Novel Redox-Dependent Esterase Activity (EC 3.1.1.2) for DJ-1: Implications for Parkinson's Disease.

Mutations the in human DJ-1 (hDJ-1) gene are associated with early-onset autosomal recessive forms of Parkinson's disease (PD). hDJ-1/parkinsonism associated deglycase (PARK7) is a cytoprotective multi-functional protein that contains a conserved cysteine-protease domain. Given that cysteine-proteases can act on both amide and ester substrates, we surmised that hDJ-1 possessed cysteine-mediated esterase activity. To test this hypothesis, hDJ-1 was overexpressed, purified and tested for activity towards 4-nitrophenyl acetate (pNPA) as µmol of pNPA hydrolyzed/min/mg·protein (U/mg protein). hDJ-1 showed maximum reaction velocity esterase activity (Vmax = 235.10 ± 12.00 U/mg protein), with a sigmoidal fit (S0.5 = 0.55 ± 0.040 mM) and apparent positive cooperativity (Hill coefficient of 2.05 ± 0.28). A PD-associated mutant of DJ-1 (M26I) lacked activity. Unlike its protease activity which is inactivated by reactive oxygen species (ROS), esterase activity of hDJ-1 is enhanced upon exposure to low concentrations of hydrogen peroxide (<10 µM) and plateaus at elevated concentrations (>100 µM) suggesting that its activity is resistant to oxidative stress. Esterase activity of DJ-1 requires oxidation of catalytic cysteines, as chemically protecting cysteines blocked its activity whereas an oxido-mimetic mutant of DJ-1 (C106D) exhibited robust esterase activity. Molecular docking studies suggest that C106 and L126 within its catalytic site interact with esterase substrates. Overall, our data show that hDJ-1 contains intrinsic redox-sensitive esterase activity that is abolished in a PD-associated mutant form of the hDJ-1 protein.

Inhibition of Urease by Disulfiram, an FDA-Approved Thiol Reagent Used in Humans.

Urease is a nickel-dependent amidohydrolase that catalyses the decomposition of urea into carbamate and ammonia, a reaction that constitutes an important source of nitrogen for bacteria, fungi and plants. It is recognized as a potential antimicrobial target with an impact on medicine, agriculture, and the environment. The list of possible urease inhibitors is continuously increasing, with a special interest in those that interact with and block the flexible active site flap. We show that disulfiram inhibits urease in Citrullus vulgaris (CVU), following a non-competitive mechanism, and may be one of this kind of inhibitors. Disulfiram is a well-known thiol reagent that has been approved by the FDA for treatment of chronic alcoholism. We also found that other thiol reactive compounds (l-captopril and Bithionol) and quercetin inhibits CVU. These inhibitors protect the enzyme against its full inactivation by the thiol-specific reagent Aldrithiol (2,2'-dipyridyl disulphide, DPS), suggesting that the three drugs bind to the same subsite. Enzyme kinetics, competing inhibition experiments, auto-fluorescence binding experiments, and docking suggest that the disulfiram reactive site is Cys592, which has been proposed as a "hinge" located in the flexible active site flap. This study presents the basis for the use of disulfiram as one potential inhibitor to control urease activity.

Chronic Psychological Distress as an Inducer of Microglial Activation and Leukocyte Recruitment into the Area Postrema.

BACKGROUND: Chronic psychological distress can cause neuroinflammation, but the involvement of leukocytes in this inflammatory response remains unclear. The area postrema (AP) is considered a neural-immune interface because it lacks a blood-brain barrier and a site for leukocyte recruitment in neuroinflammatory conditions induced by immunological insults, but its role in chronic psychological distress has not been explored. OBJECTIVE: To determine leukocyte recruitment to the AP after chronic psychological distress. METHODS: Rats were exposed to cat odor for 5 consecutive days to induce distress, and, on the 6th day, their brains were dissected to perform immunohistofluorescence studies of the AP. Immune cells were identified and quantified with CD45 and CD11b markers. The distribution of neurons and immune cells was determined using TrkA and CD45 markers, respectively. RESULTS: Distress induced a significant increase in CD45(+) and CD11b(+) cells in the AP. Three immunophenotypes were determined in the control and distress groups: CD45(+)/CD11b(-), CD45(+)/CD11b(+) and CD45(-)/CD11b(+). CD expression, morphology and fluorescence intensity enabled the identification of different immune cell types: starting from longitudinal ramified microglia (mainly in the control group) to amoeboid microglia, monocytes and lymphocytes (mostly in the distressed group). TrkA and CD45 expression in the AP revealed the proximity between soma neurons and leukocytes. Interestingly, some CD45(+) cells expressed TrkA, with increased expression in the distressed group. CONCLUSIONS: The identification of microglial activation, leukocyte recruitment and the close proximity between neurons and leukocytes in the AP after chronic psychological distress exposure suggests the AP as a site for distress-induced immune responses and engraftment of leukocytes infiltrating the CNS.

