A Multisubstrate Kinetic Mechanism of Dopamine Transport in the Nucleus Accumbens and Its Inhibition by Cocaine

Abstract: Kinetic studies of dopamine transport into suspensions of nucleus accumbens (NAcc) and effects of Na⁺ and Cl^? as cosubstrates were performed using rotating disk electrode voltammetry. To mimic chemical neurotransmission, dopamine was added as a rapid pulse, and transporter-mediated clearance of dopamine was evaluated kinetically. This paradigm was shown to approximate a zero trans entry transport experiment. Dopamine was taken up with apparent K_m and V_max values of 1.3 µM and 375 pmol/s/g wet weight, respectively. Transport exhibited apparent trans acceleration. Substitution of Na⁺ with choline or Cl^? with isethionate reduced dopamine transport with reaction orders of two and unity, respectively, accompanied by reductions in V_max with no changes in K_m. Apparent K_Na and K_Cl values were 70.0 and 92.1 mM, respectively. Dopamine transport in NAcc was found to follow a partially random, sequential mechanism in which dopamine and Na⁺ bind randomly to the transporter followed by binding of Cl^? before transport. Cocaine inhibited dopamine transport and the influences of the other substrates allosterically with an overall K_i of 0.30 µM. Thus, the general kinetic mechanism of the transport of dopamine in the NAcc is identical to that previously reported by this laboratory for dopamine transport in the striatum. However, the dopamine transporter in the NAcc is more tightly regulated by Na⁺, possesses a higher kinetic turnover rate, is four times more sensitive to cocaine than the striatal transporter, and exhibits cocaine inhibition independent of [substrate]. These findings suggest that cocaine modulates chemical signaling in NAcc differently than in striatum, providing down-regulation of function irrespective of [substrate], thereby enhancing dopaminergic signaling more robustly in the NAcc than in the striatum.     

ONTOGENETIC DEVELOPMENT OF NEOSTRIATAL DOPAMINE RECEPTORS IN THE RAT 1

Abstract— The actions of dopamine and apomorphine on the kinetic properties of striatal adenylate cyclase were investigated during ontogenesis in the rat. The maximum stimulatory effect of dopamine (5 × 10^?5 M) was constant from birth to maturity (1 to 60 days of age). In contrast, the stimulatory effect elicited by apomorphine (5 × 10^?5 M) was almost the same as that of dopamine in 6-day-old rats, but it declined during maturation reaching 50% of the initial value at 60 days of age. The apparent K_m value for dopamine did not change during development, while the K_m for apomorphine was higher in the adult than in the newborn. Apomorphine appeared to have a greater affinity than dopamine for the striatal adenylate cyclase both in adult and newborn rats.     

Release of radiolabeled dopamine,p-tyramine,andm-tyramine from rat striatal slices by some aminotetralins

The effect of several 2-aminotetralins (2ATs) on the uptake and release of [¹⁴C] dopamine and [³H]m- or [³H]p-tyramine by rat striatal slices was examined. 6,7-Dihydroxy-2AT (6,7OHAT) and 5,6-dihydroxy-2-methyl-AT (5,6OHMeAT) were the most potent uptake inhibitors as well as the most potent releasers of the three labeled amines. The 5-, 6-, and 7-hydroxy-2-N,N-dipropyl-ATs (5-, 6-, and 7OHdiPrAT) and 5,6-dihydroxy-2-N,N-dipropyl-AT (5,6OHdiPrAT) significantly inhibited the uptakes of the three labeled amines, but they released only the tyramines. The dipropyl substitution of a 2AT appeared to confer a tyraminergic specificity to its release properties. To verify this supposition, 2AT was compared to 2-N,N-dipropyl-AT (diPrAT). Although 2AT released both [³H]p-tyramine and [¹⁴C]dopamine, diPrAT released only [³H]p-tyramine. None of the compounds, however, differentiated betweenm- andp-tyramine. It was concluded that the release of tyramines could be implicated in the actions of some of the 2ATs and that the tyramines can be transported independently from dopamine.     

