Brittle-star bioluminescence functions as an aposematic signal to deter crustacean predators

The behaviour of three species of nocturnally active, visually orienting crabs (Portunus sebae, P. spinnimanus and P. ordwayi) was observed to determine whether the luminescent signals produced by Ophiopsila riisei (Echinodermata: Ophiuroidea) function as an aposematic deterrent against crustacean predators. In repeated experimental trials, crabs showed more rapid rejection of luminescent unpalatable ophiuroids than of non-luminescent controls. After five trials, unpalatable luminescent prey were rejected three times as quickly as either unpalatable or palatable non-luminescent controls. Over the course of the trials, crabs damaged fewer luminescent ophiuroids than non-luminescent controls. Furthermore, blind crabs (that could not perceive the luminescent flashes) caused significantly more damage to luminescent brittle-stars than did crabs with intact eyes (that could see the light signals). All three species of crab ophiuroids versus non-luminescent controls. Ophiopsila riisei survived the majority of crab attacks, and crabs can therefore learn to reject unpalatable luminescent prey without killing them. This suggests that individual selection may be an important mechanism for the evolution of aposematic luminescent signals in this, and probably other, luminescent species.     

Midgut proteinases in three divergent species of Coleoptera

Cysteine proteinases have been found in some families of Coleoptera and, based on this, these enzymes were supposed to be characteristic of Coleoptera. To test this hypothesis, we studied midgut homogenates of three phylogenetically distant Coleoptera species: Tenebrio molitor (Tenebrionidae) larvae, Pyrearinus termitilluminans (Elateridae) larvae, and Pheropsophus aequinoctialis (Carabidae) adults. T. molitor display two cysteine proteinases (pHo 6.8) resolved in Superose (FPLC) with Mr 31,000 and 51,000. These enzymes are inhibited by E-64 and pHMB, are activated by EDTA + cysteine and hydrolyze benzoyl-DL-arginine-β-naphthylamide. T. molitor enzymes differ from a cysteine proteinase (Mr 64,000 using Superose) present in the wheat meal ingested by the insect. The cysteine proteinases predominate in the anterior two thirds of T. molitor midgut, probably because they are unstable in the higher luminal pH observed in the posterior third of the midgut. P. termitilluminans and P. aequinoctialis do not display cysteine proteinases, although they have trypsins (Mr 15,000, 25,000 and 41,000 for P. termitilluminans; Mr 26,000, 33,000 and 52,000 for P. aequinoctialis) and chymotrypsins (Mr 38,000 and 25,000 for P. aequinoctialis and Mr 15,000 for P. termitilluminans). Our results, together with literature data, suggest that cysteine proteinases occur in the Cucujiformia ancestor, which corresponds to the ancestor of most Coleoptera which ingest seeds rich in serine proteinase inhibitors.     

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.     

Antioxidant defences in marine fish—II. Elasmobranchs

1. Tissues from four ray species and two shark species were examined concerning Superoxide dismutase and catalase content, and malonaldehyde and tocopherol concentrations.2. Livers displayed the highest Superoxide dismutase and catalase content in all species examined. Sharks exhibited higher mean enzyme contents than rays (3.6 and 2.4 nmol SOD g⁻¹ wet tissue, 185 and 120 pmol catalase g⁻¹ wet tissue).3. Tocopherol was detected in liver from all species, in kidney and in ovary from one ray and one shark species, and in shark cristaline in a range of 0.1–4.8 nmol g⁻¹ wet tissue.4. The content of antioxidant enzymes in elasmobranchs was lower than that of teleosts, and seems to follow the overall metabolic oxygen consumption or activity level from each fish major taxonomic group.5. Liver peroxidation as measured as MDA/TBARS concentration, revealed very high values (range of 1.5–4.5 μmoles g⁻¹ wet tissue), approximately one order of magnitude higher when compared with mammals.     

Characterization and expression of cytoplasmic copper/zinc superoxide dismutase (CuZn SOD) gene under temperature and hydrogen peroxide (H2O2) in rotifer Brachionus calyciflorus

