Different effects of SNP and GSNO on mitochondrial O<Stack><Subscript>2</Subscript><Superscript>.−</Superscript></Stack>/H<Subscript>2</Subscript>O<Subscript>2</Subscript> production

Sodium Nitroprusside (SNP) and S-Nitrosoglutathione (GSNO) differently affect mitochondrial H₂O₂ release at Complex-I. mM SNP increases while GSNO decreases the release induced by succinate alone or added on top of NAD-linked substrates. Stimulation likely depends on Nitric Oxide ( ^. NO) (released by SNP but not by GSNO) inhibiting cytochrome oxidase and mitochondrial respiration. Preincubations with SNP or high GSNO (10 mM plus DTE to increases its ^. NO release) induces an inhibition of the succinate dependent H₂O₂ production consistent with a ^. NO dependent covalent modification. However maximal inhibition of the succinate dependent H₂O₂ release is obtained in the presence of low GSNO (20–100 μM), but not with SNP. This inhibition appears independent of ^. NO release since μM GSNO does not affect mitochondrial respiration, or the H₂O₂ detection systems and its effect is very rapid. Inhibition may be partly due to an increased removal of O₂^.− since GSNO chemically competes with NBT and cytochrome C in O₂^.− detection.     

Negative molecular ion of deuterium D<Stack><Subscript>2</Subscript><Superscript>−</Superscript></Stack>

D₂⁻ ions produced in collisions of D⁻ ions with relative energies of 2.5–9.2 eV were detected for the first time. It is shown that the effective cross section for this reaction is no less than 1.5 × 10⁻¹⁴ cm². Along with the theoretically predicted short-lived state of negative molecular deuterium ions, a state existing for more than 1 μs was observed.     

Determination of the lifetime of D<Stack><Subscript>2</Subscript><Superscript>−</Superscript></Stack> and HD<Superscript>−</Superscript> ions

A method for determining the lifetime of unstable ions is described. The method is based on measuring the decrease in the ion beam current onto a fixed detector with increasing path length of the ion beam from the ion source to the detector. The measurements performed for D^? ₂ and HD^? molecular ions have shown that their lifetimes are 3.5 ± 0.1 and 4.4 ± 0.1 μs, respectively.     

Chronic Atmospheric NO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack> Deposition Does Not Induce NO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack> Use by <Emphasis Type="Italic">Acer saccharum</Emphasis> Marsh.

The ability of an ecosystem to retain anthropogenic nitrogen (N) deposition is dependent upon plant and soil sinks for N, the strengths of which may be altered by chronic atmospheric N deposition. Sugar maple (Acer saccharum Marsh.), the dominant overstory tree in northern hardwood forests of the Lake States region, has a limited capacity to take up and assimilate NO₃⁻. However, it is uncertain whether long-term exposure to NO₃⁻ deposition might induce NO₃⁻ uptake by this ecologically important overstory tree. Here, we investigate whether 10 years of experimental NO₃⁻ deposition (30 kg N ha⁻¹ y⁻¹) could induce NO₃⁻ uptake and assimilation in overstory sugar maple (approximately 90 years old), which would enable this species to function as a direct sink for atmospheric NO₃⁻ deposition. Kinetic parameters for NH₄⁺ and NO₃⁻ uptake in fine roots, as well as leaf and root NO₃⁻ reductase activity, were measured under conditions of ambient and experimental NO₃⁻ deposition in four sugar maple-dominated stands spanning the geographic distribution of northern hardwood forests in the Upper Lake States. Chronic NO₃⁻ deposition did not alter the V _max or K _m for NO₃⁻ and NH₄⁺ uptake nor did it influence NO₃⁻ reductase activity in leaves and fine roots. Moreover, the mean V _max for NH₄⁺ uptake (5.15 μmol ¹⁵N g⁻¹ h⁻¹) was eight times greater than the V _max for NO₃⁻ uptake (0.63 μmol ¹⁵N g⁻¹ h⁻¹), indicating a much greater physiological capacity for NH₄⁺ uptake in this species. Additionally, NO₃⁻ reductase activity was lower than most values for woody plants previously reported in the literature, further indicating a low physiological potential for NO₃⁻ assimilation in sugar maple. Our results demonstrate that chronic NO₃⁻ deposition has not induced the physiological capacity for NO₃⁻ uptake and assimilation by sugar maple, making this dominant species an unlikely direct sink for anthropogenic NO₃⁻ deposition.     

