Biomedical subjects

P Colepicolo

Publications and source records attributed to P Colepicolo.

At least 19 recordsLinked to original sources

PubMed2006

Superoxide dismutase in Cryptococcus neoformans varieties gattii, grubi, and neoformans.

Some clear dissimilarities occur among the varieties of Cryptococcus neoformans but there are few studies about the differences among individual yeast antioxidant enzymes. The total superoxide dismutase (SOD) activities and the copper, zinc-depend SOD (Cu,ZnSOD) and manganese-dependent SOD (MnSOD) isoenzymes of five reference C. neoformans strains belonged to A, B, C, AD and D serotypes (Table I) and other nine C. neoformans isolates (Table II) were determined. There were significant differences (p < 0.01 and p < 0.05) in total SOD activity among the varietie gattii (serotype C) and the other varieties. Cu,ZnSOD showed difference (p < 0.05) between A and D serotypes. These results point out a variety and serotype-independent SOD activity in C. neoformans reference strains and the other isolates that were evaluated.

Cryptococcus neoformans↗

PubMed2003

PCB-induced oxidative stress in the unicellular marine dinoflagellate Lingulodinium polyedrum.

The susceptibility of the dinoflagellate Lingulodinium polyedrum to Arochlor 1254, a commercial mixture of polychlorinated biphenyl (PCB) congeners was examined through toxicity bioassays based on cell survival and measures of oxidative balance and adaptive response to PCB stress. The highest Arochlor 1254 concentration that did not cause observed effects (NOEC) on cell growth was 100 (48 h) and 25 ppb (96 h). The concentration that caused maximum effect (MEC) was 300 ppb (48 h) and 250 ppb (microl L(-1)) (96 h). The concentrations that promoted 50% cell death (LC50) were 146 and 122 ppb after 48 and 96 h, respectively. The cell numbers were lower after 96 h of exposure than after 48 h, suggesting that neither recovery nor growth occurred. Under exposure to 120 ppb Arochlor 1254 for 48 h the oxidative damage in proteins was 121% higher than the control, as measured by reactive carbonyl levels, but no oxidative damage was found in lipids measured as malondialdehyde contents. Total superoxide dismutase (SOD) activity increased to plateau levels 146% greater than control values. The cells also exhibited increased ascorbate peroxidase (APx) activity (50%) and peridinin content (27%). No changes were observed in beta-carotene under these experimental conditions. Therefore, SOD and APx induction and increased peridinin content may be principal primary adaptive responses to an increase of reactive oxygen species in Arochlor 1254 stress as indicated by protein oxidative damage and are an early marker of Arochlor 1254 contamination.

Animals↗

PubMed2003

Induction of oxidative stress in the red macroalga Gracilaria tenuistipitata by pollutant metals.

Heavy metals are environmental pollutants that have the potential to induce severe stress-reactions in organisms on land as well as in the sea. We have studied effects of short term sublethal concentrations of copper (Cu2+) and cadmium (Cd2+) on the reactive oxygen metabolism of the marine red macroalga Gracilaria tenuistipitata. Additions of either 0.2 ppm Cu2+ or 1 ppm Cd2+ caused decreased growth (approximately 60%), increased oxidation of lipids and increased oxidative damage to proteins as shown by increased content of protein carbonyl groups. Together this strongly suggests an induction of oxidative stress. Cu2+ caused more oxidative damage than Cd2+. As a response to the increased oxidative stress, addition of Cu2+ induced the activities of catalase, ascorbate peroxidase, and superoxide dismutase. In contrast, Cd2+ only caused increased catalase activity. Ten-fold lower concentrations of the metals did not cause an increase in enzyme activity. Both heavy metals also increased the content of the antioxidants beta-carotene and lutein. The results show that Cd2+ and, to a larger extent, Cu2+ induce oxidative stress in short-term experiments and the seaweed responds by increasing the activity of the reactive oxygen metabolism.

Antioxidants↗

PubMed2002

Changes in superoxide dismutase activity and photosynthetic pigment content during growth of marine phytoplankters in batch-cultures.

