Kinetics of ammonium, nitrate and phosphorus uptake by Canna indica and Schoenoplectus validus

Canna indica L. is an upright perennial rhizomatous herb, and Schoenoplectus validus (Vahl) A. Löve and D. Löve is a tall, perennial, herbaceous sedge. The nutrient uptake kinetics of C. indica and S. validus were investigated using the modified depletion method after plants were grown for 4 weeks in simulated secondary-treated wastewater. The maximum uptake rate (I_max) and Michaelis–Menten constant (K_m) were estimated by iterative curve fitting. The I_max for NH₄N (623 μmol g⁻¹ dry root weight h⁻¹) was significantly higher than that for NO₃N (338 μmol g⁻¹ dry root weight h⁻¹) in S. validus. In contrast, no difference was observed in C. indica. The I_max values for NO₃N and NH₄N were higher in S. validus than in C. indica. A significantly lower K_m was detected for NO₃N uptake in C. indica (385 μmol L⁻¹) compared to that in S. validus (1908 μmol L⁻¹). The I_max for PO₄P did not differ between the plant species. The K_m for PO₄P was significantly higher in C. indica (157 μmol L⁻¹) than in S. validus (60 μmol L⁻¹). In conclusion, we found that S. validus preferred NH₄N over NO₃N, had greater capacity for N uptake and higher affinity for PO₄P, but C. indica had greater affinity for NO₃N. Nutrient uptake capacity is likely related to habitat preference, and is influenced by the structure of roots and rhizomes.     

Diversification of function in the haloacid dehalogenase enzyme superfamily: The role of the cap domain in hydrolytic phosphoruscarbon bond cleavage

Phosphonatase functions in the 2-aminoethylphosphonate (AEP) degradation pathway of bacteria, catalyzing the hydrolysis of the CP bond in phosphonoacetaldehyde (Pald) via formation of a bi-covalent Lys53ethylenamine/Asp12 aspartylphosphate intermediate. Because phosphonatase is a member of the haloacid dehalogenase superfamily, a family predominantly comprised of phosphatases, the question arises as to how this new catalytic activity evolved. The source of general acid-base catalysis for Schiff-base formation and aspartylphosphate hydrolysis was probed using pH-rate profile analysis of active-site mutants and X-ray crystallographic analysis of modified forms of the enzyme. The 2.9 Å X-ray crystal structure of the mutant Lys53Arg complexed with Mg²⁺ and phosphate shows that the equilibrium between the open and the closed conformation is disrupted, favoring the open conformation. Thus, proton dissociation from the cap domain Lys53 is required for cap domain–core domain closure. The likely recipient of the Lys53 proton is a water-His56 pair that serves to relay the proton to the carbonyl oxygen of the phosphonoacetaldehyde (Pald) substrate upon addition of the Lys53. The pH-rate profile analysis of active-site mutants was carried out to test this proposal. The proximal core domain residues Cys22 and Tyr128 were ruled out, and the role of cap domain His56 was supported by the results. The X-ray crystallographic structure of wild-type phosphonatase reduced with NaBH₄ in the presence of Pald was determined at 2.4 Å resolution to reveal Nε-ethyl-Lys53 juxtaposed with a sulfate ligand bound in the phosphate site. The position of the C(2) of the N-ethyl group in this structure is consistent with the hypothesis that the cap domain Nε-ethylenamine-Lys53 functions as a general base in the hydrolysis of the aspartylphosphate bi-covalent enzyme intermediate. Because the enzyme residues proposed to play a key role in PC bond cleavage are localized on the cap domain, this domain appears to have evolved to support the diversification of the HAD phosphatase core domain for catalysis of hydrolytic PC bond cleavage.     

