D; Lobo, Susana A; Saraiva, Lígia M; Golding, Bernard T; Palmer, David J; Howard, Mark J; Ferguson, Stuart J; Warren, Martin J

2011-11-08

Modified tetrapyrroles such as chlorophyll, heme, siroheme, vitamin B(12), coenzyme F(430), and heme d(1) underpin a wide range of essential biological functions in all domains of life, and it is therefore surprising that the syntheses of many of these life pigments remain poorly understood. It is known that the construction of the central molecular framework of modified tetrapyrroles is mediated via a common, core pathway. Herein a further branch of the modified tetrapyrrole biosynthesis pathway is described in denitrifying and sulfate-reducing bacteria as well as the Archaea. This process entails the hijacking of siroheme, the prosthetic group of sulfite and nitrite reductase, and its processing into heme and d(1) heme. The initial step in these transformations involves the decarboxylation of siroheme to give didecarboxysiroheme. For d(1) heme synthesis this intermediate has to undergo the replacement of two propionate side chains with oxygen functionalities and the introduction of a double bond into a further peripheral side chain. For heme synthesis didecarboxysiroheme is converted into Fe-coproporphyrin by oxidative loss of two acetic acid side chains. Fe-coproporphyrin is then transformed into heme by the oxidative decarboxylation of two propionate side chains. The mechanisms of these reactions are discussed and the evolutionary significance of another role for siroheme is examined.

Relationships between acylated ghrelin with growth hormone, insulin resistance, lipid profile, and cardio respiratory function in lean and obese men

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Hasan Matin Homaee

2011-01-01

Conclusions: Obese and lean inactive young men had different levels of acylated ghrelin, GH, insulin, insulin resistance index, cardiorespiratory function and body fat percent. Body fat percent, insulin, and GH levels appear to be best determinant factors of acylated ghrelin levels. Also, in both obese and lean young men, higher levels of cardiovascular function were associated with higher levels of acylated ghrelin.

Benfotiamine, a synthetic S-acyl thiamine derivative, has different mechanisms of action and a different pharmacological profile than lipid-soluble thiamine disulfide derivatives.

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Volvert, Marie-Laure; Seyen, Sandrine; Piette, Marie; Evrard, Brigitte; Gangolf, Marjorie; Plumier, Jean-Christophe; Bettendorff, Lucien

2008-06-12

Lipid-soluble thiamine precursors have a much higher bioavailability than genuine thiamine and therefore are more suitable for therapeutic purposes. Benfotiamine (S-benzoylthiamine O-monophosphate), an amphiphilic S-acyl thiamine derivative, prevents the progression of diabetic complications, probably by increasing tissue levels of thiamine diphosphate and so enhancing transketolase activity. As the brain is particularly sensitive to thiamine deficiency, we wanted to test whether intracellular thiamine and thiamine phosphate levels are increased in the brain after oral benfotiamine administration. Benfotiamine that is practically insoluble in water, organic solvents or oil was solubilized in 200 mM hydroxypropyl-beta-cyclodextrin and the mice received a single oral administration of 100 mg/kg. Though thiamine levels rapidly increased in blood and liver to reach a maximum after one or two hours, no significant increase was observed in the brain. When mice received a daily oral administration of benfotiamine for 14 days, thiamine derivatives were increased significantly in the liver but not in the brain, compared to control mice. In addition, incubation of cultured neuroblastoma cells with 10 muM benfotiamine did not lead to increased intracellular thiamine levels. Moreover, in thiamine-depleted neuroblastoma cells, intracellular thiamine contents increased more rapidly after addition of thiamine to the culture medium than after addition of benfotiamine for which a lag period was observed. Our results show that, though benfotiamine strongly increases thiamine levels in blood and liver, it has no significant effect in the brain. This would explain why beneficial effects of benfotiamine have only been observed in peripheral tissues, while sulbutiamine, a lipid-soluble thiamine disulfide derivative, that increases thiamine derivatives in the brain as well as in cultured cells, acts as a central nervous system drug. We propose that benfotiamine only penetrates the cells

Radiation-induced lipid peroxidation: influence of oxygen concentration and membrane lipid composition

International Nuclear Information System (INIS)

Wolters, H.; Tilburg, C.A.M. van; Konings, A.W.T.

1987-01-01

Radiation -induced lipid peroxidation phospholipid liposomes was investigated in terms of its dependence on lipid composition and oxygen concentration. Non-peroxidizable lipid incorporated in the liposomes reduced the rate of peroxidation of the peroxidizable phospholipid acyl chains, possibly by restricting the length of chain reactions. The latter effect is believed to be caused by interference of the non-peroxidizable lipids in the bilayer. At low oxygen concentration lipid peroxidation was reduced. The cause of this limited peroxidation may be a reduced number of radical initiation reactions possibly involving oxygen-derived superoxide radicals. Killing of proliferating mammalian cells, irradiated at oxygen concentrations ranging from 0 to 100%, appeared to be independent of the concentration of peroxidizable phospholipids in the cell membranes. This indicates that lipid peroxidation is not the determining process in radiation-induced reproductive cell death. (author)

Acylation of Therapeutic Peptides

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Trier, Sofie; Henriksen, Jonas Rosager; Jensen, Simon Bjerregaard

) , which promotes intestinal growth and is used to treat bowel disorders such as inflammatory bowel diseases and short bowel syndrome, and the 32 amino acid salmon calcitonin (sCT), which lowers blood calcium and is employed in the treatment of post-menopausal osteoporosis and hypercalcemia. The two...... peptides are similar in size and structure, but oppositely charged at physiological pH. Both peptides were acylated with linear acyl chains of systematically increasing length, where sCT was furthermore acylated at two different positions on the peptide backbone. For GLP-2, we found that increasing acyl...... remained optimal overall. The results indicate that rational acylation of GLP-2 can increase its in vitro intestinal absorption, alone or in combination with permeation enhancers, and are consistent with the initial project hypothesis. For sCT, an unpredicted effect of acylation largely superseded...

Double enzymatic hydrolysis preparation of heme from goose blood and microencapsulation to promote its stability and absorption.

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Wang, Baowei; Cheng, Fansheng; Gao, Shun; Ge, Wenhua; Zhang, Mingai

2017-02-15

Iron deficiency anemia (IDA) is the most common nutritional deficiency worldwide. This deficiency could be solved by preparing stable, edible, and absorbable iron food ingredients using environmentally friendly methods. This study investigated enzymatic hydrolysis and microencapsulation process of goose blood. The physicochemical properties, stabilities of the microencapsulated goose blood hydrolysate (MGBH) and a supplement for rats with IDA were also evaluated. The results showed that the synergetic hydrolytic action of neutrase and alkaline protease significantly increased the heme-releasing efficiency. The heme was then microencapsulated using sodium caseinate, maltodextrin and carboxymethyl cellulose (CMC) as the edible wall material, and the encapsulation efficiency of the product reached 98.64%. Meanwhile, favorable thermal, storage and light stabilities were observed for the microencapsulation. It was found that MGBH can significantly improve the body weight and hematological parameters of IDA Wistar rat. Copyright © 2016 Elsevier Ltd. All rights reserved.

Generation of fatty acids by an acyl esterase in the bioluminescent system of Photobacterium phosphoreum

International Nuclear Information System (INIS)

Carey, L.M.; Rodriguez, A.; Meighen, E.

1984-01-01

The fatty acid reductase complex from Photobacterium phosphoreum has been discovered to have a long chain ester hydrolase activity associated with the 34K protein component of the complex. This protein has been resolved from the other components (50K and 58K) of the fatty acid reductase complex with a purity of > 95% and found to catalyze the transfer of acyl groups from acyl-CoA primarily to thiol acceptors with a low level of transfer to glycerol and water. Addition of the 50K protein of the complex caused a dramatic change in specificity increasing the transfer to oxygen acceptors. The acyl-CoA hydrolase activity increased almost 10-fold, and hence free fatty acids can be generated by the 34K protein when it is present in the fatty acid reductase complex. Hydrolysis of acyl-S-mercaptoethanol and acyl-1-glycerol and the ATP-dependent reduction of the released fatty acids to aldehyde for the luminescent reaction were also demonstrated for the reconstituted fatty acid reductase complex, raising the possibility that the immediate source of fatty acids for this reaction in vivo could be the membrane lipids and/or the fatty acid synthetase system

Host heme oxygenase-1: Friend or foe in tackling pathogens?

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Singh, Nisha; Ahmad, Zeeshan; Baid, Navin; Kumar, Ashwani

2018-05-14

Infectious diseases are a major challenge in management of human health worldwide. Recent literature suggests that host immune system could be modulated to ameliorate the pathogenesis of infectious disease. Heme oxygenase (HMOX1) is a key regulator of cellular signaling and it could be modulated using pharmacological reagents. HMOX1 is a cytoprotective enzyme that degrades heme to generate carbon monoxide (CO), biliverdin, and molecular iron. CO and biliverdin (or bilirubin derived from it) can restrict the growth of a few pathogens. Both of these also induce antioxidant pathways and anti-inflammatory pathways. On the other hand, molecular iron can induce proinflammatory pathway besides making the cellular environment oxidative in nature. Since microbial infections often induce oxidative stress in host cells/tissues, role of HMOX1 has been analyzed in the pathogenesis of number of infections. In this review, we have described the role of HMOX1 in pathogenesis of bacterial infections caused by Mycobacterium species, Salmonella and in microbial sepsis. We have also provided a succinct overview of the role of HMOX1 in parasitic infections such as malaria and leishmaniasis. In the end, we have also elaborated the role of HMOX1 in viral infections such as AIDS, hepatitis, dengue, and influenza. © 2018 IUBMB Life, 2018. © 2018 International Union of Biochemistry and Molecular Biology.

Long-chain Acyl-CoA is not increased in Myotubes established from Type 2 Diabetic Subjects

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Just, Malene; Faergeman, Nils J; Knudsen, Jens

2006-01-01

Accumulation of intramuscular long-chain acyl-CoA esters (LCACoA) has previously in animal and human models been suggested to play an important role in lipid induced insulin resistance. The aim of this study was to examine whether myotubes established from type 2 diabetic (T2D) subjects and lean...... controls express differences in long-chain acyl-CoA esters (LCACoA) precultured under physiological conditions and during chronic exposure to palmitate (PA) and oleic acids (OA) with/without acute insulin stimulation. No significant differences were found between diabetic and control myotubes, neither...

Astroglia overexpressing heme oxygenase-1 predispose co-cultured PC12 cells to oxidative injury.

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Song, Linyang; Song, Wei; Schipper, Hyman M

2007-08-01

The mechanisms responsible for the progressive degeneration of dopaminergic neurons and pathologic iron deposition in the substantia nigra pars compacta of patients with Parkinson's disease (PD) remain unclear. Heme oxygenase-1 (HO-1), the rate-limiting enzyme in the oxidative degradation of heme to ferrous iron, carbon monoxide, and biliverdin, is upregulated in affected PD astroglia and may contribute to abnormal mitochondrial iron sequestration in these cells. To determine whether glial HO-1 hyper-expression is toxic to neuronal compartments, we co-cultured dopaminergic PC12 cells atop monolayers of human (h) HO-1 transfected, sham-transfected, or non-transfected primary rat astroglia. We observed that PC12 cells grown atop hHO-1 transfected astrocytes, but not the astroglia themselves, were significantly more susceptible to dopamine (1 microM) + H(2)O(2) (1 microM)-induced death (assessed by nuclear ethidium monoazide bromide staining and anti-tyrosine hydroxylase immunofluorescence microscopy) relative to control preparations. In the experimental group, PC12 cell death was attenuated significantly by the administration of the HO inhibitor, SnMP (1.5 microM), the antioxidant, ascorbate (200 microM), or the iron chelators, deferoxamine (400 microM), and phenanthroline (100 microM). Exposure to conditioned media derived from HO-1 transfected astrocytes also augmented PC12 cell killing in response to dopamine (1 microM) + H(2)O(2) (1 microM) relative to control media. In PD brain, overexpression of HO-1 in nigral astroglia and accompanying iron liberation may facilitate the bioactivation of dopamine to neurotoxic free radical intermediates and predispose nearby neuronal constituents to oxidative damage. (c) 2007 Wiley-Liss, Inc.

Plant fatty acyl reductases: enzymes generating fatty alcohols for protective layers with potential for industrial applications.

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Rowland, Owen; Domergue, Frédéric

2012-09-01

Primary fatty alcohols are found throughout the biological world, either in free form or in a combined state. They are common components of plant surface lipids (i.e. cutin, suberin, sporopollenin, and associated waxes) and their absence can significantly perturb these essential barriers. Fatty alcohols and/or derived compounds are also likely to have direct functions in plant biotic and abiotic interactions. An evolutionarily related set of alcohol-forming fatty acyl reductases (FARs) is present in all kingdoms of life. Plant microsomal and plastid-associated FAR enzymes have been characterized, acting on acyl-coenzymeA (acyl-CoA) or acyl-acyl carrier protein (acyl-ACP) substrates, respectively. FARs have distinct substrate specificities both with regard to chain length and chain saturation. Fatty alcohols and wax esters, which are a combination of fatty alcohol and fatty acid, have a variety of commercial applications. The expression of FARs with desired specificities in transgenic microbes or oilseed crops would provide a novel means of obtaining these valuable compounds. In the present review, we report on recent progress in characterizing plant FAR enzymes and in understanding the biological roles of primary fatty alcohols, as well as describe the biotechnological production and industrial uses of fatty alcohols. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Effect of acid whey and freeze-dried cranberries on lipid oxidation and fatty acid composition of nitrite-/nitrate-free fermented sausage made from deer meat

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Małgorzata Karwowska

2017-01-01

Full Text Available Objective This study evaluated the effect of acid whey and freeze-dried cranberries on the physicochemical characteristics, lipid oxidation and fatty acid composition of nitrite-free fermented sausage made from deer meat and pork fat. Antioxidant interactions between acid whey and cranberry compounds were also explored. Methods Four formulations of fermented venison sausage were prepared: F1 (control, F2 (with 5% liquid acid whey, F3 (with 0.06% of freeze-dried cranberries, and F4 (with 5% liquid acid whey and 0.06% of freeze-dried cranberries. Each sample was analyzed for pH, water activity (aw, heme iron content, 2-thiobarbituric acid reactive substances (TBARS value and conjugated dienes at the end of the manufacturing process and at 30 and 90 days of refrigerated storage. Fatty acid composition was measured once at the end of the manufacturing process. Results At the end of ripening, all samples presented statistically different values for a pH range of 4.47 to pH 4.59. The sum of the unsaturated fatty acids was higher, while the conjugated diene and the TBARS values were lower in sausages with freeze-dried cranberries as compared to the control sausage. The highest content of heme iron (21.52 mg/kg at day 90 was found in the sausage formulation with the addition of freeze-dried cranberries, which suggests that the addition of cranberries stabilized the porphyrin ring of the heme molecule during storage and thereby reduced the release of iron. The use of liquid acid whey in combination with cranberries appears to not be justified in view of the oxidative stability of the obtained products. Conclusion The results suggest that the application of freeze-dried cranberries can lower the intensity of oxidative changes during the storage of nitrite-free fermented sausage made from deer meat.

The mechanism of stereospecific C-H oxidation by Fe(Pytacn) complexes: bioinspired non-heme iron catalysts containing cis-labile exchangeable sites.

