Treatment with the Antipsychotic Agent,Risperidone, Reduces Disease Severity in Experimental Autoimmune Encephalomyelitis

Recent studies have demonstrated that atypical antipsychotic agents, which are known to antagonize dopamine D2 and serotonin 5-HT_2a receptors, have immunomodulatory properties. Given the potential of these drugs to modulate the immune system both peripherally and within the central nervous system, we investigated the ability of the atypical anti-psychotic agent, risperidone, to modify disease in the animal model of multiple sclerosis (MS)⁴, experimental autoimune encephalomyelitis (EAE). We found that chronic oral administration of risperidone dose-dependently reduced the severity of disease and decreased both the size and number of spinal cord lesions. Furthermore, risperidone treatment substantially reduced antigen-specific interleukin (IL)-17a, IL-2, and IL-4 but not interferon (IFN)-γ production by splenocytes at peak disease and using an in vitro model, we show that treatment of macrophages with risperidone alters their ability to bias naïve T cells. Another atypical antipsychotic agent, clozapine, showed a similar ability to modify macrophages in vitro and to reduce disease in the EAE model but this effect was not due to antagonism of the type 1 or type 2 dopamine receptors alone. Finally, we found that while risperidone treatment had little effect on the in vivo activation of splenic macrophages during EAE, it significantly reduced the activation of microglia and macrophages in the central nervous system. Together these studies indicate that atypical antipsychotic agents like risperidone are effective immunomodulatory agents with the potential to treat immune-mediated diseases such as MS.     

Noninvasive estimation of human tissue respiration with wavelet-analysis of oxygen saturation and blood flow oscillations in skin microvessels

Laser Doppler flowmetry, laser spectrophotometry of oxygen saturation, and the fluorescence determination of the NADH/FAD ratio were carried out in 30 subjects in the upper limb skin zones with and without arteriolovenular anastomoses (AVAs). It was demonstrated that the wavelet-analysis of oxygen saturation and blood flow oscillations in microvessels was an efficient approach to noninvasive estimation of the skin oxygen extraction (OE) and oxygen consumption (OC) rates. OE = (SaO₂ ? SvO₂)/SaO₂, where SaO₂ (%) and SvO₂ (%) are the oxygen saturations of arterial and venular blood, respectively. If the cardiac (A_c, perfusion units, p.u.) to respiratory rhythm amplitude (A_r, p.u.) ratio A_c/A_r ?? 1, SvO₂ = SO₂. If A_c/A_r > 1, SvO₂ = SO₂/(A_c/A_r). OC = M _nutr (SaO₂ ?? SvO₂) in p.u. · %O₂, where M _nutr is the nutritive blood flow value in p.u. M _nutr = M/SI, where SI is the shunting index of blood flow in microvessels. The perfusion, OE, and OC values were higher in the skin with AVAs than in the skin without AVAs. The perfusion and oxygen saturation values were more variable in the skin with AVAs. The oxygen diffusing from the tiniest arterioles and capillaries is the most important for tissue metabolism. The contribution of the total perfusion and the oxygen diffusion from arterioles to tissue metabolism increased under the tissue ischemia conditions.     

Ventilatory response of the diamondback water snake,Natrix rhombifera to hypoxia, hypercapnia and increased oxygen demand

Summary Simultaneous measurements of ventilatory frequency, tidal volume, O₂ uptake, CO₂ output and cardiac frequency were made in the diamondback water snake,Natrix rhombifera while breathing hypoxic (15% to 5% O₂ in N₂) or hypercarbic (2% to 10% CO₂ and 21% O₂ in N₂) gases. The snakes responded to hypoxia by increasing tidal volume and decreasing ventilatory frequency resulting in little change in ventilation (50% increase at 5% inspired O₂), or O₂ uptake and only a light increase in CO₂ output. Hypercarbia to 4.2% inspired CO₂ resulted in a slight hyperventilation but ventilation was depressed at 6.3% inspired CO₂ and became erratic at higher concentrations. The resting rate of O₂ uptake was maintained throughout hypercapnia. Heart rate increased during hypoxia and decreased during hypercapnia. Cutaneous O₂ uptake increased during extreme hypoxia (5% inspired O₂) and cutaneous CO₂ output increased during hypercapnia, probably due to changes in the body-to-ambient gas gradients (Crawford and Schultetus, 1970). Both pulmonary oxygen uptake and ventilation were dramatically increased immediately following 10–15 min experimental dives. The increased ventilation was achieved primarily through an increased tidal volume.     

