Ascorbic Reduction Research Articles

Argentophilic Hydrogen Ferrocyanide Electrodes for Robust Electrokinetic Flow in a Non-Gassing Pump

The present study reports a non-gassing and non-contaminating electrode material and its characterization for potential applications in a microfluidic device such as insulin pump. An electroosmotic pump was assembled by sandwiching a silica membrane between two flow-through argentophilic hydrogen ferrocyanide electrodes made up of ascorbate reduction of silver ferricyanide (cathode) and silver ferrocyanide (anode). This assembly generated a electroosmotic flux of 80 μL/min/cm2 with the application of 1 V across the cell. The flow increased linearly with the applied voltage. Passage of 0.7 C resulted 2.7 mL of water flow. This is equivalent to flow of 2.1 × 104 water molecules for the passage of each electron. This is the high performance electrode material reported for non-gassing, non-contaminating and low voltage electroosmotic pump in the literature with stall pressure of ∼2 kPa at 1 V applied voltage. The pump operates exclusively by electro-oxidation of hydrogen ferrocyanide at anode and electro-reduction of polymeric prussian blue analogue at cathode.

Functionalization of Silver Nanowires Surface using Ag-C Bonds in a Sequential Reductive Method.

Silver nanowires (Ag-NW) assembled in interdigitated webs have shown an applicative potential as transparent and conducting electrodes. However, upon integration in practical device designs, the presence of silver oxide, which instantaneously forms on the Ag-NW surfaces in ambient conditions, is unwanted. Here, we report on the functionalization of Ag-NWs with 4-nitrophenyl moieties through A-C bonds using a versatile two step reduction process, i.e., ascorbate reduction combined electrografting. We show that 40% of the Ag atop sites were terminated and provide high surface stability toward oxidation for more than 2 months while keeping the same intrinsic conductivity as in bulk silver.

On the Chemical Reduced Large Specific Surface Area Graphene Oxide and its Electrochemical Performances

Graphene oxide is prepared by modified Hummers method with the expanded graphite prepared from large flake graphite as raw material. The large tracts of graphene sheets prepared by ascorbic acid chemical reduction of graphite oxide are characterized by scanning electron microscope and X-ray diffraction. The electrochemical performances of graphene sheets are studied successively. The results show that large tracts of graphene sheets as an anode for lithium-ion batteries exhibits a high capacity of 1693 mAh·g-1 after initial discharge at a current density of 100 mA·g-1 and remains 426 mAh·g-1 after 100 cycles. The graphene sheets show good cycling stability even at high current density. The reversible specific capacities remains 218 mAh g-1 at the current densities of 1000 mA g-1 after 100 cycles.

Novel antioxidant capacity assay for lipophilic compounds using electron paramagnetic resonance spectroscopy.

A novel antioxidant capacity assay for lipophilic compounds was developed using electron paramagnetic resonance (EPR) spectroscopy. The assay is based on antioxidant’s scavenging ability against the tert-butoxyl radical generated photolytically from di-tert-butyl peroxide in ethyl acetate, and named the tert-butoxyl-based antioxidant capacity (BAC) assay. The radical was trapped by spin trap, 5,5-dimethyl-1-pyrroline-N-oxide, and EPR signal intensity of the spin adduct was used as a quantitative marker of radical levels. Signal intensity decreased in a dose-dependent manner in the presence of an antioxidant that competitively reacts with the radical, which was utilized to evaluate BAC values.The BAC method enabled the accurate estimation of antioxidant capacity for lipophilic materials that may counteract lipid peroxidation in biological membranes. The BAC values for quercetin and caffeic acid are 0.639 ± 0.020 and 0.118 ± 0.012 trolox equivalents, respectively, which are much smaller than values obtained by other aqueous methods such as H-ORAC and ORAC-EPR. Thus, antioxidants present in a non-aqueous environment should be evaluated using a non-aqueous system. In combination with in situ ascorbate reduction, the BAC method was capable of accurately determining the antioxidant capacity of water-insoluble materials that may be reduced in living cells.

