Subcellular distribution and activities of superoxide dismutase,catalase, glutathione peroxidase,and glutathione reductase in the southern armyworm,Spodoptera eridania

In mid-fifth-instar larvae of the southern armyworm, Spodoptera eridania, the subcellular distribution of four antioxidant enzymes—superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPOX), and glutathione reductase (GR)—were examined. Two-thirds (4.26 units ·mg protein^?1) of the SOD activity was found in the cytosol, and one-thirds (2.13 units ·mg protein^?1) in the mitochondria. CAT activity was unusually high and not restricted to the microsomal fraction where peroxisomes are usually isolated. The activity was distributed as follows: cytosol (163 units) mitochondria (125 units) and microsomes (119 units). Similar to CAT, the subcellular compartmentalization of both GPOX and GR was unusual. No activity was detected in the cytosol, but in mitochondria and microsomes, GR levels were 5.49 and 3.09 units. Although GPOX activity exhibited 14–16-fold enrichment in mitochondria and microsomes, respectively, over the 850g crude homogenate, the level was negligible (mitochondria = 1.4 × 10^?3 units; microsomes = 1.6 × 10^?3 units), indicating that this enzyme is absent. The unusual distribution of CAT has apparently evolved as an evolutionary answer to the absence of GR from the cytosol, and the lack of GPOX activity.     

Activities of enzymes that detoxify superoxide anion and related toxic oxyradicals in Trichoplusia ni

In third-, fourth-, and fifth-instar larvae of the cabbage looper moth, Trichoplusia ni, the activities of the antioxidant enzymes, superoxide dismutase (SOD*), catalase (CAT), glutathione peroxidase (GPOX), and glutathione reductase (GR) were examined using 850 g supernatants of whole-body homogenates. The enzyme activities, expressed as units mg⁻¹ protein min⁻¹ at 25°C ranged as follows: SOD, 0.67-2.13 units; CAT, 180.5-307.5 units; GPOX, none detectable; and GR, 0.40-1.19 units. There was a similar pattern of changes for SOD and CAT activities with larval ontogeny, but not for GR. The cabbage looper apparently uses SOD and CAT to form a “defensive team” effective against endogenously produced superoxide anion (O₂⪸). Glutathione may serve as an antioxidant for the destruction of any organic/lipid peroxides formed, and GSH oxidized to glutathione disulfide would be recycled by GR. Bioassays against pro-oxidant compounds exogenous sources of (O₂⪸) show high sensitivity of mid-fifth instars to the linear furanocoumarin, 8-methoxypsoralen (xanthotoxin) primarily from photoactivation (320-380 nm), and auto-oxidation of the flavonoid, quercetin. The LC₅₀s are 0.0004 and 0.0045% (w/w) concentration of xanthotoxin and quercetin, respectively. Both pro-oxidants have multiple target sites for lethal action and, in this context, the role of antioxidant enzymes is discussed.     

Antioxidant enzymes as biochemical defenses against phototoxin-induced oxidative stress in three species of herbivorous lepidoptera

Many secondary plant compounds are capable of photoactivation resulting in the production of toxic species of oxygen. One mechanism of defense for insects feeding on phototoxic plants may be the presence of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPOX), and glutathione reductase (GR). The activities of these enzymes were examined in larvae of three lepidoptera: Ostrinia nubilalis, Manduca sexta, and Anaitis plagiata. Highest levels of antioxidant enzyme activity were found in A. plagiata, a specialist feeder on Hypericum perforatum, which contains high levels of the phototoxin hypericin. Larvae of A. plagiata fed leaf discs treated with hypericin exhibited a short-term, concentration-dependent decline in enzyme activity. Longer term studies with A. palgiata fed either the photoxic H. perforatum, or the closely related but non-phototoxic H. calycinum, resulted in increased CAT and GR activity in larvae fed the phototoxic plant whereas SOD activity was not significantly different. These results suggest that CAT and GR may be inducible defenses against phototoxins.     