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Prat, Irene; Company, Anna; Postils, Verònica; Ribas, Xavi; Que, Lawrence; Luis, Josep M; Costas, Miquel

2013-05-17

A detailed mechanistic study of the hydroxylation of alkane C-H bonds using H2O2 by a family of mononuclear non heme iron catalysts with the formula [Fe(II)(CF3SO3)2(L)] is described, in which L is a tetradentate ligand containing a triazacyclononane tripod and a pyridine ring bearing different substituents at the α and γ positions, which tune the electronic or steric properties of the corresponding iron complexes. Two inequivalent cis-labile exchangeable sites, occupied by triflate ions, complete the octahedral iron coordination sphere. The C-H hydroxylation mediated by this family of complexes takes place with retention of configuration. Oxygen atoms from water are incorporated into hydroxylated products and the extent of this incorporation depends in a systematic manner on the nature of the catalyst, and the substrate. Mechanistic probes and isotopic analyses, in combination with detailed density functional theory (DFT) calculations, provide strong evidence that C-H hydroxylation is performed by highly electrophilic [Fe(V)(O)(OH)L] species through a concerted asynchronous mechanism, involving homolytic breakage of the C-H bond, followed by rebound of the hydroxyl ligand. The [Fe(V)(O)(OH)L] species can exist in two tautomeric forms, differing in the position of oxo and hydroxide ligands. Isotopic-labeling analysis shows that the relative reactivities of the two tautomeric forms are sensitively affected by the α substituent of the pyridine, and this reactivity behavior is rationalized by computational methods. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effect of irradiation and storage in the iron availability in lamb meat treated with different diets; Efeito da irradiacao e do armazenamento na disponibilidade de ferro em carne de cordeiro tratado com diferentes dietas

Energy Technology Data Exchange (ETDEWEB)

Souza, Adriana Regia Marques de; Arthur, Valter [Centro de Energia Nuclear na Agricultura (CENA/USP), Piracicaba, SP (Brazil). Lab. de Irradiacao de Alimentos e Radioentomologia]. E-mail: acornel@cena.usp.br; arthur@cena.usp.br; Canniatti-Brazaca, Solange Guidolin [Escola Superior de Agricultura Luiz de Queiroz (ESALQ/USP), Piracicaba, SP (Brazil). Dept. de Agroindustria, Alimentos e Nutricao]. E-mail: sgcbraza@esalq.usp.br; Couto, Meylene Aparecida Luzia [Escola Superior de Agricultura Luiz de Queiroz (ESALQ/USP), Piracicaba, SP (Brazil)]. E-mail: malcouto@esalq.usp.br

2008-10-15

Irradiation is an efficient method to increase the microbiological safety and to maintain the nutrients such as iron in the meat. The best absorption form, heme iron, should be preserved in order to increase the nutritional quality of stored meat. The diet can alter the nutrients contents and form in the meat. The iron is provided from the diet and it is an essential element for the metabolic processes such as oxygen transport, oxidative metabolism, and cellular growth. Meat lamb samples treated with different diets (it controls, TAC1, TAC2 and sorghum) were wrapped to vacuous, and irradiated in the doses 0, 2 and 4 kGy and stored at 4 deg C during 15 days. The values of total iron and heme iron were measured at 0 and 15 days of storage. The storage reduced the content of total iron (18.36 for 14.28 mg.100 g{sup -1}) and heme iron (13.78 for 10.52 mg.100 g{sup -1}). The diets affected the levels of total and heme iron of the meat, and the sorghum diet was the one that presented the larger content. The dose of 2 kGy was the one that affected the iron the most independently of the storage time. It was verified that the amounts of total and heme iron varied according to the storage time, irradiation doses, and lamb diets. (author)

Effects of acute creatine supplementation on iron homeostasis and uric acid-based antioxidant capacity of plasma after wingate test

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Barros Marcelo P

2012-06-01

Full Text Available Abstract Background Dietary creatine has been largely used as an ergogenic aid to improve strength and athletic performance, especially in short-term and high energy-demanding anaerobic exercise. Recent findings have also suggested a possible antioxidant role for creatine in muscle tissues during exercise. Here we evaluate the effects of a 1-week regimen of 20 g/day creatine supplementation on the plasma antioxidant capacity, free and heme iron content, and uric acid and lipid peroxidation levels of young subjects (23.1 ± 5.8 years old immediately before and 5 and 60 min after the exhaustive Wingate test. Results Maximum anaerobic power was improved by acute creatine supplementation (10.5 %, but it was accompanied by a 2.4-fold increase in pro-oxidant free iron ions in the plasma. However, potential iron-driven oxidative insult was adequately counterbalanced by proportional increases in antioxidant ferric-reducing activity in plasma (FRAP, leading to unaltered lipid peroxidation levels. Interestingly, the FRAP index, found to be highly dependent on uric acid levels in the placebo group, also had an additional contribution from other circulating metabolites in creatine-fed subjects. Conclusions Our data suggest that acute creatine supplementation improved the anaerobic performance of athletes and limited short-term oxidative insults, since creatine-induced iron overload was efficiently circumvented by acquired FRAP capacity attributed to: overproduction of uric acid in energy-depleted muscles (as an end-product of purine metabolism and a powerful iron chelating agent and inherent antioxidant activity of creatine.

Theory favors a stepwise mechanism of porphyrin degradation by a ferric hydroperoxide model of the active species of heme oxygenase.

Science.gov (United States)

Kumar, Devesh; de Visser, Samuël P; Shaik, Sason

2005-06-08

The report uses density functional theory to address the mechanism of heme degradation by the enzyme heme oxygenase (HO) using a model ferric hydroperoxide complex. HO is known to trap heme molecules and degrade them to maintain iron homeostasis in the biosystem. The degradation is initiated by complexation of the heme, then formation of the iron-hydroperoxo species, which subsequently oxidizes the meso position of the porphyrin by hydroxylation, thereby enabling eventually the cleavage of the porphyrin ring. Kinetic isotope effect studies indicate that the mechanism is assisted by general acid catalysis, via a chain of water molecules, and that all the events occur in concert. However, previous theoretical treatments indicated that the concerted mechanism has a high barrier, much higher than an alternative mechanism that is initiated by O-O bond homolysis of iron-hydroperoxide. The present contribution studies the stepwise and concerted acid-catalyzed mechanisms using H(3)O(+)(H(2)O)(n)(), n = 0-2. The effect of the acid strength is tested using the H(4)N(+)(H(2)O)(2) cluster and a fully protonated ferric hydroperoxide. All the calculations show that a stepwise mechanism that involves proton relay and O-O homolysis, in the rate-determining step, has a much lower barrier (>10 kcal/mol) than the corresponding fully concerted mechanism. The best fit of the calculated solvent kinetic isotope effect, to the experimental data, is obtained for the H(3)O(+)(H(2)O)(2) cluster. The calculated alpha-deuterium secondary kinetic isotope effect is inverse (0.95-0.98), but much less so than the experimental value (0.7). Possible reasons for this quantitative difference are discussed. Some probes are suggested that may enable experiment to distinguish the stepwise from the concerted mechanism.

Histidine at Position 195 is Essential for Association of Heme-b in Lcp1VH2

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Oetermann, Sylvia; Vivod, Robin; Hiessl, Sebastian; Hogeback, Jens; Holtkamp, Michael; Karst, Uwe; Steinbüchel, Alexander

2018-03-01

The latex clearing protein (Lcp) is the key enzyme of polyisoprene degradation in actinomycetes (Yikmis and Steinbüchel in Appl Environ Microbiol 78:4543-4551, https://doi.org/10.1128/AEM.00001-12, 2012). In this study it was shown that Lcp from Gordonia polyisoprenivorans VH2 (Lcp1VH2) harbors a non-covalently bound heme b as cofactor, which was identified by pyridine hemochrome spectra and confirmed by LC/ESI-ToF-MS. It contains iron, most likely in the Fe3+ state. We focused on the characterization of the heme-cofactor, its accessibility with respect to the conformation of Lcp1VH2, and the identification of putative histidine residues involved in the coordination of heme. A change was detectable in UV/Vis-spectra of reduced Lcp1VH2 when imidazole was added, showing that Lcp1VH2 "as isolated" occurs in an open state, directly being accessible for external ligands. In addition, three highly conserved histidines (H195, H200 and H228), presumably acting as ligands coordinating the heme within the heme pocket, were replaced with alanines by site-directed mutagenesis. The effect of these changes on in vivo rubber-mineralization was investigated. The lcp- deletion mutant complemented with the H195A variant of lcp1 VH2 was unable to mineralize poly(cis-1,4-isoprene). In vitro analyses of purified, recombinant Lcp1VH2H195A confirmed the loss of enzyme activity, which could be ascribed to the loss of heme. Hence, H195 is essential for the association of heme-b in the central region of Lcp1VH2.

Histidine at Position 195 is Essential for Association of Heme- b in Lcp1VH2

Science.gov (United States)

Oetermann, Sylvia; Vivod, Robin; Hiessl, Sebastian; Hogeback, Jens; Holtkamp, Michael; Karst, Uwe; Steinbüchel, Alexander

2018-05-01

The latex clearing protein (Lcp) is the key enzyme of polyisoprene degradation in actinomycetes (Yikmis and Steinbüchel in Appl Environ Microbiol 78:4543-4551, https://doi.org/10.1128/AEM.00001-12 , 2012). In this study it was shown that Lcp from Gordonia polyisoprenivorans VH2 (Lcp1VH2) harbors a non-covalently bound heme b as cofactor, which was identified by pyridine hemochrome spectra and confirmed by LC/ESI-ToF-MS. It contains iron, most likely in the Fe3+ state. We focused on the characterization of the heme-cofactor, its accessibility with respect to the conformation of Lcp1VH2, and the identification of putative histidine residues involved in the coordination of heme. A change was detectable in UV/Vis-spectra of reduced Lcp1VH2 when imidazole was added, showing that Lcp1VH2 "as isolated" occurs in an open state, directly being accessible for external ligands. In addition, three highly conserved histidines (H195, H200 and H228), presumably acting as ligands coordinating the heme within the heme pocket, were replaced with alanines by site-directed mutagenesis. The effect of these changes on in vivo rubber-mineralization was investigated. The lcp- deletion mutant complemented with the H195A variant of lcp1 VH2 was unable to mineralize poly( cis-1,4-isoprene). In vitro analyses of purified, recombinant Lcp1VH2H195A confirmed the loss of enzyme activity, which could be ascribed to the loss of heme. Hence, H195 is essential for the association of heme- b in the central region of Lcp1VH2.

The influence of high iron diet on rat lung manganese absorption

International Nuclear Information System (INIS)

Thompson, Khristy; Molina, Ramon; Donaghey, Thomas; Brain, Joseph D.; Wessling-Resnick, Marianne

2006-01-01

Individuals chronically exposed to manganese are at high risk for neurotoxic effects of this metal. A primary route of exposure is through respiration, although little is known about pulmonary uptake of metals or factors that modify this process. High dietary iron levels inversely affect intestinal uptake of manganese, and a major goal of this study was to determine if dietary iron loading could increase lung non-heme iron levels and alter manganese absorption. Rats were fed a high iron (1% carbonyl iron) or control diet for 4 weeks. Lung non-heme iron levels increased ∼2-fold in rats fed the high iron diet. To determine if iron-loading affected manganese uptake, 54 Mn was administered by intratracheal (it) instillation or intravenous (iv) injection for pharmacokinetic studies. 54 Mn absorption from the lungs to the blood was lower in it-instilled rats fed the 1% carbonyl iron diet. Pharmacokinetics of iv-injected 54 Mn revealed that the isotope was cleared more rapidly from the blood of iron-loaded rats. In situ analysis of divalent metal transporter-1 (DMT1) expression in lung detected mRNA in airway epithelium and bronchus-associated lymphatic tissue (BALT). Staining of the latter was significantly reduced in rats fed the high iron diet. In situ analysis of transferrin receptor (TfR) mRNA showed staining in BALT alone. These data demonstrate that manganese absorption from the lungs to the blood can be modified by iron status and the route of administration

Mitochondrial Iron Transport and Homeostasis in Plants

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Anshika eJain

2013-09-01

Full Text Available Iron (Fe is an essential nutrient for plants and although the mechanisms controlling iron uptake from the soil are relatively well understood, comparatively little is known about subcellular trafficking of iron in plant cells. Mitochondria represent a significant iron sink within cells, as iron is required for the proper functioning of respiratory chain protein complexes. Mitochondria are a site of Fe-S cluster synthesis, and possibly heme synthesis as well. Here we review recent insights into the molecular mechanisms controlling mitochondrial iron transport and homeostasis. We focus on the recent identification of a mitochondrial iron uptake transporter in rice and a possible role for metalloreductases in iron uptake by mitochondria. In addition, we highlight recent advances in mitochondrial iron homeostasis with an emphasis on the roles of frataxin and ferritin in iron trafficking and storage within mitochondria.

Moessbauer spectroscopic evidence on the heme binding to the proximal histidine in unfolded carbonmonoxy myoglobin by guanidine hydrochloride

Energy Technology Data Exchange (ETDEWEB)

Harami, Taikan, E-mail: harami.taikan@jaea.go.jp [Japan Atomic Energy Agency (Japan); Kitao, Shinji; Kobayashi, Yasuhiro [Kyoto University, Research Reactor Institute (Japan); Mitsui, Takaya [Japan Atomic Energy Agency (Japan)

2008-01-15

The unfolded heme structure in myoglobin is controversial because of no chance of direct X-ray structure analyses. The unfolding of carbonmonoxy myoglobin (MbCO) by guanidine hydrochloride (GdnHCl) was studied by the Moessbauer spectroscopy. The spectra show the presence of a sort of spectrum in the unfolded MbCO, independent on the concentration of GdnHCl from 1 to 6 M and the increase of the fraction of unfolded MbCO, depending on the GdnHCl concentration. The isomer shift of the iron of heme in the unfolded MbCO was identified to be different from that of the native MbCO as the globin structure in Mb collapses under the unfolded conditions. This result and the existing related Moessbauer data proved that the heme in the unfolded MbCO may remain coordinated to the proximal histidine.

Conversion of a heme-based oxygen sensor to a heme oxygenase by hydrogen sulfide: effects of mutations in the heme distal side of a heme-based oxygen sensor phosphodiesterase (Ec DOS)

Czech Academy of Sciences Publication Activity Database

Du, Y.; Liu, G.; Yan, Y.; Huang, D.; Luo, W.; Martínková, M.; Man, Petr; Shimizu, T.

2013-01-01

Roč. 26, č. 5 (2013), s. 839-852 ISSN 0966-0844 Institutional support: RVO:61388971 Keywords : Heme oxygenase * Heme protein * Hydrogen sulfide Subject RIV: CE - Biochemistry Impact factor: 2.689, year: 2013

LipidPioneer : A Comprehensive User-Generated Exact Mass Template for Lipidomics

Science.gov (United States)

Ulmer, Candice Z.; Koelmel, Jeremy P.; Ragland, Jared M.; Garrett, Timothy J.; Bowden, John A.

2017-03-01

Lipidomics, the comprehensive measurement of lipid species in a biological system, has promising potential in biomarker discovery and disease etiology elucidation. Advances in chromatographic separation, mass spectrometric techniques, and novel substrate applications continue to expand the number of lipid species observed. The total number and type of lipid species detected in a given sample are generally indicative of the sample matrix examined (e.g., serum, plasma, cells, bacteria, tissue, etc.). Current exact mass lipid libraries are static and represent the most commonly analyzed matrices. It is common practice for users to manually curate their own lists of lipid species and adduct masses; however, this process is time-consuming. LipidPioneer, an interactive template, can be used to generate exact masses and molecular formulas of lipid species that may be encountered in the mass spectrometric analysis of lipid profiles. Over 60 lipid classes are present in the LipidPioneer template and include several unique lipid species, such as ether-linked lipids and lipid oxidation products. In the template, users can add any fatty acyl constituents without limitation in the number of carbons or degrees of unsaturation. LipidPioneer accepts naming using the lipid class level (sum composition) and the LIPID MAPS notation for fatty acyl structure level. In addition to lipid identification, user-generated lipid m/z values can be used to develop inclusion lists for targeted fragmentation experiments. Resulting lipid names and m/z values can be imported into software such as MZmine or Compound Discoverer to automate exact mass searching and isotopic pattern matching across experimental data.

Molecular evolution of multiple-level control of heme biosynthesis pathway in animal kingdom.

Science.gov (United States)

Tzou, Wen-Shyong; Chu, Ying; Lin, Tzung-Yi; Hu, Chin-Hwa; Pai, Tun-Wen; Liu, Hsin-Fu; Lin, Han-Jia; Cases, Ildeofonso; Rojas, Ana; Sanchez, Mayka; You, Zong-Ye; Hsu, Ming-Wei

2014-01-01

Adaptation of enzymes in a metabolic pathway can occur not only through changes in amino acid sequences but also through variations in transcriptional activation, mRNA splicing and mRNA translation. The heme biosynthesis pathway, a linear pathway comprised of eight consecutive enzymes in animals, provides researchers with ample information for multiple types of evolutionary analyses performed with respect to the position of each enzyme in the pathway. Through bioinformatics analysis, we found that the protein-coding sequences of all enzymes in this pathway are under strong purifying selection, from cnidarians to mammals. However, loose evolutionary constraints are observed for enzymes in which self-catalysis occurs. Through comparative genomics, we found that in animals, the first intron of the enzyme-encoding genes has been co-opted for transcriptional activation of the genes in this pathway. Organisms sense the cellular content of iron, and through iron-responsive elements in the 5' untranslated regions of mRNAs and the intron-exon boundary regions of pathway genes, translational inhibition and exon choice in enzymes may be enabled, respectively. Pathway product (heme)-mediated negative feedback control can affect the transport of pathway enzymes into the mitochondria as well as the ubiquitin-mediated stability of enzymes. Remarkably, the positions of these controls on pathway activity are not ubiquitous but are biased towards the enzymes in the upstream portion of the pathway. We revealed that multiple-level controls on the activity of the heme biosynthesis pathway depend on the linear depth of the enzymes in the pathway, indicating a new strategy for discovering the molecular constraints that shape the evolution of a metabolic pathway.

Texture of lipid bilayer domains

DEFF Research Database (Denmark)

Jensen, Uffe Bernchou; Brewer, Jonathan R.; Midtiby, Henrik Skov

2009-01-01

We investigate the texture of gel (g) domains in binary lipid membranes composed of the phospholipids DPPC and DOPC. Lateral organization of lipid bilayer membranes is a topic of fundamental and biological importance. Whereas questions related to size and composition of fluid membrane domain...... are well studied, the possibility of texture in gel domains has so far not been examined. When using polarized light for two-photon excitation of the fluorescent lipid probe Laurdan, the emission intensity is highly sensitive to the angle between the polarization and the tilt orientation of lipid acyl...... chains. By imaging the intensity variations as a function of the polarization angle, we map the lateral variations of the lipid tilt within domains. Results reveal that gel domains are composed of subdomains with different lipid tilt directions. We have applied a Fourier decomposition method...