Dissolved cobalt speciation and reactivity in the eastern tropical North Atlantic

Recent studies highlight the role of cobalt (Co) as an important micro-nutrient with a complex scavenged type oceanic distribution. To better understand the biogeochemical cycle of Co we investigate the distribution, speciation and reactivity of dissolved Co in the eastern tropical North Atlantic in the upper 800m of the water column. For this purpose, we complement classical Co ligand titrations that require a thermodynamic equilibrium with evaluations of ligand-exchange kinetics and reducibility of potential Co(III) species. The experiments include additions of the artificial Co binding ligands dimethylglyoxime or Nioxime and detection by cathodic stripping voltammetry. We find two pools of Co compounds: a labile fraction that exchanges Co within minutes and a strong/inert fraction that does not react within a 24-h period. No intermediate, slowly exchanging fraction is observed. Detection window experiments to determine complex stability constants show that the labile Co fraction is weak and likely consists of Co(II) complexes with no detectable free Co(II) ligands. The fraction of inert Co is always highest at the depth of the chlorophyll-a maximum. Addition of the reductant ascorbate increases the fraction of Co with rapid ligand-exchange kinetics and indicates the presence of dissolved reducible Co(III). The apparent Co(III) reducibility is highest at the chlorophyll-a maximum and decreases in deeper waters. Our results are in agreement with phytoplankton and associated bacteria being a source of Co(III) species, such as vitamin B12. The presented results have important implications for our understanding of the biological availability and the marine cycle of Co.

Photoprotection mechanism in the 'Fuji' apple peel at different levels of photooxidative sunburn.

The xanthophyll cycle, flavonoid metabolism, the antioxidant system and the production of active oxygen species were analyzed in the peel of 'Fuji' apples re-exposed to sunlight after extended periods of fruit bagging treatment, resulting in different levels of photooxidative sunburn. After re-exposing bagged fruits to sunlight, the production of active oxygen species and the photoprotective capacity in apple peels were both significantly enhanced. As sunburn severity increased, the concentration of hydrogen peroxide increased, while xanthophyll cycle pool size decreased. For the key genes involved in flavonoid synthesis, expressions of MdMYB10 and MdPAL were upregulated, whereas the expressions of MdCHS, MdANS, MdFLS and MdUFGT were downregulated in sunburnt fruit peel. Correspondingly, concentrations of both quercetin-3-glycoside and cyanidin-3-galactoside decreased. Total ascorbate concentrations decreased as sunburn severity increased, with the decrease being faster for oxidized than for reduced ascorbate. Transcription levels of MdGMP, MdGME, MdGGP, MdGPP, MdGalDH and MdGalLDH, the genes involved in ascorbate synthesis, were similar in non-sunburnt and sunburnt fruit peels, whereas activities of l-galactose dehydrogenase and l-galactono-1,4-lactone dehydrogenase decreased in severely sunburnt peel. Although activities of superoxide dismutase and ascorbate peroxidase increased, the activities of monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase decreased as sunburn severity increased. In summary, the occurrence of photooxidative sunburn in 'Fuji' apple peel is closely associated with a relatively lower xanthophyll cycle pool size, reduced levels of ascorbate reduction and synthesis and reduced flavonoid synthesis. Our data are consistent with the idea that ascorbate plays a key role in protecting apple fruit from photooxidative sunburn.

Effect of red and blue LED light irradiation on ascorbate content and expression of genes related to ascorbate metabolism in postharvest broccoli

The effects of red and blue light-emitting diode (LED) lights on the senescence of broccoli (Brassica oleracea L. var. italica) after harvest were investigated. The results showed that irradiation with red LED light was effective in delaying senescence in broccoli after harvest. Under red LED light, the yellowing process was delayed, and ethylene production and reduction of ascorbate (AsA) were suppressed in broccoli after harvest. In contrast, the blue LED light treatment did not significantly affect the senescence process of broccoli after harvest. As the red light is inconvenient for customers in selecting broccoli in the supermarket, we designed a type of modified white LED light. In this modified white LED light, the ratio of blue light was decreased, while the ratio of red light was increased. Under the modified white LED light, AsA reduction in broccoli was slightly delayed on the first and second days after harvest. Moreover, the modulation of AsA reduction by the modified white LED light treatment was highly regulated at the transcriptional level. The up-regulation of the AsA biosynthetic genes (BO-VTC2 and BO-GLDH) and AsA regeneration genes (BO-MDAR1 and BO-MDAR2) contributed to the higher AsA content in the modified white LED light treatment on the first and second days after harvest. The results presented might provide new strategies to improve the nutritional quality of broccoli after harvest.

