Iron cycle disruption by heme oxygenase-1 activation leads to a reduced breast cancer cell survival.

Heme oxygenase-1 (HO-1), which catalyzes heme degradation releasing iron, regulates several processes related to breast cancer. Iron metabolism deregulation is also connected with several tumor processes. However the regulatory relationship between HO-1 and iron proteins in breast cancer remains unclear. Using human breast cancer biopsies, we found that high HO-1 levels significantly correlated with low DMT1 levels. Contrariwise, high HO-1 levels significantly correlated with high ZIP14 and prohepcidin expression, as well as hemosiderin storage. At mRNA level, we found that high HO-1 expression significantly correlated with low DMT1 expression but high ZIP14, L-ferritin and hepcidin expression. In in vivo experiments in mice with genetic overexpression or pharmacological activation of HO-1, we detected the same expression pattern observed in human biopsies. In in vitro experiments, HO-1 activation induced changes in iron proteins expression leading to an increase of hemosiderin, ROS levels, lipid peroxidation and a decrease of the growth rate. Such low growth rate induced by HO-1 activation was reversed when iron levels or ROS levels were reduced. Our findings demonstrate an important role of HO-1 on iron homeostasis in breast cancer. The changes in iron proteins expression when HO-1 is modulated led to the iron accumulation deregulating the iron cell cycle, and consequently, generating oxidative stress and low viability, all contributing to impair breast cancer progression.

Effect of surfactants and natural detergents on phosphatidylcholine synthesis in photoreceptor membranes.

The synthesis of phosphatidylcholine (PC) in rod outer segments (ROS) catalysed by lysophosphatidylcholine acyltransferase and phosphatidylethanolamine N-methyltransferase (PE N-MTase) was studied and the effects of natural (FA and lysophospholipids) and synthetic (Triton X-100, deoxycholate and CHAPS) surfactants was evaluated. In all experimental conditions used, incorporation of labelled oleate into lysophosphatidylcholine (lysoPC) was at least 40 times greater than oleate incorporation into any other lysophospholipid. Acylation of lysoPC was slightly affected by Triton X-100 and was totally inhibited in the presence of 10 mM sodium deoxycholate (NaDOC) or CHAPS. Below their critical micelle concentration (cmc) Triton X-100 and NaDOC stimulated acylation of all ROS lysophospholipids analysed. The activity of PE N-MTase was stimulated at detergent concentrations below the cmc and inhibited at concentrations above the cmc for all three detergents tested. The effect of FA with differing degree of unsaturation on PC synthesis was evaluated. Oleic acid (10 microM) inhibited methyl group incorporation into total PC, whereas from 100 microM onward, the methylating activity increased with preferential synthesis of PC. Docosahexaenoic acid, in turn, inhibited PE N-MTase activity at every concentration tested. These results suggest that PC synthesis in ROS membranes is modified by bioregulators and surfactants altering the physico-chemical state of the membrane.

Serum erythropoietin and its relation with soluble transferrin receptor in patients with different types of anaemia in a locally defined reference population.

Serum erythropoietin (Epo) and soluble transferrin receptor (sTR) were measured in a locally defined reference population (n=100): healthy volunteers (n=50); iron- deficiency anaemia (n=41) and haemolytic anaemia (n=9) (beta-thalassaemia, n = 4; autoimmune, n=5). Our data demonstrated an inverse relationship between erythroid activity and Epo levels. The regression line between Ln Epo and haemoglobin (Hb) was highly significant: P < 0.0001, r2=0.8275, Ln Epo=8.5346-0.04275 Hb, confidence limit 95%. The mean observed/predicted (O/P) ratio of Ln (Epo) was 1.01 +/- 0.11. We demonstrated that the serum Epo concentration in this particular population correlated consistently with clinical measures of erythropoietic activity. sTR, a new index of erythropoiesis, varied from 16.1 to 148 nmol/l, mean 62.0 nmol/l in the anaemic patients' group. The relationship between Ln Epo and Ln sTR was highly significant: P < 0.0001. We conclude that locally defined regression analyses are crucial for correct data interpretation and can indicate whether or not Epo production is appropriate or inappropriate. Serial determinations of sTR could help in the assessment of response to therapeutic doses of Epo.

Alternative pathways for phospholipid synthesis in different brain areas during aging.