Lipid contents of the sponge Haliclona sp.

Digital Repository Service at National Institute of Oceanography (India)

Parameswaran, P.S.; Das, B.; Kamat, S.Y.

Several fatty acids, sterols, batyl alcohol and its analogs and an N-acylated sphingosine (ceramide) have been isolated from the lipid fraction of the extract of the sponge Haliclona sp. The major sterol is found to be cholesterol (54%), followed...

On the molecular basis of the activity of the antimalarial drug chloroquine: EXAFS-assisted DFT evidence of a direct Fe–N bond with free heme in solution

International Nuclear Information System (INIS)

Macetti, Giovanni; Rizzato, Silvia; Beghi, Fabio; Presti, Leonardo Lo; Silvestrini, Lucia

2016-01-01

4-aminoquinoline antiplasmodials interfere with the biocrystallization of the malaria pigment, a key step of the malaria parasite metabolism. It is commonly believed that these drugs set stacking π···π interactions with the Fe-protoporphyrin scaffold of the free heme, even though the details of the heme:drug recognition process remain elusive. In this work, the local coordination of Fe(III) ions in acidic solutions of hematin at room temperature was investigated by extended x-ray absorption fine structure (EXAFS) spectroscopy in the 4.0–5.5 pH range, both in the presence and in the absence of the antimalarial drug chloroquine. EXAFS results were complemented by DFT simulations in polarizable continuum media to model solvent effects. We found evidence that a complex where the drug quinoline nitrogen is coordinated with the iron center might coexist with formerly proposed adduct geometries, based on stacking interactions. Charge-assisted hydrogen bonds among lateral chains of the two molecules play a crucial role in stabilizing this complex, whose formation is favored by the presence of lipid micelles. The direct Fe–N bond could reversibly block the axial position in the Fe 1st coordination shell in free heme, acting as an inhibitor for the crystallization of the malaria pigment without permanently hampering the catalytic activity of the redox center. These findings are discussed in the light of possible implications on the engineering of drugs able to thwart the adaptability of the malaria parasite against classical aminoquinoline-based therapies. (invited comment)

On the molecular basis of the activity of the antimalarial drug chloroquine: EXAFS-assisted DFT evidence of a direct Fe-N bond with free heme in solution

Science.gov (United States)

Macetti, Giovanni; Rizzato, Silvia; Beghi, Fabio; Silvestrini, Lucia; Lo Presti, Leonardo

2016-02-01

4-aminoquinoline antiplasmodials interfere with the biocrystallization of the malaria pigment, a key step of the malaria parasite metabolism. It is commonly believed that these drugs set stacking π···π interactions with the Fe-protoporphyrin scaffold of the free heme, even though the details of the heme:drug recognition process remain elusive. In this work, the local coordination of Fe(III) ions in acidic solutions of hematin at room temperature was investigated by extended x-ray absorption fine structure (EXAFS) spectroscopy in the 4.0-5.5 pH range, both in the presence and in the absence of the antimalarial drug chloroquine. EXAFS results were complemented by DFT simulations in polarizable continuum media to model solvent effects. We found evidence that a complex where the drug quinoline nitrogen is coordinated with the iron center might coexist with formerly proposed adduct geometries, based on stacking interactions. Charge-assisted hydrogen bonds among lateral chains of the two molecules play a crucial role in stabilizing this complex, whose formation is favored by the presence of lipid micelles. The direct Fe-N bond could reversibly block the axial position in the Fe 1st coordination shell in free heme, acting as an inhibitor for the crystallization of the malaria pigment without permanently hampering the catalytic activity of the redox center. These findings are discussed in the light of possible implications on the engineering of drugs able to thwart the adaptability of the malaria parasite against classical aminoquinoline-based therapies.

Fungal Morphology, Iron Homeostasis, and Lipid Metabolism Regulated by a GATA Transcription Factor in Blastomyces dermatitidis.

Directory of Open Access Journals (Sweden)

Amber J Marty

2015-06-01

Full Text Available In response to temperature, Blastomyces dermatitidis converts between yeast and mold forms. Knowledge of the mechanism(s underlying this response to temperature remains limited. In B. dermatitidis, we identified a GATA transcription factor, SREB, important for the transition to mold. Null mutants (SREBΔ fail to fully complete the conversion to mold and cannot properly regulate siderophore biosynthesis. To capture the transcriptional response regulated by SREB early in the phase transition (0-48 hours, gene expression microarrays were used to compare SREB∆ to an isogenic wild type isolate. Analysis of the time course microarray data demonstrated SREB functioned as a transcriptional regulator at 37°C and 22°C. Bioinformatic and biochemical analyses indicated SREB was involved in diverse biological processes including iron homeostasis, biosynthesis of triacylglycerol and ergosterol, and lipid droplet formation. Integration of microarray data, bioinformatics, and chromatin immunoprecipitation identified a subset of genes directly bound and regulated by SREB in vivo in yeast (37°C and during the phase transition to mold (22°C. This included genes involved with siderophore biosynthesis and uptake, iron homeostasis, and genes unrelated to iron assimilation. Functional analysis suggested that lipid droplets were actively metabolized during the phase transition and lipid metabolism may contribute to filamentous growth at 22°C. Chromatin immunoprecipitation, RNA interference, and overexpression analyses suggested that SREB was in a negative regulatory circuit with the bZIP transcription factor encoded by HAPX. Both SREB and HAPX affected morphogenesis at 22°C; however, large changes in transcript abundance by gene deletion for SREB or strong overexpression for HAPX were required to alter the phase transition.

HemeBIND: a novel method for heme binding residue prediction by combining structural and sequence information

Directory of Open Access Journals (Sweden)

Hu Jianjun

2011-05-01

Full Text Available Abstract Background Accurate prediction of binding residues involved in the interactions between proteins and small ligands is one of the major challenges in structural bioinformatics. Heme is an essential and commonly used ligand that plays critical roles in electron transfer, catalysis, signal transduction and gene expression. Although much effort has been devoted to the development of various generic algorithms for ligand binding site prediction over the last decade, no algorithm has been specifically designed to complement experimental techniques for identification of heme binding residues. Consequently, an urgent need is to develop a computational method for recognizing these important residues. Results Here we introduced an efficient algorithm HemeBIND for predicting heme binding residues by integrating structural and sequence information. We systematically investigated the characteristics of binding interfaces based on a non-redundant dataset of heme-protein complexes. It was found that several sequence and structural attributes such as evolutionary conservation, solvent accessibility, depth and protrusion clearly illustrate the differences between heme binding and non-binding residues. These features can then be separately used or combined to build the structure-based classifiers using support vector machine (SVM. The results showed that the information contained in these features is largely complementary and their combination achieved the best performance. To further improve the performance, an attempt has been made to develop a post-processing procedure to reduce the number of false positives. In addition, we built a sequence-based classifier based on SVM and sequence profile as an alternative when only sequence information can be used. Finally, we employed a voting method to combine the outputs of structure-based and sequence-based classifiers, which demonstrated remarkably better performance than the individual classifier alone

Inflammation and ER Stress Downregulate BDH2 Expression and Dysregulate Intracellular Iron in Macrophages

Directory of Open Access Journals (Sweden)

Susu M. Zughaier

2014-01-01

Full Text Available Macrophages play a very important role in host defense and in iron homeostasis by engulfing senescent red blood cells and recycling iron. Hepcidin is the master iron regulating hormone that limits dietary iron absorption from the gut and limits iron egress from macrophages. Upon infection macrophages retain iron to limit its bioavailability which limits bacterial growth. Recently, a short chain butyrate dehydrogenase type 2 (BDH2 protein was reported to contain an iron responsive element and to mediate cellular iron trafficking by catalyzing the synthesis of the mammalian siderophore that binds labile iron; therefore, BDH2 plays a crucial role in intracellular iron homeostasis. However, BDH2 expression and regulation in macrophages have not yet been described. Here we show that LPS-induced inflammation combined with ER stress led to massive BDH2 downregulation, increased the expression of ER stress markers, upregulated hepcidin expression, downregulated ferroportin expression, caused iron retention in macrophages, and dysregulated cytokine release from macrophages. We also show that ER stress combined with inflammation synergistically upregulated the expression of the iron carrier protein NGAL and the stress-inducible heme degrading enzyme heme oxygenase-1 (HO-1 leading to iron liberation. This is the first report to show that inflammation and ER stress downregulate the expression of BDH2 in human THP-1 macrophages.

Evolution of the acyl-CoA binding protein (ACBP)

DEFF Research Database (Denmark)

Burton, Mark; Rose, Timothy M; Faergeman, Nils J

2005-01-01

-CoA pool size, donation of acyl-CoA esters for beta-oxidation, vesicular trafficking, complex lipid synthesis and gene regulation. In the present study, we delineate the evolutionary history of ACBP to get a complete picture of its evolution and distribution among species. ACBP homologues were identified...... duplication and/or retrotransposition events. The ACBP protein is highly conserved across phylums, and the majority of ACBP genes are subjected to strong purifying selection. Experimental evidence indicates that the function of ACBP has been conserved from yeast to humans and that the multiple lineage...

Studies on the mechanism of pyrophosphate-mediated uptake of iron from transferrin by isolated rat-liver mitochondria

International Nuclear Information System (INIS)

Konopka, K.; Romslo, I.; Bergen Univ.

1981-01-01

1. Respiring rat liver mitochondria accumulate iron released from transferrin by pyrophosphate. The amount of iron accumulated is 1-1.5 nmol mg protein -1 h -1 , or approximately 60% of the amount of iron mobilized from transferrin. 2. The uptake declines if respiration is inhibited, substrate is depleted, or the experiments are run under anaerobic conditions. Substrate, depletion and respiratory inhibitors are less inhibitory under anaerobic conditions. 3. More than 80% of the amount of iron accumulated by aerobic, actively respiring mitochondria can be chelated by bathophenanthroline sulphonate, and with deuteroporphyrin included, up to 30% of the amount of iron accumulated is recovered as deuteroheme. Iron accumulated by respiration-inhibited mitochondria under aerobic conditions is not available for heme synthesis. 4. With time the uptake of iron increases eightfold relative to the uptake of pyrophosphate. 5. The results are compatible with a model in which ferric iron is mobilized from transferrin by pyrophosphate, ferric iron pyrophosphate is bound to the mitochondria, iron is reduced, dissociates from pyrophosphate and is taken up by the mitochondria. Ferrous irons thus formed is available for heme synthesis. (orig.) [de

IRON AND FREE RADICAL OXIDATIONS IN CELL MEMBRANES

Science.gov (United States)

Schafer, Freya Q.; Yue Qian, Steven; Buettner, Garry R.

2013-01-01

Brain tissue being rich in polyunsaturated fatty acids, is very susceptible to lipid peroxidation. Iron is well known to be an important initiator of free radical oxidations. We propose that the principal route to iron-mediated lipid peroxidations is via iron-oxygen complexes rather than the reaction of iron with hydrogen peroxide, the Fenton reaction. To test this hypothesis, we enriched leukemia cells (K-562 and L1210 cells) with docosahexaenoic acid (DHA) as a model for brain tissue, increasing the amount of DHA from approximately 3 mole % to 32 mole %. These cells were then subjected to ferrous iron and dioxygen to initiate lipid peroxidation in the presence or absence of hydrogen peroxide. Lipid-derived radicals were detected using EPR spin trapping with α-(4-pyridyl-1-oxide)-N-t-butylnitrone (POBN). As expected, lipid-derived radical formation increases with increasing cellular lipid unsaturation. Experiments with Desferal demonstrate that iron is required for the formation of lipid radicals from these cells. Addition of iron to DHA-enriched L1210 cells resulted in significant amounts of radical formation; radical formation increased with increasing amount of iron. However, the exposure of cells to hydrogen peroxide before the addition of ferrous iron did not increase cellular radical formation, but actually decreased spin adduct formation. These data suggest that iron-oxygen complexes are the primary route to the initiation of biological free radical oxidations. This model proposes a mechanism to explain how catalytic iron in brain tissue can be so destructive. PMID:10872752

Localization of acyl ghrelin- and des-acyl ghrelin-immunoreactive cells in the rat stomach and their responses to intragastric pH.

Science.gov (United States)

Mizutani, Makoto; Atsuchi, Kaori; Asakawa, Akihiro; Matsuda, Norifumi; Fujimura, Masaki; Inui, Akio; Kato, Ikuo; Fujimiya, Mineko

2009-11-01

Acyl ghrelin has a 28-amino acid sequence with O-n-octanoyl acid modification at the serine 3 position, whereas des-acyl ghrelin has no octanoyl acid modification. Although these peptides exert different physiological functions, no previous studies have shown the different localization of acyl ghrelin and des-acyl ghrelin in the stomach. Here we have developed an antibody specific for des-acyl ghrelin that does not crossreact with acyl ghrelin. Both acyl ghrelin- and des-acyl ghrelin-immunoreactive cells were distributed in the oxyntic and antral mucosa of the rat stomach, with higher density in the antral mucosa than oxyntic mucosa. Immunofluorescence double staining showed that acyl ghrelin- and des-acyl ghrelin-positive reactions overlapped in closed-type round cells, whereas des-acyl ghrelin-positive reaction was found in open-type cells in which acyl ghrelin was negative. Acyl ghrelin-/des-acyl ghrelin-positive closed-type cells contain obestatin; on the other hand, des-acyl ghrelin-positive open-type cells contain somatostatin. We measured the release of acyl ghrelin and des-acyl ghrelin in vascularly perfused rat stomach by ELISA, and the effects of different intragastric pH levels on the release of each peptide were examined. The release of des-acyl ghrelin from the perfused stomach was greater at pH 2 than at pH 4; however, the release of acyl ghrelin was not affected by intragastric pH. The present study demonstrated the differential localization of acyl ghrelin and des-acyl ghrelin in the rat stomach and their different responses to the intragastric pH.

Structural characterization of human heme oxygenase-1 in complex with azole-based inhibitors.

Science.gov (United States)

Rahman, Mona N; Vlahakis, Jason Z; Roman, Gheorghe; Vukomanovic, Dragic; Szarek, Walter A; Nakatsu, Kanji; Jia, Zongchao

2010-03-01

The development of inhibitors specific for heme oxygenases (HO) aims to provide powerful tools in understanding the HO system. Based on the lead structure (2S, 4S)-2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-4-[((4-aminophenyl)thio)methyl]-1,3-dioxolane (azalanstat, QC-1) we have synthesized structural modifications to develop novel and selective HO inhibitors. The structural study of human HO-1 (hHO-1) in complex with a select group of the inhibitors was initiated using X-ray crystallographic techniques. Comparison of the structures of four such compounds each in complex with hHO-1 revealed a common binding mode, despite having different structural fragments. The compounds bind to the distal side of heme through an azole "anchor" which coordinates with the heme iron. An expansion of the distal pocket, mainly due to distal helix flexibility, allows accommodation of the compounds without displacing heme or the critical Asp140 residue. Rather, binding displaces a catalytically critical water molecule and disrupts an ordered hydrogen-bond network involving Asp140. The presence of a triazole "anchor" may provide further stability via a hydrogen bond with the protein. A hydrophobic pocket acts to stabilize the region occupied by the phenyl or adamantanyl moieties of these compounds. Further, a secondary hydrophobic pocket is formed via "induced fit" to accommodate bulky substituents at the 4-position of the dioxolane ring. Copyright 2009 Elsevier Inc. All rights reserved.

Quantitative lipidomics reveals age-dependent perturbations of whole-body lipid metabolism in ACBP deficient mice

DEFF Research Database (Denmark)

Gallego, Sandra F; Sprenger, Richard R; Neess, Ditte

2017-01-01

The acyl-CoA binding protein (ACBP) plays a key role in chaperoning long-chain acyl-CoAs into lipid metabolic processes and acts as an important regulatory hub in mammalian physiology. This is highlighted by the recent finding that mice devoid of ACBP suffer from a compromised epidermal barrier a...

Characterization of the structure and immunostimulatory activity of a vaccine adjuvant, de-O-acylated lipooligosaccharide.