Half-sandwich Ru[9]aneS3 complexes structurally similar to antitumor-active organometallic piano-stool compounds: Preparation, structural characterization and in vitro cytotoxic activity

The preparation, structural characterization, and chemical behavior in aqueous solution of a series of new Ru[9]aneS₃ half-sandwich complexes of the type [Ru([9]aneS₃)Cl(NN)][CF₃SO₃] and [Ru([9]aneS₃)(dmso-S)(NN)][CF₃SO₃]₂ (5–15, NN = substituted bpy or 2 × 1-methylimidazole) are described. The X-ray structures of [Ru([9]aneS₃)Cl(3,3′-H₂dcbpy)][CF₃SO₃] (9) (3,3′-H₂dcbpy = 3,3′-dicarboxy-2,2′-bipyridine), [Ru([9]aneS₃)Cl(4,4′-dmobpy)][CF₃SO₃] (13) (4,4′-dmobpy = 4,4′-dimethoxy-2,2′-bipyridine), and [Ru([9]aneS₃)Cl(1-MeIm)₂][CF₃SO₃] (15) (1-MeIm = 1-methylimidazole) were also determined. The new compounds are structurally similar to anticancer-active organometallic half-sandwich complexes of formula [Ru(η⁶-arene)Cl(NN)][PF₆]. Three chloro compounds (5, 9, 15) were tested in vitro for cytotoxic activity against two human cancer cell lines in comparison with the previously described [Ru([9]aneS₃)Cl(en)][CF₃SO₃] (1, en = ethylenediamine), [Ru([9]aneS₃)Cl(bpy)][CF₃SO₃] (2), and with their common dmso precursor [Ru([9]aneS₃)Cl(dmso-S)₂][CF₃SO₃] (3). Only the ethylenediamine complex 1 showed some antiproliferative activity, ca. one order of magnitude lower than the reference organometallic half-sandwich compound RM175 that contains biphenyl instead of [9]aneS₃. This compound was further tested against a panel of human cancer cell lines (including one resistant to cisplatin).     

Protein kinase C-α negatively regulates EGF-induced PLC- activity through direct phosphorylation

Phospholipase C- is a PLC isozyme that contains a CDC25 homology domain and a pair of RA domains in addition to a conserved PLC catalytic domain. PLC- is activated by both growth factors and GPCR ligands in a distinct manner. Growth factors such as EGF stimulate PLC- in an RA2 domain-dependent manner through Ras and Rap. On the other hand, several GPCR ligands that are linked with Ga₁₂ or Ga₁₃ can activate PLC- by associating with GTP-RhoA. GTP-RhoA binds with the region in the PLC- Y domain. Gs-linked ligands such as PGE1 and adrenaline stimulate PLC- by cAMP-dependent activation of Epac and Rap2B. PLC- is important for cardiac development and function. In addition, several lines of evidence indicate that PLC- promotes cell growth in an activity-dependent or -independent manner. In particular, PLC--dependent suppression of EGF receptor downregulation contributes to its growth promoting activity. Proper regulation of PLC- activity is essential for preventing tumor formation. Our previous report indicated that EGF-dependent ubiquitination of PLC- is required for the control of PLC--dependent cell growth. Recently, we found that PLC- is phosphorylated by growth factor stimulation, and this is another mechanism of the negative regulation. PLC- is phosphorylated by PKC-α upon stimulation with growth factors such as EGF and PDGF. The EGF-induced phosphorylation of PLC- was abolished by PKC inhibitors and by the expression of the dominant negative mutant of PKC-α. Furthermore, PKC-α was found to phosphorylate PLC- directly in vitro, suggesting that PLC- is a substrate of PKC-α in cells. In addition, PLC- was co-immunoprecipitated with PKC-α in an EGF-dependent manner. Immunocytochemical studies showed that PLC- co-localized with PKC-α in the plasma membrane after EGF stimulation. In addition, inhibition of PKC activity enhanced PLC--mediated PIP₂ hydrolysis, suggesting that PKC-α negatively regulates PLC- activity. Taken together, these results suggest for the first time that PLC- is regulated by PKC-α-dependent phosphorylation.     