The three-dimensional structure of DapE from Enterococcus faecium reveals new insights into DapE/ArgE subfamily ligand specificity.

DapE is a Zn2+-metallohydrolase recognized as a drug target for bacterial control. It is a homodimer that requires the exchange of interface strands by an induced fit essential for catalysis. Identifying novel anti-DapE agents requires greater structural details. Most of the characterized DapEs are from the Gram-negative group. Here, two high-resolution DapE crystal structures from Enterococcus faecium are presented for the first time with novel aspects. A loosened enzyme intermediate between the open and closed conformations is observed. Substrates may bind to loose state, subsequently it closes, where hydrolysis occurs, and finally, the change to the open state leads to the release of the products. Mutation of His352 suggests a role, along with His194, in the oxyanion stabilization in the mono-metalated Zn2+ isoform, while in the di-metalated isoform, the metal center 2 complements it function. An aromatic-π box potentially involved in the interaction of DapE with other proteins, and a peptide flip could determine the specificity in the Gram-positive ArgE/DapE group. Finally, details of two extra-catalytic cavities whose geometry changes depending on the conformational state of the enzyme are presented. These cavities could be a target for developing non-competitive agents that trap the enzyme in an inactive state.

Clicking gallic acid into chitosan prolongs its antioxidant activity and produces intracellular Ca2+ responses in rat brain cells.

Gallic acid is a vegetable-derived and highly bioactive phenolic acid, but its antioxidant capacity is sensitive to environmental conditions. Chitosan is a biopolymer capable of exerting significant protection to various molecules, including phenolic compounds. A chitosan derivative that extends the antioxidant activity of gallic acid was synthesized by click chemistry and characterized by FT-IR, 1H NMR, and antioxidant capacity assays. Our results show that synthesized polymeric solutions and nanoparticles of N-(gallic acid) chitosan were both internalized by rat brain cells, processes that were modulated by extracellular Ca2+ and Na+. Their internalization was supported by dynamic light scattering and ζ-potential analyses, while Ca2+ imaging recordings performed in brain cells revealed the potential biological effect of N-(gallic acid) chitosan. We conclude that the synthesis of an N-(gallic acid) chitosan derivative through click chemistry is viable and may serve as strategy to prolong its antioxidant activity and to study its biological effects in vivo.

Phenolic compounds can induce systemic and central immunomodulation, which result in a neuroprotective effect.

Inflammation may negatively impact health, particularly that of the central nervous system. Phenolic compounds are bioactive molecules present in fruits and vegetables with potential anti-inflammatory effects. The purpose of the present work is to review the immunomodulatory bioactivities of phenolic compounds in the periphery and in the central nervous system. Results show that various types of phenolics are able to counter diet- or pathogen-induced systemic inflammation (among others) in various models. In vitro data show significant effects of flavonoids and phenolic acids in particular; similar bioactivities were reported in vivo, when administering them as pure compounds or from fruit and vegetable extracts that contain them. In the central nervous system, phenolics counter chronic inflammation and aggressive acute inflammatory processes, such as ischemic events, when administered preemptively and even therapeutically. We therefore conclude that the immunomodulatory potential of phenolic compounds can maintain an adequate immune response; their regular consumption should therefore be prioritized in order to maintain health. PRACTICAL APPLICATIONS: The immune response must be carefully regulated in order to avoid its deleterious effects. The present work highlights how phenolic compounds, dietary components ubiquitous in everyday diet, are able to maintain it within an adequate range. As humans are exposed to more proinflammatory stimuli (inadequate dietary pattern, mental stress, environmental pollution, chronic diseases, etc.), it becomes necessary to counter them, and consuming adequate amounts of foods that contain compounds with this ability is a rather simple strategy. Thus, the present work highlights how fruits and vegetables can help to maintain an adequate immune response that can preserve systemic health and that of the central nervous system. Furthermore, specific compounds contained in them can also be ideal candidates for additional in-depth studies, which can potentially lead to the development of potent, targeted, and safe anti-inflammatory molecules.