Magnetic Resonance Imaging (MRI) to Study Striatal Iron Accumulation in a Rat Model of Parkinson’s Disease

Abnormal accumulation of iron is observed in neurodegenerative disorders. In Parkinson’s disease, an excess of iron has been demonstrated in different structures of the basal ganglia and is suggested to be involved in the pathogenesis of the disease. Using the 6-hydroxydopamine (6-OHDA) rat model of Parkinson’s disease, the edematous effect of 6-OHDA and its relation with striatal iron accumulation was examined utilizing in vivo magnetic resonance imaging (MRI). The results revealed that in comparison with control animals, injection of 6-OHDA into the rat striatum provoked an edematous process, visible in T2-weighted images that was accompanied by an accumulation of iron clearly detectable in T2*-weighted images. Furthermore, Prussian blue staining to detect iron in sectioned brains confirmed the existence of accumulated iron in the areas of T2* hypointensities. The presence of ED1-positive microglia in the lesioned striatum overlapped with this accumulation of iron, indicating areas of toxicity and loss of dopamine nerve fibers. Correlation analyses demonstrated a direct relation between the hyperintensities caused by the edema and the hypointensities caused by the accumulation of iron.     

H2O2 production and cytochrome c peroxidase activity in mitochondria isolated from the trypanosomatid hemoflagellate Crithidia fasciculata

H₂O₂ production by coupled mitochondrial fractions from the protozoan, Crithidia fasciculata, has been measured spectrophotometrically by the formation of the stable enzyme-substrate complex with yeast cytochrome c peroxidase. H₂O₂ formation was observed with succinate, l-α-glycerophosphate, l-proline, α-ketoglutarate, and with endogenous substrate. The maximum rate of H₂O₂ generation obtained with each substrate in the presence of antimycin A was about 10% of the state 4 rate of O₂ respiration, and only 1–2% of the carbonylcyanide m-fluorophenylhydrazone-uncoupled respiratory rate. Therefore, excess O₂ uptake due to the formation of H₂O₂ cannot satisfactorily account for the low ADP:O ratios previously reported.Cytochrome c peroxidase activity was measured in mitochondrial preparations by recording the decrease in absorbance at 550 nm during the oxidation of horse heart ferrocytochrome c which was observed after addition of H₂O₂. The distribution of activity after sonic disruption of mitochondrial preparations was that expected for a soluble enzyme. The activity was proportional to the amount of enzyme protein added, and was abolished by heating at 100 °C for 3 min. Total cytochrome c peroxidase activity in mitochondrial fractions isolated from C. fasciculata was calculated to be 0.3% that of isolated yeast mitochondria, but it is suggested that the in vivo activity may be considerably higher than this estimate.     

Oxygen Inhibition of Nitrate Reductase Biosynthesis in Detached Corn Leaves via Inhibition of Total Soluble Protein Synthesis