Salicylic Acid-Mediated Alleviation of Cadmium Toxicity in Maize Leaves

The present study was carried out to investigate the effects of salicylic acid (SA) pretreatment on the activities of antioxidant enzymes and some biochemical attributes in maize (Zea mays L.) seedling leaves exposed to cadmium (Cd) stress. The Cd toxicity in maize leaves was revealed by reduction of ascorbate and cysteine concentrations. However, a remarkable increase of such non-enzymatic antioxidants concentrations was noticed on the pretreatment with SA. Cadmium- induced oxidative stress also showed a pronounced increase in hydrogen peroxide (H2O2), lipid peroxidation, electrolyte leakage (El) and proline production. The important point to be emphasized here is that the pretreatment with SA attenuated the adverse effects of Cd on these attributes. Cadmium-induced activities of some key antioxidant enzymes, peroxidase (POD) and ascorbate peroxidase (APX) was further increased on the exposure to SA. While the lower catalase (CAT) activity dues to Cd toxicity was increased by SA pretreatment.

Light-Triggered Modulation of Cellular Electrical Activity by Ruthenium Diimine Nanoswitches

Ruthenium diimine complexes have previously been used to facilitate light-activated electron transfer in the study of redox metalloproteins. Excitation at 488 nm leads to a photoexcited state, in which the complex can either accept or donate an electron, respectively, in the presence of a soluble sacrificial reductant or oxidant. Here, we describe a novel application of these complexes in mediating light-induced changes in cellular electrical activity. We demonstrate that RubpyC17 ([Ru(bpy)(2)(bpy-C17)](2+), where bpy is 2,2'-bipyridine and bpy-C17 is 2,2'-4-heptadecyl-4'-methyl-bipyridine), readily incorporates into the plasma membrane of cells, as evidenced by membrane-confined luminescence. Excitable cells incubated in RubpyC17 and then illuminated at 488 nm in the presence of the reductant ascorbate undergo membrane depolarization leading to firing of action potentials. In contrast, the same experiment performed with the oxidant ferricyanide, instead of ascorbate, leads to hyperpolarization. These experiments suggest that illumination of membrane-associated RubpyC17 in the presence of ascorbate alters the cell membrane potential by increasing the negative charge on the outer face of the cell membrane capacitor, effectively depolarizing the cell membrane. We rule out two alternative explanations for light-induced membrane potential changes, using patch clamp experiments: (1) light-induced direct interaction of RubpyC17 with ion channels and (2) light-induced membrane perforation. We show that incorporation of RubpyC17 into the plasma membrane of neuroendocrine cells enables light-induced secretion as monitored by amperometry. While the present work is focused on ruthenium diimine complexes, the findings point more generally to broader application of other transition metal complexes to mediate light-induced biological changes.

Transgenic Expression ofTobacco mosaic virusCapsid and Movement Proteins Modulate Plant Basal Defense and Biotic Stress Responses inNicotiana tabacum

Plant viruses cause metabolic and physiological changes associated with symptomatic disease phenotypes. Symptoms involve direct and indirect effects, which result in disruption of host physiology. We used transgenic tobacco expressing a variant of Tobacco mosaic virus (TMV) coat protein (CP(T42W)) or movement protein (MP), and a hybrid line (MP×CP(T42W)) that coexpresses both proteins, to study the plant response to individual viral proteins. Findings employing microarray analysis of MP×CP(T42W) plants and silenced mp×cp(T42W)* controls revealed that altered transcripts were mostly downregulated, suggesting a persistent shut-off due to MP×CP(T42W) expression. Next, we showed that MP triggered reactive oxygen species (ROS) accumulation, reduction of total ascorbate, and expression of ROS scavenging genes. These effects were enhanced when both proteins were coexpressed. MP and MP×CP(T42W) plants showed increased levels of salicylic acid (SA) and SA-responsive gene expression. Furthermore, these effects were partially reproduced in Nicotiana benthamiana when GMP1 transcript was silenced. CP(T42W) seems to be playing a negative role in the defense response by reducing the expression of PR-1 and RDR-1. MP and MP×CP(T42W) transgenic expression promoted a recovery-like phenotype in TMV RNA infections and enhanced susceptibility to Pseudomonas syringae and Sclerotinia sclerotiorum. The individual effects of viral proteins may reflect the ability of a virus to balance its own virulence.

