Norma Giusto - Academia.edu (original) (raw)
Papers by Norma Giusto
Biochimica et biophysica acta, Jan 12, 1986
Phosphatidate phosphohydrolase (EC 3.1.3.4) was detected in isolated bovine rod outer segments an... more Phosphatidate phosphohydrolase (EC 3.1.3.4) was detected in isolated bovine rod outer segments and its properties investigated. The enzyme activity was assayed using aqueously dispersed 1,2-diacyl-sn-[2-3H]glycerol 3-phosphate as substrate. The phosphatidic acid concentration was optimal at 1 mM and the estimated Km value was 6.7 X 10(-4) M. The activity was linear for 60 min with a protein concentration of 0.3 mg. A clear pH optimum was observed at 7.5. When the enzyme activity was measured using a substrate phosphatidic acid containing 15% lyso compound, the production of diacylglycerols was inhibited by about 70-75% at all concentrations studied. In rod outer segment preparations containing 0.2 mM Mg2+, further additions of the ion (0.2-2.5 mM) only produced a slight inhibition of the activity at 2.5 mM. F- (50 mM), Ca2+ (1 mM) and EDTA (50 mM) inhibited the dephosphorylation of the substrate by 80, 10 and 70%, respectively. The aqueously dispersed phosphatidic acid-dependent act...
Lipids, 1992
Sphingomyelin (SPH) content and composition in different regions of the brain were analyzed in 2.... more 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.
Experimental Gerontology, 2001
Among the morphological and biochemical changes taking place in the membranes of aged tissues, we... more Among the morphological and biochemical changes taking place in the membranes of aged tissues, we reported in previous studies on alterations in phospholipid synthesis and phospholipid-speci®c fatty acid composition. Phosphatidic acid (PA) and diacylglycerol (DAG) are central intermediates in phosphoglyceride and neutral lipid biosynthetic pathways and have also recently been implicated in signal transduction. The present paper shows the effect of aging on phosphatidate phosphohydrolase (PAPase) activiy, which operates on phosphatidic acid to synthesize diacylglycerol. Two forms of mammalian PAPase can be indenti®ed on the basis of subcellular localization and enzyme properties, one involved predominantly in lipid synthesis (PAP 1) and the other in signal transduction (PAP 2). Microsomal and cytosolic fractions of brain and liver from 3.5-month-old (adult) and 28.5-month-old (aged) rats were used. PAPase isoform activities were differentiated on the basis of N-ethylmaleimide (NEM) sensitivity and Mg 21 -dependency. Our results demonstrate that aging caused PAP 2 to increase in brain microsomal fractions but did not affect PAP 1, whereas in brain cytosolic fractions, it caused a strong decrease in PAP 1 (57%). The distribution of enzymes between microsomes and cytosol changed in aged rats with respect to adult rats, showing a translocation of PAP 1 from cytosol to microsomes. In addition, an increase in the production of monoacylglycerol (MAG) was observed in microsomes from aged brain. PAP 2 activity in liver microsomal fractions from aged rats showed no changes with respect to adult rats whereas PAP 1 S.J. Pasquare  et al. acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; TLC, thin layer chromatography; TRIS, tris[hydroxymethyl]aminomethane activity increased 228% in microsomal fractions and 76% in cytosolic fractions in this tissue. The distribution of PAP 1 activity between microsomal and cytosolic fractions in liver tissue was also affected in aged rats, indicating a translocation of this form of the enzyme from cytosolic to microsomal fractions. The production of monoacylglycerol in liver microsomes also increased, whereas there was a decrease in MAG formation from cytosolic fraction. The changes observed in the two PAPase forms in brain and liver of aged rats with respect to adult rats suggest that PA is differently utilized by the PAPase isoforms, probably generating aging-related DAGs different to those present in adults and required for speci®c cellular functions. The changes observed in liver PAP 1 from aged with respect to adult rats suggest that such changes could be related with modi®cations in lipid homeostasis induced by age-altered hormonal balance. However, PA-modi®ed utilization during aging through PAP 2 activity could be related to alterations in neural signal transduction mechanisms. q
![Research paper thumbnail of Active labeling of phosphatidylcholines by [1-14C]docosahexaenoate in isolated photoreceptor membranes](https://attachments.academia-assets.com/40853010/thumbnails/1.jpg)
](Biochimica et Biophysica Acta (BBA) - Biomembranes, 1986
~dated boyle rod ou~r ~gmen~ and pho~cep~r ~sks activdy incorporated [1-~C]docosahexaenoate (22:~... more ~dated boyle rod ou~r ~gmen~ and pho~cep~r ~sks activdy incorporated [1-~C]docosahexaenoate (22:~ into phospholipids when ivcub~ed ~ the presence ~ CoA, ATP, and Mg ~+. Abo~ 80% of the ~te~fied fatty add w~ ~ phosphatidykhd~e (PC). M~msom~ and mi~chond~ f~cfions incorporated ~ much 22:6 as rod ou~r ~gmen~, but ~ was ~s~ibu~d among va~ous phosphdipids and neural ~yce~de~ The ~da~d photoreceptor membrane ~s co~ns an acyI-CoA ~n~etase wh~h activates ~e fatty a~d and a docosahexaenoy~CoA-~sophosphatidy~h~e ac~ans~r~e acti~. The specific ~dioactiviff d PC was ~gher ~ rod outer ~gments than ~ the other subcell~ fraction~ About 2/3 ~ the ~bd ~ photoreceptor memb~ne PC w~ ~ ~s ~polyun~rated mo~c~ spe~es and 1/3 ~ hexaene~ Dipolyunsaturated PCs showed ~gh turnover ~s d 22:6 ~ ~l three subceH~ar membrane~ espe~ally ~ m~ochond~ Refin~ membranes ~ ~o ~em ~ take up free [~Cl22:6 from ~e medium by ~m~e ~ffu~on or partition ~ ~e membrane lipi& The abili~ ~ ~e membran~ ~ ~ctiv~e and e~ed~ [1-~C[22:6 ind~ates that docosahexaenoate-co~ning m~ecd~ spe~ ~ ~fina H~d~ ~du~ng ~ose ~ ph~o~ce~or membrane~ ~e su~e~ ~ acylation-deacylation ~actions ~ ~. ~ti~ Disk membranes packed in the ou~r segment of rod photoreceptor cells ~splay a continuous process of renewM, as demon~ra~d by autora~ographic and ra~ob~chemic~ ~udies [1-5]. The fipid and proton components of thee membran~ are assembled at the base of the rod outer segment, whi~ ~sks fituated at i~ apex are concomitantly shed and eng~d by the pigment epi-* To whom co~espondence shodd be adduce& Abb~afions: [~C]22:~ <Z10,1Xl6,19-[bl~C]docosahexv enome; PC, phosphadd~chofine; P~ phosphafid~hanMamine; PS, phosphafid~rine.
