Haem Biosynthesis in Isolated Human Erythroblasts

Hepatic haem biosynthesis has been well characterised. The first enzyme of the haem biosynthetic pathway, 5-aminolaevulinic acid (ALA) synthase, is rate-limiting and under negative feedback control by haem. Liver is, however, a relatively minor site of haem synthesis as 80% of haem is formed in the erythroid cells of the bone marrow. Within developing erythroid cells haem is required both for its specific complexing with globin and for the coordinate regulation of erythroblast metabolism and differentiation. The study of erythroid haem synthesis has been hampered by the heterogeneous nature of the marrow cell population, the various differentiation stages of the erythroblast population, the small sample sizes available and the lack of sensitive enzyme assays. A variety of models and techniques have been used to study erythroid haem synthesis. Accordingly, the results have been inconclusive. This work describes human bone marrow fractionation to provide four purified age-matched erythroblast cell populations. Myeloid (white) cells were removed from the sample by monoclonal antibody mediated cell lysis. The remaining cells were then fractionated by equilibrium density gradient centrifugation. These 'in vivo' human erythroblasts were used to examine the effects of normal and abnormal erythroid differentiation on the first and last enzymes of the haem pathway, ALA synthase and ferrochelatase. A sensitive radiochemical assay for ALA synthase was adopted and improved. This was then used to examine the temperature-dependent inactivation of erythroid ALA synthase. A novel radiochemical assay for ferrochelatase was developed. Both assays utilise HPLC for isolation of the radioactive product and are capable of detecting picomoles of activity. The pattern of ALA synthase and ferrochelatase activities during normoblastic erythropoiesis was established. ALA synthase activity was maximal in the most immature erythroid cells and diminished as the cells matured. Ferrochelatase activity was maximal in the intermediate erythroid cells. Hence, the development of enzyme activity appears to be sequential rather than simultaneous. On a quantitative basis, however, it is uncertain whether the development of ferrochelatase activity is limiting for haem formation. The effects of iron and haem deficiency on haem enzyme activity were studied. In iron deficient erythroblasts ALA synthase activity was significantly reduced, particularly in the most immature erythroid cells. This enzyme reduction is likely to result from consequent haem deficiency rather than iron deficiency per se as an iron replete patient with haem deficiency associated with hepatoerythropoietic porphyria also showed reduced enzyme activity. This suggests that erythroid haem synthesis is controlled in a different manner to hepatic haem synthesis - a view compatible with the recent identification of specific genes and tissue-specific isoenzymes for ALA synthase. ALA synthase and ferrochelatase activities were measured in patients with sideroblastic anaemia (SA). Haem synthesis is impaired in SA and ALA synthase is generally believed to be the site of the primary defect. ALA synthase activity was markedly impaired in 3 patients with congenital SA (CSA) and 7 patients with primary acquired SA (PASA). One patient with secondary SA (SSA) demonstrated normal activity. Ferrochelatase activity was measured in six patients (1 CSA, 5 PASA) and was found to be significantly reduced. The latter 6 patients (1 CSA, 5 PASA) were treated with haem arginate in an attempt to correct the haem deficiency and to stimulate haem enzyme activity. Haem arginate was clinically ineffective (Hb levels did not change in any of the patients). However, the study demonstrated that exogenous haem is able to enter erythroblasts and influence haem enzyme activity. In two patients (1 CSA, 1 PASA), both ALA synthase and ferrochelatase activities returned to normal. This indicates a potential role for haem arginate in the treatment of haematological disorders other than SA which may respond to stimulated haem biosynthesis. The results also suggest that the primary abnormality in these cases of CSA and PASA is not ALA synthase (or ferrochelatase) deficiency. Furthermore, the stimulatory effect of exogenous haem on enzyme activity and the reduced ALA synthase activity in haem deficiency provide strong evidence of independent regulatory mechanisms for haem biosynthesis in erythroid and hepatic tissue

