Biomedical subjects

E J Bechara

Publications and source records attributed to E J Bechara.

At least 19 recordsLinked to original sources

PubMed2001

Daily variations of antioxidant enzyme and luciferase activities in the luminescent click-beetle Pyrearinus termitilluminans: cooperation against oxygen toxicity.

Several lines of investigation have suggested an interplay between bioluminescence (BL) and oxyradical metabolism, mainly in bacteria and beetles. Although not yet confirmed, luminescent beetles seem to be challenged daily by oxidative conditions imposed by higher oxygen absorption necessary to enhance light emission for courtship (adult lampyrids and elaterids) and prey attraction (e.g. Pyrearinus termitilluminans larvae). This work reports the activities of luciferase, superoxide dismutase (SOD), catalase and dehydroascorbate reductase (DHAR) and total glutathione content at different times of the day in the bright prothorax and dim abdomen of larval Pyrearinus termitilluminans (Coleoptera: Elateridae), investigating a possible adjuvant role for luciferase in oxygen detoxification. Luciferase activity in the prothorax was shown to peak at 7 p.m., which is the time when P. termitilluminans larvae light up for prey attraction. In their habitat, P. termitilluminans larvae emit light until 8.30 p.m. However, at 8 p.m., prothorax luciferase activity achieved basal levels and total glutathione content declined to the daily lowest value, possibly resulting from hyperoxidative conditions during this time. Significant increases in the activities of total SOD (28%) and catalase (37%) were observed in the prothorax at 9 p.m., which should minimize the extent of damage from this potentially hazardous period. Prothorax total SOD (42% higher than daily average) and abdomen CuZnSOD (41%) and catalase (95%) activities showed extra peaks at 7-10 a.m., and abdomen DHAR activity was maximal (37%) earlier (4-7 a.m.). These morning increases in antioxidant enzyme activities may be associated with biological events other than bioluminescence, e.g. intense physical activity for digging tunnels and/or digestion of captured preys. These data suggest that oxyradical pathway and bioluminescence are coordinated, especially in the prothorax, to minimize the oxidative stress imposed by higher irrigation of the photocytes with O(2) when P. termitilluminans larvae emit light.

Animals↗

PubMed2001

Heteropteris aphrodisiaca O. Machado : effects of extract BST 0298 on the oxidative stress of young and old rat brains.

The plant Heteropteris aphrodisiaca O. Machado is widely employed in Brazilian popular medicine as a stimulant, a tonic and a remedy for nervous debility. Several other plant products with similar therapeutic applications may have such effects based on the antioxidant content of polyphenols (gallates, cathecols, etc.) and flavonoids. The aim of this work was to investigate the effects of Heteropteris aphrodisiaca O. Machado extracts on the antioxidant defences and oxidative stress of young and old rat brains. Tests of lipoperoxidation in vitro, comparing H. aphrodisiaca extracts with butylhydroxytoluene (BHT), a classic antioxidant, showed an efficient inhibition of lipoperoxidation (Q(1/2) = 3.3 microg/mL). Chronic treatment of young and old rats with 50 mg plant extract/kg body weight resulted in a significant increase in superoxide dismutase (SOD) activities (40%) in the brain of old rats, but no changes in catalase and glutathione peroxidase (GPX). No significant variations in antioxidant enzyme activities were observed in the brains of young rats. However, significantly lower levels of lipoperoxidation (30%) were detected in the brain of young rats upon plant extract administration, suggesting that H. aphrodisiaca extracts reduced the oxidative stress to brain lipids.

Aging↗

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↗

PubMed2001

Aerobic oxidation of aminoacetone, a threonine catabolite: iron catalysis and coupled iron release from ferritin.