Experimental eye research, Jan 10, 2015
Retina light stimulation triggers phototransduction events as well as different signaling mechani... more Retina light stimulation triggers phototransduction events as well as different signaling mechanisms in outer segments (sensorial portion) of photoreceptor cells. We have recently reported a novel light-dependent activation of diacylglycerol kinase (DAGK) and protein kinase C (PKC) at the nuclear level of photoreceptor cells. The aim of the present study was to analyze whether ex-vivo light exposure of bovine retinas also modulates insulin-related signaling pathways in nuclei from photoreceptor cells. To this end, a nuclear fraction enriched in small nuclei from photoreceptor cells (PNF) was obtained using a modified nuclear isolation protocol. In PNF obtained from bovine retinas exposed to light or darkness, the presence of insulin receptor (IR) and phosphorylated insulin receptor (pIR), the activation of Akt, p38 and extracellular signal-regulated kinase (ERK1/2) and the local action of insulin on lipid kinases were studied. Immunofluorescence (IF) and Western blot (WB) studies re...
Neurochemical Research, Jul 1, 2000
The incorporation of polar and non-polar moieties into cerebral cortex (CC) and cerebellum (CRBL)... more The incorporation of polar and non-polar moieties into cerebral cortex (CC) and cerebellum (CRBL) phospholipids of adult (3.5-month-old) and aged (21.5-month-old) rats was studied in a minced tissue suspension. The biosynthesis of acidic phospholipids through [3H]glycerol appears to be slightly increased with respect to that of zwitterionic or neutral lipids in CC of aged rats with respect to adult rats. On the contrary, the synthesis of phosphatidylcholine (PC) from [3H]choline was inhibited. However, the incorporation of [14C]serine into phosphatidylserine (PS) was higher in CC and CRBL in aged rats with respect to adult rats. The synthesis of phosphatidylethanolamine (PE) from PS was not modified during aging. Saturated ([3H]palmitic) and polyunsaturated ([3H]arachidonic) acids were incorporated successfully by adult and aged brain lipids. In addition [3H]palmitic, [3H]oleic and [3H]arachidonic acid were employed as glycerolipid precursors in brain homogenate from aged (28.5 month old) and adult (3.5 month old) rats. [3H]oleic acid incorporation into neutral lipids (NL) and [3H]arachidonic acid incorporation into PC, PE and phosphatidylinositol (PI) were increased in aged rats with respect to adult rats. Present results show the ability and avidity of aged brain tissue in vitro to incorporate unsaturated fatty acids when they are supplied exogenously. They also suggest a different handling of choline and serine by base exchange enzyme activities to synthesize PC and PS during aging.
Advances in Experimental Medicine and Biology
Neurochemical Pathology, 1983
The content and composition of retinal free fatty acids (FFA)† was determined during in vitro ano... more The content and composition of retinal free fatty acids (FFA)† was determined during in vitro anoxia. Anoxia induced a fourfold increase in FFA production, but these lipids were modified only slightly after aerobic incubation. An even greater increase in FFA occurred when BSA was present during anoxia. In the presence of BSA, the FFA pool remaining in the anoxic tissue
Membrane & cell biology, 2001
The synthesis of phosphatidylcholine (PC) in rod outer segments (ROS) catalysed by lysophosphatid... more 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 diffe...