A computer-aided quantum chemical study of the N<Stack><Subscript>15</Subscript><Superscript>−</Superscript></Stack> cluster

Ab initio (RHF, MP2) and Density Functional Theory (DFT) methods have been used to examine six isomers of the N15m cluster with the 6-31+G* basis set. Different from the known odd-numbered anionic N7m, N9m, and N11m clusters, in which the open-chain structures are the most stable species, the most stable N15m isomer is structure 1 (C1), which may be considered as a complex between the fragments cyclic N5m (D5h) and staggered N10 (D2d). The decomposition pathways of structure 2 (CS), containing two aromatic N5 rings connected by a N5 chain, and the open-chain structure 3 (C2v) were studied at the B3LYP/6-31+G* level of theory. Relative energies were refined at the level of B3LYP/6-311+G(3df,2p)//B3LYP/6-31+G*+ZPE (B3LYP/6-31+G*). The barriers for N2 and N5m (D5h) fission reactions for structure 2 are predicted to be 18.2 and 14.2 kcal x mol(-1), respectively. The corresponding N2+N3m fission barrier for structure 3 is predicted to be 11.2 kcal x mol(-1). Supplementary material is available for this article if you access the article at http://dx.doi.org/10.1007/s00894-003-0118-0. A link in the frame on the left on that page takes you directly to the supplementary material. Figure Structure 1 of the N15m cluster, showing bond distances in A and bond angles in degrees     

Estimation of denitrification potential in a karst aquifer using the <Superscript>15</Superscript>N and <Superscript>18</Superscript>O isotopes of NO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack>

A confined aquifer in the Malm Karst of the Franconian Alb, South Germany was investigated in order to understand the role of the vadose zone in denitrifiaction processes. The concentrations of chemical tracers Sr²⁺ and Cl^– and concentrations of stable isotope ¹⁸O were measured in spring water and precipitation during storm events. Based on these measurements a conceptual model for runoff was constructed. The results indicate that pre-event water, already stored in the system at the beginning of the event, flows downslope on vertical and lateral preferential flow paths. Chemical tracers used in a mixing model for hydrograph separation have shown that the pre-event water contribution is up to 30%. Applying this information to a conceptual runoff generation model, the values of ¹⁵N and ¹⁸O in nitrate could be calculated. Field observations showed the occurence of significant microbial denitrification processes above the soil/bedrock interface before nitrate percolates through to the deeper horizon of the vadose zone. The source of nitrate could be determined and denitrification processes were calculated. Assuming that the nitrate reduction follows a Rayleigh process one could approximate a nitrate input concentration of about 170 mg/l and a residual nitrate concentration of only about 15%. The results of the chemical and isotopic tracers postulate fertilizers as nitrate source with some influence of atmospheric nitrate. The combined application of hydrograph separation and determination of isotope values in ¹⁵N and ¹⁸O of nitrate lead to an improved understanding of microbial processes (nitrification, denitrification) in dynamic systems.     

Foliar N application reduces soil NO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack>-N leaching loss in apple orchards

A comparison of the effects of foliar and soil N application was made in field-grown mature fruiting Gala/M9 apple trees (Malus domestica Borkh) in 2001 and 2002 growing seasons under Pacific Northwest growing conditions in southern British Columbia, Canada. The trees, six years old at the start of the experiment, were treated: (1) with 5 g/l urea sprays supplied every two weeks (7 times) from mid May to mid August (total about 50 g N/tree/year), (2) with the same amount of N applied to the soil with the same timing and quantity as for the foliar treatment, and (3) with no N (control). Leaf color (as SPAD readings) and N concentrations (mg/g), and soil NH₄⁺-N and NO₃^–-N were measured periodically throughout the two seasons. Leached NO₃^–-N was monitored monthly via an anion exchange probe from June to October in 2001 and from May to November in 2002. Shoot length was measured in October and N concentration of one-year-old wood and roots was determined in December of each growing s eason. Soil N application significantly increased shoot length relative to control or foliar N application. Leaf color, leaf N, and N concentration of one-year-old wood and roots were similarly increased relative to control by both soil and foliar N application. These treatments also increased fruit yield relative to control. There was no significant difference in yield and fruit quality between soil and foliar N applications. Soil N application increased soil NH₄⁺-N and NO₃^–-N content in the root zone, and also increased the NO₃^– leaching loss below the root zone especially late in the growing season. Our results suggested that tree N status and yield and fruit quality could be maintained by multiple urea sprays during the growing season in apple orchards, and foliar N application will reduce the risk of soil NO₃^–-N leaching.     