The ability of phytoplankton to cope with oxidative stress is one of the main factors that influence its survival in the marine environment, when senescence conditions prevail. In a first attempt to investigate the antioxidant strategies of different phytoplanktonic groups face to oxidative stress, the superoxide dismutase (SOD; EC 1.15.1.1) activity and photosynthetic pigment content along the growth curves of the dinoflagellate Lingulodinium polyedrum (Stein) Dodge, the prasinophycean Tetraselmis gracilis (Kylin) Butcher and the diatom Minutocellus polymorphus (Hargraves and Guillard) Hasle, von Stosch and Syvertsen were evaluated in batch-cultures. Total SOD activity was determined by an indirect method involving the inhibition of cytochrome c reduction. The contents of photosynthetic pigments were analysed by HPLC using a reverse phase column (RP-18), based on a ternary gradient. A peak of total SOD activity was detected at the beginning of the T. gracilis and M. polymorphus exponential growth. In L. polyedrum and M. polymorphus, SOD activity increased approximately three times by day 17 of growth, compared to the values obtained on day 3 (exponential phase) of the growth curve. All three species of microalgae had reduced SOD activity at the end of their growth. The levels of peridinin in L. polyedrum increased about 60% by day 17 of growth compared to the values obtained at exponential phase. Tetraselmis gracilis exhibited a remarkable increase (approximately 85%) in beta-carotene concentration after 10-14 days of growth whereas the beta-carotene levels in M. polymorphus decreased about 85% along its growth curve. These findings suggest that the antioxidant response during senescence in batch-cultures differ according to the species. Induction of SOD activity may occur either in the early exponential or stationary growth phases, which is important to prevent oxidative stress triggered by a number of factors that affects growth, such as nutrient and light availability.

Journal Article↗

PubMed2001

Astaxanthin and peridinin inhibit oxidative damage in Fe(2+)-loaded liposomes: scavenging oxyradicals or changing membrane permeability?

Astaxanthin and peridinin, two typical carotenoids of marine microalgae, and lycopene were incorporated in phosphatidylcholine multilamellar liposomes and tested as inhibitors of lipid oxidation. Contrarily to peridinin results, astaxanthin strongly reduced lipid damage when the lipoperoxidation promoters-H(2)O(2), tert-butyl hydroperoxide (t-ButOOH) or ascorbate-and Fe(2+):EDTA were added simultaneously to the liposomes. In order to check if the antioxidant activity of carotenoids was also related to their effect on membrane permeability, the peroxidation processes were initiated by adding the promoters to Fe(2+)-loaded liposomes (encapsulated in the inner aqueous solution). Despite that the rigidifying effect of carotenoids in membranes was not directly measured here, peridinin probably has decreased membrane permeability to initiators (t-ButOOH > ascorbate > H(2)O(2)) since its incorporation limited oxidative damage on iron-liposomes. On the other hand, the antioxidant activity of astaxanthin in iron-containing vesicles might be derived from its known rigidifying effect and the inherent scavenging ability.

Antioxidants↗

PubMed2001

Different regulatory mechanisms modulate the expression of a dinoflagellate iron-superoxide dismutase.

Regulation of antioxidant enzymes is critical to control the levels of reactive oxygen species in cell compartments highly susceptible to oxidative stress. In this work, we studied the regulation of a chloroplastic iron superoxide dismutase (Fe-SOD) from Lingulodinium polyedrum (formerly Gonyaulax polyedra) under different physiological conditions. A cDNA-encoding Fe-SOD was isolated from this dinoflagellate, showing high sequence similarity to cyanobacterial, algal, and plant Fe-SODs. Under standard growth conditions, on a 12:12-h light-dark cycle, Lingulodinium polyedrum Fe-SOD exhibited a daily rhythm of activity and cellular abundance with the maximum occurring during the middle of the light phase. Northern analyses showed that this rhythmicity is not related to changes in Fe-SOD mRNA levels, indicative of translational regulation. By contrast, conditions of metal-induced oxidative stress resulted in higher levels of Fe-SOD transcripts, suggesting that transcriptional control is responsible for increased protein and activity levels. Daily (circadian) and metal-induced up-regulation of Fe-SOD expression in L. polyedrum are thus mediated by different regulatory pathways, allowing biochemically distinct changes appropriate to oxidative challenges.