Neurochemical research, 2000
The incorporation of polar and non-polar moieties into cerebral cortex (CC) and cerebellum (CRBL)... more The incorporation of polar and non-polar moieties into cerebral cortex (CC) and cerebellum (CRBL) phospholipids of adult (3.5-month-old) and aged (21.5-month-old) rats was studied in a minced tissue suspension. The biosynthesis of acidic phospholipids through [3H]glycerol appears to be slightly increased with respect to that of zwitterionic or neutral lipids in CC of aged rats with respect to adult rats. On the contrary, the synthesis of phosphatidylcholine (PC) from [3H]choline was inhibited. However, the incorporation of [14C]serine into phosphatidylserine (PS) was higher in CC and CRBL in aged rats with respect to adult rats. The synthesis of phosphatidylethanolamine (PE) from PS was not modified during aging. Saturated ([3H]palmitic) and polyunsaturated ([3H]arachidonic) acids were incorporated successfully by adult and aged brain lipids. In addition [3H]palmitic, [3H]oleic and [3H]arachidonic acid were employed as glycerolipid precursors in brain homogenate from aged (28.5 mont...
Journal of Lipids, 2011
This paper explores the way ceramide, sphingosine, ceramide 1-phosphate, and sphingosine 1-phosph... more This paper explores the way ceramide, sphingosine, ceramide 1-phosphate, and sphingosine 1-phosphate modulate the generation of second lipid messengers from phosphatidic acid in two experimental models of the central nervous system: in vertebrate rod outer segments prepared from dark-adapted retinas as well as in rod outer segments prepared from light-adapted retinas and in rat cerebral cortex synaptosomes under physiological aging conditions. Particular attention is paid to lipid phosphate phosphatase, diacylglycerol lipase, and monoacylglycerol lipase. Based on the findings reported in this paper, it can be concluded that proteins related to phototransduction phenomena are involved in the effects derived from sphingosine 1-phosphate/sphingosine or ceramide 1-phosphate/ceramide and that age-related changes occur in the metabolism of phosphatidic acid from cerebral cortex synaptosomes in the presence of either sphingosine 1-phosphate/sphingosine or ceramide 1-phosphate/ceramide. The present paper demonstrates, in two different models of central nervous system, how sphingolipids influence phosphatidic acid metabolism under different physiological conditions such as light and aging.
Neurochemical research, 1997
Lipid metabolism in photoreceptor rod outer segments has attracted considerable attention because... more 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.
Comparative biochemistry and physiology. B, Comparative biochemistry, 1993
1. The effect of magnesium and dl-propranolol on phosphatidate phosphohydrolase (PAPase) and diac... more 1. The effect of magnesium and dl-propranolol on phosphatidate phosphohydrolase (PAPase) and diacylglycerol lipase (DGL) activities in isolated rod outer segments (ROS) and of the former on subcellular fractions from bovine retina was investigated. 2. Mg(2+)-independent PAPase activity was found in ROS, whereas in the other subcellular fractions PAPase activities both dependent on and independent of Mg2+ were detected. 3. The membrane-bound PAPase activity was stimulated at low concentrations of Mg2+ and inhibited at higher concentrations. The soluble activity was always stimulated by the ion. 4. dl-Propranolol (1000 microM) exerted a slight stimulatory effect on PAPase in ROS whereas total PAPase activity of microsomal fraction was not affected. 5. Mg2+ (0.2 mM) stimulated DGL activity (30%) whereas it was inhibited at higher concentration. 6. DGL lipase activities, both dependent on and independent of Mg2+, were detected in subcellular fractions of bovine retina. 7. DGL properties...
Neurochemical research, 2002
In this review, changes in brain lipid composition and metabolism due to aging are outlined. The ... more In this review, changes in brain lipid composition and metabolism due to aging are outlined. The most striking changes in cerebral cortex and cerebellum lipid composition involve an increase in acidic phospholipid synthesis. The most important changes with respect to fatty acyl composition involve a decreased content in polyunsaturated fatty acids (20:4n-6, 22:4n-6, 22:6n-3) and an increased content in monounsaturated fatty acids (18:1n-9 and 20:1n-9), mainly in ethanolamine and serineglycerophospholipids. Changes in the activity of the enzymes modifying the phospholipid headgroup occur during aging. Serine incorporation into phosphatidylserine through base-exchange reactions and phosphatidylcholine synthesis through phosphatidylethanolamine methylation increases in the aged brain. Phosphatidate phosphohydrolase and phospholipase D activities are also altered in the aged brain thus producing changes in the lipid second messengers diacylglycerol and phosphatidic acid.
Experimental Gerontology, 2014
Anandamide is an endocannabinoid involved in several physiological functions including neuroprote... more Anandamide is an endocannabinoid involved in several physiological functions including neuroprotection. Anandamide is synthesized on demand and its endogenous level is regulated through its degradation, where fatty acid amide hydrolase plays a major role. The aim of this study was to characterize anandamide breakdown in physiological and pathological aging and its regulation by CB1 and CB2 receptor agonists. Fatty acid amide hydrolase activity was analyzed in an independent cohort of human cortical membrane samples from control and Alzheimer's disease patients, and in membrane and synaptosomes from adult and aged rat cerebral cortex. Our results demonstrate that fatty acid amide hydrolase activity decreases in the frontal cortex from human patients with Alzheimer's disease and this effect is mimicked by Aβ 1-40 peptide. This activity increases and decreases in aged rat cerebrocortical membranes and synaptosomes, respectively. Also, while the presence of JWH-133, a CB2 selective agonist, slightly increases anandamide hydrolysis in human controls, it decreases this activity in adults and aged rat cerebrocortical membranes and synaptosomes. In the presence of WIN55,212-2, a mixed CB1/CB2 agonist, anandamide hydrolysis increases in Alzheimer's disease patients but decreases in human controls as well as in adult and aged rat cerebrocortical membranes and synaptosomes. Although a similar profile is observed in fatty acid amide hydrolase activity between aged rat synaptic endings and human Alzheimer's disease brains, it is differently modulated by CB1/CB2 agonists. This modulation leads to a reduced availability of anandamide in Alzheimer's disease and to an increased availability of this endocannabinoid in aging.