Superoxide dismutase (SOD, EC 1.15.1.1) is an important antioxidant enzyme that protects organs from damage by reactive oxygen species (ROS). We cloned cDNA encoding SOD activated with copper/zinc (CuZn SOD) from the rotifer Brachionus calyciflorus Pallas. The full-length cDNA of CuZn SOD was 692 bp and had a 465 bp open reading frame encoding 154 amino acids. The deduced amino acid sequence of B. calyciflorus CuZn SOD showed 63.87%, 60.00%, 59.74% and 48.89% similarity with the CuZn SOD of the Ctenopharyn godonidella, Schistosoma japonicum, Drosophila melanogaster and Caenorhabditis elegans, respectively. The phylogenetic tree constructed based on the amino acid sequences of CuZn SODs from B. calyciflorus and other organisms revealed that rotifer is closely related to nematode. Analysis of the expression of CuZn SOD under different temperatures (15, 30 and 37 °C) revealed that its expression was enhanced 4.2-fold (p < 0.001) at 30 °C after 2 h, however, the lower temperature (15 °C) promoted CuZn SOD transiently (4.1-fold, p < 0.001) and then the expression of CuZn SOD decreased to normal level (p > 0.05). When exposed to H₂O₂ (0.1 mM), CuZn SOD, manganese superoxide dismutase (Mn SOD) and catalase (CAT) gene were upregulated, and in addition, the mRNA expression of CuZn SOD gene was induced instantaneously after exposure to vitamin E. It indicates that the CuZn SOD gene would be an important gene in response to oxidative and temperature stress.     

Eosinophil-mediated killing of schistosomula of Schistosoma mansoni: Oxidative requirement for enhancement by eosinophil colony stimulating factor (CSF-α) and supernatants with eosinophil cytotoxicity enhancing activity (E-CEA)

Factors which enhance eosinophil-mediated killing of antibody-coated schistosomula of Schistosoma mansoni include semipurified eosinophil colony stimulating factor (CSF-α) and eosinophil cytotoxicity enhancing activity (E-CEA) present in supernatants from cultured mononuclear cells. We have examined the mechanism of enhancement. Both actions require oxygen in order to enhance killing and do not enhance killing under anaerobic conditions (P ? 0.005). E-CEA had no detectable effect upon oxidative metabolism. In contrast to CSF-α which, in our previous studies, increased Superoxide anion productions and quantitative leukocyte iodination, E-CEA had no detectable effect upon oxidative metabolism. In order to test whether active oxygen products might mediate enhancement of killing, the effects of the addition of Superoxide dismutase and catalase were tested. Neither enzyme showed inhibition of CSF-α or E-CEA enhancement of eosinophil-mediated killing. The effects of CSF-α and E-CEA were not additive. These studies suggest that both CSF-α and E-CEA exert enhancement of killing by means of an as yet unidentified oxygen requiring process.     

Vision in click beetles (Coleoptera: Elateridae): pigments and spectral correspondence between visual sensitivity and species bioluminescence emission

Among lampyrids, intraspecific sexual communication is facilitated by spectral correspondence between visual sensitivity and bioluminescence emission from the single lantern in the tail. Could a similar strategy be utilized by the elaterids (click beetles), which have one ventral abdominal and two dorsal prothoracic lanterns? Spectral sensitivity [S(λ)] and bioluminescence were investigated in four Brazilian click beetle species Fulgeochlizus bruchii, Pyrearinus termitilluminans, Pyrophorus punctatissimus and P. divergens, representing three genera. In addition, in situ microspectrophotometric absorption spectra were obtained for visual and screening pigments in P. punctatissimus and P. divergens species. In all species, the electroretinographic S(λ) functions showed broad peaks in the green with a shoulder in the near-ultraviolet, suggesting the presence of short- and long-wavelength receptors in the compound eyes. The long-wavelength receptor in Pyrophorus species is mediated by a P540 rhodopsin in conjunction with a species-specific screening pigment. A correspondence was found between green to yellow bioluminescence emissions and its broad S(λ) maximum in each of the four species. It is hypothesized that in elaterids, bioluminescence of the abdominal lantern is an optical signal for intraspecifc sexual communication, while the signals from the prothoracic lanterns serve to warn predators and may also provide illumination in flight.     

Antioxidant responses of halophyte plant Aeluropus littoralis under long-term salinity stress

Salinity influences the agricultural production all over the world. This constrain, similar to others biotic and abiotic stresses generate the reactive oxygen species such as superoxide, hydrogen peroxide and hydroxyl radicals. In the evolution process of halophyte plants the mechanisms to detoxify ROS, such as antioxidant enzymes, have been developed. Aeluropus littoralis is a special halophyte that selected to our research, so the plants treated with NaCl at different salt concentration (0, 250, 450 and 650 mM) for a period 45 days. Leaves and roots (separately) collected and their proteins extracted for superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POD) activity assay. Meanwhile the electrolyte leakage of leaves analyzed and increased at 450 and 650 mM of NaCl concentrations. Superoxide dismutase and catalase showed same pattern for changing in enzymatic activities (increasing activity by salt stress in roots and decreasing in shoot at 450 and 650 mM stress), also peroxidase and ascorbate peroxidase activity almost increased in all stress conditions.     