Glutathione peroxidase activity in insects: A reassessment

In vertebrate species, cytotoxic H₂O₂ and other lipid or organic hydroperoxides (ROOH) formed in aerobic metabolism are removed by a selenoprotein, glutathione peroxidase (GPOX). The GPOX activity in most rat tissues ranges from 100 to 1,000 units (1 unit = 1 nmol NADPH oxidized·mg protein^?1·min^?1), except for muscles (20–30 units). In contrast, GPOX activities of two strains of the housefly (Musca domestica), cabbage looper (Trichoplusia ni), southern armyworm (Spodoptera eridania), and black swallowtail butterfly (Papilio polyxenes), were found to be in the range 2–12 units. Trivial GPOX activity was detected in the confused flour beetle (Tribolium confusum). In the earthworm (Lumbricus terrestris), banana slug (Ariolimax columbianus), and market squid (Loligo opalescens), the GPOX activity ranged from 1 to 5 units. Tissue selenium concentrations were about 500–1,000 ppb for adult M. domestica, 600 ppb in T. confusum, 32 ppb in T. ni, 17 ppb in S. eridania, and 31 ppb in P. polyxenes larvae. The form of selenium incorporated at such high levels in tissues of invertebrates such as M. domestica remains an unresolved issue. Peroxidase activity of non-selenium glutathione-S-transferase (GT) against ROOH may compensate for the low GPOX activity. Catalase (CAT) has high activity and wide subcellular distribution in insects. This may be an evolutionary adaptation to GT's inability to catalyze the reduction of H₂O₂. The GT's peroxidase and CAT activities were not assessed for other invertebrate species, and warrants an investigation due to their reported low GPOX levels.     

Glutathione S-transferase activities in phytophagous insects: Induction and inhibition by plant phototoxins and phenols

The effects of two plant phototoxins (xanthotoxin and harmine) and three plant phenols (quercetin, ellagic acid, and juglone) on detoxification enzymes were studied in the polyphagous cabbage looper, Trichoplusia ni, and the oligophagous black swallowtail, Papilio polyxenes. In P. polyxenes, glutathione S-transferase (GST) activities toward 1-chloro-2,4-dinitrobenzene (CDNB) were 1840 and 1750 nmol CDNB conjugate/mg protein/min in the cytosolic fraction of midgut and fat body, respectively. Dietary xanthotoxin (0.1% fw) increased the activity 2.5 and 2.9-fold in the midgut and fat body, respectively. Xanthotoxin-conjugating GST activity was absent in both tissues. In T. ni, GST activity, 513 nmol CDNB conjugate/mg protein/min in the cytosolic fraction of midgut, was increased almost twofold by dietary xanthotoxin and harmine. Plant phenols effectively inhibited in vitro GST and Se-independent glutathione peroxidase (GPOX) activities in a dose-dependent manner in the two species. Both GST and GPOX of P. polyxenes were 2-fold less sensitive to phenol inhibitors than T. ni. GST inhibition differed according to the nature of the inhibitor in P. polyxenes. Quercetin is competitive with CDNB and is non-competitive with respect to GSH. In contrast, inhibition by ellagic acid is non-competitive with CDNB and competitive with GSH. Juglone showed competitive inhibition with both GSH and CDNB.     

Antioxidant enzymes of the black swallowtail butterfly,Papilio polyxenes,and their response to the prooxidant allelochemical,quercetin