Nitric Oxide Participates in the Stimulatory and Neurotoxic Action of Endothelin on Rat Striatal Dopaminergic Neurons

1. Our method of real-time monitoring of dopamine release from rat striatal slices revealed that endothelin (ET)-3-induced dopamine release was inhibited by N ^G-methyl-L-arginine (L-NMMA; 1 mM), an inhibitor of nitric oxide (NO) synthase, while N ^G-methyl-D-arginine (D-NMMA; 1 mM), an inactive isomer of L-NMMA, had no effect.2. The inhibition of L-NMMA (0.1 mM) became apparent when tissues were pretreated with tetrodotoxin (1 M) for 30 min and subsequently exposed to ET-3 (4 M).3. L-NMMA (0.1 and 1 mM) dose dependently protected against ET-3-triggered hypoxic/hypoglycemic impairment of striatal responses to high K⁺.4. Thus, NO may work as a promoter in mediation of the stimulatory and neurotoxic action of ET-3 on the striatal dopaminergic system, presumably by interacting with interneurons in the striatum.     

Intraneuronal Site of Action for Imipramine in Rat Striatal Slices

: The uptake of ³H-labeled imipramine ([³H]IMI) in rat corpus striatum slices was found to be strongly temperature-dependent. The accumulation was shown to be saturable and two apparent K_m's were found: 2.2 × 10^?8 and 3.5 × 10^?7m . Once incorporated, the labeled drug was released from superfused slices by K⁺ (55 mm ) depolarization in the presence of calcium ions. Imipramine was also studied for its ability to induce the release of [³H]dopamine ([³H]DA) which had been previously accumulated by striatal slices. It was found that striatal slices superfused during 1 or 6 min with imipramine (10^?6-10^?4m ) release substantial amounts of radioactive dopamine, independently of the presence of Ca²⁺ in the medium. This release is completely abolished after reserpine pretreatment. It is proposed that imipramine enters the dopaminergic storage vesicles and displaces dopamine. An intraneuronal mechanism of action for imipramine is discussed.     

The Striatal Transporter for Dopamine in the Rat May Be Kinetically Up-Regulated Following 3 Weeks of Withdrawal from Cocaine Self-Administration

Abstract: Rotating disk electrode voltammetry was used to measure the inwardly directed V_max and K_m of dopamine with its transporter in striatal suspensions prepared from nonhandled control rats, rats that had been trained to self-administer cocaine for 20 days (at 26 mg/day per rat) via a jugular catheter and subsequently withdrawn for 3 weeks, and rats that had received saline (155 mM NaCl) via a jugular catheter on the same schedule as the rats that had received cocaine. Because a limited number of animals was available from the self-administration procedure, the velocity of dopamine transport as a function of [dopamine] was measured by incremental addition of dopamine to a given striatal preparation. In nonhandled controls the values of V_max, K_m, and turnover, observed in this experimental paradigm, were increased relative to results obtained in studies of the velocity-[dopamine] relationship where dopamine was added to suspensions, one concentration per suspension. The kinetics of the association of dopamine with the transporter were unchanged. The V_max to K_m ratios obtained in the two experiments were statistically indistinguishable, suggesting that the two types of experiments probe the same transporter. Also, the increased velocity observed in the experiment involving sequential additions to the same preparation is evidence of trans acceleration, suggesting that the movement of dopamine across the membrane is carrier mediated as opposed to being mediated via channels or pores and that the rate-limiting step in inwardly directed transport is the reorientation of the unloaded transporter from the inwardly to the outwardly facing forms. Saline-treated rats in the self-administration paradigm exhibited kinetic parameters of transport indistinguishable from those observed in nonhandled controls. In contrast, V_max and K_m of the transporter were increased in suspensions prepared from rats that self-administered cocaine and were withdrawn for 3 weeks, relative to saline-treated and nonhandled animals. Combined, these results suggest that the striatal uptake of dopamine is mediated by a transporter and that it is kinetically up-regulated following withdrawal from repeated cocaine administered in a self-administration paradigm.     