Superoxide dismutase and catalase in Azotobacter vinelandii grown in continuous culture at different dissolved oxygen concentrations

Superoxide dismutase and catalase activities were studied in Azotobacter vinelandii grown diazotrophically at different ambient oxygen concentrations in continuous culture. Activities were expressed either as specific activity or activity per cell. Specific superoxide dismutase activity increased by a factor of 1.6 with increasing oxygen concentration from about 1% to 90% air saturation of the growth medium whereas specific catalase activity increased only slightly, if at all. Since cell volumes increased in parallel to increases in the oxygen concentration cellular superoxide dismutase activities increased by a factor of 4.3 while cellular catalase activities increased by a factor of 3.3. Under all conditions only the Fe-containing form of superoxide dismutase was detected. The possible function of these enzymes in the protection nitrogenase from oxygen damage is discussed.Abbreviation SOD superoxide dismutase     

Increased spontaneous chemiluminescence from liver homogenates and isolated hepatocytes upon inhibition of o2− and h2o2 utilization

The intracellular steady-state concentrations of hydrogen peroxide or Superoxide anion were increased by inhibiting either catalase, glutathione peroxidase, or Superoxide dismutase activities. Catalase was inhibited with aminotriazole while glutathione peroxidase activity was blocked by eliminating reduced glutathione after addition of either iodoacetamide diethylmaleate or phorone. The concentration of aminotriazole that stimulated chemiluminescence in 50% (60 mM) was very similar to the K_i for catalase activity (70 mM). Cyanide, an inhibitor of both catalase and Superoxide dismutase, stimulated chemiluminescence in 50% at a concentration (0.15 mM) which is much closer from the K_i for Superoxide dismutase (0.25 mM) than from the K_i for catalase (15 μM). The Superoxide dismutase inhibitor diethyldithiocarbamate also increased chemiluminescence six- to ten-fold. Depletion of reduced glutathione stimulated spontaneous chemiluminescence when its concentration decreased below 4.5 μmol · g liver⁻¹. The results shown herein suggest that the changes in the intracellular steady-state concentration occurring after inhibition of any antioxidant enzyme are responsible for the increased spontaneous chemilumi-nescence. Spontaneous chemiluminescence from intact cells may be used as a noninvasive method for monitoring intracellular free radical metabolism.     

A trifunctional enzyme with glutathione S-transferase,glutathione peroxidase and superoxide dismutase activity

Superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione S-transferase (GST) and glutathione reductase (GR) play crucial roles in balancing the production and decomposition of reactive oxygen species (ROS) in living organisms. These enzymes act cooperatively and synergistically to scavenge ROS, as not one of them can singlehandedly clear all forms of ROS. In order to imitate the synergy of the enzymes, we designed and generated a recombinant protein, which comprises of a Schistosoma japonicum GST (SjGST) and a bifunctional 35-mer peptide with SOD and GPX activities. The engineered protein demonstrated SOD, GPX and GST activities simultaneously. This trifunctional enzyme with SOD, GPX and GST activities is expected to be the best ROS scavenger.     

Zymogram profiling of superoxide dismutase and catalase activities allows Saccharomyces and non-Saccharomyces species differentiation and correlates to their fermentation performance

Aerobic organisms have devised several enzymatic and non-enzymatic antioxidant defenses to deal with reactive oxygen species (ROS) produced by cellular metabolism. To combat such stress, cells induce ROS scavenging enzymes such as catalase, peroxidase, superoxide dismutase (SOD) and glutathione reductase. In the present research, we have used a double staining technique of SOD and catalase enzymes in the same polyacrylamide gel to analyze the different antioxidant enzymatic activities and protein isoforms present in Saccharomyces and non-Saccharomyces yeast species. Moreover, we used a technique to differentially detect Sod1p and Sod2p on gel by immersion in NaCN, which specifically inhibits the Sod1p isoform. We observed unique SOD and catalase zymogram profiles for all the analyzed yeasts and we propose this technique as a new approach for Saccharomyces and non-Saccharomyces yeast strains differentiation. In addition, we observed functional correlations between SOD and catalase enzyme activities, accumulation of essential metabolites, such as glutathione and trehalose, and the fermentative performance of different yeasts strains with industrial relevance.     