Aminoacetone (AA) is a threonine and glycine catabolite long known to accumulate in cri-du-chat and threoninemia syndromes and, more recently, implicated as a contributing source of methylglyoxal (MG) in diabetes mellitus. Oxidation of AA to MG, NH(4)(+), and H(2)O(2) has been reported to be catalyzed by a copper-dependent semicarbazide sensitive amine oxidase (SSAO) as well as by Cu(II) ions. We here study the mechanism of AA aerobic oxidation, in the presence and absence of iron ions, and coupled to iron release from ferritin. Aminoacetone (1-7 mM) autoxidizes in Chelex-treated phosphate buffer (pH 7.4) to yield stoichiometric amounts of MG and NH(4)(+). Superoxide radical was shown to propagate this reaction as indicated by strong inhibition of oxygen uptake by superoxide dismutase (SOD) (1-50 units/mL; up to 90%) or semicarbazide (0.5-5 mM; up to 80%) and by EPR spin trapping studies with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), which detected the formation of the DMPO-(*)OH adduct as a decomposition product from the DMPO-O(2)(*)(-) adduct. Accordingly, oxygen uptake by AA is accelerated upon addition of xanthine/xanthine oxidase, a well-known enzymatic source of O(2)(*)(-) radicals. Under Fe(II)EDTA catalysis, SOD (<50 units/mL) had little effect on the oxygen uptake curve or on the EPR spectrum of AA/DMPO, which shows intense signals of the DMPO-(*)OH adduct and of a secondary carbon-centered DMPO adduct, attributable to the AA(*) enoyl radical. In the presence of iron, simultaneous (two) electron transfer from both Fe(II) and AA to O(2), leading directly to H(2)O(2) generation followed by the Fenton reaction is thought to take place. Aminoacetone was also found to induce dose-dependent Fe(II) release from horse spleen ferritin, putatively mediated by both O(2)(*)(-) and AA(*) enoyl radicals, and the co-oxidation of added hemoglobin and myoglobin, which may be viewed as the initial step for potential further iron release. It is thus tempting to propose that AA, accumulated in the blood and other tissues of diabetics, besides being metabolized by SSAO, may release iron and undergo spontaneous and iron-catalyzed oxidation with production of reactive H(2)O(2) and O(2)(*)(-), triggering pathological responses. It is noteworthy that noninsulin-dependent diabetes has been frequently associated with iron overload and oxidative stress.

Acetone↗

PubMed2001

Bioflavonoid rescue of ascorbate at a membrane interface.

In aqueous solution, ascorbate potently prevents bleaching of cytochrome c on exposure to excess H2O2 or t-butyl hydroperoxide. Ascorbate failed to protect cytochrome c in the presence of liposomes of mitochondrial membranelike composition. Like the redox mediator N,N,N,'N'-tetramethyl-p-phenylenediamine (TMPD), however, the bioflavonoids epicatechin and quercetin restored the protection afforded by ascorbate in the presence of liposomes and gave further protection. The quercetin glycoside, rutin, was much less effective, as was the vitamin E analog Trolox. In the presence of liposomes, quercetin alone was relatively ineffective, but cooperated with ascorbate to extend protection synergistically. The results bear specific implications in antioxidant protection of cytochrome c and in moderation of its hydroperoxidase activities in biological membranes. The data also reveal a situation where ascorbate is without effect except in the presence of a bioflavonoid, and substantiate a possibly vital role for certain bioflavonoids in mediating electron transfer from ascorbate into a hydrophobic environment.

Animals↗

PubMed2001

Does the photochemical conversion of colchicine into lumicolchicines involve triplet transients? A solvent dependence study.

beta- and gamma-lumicolchicines are photoproducts formed by the cycloisomerization of the tropolone ring of colchicine (COL) alkaloids. The mechanism of the photoconversion, suggested to involve the triplet state, is examined here by studying the effect of the solvent polarity on the lumicolchicine photoisomer ratio. Triplet COL, detected by laser flash photolysis, is quenched by oxygen, but not by transtilbene or 1-methylnaphtalene. Neither the quantum yield of conversion of COL nor the photoproduct ratio was altered by the presence of oxygen. Likewise, energy transfer to COL from triplet acetone produced by either isobutanal/horseradish peroxidase system or tetramethyldioxetane thermolysis failed to provoke photoreaction of COL. Our data argue against the intermediacy of a COL triplet state in the photoisomerization and stress on the role of specific solvent-solute interactions in determining the partitioning of excited singlet state into the beta- and gamma-isomer formation.