Identification of protein nitrosothiols using phosphine-mediated selective reduction

Regulation of protein function by S-nitrosation of critical cysteines is known to be an important mechanism for nitric oxide signaling. Evidence for this comes from several different experimental approaches including the ascorbate-based biotin switch method. However technical problems with specificity and sensitivity of ascorbate reduction of S-nitrosothiols limit its usefulness and reliability. In the current study we report the use of triphenylphosphine ester derivatives to selectively reduce SNO bonds in proteins. After triphenylphosphine ester reduction, thiols were tagged with biotin or fluorescently labeled maleimide reagents. Importantly we demonstrate that these compounds are specific reductants of SNO in complex biological samples and do not reduce protein disulfides or protein thiols modified by hydrogen peroxide. Reduction proceeds efficiently in cell extracts and in whole fixed cells. Application of this approach allowed us to demonstrate S-nitrosation of specific cellular proteins, label S-nitrosoproteins in whole fixed cells (especially the nuclear compartment) and demonstrate S-nitrosoprotein formation in cells expressing inducible nitric oxide synthase.

Heterologous production and characterisation of two distinct dihaem-containing membrane integral cytochrome b561 enzymes from Arabidopsis thaliana in Pichia pastoris and Escherichia coli cells

Cytochrome (cyt) b561 proteins are dihaem-containing membrane proteins, belonging to the CYBASC (cytochrome-b561-ascorbate-reducible) family, and are proposed to be involved in ascorbate recycling and/or the facilitation of iron absorption. Here, we present the heterologous production of two cyt b561 paralogs from Arabidopsis thaliana (Acytb561-A, Acytb561-B) in Escherichia coli and Pichia pastoris, their purification, and initial characterisation. Spectra indicated that Acytb561-A resembles the best characterised member of the CYBASC family, the cytochrome b561 from adrenomedullary chromaffin vesicles, and that Acytb561-B is atypical compared to other CYBASC proteins. Haem oxidation–reduction midpoint potential (EM) values were found to be fully consistent with ascorbate oxidation activities and Fe3+-chelates reductase activities. The ascorbate dependent reduction and protein stability of both paralogs were found to be sensitive to alkaline pH values as reported for the cytochrome b561 from chromaffin vesicles. For both paralogs, ascorbate-dependent reduction was inhibited and the low-potential haem EM values were affected significantly by incubation with diethyl pyrocarbonate (DEPC) in the absence of ascorbate. Modification with DEPC in the presence of ascorbate left the haem EM values unaltered compared to the unmodified proteins. However, ascorbate reduction was inhibited. We concluded that the ascorbate-binding site is located near the low-potential haem with the Fe3+-chelates reduction-site close to the high-potential haem. Furthermore, inhibition of ascorbate oxidation by DEPC treatment occurs not only by lowering the haem EM values but also by an additional modification affecting ascorbate binding and/or electron transfer. Analytical gel filtration experiments suggest that both cyt b561 paralogs exist as homodimers.

Endo-inulinase Stabilization by Pyridoxal Phosphate Modification: A Kinetics, Thermodynamics, and Simulation Approach

The structural and storage and functional thermostabilization of endo-inulinase (EC 3.2.1.7) through semi-rational modification of surface accessible lysine residues by pyridoxal-5'-phosphate (PLP) and ascorbate reduction have been explored. Improved stability was observed on modifications in the absence or presence of inulin, which indicates storage or functional thermostabilization, respectively. Comparisons have been made between non-modified and modified enzyme by the determination of Tm as an indicator of structural stability, temperature-dependent half-lives (t1/2), energy barrier of the inactivation process, and thermodynamic parameters (ΔH, ΔG, and ΔS) in a storage thermostability approach. These parameters coincided well with the observed stabilization of the engineered enzyme. Moreover, relative activities with sucrose and inulin were determined for non-modified and modified endo-inulinases at different temperatures. A comparison of the sucrose-to-inulin ratios of the initial rate of hydrolysis as an indicator of substrate specificity revealed about twofold improvement in inulinase versus sucrose activity by enzyme modification. Molecular dynamics simulations and molecular docking approaches were employed to explain the observed structural and functional thermostabilization of endo-inulinase upon modification. We hypothesize the establishment of intramolecular interactions between the covalently attached PLP-Lys381 and Arg526 and Ser376 residues as a representative of modification-originated intramolecular contacts in the modified enzyme.