Manipulation of Sod1 expression ubiquitously, but not in the nervous system or muscle, impacts age-related parameters in Drosophila

Superoxide dismutase 1 (SOD1) is an important antioxidant previously shown to impact life span in Drosophila. We examined the consequences of manipulating Sod1 expression throughout the body or in the nervous system or musculature on life span and age-related locomotor impairment (ARLI) in Drosophila. Ubiquitous overexpression of SOD1 extended life span but did not substantially forestall ARLI, whereas ubiquitous knock-down of Sod1 shortened life span and accelerated ARLI. Interestingly, neither overexpression of Sod1 nor expression of Sod1 RNAi in the nervous system or muscle altered life span or ARLI. Our studies suggest that the control of reactive oxygen species by SOD1 in tissues other than the nervous system and musculature support life span and ARLI in Drosophila.     

超氧化物歧化酶(SOD)研究进展

环境胁迫使植物细胞中积累大量的活性氧,从而导致蛋白质、膜脂、DNA及其它细胞组分的严重损伤。植物体内有效清除活性氧的酶类包括超氧化物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)、过氧化氢酶(CAT)等,其中研究最深入的是SOD。利用基因工程策略增加这些物质在植物体内的含量,从而获得抗逆转基因植株。     

Superoxide radicals and antiradical defence of propionic acid bacteria

Superoxide dismutase activity was demonstrated for 6 strains of 3 propionibacteria species. Rather high level of superoxide dismutase activity found in propionibacteria was in accordance with high level of catalase activity reported for propionibacteria previously. Both these activities were shown to have cytozolic localization. For the first time peroxidase activity was detected in gel-fractionated crude cell extracts of propionibacteria. The ability to produce superoxide radicals in NADH-dependent oxidation system was revealed for three strains of the bacteria. The level of superoxide production by the membrane particles of the propionic acid bacteria correlated with the levels of superoxide dismutase and catalase activities and was the lowest for Propionibacterium shermanii. The ability to perform monovalent oxygen reduction during succinate oxidation was not revealed. The intact cells of P. globosum, P. vannielii, P. shermanii apparently did not excrete superoxide radicals into culture fluid during respiration.     

Identification and characterization of an intracellular Cu,Zn-superoxide dismutase (icCu/Zn-SOD) gene from clam Venerupis philippinarum

Superoxide dismutase (SOD, EC 1.15.1.1) represents one kind of enzyme involved in scavenging the high level of reactive oxygen species (ROS) into molecular oxygen and hydrogen peroxide. In the present study, the intracellular Cu/Zn-SOD gene (icCu/Zn-SOD) of Venerupis philippinarum (denoted as VpSOD) was identified from haemocytes by homology cloning and RACR approaches. The full-length cDNA of VpSOD consisted of 910 nucleotides with a canonical polyadenylation signal sequence AATAAA, a polyA tail, and an open-reading frame of 465 bp encoding 154 amino acids. The deduced amino acid of VpSOD shared high similarity with the icCu/Zn-SODs from other species, indicating that VpSOD should be a new member of icCu/Zn-SOD family. Several highly conserved motifs including Cu, Zn binding sites (H⁴⁶, H⁴⁸, H⁶³, H¹²⁰ for Cu binding, and H⁶³, H⁷¹, H⁸⁰, D⁸³ for Zn binding), intracellular disulfide bond and two Cu, Zn SOD signatures were also identified in VpSOD. The temporal expression of VpSOD in haemocytes after Vibrio anguillarum challenge was recorded by quantitative real-time RT-PCR. The relative expression level of VpSOD mRNA was up-regulated rapidly at 6 h post-infection and reached 18-fold of the control group. After a drastic decrease at 12 h, the expression level increased again and reached 22-fold to that in the control group at 96 h post-infection. All these results indicated that VpSOD was an acute-phase protein involved in the immune responses of V. philippinarum.     

The effect of menadione-induced oxidative stress on the in vivo reactive oxygen species and antioxidant response system of Phanerochaete chrysosporium

The antioxidant response system of Phanerochaete chrysosporium against menadione-induced oxidative stress was investigated in this study. The superoxide anion radical levels in tested menadione-supplemented conditions generally decreased over the incubation period. The level of hydrogen peroxide and the activities of NAD(P)H oxidase, superoxide dismutase (SOD) and catalase (CAT) were higher than those in the controls at all incubation times. The highest NADH and NADPH oxidase activities were determined to be 4.9- and 5.0-fold higher than those in the control, respectively in cells exposed to 0.75 mM menadione. The SOD and CAT activities increased with increasing menadione, and their highest activities were 5.4- and 5.1-fold higher than those in the control, respectively. In 0.1–0.5 mM menadione exposed cells, the lipid peroxidation levels did not change significantly when compared to each other, except 8th hour of incubation (p > 0.01). Our result shows that although menadione induces the formation of reactive oxygen species, the antioxidant response system of P. Chrysosporium is able to negate menadione-induced oxidative stress up to relatively high menadione concentrations, as 0.75 mM. These results are important to determine the effects of menadione, as a medicine, on the antioxidant response system of eukaryotic models and the resulting level of damage.     