Directory of Open Access Journals (Sweden)

Ji Eun Han

Full Text Available Lipopolysaccharide (LPS is a major component of the outer membrane of Gram-negative bacteria. LPS elicits strong immunopathological responses during bacterial infection, and the lipid A moiety of LPS is responsible for this immunostimulatory activity. Lipid A exerts its biological activity by sending signals via TLR4 present on immune cells, and TLR4 agonists have been a target for vaccine adjuvant. Previously, we demonstrated an adjuvant activity of deacylated lipooligosaccharide (dLOS to viral and bacterial antigens. In this study, we characterized the chemical structure of dLOS and evaluated its immunostimulatory activity on mouse and human immune cells in comparison with monophosphoryl lipid A (MPL. dLOS consists of the R3-type core, a glucosamine disaccharide with two phosphate groups, and two N-linked acyl groups [corrected], and two N-linked acyl groups. dLOS was similar to MPL in induction of cytokine production in mouse peritoneal macrophages, but was a more potent activator in human monocytes and dendritic cells (DCs. Results of an analysis of allogeneic T cell responses revealed that dLOS induces Th1, Th2, and Th17-type immune responses in a dose-dependent manner. The immunostimulatory activities of dLOS were completely abrogated in TLR4(-/- mice, which confirms its TLR4-dependency. These results suggest that in the presence of the core oligosaccharide, O-linked acyl groups of LPS are dispensable for activating the TLR4 signaling pathway. dLOS did not cause any pathological effects or death at 0.25, 0.5, or 1 mg per kg body weight in mice in the acute toxicity tests. This result suggests that dLOS has a low toxicity. dLOS should be considered for further development as a safe and effective adjuvant for human vaccines.

Impact of heme oxygenase-1 on cholesterol synthesis, cholesterol efflux and oxysterol formation in cultured astroglia.

Science.gov (United States)

Hascalovici, Jacob R; Song, Wei; Vaya, Jacob; Khatib, Soliman; Fuhrman, Bianca; Aviram, Michael; Schipper, Hyman M

2009-01-01

Up-regulation of heme oxygenase-1 (HO-1) and altered cholesterol (CH) metabolism are characteristic of Alzheimer-diseased neural tissues. The liver X receptor (LXR) is a molecular sensor of CH homeostasis. In the current study, we determined the effects of HO-1 over-expression and its byproducts iron (Fe(2+)), carbon monoxide (CO) and bilirubin on CH biosynthesis, CH efflux and oxysterol formation in cultured astroglia. HO-1/LXR interactions were also investigated in the context of CH efflux. hHO-1 over-expression for 3 days ( approximately 2-3-fold increase) resulted in a 30% increase in CH biosynthesis and a two-fold rise in CH efflux. Both effects were abrogated by the competitive HO inhibitor, tin mesoporphyrin. CO, released from administered CORM-3, significantly enhanced CH biosynthesis; a combination of CO and iron stimulated CH efflux. Free iron increased oxysterol formation three-fold. Co-treatment with LXR antagonists implicated LXR activation in the modulation of CH homeostasis by heme degradation products. In Alzheimer's disease and other neuropathological states, glial HO-1 induction may transduce ambient noxious stimuli (e.g. beta-amyloid) into altered patterns of glial CH homeostasis. As the latter may impact synaptic plasticity and neuronal repair, modulation of glial HO-1 expression (by pharmacological or other means) may confer neuroprotection in patients with degenerative brain disorders.

Measurement of Heme Synthesis Levels in Mammalian Cells.

Science.gov (United States)

Hooda, Jagmohan; Alam, Maksudul; Zhang, Li

2015-07-09

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-(14)C] 5-aminolevulinic acid ([(14)C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [(14)C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Prokaryotic Heme Biosynthesis: Multiple Pathways to a Common Essential Product.

Science.gov (United States)

Dailey, Harry A; Dailey, Tamara A; Gerdes, Svetlana; Jahn, Dieter; Jahn, Martina; O'Brian, Mark R; Warren, Martin J

2017-03-01

The advent of heme during evolution allowed organisms possessing this compound to safely and efficiently carry out a variety of chemical reactions that otherwise were difficult or impossible. While it was long assumed that a single heme biosynthetic pathway existed in nature, over the past decade, it has become clear that there are three distinct pathways among prokaryotes, although all three pathways utilize a common initial core of three enzymes to produce the intermediate uroporphyrinogen III. The most ancient pathway and the only one found in the Archaea converts siroheme to protoheme via an oxygen-independent four-enzyme-step process. Bacteria utilize the initial core pathway but then add one additional common step to produce coproporphyrinogen III. Following this step, Gram-positive organisms oxidize coproporphyrinogen III to coproporphyrin III, insert iron to make coproheme, and finally decarboxylate coproheme to protoheme, whereas Gram-negative bacteria first decarboxylate coproporphyrinogen III to protoporphyrinogen IX and then oxidize this to protoporphyrin IX prior to metal insertion to make protoheme. In order to adapt to oxygen-deficient conditions, two steps in the bacterial pathways have multiple forms to accommodate oxidative reactions in an anaerobic environment. The regulation of these pathways reflects the diversity of bacterial metabolism. This diversity, along with the late recognition that three pathways exist, has significantly slowed advances in this field such that no single organism's heme synthesis pathway regulation is currently completely characterized. Copyright © 2017 American Society for Microbiology.

Theory of phase equilibria and critical mixing points in binary lipid bilayers

DEFF Research Database (Denmark)

Risbo, Jens; Sperotto, Maria Maddalena; Mouritsen, Ole G.

1995-01-01

the transition is discussed in terms of the molecular properties of the lipid acyl chains. The results of the numerical model study are expected to have consequences for the interpretation of experimental measurements on lipid bilayer systems in terms of phase diagrams. (C) 1995 American Institute of Physics....

Insights on Heme Synthesis in the Malaria Parasite.

Science.gov (United States)

Nagaraj, Viswanathan A; Padmanaban, Govindarajan

2017-08-01

The malaria parasite has a functional heme-biosynthetic pathway, although it can access host hemoglobin-heme. The heme pathway is dispensable for blood stages, but essential in the mosquito stages which do not acquire hemoglobin-heme. We propose that the blood stage parasites maintain a dynamic heme pool through multiple back-up mechanisms. Copyright © 2017 Elsevier Ltd. All rights reserved.

Heme orientational disorder in human adult hemoglobin reconstituted with a ring fluorinated heme and its functional consequences

International Nuclear Information System (INIS)

Nagao, Satoshi; Hirai, Yueki; Kawano, Shin; Imai, Kiyohiro; Suzuki, Akihiro; Yamamoto, Yasuhiko

2007-01-01

A ring fluorinated heme, 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2-fluoro-7,12, 18-trimethyl-porphyrin-atoiron(III), has been incorporated into human adult hemoglobin (Hb A). The heme orientational disorder in the individual subunits of the protein has been readily characterized using 19 F NMR and the O 2 binding properties of the protein have been evaluated through the oxygen equilibrium analysis. The equilibrated orientations of hemes in α- and β- subunits of the reconstituted protein were found to be almost completely opposite to each other, and hence were largely different from those of the native and the previously reported reconstituted proteins [T. Jue, G.N. La Mar, Heme orientational heterogeneity in deuterohemin-reconstituted horse and human hemoglobin characterized by proton nuclear magnetic resonance spectroscopy, Biochem. Biophys. Res. Commun. 119 (1984) 640-645]. Despite the large difference in the degree of the heme orientational disorder in the subunits of the proteins, the O 2 affinity and the cooperativity of the protein reconstituted with 2-MF were similar to those of the proteins reconstituted with a series of hemes chemically modified at the heme 3- and 8-positions [K. Kawabe, K. Imaizumi, Z. Yoshida, K. Imai, I. Tyuma, Studies on reconstituted myoglobins and hemoglobins II. Role of the heme side chains in the oxygenation of hemoglobin, J. Biochem. 92 (1982) 1713-1722], whose O 2 affinity and cooperativity were higher and lower, respectively, relative to those of native protein. These results indicated that the heme orientational disorder could exert little effect, if any, on the O 2 affinity properties of Hb A. This finding provides new insights into structure-function relationship of Hb A

Isoporphyrin Intermediate in Heme Oxygenase Catalysis

Science.gov (United States)

Evans, John P.; Niemevz, Fernando; Buldain, Graciela; de Montellano, Paul Ortiz

2008-01-01

Human heme oxygenase-1 (hHO-1) catalyzes the O2- and NADPH-dependent oxidation of heme to biliverdin, CO, and free iron. The first step involves regiospecific insertion of an oxygen atom at the α-meso carbon by a ferric hydroperoxide and is predicted to proceed via an isoporphyrin π-cation intermediate. Here we report spectroscopic detection of a transient intermediate during oxidation by hHO-1 of α-meso-phenylheme-IX, α-meso-(p-methylphenyl)-mesoheme-III, and α-meso-(p-trifluoromethylphenyl)-mesoheme-III. In agreement with previous experiments (Wang, J., Niemevz, F., Lad, L., Huang, L., Alvarez, D. E., Buldain, G., Poulos, T. L., and Ortiz de Montellano, P. R. (2004) J. Biol. Chem. 279, 42593–42604), only the α-biliverdin isomer is produced with concomitant formation of the corresponding benzoic acid. The transient intermediate observed in the NADPH-P450 reductase-catalyzed reaction accumulated when the reaction was supported by H2O2 and exhibited the absorption maxima at 435 and 930 nm characteristic of an isoporphyrin. Product analysis by reversed phase high performance liquid chromatography and liquid chromatography electrospray ionization mass spectrometry of the product generated with H2O2 identified it as an isoporphyrin that, on quenching, decayed to benzoylbiliverdin. In the presence of H218O2, one labeled oxygen atom was incorporated into these products. The hHO-1-isoporphyrin complexes were found to have half-lives of 1.7 and 2.4 h for the p-trifluoromethyl- and p-methyl-substituted phenylhemes, respectively. The addition of NADPH-P450 reductase to the H2O2-generated hHO-1-isoporphyrin complex produced α-biliverdin, confirming its role as a reaction intermediate. Identification of an isoporphyrin intermediate in the catalytic sequence of hHO-1, the first such intermediate observed in hemoprotein catalysis, completes our understanding of the critical first step of heme oxidation. PMID:18487208

Considerations on the mechanism of action of artemisinin antimalarials: part 1--the 'carbon radical' and 'heme' hypotheses.

Science.gov (United States)

Haynes, Richard K; Cheu, Kwan-Wing; N'Da, David; Coghi, Paolo; Monti, Diego

2013-08-01

The isolation of artemisinin from the traditional medicinal herb qīng hāo (Artemisia annua), its characterization as a peroxide and preparation of the derivatives dihydroartemisinin, artemether and artesunate in the 1970s and 1980s by Chinese scientists under the umbrella of Project 523 collectively represents one of the great events in medicine in the latter third of the 20(th) Century. Artemisinins have become the most important component of chemotherapy of malaria: although used initially in monotherapy, they are now used in combination therapies or ACTs with longer half-life quinolines or arylmethanols. Nevertheless, the recent emergence of artemisinin-tolerant strains of the malaria parasite as reflected in increased clearance times of parasitaemia in patients treated with ACTs represents the greatest threat to control of malaria since resistance to chloroquine was first reported over 55 years ago. Importantly, the event brings into sharp focus the realization that relatively little is precisely understood, as opposed to widely assumed, for the mechanism of drug action of artemisinins and their synthetic peroxide analogues. Thus, we review here their antimalarial activities, the use of artemisinins in combination therapies, drug-drug interactions with the quinolines and arylmethanols, and metabolism of the artemisinins and synthetic peroxides. The mechanism of action of quinolines and arylmethanols, in particular their ability to induce redistribution of heme into the parasite cytosol, is also highlighted. This collective information is then used as a counterpoint to screen the validity of two of the prevailing hypotheses of drug action of artemisinins and synthetic peroxides, namely i. 'the C-radical hypothesis' wherein the peroxide undergoes 'bioactivation' by ferrous iron to generate C-radicals that are held to be the cytotoxic agents and ii. the 'heme hypothesis' wherein ferrous heme may generate either the same type of 'cytotoxic' C-radical, or the

Expression of Lipid Metabolism-Related Proteins Differs between Invasive Lobular Carcinoma and Invasive Ductal Carcinoma.

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Cha, Yoon Jin; Kim, Hye Min; Koo, Ja Seung

2017-01-23

We comparatively investigated the expression and clinical implications of lipid metabolism-related proteins in invasive lobular carcinoma (ILC) and invasive ductal carcinoma (IDC) of the breast. A total of 584 breast cancers (108 ILC and 476 IDC) were subjected to tissue microarray and immunohistochemical analysis for lipid metabolism-related proteins including hormone-sensitive lipase (HSL), perilipin A, fatty acid binding protein (FABP)4, carnitine palmitoyltransferase (CPT)-1, acyl-CoA oxidase 1, and fatty acid synthetase (FASN). HSL, perilipin A, and FABP4 expression (all p invasive cancers, HSL and FABP4 were highly expressed in luminal A-type ILC ( p cancers, HSL and FABP4 were more highly expressed in ILC ( p < 0.001). Univariate analysis found associations of shorter disease-free survival with CPT-1 positivity ( p = 0.004) and acyl-CoA oxidase 1 positivity ( p = 0.032) and of shorter overall survival with acyl-CoA oxidase 1 positivity ( p = 0.027). In conclusion, ILC and IDC exhibited different immunohistochemical lipid metabolism-related protein expression profiles. Notably, ILC exhibited high HSL and FABP4 and low perilipin A expression.

The Extracellular Heme-binding Protein HbpS from the Soil Bacterium Streptomyces reticuli Is an Aquo-cobalamin Binder*

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Ortiz de Orué Lucana, Darío; Fedosov, Sergey N.; Wedderhoff, Ina; Che, Edith N.; Torda, Andrew E.

2014-01-01

The extracellular protein HbpS from Streptomyces reticuli interacts with iron ions and heme. It also acts in concert with the two-component sensing system SenS-SenR in response to oxidative stress. Sequence comparisons suggested that the protein may bind a cobalamin. UV-visible spectroscopy confirmed binding (Kd = 34 μm) to aquo-cobalamin (H2OCbl+) but not to other cobalamins. Competition experiments with the H2OCbl+-coordinating ligand CN− and comparison of mutants identified a histidine residue (His-156) that coordinates the cobalt ion of H2OCbl+ and substitutes for water. HbpS·Cobalamin lacks the Asp-X-His-X-X-Gly motif seen in some cobalamin binding enzymes. Preliminary tests showed that a related HbpS protein from a different species also binds H2OCbl+. Furthermore, analyses of HbpS-heme binding kinetics are consistent with the role of HbpS as a heme-sensor and suggested a role in heme transport. Given the high occurrence of HbpS-like sequences among Gram-positive and Gram-negative bacteria, our findings suggest a great functional versatility among these proteins. PMID:25342754

Characterization of SiaA, a streptococcal heme-binding protein associated with a heme ABC transport system.

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Sook, Brian R; Block, Darci R; Sumithran, Suganya; Montañez, Griselle E; Rodgers, Kenton R; Dawson, John H; Eichenbaum, Zehava; Dixon, Dabney W

2008-02-26

Many pathogenic bacteria require heme and obtain it from their environment. Heme transverses the cytoplasmic membrane via an ATP binding cassette (ABC) pathway. Although a number of heme ABC transport systems have been described in pathogenic bacteria, there is as yet little biophysical characterization of the proteins in these systems. The sia (hts) gene cluster encodes a heme ABC transporter in the Gram positive Streptococcus pyogenes. The lipoprotein-anchored heme binding protein (HBP) of this transporter is SiaA (HtsA). In the current study, resonance Raman (rR), magnetic circular dichroism (MCD), and nuclear magnetic resonance (NMR) spectroscopies were used to determine the coordination state and spin state of both the ferric and ferrous forms of this protein. Identifiers from these techniques suggest that the heme is six-coordinate and low-spin in both oxidation states of the protein, with methionine and histidine as axial ligands. SiaA has a pKa of 9.7 +/- 0.1, attributed to deprotonation of the axial histidine. Guanidinium titration studies show that the ferric state is less stable than the ferrous state, with DeltaG(H2O) values for the oxidized and reduced proteins of 7.3 +/- 0.8 and 16.0 +/- 3.6 kcal mol-1, respectively. The reductive and oxidative midpoint potentials determined via spectroelectrochemistry are 83 +/- 3 and 64 +/- 3 mV, respectively; the irreversibility of heme reduction suggests that redox cycling of the heme is coupled to a kinetically sluggish change in structure or conformation. The biophysical characterization described herein will significantly advance our understanding of structure-function relationships in HBP.

Microsomal lipid peroxidation as a mechanism of cellular damage. [Dissertation

Energy Technology Data Exchange (ETDEWEB)

Kornbrust, D.J.