Experimental Studies on Bilirubin and Haem Biosynthesis

It has recently been shown that patients with unconjugated hyperbilirubinaemia due to Gilbert's syndrome have reduced activity of the enzyme protoporphyrinogen oxidase in circulating leucocytes. This may be explained by the further observation that unconjugated bilirubin competes with protoporphyrinogen for binding to protoporphyrinogen oxidase. In order to further investigate the effect of unconjugated hyperbilirubinaemia on porphyrin metabolism and haem biosynthesis studies have been performed in the Gunn rat which has unconjugated hyperbil irubinaemia and raised plasma bilirubin concentrations. Protoporphyrinogen oxidase activity was found to be reduced in the liver of the Gunn rat compared to heterozygous controls with normal plasma bilirubin concentrations. This reduction of protoporphyrinogen oxidase activity was not accompanied by any increase in the activity of the initial and rate-controlling enzyme of the pathway delta-aminolaevulinic acid synthase. In contrast to hepatic tissue, the activities of both protoporphyrinogen oxidase and delta-aminolaevulinic acid synthase were normal in renal tissue of the Gunn rat. Porphyrin excretion was reduced in the Gunn rat compared to that in heterozygous controls. Further studies were performed to investigate the effects of unconjugated hyperbilirubinaemia on haem biosynthesis in the brain of the Gunn rat. This was studied as the major effect of hyperbilirubinaemia is brain damage and the mechanism by which it occurs is unknown. Inherited partial enzyme deficiencies in haem biosynthesis are known to produce neurological damage. Therefore the possibility that bilirubin induced brain damage might be due to inhibition of protoporphryinogen oxidase activity and hence deficient neuronal haem synthesis, was investigated. Optimal conditions for the measurement of protoporphyrinogen oxidase activity were determined in the brain and found to be identical to those which provided optimal activity in hepatic tissue. The activity of protoporphyrinogen oxidase in brain tissue homogenates from Gunn rats was similar to that in heterozygous rats with normal bilirubin concentrations. This was also the case in neonatal Gunn rats in whom kernicterus had been induced by the displacement of bilirubin into the brain by sulphonamide treatment. Delta-aminolaevulinic acid synthase activities were also similar in the homozygous Gunn rat and heterozygous controls. These findings make it unlikely that the brain damage induced by hyperbilirubinaemia is due to inhibition of protoporphyrinogen oxidase activity and impaired neuronal haem biosynthesis. The above studies concerned the effect of disturbed haem catabolism on haem biosynthesis. The latter part of the studies extended the theme of the investigation by examining the effect of the administration of Tin-protoporphyrin, an inhibitor of haem biosynthesis, on haem biosynthesis in acute porphyria. The effect of administering Tin-protoporphyrin and haem arginate alone and in combination on succinyl acetone induced porphyria in rats was studied. This animal model proved to be unsatisfactory as the haem arginate given alone did not significantly reduce the over-production of the porphyrin precursor, delta-aminolaevulinate. This may be explained by inhibition of aminolaevulinic acid dehydratase by succinyl acetone so increasing aminolaevulinic acid overproduction in most body tissues. The effect of the haem arginate is largely confined to the liver. Studies in 1 patient during clinical attacks proved more encouraging. They showed that the coadministration of Tin-protoporphyrin with haem arginate prolonged both the biochemical response and clinical remission induced by haem arginate therapy. These findings indicate that co-administration of Tin-protoporphyrin and haem arginate may be used as prophylactic therapy in patients experiencing recurrent attacks of acute porphyria

Der Einfluss von Sn-Mesoporphyrin, einem Inhibitor der Hämoxygenase, auf myokardiales Stunning bei wachen, chronisch instrumentierten Hunden

In dieser Studie wurden die Auswirkungen der pharmakologischen Blockade der Hämoxygenase mittels Sn-mesoporphyrin auf myokardiales Stunning bei wachen, chronisch instrumentierten Hunden untersucht. Es wurden sechs Hunde der Rasse „Foxhound“ in Allgemeinanästhesie instrumentiert. Messsonden wurden eingebracht, mit deren Hilfe Herzfrequenz, arterieller Blutdruck, linksatrialer Druck, links-ventrikulärer Druck, Blutflussgeschwindigkeit im Ramus interventricularis anterior (RIVA) der linken Koronararterie und die myokardiale Wanddickenfraktion (WDF) aufgezeichnet werden konnten. Um den RIVA wurden Ballonokkluder gelegt, um kurzzeitige Ischämien im RIVA-perfundierten Myokardbereich zu induzieren. Die regionale myokardiale Perfusion konnte mit Hilfe gefärbter Mikrosphären postmortal bestimmt werden