The International Journal of Biochemistry & Cell Biology, 2014
The retinal pigment epithelium (RPE) plays an important immunological role in the retina and it i... more The retinal pigment epithelium (RPE) plays an important immunological role in the retina and it is involved in many ocular inflammatory diseases that may end in loss of vision and blindness. In this work the role of phospholipase D (PLD) classical isoforms, PLD1 and PLD2, in the inflammatory response of human RPE cells (ARPE-19) was studied. ARPE-19 cells exposed to lipopolysaccharide (LPS, 10 μg/ml) displayed increased levels of NO production and diminished mitochondrial function after 48 h of incubation. Furthermore, 24h LPS treatment strongly induced cyclooxygenase-2 (COX-2) expression and activation of extracellular signal-regulated kinase (ERK1/2). EGFP-PLDs showed the typical subcellular localization, perinuclear for PLD1 and plasma membrane for PLD2. LPS increased PLD activity by 90% with respect to the control. The presence of PLD1 inhibitor (EVJ 0.15 μM) or PLD2 inhibitor (APV 0.5 μM) reduced LPS-induced COX-2 induction but only PLD2 inhibition reduced ERK1/2 activation. Mitochondrial function was restored after inhibition of PLD2 and ERK1/2. These findings evidence the participation of PLD2 as a promoter of RPE inflammatory response through ERK1/2 and COX-2 regulation. Our results demonstrate for the first time distinctive roles of PLD isoforms in pathological conditions in RPE.
Chronobiology International, 2015
Experimental Eye Research, 2014
In this work, we describe a selective light-dependent distribution of the lipid kinase 1,2-diacyl... more In this work, we describe a selective light-dependent distribution of the lipid kinase 1,2-diacylglycerol kinase (EC 2.7.1.107, DAGK) and the phosphorylated protein kinase C alpha (pPKCα) in a nuclear fraction of photoreceptor cells from bovine retinas. A nuclear fraction enriched in small nuclei from photoreceptor cells (PNF), was obtained when a modified nuclear isolation protocol developed by our laboratory was used. We measured and compared DAGK activity as phosphatidic acid (PA) formation in PNF obtained from retinas exposed to light and in retinas kept in darkness using [γ-(32)P]ATP or [(3)H]DAG. In the absence of exogenous substrates and detergents, no changes in DAGK activity were observed. However, when DAGK activity assays were performed in the presence of exogenous substrates, such as stearoyl arachidonoyl glycerol (SAG) or dioleoyl glycerol (DOG), and different detergents (used to make different DAGK isoforms evident), we observed significant light effects on DAGK activity, suggesting the presence of several DAGK isoforms in PNF. Under conditions favoring DAGKζ activity (DOG, Triton X-100, dioleoyl phosphatidylserine and R59022) we observed an increase in PA formation in PNF from retinas exposed to light with respect to those exposed to darkness. In contrast, under conditions favoring DAGKɛ (SAG, octylglucoside and R59022) we observed a decrease in its activity. These results suggest different physiological roles of the above-mentioned DAGK isoforms. Western blot analysis showed that whereas light stimulation of bovine retinas increases DAGKζ nuclear content, it decreases DAGKɛ and DAGKβ content in PNF. The role of PIP2-phospholipase C in light-stimulated DAGK activity was demonstrated using U73122. Light was also observed to induce enhanced pPKCα content in PNF. The selective distribution of DAGKζ and ɛ in PNF could be a light-dependent mechanism that in vertebrate retina promotes selective DAG removal and PKC regulation.
International Journal of Alzheimer's Disease, 2011
The accumulation of transition metals (e.g., copper, zinc, and iron) and the dysregulation of the... more The accumulation of transition metals (e.g., copper, zinc, and iron) and the dysregulation of their metabolism are a hallmark in the pathogenesis of several neurodegenerative diseases. This paper will be focused on the mechanism of neurotoxicity mediated by iron. This metal progressively accumulates in the brain both during normal aging and neurodegenerative processes. High iron concentrations in the brain have been consistently observed in Alzheimer&amp;amp;amp;amp;amp;#39;s (AD) and Parkinson&amp;amp;amp;amp;amp;#39;s (PD) diseases. In this connection, metalloneurobiology has become extremely important in establishing the role of iron in the onset and progression of neurodegenerative diseases. Neurons have developed several protective mechanisms against oxidative stress, among them, the activation of cellular signaling pathways. The final response will depend on the identity, intensity, and persistence of the oxidative insult. The characterization of the mechanisms mediating the effects of iron-induced increase in neuronal dysfunction and death is central to understanding the pathology of a number of neurodegenerative disorders.
![Research paper thumbnail of High content of 22:6 (docosahexaenoate) and active [2-3h]glycerol metabolism of phosphatidic acid from photoreceptor membranes](https://attachments.academia-assets.com/40853004/thumbnails/1.jpg)
](Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1982
This study describes the content, fatty acid composition and [2-3H]glycerol metabolism of phospha... more This study describes the content, fatty acid composition and [2-3H]glycerol metabolism of phosphatidic acid of rod outer segment membranes from vertebrate retinas. A relatively high content of phosphatidic acid was observed in rod outer segment membranes isolated from rat, toad and bovine retinas. In bovine retinas, about 65% of the acyl groups of phosphatidic acid were composed of docosahexaenoate.