Regulation of catecholamine synthesis in rat brain synaptosomes

Abstract— Catecholamine synthesis in synaptosomal preparations of rat striatum, cortex and brain stem was investigated. The striatum had much higher activity than either the cortex or brain stem. Equilibration of labelled tyrosine between tissue and incubation medium was completed within 2 min. The apparent K_m of tyrosine hydroxylase (EC 1.14.3a) and of the overall catecholamine synthetic pathway were both approximately 5 ± 10^?6m for tyrosine. The following amines were found to inhibit striatal dopamine synthesis: dopamine, 25% inhibition at 5 ± 10^?7m ; noradrenaline, 25% inhibition at 5 ± 10^?6m ;and serotonin, 30% inhibition at 10^?5m . The catecholamine-induced inhibition of synthesis was antagonized by pre-incubation with cocaine. Increasing the potassium concentration from 5 to 55 mm caused a release of amines into the medium which was accompanied by a 40% increase in dopamine synthesis, when synthesis was measured during the first 5 min of exposure to elevated potassium. These results indicate that synaptosomal catecholamine synthesis is inhibited by increases in intra-synaptosomal amine levels, and that short-term exposure to depolarizing concentrations of potassium can increase synthesis.     

Molecular Characterization and In Situ Localization of a Mouse Retinal Taurine Transporter

Abstract: Various ocular tissues have a higher concentration of taurine than plasma. This taurine concentration gradient across the cell membrane is maintained by a high-affinity taurine transporter. To understand the physiological role of the taurine transporter in the retina, we cloned a taurine transporter encoding cDNA from a mouse retinal library, determined its biochemical and pharmacological properties, and identified the specific cellular sites expressing the taurine transporter mRNA. The deduced protein sequence of the mouse retinal taurine transporter (mTAUT) revealed >93% sequence identity to the canine kidney, rat brain, mouse brain, and human placental taurine transporters. Our data suggest that the mTAUT and the mouse brain taurine transporter may be variants of one another. The mTAUT synthetic RNA induced Na⁺- and Cl^?-dependent [³H]taurine transport activity in Xenopus laevis oocytes that saturated with an average K_m of 13.2 µM for taurine. Unlike the previous studies, we determined the rate of taurine uptake as the external concentration of Cl^? was varied, a single saturation process with an average apparent equilibrium constant (K_Cl?) of 17.7 mM. In contrast, the rate of taurine uptake showed a sigmoidal dependence when the external concentration of Na⁺ was varied (apparent equilibrium constant, K_Na+～54.8 mM). Analyses of the Na⁺- and Cl^?-concentration dependence data suggest that at least two Na⁺ and one Cl^? are required to transport one taurine molecule via the taurine transporter. Varying the pH of the transport buffer also affected the rate of taurine uptake; the rate showed a minimum between pH 6.0 and 6.5 and a maximum between pH 7.5 and 8.0. The taurine transport was inhibited by various inhibitors tested with the following order of potency: hypotaurine > β-alanine > l -diaminopropionic acid > guanidinoethane sulfonate > β-guanidinopropionic acid > chloroquine > γ-aminobutyric acid > 3-amino-1-propanesulfonic acid (homotaurine). Furthermore, the mTAUT activity was not inhibited by the inactive phorbol ester 4α-phorbol 12,13-didecanoate but was inhibited significantly by the active phorbol ester phorbol 12-myristate 13-acetate, which was both concentration and time dependent. The cellular sites expressing the taurine transporter mRNA in the mouse eye, as determined by in situ hybridization technique, showed low levels of expression in many of the ocular tissues, specifically the retina and the retinal pigment epithelium. Unexpectedly, the highest expression levels of taurine transporter mRNA were found instead in the ciliary body of the mouse eye.     

K+-Dependent Stimulation of Dopamine Synthesis in Striatal Synaptosomes Is Mediated by Protein Kinase C

Dopamine synthesis rate was measured in striatal synaptosomes. Removal of Na⁺ increased synthesis rate; this was blocked in Ca²⁺-free medium and by addition of the Ca²⁺/calmodulin inhibitor N-6-aminohexyl-5-chloro-1-naphthalenesulfonamide (W7). The increase in dopamine synthesis rate caused by the addition of the phorbol ester 12-O-tetradecanoylphorboI-13-acetate (TPA) was blocked by the protein kinase C inhibitor polymyxin B. K⁺-stimulated synthesis was unchanged in Ca²⁺-free medium or by addition of W7; it was blocked by polymyxin B. The effect of 50 mM K⁺ was additive with that of 8-Br cyclic AMP and of Na⁺ removal; the combined effect of 50 mM K⁺ and TPA was no greater than that of either alone. These results suggest that stimulation of dopamine synthesis in striatal synaptosomes by 50 mM K⁺ is mediated by protein kinase C.     