Detached first leaves of 3-day-old corn seedlings (Zea mays L. W64AxW183E) were incubated with nitrate in air or 100% O₂ in the light. Nitrate accumulation in the leaves was not depressed by O₂. NADH:nitrate reductase activity and enzyme protein, as measured with an enzyme-linked immunosorbent assay, increased in parallel during the 8 h nitrate treatment in air, but in O₂ the levels of enzyme activity and protein were depressed. NADH:nitrate reductase mRNA levels were the same in the air-and O₂-treated leaves. Total soluble protein levels in leaves were slightly depressed by O₂ and shifting from O₂ to an air environment increased the protein level. Incorporation of [³⁵S]methionine during nitrate treatment revealed that total soluble protein and nitrate reductase protein synthesis were both depressed by the O₂ environment relative to air, but both recovered when leaves were shifted from O₂ to air. Although O₂ accelerated inactivation of nitrate reductase in vitro, the in vivo inactivation rate appeared to be too low to account for the depressed level of nitrate reductase activity in O₂-treated leaves. We concluded that O₂ inhibition of nitrate reductase biosynthesis in detached corn leaves was largely due to inhibition of total soluble protein synthesis at the level of translation.     

Effects of Irradiance on the in Vivo CO(2):O(2) Specificity Factor in Tobacco Using Simultaneous Gas Exchange and Fluorescence Techniques

The effects of gas phase O₂ concentration (1%, 20.5%, and 42.0%, v/v) on the quantum yield of net CO₂ fixation and fluorescence yield of chlorophyll a are examined in leaf tissue from Nicotiana tabacum at normal levels of CO₂ and 25 to 30°C. Detectable decreases in nonphotochemical quenching of absorbed excitation occurred at the higher O₂ levels relative to 1% O₂ when irradiance was nearly or fully saturating for photosynthesis. Photochemical quenching was increased by high O₂ levels only at saturating irradiance. Simultaneous measurements of CO₂ and H₂O exchange and fluorescence yield permit estimation of partitioning of linear photosynthetic electron transport between net CO₂ fixation and O₂-dependent, dissipative processes such as photorespiration as a function of leaf internal CO₂ concentration. Changes in the in vivo CO₂:O₂ `specificity factor' (K_sp) with increasing irradiance are examined. The magnitude K_sp was found to decline from a value of 85 at moderate irradiance to 68 at very low light, and to 72 at saturating photon flux rates. The results are discussed in terms of the applicability of the ribulose bisphosphate carboxylase/oxygenase enzyme model to photosynthesis in vivo.     

Interleukin-17 (IL-17)-induced MicroRNA 873 (miR-873) Contributes to the Pathogenesis of Experimental Autoimmune Encephalomyelitis by Targeting A20 Ubiquitin-editing Enzyme

Interleukin 17 (IL-17), produced mainly by T helper 17 (Th17) cells, is increasingly recognized as a key regulator in various autoimmune diseases, including human multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Although several microRNAs (miRNAs) with aberrant expression have been shown to contribute to the pathogenesis of MS and EAE, the mechanisms underlying the regulation of abnormal miRNA expression in astrocytes upon IL-17 stimulation remain unclear. In the present study, we detected the changes of miRNA expression profiles both in the brain tissue of EAE mice and in cultured mouse primary astrocytes stimulated with IL-17 and identified miR-873 as one of the co-up-regulated miRNAs in vivo and in vitro. The overexpression of miR-873, demonstrated by targeting A20 (TNFα-induced protein 3, TNFAIP3), remarkably reduced the A20 level and promoted NF-κB activation in vivo and in vitro as well as increasing the production of inflammatory cytokines and chemokines (i.e. IL-6, TNF-α, MIP-2, and MCP-1/5). More importantly, silencing the endogenous miR-873 or A20 gene with lentiviral vector of miR-873 sponge (LV-miR-873 sponge) or short hairpin RNA (shRNA) of A20 (LV-A20 shRNA) in vivo significantly lessened or aggravated inflammation and demyelination in the central nervous system (CNS) of EAE mice, respectively. Taken together, these findings indicate that miR-873 induced by IL-17 stimulation promotes the production of inflammatory cytokines and aggravates the pathological process of EAE mice through the A20/NF-κB pathway, which provides a new insight into the mechanism of inflammatory damage in MS.     