Biochimica et biophysica acta, Jan 12, 1986
Phosphatidate phosphohydrolase (EC 3.1.3.4) was detected in isolated bovine rod outer segments an... more Phosphatidate phosphohydrolase (EC 3.1.3.4) was detected in isolated bovine rod outer segments and its properties investigated. The enzyme activity was assayed using aqueously dispersed 1,2-diacyl-sn-[2-3H]glycerol 3-phosphate as substrate. The phosphatidic acid concentration was optimal at 1 mM and the estimated Km value was 6.7 X 10(-4) M. The activity was linear for 60 min with a protein concentration of 0.3 mg. A clear pH optimum was observed at 7.5. When the enzyme activity was measured using a substrate phosphatidic acid containing 15% lyso compound, the production of diacylglycerols was inhibited by about 70-75% at all concentrations studied. In rod outer segment preparations containing 0.2 mM Mg2+, further additions of the ion (0.2-2.5 mM) only produced a slight inhibition of the activity at 2.5 mM. F- (50 mM), Ca2+ (1 mM) and EDTA (50 mM) inhibited the dephosphorylation of the substrate by 80, 10 and 70%, respectively. The aqueously dispersed phosphatidic acid-dependent act...
Lipids, 1992
Sphingomyelin (SPH) content and composition in different regions of the brain were analyzed in 2.... more 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.
Experimental Gerontology, 2001
Among the morphological and biochemical changes taking place in the membranes of aged tissues, we... more Among the morphological and biochemical changes taking place in the membranes of aged tissues, we reported in previous studies on alterations in phospholipid synthesis and phospholipid-speci®c fatty acid composition. Phosphatidic acid (PA) and diacylglycerol (DAG) are central intermediates in phosphoglyceride and neutral lipid biosynthetic pathways and have also recently been implicated in signal transduction. The present paper shows the effect of aging on phosphatidate phosphohydrolase (PAPase) activiy, which operates on phosphatidic acid to synthesize diacylglycerol. Two forms of mammalian PAPase can be indenti®ed on the basis of subcellular localization and enzyme properties, one involved predominantly in lipid synthesis (PAP 1) and the other in signal transduction (PAP 2). Microsomal and cytosolic fractions of brain and liver from 3.5-month-old (adult) and 28.5-month-old (aged) rats were used. PAPase isoform activities were differentiated on the basis of N-ethylmaleimide (NEM) sensitivity and Mg 21 -dependency. Our results demonstrate that aging caused PAP 2 to increase in brain microsomal fractions but did not affect PAP 1, whereas in brain cytosolic fractions, it caused a strong decrease in PAP 1 (57%). The distribution of enzymes between microsomes and cytosol changed in aged rats with respect to adult rats, showing a translocation of PAP 1 from cytosol to microsomes. In addition, an increase in the production of monoacylglycerol (MAG) was observed in microsomes from aged brain. PAP 2 activity in liver microsomal fractions from aged rats showed no changes with respect to adult rats whereas PAP 1 S.J. Pasquare  et al. acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; TLC, thin layer chromatography; TRIS, tris[hydroxymethyl]aminomethane activity increased 228% in microsomal fractions and 76% in cytosolic fractions in this tissue. The distribution of PAP 1 activity between microsomal and cytosolic fractions in liver tissue was also affected in aged rats, indicating a translocation of this form of the enzyme from cytosolic to microsomal fractions. The production of monoacylglycerol in liver microsomes also increased, whereas there was a decrease in MAG formation from cytosolic fraction. The changes observed in the two PAPase forms in brain and liver of aged rats with respect to adult rats suggest that PA is differently utilized by the PAPase isoforms, probably generating aging-related DAGs different to those present in adults and required for speci®c cellular functions. The changes observed in liver PAP 1 from aged with respect to adult rats suggest that such changes could be related with modi®cations in lipid homeostasis induced by age-altered hormonal balance. However, PA-modi®ed utilization during aging through PAP 2 activity could be related to alterations in neural signal transduction mechanisms. q
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1986
~dated boyle rod ou~r ~gmen~ and pho~cep~r ~sks activdy incorporated [1-~C]docosahexaenoate (22:~... more ~dated boyle rod ou~r ~gmen~ and pho~cep~r ~sks activdy incorporated [1-~C]docosahexaenoate (22:~ into phospholipids when ivcub~ed ~ the presence ~ CoA, ATP, and Mg ~+. Abo~ 80% of the ~te~fied fatty add w~ ~ phosphatidykhd~e (PC). M~msom~ and mi~chond~ f~cfions incorporated ~ much 22:6 as rod ou~r ~gmen~, but ~ was ~s~ibu~d among va~ous phosphdipids and neural ~yce~de~ The ~da~d photoreceptor membrane ~s co~ns an acyI-CoA ~n~etase wh~h activates ~e fatty a~d and a docosahexaenoy~CoA-~sophosphatidy~h~e ac~ans~r~e acti~. The specific ~dioactiviff d PC was ~gher ~ rod outer ~gments than ~ the other subcell~ fraction~ About 2/3 ~ the ~bd ~ photoreceptor memb~ne PC w~ ~ ~s ~polyun~rated mo~c~ spe~es and 1/3 ~ hexaene~ Dipolyunsaturated PCs showed ~gh turnover ~s d 22:6 ~ ~l three subceH~ar membrane~ espe~ally ~ m~ochond~ Refin~ membranes ~ ~o ~em ~ take up free [~Cl22:6 from ~e medium by ~m~e ~ffu~on or partition ~ ~e membrane lipi& The abili~ ~ ~e membran~ ~ ~ctiv~e and e~ed~ [1-~C[22:6 ind~ates that docosahexaenoate-co~ning m~ecd~ spe~ ~ ~fina H~d~ ~du~ng ~ose ~ ph~o~ce~or membrane~ ~e su~e~ ~ acylation-deacylation ~actions ~ ~. ~ti~ Disk membranes packed in the ou~r segment of rod photoreceptor cells ~splay a continuous process of renewM, as demon~ra~d by autora~ographic and ra~ob~chemic~ ~udies [1-5]. The fipid and proton components of thee membran~ are assembled at the base of the rod outer segment, whi~ ~sks fituated at i~ apex are concomitantly shed and eng~d by the pigment epi-* To whom co~espondence shodd be adduce& Abb~afions: [~C]22:~ <Z10,1Xl6,19-[bl~C]docosahexv enome; PC, phosphadd~chofine; P~ phosphafid~hanMamine; PS, phosphafid~rine.