Ru binding to RNA following treatment with the antimetastatic prodrug NAMI-A in Saccharomyces cerevisiae and in vitro

[ImH][trans-Ru(III)Cl(4)(DMSO)(Im)] (where DMSO is dimethyl sulfoxide and Im is imidazole) (NAMI-A) is an antimetastatic prodrug currently in phase II clinical trials. The mechanisms of action of this and related Ru-based anticancer agents are not well understood, but several cellular targets have been suggested. Although Ru has been observed to bind to DNA following in vitro NAMI-A exposure, little is known about Ru-DNA interactions in vivo and even less is known about how this or related metallodrugs might influence cellular RNA. In this study, Ru accumulation in cellular RNA was measured following treatment of Saccharomyces cerevisiae with NAMI-A. Drug-dependent growth and cell viability indicate relatively high tolerance, with approximately 40% cell death occurring at 6h for 450μM NAMI-A. Significant dose-dependent accumulation of Ru in cellular RNA was observed by inductively coupled plasma mass spectrometry measurements on RNA extracted from yeast treated with NAMI-A. In vitro, binding of Ru species to drug-treated model DNA and RNA oligonucleotides at pH 6.0 and 7.4 was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in the presence and absence of the reductant ascorbate. The extent of Ru-nucleotide interactions increases slightly with lower pH and significantly in the presence of ascorbate, with differences in observed species distribution. Taken together, these studies demonstrate the accumulation of aquated and reduced derivatives of NAMI-A on RNA in vitro and in cellulo, and enhanced binding with nucleic acid targets in a tumorlike acidic, reducing environment. To our knowledge, this is also the first study to characterize NAMI-A treatment of S. cerevisiae, a genetically tractable model organism.

Site-Mapping of In Vitro S-nitrosation in Cardiac Mitochondria: Implications for Cardioprotection

S-nitrosation (SNO) of mitochondrial protein cysteines can be cardioprotective. Several targets have been implicated, yet the scope and identification of specific residues has not been fully assessed. To address this, a comprehensive assessment of mitochondrial SNO-modifiable cysteines was performed to determine nitric oxide (NO) susceptible pathways and identify novel mechanisms of oxidative cardioprotection. The biotin switch assay and mass spectrometry were used on rat cardiac mitochondrial lysates treated with the nitric oxide donor, S-nitrosoglutathione, and controls (n=3) to map 83 SNO-modified cysteine residues on 60 proteins. Of these, three sites have been reported, 30 sites are new to 21 proteins previously known to be S-nitrosated but which lacked site-specific information and 50 sites were found on 39 proteins not previously implicated in SNO pathways. The SNO-modifications occurred in only a subset of available cysteines, indicating a specific targeted effect. Functional annotation and site-specificity analysis revealed a twofold greater nitric oxide-susceptibility for proteins involved in transport; including regulators of mitochondrial permeability transition suggesting SNO-regulation and a possible protective mechanism. Additionally, we identified many novel SNO-modified proteins with cardioprotective potential involved in the electron transport chain, tricarboxylic acid cycle, oxidative stress defense, fatty acid and amino acid metabolism. These findings suggest that SNO-modification may represent a novel mechanism for the regulation of oxidative phosphorylation and/or cell death. S-nitrosation of mitochondrial permeability transition-associated proteins represents an intriguing potential link to cardioprotection.