Some characteristics of photosynthetic inorganic carbon uptake of a marine macrophytic red alga

Abstract Some characteristics of photosynthetic inorganic carbon uptake by Palmaria palmata, a marine red macroalga, have been measured under physiological conditions in artificial seawater. The apparent affinity of thallus for CO₂ [K_1/2(CO₂)] at pH 8.0 and 15°C was 21.4±3.0mmol m^?3 CO₂ under air, and 25.7±70mmol m^?3 CO₂ under N₂. The corresponding values of V_max were 2.98 ± 0.42 and 3.65±0.87 mmol O₂ evolved g Chr^?1 s^?l. The apparent K_m(CO₂) of isolated ribulose bisphosphate carboxylase was determined at pH 8.0 and 30 °C to be 30.2 mmol m^?3 CO₂, and the corresponding value of V_max was 19.67 μniol CO₂ g protein^?1 s^?1. The CO₂ compensation points of the thallus were measured in artificial seawater at pH 8.0 under air and N₂, using a gas-chromatographic method. The values were relatively low, rising from 10 cm³ m^?3 at 15°C, to 35 cm³ m^?3 at 25°C, but were not affected by the O₂ concentration. The lack of an effect of O₂ on photosynthesis and on compensation point indicates that there is little photorespiratory CO₂ loss in this macroalga. The high affinity of the thallus for CO₂, and the low CO₂ compensation concentrations, are consistent with the occurrence of bicarbonate uptake in this alga.     

Secondary carbamate linker can facilitate the sustained release of dopamine from brain-targeted prodrug

To achieve the sustained release of dopamine in the brain for the symptomatic treatment of Parkinson’s disease, dopamine was conjugated to l-tyrosine, an l-type amino acid transporter 1 (LAT1)-targeting vector, using a secondary carbamate linker. The resulting prodrug, dopa-CBT, inhibited the uptake of the LAT1 substrate [¹⁴C]-l-leucine in LAT1-expressing MCF-7 cells with an IC₅₀ value of 28?µM, which was 3.5-times lower than that of the gold standard for dopamine replacement therapy, l-dopa (IC₅₀ ca. 100?µM). Despite its high affinity for LAT1, dopa-CBT was transported via LAT1 into MCF-7 cells 850-times more slowly (V_max?<?3?pmol/min/mg) than l-dopa (V_max 2.6?nmol/min/mg), most likely due to its large size compared to l-dopa. However, dopa-CBT was significantly more stable in 10% rat liver homogenate than l-dopa, releasing dopamine and l-tyrosine, an endogenous dopamine precursor, slowly, which indicates that it may serve as a dual carrier of dopamine across the blood-brain barrier selectively expressing LAT1.     

Characterization of the Human Dopamine Transporter Heterologously Expressed in BHK-21 Cells

1. cDNA of the human dopamine transporter (hDAT) was cloned into a cloning vector based on the Semliki Forest virus. Electroporation of in vitro transcribed mRNA from this plasmid into BHK-21 cells resulted in production of the transporter as measured by [³H]dopamine uptake (K _m = 2.0 ± 0.4 M), which was specifically inhibited in the presence of cocaine.2. The recombinant transporter protein exhibited an apparent molecular mass of 56 kDa, which was reduced to 50 kDa after tunicamycin treatment of the producing BHK-21 cells. Tunicamycin treatment of the electroporated cells also resulted in a decrease in transport activity with no change in the K _m value (2.1 ± 0.4 M).3. The localization of the heterologously produced transporter in the BHK cells either with or without tunicamycin treatment was studied by electron microscopic immunogold staining. The glycosylated transporter was found to be localized at the plasma membrane, whereas in the case of the unglycosylated transporter, transport to the plasma membrane was blocked.     