Experimental eye research, Jan 10, 2015
Retina light stimulation triggers phototransduction events as well as different signaling mechani... more Retina light stimulation triggers phototransduction events as well as different signaling mechanisms in outer segments (sensorial portion) of photoreceptor cells. We have recently reported a novel light-dependent activation of diacylglycerol kinase (DAGK) and protein kinase C (PKC) at the nuclear level of photoreceptor cells. The aim of the present study was to analyze whether ex-vivo light exposure of bovine retinas also modulates insulin-related signaling pathways in nuclei from photoreceptor cells. To this end, a nuclear fraction enriched in small nuclei from photoreceptor cells (PNF) was obtained using a modified nuclear isolation protocol. In PNF obtained from bovine retinas exposed to light or darkness, the presence of insulin receptor (IR) and phosphorylated insulin receptor (pIR), the activation of Akt, p38 and extracellular signal-regulated kinase (ERK1/2) and the local action of insulin on lipid kinases were studied. Immunofluorescence (IF) and Western blot (WB) studies re...
Neurochemical Research, Jul 1, 2000
The incorporation of polar and non-polar moieties into cerebral cortex (CC) and cerebellum (CRBL)... more The incorporation of polar and non-polar moieties into cerebral cortex (CC) and cerebellum (CRBL) phospholipids of adult (3.5-month-old) and aged (21.5-month-old) rats was studied in a minced tissue suspension. The biosynthesis of acidic phospholipids through [3H]glycerol appears to be slightly increased with respect to that of zwitterionic or neutral lipids in CC of aged rats with respect to adult rats. On the contrary, the synthesis of phosphatidylcholine (PC) from [3H]choline was inhibited. However, the incorporation of [14C]serine into phosphatidylserine (PS) was higher in CC and CRBL in aged rats with respect to adult rats. The synthesis of phosphatidylethanolamine (PE) from PS was not modified during aging. Saturated ([3H]palmitic) and polyunsaturated ([3H]arachidonic) acids were incorporated successfully by adult and aged brain lipids. In addition [3H]palmitic, [3H]oleic and [3H]arachidonic acid were employed as glycerolipid precursors in brain homogenate from aged (28.5 month old) and adult (3.5 month old) rats. [3H]oleic acid incorporation into neutral lipids (NL) and [3H]arachidonic acid incorporation into PC, PE and phosphatidylinositol (PI) were increased in aged rats with respect to adult rats. Present results show the ability and avidity of aged brain tissue in vitro to incorporate unsaturated fatty acids when they are supplied exogenously. They also suggest a different handling of choline and serine by base exchange enzyme activities to synthesize PC and PS during aging.
Advances in Experimental Medicine and Biology
Neurochemical Pathology, 1983
The content and composition of retinal free fatty acids (FFA)† was determined during in vitro ano... more The content and composition of retinal free fatty acids (FFA)† was determined during in vitro anoxia. Anoxia induced a fourfold increase in FFA production, but these lipids were modified only slightly after aerobic incubation. An even greater increase in FFA occurred when BSA was present during anoxia. In the presence of BSA, the FFA pool remaining in the anoxic tissue
Membrane & cell biology, 2001
The synthesis of phosphatidylcholine (PC) in rod outer segments (ROS) catalysed by lysophosphatid... more 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 diffe...
Neurochemical research, 2000
The incorporation of polar and non-polar moieties into cerebral cortex (CC) and cerebellum (CRBL)... more The incorporation of polar and non-polar moieties into cerebral cortex (CC) and cerebellum (CRBL) phospholipids of adult (3.5-month-old) and aged (21.5-month-old) rats was studied in a minced tissue suspension. The biosynthesis of acidic phospholipids through [3H]glycerol appears to be slightly increased with respect to that of zwitterionic or neutral lipids in CC of aged rats with respect to adult rats. On the contrary, the synthesis of phosphatidylcholine (PC) from [3H]choline was inhibited. However, the incorporation of [14C]serine into phosphatidylserine (PS) was higher in CC and CRBL in aged rats with respect to adult rats. The synthesis of phosphatidylethanolamine (PE) from PS was not modified during aging. Saturated ([3H]palmitic) and polyunsaturated ([3H]arachidonic) acids were incorporated successfully by adult and aged brain lipids. In addition [3H]palmitic, [3H]oleic and [3H]arachidonic acid were employed as glycerolipid precursors in brain homogenate from aged (28.5 mont...