The black swallowtail butterfly larvae, Papilio polyxenes, are specialist feeders that have adapted to feeding on plants containing high levels of prooxidant allelochemicals. Third, fourth, and fifth instar larvae were tested for their antioxidant enzyme activities, superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GPOX), using 850-g supernatants from whole-body homogenates. The overall antioxidant enzyme profile for P. polyxenes was high compared to other insects, with activities ranging as follows: SOD, 1.1–7.5; CAT, 124–343; GR, 1.0–7.5; and GPOX, 0 units. To determine whether these antioxidant enzymes were inducible, P. poly xenes larvae were given a prooxidant challenge by dipping parsley leaves (their diet in the initial studies) in solutions of quercetin, such that the leaves became coated with this prooxidant flavonoid. Mid-fifth instar larvae fed on quercetin-coated leaves were assayed for antioxidant enzyme activities as was previously done with the larvae fed the standard diet. Food consumption and quercetin intake were monitored. SOD activity was increased almost twofold at the highest quercetin concentration tested. CAT and GR activity, on the other hand, were inhibited by increased quercetin consumption, with GR activity completely inhibited at the highest quercetin concentration after 12 h of feeding. GPOX activity, not present in control insects, was also not inducible by a quercetin challenge. These studies point out the key role that the antioxidant enzymes play in insect defenses against plant prooxidants.     

How do insect herbivores cope with the extreme oxidative stress of phototoxic host plants?

Plants of the Asteraceae and Hypericaceae possess secondary compounds that induce photooxidation in insect herbivores that consume them. One of the well-established modes of action of these substances is peroxidation of membrane lipids. Some herbivores counteract these defences by avoidance of light and tissues rich in phototoxins or the ability to detoxify these secondary substances. The cytochrome P-450 polysubstrate monooxygenase systems involved, the metabolic products, and a new putative toxin pump have been described. Dietary antioxidants (β-carotene, vitamin E, ascorbate) are additional defences against phototoxicity. They reduce mortality in herbivores exposed to phototoxins and some specialist herbivores have high constitutive levels. Adapted specialist insects also have higher constitutive levels of superoxide dismutase (SOD) and respond to phototoxins in their diet by the induction of catalase (CAT), glutathione reductase (GR), and increased levels of reduced glutathione (GSH). Artificial inhibition of the enzymes SOD and CAT had little effect on phototoxicity but inhibition of GSH synthesis in herbivores enhanced photooxidative effects of administered phototoxins on lipid peroxidation. While insects have many mechanisms to overcome plant photooxidants, the Asteraceae appear to have adopted a strategy of counterattack. We suggest and provide preliminary evidence that a second group of secondary substances, the sesquiterpene lactones, occurring in the Asteraceae can attack key antioxidant defences to synergise phototoxins. © 1995 Wiley-Liss, Inc.     

Regional high affinity synaptosomal transport of choline in mouse brain — Influence of oxotremorine treatment

Kinetic parameters for high affinity [HA] uptake $\text{in vitro}$ in synaptosomes from different mouse brain regions were investigated. V_max was highest in the striatum [200 pmol.· mg protein^?1 · 4 min^?1], followed by the cortex [111 pmol · mg protein^?1 · 4 min^?1], hippocampus [63 pmol · mg protein^?1 · 4 min^?1], midbrain [21 pmol · mg protein^?1 · 4 min^?1] and, lowest, medulla oblongata [5 pmol · mg protein^?1 · 4 min^?1]. K_m was about the same in all brain regions [0.9–1.4 μM]. No sign of HA uptake was detected in synaptosomes from the cerebellum. A clear relationship between V_max for synaptosomal HA uptake of Ch $\text{in vitro}$ and apparent turnover of ACh $\text{in vivo}$ was found between the brain regions. Administration of oxotremorine [1 mg·kg^?1 i.p.] decreased V_max for HA uptake of Ch by 60% in the cortex and hippocampus, by 50% in the striatum and by 20% in the midbrain. This effect is in accordance with the previously observed marked decrease in turnover of ACh in these brain regions following oxotremorine treatment.     