A Strong Protective Action of Guttiferone-A, a Naturally Occurring Prenylated Benzophenone, Against Iron-Induced Neuronal Cell Damage

Guttiferone-A (GA) is a natural occurring polyisoprenylated benzophenone with several reported pharmacological actions. We have assessed the protective action of GA on iron-induced neuronal cell damage by employing the PC12 cell line and primary culture of rat cortical neurons (PCRCN). A strong protection by GA, assessed by the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carbox-anilide (XTT) assay, was revealed, with IC(50) values <1 µM. GA also inhibited Fe(3+)-ascorbate reduction, iron-induced oxidative degradation of 2-deoxiribose, and iron-induced lipid peroxidation in rat brain homogenate, as well as stimulated oxygen consumption by Fe(2+) autoxidation. Absorption spectra and cyclic voltammograms of GA-Fe(2+)/Fe(3+) complexes suggest the formation of a transient charge transfer complex between Fe(2+) and GA, accelerating Fe(2+) oxidation. The more stable Fe(3+) complex with GA would be unable to participate in Fenton-Haber Weiss-type reactions and the propagation phase of lipid peroxidation. The results show a potential of GA against neuronal diseases associated with iron-induced oxidative stress.

Characterization of 2′-deoxyguanosine oxidation products observed in the Fenton-like system Cu(ii)/H2O2/reductant in nucleoside and oligodeoxynucleotide contexts

Reactive oxygen species attack both base and sugar moieties in DNA with a preference among the bases for reaction at guanine. In the present study, 2'-deoxyguanosine (dG) was oxidized by a copper-mediated Fenton reaction with the reductants ascorbate or N-acetyl-cysteine, yielding oxidation on both the base and the sugar. The primary oxidized lesions observed in these studies include the 2'-deoxyribonucleosides of 8-oxo-7,8-dihydroguanosine (dOG), spiroiminodihydantoin (dSp), guanidinohydantoin (dGh), oxazolone (dZ), and 5-carboxamido-5-formamido-2-iminohydantoin (d2Ih), as well as the free base guanine. d2Ih was the major product observed in the nucleoside, single- and double-stranded oligodeoxynucleotide contexts and is proposed to arise from oxidation at C5 of guanine. Product distribution studies provide insight into the role of the reductant in partitioning of dG base oxidation along the C5 and C8 pathways.

Role of quinones in the ascorbate reduction rates of S-nitrosoglutathione

Quinones are one of the largest classes of antitumor agents approved for clinical use, and several antitumor quinones are in various stages of clinical and preclinical development. Many of these are metabolites of, or are, environmental toxins. Because of their chemical structure they are known to enhance electron transfer processes such as ascorbate oxidation and NO reduction. The paraquinones 2,6-dimethyl-1,4-benzoquinone (DMBQ), 1,4-benzoquinone, methyl-1,4-benzoquinone, 2,6-dimethoxy-1,4-benzoquinone, 2-hydroxymethyl-6-methoxy-1,4-benzoquinone, trimethyl-1,4-benzoquinone, tetramethyl-1,4-benzoquinone, and 2,3-dimethoxy-5-methyl-1,4-benzoquinone; the paranaphthoquinones 1,4-naphthoquinone, menadione, 1,4-naphthoquinone-2-sulfonate, 2-ethylsulfanyl-3-methyl-1,4-naphthoquinone and juglone; and phenanthraquinone (PHQ) all enhance the anaerobic rate of ascorbate reduction of GSNO to produce NO and GSH. Rates of this reaction were much larger for p-benzoquinones and PHQ than for p-naphthoquinone derivatives with similar one-electron redox potentials. The quinone DMBQ also enhances the rate of NO production from S-nitrosylated bovine serum albumin upon ascorbate reduction. Density functional theory calculations suggest that stronger interactions between p-benzo- or phenanthrasemiquinones and GSNO than between p-naphthosemiquinones and GSNO are the major causes of these differences. Thus, quinones, and especially p-quinones and PHQ, could act as enhancers of NO release from GSNO in biomedical systems in the presence of ascorbate. Because quinones are exogenous toxins that could enter the human body via a chemotherapeutic application or as an environmental contaminant, they could boost the release of NO from S-nitrosothiol storages in the body in the presence of ascorbate and thus enhance the responses elicited by a sudden increase in NO levels.