FadD3 is an acyl‐CoA synthetase that initiates catabolism of cholesterol rings C and D in actinobacteria

The cholesterol catabolic pathway occurs in most mycolic acid‐containing actinobacteria, such as Rhodococcus jostii RHA1, and is critical for Mycobacterium tuberculosis (Mtb) during infection. FadD3 is one of four predicted acyl‐CoA synthetases potentially involved in cholesterol catabolism. A ΔfadD3 mutant of RHA1 grew on cholesterol to half the yield of wild‐type and accumulated 3aα‐H‐4α(3′‐propanoate)‐7aβ‐methylhexahydro‐1,5‐indanedione (HIP), consistent with the catabolism of half the steroid molecule. This phenotype was rescued by fadD3 of Mtb. Moreover, RHA1 but not ΔfadD3 grew on HIP. Purified FadD3_Mtb catalysed the ATP‐dependent CoA thioesterification of HIP and its hydroxylated analogues, 5α‐OH HIP and 1β‐OH HIP. The apparent specificity constant (k_cat/K_m) of FadD3_Mtb for HIP was 7.3 ± 0.3 × 10⁵ M^?1 s^?1, 165 times higher than for 5α‐OH HIP, while the apparent K_m for CoASH was 110 ± 10 μM. In contrast to enzymes involved in the catabolism of rings A and B, FadD3_Mtb did not detectably transform a metabolite with a partially degraded C17 side‐chain. Overall, these results indicate that FadD3 is a HIP‐CoA synthetase that initiates catabolism of steroid rings C and D after side‐chain degradation is complete. These findings are consistent with the actinobacterial kstR2 regulon encoding ring C/D degradation enzymes.     

Ecosystem–atmosphere exchange of CH4 and N2O and ecosystem respiration in wetlands in the Sanjiang Plain, Northeastern China

Natural wetlands are critically important to global change because of their role in modulating atmospheric concentrations of CO₂, CH₄, and N₂O. One 4‐year continuous observation was conducted to examine the exchanges of CH₄ and N₂O between three wetland ecosystems and the atmosphere as well as the ecosystem respiration in the Sanjiang Plain in Northeastern China. From 2002 to 2005, the mean annual budgets of CH₄ and N₂O, and ecosystem respiration were 39.40 ± 6.99 g C m^?2 yr^?1, 0.124 ± 0.05 g N m^?2 yr^?1, and 513.55 ± 8.58 g C m^?2 yr^?1 for permanently inundated wetland; 4.36 ± 1.79 g C m^?2 yr^?1, 0.11 ± 0.12 g N m^?2 yr^?1, and 880.50 ± 71.72 g C m^?2 yr^?1 for seasonally inundated wetland; and 0.21 ± 0.1 g C m^?2 yr^?1, 0.28 ± 0.11 g N m^?2 yr^?1, and 1212.83 ± 191.98 g C m^?2 yr^?1 for shrub swamp. The substantial interannual variation of gas fluxes was due to the significant climatic variability which underscores the importance of long‐term continuous observations. The apparent seasonal pattern of gas emissions associated with a significant relationship of gas fluxes to air temperature implied the potential effect of global warming on greenhouse gas emissions from natural wetlands. The budgets of CH₄ and N₂O fluxes and ecosystem respiration were highly variable among three wetland types, which suggest the uncertainties in previous studies in which all kinds of natural wetlands were treated as one or two functional types. New classification of global natural wetlands in more detailed level is highly expected.     

Diurnal patterns of denitrification, oxygen consumption and nitrous oxide production in rivers measured at the whole-reach scale