THE SYNTHESIS OF GLUTAMATE BY RAT BRAIN MITOCHONDRIA

The synthesis of glutamate from 2-oxoglutarate generated by the citric acid cycle and ammonium acetate has been studied in brain mitochondria of synaptic or non synaptic origin. Non synaptic brain mitochondria synthesise glutamate at twice the rate (1.3 nmol. min^?1. mg protein^?1) of synaptic mitochondria (0.65 nmol. min^?1. mg protein^?1) when pyruvate is the precursor for 2-oxoglutarate, but at a similar rate (0.9 and 0.7 nmol. min^?1, mg protein^?1) when 3 hydroxybutyrate is the precursor. Glutamate synthesis from ammonium acetate and extramitochondrially addcd 2-oxoglutarate (5 mM) by both synaptic and nonsynaptic mitochondria was 5-fold higher (5-6nmol. min^?1. mg protein^?1) than glutamate synthesis from endogenously produced 2-oxoglutarate. In the uncoupled state (or un-coupler + oligomycin) the rate was reduced by half. (2.5-3 nmol. min^?1. mg protein^?1) as compared to mitochondria synthesising glutamate in states 3 or 4 (± oligomycin). The changes in brain mitochondrial nicotinamide nucleotide redox state have been monitored by fluorimetric, spectrophotometric and enzymatic techniques during glutamate synthesis and compared with liver mitochondria under similar conditions. On the instigation of glutamate synthesis by NH⁺₄ addition a significant NAD(P)H oxidation occurs with liver mitochondria but no detectable change occurs with brain mitochondria. Leucine (2 mM) causes a doubling of glutamate synthesis by both synaptic and non synaptic brain mitochondria with no detectable change in the NAD(P)H redox state. The results are discussed with respect to the control of glutamate synthesis by mitochondrial redox potential and the possible intramitochondrial compartmentation of this process.     

Modulation of Glutathione and Glutathione Dependent Antioxidant Enzymes in Mouse Heart Following Doxorubicin Therapy

The toxicity of the antineoplastic agent doxorubicin (DOX) has been shown to be moderated by the antioxidant enzyme glutathione peroxidase. It has been reported that acute doses of DOX can cause an inhibition of glutathione peroxidase in cardiac tissue, that may render this tissue especially susceptible to further prooxidant damage. In this study, multiple DOX treatments at a therapeutic dose were assessed for their effect on the antioxidant enzyme status of cardiac and kidney tissue. DOX was administered i.p. (5 mg/kg) once a week for two weeks to male balb/c mice. The activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPOX) and glutathione reductase (GR) were measured 1, 2 and 7 days following the second DOX treatment in both heart and kidney. Levels of reduced glutathione (GSH) were also measured in cardiac tissue at these same times. Cardiac levels of GPOX and GR showed a time-dependent decrease in activity, with 10% and 12% inhibition for GPOX and GR, respectively, at 7 days post second treatment. Cardiac levels of GSH also showed a significant decrease, approximately 15%, at 7 days post second treatment. Cardiac levels of SOD and CAT as well as kidney levels of all four antioxidant enzymes were not affected by DOX treatment. These data suggest that DOX given in a therapeutic regimen, at a therapeutic dose, can cause decreases in cardiac levels of GPOX, GR and GSH that could render the heart especially susceptible to further oxidative challenge.     

BIOSYNTHESIS OF POLY‐3‐HYDROXYBUTYRATE (PHB) IN THE TRANSGENIC GREEN ALGA CHLAMYDOMONAS REINHARDTII1

A cell‐wall deficient strain of Chlamydomonas reinhardtii P. A Dang. CC‐849 was cotransformed with two expression vectors, p105B124 and pH105C124, containing phbB and phbC genes, respectively, from Ralstonia eutropha. The transformants were selected on Tris‐acetate‐phosphate media containing 10 μg · mL^?1 Zeomycin. Upon further screening, the transgenic algae were subcloned and maintained in culture. PCR analysis demonstrated that both phbB and phbC genes were successfully integrated into the algal nuclear genome. Poly‐3‐hydroxybutyrate (PHB) synthase activity in these transgenic algae ranged from 5.4 nmol · min^?1 · mg protein^?1 to 126 nmol · min^?1 · mg protein^?1. The amount of PHB in double transgenic algae was determined by gas chromatography–mass spectrometry (GC–MS) when comparing with PHB standard. In addition, PHB granules were observed in the cytoplasm of transgenic algal cells using TEM, which indicated that PHB was synthesized in transgenic C. reinhardtii. Hence, results clearly showed that producing PHB in C. reinhardtii was feasible. Further studies would focus on enhancing PHB production in the transgenic algae and targeting the chloroplast for PHB accumulation.     