Biotransformation of sesquiterpenoids having α,β-unsaturated carbonyl groups with cultured plant cells of Marchantia polymorpha

The biotransformation of sesquiterpenoids having an α,β-unsaturated carbonyl group, such as α-santonin (1), lancerodiol p-hydroxybenzoate (2), 8,9-dehydronootkatone (3), and nootkatone (4), with cultured suspension cells of Marchantia polymorpha was investigated. It was found that the CC double bond of 1 and 2 was hydrogenated to give 1,2-dihydro-α-santonin (5) and 3,4-dihydrolancerodiol p-hydroxybenzoate (6), respectively, while the allylic position of the CC double bond of 3 and 4 was hydroxylated to give 13-hydroxy-8,9-dehydronootkatone (7) and 9-hydroxynootkatone (8), respectively.     

Alkoxy-substituted group 6 allenylidene complexes - Synthesis and properties

Bis(alkoxy)allenylidene complexes, [(CO)₅MCCC(OR′)OR], as well as mono(alkoxy)allenylidene complexes, [(CO)₅MCCC(OR′)Ph], of chromium and tungsten are accessible from propynones [HCCC(O)Ph] or propynoic acid esters [HCCC(O)OR; R = Et, (−)-menthyl, endo-bornyl] by the following reaction sequence: (a) deprotonation of the alkynes, (b) reaction with [(CO)₅M-THF] (M = Cr, W), and (c) alkylation of the resulting alkynyl metallate, [(CO)₅MCCC(O)R], with Meerwein salts. Vinylidene complexes, [(CO)₅MCC(R′)C(O)OR], are formed as a by-product by C_β-alkylation of the alkynyl metallate. Dimethylamine displaces one alkoxy substituent of the bis(alkoxy)allenylidene complexes to give dimethylamino(alkoxy)allenylidene complexes, [(CO)₅MCCC(OR)NMe₂]. The analogous reaction of dimethylamine with a mono(alkoxy)-substituted allenylidene complex affords the aminoallenylidene complex [(CO)₅CrCCC(NMe₂)Ph]. When the amine is used in large excess, the α,β-unsaturated aminocarbene complex [(CO)₅CrC(NMe₂)C(H)C(NMe₂)Ph] is additionally formed by addition of the amine across the C_αC_β-bond of the allenylidene ligand. The reaction of [(CO)₅MCCC(OEt)₂] with dimethyl ethylenediamine offers access to bis(amino)allenylidene complexes, in which C_γ is part of a five-membered heterocycle. Photolysis of bis(alkoxy)allenylidene complexes in the presence of triphenylphosphine yields tetracarbonyl- and tricarbonyl{bis(phosphine)}allenylidene complexes. Diethylaminopropyne inserts into the C_βC_γ bond of [(CO)₅MCCC(OEt)OMethyl] to give alkenylallenylidene complexes. Subsequent acid-catalyzed intramolecular cyclization affords a pyranylidene complex.     

Reduction of the lipocalin type heme containing protein nitrophorin -- sensitivity of the fold-stabilizing cysteine disulfides toward routine heme-iron reduction

The determination of the redox properties of the cofactor in heme proteins provides fundamental insight into the chemical characteristics of this wide-spread class of metalloproteins. For the preparation of the ferroheme state, probably the most widely applied reductant is sodium dithionite, which at neutral pH has a reduction potential well below the reduction potential of most heme centers. In addition to the heme iron, some heme proteins, including the nitrophorins (NPs), contain cysteinecysteine disulfide bonds. In the present study, the effect of dithionite on the disulfides of NP4 and NP7 is addressed. To gain deeper understanding of the disulfide/dithionite reaction, oxidized glutathione (GSSG), as a model system, was incubated with dithionite and the products were characterized by ¹³C NMR spectroscopy and reverse phase chromatography in combination with mass spectrometry. This revealed the formation of one equivalent each of thiol (GSH) and glutathione-S-thiosulfate (GSSO₃⁻). With this background information, the effect of dithionite on the cystines of NP4 and NP7 was studied after trapping of the thiols with para-cloromercurybenzyl sulfonate (p-CMBS) and subsequent matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) where the heterolytic cleavage of the SS bond appears with only 2 molar equivalents of the reductant. Furthermore, prolonged electrochemical reduction of NP4 and NP7 in the presence of electrochemical mediators also leads to disulfide breakage. However, due to sterical shielding of the disulfide bridges in NP4 and NP7, the cystine reduction can be largely prevented by the use of stoichiometric amounts of reductant or limited electrochemical reduction. The described disulfide breakage during routine iron reduction is of importance for other heme proteins containing cystine(s).     