1979-01-01

The NADPH/iron-dependent peroxidation of lipids in rat liver microsomes was found to be dependent on the presence of free ferrous ion and maintains iron in the reduced Fe/sup 2 +/ state. Chelation of iron by EDTA inhibited peroxidation. Addition of iron, after preincubation of microsomes in the absence of iron, did not enhance the rate of peroxidation suggesting that iron acts by initiating peroxidative decomposition of membrane lipids rather than by catalyzing the breakdown of pre-formed hydroperoxides. Liposomes also underwent peroxidation in the presence of ferrous iron at a rate comparable to intact microsomes and was stimulated by ascorbate. Carbon tetrachloride initiated lipid peroxidation in the absence of free metal ions. Rates of in vitro lipid peroxidation of microsomes and homogenates were found to vary widely between different tissues and species. The effects of paraquat on lipid peroxidation was also studied. (DC)

Heme Exporter FLVCR1a Regulates Heme Synthesis and Degradation and Controls Activity of Cytochromes P450

OpenAIRE

Vinchi, Francesca; Ingoglia, Giada; Chiabrando, Deborah; Mercurio, Sonia; Turco, Emilia; Silengo, Lorenzo; Altruda, Fiorella; Tolosano, Emanuela

2014-01-01

Background & Aims The liver has one of the highest rates of heme synthesis of any organ. More than 50% of the heme synthesized in the liver is used for synthesis of P450 enzymes, which metabolize exogenous and endogenous compounds that include natural products, hormones, drugs, and carcinogens. Feline leukemia virus subgroup C cellular receptor 1a (FLVCR1a) is plasma membrane heme exporter that is ubiquitously expressed and controls intracellular heme content in hematopoietic lineages. We inv...

Production of specific-structured lipids by enzymatic interesterification in a pilot continuous enzyme bed reactor

DEFF Research Database (Denmark)

Xu, Xuebing; Balchen, Steen; Høy, Carl-Erik

1998-01-01

Production of specific-structured lipids (interesterified lipids with a specific structure) by enzymatic interesterification was carried out in a continuous enzyme bed pilot scale reactor. Commercial immobilized lipase (Lipozyme IM) was used and investigations of acyl migration, pressure drop...

Defluoridation potential of jute fibers grafted with fatty acyl chain

Energy Technology Data Exchange (ETDEWEB)

Manna, Suvendu; Saha, Prosenjit [Materials Science Centre, IIT Kharagpur, WB 721302 (India); Roy, Debasis, E-mail: debasis@civil.iitkgp.ernet.in [Department of Civil Engineering, IIT Kharagpur, WB 721302 (India); Sen, Ramkrishna [Department of Biotechnology, IIT Kharagpur, WB 721302 (India); Adhikari, Basudam [Materials Science Centre, IIT Kharagpur, WB 721302 (India)

2015-11-30

Graphical abstract: - Highlights: • Acyl chain grafted jute has been shown to accumulate fluoride ions. • Covalent and hydrogen bonding and protonation were the contributing factors. • The process is relatively inexpensive and maintenance-free. • Acyl chain grafted jute showed higher fluoride ions accumulation than alternatives. - Abstract: Waterborne fluoride is usually removed from water by coagulation, adsorption, ion exchange, electro dialysis or reverse osmosis. These processes are often effective over narrow pH ranges, release ions considered hazardous to human health or produce large volumes of toxic sludge that are difficult to handle and dispose. Although plant matters have been shown to remove waterborne fluoride, they suffer from poor removal efficiency. Following from the insight that interaction between microbial carbohydrate biopolymers and anionic surfaces is often facilitated by lipids, an attempt has been made to enhance fluoride adsorption efficiency of jute by grafting the lignocellulosic fiber with fatty acyl chains found in vegetable oils. Fluoride removal efficiency of grafted jute was found to be comparable or higher than those of alternative defluoridation processes. Infrared and X-ray photoelectron spectroscopic evidence indicated that hydrogen bonding, protonation and C−F bonding were responsible for fluoride accumulation on grafted jute. Adsorption based on grafted jute fibers appears to be an economical, sustainable and eco-friendly alternative technique for removing waterborne fluoride.

Modulation of endotoxicity of Shigella generalized modules for membrane antigens (GMMA) by genetic lipid A modifications: relative activation of TLR4 and TLR2 pathways in different mutants.

Science.gov (United States)

Rossi, Omar; Pesce, Isabella; Giannelli, Carlo; Aprea, Susanna; Caboni, Mariaelena; Citiulo, Francesco; Valentini, Sara; Ferlenghi, Ilaria; MacLennan, Calman Alexander; D'Oro, Ugo; Saul, Allan; Gerke, Christiane

2014-09-05

Outer membrane particles from Gram-negative bacteria are attractive vaccine candidates as they present surface antigens in their natural context. We previously developed a high yield production process for genetically derived particles, called generalized modules for membrane antigens (GMMA), from Shigella. As GMMA are derived from the outer membrane, they contain immunostimulatory components, especially lipopolysaccharide (LPS). We examined ways of reducing their reactogenicity by modifying lipid A, the endotoxic part of LPS, through deletion of late acyltransferase genes, msbB or htrB, in GMMA-producing Shigella sonnei and Shigella flexneri strains. GMMA with resulting penta-acylated lipid A from the msbB mutants showed a 600-fold reduced ability, and GMMA from the S. sonnei ΔhtrB mutant showed a 60,000-fold reduced ability compared with GMMA with wild-type lipid A to stimulate human Toll-like receptor 4 (TLR4) in a reporter cell line. In human peripheral blood mononuclear cells, GMMA with penta-acylated lipid A showed a marked reduction in induction of inflammatory cytokines (S. sonnei ΔhtrB, 800-fold; ΔmsbB mutants, 300-fold). We found that the residual activity of these GMMA is largely due to non-lipid A-related TLR2 activation. In contrast, in the S. flexneri ΔhtrB mutant, a compensatory lipid A palmitoleoylation resulted in GMMA with hexa-acylated lipid A with ∼10-fold higher activity to stimulate peripheral blood mononuclear cells than GMMA with penta-acylated lipid A, mostly due to retained TLR4 activity. Thus, for use as vaccines, GMMA will likely require lipid A penta-acylation. The results identify the relative contributions of TLR4 and TLR2 activation by GMMA, which need to be taken into consideration for GMMA vaccine development. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

A New Acylated Flavonol Glycoside from Chenopodium foliosum

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Zlatina Kokanova-Nedialkova, , , , , and

2014-07-01

Full Text Available A new acylated flavonol glycoside, namely gomphrenol-3-O-( 5 '''-O-E-feruloyl-β-D-apiofuranosyl-(1→2[β-D-glucopyranosyl-(1→6]-β-D-glucopyranoside (1 was isolated from the aerial parts of Chenopodium foliosum Asch. The structure of 1 was determined by means of spectroscopic methods (1D and 2D NMR, UV, IR, and HRESIMS. Radical scavenging and antioxidant activities of 1 were established using DPPH and ABTS radicals, FRAP assay and inhibition of lipid peroxidation (LP in linoleic acid system by the ferric thiocyanate method. Compound 1 showed low activity (DPPH and ABTS or lack of activity (FRAP and LP. In combination with CCl 4, 1 reduced the damage caused by the hepatotoxic agent and preserved cell viability and GSH level, decreased LDH leakage and reduced lipid damage. Effects were concentration dependent, most visible at the highest concentration (100 µg/m L , and similar to those of silymarin .

Shigella Iron Acquisition Systems and their Regulation.

Science.gov (United States)

Wei, Yahan; Murphy, Erin R

2016-01-01

Survival of Shigella within the host is strictly dependent on the ability of the pathogen to acquire essential nutrients, such as iron. As an innate immune defense against invading pathogens, the level of bio-available iron within the human host is maintained at exceeding low levels, by sequestration of the element within heme and other host iron-binding compounds. In response to sequestration mediated iron limitation, Shigella produce multiple iron-uptake systems that each function to facilitate the utilization of a specific host-associated source of nutrient iron. As a mechanism to balance the essential need for iron and the toxicity of the element when in excess, the production of bacterial iron acquisition systems is tightly regulated by a variety of molecular mechanisms. This review summarizes the current state of knowledge on the iron-uptake systems produced by Shigella species, their distribution within the genus, and the molecular mechanisms that regulate their production.

Comparison between the availability of iron in the presence of vitamin a and β-carotene in foods and medications

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Fabiana Cristina Camargo Martini

2011-06-01

Full Text Available The objective of this work was to verify the availability of iron in the presence of vitamin A as components of foods and in combinations with medicines. The iron available was measured in the presence of vitamin A in foods - common bean (B, beef liver (Li and carrot (C - and medicines - Fer-In-Sol® (Fer (Mead Johnson, Arovit® (A (Roche and Neutrofer® (N (Sigma Pharma - as well as in combinations of both. β-carotene, vitamin A, total iron, heme and non heme iron, percentage of dialyzable iron and amount of dialyzable iron was determined. Vitamin A and β-carotene had a positive effect on the percentage of iron dialysis. Carrot and liver had a better percentage of dialyzable iron than their respective medicine at similar concentrations. Therefore, we can conclude that there has been an influence of vitamin A over the dialysis of iron, being the mixtures containing liver the ones which achieved the highest concentrations of dialyzable iron, and also that, according to the amounts needed to obtain the daily recommended intake of iron, they are good for consumption.

[Methodological aspects of evaluation of potential lipid capacity for peroxidation from the serum levels of TBA-active products during iron ion stimulation].

Science.gov (United States)

Kulikova, A I; Tugusheva, F A; Zubina, I M; Shepilova, I N

2008-05-01

The authors propose a simple and reproducible procedure for using iron ions to stimulate serum lipid peroxidation, with TBA-active products being further determined. The procedure determines the reserve of lipids that can be oxidized during oxidative stress. A combination of direct studies and correlation analysis suggests that low-density lipoproteins and very low-density lipoproteins are the major substrates for lipid peroxidation while high-density lipoproteins show a high resistance to this process. The presented procedure may be used to monitor lipid peroxidation in various conditions and upon various exposures in common laboratory practice.

Conformations of double-headed, triple-tailed phospholipid oxidation lipid products in model membranes

DEFF Research Database (Denmark)

Hermetter, Albin; Kopec, Wojciech; Khandelia, Himanshu

2013-01-01

Products of phospholipid oxidation can produce lipids with a carbonyl moiety at the end of a shortened lipid acyl tail, such as 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC). The carbonyl tail of POVPC can covalently bond to the free tertiary amine of a phosphatidylethanolamine...

Unprecedented access of phenolic substrates to the heme active site of a catalase: substrate binding and peroxidase-like reactivity of Bacillus pumilus catalase monitored by X-ray crystallography and EPR spectroscopy.

Science.gov (United States)

Loewen, Peter C; Villanueva, Jacylyn; Switala, Jacek; Donald, Lynda J; Ivancich, Anabella

2015-05-01

Heme-containing catalases and catalase-peroxidases catalyze the dismutation of hydrogen peroxide as their predominant catalytic activity, but in addition, individual enzymes support low levels of peroxidase and oxidase activities, produce superoxide, and activate isoniazid as an antitubercular drug. The recent report of a heme enzyme with catalase, peroxidase and penicillin oxidase activities in Bacillus pumilus and its categorization as an unusual catalase-peroxidase led us to investigate the enzyme for comparison with other catalase-peroxidases, catalases, and peroxidases. Characterization revealed a typical homotetrameric catalase with one pentacoordinated heme b per subunit (Tyr340 being the axial ligand), albeit in two orientations, and a very fast catalatic turnover rate (kcat  = 339,000 s(-1) ). In addition, the enzyme supported a much slower (kcat  = 20 s(-1) ) peroxidatic activity utilizing substrates as diverse as ABTS and polyphenols, but no oxidase activity. Two binding sites, one in the main access channel and the other on the protein surface, accommodating pyrogallol, catechol, resorcinol, guaiacol, hydroquinone, and 2-chlorophenol were identified in crystal structures at 1.65-1.95 Å. A third site, in the heme distal side, accommodating only pyrogallol and catechol, interacting with the heme iron and the catalytic His and Arg residues, was also identified. This site was confirmed in solution by EPR spectroscopy characterization, which also showed that the phenolic oxygen was not directly coordinated to the heme iron (no low-spin conversion of the Fe(III) high-spin EPR signal upon substrate binding). This is the first demonstration of phenolic substrates directly accessing the heme distal side of a catalase. © 2015 Wiley Periodicals, Inc.

Two Predicted Transmembrane Domains Exclude Very Long Chain Fatty acyl-CoAs from the Active Site of Mouse Wax Synthase.

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Steffen Kawelke

Full Text Available Wax esters are used as coatings or storage lipids in all kingdoms of life. They are synthesized from a fatty alcohol and an acyl-CoA by wax synthases. In order to get insights into the structure-function relationships of a wax synthase from Mus musculus, a domain swap experiment between the mouse acyl-CoA:wax alcohol acyltransferase (AWAT2 and the homologous mouse acyl-CoA:diacylglycerol O-acyltransferase 2 (DGAT2 was performed. This showed that the substrate specificity of AWAT2 is partially determined by two predicted transmembrane domains near the amino terminus of AWAT2. Upon exchange of the two domains for the respective part of DGAT2, the resulting chimeric enzyme was capable of incorporating up to 20% of very long acyl chains in the wax esters upon expression in S. cerevisiae strain H1246. The amount of very long acyl chains in wax esters synthesized by wild type AWAT2 was negligible. The effect was narrowed down to a single amino acid position within one of the predicted membrane domains, the AWAT2 N36R variant. Taken together, we provide first evidence that two predicted transmembrane domains in AWAT2 are involved in determining its acyl chain length specificity.

Gene expression profiling in Entamoeba histolytica identifies key components in iron uptake and metabolism.

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Nora Adriana Hernández-Cuevas

Full Text Available Entamoeba histolytica is an ameboid parasite that causes colonic dysentery and liver abscesses in humans. The parasite encounters dramatic changes in iron concentration during its invasion of the host, with relatively low levels in the intestinal lumen and then relatively high levels in the blood and liver. The liver notably contains sources of iron; therefore, the parasite's ability to use these sources might be relevant to its survival in the liver and thus the pathogenesis of liver abscesses. The objective of the present study was to identify factors involved in iron uptake, use and storage in E. histolytica. We compared the respective transcriptomes of E. histolytica trophozoites grown in normal medium (containing around 169 µM iron, low-iron medium (around 123 µM iron, iron-deficient medium (around 91 µM iron, and iron-deficient medium replenished with hemoglobin. The differentially expressed genes included those coding for the ATP-binding cassette transporters and major facilitator transporters (which share homology with bacterial siderophores and heme transporters and genes involved in heme biosynthesis and degradation. Iron deficiency was associated with increased transcription of genes encoding a subset of cell signaling molecules, some of which have previously been linked to adaptation to the intestinal environment and virulence. The present study is the first to have assessed the transcriptome of E. histolytica grown under various iron concentrations. Our results provide insights into the pathways involved in iron uptake and metabolism in this parasite.

Gene expression profiling in Entamoeba histolytica identifies key components in iron uptake and metabolism.

Science.gov (United States)

Hernández-Cuevas, Nora Adriana; Weber, Christian; Hon, Chung-Chau; Guillen, Nancy

2014-01-01

Entamoeba histolytica is an ameboid parasite that causes colonic dysentery and liver abscesses in humans. The parasite encounters dramatic changes in iron concentration during its invasion of the host, with relatively low levels in the intestinal lumen and then relatively high levels in the blood and liver. The liver notably contains sources of iron; therefore, the parasite's ability to use these sources might be relevant to its survival in the liver and thus the pathogenesis of liver abscesses. The objective of the present study was to identify factors involved in iron uptake, use and storage in E. histolytica. We compared the respective transcriptomes of E. histolytica trophozoites grown in normal medium (containing around 169 µM iron), low-iron medium (around 123 µM iron), iron-deficient medium (around 91 µM iron), and iron-deficient medium replenished with hemoglobin. The differentially expressed genes included those coding for the ATP-binding cassette transporters and major facilitator transporters (which share homology with bacterial siderophores and heme transporters) and genes involved in heme biosynthesis and degradation. Iron deficiency was associated with increased transcription of genes encoding a subset of cell signaling molecules, some of which have previously been linked to adaptation to the intestinal environment and virulence. The present study is the first to have assessed the transcriptome of E. histolytica grown under various iron concentrations. Our results provide insights into the pathways involved in iron uptake and metabolism in this parasite.

Tea fungus fermentation on a substrate with iron(ii-ions

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Malbaša Radomir V.

2002-01-01

Full Text Available Iron is essential element for human metabolism and it is a constituent of both heme- containing and nonheme proteins. Its deficiency can cause serious diseases, i.e. iron-deficiency anemia, with some fatal consequences. Tea fungus beverage has high nutritional value and some pharmaceutical effects. It is widely consumed allover the world and its benefits were proved a number of times. The aim of this paper was to investigate tea fungus fermentation on a substrate containing iron(II-ions and the possibility of obtaining a beverage enriched with iron. We monitored pH, iron content and also the production of L-ascorbic acid, which is very important for iron absorption in humans.

Heterogeneous electron transfer of a two-centered heme protein: redox and electrocatalytic properties of surface-immobilized cytochrome C(4).