Amino Acid Sequence↗

PubMed2001

Antioxidant modulation in response to metal-induced oxidative stress in algal chloroplasts.

To investigate adaptive responses to metal stress at the subcellular level, the oxidative balance in isolated chloroplasts was evaluated for the first time in the unicellular alga Gonyaulax polyedra exposed to the toxic metals Hg(2+), Cd(2+), Pb(2+), and Cu(2+). Different antioxidant responses were verified according to the metal and model of stress applied. Cells chronically exposed to metals exhibited high activity of the antioxidant enzymes superoxide dismutase and ascorbate peroxidase, high glutathione content, and decrease of peridinin levels, whereas no significant changes were detected for beta-carotene levels. In contrast, cells subjected to acute metal stress displayed twice as much beta-carotene but only a slight increase in superoxide dismutase and ascorbate peroxidase activities. The correlation of acute metal treatment and oxidative stress was inferred from the higher oxygen uptake and decreased reduced glutathione pool found in treated cells. In addition, increased oxidative damage to proteins and lipids occurred mainly in cells under acute stress. Pb(2+) was the most damaging toxicant, causing protein oxidation and lipid peroxidation even at chronic treatment. These results indicate that heavy metals are able to induce oxidative stress in chloroplasts of G. polyedra, particularly under acute conditions. Nevertheless, the maintenance of a high antioxidant capacity within chloroplasts seems to be an important strategy during acclimation of G. polyedra to chronic metal stress. By acting at the subcellular site, where oxidative stress is triggered, induction of such chloroplast antioxidants might be crucial for cell survival during exposure to heavy metals.

Ascorbate Peroxidases↗

PubMed2000

Peridinin as the major biological carotenoid quencher of singlet oxygen in marine algae Gonyaulax polyedra.

Carotenoids in light-harvesting proteins and reaction centers increase the overall efficiency of photosynthesis by transferring absorbed light energy to chlorophylls. Peridinin and beta-carotene were isolated from Gonyaulax polyedra in a one-step purification protocol using the preparative circular chromatography (Chromatotron), performed on silica gel under N(2) atmosphere and n-hexane/acetone 8:2 as mobile phase and characterized by extensive (1)H NMR, infrared, and electrospray ionization mass spectrometry analyses. The quenching of singlet molecular oxygen [O(2) ((1)Delta(g))] was evaluated by NIR-emission assays using singlet oxygen generated by sensitization of either perinaphthenone or methylene blue. The NIR-emission assay showed that peridinin quench as singlet oxygen (k(q) = 9.5 x 10(8) M(-1) s(-1)) 5-fold less efficiently than beta-carotene (52 x 10(8) M(-1) s(-1)). A method, based on the use of high-performance liquid chromatography with UV-VIS detection, was then developed for the sensitive quantification of peridinin (55% of total carotenoids) and beta-carotene (4.1% of total carotenoids). Thus, since peridinin is 10-fold more abundant than beta-carotene, it is expected to be the major protector against the deleterious effects of O(2) ((1)Delta(g)) in Gonyaulax polyedra.

Animals↗

PubMed1999

A new member of the leucyl aminopeptidase family purified and identified from a marine unicellular algae.

Leucyl aminopeptidase (LAP; EC 3.4.11.1) activity was purified from crude extracts of the marine unicellular algae Gonyaulax polyedra by a combination of hydrophobic interaction with phenyl sepharose, DEAE-cellulose, and mono-Q HR5/5 ion-exchange chromatography. The undenaturated protein has a molecular mass of about 110 kD and based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme appears to be composed of two possibly identical subunits of 55 kD. The identity of the protein was confirmed by a cross-reaction of the purified protein with an antibody raised against a commercial LAP. Biochemical characterization showed that the Gonyaulax enzyme was similar to most of the previously described LAPs. Gonyaulax LAP is a metallo-enzyme since EDTA and 1,10-phenathroline significantly inhibited activity. Addition of the metal ions Zn(2+), Cu(2+) inhibited 80% of LAP activity, suggesting they are not the natural cofactors of the enzyme. Other metals, such as Ca(2+), Co(2+), Mn(2+), or Mg(2+) (concentrations up to 4 mM), caused no alteration in the total activity of Gonyaulax LAP.