Regulatory links between PLC enzymes and Ras superfamily GTPases: Signalling via PLC

A growing body of work implies that links between PLC isoforms, in particular PLC, and small G-proteins from Ras superfamily could be important in regulation of a number of cellular processes. Through successful use of biochemistry and structural biology, several interactions have been characterized providing some ideas about the regulatory mechanisms. A number of signalling pathways have also been suggested that could involve direct interaction of Ras and Rho GTPases with PLC. Importantly, several studies combining cell biology and genetics have provided new insights into functions of PLC and highlighted the importance of this approach to extend further and consolidate currently incomplete picture regarding its roles in development and disease.     

CHMetal(II) axial interaction in planar complexes (metal = Cu, Pd) and implications for possible environmental effects of alkyl groups at biological copper sites

Intramolecular M(II)H–C interactions (M(II)=Cu(II), Pd(II)) involving a side chain alkyl group of planar d⁸ and d⁹ metal complexes of the N-alkyl (R) derivatives of N,N-bis(2-pyridylmethyl)amine with an N₃Cl donor set were established by structural and spectroscopic methods. The methyl group from the branched alkyl group (R = 2,2-dimethylpropyl and 2-methylbutyl) axially interacts with the metal ion with the MC and MH distances of 3.056(3)–3.352(9) and 2.317(1)–2.606(1) Å, respectively, and the M–H–C angles of 122.4–162.3°. The Cu(II) complexes showing the interaction have a higher redox potential as compared with those without it, and the ¹H NMR signals of the interacting methyl group in Pd(II) complexes shifted downfield relative to the ligand signals. Dependence of the downshift values on the dielectric constants of the solvents used indicated that the M(II)H–C interaction is mainly electrostatic in nature and may be regarded as a weak hydrogen bond. Implications for possible environmental effects of the leucine alkyl group at the type 1 Cu site of fungal laccase are also discussed.     

Study on hydrogen bonds of carboxymethyl cellulose sodium film with two-dimensional correlation infrared spectroscopy

Carboxymethyl cellulose is widely used in many industrial aspects and also in laboratory due to its good biocompatibility. However, special researches on infrared especially aiming at the hydrogen bonds structure of carboxymethyl cellulose were relatively poor. We demonstrate here a full view of infrared spectroscopy in the temperature range of 40–220 °C, mainly aiming at the hydrogen bonds in the NaCMC film. The two important transition points was defined with DSC and together with Infrared analysis, that is, 100 °C corresponding to the complete loss of water molecules and 170 °C to the starting temperature point the O6H6 being oxidized. The series of IR spectra during heating from 40 to 220 °C was analyzed by the two-dimensional correlation method. We found that the water molecules bound with CO groups and OH groups. With the evaporating of water molecules, the hydrated CO groups gradually transited into non-hydrated CO groups. As the temperature continued to increase, the intrachain hydrogen bonds were weakened and transited into weak hydrogen bonds. When the temperature was higher than 170 °C, the O6H6 groups were gradually oxidized and thus the interchain hydrogen bonds formed between CH₂COONa groups and O6H6 were weakened. In summary, we defined the main sorts of hydrogen bonds in carboxymethyl cellulose and pictured the changes of the hydrogen bonds structure during heating process, which may provide for the further application in both industry aspects and laboratory use.     