Science.gov (United States)

Monari, Stefano; Battistuzzi, Gianantonio; Borsari, Marco; Di Rocco, Giulia; Martini, Laura; Ranieri, Antonio; Sola, Marco

2009-10-15

The recombinant diheme cytochrome c(4) from the psycrophilic bacterium Pseudoalteromonas haloplanktis TAC 125 and its Met64Ala and Met164Ala variants, which feature a hydroxide ion axially bound to the heme iron at the N- and C-terminal domains, respectively, were found to exchange electrons efficiently with a gold electrode coated with a SAM of 11-mercapto-1-undecanoic acid. The mutation-induced removal of the redox equivalence of the two heme groups and changes in the net charge of the protein lobes yield two-centered protein systems with unprecedented properties in the electrode-immobilized state. The heterogeneous and intraheme electron transfer processes were characterized for these species in which the high- and low-potential heme groups are swapped over in the bilobal protein framework and experience a constrained (M64A) and unconstrained (M164A) orientation toward the electrode. The reduction thermodynamics for the native and mutated hemes were measured for the first time for a diheme cytochrome c. In the diffusing regime, they reproduce closely those for the corresponding centers in single-heme class-I cytochromes c, despite the low sequence identity. Larger differences are observed in the thermodynamics of the immobilized species and in the heterogeneous electron transfer rate constants. T-dependent kinetic measurements show that the proteins are positioned approximately 7 A from the HOOC-terminated SAM-coated electrode. Protein-electrode orientation and efficient intraheme ET enable the His,OH(-)-ligated heme A of the immobilized Met64Ala variant to carry out the reductive electrocatalysis of molecular oxygen. This system therefore constitutes a novel two-centered heme-based biocatalytic interface to be exploited for "third-generation" amperometric biosensing.

Relationship between natural and heme-mediated antibody polyreactivity

Energy Technology Data Exchange (ETDEWEB)

Hadzhieva, Maya; Vassilev, Tchavdar [Stephan Angelov Institute of Microbiology, Bulgarian Academy of Sciences, Sofia 1113 (Bulgaria); Bayry, Jagadeesh; Kaveri, Srinivas; Lacroix-Desmazes, Sébastien [Sorbonne Universités, UPMC Univ Paris 06, UMR-S 1138, Centre de Recherche des Cordeliers, F-75006 Paris (France); INSERM, UMR-S 1138, F-75006 Paris (France); Université Paris Descartes, Sorbonne Paris Cité, UMR-S 1138, F-75006 Paris (France); Dimitrov, Jordan D., E-mail: jordan.dimitrov@crc.jussieu.fr [Sorbonne Universités, UPMC Univ Paris 06, UMR-S 1138, Centre de Recherche des Cordeliers, F-75006 Paris (France); INSERM, UMR-S 1138, F-75006 Paris (France); Université Paris Descartes, Sorbonne Paris Cité, UMR-S 1138, F-75006 Paris (France)

2016-03-25

Polyreactive antibodies represent a considerable fraction of the immune repertoires. Some antibodies acquire polyreactivity post-translationally after interaction with various redox-active substances, including heme. Recently we have demonstrated that heme binding to a naturally polyreactive antibody (SPE7) results in a considerable broadening of the repertoire of recognized antigens. A question remains whether the presence of certain level of natural polyreactivity of antibodies is a prerequisite for heme-induced further extension of antigen binding potential. Here we used a second monoclonal antibody (Hg32) with unknown specificity and absence of intrinsic polyreactivity as a model to study the potential of heme to induce polyreactivity of antibodies. We demonstrated that exposure to heme greatly extends the antigen binding potential of Hg32, suggesting that the intrinsic binding promiscuity is not a prerequisite for the induction of polyreactivity by heme. In addition we compared the kinetics and thermodynamics of the interaction of heme-exposed antibodies with a panel of unrelated antigens. These analyses revealed that the two heme-sensitive antibodies adopt different mechanisms of binding to the same set of antigens. This study contributes to understanding the phenomenon of induced antibody polyreactivity. The data may also be of importance for understanding of physiological and pathological roles of polyreactive antibodies. - Highlights: • Exposure of certain monoclonal IgE antibodies to heme results in gain of antigen binding polyreactivity. • Natural polyreactivity of antibodies is dispensable for acquisition of polyreactivity through interaction with heme. • Heme-induced monoclonal IgE antibodies differ in their thermodynamic mechanisms of antigen recognition.

Age dependent accumulation of N-acyl-ethanolamine phospholipids in ischemic rat brain

DEFF Research Database (Denmark)

Moesgaard, B.; Petersen, G.; Hansen, Harald S.

2000-01-01

N-acyl-ethanolamine phospholipids (NAPE) can be formed as a stress response during neuronal injury, and they are precursors for N-acyl- ethanolamines (NAE), some of which are endocannabinoids. The levels of NAPE accumulated during post-decapitative ischemia (6 h at 37°C) were studied in rat brains...... of various age (1, 6, 12, 19, 30, and ~70 days) by the use of P NMR spectroscopy of lipid extracts. This ability to accumulate NAPE was compared with the activity of N-acyltransferase and of NAPE-hydrolyzing phospholipase D (NAPE-PLD) in brain microsomes. These two enzymes are involved in the formation...... brains NAPE accumulation could not be detected (detection limit 0.09 %)]; and 2) this age pattern of accumulation can be explained by a combination of the decreased activity of N- acyltransferase and the increased activity of NAPE-PLD during development. These results point out that it would...

Role of heme in bromine-induced lung injury

Science.gov (United States)

Lam, Adam; Vetal, Nilam; Matalon, Sadis; Aggarwal, Saurabh

2016-01-01

Bromine (Br2) gas inhalation poses an environmental and occupational hazard resulting in high morbidity and mortality. In this review, we underline the acute lung pathology (within 24 hours of exposure) and potential therapeutic interventions that may be utilized to mitigate Br2-induced human toxicity. We will discuss our latest published data, which suggests that an increase in heme-dependent tissue injury underlies the pathogenesis of Br2 toxicity. Our study was based on previous findings that demonstrated that Br2 upregulates the heme-degrading enzyme heme oxygenase-1 (HO-1), which converts toxic heme into billiverdin. Interestingly, following Br2 inhalation, heme levels were indeed elevated in bronchoalveolar lavage fluid, plasma, and whole lung tissue in C57BL/6 mice. High heme levels correlated with increased lung oxidative stress, lung inflammation, respiratory acidosis, lung edema, higher airway resistance, and mortality. However, therapeutic reduction of heme levels, by either scavenging with hemopexin or degradation by HO-1, improved lung function and survival. Therefore, heme attenuation may prove a useful adjuvant therapy to treat patients after Br2 exposure. PMID:27244263

Effect of dietary iron loading on recognition memory in growing rats.

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Murui Han

Full Text Available While nutritional and neurobehavioral problems are associated with both iron deficiency during growth and overload in the elderly, the effect of iron loading in growing ages on neurobehavioral performance has not been fully explored. To characterize the role of dietary iron loading in memory function in the young, weanling rats were fed iron-loading diet (10,000 mg iron/kg diet or iron-adequate control diet (50 mg/kg for one month, during which a battery of behavioral tests were conducted. Iron-loaded rats displayed elevated non-heme iron levels in serum and liver, indicating a condition of systemic iron overload. In the brain, non-heme iron was elevated in the prefrontal cortex of iron-loaded rats compared with controls, whereas there was no difference in iron content in other brain regions between the two diet groups. While iron loading did not alter motor coordination or anxiety-like behavior, iron-loaded rats exhibited a better recognition memory, as represented by an increased novel object recognition index (22% increase from the reference value than control rats (12% increase; P=0.047. Western blot analysis showed an up-regulation of dopamine receptor 1 in the prefrontal cortex from iron-loaded rats (142% increase; P=0.002. Furthermore, levels of glutamate receptors (both NMDA and AMPA and nicotinic acetylcholine receptor (nAChR were significantly elevated in the prefrontal cortex of iron-loaded rats (62% increase in NR1; 70% increase in Glu1A; 115% increase in nAChR. Dietary iron loading also increased the expression of NMDA receptors and nAChR in the hippocampus. These results support the idea that iron is essential for learning and memory and further reveal that iron supplementation during developmental and rapidly growing periods of life improves memory performance. Our investigation also demonstrates that both cholinergic and glutamatergic neurotransmission pathways are regulated by dietary iron and provides a molecular basis for the

Defective Pollen Wall 2 ( DPW2 ) Encodes an Acyl Transferase Required for Rice Pollen Development

Energy Technology Data Exchange (ETDEWEB)

Xu, Dawei [Shanghai Jiao Tong Univ. (China). Joint International Research Lab. of Metabolic and Developmental Sciences; Shi, Jianxin [Shanghai Jiao Tong Univ. (China). Joint International Research Lab. of Metabolic and Developmental Sciences; Rautengarten, Carsten [Univ. of Melbourne (Australia). ARC Centre of Excellence in Plant Cell Walls; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Joint BioEnergy Inst. and Biological Systems and Engineering Division; Yang, Li [Shanghai Jiao Tong Univ. (China). Joint International Research Lab. of Metabolic and Developmental Sciences; Qian, Xiaoling [Shanghai Jiao Tong Univ. (China). Joint International Research Lab. of Metabolic and Developmental Sciences; Uzair, Muhammad [Shanghai Jiao Tong Univ. (China). Joint International Research Lab. of Metabolic and Developmental Sciences; Zhu, Lu [Shanghai Jiao Tong Univ. (China). Joint International Research Lab. of Metabolic and Developmental Sciences; Luo, Qian [Shanghai Jiao Tong Univ. (China). Joint International Research Lab. of Metabolic and Developmental Sciences; An, Gynheung [Kyung Hee Univ., Yongin (Korea). Crop Biotech Inst.; Waßmann, Fritz [Univ. of Bonn (Germany). Inst. of Cellular and Molecular Botany; Schreiber, Lukas [Univ. of Bonn (Germany). Inst. of Cellular and Molecular Botany; Heazlewood, Joshua L. [Univ. of Melbourne (Australia). ARC Centre of Excellence in Plant Cell Walls; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Joint BioEnergy Inst. and Biological Systems and Engineering Division; Scheller, Henrik Vibe [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Joint BioEnergy Inst. and Biological Systems and Engineering Division; Univ. of California, Berkeley, CA (United States). Dept. of Plant and Microbial Biology; Hu, Jianping [Michigan State Univ., East Lansing, MI (United States). Dept. of Energy Plant Research Lab.; Zhang, Dabing [Shanghai Jiao Tong Univ. (China). Joint International Research Lab. of Metabolic and Developmental Sciences; Univ. of Adelaide, SA (Australia). School of Agriculture, Food and Wine; Liang, Wanqi [Shanghai Jiao Tong Univ. (China). Joint International Research Lab. of Metabolic and Developmental Sciences

2016-05-31

Aliphatic and aromatic lipids are both essential structural components of the plant cuticle, an important interface between the plant and environment. Although cross links between aromatic and aliphatic or other moieties are known to be associated with the formation of leaf cutin and root and seed suberin, the contribution of aromatic lipids to the biosynthesis of anther cuticles and pollen walls remains elusive. In this study, we characterized the rice (Oryza sativa) male sterile mutant, defective pollen wall 2 (dpw2), which showed an abnormal anther cuticle, a defective pollen wall, and complete male sterility. Compared with the wild type, dpw2 anthers have increased amounts of cutin and waxes and decreased levels of lipidic and phenolic compounds. DPW2 encodes a cytoplasmically localized BAHD acyltransferase. In vitro assays demonstrated that recombinant DPW2 specifically transfers hydroxycinnamic acid moieties, using v-hydroxy fatty acids as acyl acceptors and hydroxycinnamoyl-CoAs as acyl donors. Thus, The cytoplasmic hydroxycinnamoyl-CoA:v-hydroxy fatty acid transferase DPW2 plays a fundamental role in male reproduction via the biosynthesis of key components of the anther cuticle and pollen wall.

Altered regulation of lipid biosynthesis in a mutant of Arabidopsis deficient in chloroplast glycerol-3-phosphate acyltransferase activity

International Nuclear Information System (INIS)

Kunst, L.; Browse, J.; Somerville, C.

1988-01-01

The leaf membrane lipids of many plant species, including Arabidopsis thaliana (L.) Heynh., are synthesized by two complementary pathways that are associated with the chloroplast and the endoplasmic reticulum. By screening directly for alterations in lipid acyl-group composition, the authors have identified several mutants of Arabidopsis that lack the plastid pathway because of a deficiency in activity of the first enzyme in the plastid pathway of glycerolipid synthesis, acyl-ACP:sn-glycerol-3-phosphate acyltransferase. The lesion results in an increased synthesis of lipids by the cytoplasmic pathway that largely compensates for the loss of the plastid pathway and provides nearly normal amounts of all the lipids required for chloroplast biogenesis. However, the fatty acid composition of the leaf membrane lipids of the mutants is altered because the acyltransferases associated with the two pathways normally exhibit different substrate specificities. The remarkable flexibility of the system provides an insight into the nature of the regulatory mechanisms that allocate lipids for membrane biogenesis

Heme biosynthesis and its regulation : Toward understanding and improvement of heme biosynthesis in filamentous fungi.

NARCIS (Netherlands)

S. de Weert; P.J. Punt; Christien Lokman; C.A. van den Hondel; A.C. Franken; A.F. Ram

2011-01-01

Heme biosynthesis in fungal host strains has acquired considerable interest in relation to the production of secreted heme-containing peroxidases. Class II peroxidase enzymes have been suggested as eco-friendly replacements of polluting chemical processes in industry. These peroxidases are naturally

Heme biosynthesis and its regulation: Towards understanding and improvement of heme biosynthesis in filamentous fungi

NARCIS (Netherlands)

Franken, A.C.W.; Lokman, B.C.; Ram, A.F.J.; Punt, P.J.; Hondel, C.A.M.J.J. van den; Weert, S. de

2011-01-01

Heme biosynthesis in fungal host strains has acquired considerable interest in relation to the production of secreted heme-containing peroxidases. Class II peroxidase enzymes have been suggested as eco-friendly replacements of polluting chemical processes in industry. These peroxidases are naturally

Heme-binding plasma membrane proteins of K562 erythroleukemia cells: Adsorption to heme-microbeads, isolation with affinity chromatography

International Nuclear Information System (INIS)

Majuri, R.

1989-01-01

Heme-microbeads attached themselves to the surface of viable K562 cells in a manner inhibitable by free hemin, indicating heme-recptor interaction. The microbeads were at first evenly distributed, but after prolonged incubation at 37 deg. C they formed a cap on one pole of the cells indicating clustering of the membrane heme receptors. Membrane proteins were labeled by culturing the cells in the presence of 35 S-methionine and were then solubilized with Triton X-114. The hydrophobic proteins contained about 20% of the total bound label. The solubilized membrane proteins were subsequently adsorbed to a heme-Sepharose affinity gel. According to SDS-electrophorsis and subsequent autoradiography, the immobilized heme captures two proteins or a protein with two polypeptides of 20 000 and 32 000 daltons. The larger of these was only wekly labeled with 35 S. The same two bands were observed if the cell surface proteins were labeled with 125 I by the lactoperoxidase method and the subsequently solubilized membrane proteins were isolated with heme-Sepharose. (author)

Role of Heme and Heme-Proteins in Trypanosomatid Essential Metabolic Pathways

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Karina E. J. Tripodi

2011-01-01

Full Text Available Around the world, trypanosomatids are known for being etiological agents of several highly disabling and often fatal diseases like Chagas disease (Trypanosoma cruzi, leishmaniasis (Leishmania spp., and African trypanosomiasis (Trypanosoma brucei. Throughout their life cycle, they must cope with diverse environmental conditions, and the mechanisms involved in these processes are crucial for their survival. In this review, we describe the role of heme in several essential metabolic pathways of these protozoans. Notwithstanding trypanosomatids lack of the complete heme biosynthetic pathway, we focus our discussion in the metabolic role played for important heme-proteins, like cytochromes. Although several genes for different types of cytochromes, involved in mitochondrial respiration, polyunsaturated fatty acid metabolism, and sterol biosynthesis, are annotated at the Tritryp Genome Project, the encoded proteins have not yet been deeply studied. We pointed our attention into relevant aspects of these protein functions that are amenable to be considered for rational design of trypanocidal agents.

Malaria parasite-synthesized heme is essential in the mosquito and liver stages and complements host heme in the blood stages of infection.

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Viswanathan Arun Nagaraj

Full Text Available Heme metabolism is central to malaria parasite biology. The parasite acquires heme from host hemoglobin in the intraerythrocytic stages and stores it as hemozoin to prevent free heme toxicity. The parasite can also synthesize heme de novo, and all the enzymes in the pathway are characterized. To study the role of the dual heme sources in malaria parasite growth and development, we knocked out the first enzyme, δ-aminolevulinate synthase (ALAS, and the last enzyme, ferrochelatase (FC, in the heme-biosynthetic pathway of Plasmodium berghei (Pb. The wild-type and knockout (KO parasites had similar intraerythrocytic growth patterns in mice. We carried out in vitro radiolabeling of heme in Pb-infected mouse reticulocytes and Plasmodium falciparum-infected human RBCs using [4-(14C] aminolevulinic acid (ALA. We found that the parasites incorporated both host hemoglobin-heme and parasite-synthesized heme into hemozoin and mitochondrial cytochromes. The similar fates of the two heme sources suggest that they may serve as backup mechanisms to provide heme in the intraerythrocytic stages. Nevertheless, the de novo pathway is absolutely essential for parasite development in the mosquito and liver stages. PbKO parasites formed drastically reduced oocysts and did not form sporozoites in the salivary glands. Oocyst production in PbALASKO parasites recovered when mosquitoes received an ALA supplement. PbALASKO sporozoites could infect mice only when the mice received an ALA supplement. Our results indicate the potential for new therapeutic interventions targeting the heme-biosynthetic pathway in the parasite during the mosquito and liver stages.