FDA-approved thiol-reacting drugs that potentially bind into the SARS-CoV-2 main protease, essential for viral replication.

Emergent novel SARS-CoV-2 is responsible for the current pandemic outbreak of severe acute respiratory syndrome with high mortality among the symptomatic population worldwide. Given the absence of a current vaccine or specific antiviral treatment, it is urgent to search for FDA-approved drugs that can potentially inhibit essential viral enzymes. The inhibition of 3CLpro has potential medical application, due to the fact that it is required for processing of the first translated replicase polyproteins into a series of native proteins, which are essential for viral replication in the host cell. We employed an in silico approach to test if disulfiram, as well as its metabolites, and captopril could be used as potential antiviral drugs against COVID-19. We provide data on the potential covalent interaction of disulfiram and its metabolites with the substrate binding subsite of 3CLpro and propose a possible mechanism for the irreversible protease inactivation thought the reaction of the aforementioned compounds with the Cys145. Although, captopril is shown to be a potential ligand of 3CLpro, it is not recommended anti-COVID-19 therapy, due to the fact that it can induce the expression of the viral cellular receptor such as, angiotensin-converting enzyme ACE-2, and thus, making the patient potentially more susceptible to infection. On the other hand, disulfiram, an alcoholism-averting drug, has been previously proposed as an antimicrobial and anti-SARS and MERS agent, safe to use even at higher doses with low side effects, it is recommended to be tested for control of SARS-CoV-2 infection.Communicated by Ramaswamy H. Sarma.

Liver proteome alterations in psychologically distressed rats and a nootropic drug.

BACKGROUND: Chronic psychological distress is considered today a pandemic due to the modern lifestyle and has been associated with various neurodegenerative, autoimmune, or systemic inflammation-related diseases. Stress is closely related to liver disease exacerbation through the high activity of the endocrine and autonomic nervous systems, and the connection between the development of these pathologies and the physiological effects induced by oxidative stress is not yet completely understood. The use of nootropics, as the cognitive enhancer and antioxidant piracetam, is attractive to repair the oxidative damage. A proteomic approach provides the possibility to obtain an in-depth comprehension of the affected cellular processes and the possible consequences for the body. Therefore, we considered to describe the effect of distress and piracetam on the liver proteome. METHODS: We used a murine model of psychological stress by predatory odor as a distress paradigm. Female Sprague-Dawley rats were distributed into four experimental groups (n = 6 - 7/group) and were exposed or not to the stressor for five days and treated or not with piracetam (600 mg/kg) for six days. We evaluated the liver proteome by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D-SDS-PAGE) followed by liquid chromatography-tandem mass spectrometry (GeLC-MS/MS). Besides, we analyzed the activity of liver antioxidant enzymes, the biochemical parameters in plasma and rat behavior. RESULTS: Our results showed that distress altered a wide range of proteins involved in amino acids metabolism, glucose, and fatty acid mobilization and degradation on the way to produce energy, protein folding, trafficking and degradation, redox metabolism, and its implications in the development of the non-alcoholic fatty liver disease (NAFLD). Piracetam reverted the changes in metabolism caused by distress exposure, and, under physiological conditions, it increased catabolism rate directed towards energy production. These results confirm the possible relationship between chronic psychological stress and the progression of NAFLD, as well as we newly evidenced the controversial beneficial effects of piracetam. Finally, we propose new distress biomarkers in the liver as the protein DJ-1 (PARK7), glutathione peroxidase 1 (GPX), peroxiredoxin-5 (PRDX5), glutaredoxin 5 (GLRX5), and thioredoxin reductase 1 (TXNDR1), and in plasma as biochemical parameters related to kidney function such as urea and blood urea nitrogen (BUN) levels.