A model for control of breathing in mammals: coupling neural dynamics to peripheral gas exchange and transport

A new model for aspects of the control of respiration in mammals has been developed. The model integrates a reduced representation of the brainstem respiratory neural controller together with peripheral gas exchange and transport mechanisms. The neural controller consists of two components. One component represents the inspiratory oscillator in the pre-Bötzinger complex (pre-BötC) incorporating biophysical mechanisms for rhythm generation. The other component represents the ventral respiratory group (VRG), which is driven by the pre-BötC for generation of inspiratory (pre)motor output. The neural model was coupled to simplified models of the lungs incorporating oxygen and carbon dioxide transport. The simplified representation of the brainstem neural circuitry has regulation of both frequency and amplitude of respiration and is done in response to partial pressures of oxygen and carbon dioxide in the blood using proportional (P) and proportional plus integral (PI) controllers. We have studied the coupled system under open and closed loop control. We show that two breathing regimes can exist in the model. In one regime an increase in the inspiratory frequency is accompanied by an increase in amplitude. In the second regime an increase in frequency is accompanied by a decrease in amplitude. The dynamic response of the model to changes in the concentration of inspired O₂ or inspired CO₂ was compared qualitatively with experimental data reported in the physiological literature. We show that the dynamic response with a PI-controller fits the experimental data better but suggests that when high levels of CO₂ are inspired the respiratory system cannot reach steady state. Our model also predicts that there could be two possible mechanisms for apnea appearance when 100% O₂ is inspired following a period of 5% inspired O₂. This paper represents a novel attempt to link neural control and gas transport mechanisms, highlights important issues in amplitude and frequency control and sets the stage for more complete neurophysiological control models.     

Alteration of collagen synthesis in lung organ cultures by hyperoxic environments

Organ cultures of neo-natal rat lungs continue to accumulate collagen at a nearly linear rate for 5 days, when maintained in an atmosphere consisting of 5% CO₂ in air. Nearly 70% of the collagen synthesized in these cultures is Type I and 30%, Type III. When these cultures are maintained in 5% CO₂ + 95% O₂, the per cell accumulation of collagen increased. Although there is no significant change in the amount of radiolabeled proline incorporated, the rate of incorporation per cell is increased since there appear to be fewer cells in the O₂-treated cultures. These parameters of collagen synthesis parallel lung O₂ injury in vivo. There is a shift in the type of collagen synthesized, nearly 70% being Type III and 30%, Type I in O₂-exposed cultures. This finding is reminiscent of tissue repair after injury. Such changes in collagen may contribute to pulmonary connective tissue disorders in oxidant-induced lung injury.     

Microsomal prostaglandin E synthase-1 aggravates inflammation and demyelination in a mouse model of multiple sclerosis

Microsomal prostaglandin synthetase-1 (mPGES-1) is an inducible terminal enzyme required for prostaglandin E₂ (PGE₂) biosynthesis. In this study, we examined the role of mPGES-1 in the inflammation and demyelination observed in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). We induced EAE with myelin oligodendrocyte glycoprotein_35–55 peptide in mPGES-1-deficient (mPGES-1^−/−) and wild-type (WT) mice. First, we examined the histopathology in the early and late phases of EAE progression. Next, we measured the concentration of PGE₂ in the spinal cord and investigated the expression of mPGES-1 using immunohistochemistry. In addition, we examined the progression of the severity of EAE using an EAE score to investigate a correlation between pathological features and paralysis. In this paper, we demonstrate that WT mice showed extensive inflammation and demyelination, whereas mPGES-1^−/− mice exhibited significantly smaller and more localized changes in the perivascular area. The mPGES-1 protein was induced in vascular endothelial cells and microglia around inflammatory foci, and PGE₂ production was increased in WT mice but not mPGES-1^−/− mice. Furthermore, mPGES-1^−/− mice showed a significant reduction in the maximum EAE score and improved locomotor activity. These results suggest that central PGE₂ derived from non-neuronal mPGES-1 aggravates the disruption of the vessel structure, leading to the spread of inflammation and local demyelination in the spinal cord, which corresponds to the symptoms of EAE. The inhibition of mPGES-1 may be useful for the treatment of human MS.     