Unsaturated glycerophospholipids mediate heme crystallization: biological implications for hemozoin formation in the kissing bug Rhodnius prolixus.

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Renata Stiebler

Full Text Available Hemozoin (Hz is a heme crystal produced by some blood-feeding organisms, as an efficient way to detoxify heme derived from hemoglobin digestion. In the triatomine insect Rhodnius prolixus, Hz is essentially produced by midgut extracellular phospholipid membranes known as perimicrovillar membranes (PMVM. Here, we investigated the role of commercial glycerophospholipids containing serine, choline and ethanolamine as headgroups and R. prolixus midgut lipids (RML in heme crystallization. All commercial unsaturated forms of phospholipids, as well as RML, mediated fast and efficient β-hematin formation by means of two kinetically distinct mechanisms: an early and fast component, followed by a late and slow one. The fastest reactions observed were induced by unsaturated forms of phosphatidylethanolamine (uPE and phosphatidylcholine (uPC, with half-lives of 0.04 and 0.7 minutes, respectively. β-hematin crystal morphologies were strikingly distinct among groups, with uPE producing homogeneous regular brick-shaped crystals. Interestingly, uPC-mediated reactions resulted in two morphologically distinct crystal populations: one less representative group of regular crystals, resembling those induced by uPE, and the other largely represented by crystals with numerous sharp edges and tapered ends. Heme crystallization reactions induced by RML were efficient, with a heme to β-hematin conversion rate higher than 70%, but clearly slower (t1/2 of 9.9-17.7 minutes than those induced by uPC and uPE. Interestingly, crystals produced by RML were homogeneous in shape and quite similar to those mediated by uPE. Thus, β-hematin formation can be rapidly and efficiently induced by unsaturated glycerophospholipids, particularly uPE and uPC, and may play a role on biological heme crystallization in R. prolixus midgut.

The Lipopolysaccharide Lipid A Long-Chain Fatty Acid Is Important for Rhizobium leguminosarum Growth and Stress Adaptation in Free-Living and Nodule Environments.

Science.gov (United States)

Bourassa, Dianna V; Kannenberg, Elmar L; Sherrier, D Janine; Buhr, R Jeffrey; Carlson, Russell W

2017-02-01

Rhizobium bacteria live in soil and plant environments, are capable of inducing symbiotic nodules on legumes, invade these nodules, and develop into bacteroids that fix atmospheric nitrogen into ammonia. Rhizobial lipopolysaccharide (LPS) is anchored in the bacterial outer membrane through a specialized lipid A containing a very long-chain fatty acid (VLCFA). VLCFA function for rhizobial growth in soil and plant environments is not well understood. Two genes, acpXL and lpxXL, encoding acyl carrier protein and acyltransferase, are among the six genes required for biosynthesis and transfer of VLCFA to lipid A. Rhizobium leguminosarum mutant strains acpXL, acpXL - /lpxXL - , and lpxXL - were examined for LPS structure, viability, and symbiosis. Mutations in acpXL and lpxXL abolished VLCFA attachment to lipid A. The acpXL mutant transferred a shorter acyl chain instead of VLCFA. Strains without lpxXL neither added VLCFA nor a shorter acyl chain. In all strains isolated from nodule bacteria, lipid A had longer acyl chains compared with laboratory-cultured bacteria, whereas mutant strains displayed altered membrane properties, modified cationic peptide sensitivity, and diminished levels of cyclic β-glucans. In pea nodules, mutant bacteroids were atypically formed and nitrogen fixation and senescence were affected. The role of VLCFA for rhizobial environmental fitness is discussed.

Fluorescently labelled bovine acyl-CoA-binding protein acting as an acyl-CoA sensor: interaction with CoA and acyl-CoA esters and its use in measuring free acyl-CoA esters and non-esterified fatty acids

DEFF Research Database (Denmark)

Wadum, M.C.; Villadsen, J.K.; Feddersen, S.

2002-01-01

methods for the determination of free acyl-CoA concentrations. No such method is presently available. In the present study, we describe the synthesis of two acyl-CoA sensors for measuring free acyl-CoA concentrations using acyl-CoA-binding protein as a scaffold. Met24 and Ala53 of bovine acyl...... of ligand (excitation 387nm). Titration of FACI-24 and FACI-53 with hexadecanoyl-CoA and dodecanoyl-CoA increased the fluorescence yield 5.5-and 4.7-fold at 460 and 495nm respectively. FACI-24 exhibited a high, and similar increase in, fluorescence yield at 460nm upon binding of C14-C20 saturated...

Molecular characterization of a heme-binding protein of Bacteroides fragilis BE1.

OpenAIRE

Otto, B R; Kusters, J G; Luirink, J; de Graaf, F K; Oudega, B

1996-01-01

An iron-repressible 44-kDa outer membrane protein plays a crucial role in the acquisition of heme by the anaerobic bacterium Bacteroides fragilis. The DNA sequence of the gene encoding the 44-kDa protein (hupA) was determined. The hupA gene encodes a protein of 431 amino acid residues with a calculated molecular mass of 48,189 Da. The hupA gene is preceded by an open reading frame of 480 bp that probably encodes a protein with a calculated molecular mass of 18,073 Da. hupA and this open readi...

Implication for using heme methyl hyperfine shifts as indicators of heme seating as related to stereoselectivity in the catabolism of heme by heme oxygenase: in-plane heme versus axial his rotation.

Science.gov (United States)

Ogura, Hiroshi; Evans, John P; de Montellano, Paul R Ortiz; La Mar, Gerd N

2008-01-08

The triple mutant of the solubilized, 265-residue construct of human heme oxygenase, K18E/E29K/R183E-hHO, has been shown to redirect the exclusive alpha-regioselectivity of wild-type hHO to primarily beta,delta-selectivity in the cleavage of heme (Wang, J., Evans, J. P., Ogura, H., La Mar, G. N., and Ortiz de Montellano, P. R. (2006) Biochemistry 45, 61-73). The 1H NMR hyperfine shift pattern for the substrate and axial His CbetaH's and the substrate-protein contacts of the cyanide-inhibited protohemin and 2,4-dimethyldeuterohemin complexes of the triple mutant have been analyzed in detail and compared to data for the WT complex. It is shown that protein contacts for the major solution isomers for both substrates in the mutant dictate approximately 90 degrees in-plane clockwise rotation relative to that in the WT. The conventional interpretation of the pattern of substrate methyl hyperfine shifts, however, indicates substrate rotations of only approximately 50 degrees . This paradox is resolved by demonstrating that the axial His25 imidazole ring also rotates counterclockwise with respect to the protein matrix in the mutant relative to that in the WT. The axial His25 CbetaH hyperfine shifts are shown to serve as independent probes of the imidazole plane orientation relative to the protein matrix. The analysis indicates that the pattern of heme methyl hyperfine shifts cannot be used alone to determine the in-plane orientation of the substrate as it relates to the stereospecificity of heme cleavage, without explicit consideration of the orientation of the axial His imidazole plane relative to the protein matrix.

An ER protein functionally couples neutral lipid metabolism on lipid droplets to membrane lipid synthesis in the ER.

Science.gov (United States)

Markgraf, Daniel F; Klemm, Robin W; Junker, Mirco; Hannibal-Bach, Hans K; Ejsing, Christer S; Rapoport, Tom A

2014-01-16

Eukaryotic cells store neutral lipids such as triacylglycerol (TAG) in lipid droplets (LDs). Here, we have addressed how LDs are functionally linked to the endoplasmic reticulum (ER). We show that, in S. cerevisiae, LD growth is sustained by LD-localized enzymes. When LDs grow in early stationary phase, the diacylglycerol acyl-transferase Dga1p moves from the ER to LDs and is responsible for all TAG synthesis from diacylglycerol (DAG). During LD breakdown in early exponential phase, an ER membrane protein (Ice2p) facilitates TAG utilization for membrane-lipid synthesis. Ice2p has a cytosolic domain with affinity for LDs and is required for the efficient utilization of LD-derived DAG in the ER. Ice2p breaks a futile cycle on LDs between TAG degradation and synthesis, promoting the rapid relocalization of Dga1p to the ER. Our results show that Ice2p functionally links LDs with the ER and explain how cells switch neutral lipid metabolism from storage to consumption. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

An ER Protein Functionally Couples Neutral Lipid Metabolism on Lipid Droplets to Membrane Lipid Synthesis in the ER

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Daniel F. Markgraf

2014-01-01

Full Text Available Eukaryotic cells store neutral lipids such as triacylglycerol (TAG in lipid droplets (LDs. Here, we have addressed how LDs are functionally linked to the endoplasmic reticulum (ER. We show that, in S. cerevisiae, LD growth is sustained by LD-localized enzymes. When LDs grow in early stationary phase, the diacylglycerol acyl-transferase Dga1p moves from the ER to LDs and is responsible for all TAG synthesis from diacylglycerol (DAG. During LD breakdown in early exponential phase, an ER membrane protein (Ice2p facilitates TAG utilization for membrane-lipid synthesis. Ice2p has a cytosolic domain with affinity for LDs and is required for the efficient utilization of LD-derived DAG in the ER. Ice2p breaks a futile cycle on LDs between TAG degradation and synthesis, promoting the rapid relocalization of Dga1p to the ER. Our results show that Ice2p functionally links LDs with the ER and explain how cells switch neutral lipid metabolism from storage to consumption.

21 CFR 862.1410 - Iron (non-heme) test system.

Science.gov (United States)

2010-04-01

... characterized by pigmentation of the skin), and chronic renal disease. (b) Classification. Class I. ... diagnosis and treatment of diseases such as iron deficiency anemia, hemochromatosis (a disease associated...

Comparison of the orientational order of lipid chains in the Lα and HII phases

International Nuclear Information System (INIS)

Lafleur, M.; Cullis, P.R.; Fine, B.; Bloom, M.

1990-01-01

The orientational order profile has been determined by using deuterium nuclear magnetic resonance ( 2 H NMR) for POPE in the lamellar liquid-crystalline (L α ) and the hexagonal (H II ) phases and is shown to be sensitive to the symmetry of the lipid phase. In the H II phase, as compared to the L α phase, the acyl chains are characterized by a greater motional freedom, and the orientational order is distributed more uniformly along the lipid acyl chain. This is consistent with a change from a cylindrical to a wedge-shaped space available for the lipid chain. 2 H NMR studies of POPE dispersions containing tetradecanol or decane, both of which can induce H II phase structure, show very different behavior. Tetradecanol appears to align with the phospholipid chains and experience the L α to H II phase transition with a similar change in motional averaging as observed for the phospholipid chains themselves. In contrast, decane is apparently deeply embedded in the lipid structure and exhibits only a small degree of orientation. The L α to H II phase transition for systems containing decane leads to a dramatic increase of the motional freedom of decane which is more pronounced than that observed for the lipid chains. The presence of decane in the H II phase structure does not modify the order of the lipid chains. However, the L α phase of POPE is slightly disordered by the addition of 9 mol% decane whereas it can accommodate as much as 20 mol% tetradecanol without a significant change of order. Finally, the concept of a stretching vector associated with the lipid acyl chain has been introduced to analyze the orientational order profile obtained in the H II phase. With this model, the average order parameter of the H II phase has been calculated and found to be in good agreement with experiment

Estimation of perimortal percent carboxy-heme in nonstandard postmortem specimens using analysis of carbon monoxide by GC/MS and iron by flame atomic absorption spectrophotometry.

Science.gov (United States)

Middleberg, R A; Easterling, D E; Zelonis, S F; Rieders, F; Rieders, M F

1993-01-01

In decomposed, formalin-fixed, embalmed, exhumed, and some fire-dried cases in which normal blood is unavailable, the usual methods for determination of carboxyhemoglobin saturation frequently fail. To address these specimens, a method utilizing both gas chromatography/mass spectrometric (GC/MS) determination of carbon monoxide (CO) and flame atomic absorption spectrophotometry (FAAS) determination of iron (Fe), in the same specimen, was developed. The method is reported here, along with its application to seven pertinent forsensic death investigations. The CO analytical methodology involves acid liberation of the gas from the specimen aliquot in a headspace vial. After heating and equilibrating, a sample of the headspace vapor is injected into the GC/MS system with a gastight syringe. Quantitation is achieved by standard addition comparison utilizing the ideal gas law equation. Iron is quantified by FAAS analysis of the same aliquot used for the CO determination, following nitric acid digestion. The concentration is determined by comparison to a standard curve. A formula for determining the minimum percent carboxy-heme saturation was derived by using the ratio of the amount of CO to the amount of Fe in the aliquot analyzed. Tissue types analyzed include spleen, liver, muscle, dried blood, and unspecified decomposed tissue.

Urinary Hepcidin Levels in Iron-Deficient and Iron-Supplemented Piglets Correlate with Hepcidin Hepatic mRNA and Serum Levels and with Body Iron Status.

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Robert Staroń

Full Text Available Among livestock, domestic pig (Sus scrofa is a species, in which iron metabolism has been most intensively examined during last decade. The obvious reason for studying the regulation of iron homeostasis especially in young pigs is neonatal iron deficiency anemia commonly occurring in these animals. Moreover, supplementation of essentially all commercially reared piglets with iron entails a need for monitoring the efficacy of this routine practice followed in the swine industry for several decades. Since the discovery of hepcidin many studies confirmed its role as key regulator of iron metabolism and pointed out the assessment of its concentrations in biological fluids as diagnostic tool for iron-related disorder. Here we demonstrate that urine hepcidin-25 levels measured by a combination of weak cation exchange chromatography and time-of-flight mass spectrometry (WCX-TOF MS are highly correlated with mRNA hepcidin expression in the liver and plasma hepcidin-25 concentrations in anemic and iron-supplemented 28-day old piglets. We also found a high correlation between urine hepcidin level and hepatic non-heme iron content. Our results show that similarly to previously described transgenic mouse models of iron disorders, young pigs constitute a convenient animal model to explore accuracy and relationship between indicators for assessing systemic iron status.

Lipoproteins of slow-growing Mycobacteria carry three fatty acids and are N-acylated by apolipoprotein N-acyltransferase BCG_2070c.

Science.gov (United States)

Brülle, Juliane K; Tschumi, Andreas; Sander, Peter

2013-10-05

Lipoproteins are virulence factors of Mycobacterium tuberculosis. Bacterial lipoproteins are modified by the consecutive action of preprolipoprotein diacylglyceryl transferase (Lgt), prolipoprotein signal peptidase (LspA) and apolipoprotein N- acyltransferase (Lnt) leading to the formation of mature triacylated lipoproteins. Lnt homologues are found in Gram-negative and high GC-rich Gram-positive, but not in low GC-rich Gram-positive bacteria, although N-acylation is observed. In fast-growing Mycobacterium smegmatis, the molecular structure of the lipid modification of lipoproteins was resolved recently as a diacylglyceryl residue carrying ester-bound palmitic acid and ester-bound tuberculostearic acid and an additional amide-bound palmitic acid. We exploit the vaccine strain Mycobacterium bovis BCG as model organism to investigate lipoprotein modifications in slow-growing mycobacteria. Using Escherichia coli Lnt as a query in BLASTp search, we identified BCG_2070c and BCG_2279c as putative lnt genes in M. bovis BCG. Lipoproteins LprF, LpqH, LpqL and LppX were expressed in M. bovis BCG and BCG_2070c lnt knock-out mutant and lipid modifications were analyzed at molecular level by matrix-assisted laser desorption ionization time-of-flight/time-of-flight analysis. Lipoprotein N-acylation was observed in wildtype but not in BCG_2070c mutants. Lipoprotein N- acylation with palmitoyl and tuberculostearyl residues was observed. Lipoproteins are triacylated in slow-growing mycobacteria. BCG_2070c encodes a functional Lnt in M. bovis BCG. We identified mycobacteria-specific tuberculostearic acid as further substrate for N-acylation in slow-growing mycobacteria.

Heme oxygenase and carbon monoxide protect from muscle dystrophy.