Hydrous Manganese Oxide Doped Gel Probe Sampler for Measuring In Situ Reductive Dissolution Rates. 1. Laboratory Development

Reductive dissolution of redox-sensitive minerals such as manganese (Mn) oxides in natural sediments is an important mechanism for trace element mobilization into groundwater. A gel probe sampler has been constructed to study in situ reductive dissolution of Mn oxides. The gel consists of a polyacrylamide polymer matrix doped with hydrous Mn oxide (HMO). Gel slabs are mounted into a probe, which is designed to be inserted into the sediments. The amount of Mn released from the gel by reductive dissolution is determined by comparing the amount of Mn initially embedded into the gel with the amount remaining in the gel after exposure to conditions in the sediments or, in laboratory studies, to reducing agents. In this laboratory study, the performance of the gel probes was examined using the model reductant ascorbate and the Mn-reducing bacteria Shewanella oneidensis strain MR-1. In addition, a 1-D model was used to relate the reaction rates observed for HMO embedded in gels to those for HMO in suspension. One limitation of the HMO-doped gels for assessing microbial reduction rates is that the gels prevent direct contact between the microbes and the HMO and hence preclude enzymatic reduction at the cell surface. Nonetheless, the HMO-doped gel probes offer the possibility to establish a lower bound for Mn-reduction capacity in sediments.

Estrogen Induced Uterine Artery Endothelial Nitrosyl-Proteome: Potential Roles of Protein Nitrosylation in Uterine Blood Flow Regulation.

Estrogen is one of the major driving forces for dilating uterine arteries to facilitate the dramatic rises in uterine blood flow that parallels the exponential growth phase of fetal weight during late gestation. Estrogen-induced and pregnancy-associated uterine vasodilatations occur via enhanced endothelial nitric oxide (NO) synthase (eNOS) expression/activation thereby increasing NO production locally at the level of the uterine artery. However, it is unknown whether increased NO production by estrogen stimulation directly affects proteins and their functions in uterine arteries. Formation of S-nitrothiols via addition of NO-derived nitrosyl groups to cysteines regulates the function of a plethora of proteins. This post-translational protein modification has been named as S-nitrosylation with biological significance analogous to phosphorylation. To determine the effects of exogenous estrogen and NO treatments on protein nitrosylation and to profile the estrogen-induced nitrosyl-proteome in uterine artery endothelial cells. Primary cultured sheep uterine artery endothelial cells were treated with or without etradiol-17β (E2β, 10 nM) or a NO donor S-nitrosoglutathione (GSNO, 1 mM) for 30 min. Protein nitrosylation was measured by a modified 3-step biotin switch technique including blocking free thiols in the nitrosyl-proteomes of total protein samples by methylmethanethiosulfonate, followed by ascorbate reduction and labeling with the CyDye fluorescence tags. E2β or GSNO treated samples (Cy5 labeled, red) were mixed with equal amounts of control sample (Cy3 labeled, green) and then analyzed by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). High resolution digitalized fluorescence images of the 2D-DIGE were captured and changes in densities of nitrosyl-proteins by E2α or GSNO were quantified off-line. Protein spots of interest (based on increases or decreases in intensity) on the 2D-DIGE were picked up and identified by matrix-assisted laser desorption/ionization-time of flight (MALDI/TOF) mass spectrometry. There were >1000 nitrosylated proteins identified on 2D-DIGE. Among those, at least 25 proteins were significantly stimulated by E2β or GSNO. Interestingly, many proteins were also unexpectedly denitrosylated by estrogen or GSNO. With MALDI-TOF mass spectrometry we sequenced 100 nitrosyl-proteins of interest. We found that E2β and GSNO stimulated significantly different nitrosyl-proteomes. Some proteins were nitrosylated or denitrosylated by both E2β and GSNO, but others were only affected by one or the other. Pathway analysis of the identified nitrosyl-proteins suggested that protein nitrosylation is clearly involved in estrogen regulation of protein translation and degradation, mitochondrial redox balance, cell structure and metabolism, and ion homeostasis, etc. Our data showing estrogen stimulation of protein nitrosylation in uterine artery endothelial cells signifies a significant next step for the understanding of the biological targets of enhanced NO production by estrogen in uterine arteries that are associated with the regulation of uterine blood flow. The physiological consequences of nitrosylated/denitrosylated proteins by estrogen in uterine artery endothelial cells warrants for further investigation (NIH HL70562, HL74947 HL49210, HD38843, HL87144 and HL86939). (platform)