Xanthine Oxidase Mediates Axonal and Myelin Loss in a Murine Model of Multiple Sclerosis

Objectives

Oxidative stress plays an important role in the pathogenesis of multiple sclerosis (MS). Though reactive oxygen species (ROS) are produced by various mechanisms, xanthine oxidase (XO) is a major enzyme generating ROS in the context of inflammation. The objectives of this study were to investigate the involvement of XO in the pathogenesis of MS and to develop a potent new therapy for MS based on the inhibition of ROS.

Methods

XO were assessed in a model of MS: experimental autoimmune encephalomyelitis (EAE). The contribution of XO-generated ROS to the pathogenesis of EAE was assessed by treating EAE mice with a novel XO inhibitor, febuxostat. The efficacy of febuxostat was also examined in in vitro studies.

Results

We showed for the first time that the expression and the activity of XO were increased dramatically within the central nervous system of EAE mice as compared to naïve mice. Furthermore, prophylactic administration of febuxostat, a XO inhibitor, markedly reduced the clinical signs of EAE. Both in vivo and in vitro studies showed infiltrating macrophages and microglia as the major sources of excess XO production, and febuxostat significantly suppressed ROS generation from these cells. Inflammatory cellular infiltration and glial activation in the spinal cord of EAE mice were inhibited by the treatment with febuxostat. Importantly, therapeutic efficacy was observed not only in mice with relapsing-remitting EAE but also in mice with secondary progressive EAE by preventing axonal loss and demyelination.

Conclusion

These results highlight the implication of XO in EAE pathogenesis and suggest XO as a target for MS treatment and febuxostat as a promising therapeutic option for MS neuropathology.     

In Vivo Quantification of Inflammation in Experimental Autoimmune Encephalomyelitis Rats Using Fluorine-19 Magnetic Resonance Imaging Reveals Immune Cell Recruitment outside the Nervous System

Progress in identifying new therapies for multiple sclerosis (MS) can be accelerated by using imaging biomarkers of disease progression or abatement in model systems. In this study, we evaluate the ability to noninvasively image and quantitate disease pathology using emerging “hot-spot” ¹⁹F MRI methods in an experimental autoimmune encephalomyelitis (EAE) rat, a model of MS. Rats with clinical symptoms of EAE were compared to control rats without EAE, as well as to EAE rats that received daily prophylactic treatments with cyclophosphamide. Perfluorocarbon (PFC) nanoemulsion was injected intravenously, which labels predominately monocytes and macrophages in situ. Analysis of the spin-density weighted ¹⁹F MRI data enabled quantification of the apparent macrophage burden in the central nervous system and other tissues. The in vivo MRI results were confirmed by extremely high-resolution ¹⁹F/¹H magnetic resonance microscopy in excised tissue samples and histopathologic analyses. Additionally, ¹⁹F nuclear magnetic resonance spectroscopy of intact tissue samples was used to assay the PFC biodistribution in EAE and control rats. In vivo hot-spot ¹⁹F signals were detected predominantly in the EAE spinal cord, consistent with the presence of inflammatory infiltrates. Surprising, prominent ¹⁹F hot-spots were observed in bone-marrow cavities adjacent to spinal cord lesions; these were not observed in control animals. Quantitative evaluation of cohorts receiving cyclophosphamide treatment displayed significant reduction in ¹⁹F signal within the spinal cord and bone marrow of EAE rats. Overall, ¹⁹F MRI can be used to quantitatively monitored EAE disease burden, discover unexpected sites of inflammatory activity, and may serve as a sensitive biomarker for the discovery and preclinical assessment of novel MS therapeutic interventions.     

An Investigation of Age-Related Iron Deposition Using Susceptibility Weighted Imaging

Aim

To quantify age-dependent iron deposition changes in healthy subjects using Susceptibility Weighted Imaging (SWI).