Colchicine↗

PubMed2000

Roles of phosphate and an enoyl radical in ferritin iron mobilization by 5-aminolevulinic acid.

5-Aminolevulinic acid (ALA), a heme precursor that accumulates in acute intermittent porphyria (AIP) and lead poisoning, undergoes enolization and subsequent iron-catalyzed oxidation at neutral pH. Iron is released from horse spleen ferritin (HoSF) by both ALA-generated O(2)(.-) and enoyl radical (ALA(z.rad)), which amplifies the chain of ALA oxidation (autocatalysis). Iron chelators such as EDTA, ATP, but not citrate, and phosphate accelerate this process and ALA-promoted iron release from HoSF is faster in horse spleen isoferritins containing larger amounts of phosphate in the core. ALA (+0.377 V versus standard hydrogen electrode) is less effective in releasing iron from ferritin than are thioglycollic acid, 6-hydroxydopamine, and N,N,N', N'-tetramethyl-p-phenylenediamine. During electrochemical one electron oxidation of ALA in a nitrogen atmosphere, spin trapping experiments with 3,5-dibromo-4-nitrosobenzenesulfonic acid demonstrated the formation of a spin adduct characterized by a six line signal, indicating a secondary carbon-centered radical and attributed to a resonant ALA&z.rad; radical. Iron is also released in such anaerobic electrochemical oxidations of ALA in the presence of ferritin, suggesting that, in addition to O(2)(*-), ALA&z.rad; can promote iron mobilization from ferritin. Hence, ALA&z.rad; may amplify the metal-catalyzed oxidation of ALA, damaging ALA-accumulating cells and possibly contributing to the symptoms of porphyria.

Adenosine Triphosphate↗

PubMed2000

Modifications in heme iron of free and vesicle bound cytochrome c by tert-butyl hydroperoxide: a magnetic circular dichroism and electron paramagnetic resonance investigation.

To characterize changes to the heme and the influence of membrane lipids in the reaction of cytochrome c with peroxides, we studied the reaction of cytochrome c with tert-butyl hydroperoxide (tert-BuOOH) by magnetic circular dichroism (MCD) and direct electron paramagnetic resonance (EPR) in the presence and absence of different liposomes. Direct low-temperature (11 degrees K) EPR analysis of the cytochrome c heme iron on exposure to tert-BuOOH shows a gradual (180 s) conversion of the low-spin form to a high-spin Fe(III) species of rhombic symmetry (g = 4.3), with disappearance of a prior peroxyl radical signal (g(o) = 2.014). The conversion to high spin precedes Soret band bleaching, observable by UV/Vis spectroscopy and by magnetic circular dichroism (MCD) at room temperature, that indicates loss of iron coordination by the porphyrin ring. The presence of cardiolipin-containing liposomes delayed formation of the peroxyl radical and conversion to high-spin iron, while dicetylphosphate (DCP) liposomes accelerated these changes. Correspondingly, bleaching of cytochrome c by tert-BuOOH at room temperature was accelerated by several negatively charged liposome preparations, and inhibited by mitochondrial-mimetic phosphatidylcholinephosphatidylethanolaminecardiolipin (PCPECL) liposomes. Concomitant with bleaching, spin-trapping measurements with 5,5-dimethyl-1-pyroline-N-oxide showed that while the relative production of peroxyl, alkoxyl, and alkyl radicals was unaffected by DCP liposomes, PCPECL liposomes decreased the spin-trapped alkoxyl radical signal by 50%. The EPR results show that the primary initial change on exposure of cytochrome c to tert-BuOOH is a change to a high-spin Fe(III) species, and together with MCD measurements show that unsaturated cardiolipin-containing lipid membranes influence the interaction of tert-BuOOH with cytochrome c heme iron, to alter radical production and decrease damage to the cytochrome.