Stress factor-induced changes in the activity of antioxidant protective mechanisms in the wild type strain of <Emphasis Type="Italic">Neurospora crassa</Emphasis> and in its photoreceptor complex mutants

The effect of stress factors (changes in oxygen content, temperature, and illumination) on superoxide dismutase (SOD) and catalase activity, as well as on the content of thiol and disulfide groups, in low-molecular-weight compounds and proteins of Neurospora crassa mycelium was studied in the wild type strain and white collar-(wc-1) and white collar-2 (wc-2) mutants. Environmental stress factors induced the activation of both SOD and catalase, as well as an increase in the thiol level in the wild type strain of Neurospora crassa. In the wc-1 and wc-2 mutants, an increase in catalase activity and in the total thiol level was revealed; however, activation of superoxide dismutase was not observed. A decrease in the formation of disulfide bonds in the proteins of wc-1 and wc-2 mutants (as compared with the wild type strain) was recorded. These results indicate disrupted transduction of stress factor signals that promotes reactive oxygen species (ROS) formation in the WCC mutants.     

Chromosomal similarities and differences among four Neotropical Elateridae (Conoderini and Pyrophorini) and other related species, with comments on the NOR patterns in Coleoptera

This work deals with the comparative cytogenetic analysis of four Neotropical Elateridae species and reviews the nucleolar organizer region (NOR) patterns on Coleoptera chromosomes, for the first time. The cytogenetic characterization of Conoderus malleatus (Conoderini), Pyrearinus candelarius, Pyrophorus divergens and Pyrophorus punctatissimus (Pyrophorini) was accomplished through the study of mitotic and meiotic cells submitted to standard (Giemsa) and differential staining [silver impregnation and GC‐specific chromomycin A₃ (CMA₃) plus AT‐specific 4′‐6‐diamidino‐2‐phenylindole (DAPI) fluorochromes]. The analysis of spermatogonial cells revealed the diploid numbers: 2n = 17 in C. malleatus and 2n = 15 in P. candelarius, P. divergens and P. punctatissimus. In these species, the X0 type sex‐determination system and the acrocentric morphology of almost all chromosomes were observed. The study of meiotic cells of the four species revealed the occurrence of total synapsis between the autosomes, the presence of one terminal or interstitial chiasma in the majority of the bivalents, and the reductional behaviour and regular segregation of all chromosomes. Although the three Pyrophorini species demonstrated many similar karyotypical characteristics, there was one discrepancy, which was noted in the diplotene cells and concerns the number of bivalents with two chiasmata; P. candelarius only presented one bivalent, P. divergens showed two bivalents and P. punctatissimus exhibited up to four bivalents with two chiasmata. Testicular cells impregnated with silver nitrate demonstrated two terminal NORs located on the fourth autosomal pair of the Conoderini species and on the second autosomal pair of the three Pyrophorini representatives. Use of CMA₃/distamycin A (DA)/DAPI staining on the P. candelarius and P. punctatissimus chromosomes revealed that the CMA₃ labelled regions were coincident with the NORs. The main strategies of karyotypical differentiation that have occurred among the four Elateridae species and other related species, and the general trends of the NOR shifts during Coleoptera chromosomal evolution are discussed in this work.     

A comparative study of the components of the antioxidant defense system during growth of the mycelium of a wild-type Neurospora crassa strain and mutants, white collar-1 and white collar-2

A comparative study of the changes in the components of the antioxidant defense system (ADS), the activity of superoxide dismutase (SOD) and catalase and the level of extractable SH-groups, during the growth of wild-type and mutant (white collar-1 and white colar-2) Neurospora crassa strains was performed. Oxidative stress developing during spore germination and upon the transition to a stationary growth phase was accompanied in all strains by an increase in the level of extractable SH-groups and SOD activity, whereas the total catalase activity decreased during growth. However, in contrast to the wild-type strain, the activity of the catalase in the mutant strains wc-1 and wc-2 slightly increased upon the transition to the stationary phase. In the wc-2 mutant, SOD activity and the level of extractable SH-groups in the exponential growth phase were always lower than in the wild-type and wc-1 strains. The role of wc-1 and wc-2 genes in the level regulation of reactive oxygen species is discussed.