Biointeraction of Erythrocyte Ghost Membranes with Gold Nanoparticles Fluorescents.

The application of new technologies for treatments against different diseases is increasingly innovative and effective. In the case of nanomedicine, the combination of nanoparticles with biological membranes consists of a "camouflage" technique, which improves biological interaction and minimizes the secondary effects caused by these remedies. In this work, gold nanoparticles synthesized by chemical reduction (Turkevich ≈13 nm) were conjugated with fluorescein isothiocyanate to amplify their optical properties. Fluorescent nanoparticles were deposited onto the surface of hemoglobin-free erythrocytes. Ghost erythrocytes were obtained from red blood cells by density gradient separation in a hypotonic medium and characterized with fluorescence, optical, and electron microscopy; the average size of erythrocyte ghosts was 9 µm. Results show that the functional groups of sodium citrate (COO-) and fluorophore (-N=C=S) adhere by electrostatic attraction to the surface of the hemoglobin-free erythrocyte membrane, forming the membrane-particle-fluorophore. These interactions can contribute to imaging applications, by increasing the sensitivity of measurement caused by surface plasmon resonance and fluorescence, in the context of biological membranes.

Psychological distress and lack of PINK1 promote bioenergetics alterations in peripheral blood mononuclear cells.

Psychological distress induces oxidative stress and alters mitochondrial metabolism in the nervous and immune systems. Psychological distress promotes alterations in brain metabolism and neurochemistry in wild-type (WT) rats in a similar manner as in Parkinsonian rats lacking endogenous PTEN-induced kinase 1 (PINK1), a serine/threonine kinase mutated in a recessive forms of Parkinson's disease. PINK1 has been extensively studied in the brain, but its physiological role in peripheral tissues and the extent to which it intersects with the neuroimmune axis is not clear. We surmised that PINK1 modulates the bioenergetics of peripheral blood mononuclear cells (PBMCs) under basal conditions or in situations that promote oxidative stress as psychological distress. By using an XF metabolic bioanalyzer, PINK1-KO-PBMCs showed significantly increased oxidative phosphorylation and basal glycolysis compared to WT cells and correlated with motor dysfunction. In addition, psychological distress enhanced the glycolytic capacity in PINK1-KO-PBMCs but not in WT-PBMCs. The level of antioxidant markers and brain-derived neurotrophic factor were altered in PINK1-KO-PBMCs and by psychological distress. In summary, our data suggest that PINK1 is critical for modulating the bioenergetics and antioxidant responses in PBMCs whereas lack of PINK1 upregulates compensatory glycolysis in response to oxidative stress induced by psychological distress.

Psychological Stress Phenocopies Brain Mitochondrial Dysfunction and Motor Deficits as Observed in a Parkinsonian Rat Model.

Psychological distress is a public health issue as it contributes to the development of human diseases including neuropathologies. Parkinson's disease (PD), a chronic, progressive neurodegenerative disorder, is caused by multiple factors including aging, mitochondrial dysfunction, and/or stressors. In PD, a substantial loss of substantia nigra (SN) neurons leads to rigid tremors, bradykinesia, and chronic fatigue. Several studies have reported that the hypothalamic-pituitary-adrenal (HPA) axis is altered in PD patients, leading to an increase level of cortisol which contributes to neurodegeneration and oxidative stress. We hypothesized that chronic psychological distress induces PD-like symptoms and promotes neurodegeneration in wild-type (WT) rats and exacerbates PD pathology in PINK1 knockout (KO) rats, a well-validated animal model of PD. We measured the bioenergetics profile (oxidative phosphorylation and glycolysis) in the brain by employing an XF24e Seahorse Extracellular Flux Analyzer in young rats subjected to predator-induced psychological distress. In addition, we analyzed anxiety-like behavior, motor function, expression of antioxidant enzymes, mitochondrial content, and neurotrophic factors brain-derived neurotrophic factor (BDNF) in the brain. Overall, we observed that psychological distress diminished up to 50% of mitochondrial respiration and glycolysis in the prefrontal cortex (PFC) derived from both WT and PINK1-KO rats. Mechanistically, the level of antioxidant proteins, mitochondrial content, and BDNF was significantly altered. Finally, psychological distress robustly induced anxiety and Parkinsonian symptoms in WT rats and accelerated certain symptoms of PD in PINK1-KO rats. For the first time, our collective data suggest that psychological distress can phenocopy several aspects of PD neuropathology, disrupt brain energy production, as well as induce ataxia-like behavior.