Animals↗

PubMed2000

Chemiluminescent aldehyde and beta-diketone reactions promoted by peroxynitrite.

Peroxynitrite is shown here to promote the aerobic oxidation of isobutanal (IBAL) and 3-methyl-2,4-pentanedione (MP) in a pH 7.2 phosphate buffer into acetone plus formate and biacetyl plus acetate, respectively. These products are expected from dioxetane intermediates, whose thermolysis is known to be chemiluminescent (CL). Accordingly, the extent of total oxygen uptake by IBAL at different concentrations parallels the corresponding CL maximum intensities. The pH profile based on oxygen uptake data for the MP reaction matches the titration curve of peroxynitrous acid (pK(a) approximately 7), indicating that peroxynitrite anion is the oxidizing agent. Energy transfer studies with IBAL and the 9, 10-dibromoanthracene-2-sulfonate ion, a triplet carbonyl detector, indicates that triplet acetone (tau = 19 micros) is the energy donor. It is postulated that IBAL- or MP-generated triplet carbonyls are produced by the thermolysis of dioxetane intermediates, which are formed by the cyclization of alpha-hydroperoxide intermediates produced by insertion of dioxygen into the IBAL or MP enolyl radicals, followed by their reduction. Accordingly, EPR spin-trapping studies with 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS) and 2-methyl-2-nitrosopropane (MNP) revealed the intermediacy of carbon-centered radicals, as expected for one-electron abstraction from the enol forms of IBAL or MP by peroxynitrite. The EPR data obtained with IBAL also reveal formation of the isopropyl radical produced by competitive nucleophilic addition of ONOO(-) to IBAL, followed by homolytic cleavage of this adduct and beta-scission of the resulting Me(2)CHCH(O(-))O(*). Superstoichiometric formation of fragmentation products from IBAL or MP attests to the prevalence of an autoxidation chain reaction, here proposed to be initiated by one-electron abstraction by ONOO(-) from the substrate. This work reveals the potential role of peroxynitrite as a generator of electronically excited species that may contribute to deleterious and pathological processes associated with excessive nitric oxide and aldehyde production.

Aldehydes↗

PubMed2000

Iron mobilization by succinylacetone methyl ester in rats. A model study for hereditary tyrosinemia and porphyrias characterized by 5-aminolevulinic acid overload.

Accumulation of 5-aminolevulinic acid (ALA) is an event characteristic of porphyrias that may contribute to their pathological manifestations. To investigate effects of ALA independent of porphyrin accumulation we treated rats with the methyl ester of succinylacetone, an inhibitor of 5-aminolevulinic acid dehydratase that accumulates in the porphyric-like syndrome hereditary tyrosinemia. Acute 2-day treatment of fasted rats with succinylacetone methyl ester (SAME) promoted a 27% increase in plasma ALA. This increase in plasma ALA was accompanied by augmentation of the level of total nonheme iron in liver (37%) and brain (20%). Mobilization of iron was also indicated by 49% increase in plasma iron and a 77% increase in plasma transferrin saturation. Liver responded with a mild (12%) increase in ferritin. Under these acute conditions, some indications of oxidative stress were evident: a 15% increase in liver reactive protein carbonyls, and a 42% increase in brain subcellular membrane TBARS. Brain also showed a 44% increase in CuZnSOD activity, consistent with observations in treatment with ALA. Overall, the data indicate that SAME promotes ALA-driven changes in iron metabolism that could lead to increased production of free radicals. The findings support other evidence that accumulation of ALA in porphyrias and hereditary tyrosinemia may induce iron-dependent biological damage that contributes to neuropathy and hepatoma.