Estudios sobre la síntesis enzimática de porfobilinógeno a partir del precursor delta aminolevúlico : Aminolevúlico dehidrasa

Fil: Bustos, Norma Lidia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Effect of isothiocyanates, BITC and PEITC, on stress protein accumulation, protein aggregation and aggresome-like structure formation in Xenopus A6 kidney epithelial cells

The final publication is available at Elsevier via https://doi.org/10.1016/j.cbpc.2017.10.011 © 2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/Numerous studies have elucidated the health benefits of organosulfur compounds, known as isothiocyanates (ITCs), derived from cruciferous vegetables. As electrophiles, ITCs have the ability to directly bind and modify thiol-containing compounds such as glutathione and cellular protein, including tubulin. While the biochemical effects of ITCs have been well characterized, less information is available regarding their effects on the accu- mulation of stress-inducible heme oxygenase-1 (HO-1), heat shock proteins (HSPs) and the possible formation of aggregated protein due to thiol modification. The present study has examined the effect of the ITCs, benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC), on the accumulation of HO-1, HSP70 and HSP30 in Xenopus laevis A6 kidney epithelial cells. Immunoblot analysis revealed that both BITC and PEITC induced the accumulation of HO-1 and HSP70 whereas HSP30 levels were enhanced only in cells treated with BITC. Immunocytochemistry determined that ITC treatment induced F-actin disorganization and membrane ruffling and enhanced accumulation of HO-1 in the cytoplasm. Additionally, BITC induced enhanced levels of ubiqui- tinated protein, aggregated protein, and the collapse and fragmentation of microtubules. In comparison, treat- ment of cells with the proteasomal inhibitor, MG132, induced the accumulation of all three stress proteins, aggregated protein and aggresome-like structures. Finally, cells pretreated with BITC inhibited the formation of MG132-induced aggresome-like structures in the perinuclear region. This latter finding suggests that BITC-in- duced microtubule fragmentation may impede the movement of aggregated protein via microtubules and their subsequent coalescence into aggresome-like structures in the perinuclear region.Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery grant (RGPIN/2014- 04376

No- and co-mediated inhibition of mitochondrial respiration in activated macrophages.

Nitric oxide (NO) is a gaseous signalling molecule that is synthesised by nitric oxide synthases (NOSs) and has a variety of physiological and pathophysiological roles. Some of its physiological functions are primarily mediated via the activation of soluble guanylate cyclase. However, NO is also able to potently inhibit mitochondrial respiration at complex IV of the electron transport chain (ETC) in a manner that is reversible and in competition with oxygen (O2). Following prolonged exposure to NO, the ETC undergoes an NO-dependent modification at complex I, which results in a persistent inhibition of respiration. Activated macrophages were found to produce NO, via upregulation of the inducible isoform of NOS (iNOS), in sufficient quantities to inhibit respiration. This inhibition was initially reversible but became persistent with time. In addition, haem oxygenase-1 (HO-1) is upregulated in these cells, producing carbon monoxide (CO), which is also known to inhibit complex IV. The ability of exogenous and endogenous CO to inhibit respiration was investigated. Although less potent than NO, CO was shown to cause significant inhibition of respiration, particularly under hypoxic (1% O2) conditions. Furthermore, hypoxia was found to attenuate significantly the synthesis of NO from iNOS but not CO from HO-1. The consequences of inhibition of respiration were investigated under hypoxia, where the availability of O2 may be limiting for O2-dependent cellular processes. Specifically, the effects of respiratory inhibition were investigated with regards to the stability of hypoxia-induced hypoxia inducible factor la (HTFla). Hypoxia resulted in the stabilisation of HIFla protein, a phenomenon that was prevented by inhibition of the ETC at various complexes. This destabilisation of HIFla was found to be due to a redistribution of intracellular O2 from mitochondrial consumption, resulting in an increased intracellular O 2 concentration and the reactivation of the (-dependent degradation of HIFla protein