Maternal separation induces retinal and peripheral blood mononuclear cell alterations across the lifespan of female rats.

Early life stress alters the function and feedback regulation of the hypothalamic-pituitaryadrenal (HPA) axis, and can contribute to neuroinflammation and neurodegeneration by modifying peripheral blood mononuclear cell (PBMC) activity. The retina, as part of the nervous system, is sensitive to immune changes induced by stress. However, the consequences of stress experienced at an early age on retinal development have not yet been elucidated. Here we aimed to evaluate the impact of maternal separation (MatSep) across three stages of the lifespan (adolescent, adult, and aged) on the retina, as well as on progression through the cell cycle and mitochondrial activity in PBMCs from female Wistar rats. Newborn pups were separated from their mother from postnatal day (PND) 2 until PND 14 for 3 h/day. Retinal analysis from the MatSep groups showed architectural alterations such as a diminished thickness of retinal layers, as well as increased expression of proinflammatory markers DJ-1, Iba-1, and CD45 and the gliotic marker GFAP. Additionally, MatSep disrupted the cell cycle and caused long-term increases in mitochondrial activity in PBMCs from adolescent and adult rats. Changes in the cell cycle profile of the PBMCs from aged MatSep rats were undetected. However, these PBMCs exhibited increased sensitivity to H2O2-induced oxidative stress in vitro. Therefore, these results suggest that early life stress can have long-term effects on retinal structure and function, possibly elicited by neonatal immune preconditioning.

Interaction of N-succinyl-diaminopimelate desuccinylase with flavonoids.

DapE is an enzyme that belongs to the meso-diaminopimelate/Lysine pathway. It is recognized as an antimicrobial target, hence compounds that inhibit its catalytic activity are required. The principal features considered in the selection of potential inhibitors for this enzyme are compounds containing metal binding groups that could block access of the substrate to the Zinc metal centers and/or block the assembly of the oxyanion hole. We show the interaction of DapE from Enterococcus faecium, Staphylococcus aureus, Klebsiella aerogenes, Pseudomonas aeruginosa and Escherichia coli with flavonoids: quercetin, catechin, luteolin, rutin and hesperidin. Flavonoids contain several oxygen atoms distributed along their structure in a pattern that may be considered for the development of new antibiotics. Docking experiments suggest that these compounds containing metal binding groups that interact with metal centers of DapE and binding experiments indicate that glycoside flavonoids are preferred by DapE.

Descelularización y regeneración de tráquea porcina: un acercamiento a la ingeniería tisular / Decellularization and Regeneration of Porcine Trachea: an Approach to Tissue Engineering

Resumen Antecedentes: la ingeniería tisular permite obtener órganos como injertos a partir de tejidos descelularizados, regenerados con células autólogas. Objetivo: descelularizar y regenerar tráqueas porcinas. Material y métodos: se descelularizaron tráqueas porcinas colocándolas cada una en el epiplón de cuatro cerdos Yorkshire para su regeneración in vivo. Una tráquea desce-lularizada con tritón (DT), descelularizada con desoxicolato (DD), descelularizada con desoxicolato y reforzada con un polímero y células epiteliales (DDR), y una nativa crio-preservada (NC). Después de 8 días se obtuvieron la DD, NC y DDR; y al día 15, la DT. Se las evaluó mecánica e histológicamente, se realizó el análisis casuístico. Resultados: las tráqueas descelularizadas conservaron la integridad del cartílago, sin diferencias mecánicas, excepto la DDR con mayor rigidez. Las tráqueas regeneradas presentaron menor rigidez, excepto la DDR que además perdió el epitelio y la vascula-ridad. Las DT, DD mostraron epitelio no respiratorio, fibrosis y vasculogénesis con in-flamación. Conclusiones: las matrices conservaron sus características mecánicas. La regenera-ción in vivo ofrece ventajas como la esterilidad, interacción celular, nutrientes; es senci-llo, factible y económico, pero no hay control del crecimiento celular y vascularización, y los tejidos presentaron alteraciones mecánicas e histológicas. El polímero impidió la re-epitelialización y revascularización. Este estudio abre la posibilidad de mejorar las me-todologías de ingeniería tisular aplicadas al tejido traqueal.