Morphological and biochemical changes take place in the membrane of aged brain. In particular, studies on aged rats report alterations in brain phospholipid synthesis and in phospholipid-specific fatty acid composition. However, no significant changes in main phospholipid class content have been reported in aged brain, possibly owing to alterations in the alternative pathways for phospholipid synthesis during aging. Therefore, the present study was designed to determine the effect of aging on the enzyme activities responsible for phospholipid synthesis by alternative pathways. Indifferent brain areas of adult (3.5-month-old) and aged (28.5-month-old) rats we examined: 1) the activity of base exchange enzymes, which is a calcium-dependent, energy-independent and calcium stimulated enzymatic pathway; 2) phosphatidylethanolamine (PE) synthesis by phosphatidylserine decarboxylase activity (PSD); 3) phosphatidylcholine (PC) synthesis by transfer of methyl groups to endogenous PE by phosphatidylethanolamine N-methyltransferase activity (PEMT); 4) the synthesis of phosphatidylglycerol (PG) through phospholipase D (PLD) activity. Because the dependence on and the stimulation by calcium of base-exchange reactions is a well known mechanism and alterations in calcium levels in rat brain have been reported, we decided to investigate PS synthesis in the presence of endogenous and exogenous calcium (2.5mM). PS synthesis increased in cerebral cortex (CC) and cerebellum (CRBL) of aged rats with respect to adult rats in basal conditions (without the addition of exogenous calcium), but more significant changes were observed in serine base exchange activity during aging when exogenous calcium was added. PEMT activity in aged CC increased by 100%, the principal modification being observed in the first methylated product of the sequential reaction. Furthermore, the transphosphatidyl reaction was higher in aged brain as indicated by the increased PG synthesis. Our findings allow us to conclude that age affects some alternative pathways for phospholipid synthesis in the central nervous system, and indicate the presence of a compensatory mechanism to provide a pool of phospholipid classes for the maintenance of cellular membrane lipid composition during aging.

Effect of light on phosphatidate phosphohydrolase activity of retina rod outer segments: the role of transducin.

The aim of the present paper is to evaluate the modulation of phosphatidate phosphohydrolase (PAPase) and diacylglyceride lipase (DGL) activities in bovine rod outer segment (ROS) under dark and light conditions and to evaluate the role of transducin (T) in this phenomenon. In dark-adapted ROS membranes exposed to light, PAPase activity is inhibited by 20% with respect to the activity found under dark conditions. To determine whether the retinal G protein, T, participates in the regulation of PAPase activity in these membranes, the effects of GTPgammaS and GDPbetaS on enzyme activity were examined. Under dark conditions in the presence of GTPgammaS, which stabilizes T in its active form (Talpha + Tbetagamma), enzyme activity was inhibited and approached control values under light conditions. GDPbetaS, on the other hand, which stabilizes the inactive state of T (Talphabetagamma), stimulated PAPase activity by 36% with respect to control light conditions. ADP-ribosylation by cholera and pertussis toxin was also studied. In ADP-rybosilated ROS membranes with pertussis toxin under dark conditions, PAPase activity was 36% higher than the activity found under control light conditions. ADP-ribosylation by CTx, on the other hand, inhibited PAPase activity by 22%, with respect to dark control conditions, mimicking light effect. The effects of GTPgammaS and GDPbetaS and conditions of ADP-ribosylation by PTx and CTx on DGL activity were similar to those of PAPase activities. Based on NEM sensitivity we have also demonstrated that the PAPase present in ROS is the PAP 2 isoform. Our findings therefore suggest that light inhibition of PAP 2 in ROS is a transducin-mediated mechanism.

Light activation of phosphatidylethanolamine N-methyltransferase in rod outer segments and its modulation by association states of transducin.

Phosphatidylethanolamine N-Methyltransferase (PE N-MTase) is the enzyme responsible for the synthesis of phosphatidylcholine from phosphatidylethanolamine by successive transfer of methyl groups. This enzyme is present in bovine rod outer segments (ROS) and it is the only pathway for the synthesis of phosphatidylcholine in the outer segment of rod photoreceptor cells. In dark-adapted ROS membranes PE N-MTase activity is stimulated by 100% when ROS membranes are incubated under light condition. To determine whether the retinal G protein, transducin (Gt), intervenes in the regulation of PE N-MTase in these membranes, the effects of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) and guanosine 5'-O-(2-thiodiphosphate (GDPbetaS) on the enzyme activity were examined. In dark, GTPgammaS which induces dissociation of Gt, stimulates the enzyme activity mimicking the stimulation by light. On the contrary, GDPbetaS stabilizes the inactive state of Gt, inhibiting the stimulation by light of PE N-MTase without affecting basal activities. In addition, adenosine 5'-diphosphate (ADP)-ribosylation by cholera and pertussis toxin was studied. ADP-ribosylation of ROS membrane with pertussis toxin, which stabilizes transducin in its inactive state, prevents the light-induced increase in PE N-MTase activity. On the contrary ADP-ribosylation with cholera toxin stimulates the enzyme activity. Our findings therefore suggest that light-stimulated effect of PE N-MTase activity is transducin-mediated.