Molecular Weight and Subunit Composition of the Sensitive to GABA-Ergic Ligands Cl<Superscript>−</Superscript>/HCO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack>-Stimulated Mg<Superscript>2+</Superscript>-ATPase from Plasma Membrane of Rat Brain

The molecular weight and subunit composition of Cl-,HCO3(-)- and picrotoxin-stimulated Mg2+-ATPase from rat brain plasma membrane solubilized in sodium deoxycholate were studied by gel filtration chromatography. The enzyme activity eluted from a Sephacryl S-300 column in a single peak associated with a protein of molecular weight approximately 300 kD and a Stokes radius of 5.4 nm. The enzyme-enriched fraction, concentrated and denatured by SDS, migrated through a Sephacryl S-200 column as three peaks with molecular weights of approximately 57, 53, and 45 kD. SDS-PAGE also showed three major protein bands with molecular weights of about 57, 53, and 48 kD. The molecular weight and subunit composition of the Cl- and HCO3(-)-stimulated Mg2+-ATPase from neuronal membrane of rat brain are similar with the molecular properties of GABA(A)-benzodiazepine receptor complex from mammalian brain but are different from those of P-type transport ATPases.     

Characteristics of DOC Exported from Northern Hardwood Forests Receiving Chronic Experimental NO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack> Deposition

Abstract Sugar maple (Acer saccharum Marsh.)-dominated northern hardwood forests of the Great Lakes Region commonly receive elevated levels of atmospheric nitrate (NO₃⁻) deposition, which can alter belowground carbon (C) cycling. Past research has demonstrated that chronic experimental NO₃⁻ deposition (3 g N m⁻² y⁻¹ above ambient) elicits a threefold increase in the leaching loss of dissolved organic carbon (DOC). Here, we used DOC collected from tension-cup lysimeters to test whether increased DOC export under experimental NO₃⁻ deposition originated from forest floor or mineral soil organic matter (SOM). We used DOC radiocarbon dating to quantify C sources and colorimetric assays to measure DOC aromaticity and soluble polyphenolic content. Our results demonstrated that DOC exports are primarily derived from new C (<50-years-old) in the forest floor under both ambient and experimental NO₃⁻ deposition. Experimental NO₃⁻ deposition increased soluble polyphenolic content from 25.03 ± 4.26 to 49.19 ± 4.23 μg phenolic C mg DOC⁻¹, and increased total aromatic content as measured by specific UV absorbance. However, increased aromatic compounds represented a small fraction (<10%) of the total observed increased DOC leaching. In combination, these findings suggest that experimental NO₃⁻ deposition has altered the production or retention as well as phenolic content of DOC formed in forest floor, however exact mechanisms are uncertain. Further elucidation of the mechanism(s) controlling enhanced DOC leaching is important for understanding long-term responses of Great Lakes forests to anthropogenic N deposition and the consequences of those responses for aquatic ecosystems.     

Structures and stability of N<Subscript>13</Subscript><Superscript>+</Superscript> and N<Subscript>13</Subscript><Superscript>−</Superscript> clusters

The structures and stabilities of eleven N₁₃ ⁺ and N₁₃ ^– isomers have been investigated with second-order Møller–Plesset (MP2) and density functional theory (DFT) methods. Five N₁₃ ⁺ isomers and six N₁₃ ^– isomers are all reasonable local minima on their potential energy hypersurfaces. The most stable N₁₃ ⁺ cation is structure C-2 with C_2v symmetry, which contains a pentazole ring and two N₄ open chains. It is different from those of the N₇ ⁺ and N₉ ⁺ clusters, but similar to the N₁₁ ⁺ cluster. Meanwhile, the most stable N₁₃ ^– structure A-2 is composed of a pentazole ring and a six-membered ring connected by two nitrogen atoms. It is not only different from those of the N₇ ^– and N₉ ^– clusters, but also from the N₁₁ ^– cluster. The decomposition pathways of structures C-2 and A-2 were investigated at the B3LYP/(aug)-cc-pVDZ level. From the barrier heights of the structures C-2 and A-2 decomposition processes, it is suggested that C-2 is difficult to observe experimentally and A-2 may be observed as a short-lived species. Figure Optimized geometrical parameters of N₁₃ ⁺ isomer C-2      