Journal of Lipids, 2011
This paper explores the way ceramide, sphingosine, ceramide 1-phosphate, and sphingosine 1-phosph... more This paper explores the way ceramide, sphingosine, ceramide 1-phosphate, and sphingosine 1-phosphate modulate the generation of second lipid messengers from phosphatidic acid in two experimental models of the central nervous system: in vertebrate rod outer segments prepared from dark-adapted retinas as well as in rod outer segments prepared from light-adapted retinas and in rat cerebral cortex synaptosomes under physiological aging conditions. Particular attention is paid to lipid phosphate phosphatase, diacylglycerol lipase, and monoacylglycerol lipase. Based on the findings reported in this paper, it can be concluded that proteins related to phototransduction phenomena are involved in the effects derived from sphingosine 1-phosphate/sphingosine or ceramide 1-phosphate/ceramide and that age-related changes occur in the metabolism of phosphatidic acid from cerebral cortex synaptosomes in the presence of either sphingosine 1-phosphate/sphingosine or ceramide 1-phosphate/ceramide. The present paper demonstrates, in two different models of central nervous system, how sphingolipids influence phosphatidic acid metabolism under different physiological conditions such as light and aging.
Neurochemical research, 1997
Lipid metabolism in photoreceptor rod outer segments has attracted considerable attention because... more 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.
Comparative biochemistry and physiology. B, Comparative biochemistry, 1993
1. The effect of magnesium and dl-propranolol on phosphatidate phosphohydrolase (PAPase) and diac... more 1. The effect of magnesium and dl-propranolol on phosphatidate phosphohydrolase (PAPase) and diacylglycerol lipase (DGL) activities in isolated rod outer segments (ROS) and of the former on subcellular fractions from bovine retina was investigated. 2. Mg(2+)-independent PAPase activity was found in ROS, whereas in the other subcellular fractions PAPase activities both dependent on and independent of Mg2+ were detected. 3. The membrane-bound PAPase activity was stimulated at low concentrations of Mg2+ and inhibited at higher concentrations. The soluble activity was always stimulated by the ion. 4. dl-Propranolol (1000 microM) exerted a slight stimulatory effect on PAPase in ROS whereas total PAPase activity of microsomal fraction was not affected. 5. Mg2+ (0.2 mM) stimulated DGL activity (30%) whereas it was inhibited at higher concentration. 6. DGL lipase activities, both dependent on and independent of Mg2+, were detected in subcellular fractions of bovine retina. 7. DGL properties...
Neurochemical research, 2002
In this review, changes in brain lipid composition and metabolism due to aging are outlined. The ... more In this review, changes in brain lipid composition and metabolism due to aging are outlined. The most striking changes in cerebral cortex and cerebellum lipid composition involve an increase in acidic phospholipid synthesis. The most important changes with respect to fatty acyl composition involve a decreased content in polyunsaturated fatty acids (20:4n-6, 22:4n-6, 22:6n-3) and an increased content in monounsaturated fatty acids (18:1n-9 and 20:1n-9), mainly in ethanolamine and serineglycerophospholipids. Changes in the activity of the enzymes modifying the phospholipid headgroup occur during aging. Serine incorporation into phosphatidylserine through base-exchange reactions and phosphatidylcholine synthesis through phosphatidylethanolamine methylation increases in the aged brain. Phosphatidate phosphohydrolase and phospholipase D activities are also altered in the aged brain thus producing changes in the lipid second messengers diacylglycerol and phosphatidic acid.
Experimental Gerontology, 2014
Anandamide is an endocannabinoid involved in several physiological functions including neuroprote... more Anandamide is an endocannabinoid involved in several physiological functions including neuroprotection. Anandamide is synthesized on demand and its endogenous level is regulated through its degradation, where fatty acid amide hydrolase plays a major role. The aim of this study was to characterize anandamide breakdown in physiological and pathological aging and its regulation by CB1 and CB2 receptor agonists. Fatty acid amide hydrolase activity was analyzed in an independent cohort of human cortical membrane samples from control and Alzheimer's disease patients, and in membrane and synaptosomes from adult and aged rat cerebral cortex. Our results demonstrate that fatty acid amide hydrolase activity decreases in the frontal cortex from human patients with Alzheimer's disease and this effect is mimicked by Aβ 1-40 peptide. This activity increases and decreases in aged rat cerebrocortical membranes and synaptosomes, respectively. Also, while the presence of JWH-133, a CB2 selective agonist, slightly increases anandamide hydrolysis in human controls, it decreases this activity in adults and aged rat cerebrocortical membranes and synaptosomes. In the presence of WIN55,212-2, a mixed CB1/CB2 agonist, anandamide hydrolysis increases in Alzheimer's disease patients but decreases in human controls as well as in adult and aged rat cerebrocortical membranes and synaptosomes. Although a similar profile is observed in fatty acid amide hydrolase activity between aged rat synaptic endings and human Alzheimer's disease brains, it is differently modulated by CB1/CB2 agonists. This modulation leads to a reduced availability of anandamide in Alzheimer's disease and to an increased availability of this endocannabinoid in aging.