Phosphorylation of rod outer segment proteins modulates phosphatidylethanolamine N-methyltransferase and phospholipase A2 activities in photoreceptor membranes.

The activities of enzymes involved in lipid metabolism--phospholipase A2 (PLA2) and phosphatidylethanolamine N-methyltransferase (PE N-MTase)--were found to be differently affected by pre-incubation of rod outer segments (ROS) under protein phosphorylating or dephosphorylating conditions. Exposure to cAMP-dependent protein kinase (PKA), under dark or light conditions, produced a significant increase in PE N-MTase activity, whereas PLA2 activity decreased. Under standard protein kinase C (PKC) phosphorylating conditions in light, PE N-MTase activity was stimulated and PLA2 activity was not affected. When the assays were performed in the dark, both enzymatic activities were unaffected when compared to the corresponding controls. Incubation of ROS membranes in light in the presence of PKC activators phorbol 12,13-dibutyrate (PDBu) and dioctanoylglycerol (DOG) resulted in the same pattern of changes in enzyme activities as described for standard PKC phosphorylating condition. Pre-incubation of membranes with the PKC inhibitor H-7 reduced the stimulation of PDBu on PE N-MTase activity, and had no effect on PLA2 activity in ROS membranes incubated with the phorbol ester. Pre-treatment of isolated ROS with alkaline phosphatase resulted in decreased PE N-MTase activity and produced a significant stimulation of PLA2 activity under dark as well as under light conditions when compared to the corresponding controls. These findings suggest that ROS protein phosphorylation and dephosphorylation modulates PE N-MTase and PLA2 activities in isolated ROS, and that these activities are independently and specifically modulated by particular kinases. Furthermore, dephosphorylation of ROS proteins has the opposite effect to that produced by protein phosphorylation on the enzymes studied.

Can phosphorylation and dephosphorylation of rod outer segment membranes affect phosphatidate phosphohydrolase and diacylglycerol lipase activities?

Phosphatidate phosphohydrolase (PAPase) and diacylglycerol lipase (DGL) enzymatic activities were found to be differently affected by preincubation of rod outer segments (ROS) under protein phosphorylation or dephosphorylation conditions in darkness or in light. Under protein kinase C (PKC) phosphorylation conditions, PAPase and DGL were inhibited in darkness and in light. The inhibitory effect on PAPase and DGL activities by PKC phosphorylation in the presence of light was more pronounced when the activities were compared with the activities in control membranes determined in the presence of EGTA. The addition of PKC activators such as phorbol-12,13-dibutyrate and dioctanoylglycerol (DOG) instead of DG produced the same pattern of changes in enzymatic activities. Pretreatment of ROS membranes with cAMP-dependent protein kinase (PKA) produced a significant increase in both enzymatic activities in the presence of light. No changes were observed when ROS proteins were phosphorylated by PKA in the dark. Dephosphorylation of ROS membranes with alkaline phosphatase resulted in a decrease in PAPase activity that was more marked under light than under dark conditions. DGL activity was not modified under dephosphorylation conditions. These findings suggest that the metabolization of phosphatidic acid in isolated ROS is differently affected by protein phosphorylation and dephosphorylation reactions.

Lipid metabolism in photoreceptor membranes: regulation and mechanisms.

Lipid metabolism in photoreceptor rod outer segments has attracted considerable attention because of its importance in providing the appropriate environment for supporting an efficient phototransduction mechanism. Recent studies suggest that lipid metabolism in these membranes is involved in the generation of second messengers and in signal transduction mechanisms. Phospholipid turnover is tightly regulated by phosphorylation-dephosphorylation reactions and light, and provides, in turn, with molecules capable of activating protein kinases and cellular processes such as membrane fusion or light-adaptation. These findings suggest that photoreceptor membrane lipids are more than just important structural components of the visual cell rod outer segment.

Phosphatidylethanolamine N-methyltransferase activity in isolated rod outer segments from bovine retina.