Aminolevulinic Acid↗

PubMed2000

Direct determination of the kinetics of oxygen diffusion to the photocytes of a bioluminescent elaterid larva, measurement of gas- and aqueous-phase diffusional barriers and modelling of oxygen supply.

We describe the development and use of a direct kinetic technique to determine the time taken for oxygen to diffuse from the external environment into the light-producing cells (photocytes) in the prothorax of bioluminescent larvae of Pyrearinus termitilluminans. This was achieved by measuring the time course of the pseudoflash induced through sequential anoxia followed by normoxia. We have also determined the separate times taken for this oxygen diffusion in gaseous and tissue (predominantly aqueous) phases by using helium and nitrogen as the carrier gas. Of the total time taken for diffusion, that in the gas phase required 613+/-136 ms (mean +/- s.e. m., N=5) whilst that in the aqueous phase required 1313+/-187 ms. These values imply pathlengths of diffusion in the gaseous and aqueous phases of 4.80x10(-)(3)+/-0.53x10(-)(3) and 8. 89x10(-)(5)+/-0.61x10(-)(5 )m, respectively. In addition, the pathlength of gas-phase diffusion was used to derive a parameter relating to the tortuosity of the tracheal system. These values, together with those obtained upon bioluminescent oxygen consumption, have been used to model oxygen supply to the photocyte. From these studies, it would also appear that the modulation of tracheolar fluid levels might be a significant mechanism of control of tissue oxygen levels in at least some insects.

Animals↗

PubMed2000

Luciferase and urate may act as antioxidant defenses in larval Pyrearinus termitilluminans (Elateridae: Coleoptera) during natural development and upon 20-hydroxyecdysone treatment.

Insects show unique adaptations to cope with oxidative challenges during larval development, metamorphosis and adulthood. Our previous findings suggested that bioluminescence may act as an auxiliary oxygen-detoxifying mechanism in larvae of Pyrearinus termitilluminans (Elateridae: Coleoptera). We now study the antioxidant status in larval P. termitilluminans, evaluated in terms of levels of chemical and enzymatic antioxidant defenses, as compared to luciferase activity in the prothorax (intensely bright) and abdomen (dim) of the larvae, during natural- and 20-hydroxyecdysone (20-HE)-induced development. In the prothorax, relative total SOD activities in small (< 1 cm), medium (1-2 cm) and large (> 2 cm) larvae were 1.00:0.53:0.32. Catalase activity also decreased with development (1.00:0.69:0.55). In contrast, prothorax luciferase activities and urate content increased with ratios of 1.0:2.2:2.5 and 1:15:97, respectively. No increases were found in the level of prothorax lipid and protein oxidation. In the abdomen, luciferase activity decreased markedly with development (1.00:0.33:0.17), as did other antioxidant enzymes, including dehydroascorbate reductase (1.00:0.59:0.17) and levels of lipid peroxidation products and protein carbonyls. Similar variations were observed in antioxidant enzyme activities when the larvae were treated with 20-HE, except for prothorax catalase. As observed in natural larval growth, luciferase activity was augmented (two-fold in prothorax) upon steroid treatment, and the levels of thiobarbituric acid-reactive substances were magnified in both segments. The increase of luciferase activity and a higher urate content in the prothorax during larval development may reflect metabolic adaptations to keep levels of oxyradicals low in order to compensate for decreased antioxidant enzyme activities.

Animals↗

PubMed1999

Cloning, sequence analysis, and expression of active Phrixothrix railroad-worms luciferases: relationship between bioluminescence spectra and primary structures.