Abstract Introduction: tissue engineering makes it possible to obtain organs as grafts from de-cellularized tissues, regenerated with autologous cells.Objective: decellularize and regenerate porcine tracheas.ARTÍCULO ORIGINAL | Respirar, 2023; 15(3): 188-199 | ISSN 2953-3414 | https://doi.org/10.55720/respirar.15.3.5RECIBIDO: 9 agosto 2023ACEP TADO: 31 agosto 2023 Elisa Barrera-Ramírezhttps://orcid.org/0000-0002-2778-0882Rubén Efraín Garrido-Cardonahttps://orcid.org/0000-0001-6083-5403Alejandro Martínez-Martínezhttps://orcid.org/0000-0003-3448-910XLuis Fernando Plenge-Tellecheahttps://orcid.org/0000-0002-1619-5004Edna Rico-Escobarhttps://orcid.org/0000-0002-0933-0220Esta revista está bajo una licencia de Creative Commons Reconocimiento 4.0 Internacional. Respirar 2023; 15 (3): 189ARTÍCULO ORIGINAL / E. Barrera-Ramírez, R.E. Garrido-Cardona, A. Martínez-Martínez, L.F. Plenge-Tellechea, E. Rico-EscobarDescelularización y regeneración de tráqueaISSN 2953-3414Materials and Methods: Porcine tracheas were decellularized by placing each one in the omentum of four Yorkshire pigs for regeneration in vivo. A trachea decellularized with triton (DT), decellularized with deoxycholate (DD), decellularized with deoxycho-late and reinforced with a polymer, and epithelial cells (DDR), and a cryopreserved na-tive (NC). After 8 days, the DD, NC and DDR were obtained; and on day 15, the DT. The evaluation was mechanically and histologically, performing the case analysis.Results: the decellularized tracheas preserved the integrity of the cartilage, with no me-chanical differences, except for the DDR with greater rigidity. The regenerated trache-as presented less rigidity, except the DDR, which also lost the epithelium and vascular-ity. The DT, DD showed non-respiratory epithelium, fibrosis and vasculogenesis with inflammation.Conclusions: the matrices retained their mechanical characteristics, in vivo regenera-tion offers advantages such as sterility, cell interaction, nutrients; it is simple, feasible and economical, but there is no control of cell growth and vascularization, and the tis-sues presented mechanical and histological alterations. The polymer prevented re-epi-thelialization and revascularization. This study opens the possibility of improving tissue engineering methodologies applied to tracheal tissue.

Blood mononuclear cells as speculum of emotional stress analyzed by synchrotron infrared spectroscopy and a nootropic drug.

Chronic psychological stress is an important public health issue which generates behavioral changes, anxiety, immunosuppression and oxidative damage. Piracetam is a cognitive enhancer, at cellular level it protects from oxidative stress. The aim of this study was to evaluate the effect of psychological stress and of piracetam on circulating mononuclear cells by analyzing the biochemical spectrome using Synchrotron Radiation Fourier Transform Infrared Microspectroscopy (SR-µFTIR). Rats were exposed for five days to a stressor (cat odor) under oral administration of piracetam (600 mg/kg). SR-µFTIR analysis showed a decrease in bands associated to the lipids region (2852 cm-1, 2923 cm-1 and 2962 cm-1) and an increase absorption of the amide I band (1654 cm-1) under stress conditions. The principal component analysis showed increase oxidation of lipids (decrease of 3010 cm-1, 2923 cm-1 and 2852 cm-1 bands) as well as proteins denaturation (increase of 1610 cm-1 and 1690 cm-1 bands) under stress. Piracetam provided protection to polyunsaturated lipids (p ≤ 0.001) and lipids/proteins ratio (p ≤ 0.001). Behaviorally, this drug diminished fear and anxiety in stressed animals by the plus maze test (p ≤ 0.002). However, this drug induced oxidative stress in mononuclear cells from unstressed animals and altered their behavior.