Fine structure characterization of amylopectins from grain amaranth starch

The aim of this study was to determine the fine structure of amylopectin from grain amaranth. Amaranthus amylopectin was hydrolyzed with α-amylase, and single clusters and a group of clusters (domain) were isolated by methanol precipitation. The domain and the clusters were treated with phosphorylase a and then β-amylase to remove all external chains, whereby the internal structure was obtained. The ,β-limit dextrins were analyzed on Sepharose CL 6B. The average DP (degree of polymerization) and peak-DP values of fractions of clusters were 57 and 82, respectively; the values of the domain were 137 and 309, respectively. The unit chain length profiles were analyzed by high-performance anion-exchange chromatography with pulsed amperometric detector (HPAEC–PAD). The results showed that the domain fraction contained 2.2 clusters, and single clusters were composed of 13 chains. The ,β-limit dextrins of the clusters were further hydrolyzed with α-amylase to characterize their building block composition. The average DP of the branched blocks was 11 and they contained on average 2.5 chains. Their average chain length, internal chain length, and degree of branching were approximately 4.3, 2.8, and 14, respectively. A cluster consisted of 6 branched blocks, and the internal chain length between the blocks was 6.8.     

In vivo real-time measurement of superoxide anion radical with a novel electrochemical sensor

The dynamics of superoxide anion (O₂⁻) in vivo remain to be clarified because no appropriate method exists to directly and continuously monitor and evaluate O₂⁻ in vivo. Here, we establish an in vivo method using a novel electrochemical O₂⁻ sensor. O₂⁻ generated is measured as a current and evaluated as a quantified partial value of electricity (Q_part), which is calculated by integration of the difference between the baseline and the actual reacted current. The accuracy and efficacy of this method were confirmed by dose-dependent O₂⁻ generation in xanthine–xanthine oxidase in vitro in phosphate-buffered saline and human blood. It was then applied to endotoxemic rats in vivo. O₂⁻ current began to increase 1 h after lipopolysaccharide, and Q_part increased significantly for 6 h in endotoxemic rats, in comparison to sham-treated rats. These values were attenuated by superoxide dismutase. The generation and attenuation of O₂⁻ were indirectly confirmed by plasma lipid peroxidation with malondialdehyde, endothelial injury with soluble intercellular adhesion molecule-1, and microcirculatory dysfunction. This is a novel method for measuring O₂⁻ in vivo and could be used to monitor and treat the pathophysiology caused by excessive O₂⁻ generation in animals and humans.     

Biphasic effect of nitric oxide on the cardiac voltage-dependent anion channel

Nitric oxide (NO) effects on the cardiac mitochondrial voltage-dependent anion channel (VDAC) are unknown. The effects of exogenous NO on VDAC purified from rat hearts were investigated in this study. When incorporated into lipid bilayers, VDAC was inhibited directly by an NO donor, PAPA NONOate, in a concentration-dependent biphasic manner. This was prevented by an NO scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. The effect paralleled that of NO in delaying the opening of the mitochondrial permeability transition (PT) pore. These biphasic effects on the cardiac VDAC and the mitochondrial PT pore reveal a tandem impact of NO on the two mitochondrial entities.     

The effect of hormone therapy on olfactory sensitivity is dependent on apolipoprotein E genotype

Patients with Alzheimer's disease (AD) show a deficit in olfactory threshold sensitivity. The Apolipoprotein E (ApoE) 4 allele is associated with increased risk of AD and earlier symptom onset. Hormone therapy (HT) may exert neuroprotective effects on brain areas affected by AD. The current study investigated the effect of HT on performance on an olfactory threshold test in 4 positive and 4 negative non-hysterectomized, non-demented, elderly females and AD patients. Among the non-demented participants, 4 positive females who had received HT performed 1) significantly better than those without HT, and 2) at levels similar to those of 4 negative females. In contrast, those without HT who were 4 positive performed significantly worse than those who were 4 negative. HT had no effect on performance in AD patients regardless of 4 status. These results suggest that HT may offer protection against loss of olfactory function in 4 positive individuals in preclinical stages of AD. Future research is warranted in order to investigate further the neuroprotective role of HT on sensory and cognitive functions in non-demented aging individuals.     