Expression and Developmental Regulation of the Cystine/Glutamate Exchanger (x<Stack><Subscript>c</Subscript><Superscript>−</Superscript></Stack>) in the Rat

The cystine/glutamate exchanger (antiporter x_c⁻) is a membrane transporter involved in the uptake of cystine, the rate-limiting amino acid in the synthesis of glutathione. Recent studies suggest that the antiporter plays a role in the slow oxidative excitotoxity and in the pathological effects of β-N-oxalylamino-l-alanine, the molecule responsible for neurolathyrism, a neurotoxic upper motor neuron disease. The mouse cystine/glutamate exchanger has been cloned and showed to be composed of two distinct proteins, one of which being a novel protein, named xCT, of 502 amino acids and 12 putative trans-membrane domains. We have generated and purified a polyclonal antibody to mouse xCT and studied its expression in rat brain and in different cultured cells (astrocytes, fibroblasts and neurons) using Western blot and immunocytochemical techniques. Expression of xCT was also studied in rat brain and muscle at different developmental stages. Parallel experiments were carried out with antibodies to the heavy chain of 4F2 surface antigen, the non-specific subunit of the antiporter x_c⁻. xCT antibody detected in all cell and tissue extracts a specific band of about 40 kDa. Subcellular fractionation demonstrated that xCT is concentrated mainly in the microsomal-mitochondrial fraction, in accord with its structure as transmembrane protein. Immunocytochemical analysis showed a strong staining in all cells examined, included neurons. Furthermore, both xCT and the heavy chain of 4F2 surface antigen increased in the brain during development, reaching the highest expression in adulthood. The study of the expression and developmental profile of xCT represents a first step towards a better characterization of its biochemical properties and function, which in turn may help to understand the relative contribution of the x_c⁻ antiporter in the pathogenesis of certain neurodegenerative diseases.     

Flow of Deposited Inorganic N in Two Gleysol-dominated Mountain Catchments Traced with <Superscript>15</Superscript>NO<Subscript>3</Subscript><Superscript>−</Superscript> and <Superscript>15</Superscript>NH<Subscript>4</Subscript><Superscript>+</Superscript>

In two mountain ecosystems at the Alptal research site in central Switzerland, pulses of ¹⁵NO₃ and ¹⁵NH₄ were separately applied to trace deposited inorganic N. One forested and one litter meadow catchment, each approximately 1600 m², were delimited by trenches in the Gleysols. K¹⁵NO₃ was applied weekly or fortnightly over one year with a backpack sprayer, thus labelling the atmospheric nitrate deposition. After the sampling and a one-year break, ¹⁵NH₄Cl was applied as a second one-year pulse, followed by a second sampling campaign. Trees (needles, branches and bole wood), ground vegetation, litter layer and soil (LF, A and B horizon) were sampled at the end of each labelling period. Extractable inorganic N, microbial N, and immobilised soil N were analysed in the LF and A horizons. During the whole labelling period, the runoff water was sampled as well. Most of the added tracer remained in both ecosystems. More NO₃⁻ than NH₄⁺ tracer was retained, especially in the forest. The highest recovery was in the soil, mainly in the organic horizon, and in the ground vegetation, especially in the mosses. Event-based runoff analyses showed an immediate response of ¹⁵NO₃⁻ in runoff, with sharp ¹⁵N peaks corresponding to discharge peaks. NO₃⁻ leaching showed a clear seasonal pattern, being highest in spring during snowmelt. The high capacity of N retention in these ecosystems leads to the assumption that deposited N accumulates in the soil organic matter, causing a progressive decline of its C:N ratio.     