Phosphatidylcholine (PC) can be synthesized in isolated rod outer segments from bovine retina by successive transfer of methyl groups from S-adenosyl-L-methionine (SAM) to phosphatidylethanolamine (PE), with the intermediate formation of phosphatidyl-N-monomethylethanolamine (PMME) and phosphatidyl-N,N-dimethylethanolamine (PDME). This reaction is time-protein-and SAM concentration-dependent. Phosphatidylethanolamine N-methyltransferase (PE N-MTase) has two pH optima, 8.5 and 10, at low (10 microM) and high (200 microM) SAM concentrations and requires magnesium ions for full activity. When ROS membranes were incubated at 5 to 200 microM SAM concentrations at pH 8.5 or pH 10, the major methylated product was PMME, followed by PC and PDME. The apparent Kms for SAM at pH 8.5 and at pH 10 were similar (37 and 38 microM, respectively). The Vmax was 13 pmol h-1 (mg protein)-1 at pH 8.5 and 12.50 pmol h-1 (mg protein)-1 at pH 10. Pulse-chase experiments demonstrated a precursor-product relationship with [3H]PC as the end product. The level of PE N-Mtase activity in the purified ROS preparation obtained from crude ROS fractions by discontinuous sucrose gradient centrifugation, was as high as 65% of the level found in the microsomal fraction obtained from the remainder of the retinas. The presence of microsomal and mitochondrial marker enzymes, however, was minimal in the ROS preparation. The radioactivity incorporated into ROS PC was measured in an upper and lower band of PC obtained by two-dimensional TLC. We found that the amount of [methyl-3H] groups incorporated into the upper PC band was 2.5-fold greater than that incorporated into the lower one. The fatty acid composition of the upper band was very different from that of the lower band, the former being enriched in very long-chain polyunsaturated fatty acids and the latter in saturated fatty acids. Phosphatidyl-ethanolamine N-methyltransferase activity increased in the presence of exogenous phospholipid substrates. PDME being augmented ten-fold and PC eight-fold when the incubations were carried out in the presence of PMME and PDME, respectively. At a 2 mM concentration, S-adenosyl-L-homocysteine (SAH) inhibited the methyl groups' incorporation into the endogenous phospholipids by 40%. When ROS membranes were selectively depleted of soluble or peripheral and soluble proteins, the PE N-MTase activity remained mainly associated to the membrane, suggesting that this enzyme (s) is an intrinsic membrane protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of aging on the content, composition and synthesis of sphingomyelin in the central nervous system.

Sphingomyelin (SPH) content and composition in different regions of the brain were analyzed in 2.5, 21.5 and 26.5-month-old rats. SPH content increased in the cerebral hemispheres, cerebellum and medulla oblongata plus pons as age increased. The highest SPH content was observed in 26.5-month-old rats, with values increasing by 1.74, 2.75 and 0.88-fold, respectively, over 2.5-month-old rats. The SPH fatty acid composition of brains from aged rats was markedly different from that of adult rats. Between 2.5 and 26.5 months of age the monoenoic/saturated fatty acid ratio increased from 0.22, 0.30 and 0.54 to 0.54, 0.68 and 1.03 in cerebral hemispheres, cerebellum and medulla oblongata plus pons, respectively. The percentage and content of fatty acids longer than 22 carbon atoms esterified to SPH increased with age from 18, 26 and 44 to 48, 52 and 62 mole % in cerebral hemispheres, cerebellum and medulla oblongata plus pons in 26.5-month-old rats. In subcortical white matter from aged rats, monoenoic 22-26 carbon atom fatty acids increased more than the saturated ones in 21.5-month-old rats relative to 2.5-month-old rats. In vitro synthesis of SPH from [3H]choline and [3H]palmitic acid in cerebral cortex and cerebellum showed no significant differences between adult rats and those 21.5 months of age. In cerebellum and in cerebral cortex, [14C]serine incorporation increased in aged rats. The results suggest that aging induces increases in both SPH content and in the monoenoic/saturated fatty acid ratio. These increases are quantitatively different in all brain regions analyzed.

Isolation and characterization of acetylcholine receptor membrane-associated (nonreceptor v2-protein) and soluble electrocyte creatine kinases.

Creatine kinase has been identified as a most prominent component of Torpedo electric organ and a minority constituent of the acetylcholine receptor (AChR) membranes obtained therefrom. Purification by low temperature ethanol extraction, precipitation of the Mg2+-enzyme complex, and mercurial-agarose chromatography yield preparations of soluble kinase with specific activities greater than 550 units/mg protein. Retention times in ion-exchange high performance liquid chromatography, electrophoretic behavior, immunochemical properties, tryptic mapping, and amino acid composition enable the comparison of creatine kinase isoenzymes. The denatured subunits of the predominant species have pI values of 6.3-6.8 and Mr = 40,000-42,000 characteristic of the so-called v2 proteins and show cross-reactivity with antibodies against the BB ("brain" type) creatine kinase. The MM ("muscle" type) antigens could be detected in the total electrocyte, but not in the AChR membranes; they have a slightly lower molecular weight and higher pI. The in situ membrane association of the BB isoenzyme is confirmed by immunocytochemistry. The apparent Km values for the substrate creatine phosphate are 2.2 mM for the AChR membrane-associated enzyme and 2.5 mM for the muscle form. The apparent Km values for Mg2+-ADP are 0.54 and 0.22 mM, respectively. Thus, a 2-fold higher affinity in the binding of ADP to the binary enzyme-creatine-P complex results from membrane association.