1. Denitrification, net oxygen consumption and net nitrous oxide flux to the atmosphere were measured in three small rivers (discharge approximately 2–27 m³ s^?1) at the whole reach scale during Spring and Summer, 2002. Two of these rivers (Iroquois River and Sugar Creek in north‐west Indiana – north‐east Illinois, U.S.A.) drained agricultural catchments and the other (Millstone River in central New Jersey, U.S.A.) drained a mixed suburban–agricultural catchment. 2. Denitrification, oxygen consumption and N₂O flux were measured based on net changes in dissolved gas concentrations (N₂, O₂, and N₂O) during riverine transport, correcting for atmospheric exchange. On each date, measurements were made during both light and dark periods. 3. Denitrification rates in these rivers ranged from 0.31 to 15.91 mmol N m^?2 h^?1, and rates within each river reach were consistently higher during the day than during the night. This diurnal pattern could be related to cyclic patterns of nitrification driven by diurnal variations in water column pH and temperature. 4. Oxygen consumption ranged from 2.56 to 241 mmol O₂ m^?2 h^?1. In contrast to denitrification, net oxygen consumption was generally higher during the night than during the day. 5. River water was consistently supersaturated with N₂O, ranging from 102 to 209% saturated. Net flux of N₂O to the atmosphere ranged from 0.4 to 60 μmol N m^?2 h^?1. Net flux of N₂O was generally higher at night than during the day. The high flux of N₂O from these rivers strengthens the argument that rivers are an important contributor to anthropogenic emissions of this greenhouse gas.     

Effects on Mitochondrial K Flux of pH,K Concentration,and N-Ethyl Maleimide

Based on published evidence that cation transport in mitochondria is not significantly dependent on a membrane potential, it is suggested that the process of mitochondrial cation transport may be nonelectrogenic. These experiments focused on the possibility that K⁺ flux into rat liver mitochondria may be directly coupled, via an energy-linked carrier mechanism, to OH^? influx or H⁺ efflux. The dependence of the unidirectional K⁺ influx on the external K⁺ concentration indicates involvement of a saturable mechanism. Increasing the external pH from 7.0 to 8.0 increases the apparent V_max of the K⁺ influx without significantly altering the apparent K_m for K⁺. The pH dependence is greater in the presence of N-ethyl maleimide, a known inhibitor of the mitochondrial P_i/OH^? exchange mechanism. N-Ethyl maleimide decreases the apparent V_max at pH 7.0 and increases it at pH 8.0. Evidence indicates that both N-ethyl maleimide and a high external P_i concentration may stimulate the K⁺ influx at alkaline external pH (8.0) by preventing net exchanges between endogenous P_i and external OH^?. An apparent first-order dependence of the K⁺ influx on the external OH^? concentration is observed in the presence of N-ethyl maleimide. These results are consistent with a possible role of external OH^? as a cosubstrate of the K⁺ transport mechanism.     

Sulphate transport in the cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. leopoliensis) PCC 7942

Synechococcus R-2 (PCC 1942) actively accumulates sulphate in the light and dark. Intracellular sulphate was 1.35 ± 0.23 mol m^?3 (light) and 0.894 ± 0.152 mol m^?3 (dark) under control conditions (BG-11 media: pH_o, 7.5; [SO₄^2?]_o, 0.304 mol m^?3). The sulphate transporter is different from that found in higher plants: it appears to be an ATP-driven pump transporting one SO₄^2?/ATP [ΔμSO₄^2?_i,o=+ 27.7 ± 0.24 kJ mol^?1 (light) and + 24 ± 0.34 kj mol^?1 (dark)]. The rate of metabolism of SO₄^2?at pH_o, 7.5 was 150 ± 28 pmol m^?2 s^?1 (n = 185) in the light but only 12.8 ± 3.6 pmol m^?2 s^?1 (n = 61) in the dark. Light-driven sulphate uptake is partially inhibited by DCMU and chloramphenicol. Sulphate uptake is not linked to potassium, proton, sodium or chloride transport. The alga has a constitutive over-capacity for sulphate uptake [light (n= 105): K_m= 0.3 ± 0.1 mmol m^?3, V_max, = 1.8 ± 0.6 nmol m^?2 s^?1; dark (n= 56): K_m= 1.4 ± 0.4 mmol m^?3, V_max= 41 ± 22 pmol m^?2 s^?1]. Sulphite (SO₃^2?) was a competitive inhibitor of sulphate uptake. Selenate (SeO₄^2?) was an uncompetitive inhibitor.     