Animals↗

PubMed1999

Acute and chronic effects of toxic metals on viability, encystment and bioluminescence in the dinoflagellate Gonyaulax polyedra.

Toxicity bioassays based on survival were carried out with cells of the marine dinoflagellate Gonyaulax polyedra exposed to mercury (Hg2+ ), cadmium (Cd2+), lead (Pb2+) and copper (Cu2+). The toxicity scale of these metals found was Hg2+ > Cu2+ > Cd2+ > Pb2+. Cells exposed to metals promptly underwent encystment, which is an important strategy for surviving metal exposure. Following 48 h exposure to Cu2+, complete excystment occurred within 96 h after reinoculation of cells in fresh metal-free media, and with Pb2+ partial recovery occurred in that time. Bioluminescence was affected by the metals in a dose-dependent manner primarily by increasing the frequency of flashing, but the glow emission was also altered with acute Cu2+ and Pb2+ treatments. Several physiological processes in G. polyedra are under circadian control. Chronic exposures to metals caused no substantial alterations in the circadian rhythm of bioluminescence glow, indicating that the biological clock of this dinoflagellate is not sensitive to these metals at the concentrations tested.

Animals↗

PubMed1998

Response of superoxide dismutase to pollutant metal stress in the marine dinoflagellate Gonyaulax polyedra.

The response of superoxide dismutase (SOD) activity in the marine dinoflagellate Gonyaulax polyedra to chronic (5.0 ppb Hg, 0.5 ppm Cd, 2.0 ppm Pb and 0.1 ppm Cu, during 30 days) and acute (10.0 ppb Hg, 1.0 ppm Cd, 5.0 ppm Pb and 0.25 ppm Cu, during 48 hours) exposure to metals was investigated. Under chronic exposure to Hg, Cd, Pb, and Cu, total SOD activity of metal-treated cells increased during the first day of exposure to plateau levels of 134, 148, 127, and 139% of control values respectively. Under acute metal exposure, SOD activity increases were of similar magnitude but much more rapid (within several hours) and of shorter duration. In addition, assays for oxidative damage to lipids revealed high levels of lipid peroxidation in cells kept in either chronic or acute exposure to metals reaching values 2-fold greater than the control group. Changes in SOD activity were dependent on the metal, its concentration, and the time of exposure. Non-denaturing polyacrylamide gels revealed induction of Fe-SOD and Mn-SOD but not Cu-Zn-SOD isoforms in cells kept under acute exposure to metals. These results suggest that oxidative stress may be an important mediator of metal toxicity in algal systems, with SOD providing antioxidant protection.

Animals↗

PubMed1996

The effect of light on the biosynthesis of beta-carotene and superoxide dismutase activity in the photosynthetic alga Gonyaulax polyedra.

Daily oscillations of both beta-carotene and superoxide dismutase (SOD) activity are related to the intracellular control of reactive oxygen species (ROS). It is well established that ROS are present in all aerobic cells. We studied the marine dinoflagellate Gonyaulax polyedra which has been extensively used as a model to understand the biological clock at the molecular level. beta-Carotene, besides suppressing singlet molecular oxygen (1O2), may act as a photoreceptor pigment in many photosynthetic cells. The levels of beta-carotene during the day phase were shown to be twice as high as during the night phase. The dose-response curve for light-induced carotenoid synthesis was linear for up to 45 min of light exposure, after which night phase cells contained the same levels of beta-carotene as day phase cells. Cells exposed to light pulses at different times during the dark period displayed the highest beta-carotene induction in the middle of the night. SOD activity of cell-free extracts of G. polyedra was three to four times higher during the day. This rhythm continued in cells kept in constant light, indicating that the regulation can be attributed to the cellular circadian clock. No-denaturing polyacrylamide gels revealed the presence of several SOD isoenzymes in G. polyedra, including CuZnSOD and MnSOD. Furthermore, G. polyedra SOD cross-reacts with a polyclonal antibody raised against SOD. In addition to being gene regulated by ROS concentration, G. polyedra SOD expression seems also to be under the control of the biological clock.