An optical material for the detection of trace S<Subscript>2</Subscript>O<Subscript>3</Subscript><Superscript>2−</Superscript> in milk based on a copper complex

A novel S₂O₃ ^2? luminescent sensor (Cu²⁺-p-CPIP) was developed and the presence of S₂O₃ ^2? caused an obvious fluorescence enhancement at 420 nm upon excitation at 330 nm, which could be distinguished with the naked eye under a UV lamp. Remarkably, the compound exhibited excellent selective and sensitive response to S₂O₃ ^2? over other common anions with a micromolar limit of detection (0.442 μM) in DMSO/H₂O (v/v, 1:1) buffer. The absorbance intensity and the color of Cu²⁺-p -CPIP solution changed gradually with the increase of S₂O₃ ^2? concentration. The proposed method was applied to the determination of S₂O₃ ^2? in milk samples and the recoveries were 97.5–105%. The preparation of Cu²⁺-p -CPIP exhibited the quick, simple and facile advantages. The results showed that Cu²⁺-p -CPIP can be a good candidate for simple, rapid and sensitive colorimetric detection of S₂O₃ ^2? in aqueous solution.     

Does Atmospheric NO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack> Deposition Alter the Abundance and Activity of Ligninolytic Fungi in Forest Soils?

Although field studies have demonstrated an ecosystem-specific effect of experimental atmospheric nitrogen (N) deposition on litter decomposition, a mechanistic understanding of how ligninolytic microbial communities respond to atmospheric deposition is lacking. Because high levels of inorganic N suppress lignin decomposition by some basidiomycetes, it is plausible that the abundance and activity of these key microorganisms underlies differential ecosystem responses of decomposition to atmospheric N deposition. We hypothesize that: (a) atmospheric N deposition will cause an ecosystem-specific reduction in basidiomycete activity and abundance with greatest decreases in ecosystems with lignin-rich forest litter and (b) the abundance of lignin degrading basidiomycetes will be positively correlated with ligninolytic enzyme activity. To test these hypotheses, we measured the effects of experimental N deposition on the potential activity of phenol oxidase enzymes, and the abundance of basidiomycete genes encoding laccase, a primary phenol oxidase enzyme, in three hardwood forests spanning a range of leaf litter lignin content. The black oak-white oak (BOWO) contains high lignin litter, the sugar maple-basswood (SMBW) has low lignin litter, and the sugar maple-red oak (SMRO) is intermediate. An ecosystem by N deposition interaction significantly influenced phenol oxidase activity in the surface soil (P = 0.05), where phenol oxidase activity decreased with increasing experimental N deposition in the BOWO ecosystem. No consistent response to N deposition was evident for surface soil phenol oxidase activity within either the SMRO or SMBW ecosystem. This interaction did not influence laccase gene abundance. Instead, basidiomycete laccase gene abundance was reduced by experimental N deposition (main effect) in surface soil. There was only a weak correlation between basidiomycete laccase gene abundance and potential phenol oxidase enzyme activity, suggesting that the abundance of organisms possessing laccase genes may not control phenol oxidase activity in soil. Our results suggest that the regulation of laccase gene expression may mediate the decomposition response to atmospheric N deposition.     

Quantitative targeted metabolomics for 15d-deoxy-Δ<Superscript>12, 14</Superscript>-PGJ<Subscript>2</Subscript> (15d-PGJ<Subscript>2</Subscript>) by MALDI-MS

Prostaglandins (PGs) are lipid mediators that may play important roles in cancer, immunomodulation, and neurodegeneration. So, the quantitative analysis of PGs will therefore be important in order to understand the natural history of a range of diseases and may be used as a tool in the development of new biotherapeutics. However, such an analysis is problematic because of the small quantities of PGs present in the body. Here, we developed a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-MS)-based analytical method for rapid and easy quantification of the ketone-containing PGs (15d-PGJ₂ etc.) as a targeted metabolomics platform. The chemical derivatization with Girard’s reagent P (GP) provided a good linearity (R² > 0.99) between peak area and quantity of 15d-PGJ₂ and highly improved sensitivity (limit of quantitation, LOQ: 1.25 pmol on a spot in MALDI plate). Finally, we utilized this method to directly characterize the interaction between peroxisome proliferatoractivated receptor gamma-ligand binding domain (PPARγ-LBD) and 15d-PGJ₂. The 15d-PGJ₂ was enriched by PPARγ-LBD and also the binding level of the ligand was dropped considerably by the treatment of PPARγ agonist such as rosiglitazone (about 0.61-fold reduction). Taken together, our MALDI-MS-based targeted metabolomics method for ketone-containing PGs may be applicable to elucidate the protein-metabolite interactions and to identify natural ligands for drug candidates.     