A review of the Cambrian biostratigraphy of South Australia

Cambrian rocks in South Australia occur in the Stansbury, Arrowie, eastern Officer and Warburton Basins. The succession in the Stansbury and Arrowie Basins can be divided into three sequence sets (supersequences), 1, 2 and 3. Sequence set 1 can be divided into five third-order sequences: 1.0, 1.1A, 1.1B, 1.2 and 1.3. Trilobites from the Stansbury and Arrowie Basins are restricted largely to the lower part of the succession. Four trilobite zones are recognized: Abadiella huoi (latest Atdabanian–earliest Botoman), Pararaia tatei, Pararaia bunyerooensis and Pararaia janeae Zones (all Botoman). Trilobites higher in the succession are known from only a few horizons and in part correlate with the upper Lower Cambrian Lungwangmiaoan Stage of China, equivalent to the top Toyonian. Pagetia sp. has been reported in the Coobowie Formation of the Stansbury Basin, thus suggesting an early Middle Cambrian age.The Cambrian faunas of the Warburton Basin range in age from early Middle Cambrian (Late Templetonian) to very Late Cambrian, although the richest faunal assemblages are late Middle Cambrian (Ptychagnostus punctuosus to Goniagnostus nathorsti Zones). Conodonts, including Cordylodus proavus, occur in a Datsonian fauna.The Arrowie Basin contains the most complete and best studied archaeocyath succession in the Australia–Antarctica region. The Warriootacyathus wilkawillensis, Spirillicyathus tenuis and Jugalicyathus tardus Zones from the lower Wilkawillina Limestone (Arrowie Basin) and equivalents are correlated with the Atdabanian. Botoman archaeocyathids occur higher in the Wilkawillina Limestone. The youngest (Toyonian) archaeocyath fauna in Australia occurs in the Wirrealpa Limestone (Arrowie Basin).Brachiopods and molluscs of the Arrowie and Stansbury Basins can be divided into four biostratigraphic assemblages. Several informal Early Cambrian SSF biostratigraphic assemblages are recognized. Probable tabulate-like corals occur in the Botoman Moorowie Formation. Seven informal acritarch assemblages occur in the Early Cambrian of the Stansbury and Arrowie Basins. Trace fossils may mark the Precambrian–Cambrian boundary. Only two of several tuffaceous horizons from the Stansbury and Arrowie Basins have been dated (i) a date of 522.0 ± 2.1 Ma from the Heatherdale Shale of the Stansbury Basin, about 400 m above latest Atdabanian archaeocyathids and (ii) a date of 522.0 ± 1.8 Ma from the lower part of the Billy Creek Formation in the Arrowie Basin. Neither date is regarded as reliable.     

Spectroscopic characterization of cytochrome P450 Compound I

The cytochrome P450 protein-bound porphyrin complex with the iron-coordinated active oxygen atom as Fe(IV)O is called Compound I (Cpd I). Cpd I is the intermediate species proposed to hydroxylate directly the inert carbon–hydrogen bonds of P450 substrates. In the natural reaction cycle of cytochrome P450 Cpd I has not yet been detected, presumably because it is very short-lived. A great variety of experimental approaches has been applied to produce Cpd I artificially aiming to characterize its electronic structure with spectroscopic techniques. In spite of these attempts, none of the spectroscopic studies of the last decades proved capable of univocally identifying the electronic state of P450 Cpd I. Very recently, however, Rittle and Green [9] have shown that Cpd I of CYP119, the thermophillic P450 from Sulfolobus acidocaldarius, is univocally a Fe(IV)O–porphyrin radical with the ferryl iron spin (S = 1) antiferromagnetically coupled to the porphyrin radical spin (S′ = 1/2) yielding a S_tot = 1/2 ground state very similar to Cpd I of chloroperoxidase from Caldariomyces fumago. In this mini-review the efforts to characterize Cpd I of cytochrome P450 by spectroscopic methods are summarized.     