Animals↗

PubMed1996

Imaging oscillations in Gonyaulax: a chloroplast rhythm of nitrate reductase visualized by immunocytochemistry.

Gonyaulax polyedra is a unicellular marine photosynthetic dinoflagellate known to display numerous circadian rhythms, including bioluminescence, motility, cell division and several chloroplast-related rhythms. Due to this, Gonyaulax has become a widely used model organism for studying the cellular biological clock. In this work we describe another rhythm for Gonyaulax cells also associated with the cell's chloroplasts, a rhythm in localization of the enzyme nitrate reductase (NR). A polyclonal antibody was raised against NR purified from G. polyedra cells and used as a probe in immunogold labelling experiments on cell thin sections, comparing day- and night-phase cells. The enzyme localizes to chloroplasts in day-phase cells, while the enzyme is active, and is largely absent in night-phase cells. Counts of gold particle distribution in day- versus night-phase cells show an approximate three-fold increase in enzyme labelling in day-phase plastids. These results closely approximate the four-fold differences shown for NR activity between day and night Gonyaulax cells by biochemical studies. We conclude from the diurnal difference in labelling that NR is localized in Gonyaulax chloroplasts during the day phase and is absent (broken down) in night-phase cells. Thus NR in Gonyaulax is compartmentalized in the chloroplasts and is therefore subject to similar circadian control mechanisms exhibited for other plastid rhythms.

Animals↗

PubMed1995

Post-implantation mouse embryos have the capability to generate and release reactive oxygen species.

The capability of the mouse embryo to generate reactive oxygen species (ROS) was examined. Post-implantation embryos were carefully harvested on Day 8 of pregnancy and the production of ROS was quantified using luminol-sensitized chemiluminescence. The embryos were stimulated with either phorbol myristate acetate (PMA) or all-trans-retinal (retinal) and the reaction kinetics were followed over 10 min. ROS secretion was directly proportional to the number of embryos and was suppressed 56% by superoxide dismutase (SOD), 25% by mannitol and as little as 16% by catalase. Embryos deprived of trophoblast showed no light emission suggesting that the source of ROS generation is the trophoblast. Dihydronicotinamide adenine dinucleotide (NADH)-dependent oxidase activity in the plasma membrane of the trophoblast surface was demonstrated by cytochemical methods. The release of ROS into the extracellular medium during the phagocytic process has been related to the cytolytic effect exhibited by these molecules and, perhaps by this means, the trophoblast can play an active role in the phagocytosis of maternal cells during the process of embryo implantation.

Animals↗

PubMed1995

Circadian oscillation of nitrate reductase activity in Gonyaulax polyedra is due to changes in cellular protein levels.

A circadian rhythm in the activity of nitrate reductase (NR; EC 1.6.6.1) isolated from the marine dinoflagellate Gonyaulax polyedra is shown to be attributable to the daily synthesis and destruction of the protein. The enzyme was purified in three steps: gel filtration on S-300 Sephacryl, an Affigel-Blue column, and a diethylaminoethyl ion-exchange column. Undenatured protein shows a molecular mass of about 310 kD; based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme appears to be composed of six possibly identical subunits. The amino acid composition of the G. polyedra NR is very similar to that reported for the NR of barley leaves, Chlorella vulgaris, and Ankistrodesmus braunii. The experiments reported indicate that the cellular expression of NR is under circadian control. In extracts of cells grown under either constant dim light or a light-dark cycle, the activity of NR exhibits a daily rhythm, peaking at midday phase, as does photosynthesis. Staining with affinity-purified polyclonal antibodies, raised in rabbits against purified NR, shows that the amount of protein changes by a factor of about 10, with the maximum occurring in midday phase.