NO<Subscript>3</Subscript><Superscript>−</Superscript>-Driven SO<Subscript>4</Subscript><Superscript>2−</Superscript> Production in Freshwater Ecosystems: Implications for N and S Cycling

Massive anthropogenic acceleration of the global nitrogen (N) cycle has stimulated interest in understanding the fate of excess N loading to aquatic ecosystems. Nitrate (NO₃ ⁻) is traditionally thought to be removed mainly by microbial respiratory denitrification coupled to carbon (C) oxidation, or through biomass assimilation. Alternatively, chemolithoautotrophic bacterial metabolism may remove NO₃ ⁻ by coupling its reduction with the oxidation of sulfide to sulfate (SO₄ ²⁻). The NO₃ ⁻ may be reduced to N₂ or to NH₄ ⁺, a form of dissimilatory nitrate reduction to ammonium (DNRA). The objectives of this study were to investigate the importance of S oxidation as a NO₃ ⁻ removal process across diverse freshwater streams, lakes, and wetlands in southwestern Michigan (USA). Simultaneous NO₃ ⁻ removal and SO₄ ²⁻ production were observed in situ using modified “push-pull” methods in nine streams, nine wetlands, and three lakes. The measured SO₄ ²⁻ production can account for a significant fraction (25–40%) of the overall NO₃ ⁻ removal. Addition of ¹⁵NO₃ ⁻ and measurement of ¹⁵NH₄ ⁺ production using the push–pull method revealed that DNRA was a potentially important process of NO₃ ⁻ removal, particularly in wetland sediments. Enrichment cultures suggest that Thiomicrospira denitrificans may be one of the organisms responsible for this metabolism. These results indicate that NO₃ ⁻-driven SO₄ ²⁻ production could be widespread and biogeochemically important in freshwater sediments. Removal of NO₃ ⁻ by DNRA may not ameliorate problems such as eutrophication because the N remains bio-available. Additionally, if sulfur (S) pollution enhances NO₃ ⁻ removal in freshwaters, then controls on N processing in landscapes subject to S and N pollution are more complex than previously appreciated. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

α-lipoic acid induces apoptosis in human colon cancer cells by increasing mitochondrial respiration with a concomitant O<Subscript>2</Subscript><Superscript>−.</Superscript>-generation

The antioxidant -lipoic acid (ALA) has been shown to affect a variety of biological processes associated with oxidative stress including cancer. We determined in HT-29 human colon cancer cells whether ALA is able to affect apoptosis, as an important parameter disregulated in tumour development. Exposure of cells to ALA or its reduced form dihydrolipoic acid (DHLA) for 24 h dose dependently increased caspase-3-like activity and was associated with DNA-fragmentation. DHLA but not ALA was able to scavenge cytosolic O₂^–. in HT-29 cells whereas both compounds increased O₂^{– .}-generation inside mitochondria. Increased mitochondrial O₂^{– .}-production was preceded by an increased influx of lactate or pyruvate into mitochondria and resulted in the down-regulation of the anti-apoptotic protein bcl-X_L. Mitochondrial O₂^–.-generation and apoptosis induced by ALA and DHLA could be prevented by the O₂^{– .}-scavenger benzoquinone. Moreover, when the lactate/pyruvate transporter was inhibited by 5-nitro-2-(3-phenylpropylamino) benzoate, ALA- and DHLA-induced mitochondrial ROS-production and apoptosis were blocked. In contrast to HT-29 cells, no apoptosis was observed in non-transformed human colonocytes in response to ALA or DHLA addition. In conclusion, our study provides evidence that ALA and DHLA can effectively induce apoptosis in human colon cancer cells by a prooxidant mechanism that is initiated by an increased uptake of oxidizable substrates into mitochondria.     