Phrixothrix railroad-worms emit yellow-green light through 11 pairs of lateral lanterns along the body and red light through two cephalic lanterns. The cDNAs for the lateral lanterns luciferase of Phrixothrix vivianii, which emit green light (lambda max= 542 nm), and for the head lanterns of P. hirtus, which emit the most red-shifted bioluminescence (lambda max= 628 nm) among luminescent beetles, were cloned. Positive clones which emitted green (PvGR: lambda max= 549 nm) and red (PhRE: lambda max= 622 nm) bioluminescence were isolated. The lucifereases coded by PvGR (545 amino acid residues) and PhRE (546 amino acid residues) cDNAs share 71% identity. PvGR and PhRE luciferases showed 50-55% and 46-49% identity with firefly luciferases, respectively, and 47-49% with click-beetle luciferases. PhRE luciferase has some unique residues which replace invariant residues in other beetle luciferases. The additional residue Arg 352 in PhRE, which is deleted in PvGR polypeptide, seems to be another important structural feature associated with red light production. As in the case of other railroad-worms and click-beetle luciferases studied, Phrixothrix luciferases do not undergo the typical red shift suffered by firefly luciferases upon decreasing pH, a property which might be related to the many amino acid residues shared in common between railroad-worm and click-beetle luciferase.

Amino Acid Sequence↗

PubMed1999

Superoxide dismutase, catalase and glutathione peroxidase activities in the lymphoid organs and skeletal muscles of rats treated with dexamethasone.

The effect of dexamethasone (a synthetic glucocorticoid) on the activity of antioxidant enzymes (superoxide dismutase (SOD), catalase and glutathione peroxidase) of the lymphoid organs (mesenteric lymph nodes (MLN), spleen and thymus) was investigated. For comparison with non-immune tissues, skeletal muscles (soleus and gastrocnemius (GC) were also studied. As an indication of the occurrence of lipid peroxidation, the content of thiobarbituric acid reactant substances (TBARs) was also determined. Dexamethasone treatment decreased the TBARs content of the lymphoid organs and raised it in the GC and soleus muscles. The activity of Cu/Zn-SOD was reduced in all tissues. However, the activity of Mn-SOD was decreased in the MLN and soleus muscle only. The activity of catalase was reduced in the MLN and thymus and raised in the spleen and GC and soleus muscles. The imposed treatment raised the activity of GPX in the MLN, thymus and spleen and reduced it in GC and soleus muscles. These data led us to postulate that the mechanism for the therapeutic effect of glucocorticoids as antiinflammatory and immunosuppressive agents might include modification of antioxidant enzyme activities.

Animals↗

PubMed1999

Trapping of free radicals with direct in vivo EPR detection: a comparison of 5,5-dimethyl-1-pyrroline-N-oxide and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide as spin traps for HO* and SO4*-.

To spin trap hydroxyl radical (HO*) with in vivo detection of the resultant radical adducts, the use of two spin traps, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) (10 mmol/kg) has been compared. In mice treatment with 5-aminolevulinic acid and Fe3+ resulted in detection of adducts of hydroxyl radicals (HO*), but only with use of DEPMPO. Similarly, 'HO* adducts' generated via nucleophilic substitution of SO4*- adducts formed in vivo could be observed only when using DEPMPO as the spin trap. The reasons for the differences observed between DEPMPO and DMPO are likely due to different in vivo lifetimes of their hydroxyl radical adducts. These results seem to be the first direct in vivo EPR detection of hydroxyl radical adducts.

Animals↗

PubMed1999

Diphenylacetaldehyde-generated excited states promote damage to isolated rat liver mitochondrial DNA, phospholipids, and proteins.

This work studies damage to rat liver mitochondrial protein, lipid, and DNA caused by electronically excited states generated by cytochrome c-catalyzed diphenylacetaldehyde enol oxidation to triplet benzophenone. The extension of lipid peroxidation was estimated by production of thiobarbituric acid-reactive substances and by formation of Schiff bases with membrane proteins, evaluated by SDS-polyacrylamide gel electrophoresis. Concomitant with DPAA-driven mitochondrial permeabilization, extensive mtDNA fragmentation occurred and DNA adducts with aldehydes-products of fatty acid oxidation-were observed. The degree of lipid peroxidation and mtDNA alterations were significantly decreased by butylated hydroxytoluene, a potent peroxidation chain breaker. The lipid peroxidation process was also partially inhibited by the bioflavonoid rutin and urate totally prevented the mitochondrial transmembrane potential collapse. In all cases, the mitochondrial damage was dependent on the presence of phosphate ions, a putative bifunctional catalyst of carbonyl enolization. These data are consistent with the notion that triplet ketones may act like alkoxyl radicals as deleterious reactive oxygen species on biologic structures. Involvement of singlet dioxygen formed by triplet-triplet energy transfer from benzophenone in the model reaction with DPAA/cytochrome c in the presence of DCP liposomes was suggested by quenching of the accompanying chemiluminescence upon addition of histidine and lycopene.