Materials and Methods

In total, 143 healthy volunteers were enrolled. All underwent conventional MR and SWI sequences. Subjects were divided into eight groups according to age. Using phase images to quantify iron deposition in the head of the caudate nucleus and the lenticular nucleus, the angle radian value was calculated and compared between groups. ANOVA/Pearson correlation coefficient linear regression analysis and polynomial fitting were performed to analyze the relationship between iron deposition in the head of the caudate nucleus and lenticular nucleus with age.

Results

Iron deposition in the lenticular nucleus increased in individuals aged up to 40 years, but did not change in those aged over 40 years once a peak had been reached. In the head of the caudate nucleus, iron deposition peaked at 60 years (p<0.05). The correlation coefficients for iron deposition in the L-head of the caudate nucleus, R-head of the caudate nucleus, L-lenticular nucleus and R-lenticular nucleus with age were 0.67691, 0.48585, 0.5228 and 0.5228 (p<0.001, respectively). Linear regression analyses showed a significant correlation between iron deposition levels in with age groups.

Conclusions

Iron deposition in the lenticular nucleus was found to increase with age, reaching a plateau at 40 years. Iron deposition in the head of the caudate nucleus also increased with age, reaching a plateau at 60 years.     

Redox Cycling of Iron Supports Growth and Magnetite Synthesis by Aquaspirillum magnetotacticum

Under anaerobic conditions and in the absence of alternative electron acceptors, growth of the magnetic bacterium Aquaspirillum magnetotacticum MSI was iron concentration dependent. Weak chelation of the iron (with quinate, oxalate, or 2,3-dihydroxybenzoate) enhanced growth, whereas strong chelation (with EDTA, citrate, or nitrilotriacetic acid) retarded the growth of strain MSI relative to that of controls lacking chelators. Growth was proportional to the percentage of unchelated iron in medium containing EDTA in various molar ratios to iron. Addition of the respiratory inhibitors antimycin A (5 μM), NaCN (10 mM), and NaN₃ (10 mM) inhibited growth with Fe(III) or NO₃^- as the terminal electron acceptor. Growth with O₂ and NO₃^- was inhibited by 2-heptyl-4-hydroxyquinolone-N-oxide (HOQNO) but not with 2 mM Fe(III). Under strongly reducing conditions, strain MS1 survived but grew poorly and became irreversibly nonmagnetic. Growth and iron reduction in anaerobic cultures were stimulated by the provision of small amounts of O₂ or H₂O₂. Slow infusion of air to cultures which had reduced virtually all of the Fe(III) in the medium (2 mM) supported a high rate of iron reoxidation (relative to killed controls) and growth in proportion to the amount of iron reoxidized. Oxygen consumption by iron-reducing cultures was predominantly biological, since NaCN and HOQNO both inhibited consumption. Inhibition of oxygen consumption (and iron reoxidation) by the addition of ferrozine and the inhibition of iron oxidation (and oxygen consumption) by the addition of HOQNO suggest that iron oxidation by strain MS1 is an aerobic respiratory process, perhaps tied to energy conservation. Iron oxidation was also necessary for magnetite synthesis, since in microaerobic denitrifying cultures, sequestration of reduced iron by ferrozine present in 10-fold molar excess to the available iron resulted in loss of magnetism and a severe drop in the average magnetosome number of the cells.     

Effects of macrophyte-associated nitrogen cycling bacteria on denitrification in the sediments of the eutrophic Gonghu Bay, Taihu Lake