Toxicity of copper hydroxide nanoparticles, bulk copper hydroxide, and ionic copper to alfalfa plants: A spectroscopic and gene expression study.

Bulk Cu compounds such as Cu(OH)2 are extensively used as pesticides in agriculture. Recent investigations suggest that Cu-based nanomaterials can replace bulk materials reducing the environmental impacts of Cu. In this study, stress responses of alfalfa (Medicago sativa L.) seedlings to Cu(OH)2 nanoparticle or compounds were evaluated. Seeds were immersed in suspension/solutions of a Cu(OH)2 nanoform, bulk Cu(OH)2, CuSO4, and Cu(NO3)2 at 25 and 75 mg/L. Six days later, the germination, seedling growth, and the physiological and biochemical responses of sprouts were evaluated. All Cu treatments significantly reduced root elongation (average = 63%). The ionic compounds at 25 and 75 mg/L caused a reduction in all elements analyzed (Ca, K, Mg, P, Zn, and Mn), excepting for S, Fe and Mo. The bulk-Cu(OH)2 treatment reduced K (48%) and P (52%) at 75 mg/L, but increased Zn at 25 (18%) and 75 (21%) mg/L. The nano-Cu(OH)2 reduced K (46%) and P (48%) at 75 mg/L, and also P (37%) at 25 mg/L, compared with control. Confocal microscopy images showed that all Cu compounds, at 75 mg/L, significantly reduced nitric oxide, concurring with the reduction in root growth. Nano Cu(OH)2 at 25 mg/L upregulated the expression of the Cu/Zn superoxide dismutase gene (1.92-fold), while ionic treatments at 75 mg/L upregulated (∼10-fold) metallothionein (MT) transcripts. Results demonstrated that nano and bulk Cu(OH)2 compounds caused less physiological impairments in comparison to the ionic ones in alfalfa seedlings.

Lead toxicity in alfalfa plants exposed to phytohormones and ethylenediaminetetraacetic acid monitored by peroxidase, catalase, and amylase activities.

This manuscript describes the toxicity of lead in alfalfa plants treated with ethylenediaminetetraacetic acid (EDTA) and the phytohormones indole-3-acetic-acid (IAA), gibberellic acid (GA), and kinetin (KN), on catalase (CAT), ascorbate peroxidase (APOX), and total amylase activity (TAA). In all cases Pb was used at 40 mg/L; EDTA at 0.2 mM (equimolar to Pb); and IAA, GA, and KN at 1, 10, and 100 microM, respectively. An experiment containing Pb at 40 mg/L, 0.2 mM EDTA, and IAA and KN at 100 microM each was performed to determine changes in TAA. A control (plain nutrient solution) also was used for comparison. In all cases the treatments were performed in triplicate. Standard procedures were followed to determine the activity of the respective enzymes. After 10 d of exposure to the treatments, the leaves were harvested, homogenized, and centrifuged, and the supernatants were analyzed for CAT, APOX, and TAA. All determinations were performed in triplicate. The results demonstrated that CAT was reduced significantly (p < 0.05) by all treatments containing Pb, IAA, and GA at 10 and 100 microM. However, only the treatments Pb/EDTA/KN at 1, 10, and 100 microM reduced the APOX. The TAA in leaves of alfalfa plants was increased significantly (p < 0.05) by all treatments. Overall, the results suggest that the CAT tests showed no lead toxicity to the alfalfa seedlings. However IAA at 10 and 100 muM revealed toxicity to the CAT enzyme. In addition, the APOX tests exhibited no toxicity to the peroxidase enzyme with the exception of Pb/EDTA/KN treatments. Finally, the TAA tests showed high Pb/EDTA/phytohormone toxicity to the amylase enzyme in alfalfa seedlings.

Role of ethylenediaminetetraacetic acid on lead uptake and translocation by tumbleweed (salsola kali L.).