The International Journal of Biochemistry & Cell Biology, 2014
The retinal pigment epithelium (RPE) plays an important immunological role in the retina and it i... more The retinal pigment epithelium (RPE) plays an important immunological role in the retina and it is involved in many ocular inflammatory diseases that may end in loss of vision and blindness. In this work the role of phospholipase D (PLD) classical isoforms, PLD1 and PLD2, in the inflammatory response of human RPE cells (ARPE-19) was studied. ARPE-19 cells exposed to lipopolysaccharide (LPS, 10 μg/ml) displayed increased levels of NO production and diminished mitochondrial function after 48 h of incubation. Furthermore, 24h LPS treatment strongly induced cyclooxygenase-2 (COX-2) expression and activation of extracellular signal-regulated kinase (ERK1/2). EGFP-PLDs showed the typical subcellular localization, perinuclear for PLD1 and plasma membrane for PLD2. LPS increased PLD activity by 90% with respect to the control. The presence of PLD1 inhibitor (EVJ 0.15 μM) or PLD2 inhibitor (APV 0.5 μM) reduced LPS-induced COX-2 induction but only PLD2 inhibition reduced ERK1/2 activation. Mitochondrial function was restored after inhibition of PLD2 and ERK1/2. These findings evidence the participation of PLD2 as a promoter of RPE inflammatory response through ERK1/2 and COX-2 regulation. Our results demonstrate for the first time distinctive roles of PLD isoforms in pathological conditions in RPE.
Chronobiology International, 2015
Experimental Eye Research, 2014
In this work, we describe a selective light-dependent distribution of the lipid kinase 1,2-diacyl... more In this work, we describe a selective light-dependent distribution of the lipid kinase 1,2-diacylglycerol kinase (EC 2.7.1.107, DAGK) and the phosphorylated protein kinase C alpha (pPKCα) in a nuclear fraction of photoreceptor cells from bovine retinas. A nuclear fraction enriched in small nuclei from photoreceptor cells (PNF), was obtained when a modified nuclear isolation protocol developed by our laboratory was used. We measured and compared DAGK activity as phosphatidic acid (PA) formation in PNF obtained from retinas exposed to light and in retinas kept in darkness using [γ-(32)P]ATP or [(3)H]DAG. In the absence of exogenous substrates and detergents, no changes in DAGK activity were observed. However, when DAGK activity assays were performed in the presence of exogenous substrates, such as stearoyl arachidonoyl glycerol (SAG) or dioleoyl glycerol (DOG), and different detergents (used to make different DAGK isoforms evident), we observed significant light effects on DAGK activity, suggesting the presence of several DAGK isoforms in PNF. Under conditions favoring DAGKζ activity (DOG, Triton X-100, dioleoyl phosphatidylserine and R59022) we observed an increase in PA formation in PNF from retinas exposed to light with respect to those exposed to darkness. In contrast, under conditions favoring DAGKɛ (SAG, octylglucoside and R59022) we observed a decrease in its activity. These results suggest different physiological roles of the above-mentioned DAGK isoforms. Western blot analysis showed that whereas light stimulation of bovine retinas increases DAGKζ nuclear content, it decreases DAGKɛ and DAGKβ content in PNF. The role of PIP2-phospholipase C in light-stimulated DAGK activity was demonstrated using U73122. Light was also observed to induce enhanced pPKCα content in PNF. The selective distribution of DAGKζ and ɛ in PNF could be a light-dependent mechanism that in vertebrate retina promotes selective DAG removal and PKC regulation.
International Journal of Alzheimer's Disease, 2011
The accumulation of transition metals (e.g., copper, zinc, and iron) and the dysregulation of the... more The accumulation of transition metals (e.g., copper, zinc, and iron) and the dysregulation of their metabolism are a hallmark in the pathogenesis of several neurodegenerative diseases. This paper will be focused on the mechanism of neurotoxicity mediated by iron. This metal progressively accumulates in the brain both during normal aging and neurodegenerative processes. High iron concentrations in the brain have been consistently observed in Alzheimer&amp;amp;amp;amp;amp;#39;s (AD) and Parkinson&amp;amp;amp;amp;amp;#39;s (PD) diseases. In this connection, metalloneurobiology has become extremely important in establishing the role of iron in the onset and progression of neurodegenerative diseases. Neurons have developed several protective mechanisms against oxidative stress, among them, the activation of cellular signaling pathways. The final response will depend on the identity, intensity, and persistence of the oxidative insult. The characterization of the mechanisms mediating the effects of iron-induced increase in neuronal dysfunction and death is central to understanding the pathology of a number of neurodegenerative disorders.
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1982
This study describes the content, fatty acid composition and [2-3H]glycerol metabolism of phospha... more This study describes the content, fatty acid composition and [2-3H]glycerol metabolism of phosphatidic acid of rod outer segment membranes from vertebrate retinas. A relatively high content of phosphatidic acid was observed in rod outer segment membranes isolated from rat, toad and bovine retinas. In bovine retinas, about 65% of the acyl groups of phosphatidic acid were composed of docosahexaenoate.