The handling of iron by erythroid and erythrophagocytic cells /

Iron is not a trace element in mammalian physiology. Using textbook values for blood volume (5.5 L), red blood cell (RBC) count (5 million/muL), and a lifespan of 120 days for red blood cells, the equilibrium value for the erythrocyte generation/death rate in the average adult male human is over 2 million/sec. It follows that the amount of iron required for hemoglobin synthesis in one day amounts to about 25 mg. Virtually every atom of that 25 mg is recycled by macrophages of the reticuloendothelial system (RES) that provide iron to the plasma for its subsequent delivery back to the erythron (with a small fraction going to other tissues). In light of the certain toxicity of unprotected iron, both erythroid precursors and RES macrophages perform remarkable tasks in handling such copious amounts of the catalytic metal. In my studies, I have examined specific aspects of iron metabolism in these two tissues.Iron is taken up by nearly every cell through a mechanism of receptor-mediated endocytosis, whereby the plasma iron binding protein transferrin (Tf) binds to its cognate receptor (TfR) on the cell surface, followed by internalization of the complex into a membrane bound organelle. Subsequent to endocytosis, the endosome is acidified by a v-ATPase proton pump, facilitating the release of iron from Tf. Through an unknown mechanism, iron is targeted to the inner membrane of the mitochondria, where the enzyme that inserts Fe2+ into protoporphyrin IX, ferrochelatase, resides. Although it has been demonstrated that the divalent metal transporter, DMT1, is responsible for the egress of reduced Fe from the vesicle, the immediate fate of the iron atoms after their transport across the vesicular membrane remains unknown. Therefore, we have investigated the uptake of iron in reticulocytes, cells that are taking up large amounts of iron for the synthesis of hemoglobin. Through both biochemical and imaging techniques, we have demonstrated that iron is transferred via a direct interorganellar relation between the endosome and mitochondria.The "haemoglobin-deficit" (hbd) mouse has an erythroid-specific mutation which is responsible for its microcytic, hypochromic phenotype. Previous studies have shown that these mice have normal dietary iron acquisition and normal to elevated serum iron levels. We therefore investigated the handling of iron in reticulocytes from these animals to determine whether the mutated gene possibly plays a role in the trafficking of transferrin-iron-containing organelles. A systematic examination of the steps in the transferrin pathway revealed that the intracellular trafficking of the protein is compromised in the hbd mice.The rapid turnover of iron by macrophages of the RES requires heme oxygenase-1 (HO-1), which catalyzes the rate-limiting step in heme degradation. This highly inducible enzyme, besides its major role in erythrocyte iron recycling, has been demonstrated to confer astonishing cytoprotectivity to cells and tissues in which its expression is elevated (either through chemical induction or genetic manipulation). In addition to, reportedly protective, carbon monoxide and biliverdin, the HO-1 catalyzed reaction releases ferrous iron, which itself is a potent pro-oxidant. Also, it is unlikely that there exists a significant amount of free heme in most tissues (i.e., non-erythroid, non-erythrophagocytic), to provide significant amounts of substrate to this enzyme. Hence, it is tempting to speculate that the mechanism of heme oxygenase cytoprotection is removed from its function of heme catabolism. Therefore, we investigated whether increased expression of heme oxygenase will, in and of itself, alter iron metabolism in cultured cells. My experiments show that in the absence of exogenous hemin, elevation of HO-1 protein levels does not have any effect on cellular iron metabolism in cultured cells

Unravelling novel modes of antimicrobial action

Dissertation presented to obtain the Ph.D. degree in Biochemistry at the Instituto de Tecnologia Química e Biológica, Universidade Nova de LisboaThe work presented in this thesis aimed at unravelling novel modes of antimicrobial action through: i) the study of Staphylococcus aureus defences against nitric oxide, an antimicrobial weapon of the innate immune system, namely by performing the biochemical characterisation of S. aureus NO-detoxifying flavohaemoglobin and analysing the conditions under which the enzyme is operative; ii) the identification of the mechanisms beyond the antibacterial activity of azole antibiotics towards S. aureus that were shown to include the increase of endogenous reactive oxygen species triggered by the binding of imidazoles to flavohaemoglobin; and iii) the discovery of a novel type of bactericides, the carbon monoxide-releasing molecules, and identification of its potential cellular targets via analysis of the transcriptional response of Escherichia coli to CORM-2, a carbon monoxide-releasing molecule known to mimic the physiological function of carbon monoxide.(...