Amino Acids↗

PubMed1995

Diurnal rhythm of beta-carotene in photosynthetic alga Gonyaulax polyedra.

Carotenoids comprise one of the most widespread classes of pigments found in nature. Polyene pigments from the dinoflagellate Gonyaulax polyedra were extracted every hour over a 24 hour period and the levels of beta-carotene during the day-phase were found to be twice as high as during the night-phase period. Using the thermal decomposition of 1,4-dimethylnaphthalene endoperoxide, the overall quenching ability of beta-carotene and G. polyedra carotenoids of singlet oxygen were found to be 2.04 x 10(9) M-1 s-1 and 4.75 x 10(9) M-1 s-1 respectively. Such a high quenching rate constant suggest a possible role for carotenoids as effective singlet oxygen quenchers in G. polyedra. The dose response curve for light-induced carotenoid synthesis shows a linear response up to 45 minutes of light exposure, after which night phase cells contain the same levels of beta-carotene as day-phase cells. Cells exposed to light pulses at different times during the night period display the highest beta-carotene induction in the middle of the night.

Animals↗

PubMed1994

Aspects of the release of superoxide by leukocytes, and a means by which this is switched off.

Although great progress has been made in understanding the respiratory burst of leukocytes that produce superoxide (O2-), it is possible that a component or components, might have been overlooked. Furthermore, O2- production and its sequels, though cardinal in bactericidal action, might ultimately be damaging to the host's own cells. It is important, therefore, that a biologic mechanism exist to turn off O2- production by stimulated leukocytes. This article offers evidence that methoxatin (PQQ), a redox-cycling orthoquinone, might be involved in O2- production by leukocytes. This is based on the fact that inhibitors of O2- production, such as diphenylene iodonium (DPI) and 4,5-dimethylphenylene diamine (DIMPDA), were shown to sequester PQQ in leukocytes, i.e., to form adducts with that substance. Addition of PQQ to cells blocked with the inhibitors partially restored O2- release. With respect to turning off cellular O2- release, a factor was observed to be released to the medium by old macrophages (14 days old, but not by those less than 7 days old). Such conditioned medium, when added to stimulated neutrophils or macrophages, blocked O2- release. This factor was sensitive to proteases, exhibited molecular sizes of 3 and 11 kDa, and its action was independent of the nature of the stimulus applied to the leukocytes. It was partially purified by column (sizing) chromatography and HPLC. It seems to be a general modulator of the release of reactive oxygen species by phagocytes and is irrespective of phagocytic cellular type, or species from which the cells were derived.

Chromatography, High Pressure Liquid↗

PubMed1993

Leukocyte-deactivating factor from macrophages: partial purification and biochemical characterization. A novel cytokine.

A deactivating factor (MDF) is released from granuloma-like lesions of mice (giant and epithelioid macrophages) to the surrounding medium. Test cells incubated in the presence of MDF display dramatic inhibition of superoxide anion (O2-) release when stimulated. This failure to manifest O2 release is observed whether PMA, all-transretinal, or fMet-Leu-Phe is the stimulating agent. MDF acts on different cell types from different species; mouse macrophages as well as guinea pig, human, and mouse neutrophils. Such results suggest that it is a universal regulatory cytokine with high affinity for phagocytic lineages. The factor was subjected to various purification methods: ultrafiltration, gel chromatography, and reversed phase HPLC. A crude preparation that resulted from conditioning of medium by old macrophages (MCM) shows two peaks of activity when subjected to gel filtration. These correspond to molecular weights for the active principle of 3 and 11 kD. When the factor was obtained by extraction of the same cells after washing and sonication, only the former peak was seen. Fractions corresponding to a MW of 3 kD from several preparations were combined and subjected to HPLC. MDF activity then appeared in a single fraction. MDF is thus putatively a modulator of the cidal activity of phagocytic cells that utilize release of reactive oxygen species for cytocidal activity.

Animals↗