Science.gov (United States)

Chan, Mun Chun; Ziegler, Olivia; Liu, Laura; Rowe, Glenn C; Das, Saumya; Otterbein, Leo E; Arany, Zoltan

2016-11-28

Duchenne muscle dystrophy (DMD) is one of the most common lethal genetic diseases of children worldwide and is 100% fatal. Steroids, the only therapy currently available, are marred by poor efficacy and a high side-effect profile. New therapeutic approaches are urgently needed. Here, we leverage PGC-1α, a powerful transcriptional coactivator known to protect against dystrophy in the mdx murine model of DMD, to search for novel mechanisms of protection against dystrophy. We identify heme oxygenase-1 (HO-1) as a potential novel target for the treatment of DMD. Expression of HO-1 is blunted in the muscles from the mdx murine model of DMD, and further reduction of HO-1 by genetic haploinsufficiency worsens muscle damage in mdx mice. Conversely, induction of HO-1 pharmacologically protects against muscle damage. Mechanistically, HO-1 degrades heme into biliverdin, releasing in the process ferrous iron and carbon monoxide (CO). We show that exposure to a safe low dose of CO protects against muscle damage in mdx mice, as does pharmacological treatment with CO-releasing molecules. These data identify HO-1 and CO as novel therapeutic agents for the treatment of DMD. Safety profiles and clinical testing of inhaled CO already exist, underscoring the translational potential of these observations.

Iron and Zinc Complexes of Bulky Bis-Imidazole Ligands : Enzyme Mimicry and Ligand-Centered Redox Activity

NARCIS (Netherlands)

Folkertsma, E.

2016-01-01

The research described in this thesis is directed to the development of cheap and non-toxic iron-based homogeneous catalysts, using enzyme models and redox non-innocent ligands. Inspired by nature, the first approach focuses on the synthesis of structural models of the active site of non-heme iron

A rapid, simple method for obtaining radiochemically pure hepatic heme

International Nuclear Information System (INIS)

Bonkowski, H.L.; Bement, W.J.; Erny, R.

1978-01-01

Radioactively-labelled heme has usually been isolated from liver to which unlabelled carrier has been added by long, laborious techniques involving organic solvent extraction followed by crystallization. A simpler, rapid method is devised for obtaining radiochemically-pure heme synthesized in vivo in rat liver from delta-amino[4- 14 C]levulinate. This method, in which the heme is extracted into ethyl acetate/glacial acetic acid and in which porphyrins are removed from the heme-containing organic phase with HCl washes, does not require addition of carrier heme. The new method gives better heme recoveries than and heme specific activities identical to, those obtained using the crystallization method. In this new method heme must be synthesized from delta-amino[4- 14 C]levulinate; it is not satisfactory to use [2- 14 C]glycine substrate because non-heme counts are isolated in the heme fraction. (Auth.)

Heme-containing enzymes and inhibitors for tryptophan metabolism.

Science.gov (United States)

Yan, Daojing; Lin, Ying-Wu; Tan, Xiangshi

2017-09-20

Iron-containing enzymes such as heme enzymes play crucial roles in biological systems. Three distinct heme-containing dioxygenase enzymes, tryptophan 2,3-dioxygenase (TDO), indoleamine 2,3-dioxygenase 1 (IDO1) and indoleamine 2,3-dioxygenase 2 (IDO2) catalyze the initial and rate-limiting step of l-tryptophan catabolism through the kynurenine pathway in mammals. Overexpression of these enzymes causes depletion of tryptophan and the accumulation of metabolic products, which contributes to tumor immune tolerance and immune dysregulation in a variety of disease pathologies. In the past few decades, IDO1 has garnered the most attention as a therapeutic target with great potential in cancer immunotherapy. Many potential inhibitors of IDO1 have been designed, synthesized and evaluated, among which indoximod (d-1-MT), INCB024360, GDC-0919 (formerly NLG-919), and an IDO1 peptide-based vaccine have advanced to the clinical trial stage. However, recently, the roles of TDO and IDO2 have been elucidated in immune suppression. In this review, the current drug discovery landscape for targeting TDO, IDO1 and IDO2 is highlighted, with particular attention to the recent use of drugs in clinical trials. Moreover, the crystal structures of these enzymes, in complex with inhibitors, and the mechanisms of Trp catabolism in the first step, are summarized to provide information for facilitating the discovery of new enzyme inhibitors.

Iron absorption from adequate Filipino meals

International Nuclear Information System (INIS)

Trinidad, T.P.; Madriaga, J.R.; Valdez, D.H.; Cruz, E.M.; Mallillin, A.C.; Sison, C.C.; Kuizon, M.D.

1991-01-01

Iron absorption from adequate Filipino meals representing the three major island groups of the Philippines (Luzon, Visayas and Mindanao) was studied using double isotope extrinsic tag method. Mean iron absorption of the one-day meal for Metro Manila was 6.6 ± 1.26%, Central Visayas, 6.3 ± 1.15% and Southern Mindanao, 6.4 ± 1.19%. Comparison between meals (breakfast, lunch, dinner) for each region as well as one-day meal for the three regions showed no significant differences (P > .01). Correlation tests done between iron absorption and the following iron enhancers: ascorbic acid, amount of fish, meat or poultry and inhibitors: phytic acid and tannic acid did not give significant results. The overall bar x of 6.4 ± 1.20% may be used as the non-heme iron absorption level from an adequate Filipino meal. This value can be considered as one of the bases for arriving at recommended dietary allowances for iron among Filipinos instead of the 10% iron absorption assumed in 1976

Human heme oxygenase-1 gene transfer lowers blood pressure and promotes growth in spontaneously hypertensive rats.

Science.gov (United States)

Sabaawy, H E; Zhang, F; Nguyen, X; ElHosseiny, A; Nasjletti, A; Schwartzman, M; Dennery, P; Kappas, A; Abraham, N G

2001-08-01

Heme oxygenase (HO) catalyzes the conversion of heme to biliverdin, with release of free iron and carbon monoxide. Both heme and carbon monoxide have been implicated in the regulation of vascular tone. A retroviral vector containing human HO-1 cDNA (LSN-HHO-1) was constructed and subjected to purification and concentration of the viral particles to achieve 5x10(9) to 1x10(10) colony-forming units per milliliter. The ability of concentrated infectious viral particles to express human HO-1 (HHO-1) in vivo was tested. A single intracardiac injection of the concentrated infectious viral particles (expressing HHO-1) to 5-day-old spontaneously hypertensive rats resulted in functional expression of the HHO-1 gene and attenuation of the development of hypertension. Rats expressing HHO-1 showed a significant decrease in urinary excretion of a vasoconstrictor arachidonic acid metabolite and a reduction in myogenic responses to increased intraluminal pressure in isolated arterioles. Unexpectedly, HHO-1 chimeric rats showed a simultaneous significant proportionate increase in somatic growth. Thus, delivery of HHO-1 gene by retroviral vector attenuates the development of hypertension and promotes body growth in spontaneously hypertensive rats.

Cyanide binding to human plasma heme-hemopexin: A comparative study

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Ascenzi, Paolo, E-mail: ascenzi@uniroma3.it [Laboratorio Interdipartimentale di Microscopia Elettronica, Universita Roma Tre, Roma (Italy); Istituto Nazionale di Biostrutture e Biosistemi, Roma (Italy); Leboffe, Loris [Istituto Nazionale di Biostrutture e Biosistemi, Roma (Italy); Polticelli, Fabio [Dipartimento di Biologia, Universita Roma Tre, Roma (Italy)

2012-11-16

Highlights: Black-Right-Pointing-Pointer Cyanide binding to ferric HHPX-heme-Fe. Black-Right-Pointing-Pointer Cyanide binding to ferrous HHPX-heme-Fe. Black-Right-Pointing-Pointer Dithionite-mediated reduction of ferric HHPX-heme-Fe-cyanide. Black-Right-Pointing-Pointer Cyanide binding to HHPX-heme-Fe is limited by ligand deprotonation. Black-Right-Pointing-Pointer Cyanide dissociation from HHPX-heme-Fe-cyanide is limited by ligand protonation. -- Abstract: Hemopexin (HPX) displays a pivotal role in heme scavenging and delivery to the liver. In turn, heme-Fe-hemopexin (HPX-heme-Fe) displays heme-based spectroscopic and reactivity properties. Here, kinetics and thermodynamics of cyanide binding to ferric and ferrous hexa-coordinate human plasma HPX-heme-Fe (HHPX-heme-Fe(III) and HHPX-heme-Fe(II), respectively), and for the dithionite-mediated reduction of the HHPX-heme-Fe(III)-cyanide complex, at pH 7.4 and 20.0 Degree-Sign C, are reported. Values of thermodynamic and kinetic parameters for cyanide binding to HHPX-heme-Fe(III) and HHPX-heme-Fe(II) are K = (4.1 {+-} 0.4) Multiplication-Sign 10{sup -6} M, k{sub on} = (6.9 {+-} 0.5) Multiplication-Sign 10{sup 1} M{sup -1} s{sup -1}, and k{sub off} = 2.8 Multiplication-Sign 10{sup -4} s{sup -1}; and H = (6 {+-} 1) Multiplication-Sign 10{sup -1} M, h{sub on} = 1.2 Multiplication-Sign 10{sup -1} M{sup -1} s{sup -1}, and h{sub off} = (7.1 {+-} 0.8) Multiplication-Sign 10{sup -2} s{sup -1}, respectively. The value of the rate constant for the dithionite-mediated reduction of the HHPX-heme-Fe(III)-cyanide complex is l = 8.9 {+-} 0.8 M{sup -1/2} s{sup -1}. HHPX-heme-Fe reactivity is modulated by proton acceptor/donor amino acid residue(s) (e.g., His236) assisting the deprotonation and protonation of the incoming and outgoing ligand, respectively.

Effect of Ring Size in ω-Alicyclic Fatty Acids on the Structural and Dynamical Properties Associated with Fluidity in Lipid Bilayers.

Science.gov (United States)

Poger, David; Mark, Alan E

2015-10-27

Fatty acids containing a terminal cyclic group such as cyclohexyl and cycloheptyl are commonly found in prokaryotic membranes, especially in those of thermo-acidophilic bacteria. These so-called ω-alicyclic fatty acids have been proposed to stabilize the membranes of bacteria by reducing the fluidity in membranes and increasing lipid packing and lipid chain order. In this article, molecular dynamics simulations are used to examine the effect of 3- to 7-membered cycloalkyl saturated and unsaturated (cyclopent-2-enyl and phenyl) rings in ω-alicyclic fatty acyl chains on the structure (lipid packing, lipid chain order, and fraction of gauche defects in the chains) and dynamics (lateral lipid diffusion) of a model lipid bilayer. It was found that ω-alicyclic chains in which the ring was saturated reduced lipid condensation and lowered chain order which would be associated with enhanced fluidity. However, this effect was limited. The lateral diffusion of the lipids diminished as the ring size increased. In particular, ω-cyclohexyl and ω-cycloheptyl acyl tails led to a decrease in lipid diffusion. In contrast, ω-alicyclic acyl chains that contain an unsaturated ring promoted membrane fluidity both in terms of changes in membrane structure and lipid diffusion. This may indicate that saturated and unsaturated terminal rings in ω-alicyclic fatty acids fulfill alternative functions within membranes. Overall, the simulations suggest that ω-alicyclic fatty acids in which the terminal ring is saturated might protect the membrane of thermo-acidophilic bacteria from high-temperature and low-pH conditions through a "dynamical barrier" that would limit lipid diffusion and transmembrane diffusion of undesired ions and molecules.

Evolutionary view of acyl-CoA diacylglycerol acyltransferase (DGAT, a key enzyme in neutral lipid biosynthesis

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Margis-Pinheiro Marcia

2011-09-01

Full Text Available Abstract Background Triacylglycerides (TAGs are a class of neutral lipids that represent the most important storage form of energy for eukaryotic cells. DGAT (acyl-CoA: diacylglycerol acyltransferase; EC 2.3.1.20 is a transmembrane enzyme that acts in the final and committed step of TAG synthesis, and it has been proposed to be the rate-limiting enzyme in plant storage lipid accumulation. In fact, two different enzymes identified in several eukaryotic species, DGAT1 and DGAT2, are the main enzymes responsible for TAG synthesis. These enzymes do not share high DNA or protein sequence similarities, and it has been suggested that they play non-redundant roles in different tissues and in some species in TAG synthesis. Despite a number of previous studies on the DGAT1 and DGAT2 genes, which have emphasized their importance as potential obesity treatment targets to increase triacylglycerol accumulation, little is known about their evolutionary timeline in eukaryotes. The goal of this study was to examine the evolutionary relationship of the DGAT1 and DGAT2 genes across eukaryotic organisms in order to infer their origin. Results We have conducted a broad survey of fully sequenced genomes, including representatives of Amoebozoa, yeasts, fungi, algae, musses, plants, vertebrate and invertebrate species, for the presence of DGAT1 and DGAT2 gene homologs. We found that the DGAT1 and DGAT2 genes are nearly ubiquitous in eukaryotes and are readily identifiable in all the major eukaryotic groups and genomes examined. Phylogenetic analyses of the DGAT1 and DGAT2 amino acid sequences revealed evolutionary partitioning of the DGAT protein family into two major DGAT1 and DGAT2 clades. Protein secondary structure and hydrophobic-transmembrane analysis also showed differences between these enzymes. The analysis also revealed that the MGAT2 and AWAT genes may have arisen from DGAT2 duplication events. Conclusions In this study, we identified several DGAT1 and DGAT2

Evolutionary view of acyl-CoA diacylglycerol acyltransferase (DGAT), a key enzyme in neutral lipid biosynthesis.

Science.gov (United States)

Turchetto-Zolet, Andreia C; Maraschin, Felipe S; de Morais, Guilherme L; Cagliari, Alexandro; Andrade, Cláudia M B; Margis-Pinheiro, Marcia; Margis, Rogerio

2011-09-20

Triacylglycerides (TAGs) are a class of neutral lipids that represent the most important storage form of energy for eukaryotic cells. DGAT (acyl-CoA: diacylglycerol acyltransferase; EC 2.3.1.20) is a transmembrane enzyme that acts in the final and committed step of TAG synthesis, and it has been proposed to be the rate-limiting enzyme in plant storage lipid accumulation. In fact, two different enzymes identified in several eukaryotic species, DGAT1 and DGAT2, are the main enzymes responsible for TAG synthesis. These enzymes do not share high DNA or protein sequence similarities, and it has been suggested that they play non-redundant roles in different tissues and in some species in TAG synthesis. Despite a number of previous studies on the DGAT1 and DGAT2 genes, which have emphasized their importance as potential obesity treatment targets to increase triacylglycerol accumulation, little is known about their evolutionary timeline in eukaryotes. The goal of this study was to examine the evolutionary relationship of the DGAT1 and DGAT2 genes across eukaryotic organisms in order to infer their origin. We have conducted a broad survey of fully sequenced genomes, including representatives of Amoebozoa, yeasts, fungi, algae, musses, plants, vertebrate and invertebrate species, for the presence of DGAT1 and DGAT2 gene homologs. We found that the DGAT1 and DGAT2 genes are nearly ubiquitous in eukaryotes and are readily identifiable in all the major eukaryotic groups and genomes examined. Phylogenetic analyses of the DGAT1 and DGAT2 amino acid sequences revealed evolutionary partitioning of the DGAT protein family into two major DGAT1 and DGAT2 clades. Protein secondary structure and hydrophobic-transmembrane analysis also showed differences between these enzymes. The analysis also revealed that the MGAT2 and AWAT genes may have arisen from DGAT2 duplication events. In this study, we identified several DGAT1 and DGAT2 homologs in eukaryote taxa. Overall, the data show that

Synthetic heme/copper assemblies: toward an understanding of cytochrome c oxidase interactions with dioxygen and nitrogen oxides.

Science.gov (United States)

Hematian, Shabnam; Garcia-Bosch, Isaac; Karlin, Kenneth D

2015-08-18

Our long-time niche in synthetic biological inorganic chemistry has been to design ligands and generate coordination complexes of copper or iron ions or both, those reacting with dioxygen (O2) or nitrogen oxides (e.g., nitric oxide (NO(g)) and nitrite (NO2(-))) or both. As inspiration for this work, we turn to mitochondrial cytochrome c oxidase, which is responsible for dioxygen consumption and is also the predominant target for NO(g) and nitrite within mitochondria. In this Account, we highlight recent advances in studying synthetic heme/Cu complexes in two respects. First, there is the design, synthesis, and characterization of new O2 adducts whose further study will add insights into O2 reductive cleavage chemistry. Second, we describe how related heme/Cu constructs reduce nitrite ion to NO(g) or the reverse, oxidize NO(g) to nitrite. The reactions of nitrogen oxides occur as part of CcO's function, which is intimately tied to cellular O2 balance. We had first discovered that reduced heme/Cu compounds react with O2 giving μ-oxo heme-Fe(III)-O-Cu(II)(L) products; their properties are discussed. The O-atom is derived from dioxygen, and interrogations of these systems led to the construction and characterization of three distinctive classes of heme-peroxo complexes, two high-spin and one low-spin species. Recent investigations include a new approach to the synthesis of low-spin heme-peroxo-Cu complexes, employing a "naked" synthon, where the copper ligand denticity and geometric types can be varied. The result is a collection of such complexes; spectroscopic and structural features (by DFT calculations) are described. Some of these compounds are reactive toward reductants/protons effecting subsequent O-O cleavage. This points to how subtle improvements in ligand environment lead to a desired local structure and resulting optimized reactivity, as known to occur at enzyme active sites. The other sector of research is focused on heme/Cu assemblies mediating the redox