Stimulation of caffeine metabolism in the rat by 3-methylcholanthrene

Groups of adult male rats treated with 3-methylcholanthrene, phenobarbital or vehicles alone, were administered caffeine either orally or intravenously. Serum caffeine concentrations were measured by radioimmunoassay. In vehicle and phenobarbital pretreated animals, caffeine elimination kinetics were non-linear. In control animals, the $\text{in}$$\text{vivo}$ apparent Km was 8 μg·ml^?1 (40 μM) and the apparent Vmax was 0.1 μg·ml^?1·min^?1 (0.5 μM·min^?1). Phenobarbital pretreatment did not change the apparent Km but slightly increased the apparent Vmax. 3-Methylcholanthrene pretreatment dramatically altered the elimination kinetics of caffeine, whether caffeine was given orally or intravenously. The elimination of caffeine from serum of 3-methylcholanthrene pretreated rats was first order with a $\text{t}\text{1}\text{2}$ of approximately 14 minutes.Our results are consistent with the proposed involvement of the cytochromes P-450 monooxygenase system in the elimination of caffeine. In addition, our results suggest that caffeine is a moderately poor substrate for the cytochromes P-450 present in control and phenobarbital-pretreated rats, but a particularly good substrate for the form(s) induced by 3-methylcholanthrene.     

Antioxidant enzyme activities in subcellular fractions of larvae of the black swallowtail butterfly,Papilio polyxenes

The black swallowtail butterfly, Papilio polyxenes, larvae are specialized feeders of pro-oxidant rich plants of Apiaceae and Rutaceae. An important defense against toxic forms of oxygen species generated by ingestion of the pro-oxidants, are the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), GSH-dependent glutathione peroxidases (selenium-dependent glutathione peroxidase [GPOX] and peroxidase activity of selenium-independent glutathione-S-transferase [GT_px]), and glutathione reductase (GR). The subcellular distribution of these enzymes in black swallowtail larvae was investigated and was found to resemble the patterns described for larvae of two other lepidopteran species: the southern armyworm, Spodoptera eridania, and the cabbage looper, Trichoplusia ni. The confinement of SOD in the cytosol and mitochondria was typically eukaryotic, but the relative proportion (1:1) was markedly different from the mammalian pattern (4:1; cytosol:mitochondria). The most obvious difference between the black swallowtail and other lepidoptera as a group, and mammalian species, is in very wide intracellular distributions of CAT, GTpx, and GR in insect species. Insects possess very low levels of a GPOX-like activity which reduces both H₂O₂ and organic peroxides. Consequently, insects have elaborate activities with a wide subcellular distribution of both CAT which decomposes H₂O₂, and GTpx which decomposes organic peroxides. The reduction of peroxides is dependent on GSH, which in this process is oxidized to GSSG. GR which reduces GSSG to GSH is also of wide subcellular distribution, analogous to the distribution pattern of GTpx.     

GLYOXYLATE CYCLE ENZYME ACTIVITIES IN THE CYANOBACTERIUM ANACYSTIS NIDULANS1

The enzymes of the glyoxylate cycle, isocitrate lyase (EC.4.1.3.1) and malate synthase (EC.4.1.3.2), were measured in cell-free extracts from the cyanobacterium Anacystis nidulans Drouet during photoautotrophic growth in medium aerated with ordinary air (0.03% CO₂). Isocitrate lyase had an average specific activity of 112 nmoles·min^?1·mg protein^?1 whereas malate synthase had an average specific activity of 12.5 nmoles·min^?1·mg protein^?1. Unpurified isocitrate lyase showed classical Michaelis kinetics with a K_m of 8 mM. Isocitrate lyase activity was strongly inhibited by numerous cellular metabolites at 10 mM concentration. The previously reported low specific activity for isocitrate lyase may be due to metabolite inhibition caused by growth in high CO₂ concentrations. The activities reported for isocitrate lyase and malate synthase suggest the operation of the glyoxylate cycle in Anacystis nidulans under CO₂-limiting growth conditions.     