Aldehydes↗

PubMed1999

Measurement of oxygen partial pressure, its control during hypoxia and hyperoxia, and its effect upon light emission in a bioluminescent elaterid larva.

This study investigates the respiratory physiology of bioluminescent larvae of Pyrearinus termitilluminans in relation to their tolerance to hypoxia and hyperoxia and to the supply of oxygen for bioluminescence. The partial pressure of oxygen (P(O2)) was measured within the bioluminescent prothorax by in vivo electron paramagnetic resonance (EPR) oximetry following acclimation of larvae to hypoxic, normoxic and hyperoxic (normobaric) atmospheres and during periods of bioluminescence (during normoxia). The P(O2) in the prothorax during exposure to an external P(O2) of 15.2, 160 and 760 mmHg was 10.3+/-2.6, 134+/-0.9 and 725+/-73 mmHg respectively (mean +/- s.d., N=5; 1 mmHg=0.1333 kPa). Oxygen supply to the larvae via gas exchange through the spiracles, measured by determining the rate of water loss, was also studied in the above atmospheres and was found not to be dependent upon P(O2). The data indicated that there is little to no active control of extracellular tissue P(O2) within the prothorax of these larvae. The reduction in prothorax P(O2) observed during either attack-response-provoked bioluminescence or sustained feeding-related bioluminescence in a normoxic atmosphere was variable, but fell within the range 10-25 mmHg. The effect of hypoxic atmospheres on bioluminescence was measured to estimate the intracellular P(O2) within the photocytes of the prothorax. Above a threshold value of 50-80 mmHg, bioluminescence was unaffected by P(O2). Below this threshold, an approximately linear relationship between P(O2) and bioluminescence was observed. Taken together with the extracellular P(O2) measurements, this suggests that control of P(O2) within the photocyte may occur. This work establishes that EPR oximetry is a valuable technique for long-term measurement of tissue P(O2) in insects and can provide valuable insights into their respiratory physiology. It also raises questions regarding the hypothesis that bioluminescence can have a significant antioxidative effect by reduction of prothorax P(O2 )through oxygen consumption.

Animals↗

PubMed1999

Cloning and molecular characterization of the cDNA for the Brazilian larval click-beetle Pyrearinus termitilluminans luciferase.

The larval click-beetle Pyrearinus termitilluminans elicits the phenomenon of luminous termite mounds in the central-west region of Brazil. The bioluminescence (BL) spectrum of this larva (lambda max = 534 nm) is one of the most blue-shifted reported among known luminescent Coleoptera. We have isolated mRNA from larval thoracic lanterns and constructed a cDNA library into a lambda ZAP II vector. An expression library was obtained after excision of the pBluescript plasmid. This library was screened by photodetection and one clone that emitted green BL (lambda max = 538 nm) was isolated. The 2.2 kb cDNA insert includes a 543 residue open reading frame showing 82% homology with the luciferase isoenzymes of Pyrophorus plagiophthalamus (Coleoptera: Elateridae). As expected, the region between residues 223 and 247 that contains the putative active site for BL color determination showed a higher degree of homology among click-beetle luciferases that elicit closer BL colors. The in vitro BL spectrum of recombinant P. termitilluminans luciferase also peaks at 538 nm and, as in the case of native enzyme, does not show any bathochromic shift upon decreasing the pH.

Amino Acid Sequence↗