Nitrogen dioxide solubility and permeation in lipid membranes

Nitrogen dioxide (NO₂) is an important oxidant molecule in biology that is produced by several biological processes, and it is also an important air pollutant. It can oxidize proteins and lipids with important consequences on their biological functions. Despite its relevance, the interaction of NO₂ with the cell barrier, the lipid membrane, is poorly understood. For instance, can lipid membranes limit NO₂ diffusion? To estimate the permeability of lipid membranes to NO₂ it is necessary to learn more about its solubility in the lipid phase. However, experimental data on NO₂ solubility is very limited. To improve our knowledge on this matter, we used a mixed approach consisting in calculating the solubility of NO₂ and related diatomic and triatomic gases (NO, O₂, CO₂, etc.) in different solvents using quantum calculations and Tomasi’s Polarizable Continuum Model and validating and correcting these results using experimental data available for the related gases. This approach led to an estimated partition coefficient for NO₂ of 2.7 between n-octanol and water, and 1.5 between lipid membranes and water, meaning that NO₂ is a moderately hydrophobic molecule (less than NO, more than CO₂). Based on the solubility-diffusion permeability theory, the permeability coefficient was estimated to be 5 cm s⁻¹, up to 4000 times higher than that of peroxynitrous acid. It is concluded that lipid membranes are not significant barriers to NO₂ transport.     

Decreased S-nitrosation of peptide thiols in the membrane interior

It has been proposed that autoxidation of nitric oxide (NO) stimulates S-nitrosation of thiols located in the hydrophobic milieu. We tested whether thiols located in hydrophobic membranes undergo enhanced S-nitrosation in the presence of NO/O₂. The transmembrane cysteinyl peptides C₄ (AcNH-KKACALA(LA)₆KK-CONH₂) and C₈ (AcNH-KKALALACALA(LA)₃KK-CONH₂) were incorporated into dilauroylphosphatidylcholine bilayers; their location in the membrane was determined by EPR spin labeling. The peptides, C₈ and C₄, and glutathione (GSH; 300 μM) were treated with a NO donor, DEA-NONOate, and nitrosothiol formation was determined under various O₂ levels. Surprisingly, the more hydrophobic cysteinyl peptide, C₈, did not yield any S-nitrosated product compared to GSH in the aqueous phase or C₄ peptide in the liposomes in the presence of NO/O₂. These data suggest that thiols located deeply in the hydrophobic core of the membrane may be less likely to undergo S-nitrosation in the presence of NO/O₂.     

Production of superoxides and nitric oxide generation in haemocytes of mussel Mytilus galloprovincialis (Lmk.) after exposure to cadmium: A possible involvement of Na/H exchanger in the induction of cadmium toxic effects

The present study investigates cadmium (Cd) ability to enhance superoxides (O₂⁻) and nitric oxide (NO) production (as nitrites) in haemocytes of mussel Mytilus galloprovincialis as well as the possible involvement of Na⁺/H⁺ exchanger (NHE) in the induction of NADPH oxidase and NO synthase activity. PMA, a well-known PKC-mediated NADPH oxidase as well as NO synthase stimulator was also used, in order to verify Cd effects on both O₂⁻ and NO generation. According to the results of the present study, micromolar concentrations of Cd (0.05, 5, 10 and 50 μM) seemed to enhance O₂⁻ and NO generation in haemocytes of mussels. Moreover, O₂⁻ and NO generation in haemocytes exposed to Cd could be enhanced by its ability to induce reactive oxygen species (ROS) but respiratory burst activation as well. Inhibition of NO synthase with 10 μM l-NAME, significantly attenuated Cd ability to enhance O₂⁻ production and diminished NO generation, thus leading to the suggestion that Cd toxic effects, started at concentration of 50 μM, could enhance NADPH oxidase and NO synthase stimulation in haemocytes of mussels. NHE seems to play a regulatory role in the induction of either O₂⁻ or NO generation in haemocytes exposed to the metal, since its inhibition with the use of 10 μM EIPA significantly decrease both O₂⁻ and NO production. The involvement of NHE in the induction of O₂⁻ and NO generation, probably via PKC-mediated NADPH oxidase and NO synthase activation, is likely to be crucial to haemocytes exposed to heavy metals, such as Cd.