Integrated Elodea nuttallii-immobilized nitrogen cycling bacteria (INCB) technology was used for ecological restoration in the eutrophic Gonghu Bay, Taihu Lake. Sediment denitrification was investigated through microcosm incubations with four different treatments: bare sediment core as control without restoration, sediment + E. nuttallii, sediment + E. nuttallii + INCB, and sediment + INCB. The sediments with E. nuttallii-INCB assemblage (E-INCB) had the highest denitrification rates among all the treatments, and the E-INCB increased the denitrification rate by 162% in the sediments. The presence of macrophytes yielded a penetration depth of O₂ to more than 20 mm below the sediment–water interface (SWI), while the depth was only 4 mm in the sediments without macrophytes. The quantity of denitrifier in E-INCB sediments (within ~2 cm below the SWI) showed a significant increasing trend during one-month incubation, which was one order of magnitudes higher than that in the sediments without INCB. Macrophytes caused deeper O₂ penetration and increased oxic-anoxic interface, which could stimulate the coupled nitrification–denitrification. The high denitrification rate of the E-INCB treatment may result from the increased inorganic nitrogen content in the vicinity of the SWI, causing more nitrate to reach the anoxic denitrification zone. The results showed that E-INCB assemblage could increase benthic N removal by stimulating denitrification via combined O₂ penetration and enhanced microbial N cycling processes. E-INCB might be used as a potential restoration method for controlling fresh water system eutrophication.     

Nitrite photoreduction in vivo is inhibited by oxygen

It was hypothesized previously that an O₂ inhibition of NO₂⁻ photoreduction would reflect a competition between O₂ and NO₂⁻ for electrons from ferredoxin at the site of plastid nitrite reductase. In order to test this in vivo, intact spinach (Spinacia oleracea L.) leaf chloroplast and mesophyll cell isolates held in high light were aerated with streams of 20% O₂/80% N₂ (250 micromolar O₂ in aqueous solution) or, alternatively, streams of 100% N₂. Bicarbonate plus CO₂ and NO₂⁻ were supplied to reaction mixtures at levels just sufficient to promote maximal assimilations of CO₂ and NO₂⁻. In chloroplast isolates, there was a 9 to 30% O₂ inhibition of NO₂⁻ reduction while there were high rates of CO₂ fixation. In spinach and soybean (Glycine max) leaf cell isolates, NO₂⁻ photoreduction rates were 10 to 55% inhibited by O₂ at near ambient levels. It is possible that O₂ may compete, albeit weakly, with NO₂⁻ (nitrite reductase) for equivalents derived from reduced ferredoxin. Also, O₂ may oxidize sulfhydryl groups on nitrite reductase which are involved in substrate binding and/or activation.     

Spontaneous Chemiluminescence of Soybean Embryonic Axes during Imbibition

Isolated soybean (Glycine max L. var Hood) embryonic axes have a spontaneous chemiluminescence (about 150 counts per minute per embryo) that increases showing two phases, upon water imbibition. The first photoemission burst was measured between 0 and 7 hours of imbibition with a maximum of about 350 counts per minute per embryo after 2 hours. The second photoemission phase, between 7 and 30 hours, increased from about 220 to 520 counts per minute per embryo. Both chemiluminescence phases were inhibited by infused butylated hydroxyanisole while only the second phase was inhibited by infused salicylhydroxamic acid. On the basis of the sensitivity of the lipoxygenase reaction to both inhibitors (about 90%), the first burst is tentatively assigned to oxy-radicals mobilized upon water uptake by the embryonic axes, and the second phase is tentatively identified as due to lipoxygenase activity. The in vivo lipoxygenase activity of the embryonic axes was estimated by both the fraction of total oxygen uptake that was inhibited by butylated hydroxyanisole and by the fraction of photoemission that was inhibited by butylated hydroxyanisole and by salicylhydroxamic acid. Both approaches indicated marked increases (5-fold and 12-fold, respectively) of lipoxygenase activity between 2 and 30 hours of imbibition. The measured chemiluminescence per O₂ uptake ratio (the experimental quantum yield) for the lipoxygenase reaction (3.3 × 10⁻¹⁴ counts per O₂ molecule) was used to estimate the O₂ uptake due to lipoxygenase activity from the photoemission of the embryonic axes after 30 hours of imbibition. The value (0.54 microliters per minute per axis) was close to the butylated hydroxyanisole-sensitive O₂ uptake (1.2 microliters O₂ per minute per axis) of the same embryonic axes. Chemiluminescence may afford a noninvasive assay for lipoxygenase activity in intact plant tissues.