GLUTATHIONE PEROXIDASE LEVELS IN BRAIN

Glutathione peroxidase activity in brains of various animals was examined. Enzyme activity was low, approximately 10 nmol of glutathione oxidized min^?1 mg protein^?1 or less. This result suggests that brain tissues contain insufficient glutathione peroxidase activity to provide protection from peroxidative damage and that an alternative mechanism may be present.     

NAD-Independent Lactate and Butyryl-CoA Dehydrogenases of Clostridium acetobutylicum P262

Clostridium acetobutylicum P262 cells that were growing on lactate and acetate had an NAD-independent lactate dehydrogenase (iLDH) activity of 200 nmol mg protein⁻¹ min⁻¹. Ammonium sulfate precipitation and DEAE cellulose caused a 35-fold purification. Gel filtration indicated that the iLDH had a molecular weight of approximately 55 kDa, but two bands were always observed. Phenyl sepharose could not separate the two proteins, and hydroxyapatite caused a complete loss of activity. The semi-purified iLDH had a V_max of 13,000 nmol mg protein⁻¹ min⁻¹ and a K _m value of 3.5 mM for D-lactate. The V_max and K _m values for L-lactate were 300 nmol mg protein⁻¹ min⁻¹ and 0.7 mM. The iLDH had a pH optimum of 7.5, was not activated by fructose-1,6-bisphosphate (FDP), and could be coupled to either 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) or dichlorophenol-indophenol (DCPIP), but not methyl viologen (MV) or benzyl viologen (BV). The iLDH did not have strong absorbance between 500 and 300 nm, and trichloroacetic acid or acid ammonium sulfate extracts had virtually no fluorescence at 450 nm. The crude extracts also had MTT-linked butyryl-CoA dehydrogenase activity (60 nmol mg protein⁻¹ min⁻¹). The NAD-independent butyryl-CoA dehydrogenase eluted from DEAE-cellulose as two fractions. The yellow fraction was extremely unstable, but the green fraction could be stored for short periods of time at 5°C. The green-colored butyryl-CoA dehydrogenase had strong absorption at 450 nm, and gel filtration indicated that it had a molecular weight of 90 kDa. The NAD-independent butyryl-CoA dehydrogenase could be coupled to MTT, DCPIP, or MV, but not BV. Because the NAD-independent lactate and butyryl-CoA dehydrogenase could both be linked to low potential carriers, these two enzymes may function as oxidation-reduction system in vivo. Received: 24 July 1996 / Accepted: 10 September 1996     

Protective action of indole‐3‐acetic acid on induced hepatocarcinoma in mice

In this study, we report the protective effects of IAA on diethylnitrosamine (DEN)‐induced hepatocarcinogenesis. BALB/c mice received daily IAA at 50 (T₅₀), 250 (T₂₅₀), and 500 (T₅₀₀) mg Kg^?1 per body mass by gavage for 15 days. At day 15, animals were administered DEN and sacrificed 4 h later. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed in sera. In addition, hepatomorphologic alterations, activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR), gene expression of antioxidant enzymes and DNA integrity were evaluated in the liver. IAA administration did not show any alterations in any of the parameters available, except for a reduction of the gene expression for antioxidant enzymes by 55, 56, 27, and 28% for SOD, CAT, GPx, and GR upon T₅₀₀, respectively compared with the control. Several hepatic alterations were observed by DEN exposure. Moreover, IAA administration at 3 doses was shown to provide a total prevention of the active reduction of CAT and GR induced by DEN exposure compared with the control. IAA at T₅₀₀ was shown to give partial protection (87, 71, 57, and 90% for respectively SOD, CAT, GPx, and GR) on the down‐regulation of the enzymes induced by DEN and this auxin showed a partial protection (50%) on DEN‐induced DNA fragmentation for both parameters when compared to DEN alone. This work showed IAA hepatocarcinogenesis protection for the first time by means of a DEN‐protective effect on CAT and GR activity, and by affecting antioxidant gene expression and DNA fragmentation. Copyright © 2008 John Wiley & Sons, Ltd.     