Tumbleweed plants (Salsola kali L.) grown in agar and liquid media demonstrated a high capacity to accumulate Pb in their different parts without affecting biomass. Whereas shoot elongation and biomass were not significantly affected by high tissue concentrations of Pb, root growth was significantly affected relative to controls. Roots, stems, and leaves demonstrated Pb concentrations of 31,000, 5,500, and 2,100 mg/kg dry weight, respectively, when plants were grown in the agar medium containing 80 mg Pb/L. Application of ethylenediaminetetraacetic acid (EDTA) to Pb-contaminated media dramatically reduced the total acquisition of Pb from both types of media. However, EDTA significantly increased the translocation of Pb from roots to the aerial parts, as evidenced by a multifold increase (23- and 155-fold for agar and liquid media, respectively) in the translocation concentration factor. The concentration of the antioxidant thiol compounds significantly increased (p < 0.05) in plants grown with uncomplexed Pb treatments relative to control plants. Scanning-electron microscopy and electron dispersive x-ray spectroscopic evaluation of leaf samples demonstrated an interesting pattern of Pb translocation in the presence or absence of EDTA. Large Pb crystals were found across the leaf tissues (palisade, spongy parenchyma, and conducting tissues) in the absence of EDTA. Lead nanoparticles also were seen when plants were grown in Pb-EDTA solution. Ultramicroscopic features of tumbleweed provide clear evidence for the unrestricted conduction of Pb from the root to the aerial parts, and this property makes the plant a good candidate for phytoremediation.

Biochemical and spectroscopic studies of the response of Convolvulus arvensis L. to chromium(III) and chromium(VI) stress.

The objective of the present study was to determine the oxidative stress caused by hexavalent chromium (Cr[VI]), the chromium (Cr) uptake, and the Cr speciation in Convolvulus arvensis L. plants grown in hydroponics media containing either Cr(VI) or Cr(III). The results demonstrated that C. arvensis plants exposed to Cr(VI) concentrations ranging from 0 to 40 mg/L expressed higher ascorbate peroxidase specific activity in roots than in shoots. On the other hand, catalase activity monitored in plants exposed to 2 mg/L of Cr(VI) for 24 h increased in roots after a few hours of exposure. However, catalase activity in shoots revealed a decrement almost immediately after treatment was initiated. The results from x-ray absorption spectroscopic studies indicated that the oxidation state of the supplied Cr(III) remained the same in plant tissues. The supplied Cr(VI), however, was reduced to the trivalent form in plant tissues. The results of inductively coupled plasma/optical emission spectroscopy demonstrated that after 5 d, the roots of plants exposed to 40 mg/L of Cr(III) or Cr(VI) accumulated approximately 25,000 and 3,500 mg/kg dry weight of Cr, respectively. Nevertheless, shoots concentrated 1,500 and 2,000 mg/kg dry weight of Cr from Cr(III) and Cr(VI), respectively, which indicated that Cr moved faster into C. arvensis plants when supplied as Cr(VI).

Investigations of Sulfur Chemical Status with Synchrotron Micro Focused X-ray fluorescence and X-ray Absorption Spectroscopy.

Sulfur (S) is an essential macronutrient for all living organisms. A variety of organic and inorganic S species with oxidation states ranging from -2 to +6 exist. Today few spectroscopic and biochemical methods are used to investigate sulfur oxidation state and reactivity in biological samples. X-ray absorption near edge spectroscopy (XANES) is a very well suited spectroscopic technique to probe the oxidation state and the surrounding chemical environment of sulfur. Microspectroscopy beamlines, operating at almost all synchrotron facilities, allow the combination of XANES with X-ray fluorescence mapping (µXRF). Using this approach distribution maps of S in complex biological samples (intact parts of tissue, or individual cells) can be obtained using µXRF and its oxidation state can be probed in-situ (µXANES). Moreover, µXRF mapping at specific energies enables for chemical contrast of S at different oxidation states without the need of staining chemicals. This review introduces the basic concepts of synchrotron µXRF and µXANES and discusses the most recent applications in life science. Important methodological and technical issues will be discussed and results obtained in different complex biological samples will be presented.