On the generation of OH<Superscript>·</Superscript> radical species from H<Subscript>2</Subscript>O<Subscript>2</Subscript> by Cu(I) amyloid beta peptide model complexes: a DFT investigation

According to different studies, the interaction between amyloid β-peptide (Aβ) and copper ions could yield radical oxygen species production, in particular the highly toxic hydroxyl radical OH^· that is suspected to contribute to Alzheimer’s disease pathogenesis. Despite intensive experimental and computational studies, the nature of the interaction between copper and Aβ peptide, as well as the redox reactivity of the system, are still matter of debate. It was proposed that in Cu(II) → Cu(I) reduction the complex Cu(II)–Aβ could follow a multi-step conformational change with redox active intermediates that may be responsible for OH^· radical production from H₂O₂ through a Fenton-like process. The purpose of this work is to evaluate, using ab initio Density Functional Theory computations, the reactivity of different Cu(I)–Aβ coordination modes proposed in the literature, in terms of OH^· production. For each coordination model, we considered the corresponding H₂O₂ adduct and performed a potential energy surface scan along the reaction coordinate of O–O bond dissociation of the peroxide, resulting in the production of OH^· radical, obtaining reaction profiles for the evaluation of the energetic of the process. This procedure allowed us to confirm the hypothesis according to which the most populated Cu(I)–Aβ two-histidine coordination is not able to perform efficiently H₂O₂ reduction, while a less populated three-coordinated form would be responsible for the OH^· production. We show that coordination modes featuring a third nitrogen containing electron-donor ligand (an imidazole ring of an histidine residue is slightly favored over the N-terminal amine group) are more active towards H₂O₂ reduction.     

Transport of nitrogen and zinc to rhodes grass by arbuscular mycorrhiza and roots as affected by different nitrogen sources (NH<Subscript>4</Subscript><Superscript>+</Superscript>-N and NO<Subscript>3</Subscript><Superscript>−</Superscript>-N)

We investigated the effect of mineral nitrogen forms on transfer of nitrogen (N) and zinc (Zn) from attached compartments to rhodes grass (Chloris gayana) colonised with arbuscular mycorrhizal fungi (AMF). After being pre-cultivated in substrates with adequate nutrient supply and either AMF inoculated (+AM) or left non-inoculated (?AM), rhodes grass was positioned adjacent to an outer compartment holding a similar substrate but applied with labelled nitrogen (¹⁵N) either as ammonium (NH₄ ⁺) or nitrate (NO₃ ^?), and a high supply of Zn (150 mg kg^?1 DS). Plant roots together with fungal mycelium were either allowed to explore the outer compartment (with root access) or only mycorrhizal hyphae were allowed (without root access). Within each access treatment, biomasses of rhodes grass were not significantly affected by AMF inoculation or N form. AMF contribution to plant ¹⁵N uptake was about double in NH₄ ⁺ compared with NO₃ ^?-supplied treatments while the mycorrhizal influence on plant Zn uptake was insignificant. Without root access, the shoot ¹⁵N/Zn concentration ratio was up to ten-fold higher in +AM than –AM treatments and this ratio increase was clearly more pronounced in NH₄ ⁺ than NO₃ ^?-supplied treatments. In conclusion, rhodes grass in symbiosis with the tested AMF acquired more N when supplied with ammonium. Moreover, there is clear indication that although the AMF have transported both nutrients (N and Zn), N was preferentially transferred as compared to Zn. We confirmed that, while rhodes grass is not able to prevent excessive Zn uptake via roots under conditions of high Zn, mycorrhiza is able to avoid excessive Zn supply to the host plant when the fungus alone has access to contaminated patches.     

Association of β<Subscript>2</Subscript>-microglobulin with the α<Subscript>3</Subscript> domain of H-2D<Superscript>b</Superscript> heavy chain

MHC class I molecules are heterotrimeric complexes composed of heavy chain, ₂-microglobulin (₂m) and short peptide. This trimeric complex is generated in the endoplasmic reticulum (ER), where a peptide loading complex (PLC) facilitates transport from the cytosol and binding of the peptide to the preassembled ER resident heavy chain/₂m dimers. Association of mouse MHC class I heavy chain with ₂m is characterized by allelic differences in the number and/or positions of amino acid interactions. It is unclear, however, whether all alleles follow common binding patterns with minimal contributions by allele-specific contacts, or whether essential contacts with ₂m are different for each allele. While searching for the PLC binding site in the ₃ domain of the mouse MHC class I molecule H-2D^b, we unexpectedly discovered a site critical for binding mouse, but not human, ₂m. Interestingly, amino acids in the corresponding region of another MHC class I heavy chain allele do not make contacts with the mouse ₂m. Thus, there are allelic differences in the modes of binding of ₂m to the heavy chain of MHC class I.