METHYLAMINE UPTAKE IN THE GREEN ALGA CHLORELLA PYRENOIDOSA1

Methylamine uptake in nitrogen-starved Chlorella pyrenoidosa Beij. follows Michaelis-Menten kinetics: maximum uptake is about 1.6 nmol μl^?1· cells · min^?1, half-saturation occurs at 4 μM methylamine, and the slope in the range where uptake is proportional to concentration is 0.4 nmol μl^?1· min^?1·μM^?1. In cells grown in the presence of a non-limiting nitrogen concentration, methylamine uptake is directly proportional to concentration up to at least 0.5 mM, and the slope is 1/500 that for starved cells. Similar uptake kinetics have been reported for Penicillium chrysogenum and attributed to an inducible “ammonium permease.” Apparently, a similar permease occurs in algae.     

28-Homobrassinolide potential for oxidative interface in <Emphasis Type="Italic">Brassica juncea</Emphasis> under temperature stress

Exploration of scavenging potential of 28-homobrassinolide (28-homoBL) in mitigating the oxidative stress caused by free radicals (·O₂ ^?, H₂O₂, ·NO, OH^?) produced due to temperature stress (4, 44 °C) in Brassica juncea L. was made in the present research. Brassica juncea var. RLC-1 seeds were given pre-sowing soaking of different concentrations of 10^?9 M 28-homoBL for 8 h. Seeds were sown in bedded petri plates lined with 10 No. What’s man filter paper under controlled laboratory conditions. Temperature of 4 and 44 °C, taken as low- and high-temperature stress, suppressed membrane stability and overall growth of the seedlings, while cell death was triggered. Accumulation of malondialdehyde (MDA), hydrogen peroxide (H₂O₂), and nitric oxide (NO) was boosted which resulted in enhanced oxidative stress on the 10th day after sowing. Activity level of antioxidant enzymes viz. superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPOX), and ascorbate peroxidase (APOX) was enhanced which was ensued for up-regulation of total antioxidant potential in 10-day-old plants exposed to negative effect of temperature stress. Priming treatment of 28-homoBL at seed level helped in maintaining the growth of seedlings to higher level as compared to only stressed as well as from control double distilled water-raised seedlings. 10^?9 M 28-HBL found to be the best in enhancing the enzymatic activities of SOD, CAT, GPOX, and APOX and thus maintained antioxidant potential at higher level which accounted for alleviating oxidative stress caused due to extreme temperature stress. Dead cell formation reduced significantly in 28-homoBL-treated plants, membrane stability was upturned, while production of MDA, H₂O_2, and NO was under control. These results suggested and try to establish 28-homoBL as effective stress protector for B. juncea particularly from the oxidative damage induced by extreme temperatures.     

NADPH-cytochrome P450 reductase.

The rate of reduction of cytochrome P450 in hepatic microsomes in the presence of NADPH has been measured with a dual wavelength stopped-flow spectrophotometer. The results obtained, with microsomes prepared from phenobarbital-pretreated rats, indicate that the reduction process is biphasic and most probably composed of two concurrent first-order reactions. The rate constant for the reduction of cytochrome P450 in the fast phase in the presence of ethylmorphine is 1.74 s^?1. Since approximately 50% or more of the cytochrome P450 is reduced in the fast phase under these conditions, the rate of reduction of cytochrome P450 is approximately 150 nmol min^?1 (mg of protein)^?1. Under similar conditions the rate of ethylmorphine N-demethylation is 8.6 nmol min^?1 (mg of protein)^?1. Thus the rate-limiting step in ethylmorphine N-demethylation cannot be the introduction of the first electron into cytochrome P450 by NADPH-cytochrome P450 reductase.