Reduction of extracellular dehydroascorbic acid by K562 cells

K562 erythroleukaemic cells produced ascorbate when incubated with dehydroascorbic acid. The reduction depended on the number of cells and on the concentration of dehydroascorbic acid. The observed rate consists of a high affinity (apparent) K_m 7 μM , V_max 3·25 pmol min^?1 (10⁶ cells)^?1 and a low affinity component, which was non-saturable up to 1 mM of DHA (rate increase of 0·1 pmol min^?1 (10⁶ cells)^?1 (1 μM of DHA^?1). The rate was dependent on temperature and was stimulated by glucose and inhibited by phloretin, N-ethylmaleimide, parachloro-mercuribenzoate and thenoyltrifluoroacetone. Although uptake of DHA proceeded at a higher rate than its extracellular reduction, the generation of extracellular ascorbate from DHA cannot be accounted for by intracellular reduction and the release of ascorbate, since the latter was not linear with time and had an initial rate of approximately 3 pmol min^?1 (10⁶ cells^?1). At a concentration of DHA of 100 μM this is 25 per cent of the observed reduction.     

Phenoloxidase activity and thermostability of Cancer pagurus and Limulus polyphemus hemocyanin

The intrinsic and inducible o-diphenoloxidase (o-diPO) activity of Cancer pagurus hemocyanin (CpH) and Limulus polyphemus hemocyanin (LpH) were studied using catechol, l-Dopa and dopamine as substrates. The kinetic analysis shows that dopamine is a more specific substrate for CpH than catechol and l-Dopa (K_m value of 0.01 mM for dopamine versus 0.67 mM for catechol, and 2.14 mM for l-Dopa), while k_cat is highest for catechol (2.44 min^? 1 versus 0.67 min^? 1 for l-Dopa and 0.71 min^? 1 for dopamine). On treatment with 4 mM sodium dodecyl sulfate (SDS) or by proteolysis the o-diPO activity of CpH increases about twofold. In contrast, native LpH shows no o-diPO activity, and exhibits only a slight activity after incubation with SDS. Neither CpH nor LpH show intrinsic mono-PO activity with l-tyrosine and tyramine as substrates. To explore the possible correlation between the degree of PO activity and protein stability of arthropod hemocyanins, the thermal stability of CpH and LpH was investigated by differential scanning calorimetry and Fourier transform infrared spectroscopy. CpH is found to be less thermostable (T_m ~ 80 °C), suggesting that the dicopper active sites are more accessible, thereby allowing the hemocyanin to show PO activity in the native state. The LpH, on the other hand, is more thermostable (T_m ~ 92 °C), suggesting the existence of a correlation between the thermal stability and the intrinsic PO activity of arthropod hemocyanins.     

Gross nitrous oxide production drives net nitrous oxide fluxes across a salt marsh landscape

Sea level rise will change inundation regimes in salt marshes, altering redox dynamics that control nitrification – a potential source of the potent greenhouse gas, nitrous oxide (N₂O) – and denitrification, a major nitrogen (N) loss pathway in coastal ecosystems and both a source and sink of N₂O. Measurements of net N₂O fluxes alone yield little insight into the different effects of redox conditions on N₂O production and consumption. We used in situ measurements of gross N₂O fluxes across a salt marsh elevation gradient to determine how soil N₂O emissions in coastal ecosystems may respond to future sea level rise. Soil redox declined as marsh elevation decreased, with lower soil nitrate and higher ferrous iron in the low marsh compared to the mid and high marshes (P < 0.001 for both). In addition, soil oxygen concentrations were lower in the low and mid‐marshes relative to the high marsh (P < 0.001). Net N₂O fluxes differed significantly among marsh zones (P = 0.009), averaging 9.8 ± 5.4 μg N m^?2 h^?1, ?2.2 ± 0.9 μg N m^?2 h^?1, and 0.67 ± 0.57 μg N m^?2 h^?1 in the low, mid, and high marshes, respectively. Both net N₂O release and uptake were observed in the low and high marshes, but the mid‐marsh was consistently a net N₂O sink. Gross N₂O production was highest in the low marsh and lowest in the mid‐marsh (P = 0.02), whereas gross N₂O consumption did not differ among marsh zones. Thus, variability in gross N₂O production rates drove the differences in net N₂O flux among marsh zones. Our results suggest that future studies should focus on elucidating controls on the processes producing, rather than consuming, N₂O in salt marshes to improve our predictions of changes in net N₂O fluxes caused by future sea level rise.